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THE INDIAN JOURNAL OF VETERINARY SCIENCES AND BIOTECHNOLOGY Vol. 14 No. 2 Oct. - Dec. 2018 NAAS Rating : 4.47 ISSN : 2394 - 0247 Print and online ISSN 2395- 1176 EDITOR-IN-CHIEF Dr. R.S. Dhanotiya, Professor of Veterinary Biochemistry (Rtd) College of Veterinary Science & A.H. Mhow EDITORIAL BOARD Dr. A.J. Dhami Prof. & Head, AGRO, Vet. College, AAU, Anand ajdhami@aau.in Dr. Mahesh Chander Professor, Division of Veterinary Extension IVRI, Izatnagar drmahesh.chander@gmail.com Dr. K.M. Panchal Prof & Head, Anatomy, Vet. College, AAU, Anand kmpanchal@aau.in Dr. B.P. Brahmkshatri Prof. & Head, AGB/Biotech, Vet. College, NAU, Navsari bpbkhatri@gmail.com; bpb@nau.in Dr. M.K. Awasthi Prof. Gynaec, Vet. College, Anjora, Durg (CG) awasthimk1963@rediffmail.com Dr. R.K. Bagherwal Prof. Medicine, Vet. College Mhow rkb_vet@yahoo.com Dr. S.K. Jhala Assoc. Prof Surgery, Vet. College, Navsari drshivrajjhala@gmail.com Dr. Vishal S. Suthar Asstt. Prof. Gynaecology, Kandhenu University, Gandhinagar, vsuthar28@gmail.com Dr. A.C. Patel Assoc. Prof. AGB, Vet. College, AAU, Anand aashishvet@gmail.com Dr. S.K. Maiti Prof. & Head, TVCC, College of Veterinary Science & AH Anjora, Durg. maitisushil_kumar@rediffmail.com Dr. Amit Kumar Associate Professor, DUVASU, Mathura balan74@gmail.com Dr. M. Chellapandian Veterinary College & Research Institute, Tirnelveli mcpandian69@gmail.com Dr. B.C. Das Vet. and AH Extension, College of Vet. Sc. and AH OUAT, Bhubneswar drbdas1@rediffmail.com Dr. Alok Kumar Dixit Department of Veterinary Parasitology College of Veterinary Science & A. H., Rewa (M.P.). alokdixit7@yahoo.com Dr. D.J. Ghodasara Prof. Pathology, Vet. College, AAU, Anand dinghodasara@gmail.com Dr. S. Selvaraju ICAR-National Fellow & Principal Scientist, NIANP, Bangalore selvarajuars@gmail.com Dr. U.V. Ramani Asstt. Prof. Biotechnology, Vet. College, Navsari uvramani@nau.in Dr. K.S. Christi Sr. Lecturer, School of Biological and Chemical Sciences, Fiji Island ketan.christi@usp.ac.fj; Christi_k@usp.ac.fj Dr. Bharat B. Bhanderi Asstt. Pof. Microbiology, Vet. College, AAU, Anand bbbhanderi@gmail.com Dr. Vipin Gupta Assistant Professor, Public Health, Vet. College, Mhow drvipin80@gmail.com Prakash Kumar Rathod Department of Extension, College of Veterinary, Sci.& A.H. Bider prakashkumarkr@gmail.com Dr. D.R. Barvalia Vet Practitioner, Ontario, Canada barvalia@gmail.com Dr. Shiv Kumar Sharma Associate Professor, Vet. Medicine, RAJUVAS, Udaipur, Rajasthan drshivsharmavet@rediffmail.com All Correspondence Regarding THE INDIAN JOURNAL OF VETERINARY SCIENCES AND BIOTECHNOLOGY should be made with the Editor-in-Chief only. Note : Neither the Editorial Board nor the publisher in any way is responsible for the views and data in the contributed papers individually or collectively. Online Publication : www.myresearchjournals.com website : www.ijvsbt.org THE INDIAN JOURNAL OF VETERINARY SCIENCES & BIOTECHNOLOGY Vol. 14 No. 2 Oct. - Dec. 2018 ISSN : 2394 - 0247 Print and Online ISSN 2395 – 1176 Contents ❖ Seminal Attributes, Freezability and their Interrelationships in Zebu Cattle and Buffalo Bulls from Central Gujarat P.K. Pathak, A.J. Dhami and D.V. Chaudhari 01-08 ❖ Study on Blood Biochemical Profile in Relation to Age and Scrotal Biometry in Adolescent Surti Bucks D.V. Chaudhari, A.J. Dhami, C.P. Parmar, J.A. Patel and M.M. Pathan 09-13 ❖ Gross Anatomy of Cerebral Ventricles and Septum Pellucidum of Brain of Surti Buffalo (Bubalus bubalis) Alka Suman and Sweta Pandya 14-17 ❖ Stress enzyme Level during different seasons in Pandharpuri Buffalo R.B. Ambade, S.H. Dalvi, M.M. Gatne, V.D. Dighe, A.Y. Doiphode, and B.N. Ramteke 18-21 ❖ Histomorphological and Histochemical Studies Shabir Ahmad Malik, Rajesh Rajput, 22-27 Mohd Rafiq, Uiase Bin Farooq and on Esophagus in Gaddi Sheep (Ovis aries) Harishbhai Gori ❖ Studies on Period of Oviposition and Hatching of Eggs in Hyalomma anatolicum anatolicum L. Prasad, R.K. Bagherwal, A.K. Jayraw, N. Rajput, N. Yadav, Veena and P. Thakur 28-29 ❖ Biochemical alterations in Horses Infected with Theileria equi T.M. Vidhyalakshmi, S.K. Raval, P.V. Parikh and P.V. Patel 30-33 ❖ Effect of Supplementation of Monensin Prathviraj. Shrikant Kulkarni, Sodium on Rumen Metabolism and Milk Yield N.M. Soren, K.B. Sathisha, in Early Lactating Buffalo (Bubalus Bubalus) Srinivas Reddy Bellur, Prashant Bellundagi and B. Ramachandra 34-38 ❖ Ultrasonographic Fetometry and Fetal Structures during Early Pregnancy in Surti Buffaloes Mitesh Gaur and G.N. Purohit 39-43 ❖ MTNR-1A Gene Polymorphism in Two Tropical Sheep breeds of South India C.R. Vibha, Nagaraja Ramakrishnappa, 44-48 G.S. Naveen Kumar, R. Jayashree, B.M. Ravindranath ❖ Comparative evaluation of Egg Quality Parameters of Kadaknath and Vanaraja Chicken in Intensive Farming System S.S. Pathak, U.Tamuli, S. Khargharia, 49-51 G. Bordoloi, L. S. Khuman, P. Chabukdahara, A. Gogoi, P. Rajbongshi, K. Saikia, K. Boruah THE INDIAN JOURNAL OF VETERINARY SCIENCES AND BIOTECHNOLOGY Vol. 14 No. 2 - Contents ❖ Pathological Studies on Acute Marek’s Disease in Native and Crossbred Chickens of Organized Poultry Farm Kinnari R. Makwana, B.P. Joshi, D.J. Ghodasara, C.J. Dave, Monika P. Patel 52-57 ❖ In vitro efficacy of Cypermethrin against Hyalomma anatolicum anatolicum L. Prasad, R.K. Bagherwal, A.K. Jayraw, N. Rajput, N. Yadav, M. Shakya and P. Thakur 58-59 ❖ Effect of Garlic (Allium sativum) Extract on Recovery and Conception Rate in Infectious Repeat Breeder Crossbred Cows Anavil Bhardwaz, S.P. Nema, S.S. Mahour, Daljeet Chabbra, N. Rajput and K. Sudarshan 60-63 ❖ Osteodystrophia Fibrosa in a Goat A case report Tejaswini K.G, Usha Narayana Pillai, 64-65 Madhavan Unny N. and Sudheesh S. Nair ❖ Late-Term Abortion and Retention of Placenta in Donkey (Equus acinus): A Report of Two Cases S.C. Parmar, J.A. Patel, D.V. Chaudhari, K.K. Hadiya and A.J. Dhami 66-68 ❖ Enamel Hypoplasia In Canine Distemper Recovered Pup : A Case Report V. Vijayanand, M. Shiju Simon, A. Methai, S. Kavitha and T. Sathiamoorthy 69-70 ❖ Therapeutic Management of Renal Dysfunction Associated with Trypanosomiasis in a Horse - A Case Report T.M. Vidhyalakshmi, S.K. Raval, G.M. Akshatha, P.V. Parikh and J.M. Kathri 71-73 ❖ Cerebral Babesiosis in a Gir Bullock and its Successful Therapeutic Management J.B. Solanki, B.J. Thakre, N. Kumar, D.C. Patel and Y.G. Patel 74-75 ❖ Hydrocephalic Monster in a non Descript Buffalo A.D. Patil, C.K. Lakde, S.K. Sahatpure 76-77 Chandini and Kamaljit ❖ Prevalence of Bunostomum trigonocephalum infection in Sheep and Goats in Madhya Pradesh Ravi Kumar Khare, A.K. Dixit, R. Kumar, G. Das, D. Bhinsara, Subhamoy Ghosh, Rohit Singh, D. Chandra, M. Sankar 78-81 The Indian Journal of Veterinary Sciences & Biotechnology (2018) Volume 14, Issue 2, 01-08 ISSN (Print) : 2394-0247 : ISSN (Print and online) : 2395-1176, abbreviated as IJVSBT 10.21887/ijvsbt.14.2.1 Seminal Attributes, Freezability and their Interrelationships in Zebu Cattle and Buffalo Bulls from Central Gujarat P.K. Pathak, A.J. Dhami* and D.V. Chaudhari Department of Veterinary Gynaecology & Obstetrics College of Veterinary Science & Animal Husbandry Anand Agricultural University, Anand-388 001, Gujarat, India Abstract Publication Info Article history: Received : 22-08-2018 Accepted : 20-09-2018 Published : 17-10-2018 Key Words: Gir, Surti, Murrah, Seminal attributes, Freezability, Interrelationships. *Corresponding author: ajdhami@aau.in This work is licensed under the Creative Commons Attribution International License (http:// creativecommons.org/licenses / by/4.0/P), which permits unrestricted use, distribution and reproduction in any medium, provided the original work is properly cited. Copyright @: 2018 by authors and SVSBT. A study was carried out on nine healthy mature breeding bulls (3 each of Gir, Surti and Murrah breed) to evaluate their fresh and frozen semen quality and their interrelationships. The ejaculates immediately after collection were evaluated for routine physicomorphological attributes, including HOS test. The ejaculates (n=72) having >75% initial motility were diluted @ 80 million sperm/ml using TFYG extender and the French mini straws filled were frozen in liquid nitrogen vapour using a programmable biofreezer. Thawing of straws was done at 37°C for 30 sec and assessed for freezability by conventional technique. All the cattle and buffalo bulls donated consistently normal thick creamy yellow and thick milky white semen, respectively. In Gir, Suti and Murrah bulls (n=24 ejaculate each) the seminal attributes such as ejaculate volume (6.69±0.17, 3.12±0.10 and 3.96±0.16 ml, p<0.01); initial motility (80.21±0.88, 84.58±0.60 and 84.38±0.76 %, p<0.01); total sperm output/ejaculate (9013.85±265.32, 3935.49±259.63 and 5366.48±332.99 million, p<0.01) and live sperm (84.71±0.83, 86.17±0.78 and 86.79±0.79 %, p<0.05) differed significantly. The mean percentages of post-thaw motile sperm (53.29±1.56, 58.33±1.43 and 59.58±1.20, p<0.01); live sperm (59.00±1.95, 67.00±1.59 and 68.42±1.66 %, p<0.01); and HOS reactive sperm (48.25±0.78, 44.21±1.29 and 51.54±1.29 %, p<0.01) in Gir, Surti and Murrah bulls semen also differed significantly. The variation among the bulls was significant for buffalo breeds in most of their fresh seminal attributes, except HOST, and for post-thaw motility, but not among Gir bulls. The important seminal attributes like motility, live sperm and HOS reactive sperm of fresh and frozen-thawed semen were significantly and positively interrelated in all three breeds of bulls (r = 0.40 to 0.81, p<0.05 to 0.01), suggesting that motility and HOST of fresh semen were good predictors of freezability of bovine semen. Introduction Male fertility is an important factor in bovine reproduction, since a single bull is generally bred Indian J. Vet Sci. Biotech (2018) Vol. 14 No. 2 to numerous cows, particularly through AI. Therefore semen analysis is the most valuable diagnostic tool to evaluate male fertility potential (Patel et al., 2012). However, no single test or 1 combinations of tests have been proved to be totally reliable for accurate prediction of semen quality in relation to fertility. Most of the tests that are used for evaluation of semen are based on physical characteristics of spermatozoa. The correlations of these physical attributes with fertility are highly variable and relatively poor (Dhami et al., 1990; Shelke and Dhami, 2001; Tiwari et al., 2009). There are numerous factors that may affect the motility, plasma membrane integrity, morphology and viability of semen. Hypo-osmotic swelling (HOS) test is used to evaluate sperm plasma membrane integrity as in vitro fertility test as it is of fundamental importance in the fertilization process (Jeyendran et al., 1984; Lodhi et al., 2008). The evaluation of interrelationships of spermatozoal attributes of fresh and cryopreserved bovine semen would help to select a few most valid simple traits of fresh semen to predict freezability and even fertility of such ejaculates, instead of going through a plethora of time consuming unpredictable cumbersome tests. Hence, this study was planned to evaluate the comparative physico-morphological and functional attributes of fresh and frozen-thawed semen of cattle and buffalo bulls and their interrelationships in order to select the test(s) that are predictive of semen freezability. Materials and Methods This investigation included nine healthy mature breeding bulls (3 each of Gir, Surti and Murrah breed), aged 5-8 years, stationed at Sperm Station of the College of Veterinary Science, AAU, Anand-388 001 during September 2017 to May 2018. All these bulls were in good health and under optimal veterinary care. They were maintained in nearly identical nutritional and managerial conditions throughout the period of study with twice a week semen collection schedule. Semen was collected using artificial vagina from each bull in the morning hours between 7.30 and 8.30 am over a dummy buffalo bull. Immediately after collection, the tubes containing semen were placed in a water-bath maintained at 34ºC and evaluated for various physico-morphological attributes (Salisbury et al., 1978) including hypo-osmotic swelling (HOS) test (Jeyendran et al., 1984). In all, 72 semen ejaculates from 9 bulls (8 ejaculates/bull) were Indian J. Vet Sci. Biotech (2018) Vol. 14 No. 2 studied at weekly intervals. Following evaluation, the ejaculates were extended at 32-35ºC with Tris-citric acid-fructoseegg yolk-glycerol (TFYG) diluent keeping 80 million sperm per ml. The extended semen was filled and sealed in French mini straws by using IS4 machine (IMV, France). The straws were gradually cooled to 5ºC over 60-90 minutes in a cold handling cabinet and then equilibrated for 4 hrs. The freezing of straws was carried out in liquid nitrogen vapour using a programmable bio-freezer (Digicool, IMV, France). The straws were thawed in water bath at 37°C for 30 sec, and were assessed for sperm motility, viability, morphology and HOS test. The seminal attributes of fresh and frozen-thawed sperm traits were analyzed statistically using completely randomized design to derive mean ± SEs and ANOVA. The mean differences among bulls and among breeds were statistically tested by employing SPSS version 20.00. The Pearson correlations of fresh and frozen thawed sperm parameters were also worked out (Snedecor and Cochran, 1994). Results and Discussion All the bulls of cattle (Gir) and buffalo (Surti and Murrah) breeds donated consistently normal thick creamy yellow and thick milky white semen, respectively. This was in accordance with many previous reports on different breeds (Rana and Dhami, 2004; Chaudhari et al., 2014; Bhakat et al., 2015; Chaudhary et al., 2017a). The breedwise means (±SE) of various seminal attributes observed in freshly ejaculated and frozen-thawed semen of Gir, Surti and Murrah bulls are presented in Table 1 and 2. Ejaculate Volume, Mass Activity and Sperm Concentration The mean values of ejaculate volume and sperm output per ejaculate were significantly (p<0.01) higher in Gir bulls than Surti and Murrah buffalo bulls, however, the latter two breeds did not differ significantly (Table 1). The individual bull variation was significant among the buffalo breeds, but not in cattle. However, the mass activity scores and sperm concentration/ml neither varied significantly between breeds nor between bulls within the breed, except sperm concentration in Surti bulls. The absolute values and significantly higher ejaculate volume and 2 Table 1: Comparative sperm output in Gir cattle and Surti and Murrah buffalo bulls under middle Gujarat climate (Mean ± SE) Seminal attribute Breed of bull Gir (n=24) Surti (n=24) Murrah (n=24) Ejaculate volume (ml) 6.69y±0.17 3.12x±0.10 3.96x±0.16 Mass activity (score 0-5) 3.75±0.06 3.63±0.08 3.63±0.08 Sperm concentration (million/ml) 1348±21.85 1246±58.40 1343±42.93 Sperm Output/Ejaculate (million) 9014y±265.3 3936x±259.6 5366x±332.9 Means bearing uncommon superscripts within the row differ significantly (p<0.05). Table 2: Mean (±SE) sperm quality parameters of fresh and frozen-thawed semen of Gir cattle and Surti and Murrah buffalo bulls Seminal attribute Gir (n=24) Surti (n=24) Murrah (n=24) Fresh 80.21x±0.88 84.58y±0.60 84.38y±0.76 Post-thaw 53.29x±1.56 58.33y±1.43 59.58y±1.20 Fresh 84.71±0.83 86.17±0.78 86.79±0.79 Post-thaw 59.00x±1.95 67.00y±1.59 68.42y±1.66 Total sperm abnormality Fresh (%) Post-thaw 4.00±0.16 3.63±0.12 3.79±0.17 7.04±0.37 6.75±0.31 7.33±0.26 HOS reactive sperm (%) Fresh 82.54±0.91 82.63±0.73 83.50±1.02 48.25x±0.78 44.21y±1.29 51.54z±1.29 Sperm motility (%) Live sperm (%) Stage Post-thaw Means bearing uncommon superscripts within the row differ significantly between breeds (p<0.05). sperm output obtained for cattle bulls than buffalo bulls were in harmony with those reported by Dhami and Sahni (1994) and Dhami et al. (2001), Chowdhury et al. (2013), Bhakat et al. (2015) and Chaudhary et al. (2017a). Further, the findings on mass activity were in accordance with those of Chaudhary et al. (2017a), while Shelke and Dhami (2001) and Rana and Dhami (2004) recorded higher mass activity in cattle than the buffalo semen. Like present trend of nonsignificantly higher sperm concentration per ml and significantly higher total sperm output in Gir cattle than buffalo semen has been documented earlier by Shelke and Dhami (2001), Rana and Dhami (2004), Chowdhary et al. (2013) and Chaudhary et al. (2017a), while Dhami and Sahni (1994) and Bhakat et al. (2015) reported higher Indian J. Vet Sci. Biotech (2018) Vol. 14 No. 2 values in Murrah than HF or Karan Fries bulls. Patel et al. (2012) reported significantly (p<0.01) higher mean sperm concentration per ml in Jafarabadi and Mehsana buffalo bulls than the crossbred bulls, but the total sperm output per ejaculate did not differ between them. The ejaculate volume of bull semen depends upon the body/scrotal size and weight, apart from general & genital health and quality of ejaculatory thrust by the bull. Ejaculate volume together with sperm concentration and motility are of great importance in frozen semen production for wider application in AI industry. Higher the motility and sperm concentration greater will be the swirls, with faster waves and eddies giving better mass activity score, which reflects the initial quality of semen. The differences 3 in ejaculate volume and sperm concentration in bovine and bubaline species could be due to variation between breeds, individual, age, libido, climate, testicular health, accessory sex glands function, and frequency and method of semen collection etc. Sperm Motility and Viability of Fresh and Frozen-Thawed Semen The percentages of motility and live sperm in both fresh and frozen-thawed semen were significantly (p<0.01) higher in buffalo bulls of both the breeds than the Gir bulls (Table 2), and the bull variation was significant only in buffalo breeds. Dhami and Sahni (1994), Bhakat et al. (2015) and Chaudhary et al. (2017a) reported similar higher initial sperm motility and viability in Murrah or Surti buffalo than Friesian, Gir or Karan Fries bulls’ semen. On the contrary, Shelke and Dhami (2001), Rana and Dhami (2004) and Chowdhury et al. (2013) recorded higher sperm motility in fresh and frozen-thawed semen of cow bulls than buffalo bulls. The present findings on significantly higher live sperm per cent reported in buffalo breeds than in Gir breed concurred with Shelke and Dhami (2001) and Chaudhary et al. (2017a). Chowdhury et al. (2013) however reported comparatively lower post-thaw motility and viability in both cattle and buffalo semen as compared to present observations. The individual sperm motility and viability are an essential parameters for assessment of semen quality and freezability, and can yield a reliable picture of semen potency, because they give clue concerning acceptance or rejection of the ejaculate for advance processing & use, and both are positively correlated with freezability of semen sample (Shelke and Dhami, 2001; Rana and Dhami, 2004; Patel et al., 2012; Chaudhary et al., 2017a,b). Sperm motility is essential during their transportation in the oviduct and oocyte penetration. However, it swings between breeds, species, individuals, age groups, seasons and the evaluation techniques employed. The variation in post-thaw motility and viability of sperms may be due to variation in initial quality of semen, extender, equilibration and freezing-thawing protocol used, and the stain and staining technique followed in different studies. Indian J. Vet Sci. Biotech (2018) Vol. 14 No. 2 Sperm Abnormalities and HOST of Fresh and Frozen-Thawed Semen The percentages of total abnormal sperms in both fresh and frozen-thawed semen of Gir cattle and Surti and Murrah buffalo bulls were quite low, and did not differ statistically between them. The bull variation was significant only for fresh sperm abnormalities in Gir breed. The mean post-thaw HOS reactive sperm per cent were significantly (p<0.01) lower in Surti bulls compared to Murrah buffalo, but no such difference was seen in fresh semen between cattle and buffalo breeds. The post-thawed sperms of Murrah buffalo bulls were better HOS resistant, while Surti buffalo sperms showed greater fragility. Similar trend of non-significantly higher sperm abnormalities in Gir than Surti bulls (Chaudhary et al., 2017a), or in HF than Murrah bulls (Dhami and Sahni, 1994), in Jafarabadi & Mehsana buffalo than crossbred bulls (Patel et al., 2012) and in Karan Fries than Murrah (Bhakat et al., 2015) has been reported in fresh and/or frozenthawed semen, while Rana and Dhami (2004) noted identical and much higher total sperm abnormalities in Gir and Jafarabadi bulls (2223% in fresh and 32-33% in frozen-thawed semen). In contrast, Shelke and Dhami (2001) and Chowdhury et al. (2013) reported greater sperm abnormalities in buffalo than cow bulls. For semen sample to be accepted for use in AI, it should have less than 20 per cent total sperm abnormalities. This is because only the live and morphologically normal sperm can drift in the forward direction to reach the site of fertilization in the oviduct after being deposited in the reproductive tract of the female animal in estrus. The present HOS reactive sperms in fresh semen were in accordance with those reported by different workers either in cattle or buffalo bulls (Lodhi et al., 2008; Chaudhary et al., 2017a; Kapadiya et al., 2018) without species variation. However, several authors reported significantly lower HOS reactive sperms in cattle than buffalo bulls semen (Rana and Dhami, 2004; Chowdhury et al., 2013; Bhakat et al., 2015) with much lower absolute values of 40 to 60 per cent only, while other recorded value around 80 per cent (Tiwari et al., 2009) in buffalo semen. Brahmkshatri (1995) found significantly higher post-thaw HOS reactive sperm in Murrah than crossbred bulls 4 (52 vs 41 %) using distilled water, while Prasad et al. (1999), Rana and Dhami (2004), Zubair et al. (2013) and Chaudhary et al. (2017 b) documented very low value (24 to 28 %) in Gir, Surti, Jafarabadi and crossbred bulls. Assessment of sperm membrane function appears to be a significant marker for the fertilizing capacity of spermatozoa, since it is involved in metabolic changes with the surrounding medium and in the process of capacitation, acrosome reaction and fusion with the oocyte membrane (Brahmkshatri, 1995). Greater post-thaw HOS reactivity is generally positively correlated with better sperm longevity and fertility. Interrelationships of Sperm Traits of Fresh & Frozen-Thawed Semen The ejaculate volume in Gir bull semen (Table 3) had highly significant (p<0.01) positive correlations only with post-thaw motility (r=0.62) and live sperm (r=0.54) and negative correlation with post-thaw abnormal sperm (-0.71). Sperm concentration/ml had highly significant (p<0.01) positive correlations with mass activity (0.54), live sperm (0.43) and post-thaw HOS reactive sperm (0.52). Mass activity score revealed significant (p<0.01) positive correlations with motile (0.47), live (0.70) and HOS reactive (0.60) sperm in fresh ejaculates, and with post-thaw HOS reactive sperm (0.41). Initial sperm motility had significant (p<0.01) correlations with initial live (0.78), abnormal (-0.71) and HOS reactive (0.72) sperm. The HOS reactive sperm in the fresh semen showed significant (p<0.01) correlations with live (0.71) and abnormal (-0.58) sperm per cent in fresh semen and with postthaw HOS reactive sperm (0.62). The post-thaw motility had significant correlations with postthaw live (0.71) and abnormal (-0.77) sperms. Among Surti buffalo bull semen (Table 4), ejaculate volume showed highly significant (p<0.01) positive correlations only with post-thaw sperm motility (r=0.63) and live sperm (r=0.66) per cent and negative correlation with post-thaw HOS reactive (r= -0.41) and abnormal (r= -0.52) sperm. Sperm concentration per ml had similar highly significant (p<0.01) positive correlations with post-thaw sperm motility (0.59) and live sperm (0.48) per cent and negative correlation with post-thaw HOS reactive sperm (-0.41). Mass activity score revealed significant (p<0.01) positive Indian J. Vet Sci. Biotech (2018) Vol. 14 No. 2 correlations with initial motile (0.61), live (0.76) and HOS reactive (0.77) sperm, and with postthaw HOS reactive sperm (0.48). The HOS reactive sperm in the fresh semen showed significant (p<0.01) correlations with live (0.79) and abnormal (-0.43) sperm in fresh semen and with post-thaw HOS reactive (0.69) and live (0.49) sperm. Initial live sperm had negative correlation with abnormal sperm in fresh semen (-0.52), and with post-thawed HOS reactive (0.64), live (0.47) and abnormal (-0.53) sperm. Post-thaw motility had significant positive correlations with post-thaw live (0.81) and HOS reactive (0.52) sperm and negative correlation with abnormal sperm (-0.62). Post-thaw HOS reactive sperm showed significant correlations with post-thaw live (0.55) and abnormal (-0.52) sperms. Almost similar correlations were also noted among Murrah bulls’ semen (Table 4). The present correlation findings in ox and buffalo bull semen corroborated well with many previous reports, particularly of Brahmkshatri (1995), Prasad et al. (1999), Shelke and Dhami (2001), Lodhi et al. (2008), Patel et al. (2012), Zubair et al. (2013), Chaudhari et al. (2014) and Chaudhary et al. (2017b). Dhami and Sahni (1994) found all the seminal attributes, except volume, to be significantly interrelated in Murrah and HF bulls (r = 0.32 to 0.86). Patel et al. (2012) found significant (p<0.01) positive correlation for sperm motility and HOS test. Rana and Dhami (2004) found significant (p<0.01) interrelationships for percentages of motile, live, abnormal spermatozoa and HOS reactive sperms in fresh and post-thawed semen of bovine and bubaline species (r = 0.17 to 0.90). Patel and Siddiquee (2013) found positive correlations of ejaculate volume with mass motility and sperm concentration, and mass motility was positively correlated with motility and live sperm count in fresh semen of Kankrej bulls. The present findings and those of others suggested that these traits could be of practical utility in routine semen evaluation to predict semen quality, freezability and fertility. Under the conditions of the present study, it is inferred that assessment of sperm motility and HOS test could be a valuable and practical tool to know the functional capacity of fresh and cryopreserved bull and buffalo spermatozoa. 5 Table 3: Correlation (r) matrix of sperm quality parameters of fresh and frozen-thawed semen of Gir bulls Trait Ejacul Sp Mass Initial Volume Count Activity Motile Initial HOST Initial Live Sp Count -0.07 --- M Activity -0.02 0.54** --- Fr Motility 0.23 0.34 0.47* Fr HOST 0.26 0.30 0.60** 0.72** --- Fr Live Sp 0.07 0.43* 0.70** 0.78** 0.71** Fr Abn Sp -0.13 -0.19 -0.28 -0.71** -0.58** -0.60** PT Motility 0.62 ** Initial Abn PT Motile PT HOST PT Live --- 0.03 -0.10 0.40 * ----- 0.22 0.06 -0.33 --- PT HOST 0.12 0.52** 0.41* 0.39 0.62** 0.33 -0.39 0.24 --- PT Live Sp 0.54** 0.07 -0.29 0.16 0.06 0.02 -0.01 0.71** 0.08 --- PT Abn Sp -0.71** 0.19 0.10 -0.25 -0.17 -0.01 0.22 -0.77** -0.01 -0.78** N = 24; Fr = Fresh/Initial, PT = Post-thaw; *Significant at p<0.05 level; **Significant at p<0.01 level. Table 4: Correlation (r) matrix of quality parameters of fresh and frozen-thawed semen of Surti and Murrah buffalo bulls Trait Ejacul Sp Mass Fresh Volume Count Activity Motile Fresh HOST Fresh Live Fresh PT PT Abn Motile HOST PT Live PT Abn Correlations: Surti buffalo semen -- 0.36 0.00 0.26 -0.14 -0.19 -0.07 0.63** -0.41* 0.66** -0.52** Sp Count 0.33 -- 0.10 0.39 -0.31 -0.16 -0.06 0.59** -0.41* 0.48* -0.33 M Activity -0.17 0.37 -- 0.61** 0.77** 0.76** -0.45* -0.12 0.48* -0.22 0.37 - 0.36 0.32 -0.23 0.34 0.10 0.22 0.02 Ej. Volume Fr Motility 0.22 0.31 Fr HOST 0.06 -0.05 Fr Live Sp -0.11 -0.21 Fr Abn Sp 0.43* 0.25 0.46 0.60 * ** 0.35 -- 0.34 -0.03 0.61** -- -0.27 0.10 -0.38 -0.62** ** ** 0.24 -0.03 .54 PT HOST -0.11 0.17 0.53** PT Live Sp 0.30 -0.04 PT Abn Sp -0.45* -0.25 PT Motility 0.60 0.79 ** -0.43 * -0.40 -0.52** -0.29 -- -0.05 0.69 ** 0.49 * -0.41* 0.64** 0.47* -0.53** -0.22 -0.07 ** ** 0.52 0.81 -0.06 -0.62** 0.36 0.11 0.12 -- 0.27 0.70** 0.37 -0.33 0.29 -- -0.40* 0.34 -0.07 -0.07 0.01 0.65** 0.07 -- -0.72** 0.00 -0.61** -0.19 0.02 -0.01 -0.74** -0.20 -0.74** -- 0.55** -0.52** Correlations: Murrah buffalo semen N = 24; Fr = Fresh/Initial; PT = Post-thaw; *Significant at p<0.05 level; **Significant at p<0.01 level. Indian J. Vet Sci. Biotech (2018) Vol. 14 No. 2 6 Conclusion The bulls of Gir, Surti and Murrah breeds perform well in middle Gujarat climate in terms of ejaculate quality and freezability. In this region, the buffalo bulls produce good quality semen than cattle. The significant positive correlations between mass activity and progressive motility (%) as well as between HOST score and progressive motility for all three breeds, suggest that motility estimation and HOST in fresh and post-thawed semen can be adopted for routine assessment of semen quality. Acknowledgements We thank the Dean of the College and authorities of Anand Agricultural University, Anand for the facilities provided for this work. Conflict of Interest Authors declare that they have no conflict of interest. References: Bhakat, M., Mohanty, T.K., Singh, S., Gupta, A.K., Chakravarty, A.K. and Singh, P. (2015). Influence of semen collector on semen characteristics of Murrah buffalo and crossbred bulls. Adv. Anim. Vet. Sci., 3(4): 253-258. Brahmkshatri, B.P. (1995). Zona free Hamster egg penetration bioassay to assess fertility of bovine semen. Ph. D. Thesis, Tamil Nadu University of Veterinary Sciences, Madras, India. Chaudhari, D.V., Dhami, A.J. and Patel, A.C. (2014). Norms and interrelationships of quality attributes of fresh, refrigerated and cryopreserved buffalo semen. Indian J. Field Vets., 10(2): 93-99. Chaudhary, P.J., Dhami, A.J., Chaudhari, D.V. and Parmar, S.C. (2017a). Freezability of cattle and buffalo semen and association of fresh and frozen-thawed sperm quality parameters. Intl. J. Curr. Microbiol. & Appl. Sci., 6(12): 1445-1454. Chaudhary, P.J., Dhami, A.J., Chaudhari, D.V., Hadiya, K.K. and Patel, J.A. (2017b). Comparative study of Gir cattle and Surti buffalo bulls semen under middle Gujarat climate. Indian J. Vet. Sci. & Biotech., 13(1): 56-61. Chowdhury, S., Das, S., Gupta, T., Sana, D. and Bose, S. (2013). A comparative study with Murrah buffalo and indigenous Gir spermatozoa to hypoosmotic swelling test. Explor. Anim. Med. Res., 3(2): 159-164. Indian J. Vet Sci. Biotech (2018) Vol. 14 No. 2 Dhami, A.J. and Sahni, K.L. (1994). Comparative appraisal of physico-morphological and enzymatic attributes of semen and their interrelationships in ox and buffalo. J. Applied Anim. Res., 5(1): 1320. Dhami, A.J., Panchal, M.T. and Kodagali, S.B. (1990). Comparative biochemical and enzymatic study of Jersey, crossbred and Surti buffalo semen with reference to freezability and fertility. Indian J. Anim. Res., 24(1): 1-8. Dhami, A.J., Shelke, V.B., Dave, R.R. and Kavani, F.S. (2001). Scrotal biometry, sexual behaviour and semen production in breeding bulls of different breeds – A comparative study. Indian J. Dairy Sci., 54: 255-260. Jeyendran, R.S., Van der Ven, H.H., Perez-Pelaez, M., Crabo, B.G. and Zaneveld, L.J.D. (1984). Development of an assay to assess the functional integrity of the human sperm membrane and its relationship to other semen characteristics. J. Reprod. Fertil., 70: 219-228. Kapadiya, P.S., Nakhashi, H.C., Chauhan, P.M., Sutaria, T.V., Suthar, B.N. and Sharma, V. K. (2018). Studies on cer tain physical and biochemical attributes of Kankrej bull semen. Int. J. Curr. Microbiol. Appl. Sci., 7: 4574-4581. Lodhi, L.A., Zubair, M., Qureshi, Z.I., Ahmad, I. and Jamil, H. (2008). Correlation between hypo-osmotic swelling test and various conventional semen evaluation parameters in fresh Nili-Ravi buffalo and Sahiwal cow bull semen. Pakistan Vet. J., 28(4): 186-188. Patel, B.R. and Siddiquee, G.M. (2013). Physical and morphological characteristics of Kankrej bull semen. Vet. World, 6(7): 405-408. Patel, J.B., Dhami, A.J. and Patel, P.A. (2012). Comparative evaluation of physico-morphological attributes of semen of Jafarabadi, Mehsana (Bubalus bubalis) and crossbred (HF x Kankrej, Bos indicus) bulls. Indian J. Field Vets., 7(3): 17. Prasad, J.K., Satish Kumar, Greesh Mohan, Uma Shanker and Agarwal, S.K. (1999). Hypo-osmotic swelling test and its response in fresh and freezethawed semen. Indian J. Anim. Sci., 69(10): 766769. Rana, C.M. and Dhami, A.J. (2004). Physical attributes, intact acrosome, HOS test and freezability of semen of Gir and Jafarabadi bulls. Indian Vet. J., 81(4): 406-410. 7 Salisbury, G.W., Van Demark, N.L. and Lodge, J.R. (1978). Physiology of reproduction and artificial insemination of cattle. 2nd edn. W.H. Freeman & Co., San Francisco. Tiwari, M., Prasad, R.B. and Gupta, H.P. (2009). Physico-morphology and in-vitro fertility of semen/ spermatozoa of Tarai buffalo semen. Indian J. Anim. Physiol., 1: 11-14. Shelke, V.B. and Dhami, A.J. (2001). Comparative evaluation of physico-morphological attributes and freezability of semen of Gir cattle (Bos indicus) and Jafarabadi buffalo (Bubalus bubalis) bulls. Indian J. Anim. Sci., 71: 319-324. Zubair, M., Lodhi, L.A., Ahmad, E. and Muhammad, G. (2013). Hypo osmotic swelling test as screening for evaluation of semen of bull. J. Entomol. Zool. Studies, 1(6): 124-128. Snedecor, G.W. and Cochran, W.G. (1994). Statistical Methods. 14th edn. Oxford and IBH Publishing House, New Delhi, India. Indian J. Vet Sci. Biotech (2018) Vol. 14 No. 2 ❏ 8 The Indian Journal of Veterinary Sciences & Biotechnology (2018) Volume 14, Issue 2, 09-13 ISSN (Print) : 2394-0247 : ISSN (Print and online) : 2395-1176, abbreviated as IJVSBT 10.21887/ijvsbt.14.2.2 Study on Blood Biochemical Profile in Relation to Age and Scrotal Biometry in Adolescent Surti Bucks D.V. Chaudhari, A.J. Dhami*, C.P. Parmar, J.A. Patel and M.M. Pathan Department of Animal Reproduction Gynaecology & Obstetrics College of Veterinary Science & AH, Anand Agricultural University, Anand-388001, India Publication Info Article history: Received : 10-08-2018 Accepted : 08-09-2018 Published : 17-10-2018 Key Words: Sur ti buck, Adolescence, Scrotal biometr y, Blood biochemistry, Maturity. *Corresponding author: ajdhami@aau.in This work is licensed under the Creative Commons Attribution International License (http:// creativecommons.org/licenses /by/4.0/P), which permits unrestricted use, distribution and reproduction in any medium, provided the original work is properly cited. Copyright @: 2018 by authors and SVSBT. Abstract The study was undertaken on 11 Surti male kids of identical age and birth weight from 14 weeks of age till puberty and sexual maturity up to 47 weeks (12 months) of age. The scrotal biometry, i.e., length, width, circumference (cm) and volume (cm3) were recorded using standard procedures at 3 weeks intervals. The mean weight of animals at birth, 14 and 35 weeks of age was 1.53±0.05, 9.86±0.61 kg and 17.84±1.09 kg, respectively and thereafter it did not change much till 47 weeks of age. Similarly, the mean values of scrotal length, width, circumference (cm) and scrotal volume (cm3) at 14 weeks of age were 2.89±0.22, 2.05±0.17, 8.82±0.72 and 21.36±0.93, respectively, which then gradually increased with an advancing age till 35 weeks of age reaching 10.65±0.30, 7.55±0.24, 19.45±0.65 and 200.45±16.67, respectively, which later became almost stable. At puberty, the average age and body weight were 27.00±0.75 weeks and 15.16±0.56 kg, and at sexual maturity 38.18±0.90 weeks and 19.61±0.93 kg, respectively. Great individual variation was noted in all the biometric traits among bucks studied. The blood plasma profile studied at bimonthly intervals revealed significant increase in total protein and ALT, and decrease in cholesterol and cobalt concentrations with advancing age. However, the AST and macro-micro minerals did not vary, suggestive of acquiring adult profile by 6 months of age. Based on the age, scrotal biometry, semen quality and blood biochemistry, it could be inferred that the Surti bucks attained full reproductive potential at around 910 months of age with stable blood plasma profile. Introduction Goats often termed as the “poor man’s cows” are primarily kept for household income and food security of downtrodden rural mass. Surti goat is a medium sized dual purpose goat breed found in the middle and south Gujarat and adjoining areas of Maharashtra. Surti goats are famous for their fertility, prolificacy, meat and milk quality as well as adaptability to the hot humid condition. Age of onset of puberty and male fertility are important factors in caprine reproduction since Indian J. Vet Sci. Biotech (2018) Vol. 14 No. 2 numerous does are generally bred to a single buck. Semen quality, fertility and scrotal measurements are of paramount importance to achieve breeding success (Hoflack et al., 2006). The increased socio-economic importance of goats and the increased requirements for proper goat husbandry, demand best breeding bucks for profitable goat production. There is need to establish measurable criteria for judging breeding soundness and guiding selection of males for breeding to facilitate 9 effective genetic improvement of goat breeds (Giri et al., 1994 and Gogoi et al., 2005). Scrotal circumference is an indirect measurement of testicular size and onset of active spermatogenesis (Bongso et al., 1982). Scrotal biometry during the period of adolescence can provide fair indication to find out the age at which the male goats can be used for breeding purpose (Jadav, 2008). The growth phase of adolescent bucks is also associated with alterations in blood biochemical profile. The paucity of literature on these aspects in Surti goats from birth till attainment of puberty and sexual maturity prompted us to study the same, with an objective to determine the suitable age for their possible commencement in breeding and to determine the influence of age and scrotal biometry on blood biochemical profile in growing Surti bucks. Goa with the help of Chemistry Analyzer (Nova 2021, Analytical Technologies Pvt. Ltd., Vadodara). The micro-minerals, viz., zinc, iron, copper, cobalt and manganese were determined in tri-acids wet digested plasma samples on ICP-OES (Optical Emission Spectrometer; Model Optima 7000 DV; Perkin-Elmer, USA) machine against standard curves at the Micro-Nutrient Research Project (ICAR) of the University. Sexual behaviour of bucks was observed and semen was collected in AV on a dummy goat from the friendly/ cooperative bucks from 7 months till 12 months of age. Data were analyzed statistically for ANOVA and DMRT by using SPSS software version 20.00. Materials and Methods The average birth weight recorded was 1.53±0.05 kg for 11 Surti male kids selected for the study. The live weight of these kids at the age of 14 weeks was 9.86±0.61 kg, which gradually increased with an advancing age till 35 weeks of age (17.84±1.09 kg), and thereafter it did not vary much till the record period of 47 weeks of age. Similarly, the mean values of scrotal length, width, circumference (cm) and scrotal volume (cm3) at 14 weeks of age were 2.89±0.22, 2.05±0.17, 8.82±0.72 and 21.36±0.93, respectively. These values then gradually increased with an advancing age till 35 weeks of age reaching 10.65±0.30, 7.55±0.24, 19.45±0.65 and 200.45±16.67, respectively, which later remained statistically almost stable till 47 weeks of age, except scrotal volume, which showed increasing trend till 38 weeks of age and then did not show significant increase (Table 1). Moreover, great individual variation was also noted in all the biometric traits among bucks studied. This study included 11 Surti male kids of identical age and birth weight born during 10 to 19 September 2016 at Surti Goat Breeding Farm, Ramana Muvada, Gujarat. The study plan was approved by the Institutional Animal Ethics Committee. The actual study was undertaken from 14 weeks of age till puberty and sexual maturity and beyond, up to 47 weeks of age. The initial work up to 6 months of age was carried out at Ramana Muvada and thereafter the pubertal bucks were transferred to Dept of Gynaecololgy, Veterinary College, Anand to study the scrotal biometry, sexual behaviour and blood profile. The live weight was recorded using digital weighing platform and scrotal biometry, i.e., length, width, circumference (cm) and volume (cm3) were recorded using standard procedures i.e., by using Vernier calliper, scrotal (tailor’s) tape and water displacement technique, respectively (Hahn et al., 1969), at 3 weeks intervals starting from 14 weeks till 47 weeks of age. Bi-monthly blood samples were also collected from Jugular veins of these animals at the age of 6, 8, 10 and 12 months to study plasma biochemical, enzymatic and mineral constituents. Plasma samples stored at -20°C till estimation of total protein, cholesterol, serum ALT-AST and macro-minerals, calcium, phosphorus and magnesium by using standard procedures and assay kits procured from Coral/Crest Biosystems, Indian J. Vet Sci. Biotech (2018) Vol. 14 No. 2 Results and Discussion Age, Body Weight and Scrotal Biometry These observations on scrotal/testicular measurements with advancing age were in agreement with the reports of earlier workers (Bilaspuri and Singh, 1992; Kakoty, 1999; Gogoi et al., 2005; Jadav, 2008; Akpa et al., 2013) in different breeds of goat. The variations between studies could be attributed to difference in the breed, nutritional management, climate/season, body weight of bucks and individual variation. The significant age X buck interaction observed in our study indicated that the rate of increase 10 Table 1: Average scrotal biometry of Surti male kids in relation to age and body weight (Mean ± SE) Scrotal volume (cm3) 2.05±0.17a Scrotal circumference (cm) 8.82±0.72a 4.05±0.28b 2.30±0.28ab 8.42±0.49a 24.09±1.13a 11.87±0.74ab 4.40±0.34b 2.92±0.32b 11.20±0.80b 28.64±1.55a 23 12.40±0.76ab 5.74±0.39c 4.45±0.33c 12.48±0.75bc 56.91±7.20ab 26 12.88±0.82b 7.24±0.39d 5.55±0.34d 14.35±0.86c 84.18±12.60bc 29 15.49±1.01c 8.67±0.28e 6.35±0.31e 16.91±0.84d 117.73±14.73c 32 16.57±1.08cd 10.41±0.31f 7.27±0.27f 18.64±0.71de 172.73±15.13d 35 17.84±1.09cde 10.65±0.30f 7.55±0.24f 19.45±0.65e 200.45±16.67de 38 19.18±1.06de 10.97±0.36f 7.77±0.23f 20.14±0.65e 229.09±15.91ef 41 19.47±1.02e 11.19±0.32f 7.98±0.20f 20.71±0.60e 237.82±14.23f 44 19.65±0.98e 11.26±0.37f 7.95±0.22f 20.59±0.60e 239.00±14.41f 47 19.98±0.92e 11.17±0.37f 7.94±0.19f 20.58±0.56e 245.45±13.22f Age (wk) Body wt (kg) Scrotal length (cm) Scrotal width (cm) 14 9.86±0.61a 2.89±0.22a 17 11.46±0.70ab 20 21.36±0.93a Means bearing uncommon superscripts within the column differ significantly (p<0.05). in the scrotal dimensions with advancing age was not uniform in different bucks. In our study, for the eleven adolescent Surti bucks studied, the average age and body weight at puberty were 27.00±0.75 weeks (6.5 months) and 15.16±0.56 kg, respectively, and those at sexual maturity with stable scrotal biometry and semen quality were observed to be 38.18±0.90 weeks (9 months) and 19.61±0.93 kg, respectively. There was a vast variation in individual bucks in respect of age and body weight at attaining puberty and sexual maturity. All the scrotal measurements were significantly affected by age and body weight. This observation suggested that bucks with heavier body mass might possess larger testicular size, which may invariably result into a good reproductive capability and improve the fertility of the animal (Akpa et al., 2013). It could be inferred from the body weight and scrotal biometry that the highest reproductive potential in Surti bucks is attained at around 910 months of age. Blood Biochemical Profile of Adolescent Bucks The blood plasma profile of certain biochemical, enzymatic and mineral constituents Indian J. Vet Sci. Biotech (2018) Vol. 14 No. 2 studied in adolescent Surti bucks at bimonthly intervals from 6 to 12 months of age (Table 2) revealed significant increase in the concentrations of total protein and ALT, and decrease in the concentrations of cholesterol and cobalt with advancing age. However, the other constituents mainly AST and macro-and micro-minerals did not differ much. These findings suggested that Surti bucks studied acquired the blood profile of mature or adult animals by 6 months of age. Jadav (2008) recorded similar variations in blood plasma profile of protein, cholesterol and minerals in Surti bucks of different age groups, i.e., 3, 6 and 9 months and mature ones. Total protein reported in present study was similar with previous findings of Sorathiya et al. (2016), Hassan et al. (2013), Mahore et al. (2013). The level of cholesterol was similar to that reported earlier (Mahore et al., 2013). According to Zubcic (2001) and Kaneko et al. (1997) cholesterol is not affected by feeding system and it shows an increasing trend after puberty. In the present study also the level of serum cholesterol increased after six to eight months of age. Calcium and magnesium did not vary significantly (Pandey et al., 2006). ALT is an 11 Table 2: Blood biochemical profile of adolescent Surti bucks till sexual maturity Total protein, g/dl 23 wk (n=11) 6.72±0.12a Age of bucks 32 wk (n=11) 41 wk (n=11) a 6.74±0.09 7.38±0.08b 47 wk (n=11) 7.64±0.11b Cholesterol, mg/dl 108.71±3.40a 100.56±1.03b 103.14±1.68ab 106.70±2.65ab AST, U/L 104.39±1.05 103.90±1.10 102.79±1.53 103.34±1.78 ALT, U/L a ab b Plasma Profile 34.67±1.15 36.51±1.04 40.42±0.83 38.66±0.84ab Calcium, mg/dl 9.18±0.19 8.93±0.18 9.16±0.21 8.99±0.34 Phosphorus, mg/dl 5.27±0.19 5.13±0.28 5.15±0.28 4.98±0.34 Magnesium, mg/dl 2.72±0.12 2.68±0.10 2.72±0.12 2.76±0.23 Zinc, ppm 1.20±0.03 1.21±0.04 1.22±0.03 1.17±0.03 Iron, ppm 2.41±0 20 2.28±0.10 2.40±0.06 2.46±0.01 Copper, ppm 1.09±0.03 1.12±0.03 1.08±0.03 1.09±0.05 Cobalt, ppm b 0.72±0.04 b 0.66±0.04 b 0.64±0.05 0.46±0.02a Manganese, ppm 0.07±0.01 0.07±0.01 0.06±0.01 0.06±0.01 Means bearing uncommon superscripts within the row differ significantly (p<0.05). enzyme found in the highest amount in liver and typically used to detect liver injury. AST is an enzyme abundantly found in liver and heart muscles and plays an important role in amino acid metabolism. The ALT and AST were in accordance with other reports (Kiran et al., 2012; Shaikat et al., 2013). Elitok (2012) studied blood biochemical parameters in different age groups and concluded that age did not affect on these parameters and concluded that blood biochemical parameters approach to normal adult range after 6-8 months of age. Conclusions The findings of the study showed that the adolescent Surti male kids attain puberty and sexually maturity at around 27.00±0.75 and 38.18±0.90 weeks of age, and 15.16±0.56 and 19.61±0.93 kg body weight, with scrotal circumference of 16.91±0.84 and 20.14±0.65 cm, and scrotal volume 117.73±14.73 & 229.09±15.91 cm3, respectively. The blood plasma profile studied at bimonthly intervals from 6 to 12 months of age showed significant increase in the concentration of total protein and ALT, and decrease in cholesterol and cobalt with advancing age. However, the AST and macro-micro minerals did not vary, suggestive of acquiring adult profile by 6 months of age. Indian J. Vet Sci. Biotech (2018) Vol. 14 No. 2 Acknowledgements We thank the Dean of Veterinary Faculty and Authorities of Anand Agricultural University, Anand for the fund and facilities provided to carry out this work. Conflict of Interest Authors declare that they have no conflict of interest. References: Akpa, G.N., Ambali, A.L. and Suleiman, I.O. (2013). Body conformation, testicular and semen characteristics as influenced by age, hair type and body condition of Red Sokoto goat. New York Sci. J., 6(7): 44-58. Bilaspuri, G.S. and Singh, K. (1992). Developmental changes in body weight and testicular characteristics in Malabari goat kids. Theriogenology, 37(2): 507-520. Bongso, T.A., Jainudeen, M.R. and Siti Zahrah, A. (1982). Relationship of scrotal circumference to age, body weight and onset of spermatogenesis in goats. Theriogenology, 18: 513-524. Elitok, B. (2012). Reference Values for Hematological and Biochemical Parameters in Saanen Goats Breeding in Afyonkarahisar Province. Kocatepe Vet. J., 5(1): 7-11. 12 Giri, S.C., Mohanty, B.N., Ray, S.K.H. and Mohanty, D.N. (1994). Biometry of scrotum and testicles in Black Bengal and Ganjam breed of bucks with relation to fertility. Indian Vet. J., 71: 561-564. Kaneko, J.J., Harvey, J.W. and Bruss, M.L. (1997). Clinical Biochemistry of Domestic Animals. Academic Press, Inc., San Diego, London, Boston, New York, Sydney. Gogoi, Chandan, Deka, B.C., Biswas, R.K. Nath, K.C., Baruah, P.M. and Sarmah, B.C. (2005). Testicular biometry in Beetal x Assam bucks during Adolescence. Indian J. Small Rum., 11(1): 24-27. Kiran, S., Bhutta, A.M., Khan, B.A., Durrani, S., Ali, A., Ali, M. and Iqbal, F. (2012). Effect of age and gender on some blood biochemical parameters of apparently healthy small ruminants from Southern Punjab in Pakistan. Asian Pac. J. Trop. Biomed., 2(4): 304-306. Hahn, J., Foote, R. H., & Seidel, G. E. (1969). Testicular growth and related sperm output in dairy bulls. J. Anim. Sci., 29(1): 41-47. Hassan, D.I., Musa-Azara, I.S., Mohammed, J. and Zanwa, I.A. (2013). Influence of age, sex and season on hematology and serum chemistry of Red Sokoto goats in Lafia, Nasarawa state Nigeria. Int. J. Agric. Sci & Vet. Med., 1(4): online journal ISSN 2320-3730 www.ijasvm.com Hoflack, G., Van Soom, A., Maes, D., De Kruif, A., Opsomer, G. and Duchateau, L. (2006). Breeding soundness and libido examination of Belgian Blue and Holstein Friesian artificial insemination bulls in Belgium and The Netherlands. Theriogenology, 66: 207-216. Jadav, P.V. (2008). Blood biochemical and hormonal profiles in relation to different age groups in Surti bucks with semen studies. M.V.Sc. thesis, Anand Agricultural University, Gujarat, India. Kakoty, D. (1999). Studies on body measurements, testicular biometry, age at puberty, sexual behaviour and semen characteristics in Beetal X Assam local male kids. M.V.Sc. thesis, Assam Agricultural University, Khanapara, Guwahati, India Indian J. Vet Sci. Biotech (2018) Vol. 14 No. 2 Mahore, J. and Mahanta, S.K. (2013). Certain haematological and biochemical parameters in local Bundelkhandi goats. The Indian J. Small Rumin., 19(1): 36-39. Pandey, V., Sareen, M., Moolchandani, A. and Singh, R. (2006). Age related changes in serum mineral and electrolyte profile in Marwari goats. Indian J. Anim. Sci., 76(9): 694-696. Shaikat, A.H., Hassan, M.M., Khan, S.A., Islam, M.N., Hoque, M.A. Bari, M.S. and Hossain, M.E. (2013). Haemato-biochemical profiles of indigenous goats (Capra hircus) at Chittagong, Bangladesh. Veterinary World, 6(10): 789-793. Sorathiya, L. and Fulsoundar, A. (2016). Haematobiochemical profiles, body condition and FAMACHA scores at various ages and their interrelations in Surti goats. Indian J. Anim. Prod. Mgmt., 32(3-4): 206-210. Zubcic, D. (2001). Some biochemical parameters in the blood of grazing Germanim proved fawn goats from Istria, Croatia. Veterinarski Arhiv., 71(5): 237-244. ❏ 13 The Indian Journal of Veterinary Sciences & Biotechnology (2018) Volume 14, Issue 2, 14-17 ISSN (Print) : 2394-0247 : ISSN (Print and online) : 2395-1176, abbreviated as IJVSBT 10.21887/ijvsbt.14.2.3 Gross Anatomy of Cerebral Ventricles and Septum Pellucidum of Brain of Surti Buffalo (Bubalus bubalis) Alka Suman* and Sweta Pandya Department of Veterinary Anatomy and Histology College of Veterinary Science and AH, Anand Agriculture University, Anand-388001 Publication Info Article history: Received : 25-06-2018 Accepted : 20-07-2018 Published : 17-10-2018 Key Words: Biometry, Topography, Cerebral ventricle, Septum pellucidum, Surti buffalo. *Corresponding author: dralkasuman@gmail.com This work is licensed under the Creative Commons Attribution International License (http:// creativecommons.org/licenses /by/4.0/P), which permits unrestricted use, distribution and reproduction in any medium, provided the original work is properly cited. Abstract The objective of the present work was to study the cerebral ventricles and its associated parts of brain of Surti buffalo (n=12), The cerebral ventricles included two lateral ventricles and third ventricle. The lateral ventricles were curved cavities presented large central part and three horns within the cerebral hemispheres. The third ventricle was an annular space on the periphery of inter-thalamic adhesion between two thalami. The mean length and width of central part of lateral ventricle were 4.78±0.07 and 1.49±0.07 cm, respectively. The septum pellucidum was thin translucent membrane of two laminae which separates two lateral ventricles. The mean values of length and height of septum pellucidum were 3.6±0.12 and 0.52±0.04 cm, respectively. The cavum septum pellucidum was a small space found between two laminae of septum pellucidum. Copyright @: 2018 by authors and SVSBT. Introduction The ventricular system of the brain is a set of four interconnected cavities (ventricles), where the cerebrospinal fluid (CSF) is produced from the choroid plexus which bathes the central nervous system (Crisan and Chawla, 2016). The ventricular system is composed of the two largest lateral ventricles in the cerebrum; the third ventricle is in the diencephalon of the forebrain between the right and left thalamus; and the fourth ventricle is located at the back of the pons and upper half of the medulla oblongata of the hind brain (Mortazavi et al., 2014). The ventricles Indian J. Vet Sci. Biotech (2018) Vol. 14 No. 2 are concerned with the production and circulation of cerebrospinal fluid. The Surti is a breed of water buffalo found in the Charottar tract of Gujarat (Banerjee, 2014). There is no data established on the topography and morphometry of cerebral ventricles and septum pellucidum of brain of these animals. Hence, the present work was aimed to study the cerebral ventricles and its associated parts of brain of Surti buffalo. Materials and Methods The study was carried out at the Department of Veterinary Anatomy & Histology, College of 14 Veterinary Science and A.H., Anand, Gujarat. The materials required for the study were collected from normal healthy adult Surti buffaloes (n=12) immediately after slaughter from the abattoir of Ahmedabad Municipal Corporation. The samples were preserved in 10 % neutral buffered formalin at least for 24 hours. The measurements like length and width of various cerebral ventricles and septum pellucidum of brain were taken with the digital Vernier callipers, non-stretchable thread and scale. The means, standard errors and coefficients of variance were worked out (Snedcor and Cochran, 1994). Result and Discussion Lateral Ventricles The ventricular system was very well developed in the brain of Surti buffalo. There were four cavities, which were ventricles of brain, namely two laterals, third and fourth ventricle. Two lateral cavities were present in cerebral hemisphere, one circular groove like cavity was present surrounding the thalamus and the fourth one was present between the cerebellum above and medulla oblongata below. The lateral ventricles were curved cavities within the cerebral hemisphere. Each cavity presented a large central part and three horns. The central part of both the lateral ventricles was separated by septum pellucidum (Fig. 1). The anterior horn passed through the olfactory tract and reached up to olfactory bulb. The posterior horn was directed laterally, turned downward and forward and ended in piriform lobe. The roof of ventricle was formed by corpus callosum. The floor was formed by caudate nucleus on anterolateral aspect and by fornix on posteromedial aspect. The choroid Fig. 1 : Coronal section of brain of Surti buffalo showing (LV) Lateral ventricle, (SP) Septum pellucidum, (CC) Corpus callosum, (F) Fornix and (CN) Caudate nucleus Indian J. Vet Sci. Biotech (2018) Vol. 14 No. 2 plexuses were present in the groove formed by the caudate nucleus and fornix in the central cavity of lateral ventricle (Fig. 2). The foramen Monro was present between the fornix and thalamus dorsally, through which the lateral ventricles opened in the third ventricle (Fig. 4). These findings match well with the reports of Trotter and Lumb (1962) in bovine, Getty (1975) in horse, Lignereux et al. (1987) in Friesian cow, Lignereux et al. (1991) in ewe, Hagenlocher et al. (2013) in mammals and Akers and Denbow (2013) in domestic animals. The overall mean value of the length of the lateral ventricle was 4.78±0.07 cm (range 4.54.85 cm), while the width of central part of lateral ventricle was 1.49±0.07 cm (range 1.25-1.8 cm). Malik et al. (1978) studied cerebral ventricles of goat and reported that each of the lateral ventricles measured 5.46 cm and 1.25 cm in greatest length and transverse linear Fig. 2 : Mid sagittal section of brain of Surti buffalo showing (LV) Lateral ventricle, (CC) Corpus callosum, (CN) Caudate nucleus, (H) Hippocampus, (F) Fornix, (CP) Choroid plexus, (SP) Septum Pellucidum, (Ge) Genu and (Sp) Splenium Fig. 3 : Mid Sagittal section of brain of surti buffalo showing (3V) Third ventricle, (ER) Epiphyseal Recess, (OR) Optic Recess, (IR) Infundibular Recess, and (CA) Cerebral aqueduct. 15 Fig. 4 : Photograph showing (T) Thalamus, (3V) Third Ventricle, (IVF) Interventricular Foramen , (AC) Anterior Commissure, (F) Fornix and (PG) Pineal Gland Fig. 5 : Photograph showing (SP) Septum pellucidum, (CSP) Cavum septum pellucidum (LV) Lateral ventricle and (CP) Choroid plexus measurement. They further mentioned that each ventricle presented three parts rostral horn, body and temporal horn. The present results are lower in the length and higher in the width than the lateral ventricles of goat. end of corpus callosum, the splenium (Fig. 2). The cavum septum pellucidum is a closed triangular space between two laminae of the septum pellucidum. It lacks cerebrospinal fluid and choroid plexuses. The two leaves of septum pellucidum were apart from each other with little distance (Fig. 5). These findings are parallel with the observations of Trotter and Lumb (1962) and Srinivasan (2012) in bovine, Getty (1975) in horse, Sarwar (1989) and Sartori et al. (2015) in human, Lucy et al. (2008) in goat, who all mentioned that the septum pellucidum is a median partition which separates the lateral ventricles and remains attached to the corpus callosum dorsally and fornix ventrally, but they did not mention anything about cavum septum pellucidum in animals. However, Sarwar (1989) in human reviewed that the cavum septum pellucidum is not a part of subarachnoid space nor is a part of ventricular system. Third Ventricle The third ventricle was an annular space on the periphery of inter-thalamic adhesion between two thalami. Two lateral ventricles opened into it through foramen Monro or inter-ventricular foramen (Fig. 4), while the third ventricle continued caudally with the fourth ventricle through aqueduct of sylvius. The cavity of third ventricle presented three recesses; the epiphyseal recess presented in the stalk of the pineal body, the infundibular recess which extended through the infundibulum to the pituitary body, and the optic recess above optic chiasms (Fig. 3). These findings match well with the reports of Trotter and Lumb (1962) in bovine, Getty (1975) in horse, and Thomas and Joanna (2002) in farm animals. Septum Pellucidum Septum pellucidum was thin translucent membrane of two laminae which separates the two lateral ventricles. The dorsal surface of it attached with corpus callosum, while ventral surface was attached with fornix (Fig. 1). The septum starts from the cranial parts of the corpus callosum, the genu and it ends at caudal Indian J. Vet Sci. Biotech (2018) Vol. 14 No. 2 The overall mean length of the septum pellucidum was 3.6±0.12 cm (range 3.0 to 4.3 cm) and height 0.52±0.04 cm (range 0.3 to 0.65 cm). Lignereux et al. (1987) measured the height of septum pellucidun from two different levels, at the inter-venticular foramen and above the hippocampus, which was 2.5 to 9 mm and 0 to 5-6 mm in Friesian cow. The values of length and width are higher in present study on Surti buffalo than the Friesian cow. 16 Acknowledgment We thank the authorities of Ahmedabad Municipal Corporation Slaughter-house for providing brain specimen of adult Surti buffaloes, and Dean, College of Veterinary Science & AH, AAU, Anand, for extending all the facilities to carry out this study. Conflict of Interest All authors declare no conflict of interest. References: Akers, R.M. and Denbow, D.M. (2013). Anatomy and Physiology of Domestic Animals. Wiley Blackwell, USA, pp. 241-264. Banerjee, G.C. (2014). Buffalo. In: A Text Book of Animal Husbandry. 8th ed., Oxford & IBH Publishing House, New Delhi, India, pp. 694-727. Crisan, E. and Chawla, J. (2016). Ventricle of Brain. Drugs and disease. https://emedicine. medscape.com/article/1923254-overview ewe (Ovis aries l.) conformation, relations and stereotaxic topography. Acta Anatomica, 141: 8284. Lucy, K.M., Harshan, K.R., Chungarth, J.J., & Ashok, N. (2008). Prenatal development of the ventricular system of brain in goats! Tamilnadu J. Vet. Anim. Sci., 4 (1): 5-12. Malik, M.R., Shrivastava, A.M. and Parmar, M.L. (1978). Cerebral ventricles of goat. Indian J. Anim. Sci., 48(3): 194-197. Mortazavi, M.M., Adeeb, N., Griessenauer, C.J., Sheikh, H., Shahidi, S., Tubbs, R.I. and Tubbs, R.S. (2014). The ventricular system of the brain: a comprehensive review of its history, anatomy, histology, embryology, and surgical considerations. Childs Nerv Syst.,30: 19-35. Sartori, P., Anaya, V., Montenegro, Y., Cayo, M. and Barba, G. (2015). Anatomical variations of septum pellucidum. Rev. Argent. Radiol., 79(2): 80-85. Sarwar, M. (1989). The Septim pellucidum. Am. J. Neuroradiol., 10: 989-1005. Getty. R. (1975). Sisson and Grossman’s: The Anatomy of the Domestic Animal. Vol. 2, 5th ed., W.D. Saunders Company, Philadelphia, pp. 1211. Snedecor, G.W. and Cochran, W.G. (1994). Statistical Methods. 14th edn, The Iowa State Univ. Press, Ames, Iowa, USA. Hagenlocher, C., Walentek, P. and Muller, C. (2013). Ciliogenesis and cerebrospinal fluid flow in the developing xenopus brain are regulated by fox. Cilia, 2: 12. Srinivasan, P. (2012). Veterinary Anatomy of Ox. BioGreen Books, New Delhi, India, pp. 9-15. Lignereux, Y., Fargeas, J., Marty, H. and Benard, P. (1987). Cerebral ventricles of the Friesian cow (Bos taurus L). Anatomy, 128: 89-92. Lignereux, Y., Regodon, S., Marty, H., Franco, A. and Bubien, A. (1991). Encephalic ventricles of the Thomas, C. and Joanna, M.B. (2002). Clinical Anatomy and Physiology for Veterinary Technicians. 2nd edn., W.B. Saunders Company. Philadelphia, pp. 63-68. Trotter, D.M. and Lumb, J.W. (1962). Bovine Anatomy. 2nd edn., Burgess Publishing Company, pp. 203220. ❏ Indian J. Vet Sci. Biotech (2018) Vol. 14 No. 2 17 The Indian Journal of Veterinary Sciences & Biotechnology (2018) Volume 14, Issue 2, 18-21 ISSN (Print) : 2394-0247 : ISSN (Print and online) : 2395-1176, abbreviated as IJVSBT 10.21887/ijvsbt.14.2.4 Stress enzyme Level during different seasons in Pandharpuri Buffalo R.B. Ambade1, S.H. Dalvi2, M.M. Gatne3, V.D. Dighe4, A.Y. Doiphode5, and B.N. Ramteke6 1,2. Department of Veterinary Biochemistry, 3. Department of Veterinary Pharmacology and Toxicology, 4. NIRRH, 6. Department of Animal nutrition, BVC, Parel, Mumbai – 12 5. Department of Animal Genetics and Breeding, KNPVC, Shirwal Abstract Publication Info Article history: Received : 15-06-2018 Accepted : 10-07-2018 Published : 17-10-2018 Key Words: Stress enzymes, SOD, Catalase, GPx, THI, Seasons, Pandharpuri buffalo *Corresponding author: ambaderb@gmail.com This work is licensed under the Creative Commons Attribution International License (http:// creativecommons.org/licenses /by/4.0/P), which permits unrestricted use, distribution and reproduction in any medium, provided the original work is properly cited. The aim of the present study was to determine the stress enzyme level during different seasons in Pandharpuri Buffalo. The study was conducted on 10 healthy Pandharpuri buffaloes more than 2 years of age. All the animals were maintained under standard feeding and management practices. Temperature Humidity Index (THI) was calculated from meteorological data for the different seasons. Five ml blood was collected from each buffalo aseptically from jugular vein once in the Month of May, August and December. Serum was separated by centrifugation from each blood sample and was used for estimation of activity of stress enzymes. Mean ± S.E. THI was 76.97 ± 0.38, 73.32 ± 0.38 and 68.81 ± 0.49 for summer, rainy and winter season, respectively. Mean ± S.E. activity of SOD (U/ mg of protein), CAT and GPx was 52.41± 1.49, 39.55± 1.92 and 32.54± 0.70; 61.84 ± 2.23, 43.98 ± 3.57 and 29.304 ± 1.51 and 0.374 ± 0. 05, 0.232 ± 0.01 and 0.128 ± 0.20 during summer, rainy and winter seasons, respectively. All the serum antioxidant enzymes were found to be significantly (P < 0.05) higher in the summer season compared to the rainy and winter seasons. Copyright @: 2018 by authors and SVSBT. Introduction Livestock plays an important role in rural economy through its contribution to food, employment generation and drought power. Buffalo has been the mainstay of rural economy in the Indian subcontinent and South East Asian countries. Most of the domestic animals are able to maintain equilibrium between the heat production and heat loss under thermoneutral zones. Heat stress has many detrimental effects on livestock (Marai and Habeeb, 2010). High ambient temperature, relative humidity and radiant energy compromise this ability of animals Indian J. Vet Sci. Biotech (2018) Vol. 14 No. 2 to dissipate heat. As a part of defence against the menace of reactive oxygen species, the body employs antioxidants to quench these free radicals. The antioxidant enzymes superoxide dismutase (SOD), catalase and glutathione peroxidase (GPx) act by scavenging both intracellular and extracellular superoxide radical and preventing lipid peroxidation of plasma membrane. The activities of antioxidant enzymes and lipid peroxidation alter signiûcantly during oxidative stress. So, they can be used as markers of oxidative stress. Glutathione peroxidase functions in cellular redox reactions to protect the cell membrane from oxidative damage caused 18 by free radicals. Hence the present study was undertaken to assess the influence of seasons on antioxidant enzymes in Pandharpuri Buffaloes at their habitat tract. Materials and Methods: The present study was conducted at Department of Veterinary Biochemistry, K.N.P. College of Veterinary Science, Shirwal and Bombay Veterinary College, Parel, Mumbai400012. The experiment was conducted on 10 apparently healthy Pandharpuri buffaloes above two years of age maintained under loose housing condition at LIRD Farm, K.N.P. College of Veterinary Science, Shirwal. The meteorological variables like temperature (dry bulb and wet bulb) and relative humidity were recorded for the Month of May, August and December and were used for calculation of temperature humidity index by formula of Mader et al., (2006). Five ml of blood samples were collected from 10 apparently healthy Pandharpuri buffaloes from jugular vein at morning hours once in the Month of May, once in Month of August and once in Month of December. Clear serum samples were separated by centrifugation and were immediately transported in ice box by road within five hours to Bombay Veterinary College Mumbai for further investigations. The activities of stress enzymes viz, Superoxide dismutase (SOD), Catalase (CAT) and Glutathione peroxidise (GPx) was measured by the method of Marklund and Marklund, (1974); Sinha (1972) and by Assay Kit (Sigma-AldrichCatalog Number CGP1), respectively. The data were analysed using Completely Randomized Design as per standard statistical method (Snedecor and Cochran, 1994). Results and Discussion: Mean ± S.E. of THI, SOD, CAT and GPx during different seasons presented in Table 1 reveals that there was a gradual increase in all the parameters from winter to rainy and summer season. There was significant difference among the three seasons in all the parameters studied. Table 1: Mean ± S.E. of THI, SOD, CAT and GPx during different seasons. Season THI Summer Rainy Winter 76.97 a ± 0.38 73.32 b ± 0.38 68.81c ± 0.49 SOD (U/ mg of protein) 52.41 a± 1.49 39.55 b± 1.92 32.54 c ± 0.70 CAT(U/ mg of protein) 61.84 a ± 2.23 43.98 b ± 3.57 29.30 c ± 1.51 GPx (U/ mg of protein) 0.374 a ± 0. 051 0.232 b ±0.012 0.128 c ± 0.20 Means with different superscripts differ significantly (P<0.5) Temperature humidity index (THI) Superoxide dismutase (SOD) Mean values of temperature humidity index (THI) during different seasons of the study period was recorded significantly (P<0.5) higher in summer as compared to winter and rainy season and differ significantly from each other. Our observations closely resembles with reports of Dikmen and Hansen, (2008) and Al-Samawi et al. (2014). Heat stress has adverse effects on reproductive performances of cattle and buffaloes. The Higher ambient temperature during the summer has been associated with reduced fertility in dairy cattle through its deleterious impact on oocyte maturation and early embryo development (Das et al., 2013 and Chandrabhan, et al., 2012) In the present study, serum SOD activities recorded in summer season was significantly (P<0.05) higher compared to winter and rainy season. SOD catalyses the dismutation of O2into oxygen and hydrogen peroxide (H2O2). The dismutation of O2- results into increased level of H2O2. Our results corroborate with the findings of Megahed et al, (2008) who reported significantly increasing trends in the serum SOD activities in she Egyptian buffaloes during summer as compared to winter season and with the findings of Ganaie et al., (2013) who revealed increased plasma SOD activities in Murrah buffaloes due to oxidative stress during advanced pregnancy and Bernabucci et al., (2002) in dairy cows. Indian J. Vet Sci. Biotech (2018) Vol. 14 No. 2 19 Catalase (CAT) In the present study, serum catalase activities recorded in summer season was significantly (P<0.05) higher as compared to the winter and rainy season. The increased activity of catalase during summer may be due to enhanced production of H2O2 as a result of increased activity of superoxide dismutase observed in the present study during summer season. Similar findings were also reported by Kumar et al. (2007) in cattle and buffaloes, Ganaie et al. (2013) and Lallawmkimi et al., (2009) in Murrah buffaloes. The thermal stress stimulates excessive production of reactive oxygen species (ROS), such as superoxide anion (O2-), hydroxyl ion (OH) and hydrogen peroxide (H2O2), which are continuously produced in the course of normal aerobic metabolism and these free radicals can damage healthy cells. Glutathione peroxidise (U/ mg of protein): In the present study, the serum GPx activities recorded in summer season was significantly (P<0.05) higher compared to the winter season. A significant increase in GPx concentration in summer was observed which indicates increased production of the free radical in hot dry as well as in hot humid season. Our studies are in close resemblance with the reports of Lallawmkimi (2009) in summer season in Murrah buffalo calves, heifers and lactating buffaloes. Conclusion During summer season, there is an increase in the production of different antioxidants enzymes (SOD, CAT and GPx) in Pandharpuru buffalo. All the serum antioxidant enzymes were found to be significantly (P < 0.05) higher in the summer season compared to the rainy and winter seasons. This increased concentration of theses enzymes might help the Pandharpuri buffalo to adapt and acclimatize themselves to the changing environmental condition of western region of Maharashtra. Acknowledgement The authors are grateful to the Associate Dean, K. N. P. College of Veterinary Science, Shirwal- 412801 for his permission to collect the blood samples from Pandharpuri buffalo from LIRD unit KNPVC, Shirwal. Indian J. Vet Sci. Biotech (2018) Vol. 14 No. 2 Conflict of Interest All authors declare no conflict of interest. References: Al-Samawi, K. A., Al-Hassan, M. J. and Swelum, A. A. (2014). Thermoregulation of female Aardi goats exposed to environmental heat stress in Saudi Arabia. Indian J. Anim. Res., 48(4): 344-349 Bernabucci, U., Ronchi B., Lacetera, N. and Nardone, A. (2002). Markers of oxidative status in plasma and erythrocytes of transition dairy cows during hot season. J. Dairy Sci., 85: 2173-2179. Chandrabhan, Singh, S.V., Hooda, O.K., Upadhyay, R.C., Baliyan Beenam and Vaidya, M. (2012). Influence of temperature variability on physiological, hematological and biochemical profile of growing and adult Sahiwal cattle. J. Environ. Res. Dev.,7: 986-994. Das, K., Singh, J., Singh, G. and Nayan, V. (2013) Effect of heat stress alleviation on plasma protein, metabolites and lipid profile in lactating Nili-Ravi buffaloes under tropical climate. Indian J. Anim. Sci., 83(5): 86-89. 27. Dikmen, S. and Hansen, P.J. (2008) : -Is the temperature-humidity index the best indicator of heat stress in lactating dairy cows in a subtropical environment? Journal of Dairy Science 92: 109116. Ganaie, A.H., Shanker, G., Bumla, N.A., Ghasura, R.S., Mir, N.A., Wani, S.A. and Dudhatra, G.B. (2013) Biochemical and physiological changes during thermal stress in bovines. J. Vet. Sci. Technol., 4(126): 126-132. 3. Kumar, A., Kumar, P. and Singh, S.V. (2007). Oxidative stress markers profile in erythrocytes of natural and heat exposed cattle and buffalos. Indian. J. Dairy. Sci., 60: 114-118. Lallawmkimi, C. M. (2009). Impact of thermal stress and vitamin-E supplementation on Heat shock protein 72 and antioxidant enzymes in Murrah buffaloes. Ph.D. Thesis submitted to NDRI, deemed University, Karnal (Haryana), India. Marklund, S. and Marklund, G. (1974,) Involvement of the superoxide anion radical in the autooxidation of pyrogallol and a convenient assay for superoxide dismutase. Eur. J. Biochem.47: 469474. Mader, T. L., Davis, M. S. and Brown-Brand, T. (2006). Environmental factors influencing heat stress in feedlot cattle. J. Anim. Sci., 84: 712–719. 20 Marai, I. F. M. and Habeeb, A. A. M. (2010). Buffalo’s biological functions as affected by heat stress — a review. Livestock Science,127: 89-109. Megahed, G. A., Anwar, M. M., Wasfy, S. I. and Hammadeh, M. E. (2008). Influence of heat stress on the cortisol and oxidant-antioxident balance during oestrous phase in buffalo-cows (Bubalus bubalis): Thermo-protective role of antioxidant treatment. Reproduction of Domestic Animals., 43: 672-677. Indian J. Vet Sci. Biotech (2018) Vol. 14 No. 2 Sinha, A.K. (1972): Colorimetric assay of catalase. Analytical Biochemistry, 47(2):389 - 394 Snedecor, G.W. and Cochran, W.G. (1994). Statistical Methods. 8th Edn. Iowa State University Press, United States of America. Sigma-Aldrich (Catalog Number CGP1): Glutathione Peroxidase Cellular Activity Assay Kit, www. sigma-aldrich.com ❏ 21 The Indian Journal of Veterinary Sciences & Biotechnology (2018) Volume 14, Issue 2, 22-27 ISSN (Print) : 2394-0247 : ISSN (Print and online) : 2395-1176, abbreviated as IJVSBT 10.21887/ijvsbt.14.2.5 Histomorphological and Histochemical Studies on Esophagus in Gaddi Sheep (Ovis aries) Shabir Ahmad Malik1*, Rajesh Rajput1, Mohd Rafiq2, Uiase Bin Farooq2 and Harishbhai Gori3 1 Department of Anatomy & Histology, 2Department of Surgery & Radiology DGCN College of Veterinary and Animal Sciences, CSKHPKV, Palampur-176061 3 Department of Anatomy and Histology, College of Veterinary & AH, AAU, Anand-388001 Publication Info Article history: Received : 19-06-2018 Accepted : 19-07-2018 Published : 17-10-2018 Key Words: Esophagus, Histology, Histochemistry, Gaddi sheep. *Corresponding author: malikshabir21@gmail.com This work is licensed under the Creative Commons Attribution International License (http:// creativecommons.org/licenses /by/4.0/P), which permits unrestricted use, distribution and reproduction in any medium, provided the original work is properly cited. Copyright @: 2018 by authors and SVSBT. Abstract The present work was conducted to study the histoarchitecture and histochemical characteristics of esophagus in six adult Gaddi sheep. Lamina epithelialis consisted of keratinized stratified squamous epithelium with four functional regions: stratum corneum, stratum granulosum, stratum spinosum and stratum basale. In the stratum spinosum layer, the cell nuclei appeared polygonal whereas in the stratum corneum the cell nuclei were flattened and condensed. The stratum spinosum was the thicker layer and stratum granulosum was a thin layer that contained basophilic keratohyalin granules. Pyknotic cells were observed towards the luminal side of stratum corneum. Blood capillaries and lymphoid aggregations in the form of dark stained cells were present in the connective tissue of lamina propria. Connective tissue of lamina propria layer was denser than the same of the submucosa. The tunica muscularis consisted of striated muscle cells throughout the length of esophagus. Stratum corneum of the stratified epithelium of esophagus showed strong periodic acid-Schiff reaction indicating accumulation of glycogen whereas the cells of the basal layer lacked glycogen. The intercellular spaces in the upper layers of stratum spinosum of the epithelium contained acidic mucopolysaccharides as indicated by their reactivity to alcian blue stain. Introduction The esophagus is a relatively simple organ that is evolved to transport food and liquids through the thoracic cavity. It is only part of the gastrointestinal tract that lacks any metabolic, digestive or absorptive function. The degree of keratinization of the stratified squamous epithelium lining the esophagus varies with the species which is usually non keratinized in carnivores, slightly keratinized in pigs, more so in horses, and keratinized to a high degree in ruminants (Eurell and Frappier, 2006). Literature Indian J. Vet Sci. Biotech (2018) Vol. 14 No. 2 is available on the microscopic structure of the esophagus in cow, sheep, dog (Goetsch, 1910), buffalo (Gupta and Sharma, 1991) and goat (Kumar et al., 2009). The present investigation was aimed to study the histological and histochemical characteristics of esophagus in Gaddi sheep considering the economic importance of this Himalyan breed of sheep. Materials and Methods Esophagus of six adult Gaddi sheep of either sex were collected from local slaughter houses. Tissue specimens from different regions of 22 esophagus were fixed in 10% neutral buffered formalin. The tissues were processed by routine paraffin embedding technique (Luna, 1968) and paraffin sections of 5 to 7 μ were cut. The sections were stained with haematoxylin and eosin for routine histology, Masson’s trichome for collagen fibres, Gomori’s for reticular fibres and Verhoeff’s technique for elastic fibres. For histochemical studies, the sections were stained for carbohydrates by PAS stain, acid mucopolysaccharides by Alcian blue method and fat by Sudan Black B (Luna, 1968). The micrometrical data obtained were analyzed statistically using independent samples ‘T’ test (SPSS Statistics-17.0). Results were expressed as means and standard error of mean. Results and Discussion The mean length of esophagus was 40.6 ± 0.64 cm (range: 35-45). The mean diameter of esophagus was 1.28 ± 0.03 cm (range: 1.101.70) in the proximal region, 1.58 ± 0.04 cm (1.30-2.00) in the middle region and 2.01 ± 0.05 cm (1.50-2.40) in the distal region. Nickel et al. (1979) had reported that in sheep, the esophagus was 45 cm long, and its diameter increased from 1.8 cm at the pharynx to 2.5 cm at the cardia. Islam et al. (2008) observed the length of esophagus of Black Bengal goat as 45-50 cm long. The wall of esophagus in Gaddi sheep was found to be composed of four distinct layers, i.e. tunica mucosa, tunica submucosa, tunica muscularis and tunica adventitia/serosa. Tunica mucosa of esophagus was characteristically thrown into longitudinal folds that were composed Fig.1:Microphotograph of esophagus showing lamina epithelialis (LE), lamina propria mucosa (LPM), lamina muscularis mucosa (LMM), tunica submucosa (TS) and tunica muscularis (M). H & E x 40 of lamina epithelialis, lamina propria and lamina muscularis mucosa (Fig. 1). Mucosal folds were identified by their core of lamina propria. Lamina epithelialis was a multilayered epithelial structure composed of keratinized stratified squamous epithelium (Fig. 2). The lamina epithelialis was composed of four tightly adherent layers of epithelium which were organized into distinct vertical zones by stages of development. From the innermost to outermost the layers were: stratum basale, stratum spinosum, stratum granulosum and stratum corneum. However, the presence of stratum granulosum was not a consistent finding (Fig. 2 & 3). The innermost layer of the esophagus was the stratum basale. It was a single layer of cuboidal to columnar cells that rested on the basement membrane below. The basal layer was a very thin layer and the cells had nuclei that were deeply basophilic. The basal layer of epithelium was deeply indented by connective tissue papillae. In oblique sections through the epithelium, these connective tissue papillae looked like islands apparently surrounded by epithelium (Fig. 3). Kumar et al. (2009) observed that the nuclei of stratum basale were strongly basophilic due to condensation of darkly stained chromatin material throughout the nucleoplasm and also contained eccentric nucleoli. It has been reported that the basal layer is the location for cell renewal. As new cells are produced they gradually lose contact with the basement membrane and migrate upwards as they differentiate and change shape (Jankowski et al., Fig. 2: Different layers of the stratified epithelium. Stratum basale (SB), stratum spinosum (SS), stratum granulosum (SG) and stratum corneum. H & E x 200 Indian J. Vet Sci. Biotech (2018) Vol. 14 No. 2 Fig.3:Microphotograph of esophagus showing stratum corneum, stratum spinosum and upper layers of stratum spinosum. Van Gieson’s stain x 200 23 1992). Cells of the basal cell layers lacked glycogen as indicated by their non-reactivity to Periodic Acid-Schiff. The cells mature as they migrate towards the surface. Accumulation of cytoplasmic glycogen starts above the basal zone and is considered a marker of maturation (Jankowski et al., 1992). Fig. 4 : Microphotograph showing lamina epithelialis (LE), Lamina propria (LP), lamina muscularis (LM) and tunica submucosa (TS). Van Gieson’s stain x 100 Fig. 5 : Collagen fibres in lamina propria mucosa (LPM) and tunica submucosa. Also showing lamina muscularis mucosae (LMM) and lamina epithelialis (LE). Mason’s trichome x 40 Fig. 6 : Reticular fibres in the lamina propria mucosae (LPM) and tunica submucosa (TS). Also showing lamina muscularis mucosae (LMM). Gomori’s reticulum x 40 Fig. 7 : Microphotograph of esophagus showing abundant blood vessels in the tunica submucosa. H & E x 40 Fig. 8 : Microphotograph showing tunica muscularis and tunica serosa (S) of the esophagus. Mason’s trichome x 40 Fig. 9 : Periodic acid Schiff (PAS) reactivity seen in the reaction in stratum corneum. Periodic-acid-Schiff x 100 Fig. 10 : Microphotograph showing Alcian blue reactivity in the stratum corneum and intercellular spaces of upper layers of stratum spinosum. Alcian Blue (pH 2.5) x 100 The stratum spinosum was the thicker layer of the esophagus. It contained several layers of polygonal shaped cells, and as they get closer to the upper layer, became flattened. The nuclei of these polygonal cells were round but became increasingly flattened and spindle shaped in the upper layers. The nuclei of these cells were less basophilic compared to the nuclei of stratum basale. Cells of stratum spinosum had prominent cell to cell junctions that appeared as spiky membrane projections. In the upper layers the cell boundaries were clearly distinct (Fig. 3). Mild Periodic Acid-Schiff reactivity was seen in the upper layers of stratum spinosum. However moderate Alcian blue reactivity was seen in the intercellular ground substance indicating presence of acidic mucopolysaccharides (Fig. 9 & 10). Also mild Sudan Black B reactivity indicating presence of lipids was observed in the lower layer of stratum spinosum. Moderate Sudan Black B reactivity was observed in the intercellular matrix. Indian J. Vet Sci. Biotech (2018) Vol. 14 No. 2 24 Kumar et al., (2009) observed that superficial layers of stratum spinosum except cervical region were comprised of varying rows of irregular shaped nuclei having small aggregations of chromatin material throughout the nucleoplasm and with centric /eccentric nucleolus in goat esophagus. The cells of the stratum granulosum contained 3 to 5 layers of flattened spindle shaped cells with cytoplasm containing basophilic keratohyalin granules (Fig. 2). The shape of the nuclei varied from round to elongated. Stratum granulosum, however, was not apparent in all regions of the esophagus. In the superficial layer of stratum corneum the cells were polygonal with abundant eosinophilic cytoplasm and pyknotic nucleus (Fig. 2). Stratum corneum showed strong reactivity for Periodic Acid-Schiff indicating presence of neutral polysaccharides like glycogen (Fig. 9). Acidic mucopolysaccharides were observed in the stratum corneum which reacted strongly to Alcian Blue (pH 2.5) (Fig. 10). Lipids were also observed in stratum corneum as indicated by moderate Sudan Black B reactivity. Meyer and Schnapper (2014) reported that the keratinization of epithelium plays a very important role in the mechanical stability of the epithelial cells and cells layers in the mammalian esophagus and also emphasized the role of these keratins in the esophageal epithelium being of specific interest owing to the varying feed qualities and mechanical loads of different nutritious groups, which have to be encountered. The thickness of epithelium in the thoracic region was higher (443.7 ± 17.30 µm) compared to cervical region (398.3 ± 16.72 µm). Kumar et al., (2009) measured the thickness of epithelium in the cervical region as 337.2 ± 34.4 µm and in the thoracic region as 417.5 ± 42.1 µm in goat. Lamina propria consisted of dense network of collagen and reticular fibres. Blood capillaries and lymphoid aggregations in the form of dark stained cells were present in the connective tissue of lamina propria. These lymphocytes and blood vessels were located closer to the lamina muscularis mucosae. The connective tissue of lamina propria formed connective tissue papillae into the basal layers of stratified squamous epithelium. Connective tissue of lamina propria was denser than the connective tissue of the submucosa. The subepithelial connective tissue had dense arrangement of collagen fibres compared to the layers below (Fig. 1 & 4). Lamina muscularis mucosa consisted of interrupted longitudinally oriented smooth muscle bundles (Fig. 4). Lamina muscularis mucosa was present throughout the length of esophagus. In contrast the lamina muscularis mucosae was as thick as outer layer of tunica muscularis (Eurell and Frappier, 2006) and was divided into an inner circular and an outer longitudinal layer of smooth muscles in pig (Sloss, 1954). Jamdar and Ema (1982) had reported that the lamina muscularis mucosa consisted of few, thin, scattered strands of smooth muscle, identifiable only in the caudal part of esophagus, being located deep in the submucosal glands in camel. The mean thickness of epithelium, lamina propria and tunica submucosa was significantly higher in thoracic region of esophagus (Table 1). Table 1: Micrometrical parameters (µm) of esophagus in the cervical and thoracic region in Gaddi sheep Parameters Stratum corneum Epithelium# Lamina propria Lamina muscularis Tunica submucosa Circular muscle layer Longitudinal muscle layer Tunica Muscularis Measurement (µm, Mean ± SE) Cervical region Thoracic region a 152 ± 11.3 201.0b ± 10.7 398.3a ± 16.72 443.7b ± 17.30 a 157.5 ± 9.04 215.3b ± 10.14 102.5 ± 4.39 128.8 ± 7.62 467.5a ± 25.80 645.5b ± 46.93 993.3a ± 31.62 1206.7b ± 43.95 243.3 ± 11.68 278.0 ± 13.23 1236.6a ± 43.50 1484.7b ± 57.18 Values with different superscripts within the row (a, b) vary significantly (p<0.05). # Epithelium includes stratum corneum Indian J. Vet Sci. Biotech (2018) Vol. 14 No. 2 25 Tunica submucosa was a relatively thick layer of loose connective tissue and was composed of loosely interwoven collagenous, reticular fibres, few elastic fibres, fibrocytes, lymphocytes and blood vessels (Fig. 5 & 7). Submucosal glands were not observed in any region of the esophagus. Connective tissue of the submucosa was more fibrous and less cellular than the connective tissue of the lamina propria where large number of lymphocytes were observed. The submucosa was a highly vascular tunic containing many longitudinally oriented blood vessels (Fig. 7). Loose connective tissue allowed forming of longitudinal folds in the mucosa of relaxed esophagus (Eurell and Frappier, 2006.) Naghani and Andi (2012) reported presence of abundant submucosal glands throughout the length of the esophagus in one humped camel. Islam et al. (2008) reported presence of submucosal glands only in proximal region of esophagus in Black Bengal goat. It has been observed that the thickness of the stratified epithelium or lamina propria was higher in those species where esophageal glands were absent and also in animals which depend on coarse feed especially vegetable fodders (Goetsch, 1910). Tunica muscularis was composed of two layers, outer longitudinal layer and inner circular layer. Collagen and reticular fibres separated the two muscle layers from each other. The tunica muscularis consisted of striated muscle throughout the length of esophagus (Fig. 5, 7 & 8). The thickness of tunica muscularis was significantly higher in the thoracic region (Table 1). Nickel et al. (1979) had reported that the two layers of the tunica muscularis cross obliquely, then spiral and finally form an inner circular and an outer longitudinal layer. Tunica muscularis had been reported to be composed of entirely striated muscles towards upper half of esophagus while in lower part smooth muscles appeared intermingled and constituted inner layer towards cardia in pig (Sloss, 1954). However, completely striated muscles had been reported in buffalo calves (Gupta and Sharma, 1991). The entirely striated muscle might allow regurgitation to chew cud. Tunica adventitia was an external fibrous layer that covered the esophagus in the cervical Indian J. Vet Sci. Biotech (2018) Vol. 14 No. 2 part of esophagus (Fig. 8). It was composed of a loose connective tissue layer containing blood capillaries and collagen fibres as reported in buffalo calves (Gupta and Sharma, 1991). The thoracic part was invested by a serosa supported by connective tissue fibres. Acknowledgement This study was partially supported by research fellowship from Indian council of agricultural research (ICAR). The support and cooperation of the faculty members and support staff of the Department of Veterinary Anatomy and Histology, College of Veterinary and Animal Sciences, CSKHPKV, Palampur is thankfully acknowledged. Conflict of interest The authors declare no conflict of interest. References: Eurell, J.A. and Frappier, B.L. (2006). Dellmann’s Text book of Veterinary Histology. 3rd edn, Blackwell Publishing Limited. pp 190. Goetsch, E. (1910). The structure of the mammalian esophagus. Am. J. Anat., 10: 1-40. Gupta, S.K and Sharma, D.N. (1991). Regional histology of the esophagus of buffalo calves. Indian J. Anim.Sci., 61: 722-724. Islam, M.S., Awal, M.A., Quasem, M.A., Asaduzzaman, M. and Das, S.K. (2008). Histology of esophagus of Black Bengal goat. Bangladesh. J. Vet. Med., 3(2): 152-154. Jamdar, M.N. and Ema, A.N. (1982). The submucosal glands and the orientation of the musculature in the esophagus of the camel. J. Anat., 135(1): 165-171. Jankowski, J., Coghill, G., Tregaskis, B., Hopwood, D. and Wormsley, K.G. (1992). Epidermal growth factor in the esophagus. Gut., 33: 1448-1453. Kumar, P., Mahesh, R and Kumar, P. (2009). Histological architecture of esophagus of goat (Capra hircus). Haryana vet., 48: 29-32. Luna, L.G. (1968). Manual of Histologic Staining Methods of the Armed Forces Institute of Pathology. 3rd edn., McGraw Hill Book Co., New York, pp. 368. Meyer, W. and Schnapper, A. (2014). Keratinization of the esophageal epithelium of domesticated mammals. Acta Histochemica, 116(1): 235-242. 26 Naghani, S.E. and Andi, A.M. (2012). Some histological and histochemical study of the esophagus in onehumped camel. Glob. Vet., 8(2): 124-127. Sloss, M.W. (1954). The microscopic anatomy of the digestive tract of Sus Scrofa Domestica. Am. J. Vet. Res., 57: 578-593. Nickel, R., Schummer. A. and Seiferle, E. (1979). The Viscera of the Domestic Mammals. 2nd edn, Verlag Paul Parey, Berlin, Hamburg. pp: 81-85. ❏ Indian J. Vet Sci. Biotech (2018) Vol. 14 No. 2 27 The Indian Journal of Veterinary Sciences & Biotechnology (2018) Volume 14, Issue 2, 28-29 ISSN (Print) : 2394-0247 : ISSN (Print and online) : 2395-1176, abbreviated as IJVSBT 10.21887/ijvsbt.14.2.6 Studies on Period of Oviposition and Hatching of Eggs in Hyalomma anatolicum anatolicum L. Prasad*, R.K. Bagherwal, A.K. Jayraw1, N. Rajput2, N. Yadav, Veena and P. Thakur Department of Veterinary Medicine, Department of Veterinary Parasitology1, Department of Veterinary Pharmacology and Toxicology2 College of Veterinary Science and Animal Husbandry, Mhow Nanaji Deshmukh Veterinary Science University, Jabalpur (MP), India Abstract Publication Info Article history: Received : 03-07-2018 Accepted : 29-07-2018 Published : 17-10-2018 Key Words: Hyalomma anatolicum anatolicum, period, oviposition and hatching, eggs The engorged Hyalomma anatolicum anatolicum females were collected from healthy cattle of college farm as well as nearby private dairy farms and villages of Mhow to evaluate the period of oviposition and hatching of eggs of H. a. anatolicum under laboratory conditions. The present study recorded the period of oviposition and hatching of eggs (Mean ± SE) as 9.4 ± 0.54 and 21.9 ± 0.69 days, respectively. *Corresponding author: dr.l.p9355@gmail.com This work is licensed under the Creative Commons Attribution International License (http:// creativecommons.org/licenses /by/4.0/P), which permits unrestricted use, distribution and reproduction in any medium, provided the original work is properly cited. Copyright @: 2018 by authors and SVSBT. Introduction Hyalomma anatolicum anatolicum is a widely distributed multi-host tick infesting cattle, buffaloes, sheep and goats responsible for transmitting Theileria annulata, T. buffeli and T. lestocardi ( T. hirci ) in India (Ghosh and Azhahianambi, 2007). Depending on atmospheric conditions, variation is observed in period of oviposition and hatching of eggs. The period of oviposition and hatching of eggs varies upon Indian J. Vet Sci. Biotech (2018) Vol. 14 No. 2 acaricide application and using anti-tick vaccine. Hence, owing to scanty literature pertaining to Hyalomma anatolicum anatolicum compared to Rhipicephalus (Boophilus) microplus. The present study was designed to know the normal of period oviposition and hatching of eggs in case of H. a. anatolicum. Materials and Methods Engorged H. a. anatolicum females dropped from healthy cattle were collected from the 28 College farm as well as private dairy farms and villages in and around Mhow. The collected ticks were processed for preparing permanent slides as per the method described by Bhatia et al. (2010) for their morphological identification. The male and female ticks were identified based on the characters described by Geeverghese and Dhanda (1987). The engorged live female ticks were identified based on the longirostrate mouth parts, first coxa with two equal spurs, longer than wide scutum with a narrowly rounded posterior margin. The males were identified based on the characters like, small elongated body, elongate adanal shields, subanal shields situated away from the mid axis. Further, the collected live ticks were observed under stereoscope and based on long mouth parts and first coxa with two equal spurs, subsequently each engorged female H. a. anatolicum was placed in a test tube and was closed with a piece of cloth and rubber band. These tubes were transferred in a desiccator having saturated potassium hydroxide solution at the base to maintain 80-85% relative humidity (Solomon, 1951). After closing the desiccator, it was placed in an incubator at 28±1°C and 85±5% relative humidity (RH). The tubes were examined periodically to check laying of eggs and after completion of the oviposition, the dead females were removed from the glass tubes in order to avoid the fungal growth on the dead ticks and subsequent contamination of the eggs and freshly laid eggs were kept in BOD incubator at 28±1°C and 85±5% RH in order to record the period of hatching of eggs. Results and Discussion Period of Oviposition The present study recorded the period of oviposition (Mean ± S.E.) as 9.4 ± 0.54 days at temperature of 28 ± 1°C and 85 ± 5% relative humidity. These findings are in agreement with the findings of Bagherwal and Sisodia (1989), as they have reported the oviposition period as 7 – 11 days in case of H. a.anatolicum at a temperature of 29oC and a relative humidity of 85%. Further, Ghosh and Azhahianambi (2007) also recorded oviposition period as 10 - 12.2 days at a temperature of 28°C and 85% relative humidity. Durrani and Shakoori (2009) also reported the oviposition period as 8 – 10 days at 30oC and 85 ± 5 % relative humidity. Anusha Indian J. Vet Sci. Biotech (2018) Vol. 14 No. 2 (2014) recorded oviposition period of H. a. anatolicumas 10 - 12 days at 35 - 38°C and 85% relative humidity. Period of hatching of eggs The current study recorded the period of hatching of eggs (Mean ± S.E.) as 21.9 ± 0.69 days at 28 ± 1°C and 85 ± 5% relative humidity, which is in line with the findings of Bagherwal and Sisodia (1989) who reported the period of hatching as 18 - 29 days. Similarly, Durrani and Shakoori (2009) recorded period of hatching as 15 - 25 days during spring season in Pakistan. Acknowledgement Authors are thankful to the Honourable Vice Chancellor, NDVSU, Jabalpur and Dean, College of Veterinary Science and Animal Husbandry, Mhow for providing the necessary facilities to conduct the research work. Conflict of Interest All authors declare that there is no conflict of interest amongst us. References: Anusha, M. (2014). PCR evaluation for detection of Theileriaannulata in Hyalomma anatolicum anatolicum ticks in Rayalaseema region of Andhra Pradesh. Thesis submitted to Sri Venkateswara Veterinary University, Tirupati. Bagherwal, R.K. and Sisodia, R.S. (1989). Biology and life cycle of tick (Hyalomma anatolicum anatolicum) in and around Mhow (Indore) M.P. Gujarat Vet. J., 23: 13-15. Bhatia, B.B., Pathak, K.M.L. and Juyal, P.D. (2010). Textbook of Veterinary Parasitology, Kalyani Publishers, Ludhiana, pp.: 641 – 642. Durrani, A.J. and Shakoori, A.R. (2009).Study on ecological growth conditions of cattle Hyalommaticks in Punjab, Pakistan. Iranian J. Parasitol, 4: 19-25. Geeverghese, G., and Dhanda, V. (1987). The Indian Hyalomma ticks (ixodoidea: ixodidae). ICAR, New Delhi, 3159. Ghosh,S, and Azhahianambi, P. (2007).Laboratory rearing of Theileriaannulata -free Hyalomma anatolicum anatolicum ticks. Exp. Appl. Acarol. 43: 137-146. Solomon, M.E. (1951). Control of humidity with potassium hydroxide, sulphuric acid or other solutions.Bulletin of Entomological Research, 42: 543-554. ❏ 29 The Indian Journal of Veterinary Sciences & Biotechnology (2018) Volume 14, Issue 2, 30-33 ISSN (Print) : 2394-0247 : ISSN (Print and online) : 2395-1176, abbreviated as IJVSBT 10.21887/ijvsbt.14.2.7 Biochemical alterations in Horses Infected with Theileria equi T.M. Vidhyalakshmi¹, S.K. Raval¹*, P.V. Parikh² and P.V. Patel3 1 Dept. of Veterinary Medicine, ² Dept. of Surgery & Radiology, 3Dept. of Veterinary Parasitology College of Veterinary Science and Animal Husbandry, Anand Agricultural University, Anand-388001, Gujarat, India Publication Info Article history: Received : 04-09-2018 Accepted : 29-09-2018 Published : 17-10-2018 Key Words: Serum biochemistry, Equine merozoite antigen, PCR, Standard assay kits, Theileria equi. *Corresponding author: skraval23@rediffmail.com This work is licensed under the Creative Commons Attribution International License (http:// creativecommons.org/licenses /by/4.0/P), which permits unrestricted use, distribution and reproduction in any medium, provided the original work is properly cited. Copyright @: 2018 by authors and SVSBT. Abstract Equine theileriosis, an OIE listed disease caused by Theileria equi is an economically important haemoprotozoan disease of horses in tropical and subtropical parts of the world. The aim of the present study was to monitor the biochemical alterations in order to find out the severity of the disease. Standard assay kits were used to study the biochemical parameters of horses positive for theileriosis in comparison with healthy ones. Out of 75 serum samples, 25 samples were collected from horses positive for theileriosis and 50 from healthy horses. The serum levels of GPT (32.00±2.30 U/L), GOT (350.84±27.87 U/L), GGT (20.95±0.92 U/L), LDH (823.74±102.93 U/L), total protein (8.11±0.37 g/dl), globulin (3.84±0.40 g/dl), BUN (46.26±5.27 mg/dl) and creatinine (1.33±0.18 mg/dl) were found to be significantly increased (p<0.01) in infected horses compared to the healthy group. Nonsignificant elevation (p>0.05) in total bilirubin (1.08±0.14 mg/dl), unconjugated bilirubin (0.57±0.11 mg/dl) and glucose (102.57±30.41 mg/dl) as well as non-significant reduction (p>0.05) in conjugated bilirubin (0.42±0.04 mg/dl), albumin (4.26±0.24 g/ dl) and A/G ratio (1.55±0.30) were also observed in infected group. There was no significant alteration (p>0.05) in the level of serum calcium (12.32±0.36 mg/dl) and phosphorus (4.53±0.33 mg/dl), as compared to the healthy group. Significant elevation of GOT, GPT, GGT, BUN and creatinine revealed the involvement of liver and kidney functions in T. equi infected horses. Introduction Equine piroplasmosis caused by Theileria equi is a tick-transmitted haemoprotozoan disease of equids (horse, pony, donkey, and mule) occurring in most tropical and subtropical regions of the world (Boldbaatar et al., 2005) as well as in temperate zones, and poses a serious threat to equine health. The economic importance of this disease is concerned with weakness and inability of horse to work, cost of treatment and deaths (Salib et al., 2013). Due to the carrier status of infection, the draught ability of these Indian J. Vet Sci. Biotech (2018) Vol. 14 No. 2 animals gets lowered and poor farmers suffer economically (Kumar et al., 2013). The aim of the present study was therefore to explore biochemical alterations indicating extent of involvement of various organs which may contribute to the future diagnostic as well as therapeutic approaches in cases of equine theileriosis. Materials and Methods The study population included working as well as racing horses presented to the Teaching Veterinary Clinical Complex of the College as 30 well as horses which participated in the horse shows conducted at 3 different province of the state. Consent from the owners of the horses (n=75) was obtained for blood sample collection and the samples were collected as per the standard procedure without any stress or harm to the horses. The work primarily included evaluation of alterations in the serum biochemical parameters of horses positive for Theileria equi (25) in comparison with healthy horses (50). About 9 ml of blood was collected from each horse into serum clotting accelerator vials followed by centrifugation at 3000-3500 rpm for 15-30 minutes. Serum was separated and stored at 20°C until further use for biochemical analysis by using standard assay kits of Coral Clinical Systems, Goa, India, with the help of the clinical chemistry analyzer (Model: Photometer 5010, Robert Riele GmbH & Co. KG, Germany). The biochemical parameters evaluated were total protein, albumin, globulin, glucose, creatinine, blood urea nitrogen (BUN), total bilirubin, direct bilirubin, conjugated bilirubin, unconjugated bilirubin, aspartate amino-transferase (GOT), alanine amino-transferase (GPT), gamma glutamyl transferase (GGT), lactate dehydrogenase (LDH), calcium and phosphorus. The data were analysed statistically using students “t test to compare the differences between healthy and infected group (Snedecor and Cochran, 1994). Results and Discussion The serum biochemical profile of theileriosis positive and healthy control horses is presented in Table 1. The mean values of GPT, GOT and GGT of T. equi infected horses were 32.00±2.30 U/L, 350.84±27.87 U/L and 20.94±0.92 U/L, respectively, whereas the corresponding values in healthy group were 16.13±0.91 U/L, 168.66±10.34 U/L and 12.46 ±0.54 U/L. The levels of all three enzymes were increased highly significantly (p<0.01) in infected than healthy horses (Table 1). These findings agreed with Hailat et al. (1997), Camacho et al. (2005), Alsaad et al. (2010), Ibrahim et al. (2011) and Salib et al. (2013), who reported significant increase in serum GOT, GPT and GGT in T. equi infected horses. Significantly increased (p<0.01) levels of liver enzymes GPT, GOT and GGT could be the result of centrilobular degeneration and necrosis of hepatocytes. The mean value of Table 1: Alteration in the serum biochemical profile of T. equi infected horses in comparison to healthy horses Parameters T. equi positive (n=25) 32.00±2.30** 350.84±27.87** 20.95±0.92** 823.74±102.93** 8.11±0.37** 4.26±0.24 3.84±0.40** 1.55±0.30 1.08±0.14 0.42±0.04 Healthy control (n=50) 16.13±0.92 168.67±10.34 12.46±0.55 478.83±26.46 7.06±0.12 4.40±0.11 2.67±0.11 1.93±0.13 0.92±0.07 0.54±0.05 SGPT (U/L) SGOT (U/L) GGT (U/L) LDH (U/L) Total protein (g/dl) Albumin (g/dl) Globulin (g/dl) A/G ratio Total Bilirubin (mg/dl) Conju Bilirubin (mg/dl) Unconjugated 0.57±0.11 0.34±0.07 Bilirubin (mg/dl) Glucose (mg/dl) 102.57±30.41 82.93±6.47 BUN (mg/dl) 46.26±5.27** 19.09±1.32 Creatinine (mg/dl) 1.33±0.18** 0.80±0.05 Calcium (mg/dl) 12.32±0.36 12.35±0.21 Phosphorus (mg/dl) 4.53±0.33 4.37±0.25 *Significant (p<0.05); **highly significant (p<0.01). Indian J. Vet Sci. Biotech (2018) Vol. 14 No. 2 31 mean value of LDH of T. equi infected horses (823.74±102.93 U/L) was also found to be significantly increased (p<0.01) as compared to healthy group (478.83±26.46 U/L). This observation agreed well with Camacho et al. (2005) and could be due to hemolytic anemia. The mean values of total protein, albumin, globulin and A/G ratio of T. equi infected horses were 8.11±0.37 g/dl, 4.26±0.24g/dl, 3.84±0.40 g/ dl and 1.55±0.30, respectively, whereas in healthy group the corresponding values were 7.06±0.12 g/dl, 4.40±0.11 g/dl, 2.67±0.11 g/dl and 1.93±0.13, respectively. Total protein and globulin were found to be increased (p<0.01) significantly in infected group compared to healthy group, whereas albumin and A/G ratio were decreased non-significantly (Table 1). The present findings disagreed with the observations of Hailat et al. (1997), Alsaad et al. (2010) and Salib et al. (2013), who reported decreased total serum protein, but agreed with Takeet et al. (2009) and Ibrahim et al. (2011), who reported increase in both total protein as well as globulin with decreased A/G ratio. Zobba et al. (2008) and GraciaBocanegra et al. (2013) however reported hypoalbuminemia. A significant increase in total protein could be observed as a result of increased value of globulin fraction as albumin showed non-significant change, and/or it may be due to chronic inflammatory disorder of the liver such as cirrhosis. The mean values of total bilirubin, conjugated bilirubin and unconjugated bilirubin of T. equi infected horses were 1.08±0.14 mg/dl, 0.42±0.04 mg/dl and 0.57±0.11 mg/dl, respectively, whereas in healthy group the values were 0.92±0.07 mg/ dl, 0.54±0.05 mg/dl and 0.34±0.07 mg/dl, respectively. Increase in bilirubin in infected horses as compared to healthy horses was nonsignificant. Hailat et al. (1997), Camacho et al. (2005), Zobba et al. (2008), Alsaad et al. (2010), Ibrahim et al. (2011), Gracia-Bocanegra et al. (2013) and Salib et al. (2013) reported elevated total bilirubin, whereas Takeet et al. (2009) reported decreased levels of conjugated bilirubin and Salib et al. (2013) reported increase in unconjugated bilirubin. The increase in bilirubin may be due to hemolytic anemia. When RBCs are destructed, heme part of hemoglobin is converted into biliverdine by oxygenase enzyme Indian J. Vet Sci. Biotech (2018) Vol. 14 No. 2 and consequently biliverdine is converted into bilirubin by reductase enzyme. The mean value of glucose level of T. equi infected horses was 102.57±30.41 mg/dl, whereas in healthy horses it was 82.93±6.47 mg/ dl, which agreed with Ibrahim et al. (2011). The increase in glucose level in infected horses may be due to stress or increased cortisol level or due to increased glucose mobilization. The mean values of BUN and creatinine of T. equi infected horses were 46.26±5.27 mg/dl and 1.33±0.18 mg/dl, whereas in healthy group the values were 19.09±1.32 mg/dl and 0.80±0.05 mg/dl. There was significant increase in BUN and creatinine values (p<0.01) in infected horses and concurred well with the reports of Ibrahim et al. (2011) and Camacho et al. (2005). Increase in BUN was also reported by Takeet et al. (2009) and Alsaad et al. (2010). An increased level of BUN and creatinine indicate indirect damage of renal tissues. The mean serum calcium and phosphorus levels of T. equi infected horses were 12.32±0.36 mg/dl and 4.53±0.33 mg/dl, whereas in healthy group the values were 12.35±0.21 mg/dl and 4.37±0.25 mg/dl. There was no significant change in calcium and phosphorus levels (p>0.05) in infected group compared to healthy one (Table 1). These findings agreed with the observations of Garba et al. (2012), but disagreed with Zobba et al. (2008), Takeet et al. (2009) and Ibrahim et al. (2011), who reported altered serum calcium and/or phosphorus levels in infected horses. In brief significant elevation of serum GOT, GPT, GGT, LDH, BUN and creatinine suggested the involvement of liver and kidney functions in T. equi infected horses compared to healthy ones. Acknowledgements This work was supported by faculty of Veterinary Medicine, Veterinary Pharmacology and Animal Genetics & Breeding, College of Veterinary Science and Animal Husbandry, AAU, Anand. References: Alsaad, K.M., Alsaad, E.A. and Al-Derawie, H.A. (2010). Clinical and diagnostic study of equine babesiosis in drought horses in some areas of 32 Basrah province. Res. J. Anim. Sci., 4(1): 16-22. Boldbaatar, D., Xuan, X., Battsetseg, B., Igarashi, I., Battur, B., Batsukh, Z., Bayambaa, B. and Fujisaki, K. (2005). Epidemiological study of equine piroplasmosis in Mongolia. Vet. Parasitol., 127: 29-32. Camacho, A.T., Guitian, F.J., Pallas, E., Gestal, J.J., Olmeda, A.S., Habela, M.A., Telford, S.R. and Speilman, A. (2005). Theileria (Babesia) equi and Babesia caballi infections in horses in Galicia, Spain. Trop. Anim. Health. Prod., 37: 293-302. Garba, U.M., Sackey, A.K.B., Agbede, R.I.S., Tekdek, L.B. and Bisalla, M. (2012). Plasma total protein, serum calcium and inorganic phosphate levels in Nigerian horses with natural piroplasmosis. J. Phys. Pharm. Adv., 2: 117-121. Garcia-Bocanegra, I., Arenas-Montes, A., Hernandez, E., Adaszek, L., Carbonero, A., Almeria, S., Jaen-Tellez, J.A., Gutierrez-Palomino, P. and Arenas, A. (2013). Seroprevalence and risk factors associated with Babesia caballi and Theileria equi infection in equids. Vet. J., 195(2): 137-138. Hailat, N.Q., Lafi, S.Q., Al-Darraji, A.M. and Al-Ani, F.K. (1997). Equine babesiosis associated with strenuous exercise: clinical and pathological studies in Jordan. Vet. Parasitol., 69(1-2): 1-8. Indian J. Vet Sci. Biotech (2018) Vol. 14 No. 2 Ibrahim, A.K., Gamil, I.S., Abd-El baky, A.A., Hussein, M.M. and Tohamy, A.A. (2011). Comparative molecular and conventional detection methods of Babesia equi in Egyptian equine. Global Veterinaria, 7: 201-210. Kumar, Y., Malhotra, D. V., Nichani, A. K., Kumar, A., Dhar, S. and Kumar, S. (2013). Immunokinetics of Theileria equi specific antibodies: a comparison in serial and single dilution ELISA antibody end titres. Turk. J. Vet. Anim. Sci., 37: 429-433. Salib, F.A., Youssef, R.R., Rizk, L.G. and Said, S.F. (2013). Epidemiology, diagnosis and therapy of Theileria equi infection in Giza, Egypt. Vet. World, 6: 76-82. Snedecor, G.W. and Cochran, W.G. (1994). Statistical Methods. 6th ed., Oxford and IBH Publishing Company, Calcutta, India. Takeet, M.I., Adeleye, A.I., Adebayo, O.O. and Akande, F.A. (2009). Haematology and serum biochemical alteration in stress induced equine theileriosis. A case report. Sci. World, 4: 19-21. Zobba, R., Ardu, M., Niccolini, S., Chessa, B., Manna, L., Cocco, R. and Parapaglia, M.L.P. (2008). Clinical and laboratory findings in equine piroplasmosis. J. Eq. Vet. Sci., 28: 301-308. ❏ 33 The Indian Journal of Veterinary Sciences & Biotechnology (2018) Volume 14, Issue 2, 34-38 ISSN (Print) : 2394-0247 : ISSN (Print and online) : 2395-1176, abbreviated as IJVSBT 10.21887/ijvsbt.14.2.8 Effect of Supplementation of Monensin Sodium on rumen metabolism and Milk Yield in Early Lactating Buffalo (Bubalus Bubalus) Prathviraj*. Shrikant Kulkarni**, N. M. Soren***, Sathisha K B**, Srinivas Reddy Bellur**, Prashant Bellundagi and Ramachandra B*, **Department of Veterinary Physiology and Biochemistry and *Animal Nutrition Veterinary College, Nandi Nagar, Bidar-585401, Karnataka Animal Nutrition Division ***ICAR-National Institute of Animal Nutrition and Physiology Publication Info Article history: Received : 20-08-2018 Accepted : 15-09-2018 Published : 17-10-2018 Key Words: TVFA, Acetate/Propionate ratio, buffalo, Monensin sodium *Corresponding author: hprithvi@gmail.com This work is licensed under the Creative Commons Attribution International License (http:// creativecommons.org/licenses /by/4.0/P), which permits unrestricted use, distribution and reproduction in any medium, provided the original work is properly cited. Abstract The study was carried out to assess the effect of monensin sodium supplementation on rumen fermentation metabolites and milk yield in early lactating buffaloes. Twelve buffaloes in their 2nd week of lactation were selected. Control group was fed on standard ration whereas the treatment group was supplemented with monensin sodium @ 200 mg/head/day in addition to standard ration. Rumen liquor and blood sample was collected at 2nd and 12th week of lactation. Total and individual volatile fatty acids concentration was estimated by gas chromatography. Monensin sodium (P<0.05) decreases acetate and increases propionate concentration and decreases the ratio of acetate to propionate in the rumen liquor without altering the total volatile fatty acid concentration in experimental period. Supplemented buffaloes yielded 8.22 per cent more milk than the control. Copyright @: 2018 by authors and SVSBT. Introduction India has 105.1 millions buffaloes which is approximately 56.7 per cent of the total world buffalo population. They contribute 47.85 million MT of milk amounting to 55% of total milk produced in India. Although buffaloes are better converter of poor quality fibrous feeds into milk, there are few reports on low milk yield, poor reproductive performance and low growth rate Indian J. Vet Sci. Biotech (2018) Vol. 14 No. 2 because of poor feeding practices, irregular and inadequate availability of quality feedstuffs. Lactating dairy animals have high demand for energy and protein for milk synthesis. Mobilizing energy and protein from body tissues stores and repartition of nutrients away from extra mammary tissues are the primary alternatives source of sufficient nutrients for milk production during early lactation. Excessive utilization of body 34 reserves, especially fat, can subject lactating animals to negative energy balance leading to a series of metabolic disorders and consequent production losses (Fourichon et al., 1999). Several methods of modifying ruminal fermentation have been developed by various researchers to enhance feed utilization in cattle and buffaloes. Fermentation modifiers are used in feed to manipulate rumen fermentation for better feed utilization and improved milk production. Monensin is one of the fermentation modifiers that have been extensively used in dairy and beef cattle to improve the feed utilization and productive response (Schelling, 1984). The aim of the present study was to assess changes in concentration of the rumen fermentation metabolites (TVFA) and milk yield in early lactating buffaloes by supplementing monensin sodium @ 200mg/day/ animal. Materials and Methods Twelve apparently healthy local non descript buffaloes (Av. body weight 362.5±12.5) in their 2nd week of third lactation were selected from an organized private dairy farm in Bidar district of Karnataka state, India. They were randomly divided into two equal groups of six animals each and one group served as control and the other treatment group. Control group was fed on standard ration comprising of concentrate, green fodder (Napier grass; Pennisetum purpureum) and dry fodder (Jowar stover; Sorghum bicolor) as per Paul et al. (2002) to meet the nutrient requirements.. The concentrate, napier fodder and jowar stover contained 18.66, 9.7 and 3.7% crude protein, respectively. The treatment group also received standard ration as that of control group and in addition, they were supplemented with monensin @ 200 mg per head per day. Third week of lactation was adaptation period for treatment group, where they received monensin gradually in increasing dose from 50 mg per day to 150 mg per day/head. Monensin sodium was uniformly mixed in the concentrate mixture and was supplemented in divided dose i.e. 100 mg in the morning and 100 mg in the evening daily during the experimental period i.e. from 4th to 12th week of lactation. Rumen liquor samples were collected from all the buffaloes during the 2nd and 12th week of lactation by means of stomach tube attached to a suction pump. The rumen liquor Indian J. Vet Sci. Biotech (2018) Vol. 14 No. 2 samples were filtered by four layers of cheese cloth and 800 µl was transferred into microcentrifuge tube and 200 µl of 20% metaphosphoric acid was added and centrifuged at 5000 rpm for 20 minutes. The supernatant was collected and stored at -20oC for TVFA analysis. The concentration of individual volatile fatty acids viz., acetate, propionate, butyrate, isobutyrate, isovalerate and valerate were estimated by gas chromatography (Agilent; Model 7890A GC System) using flame ionization detector, programmable temperature vaporizer injector and capillary column (Agilent J&W DB-WAX GC Column 40 m × 0.18 mm × 0.18 µm). The Ruminal liquor samples were injected by an automatic injector at an injection volume of 1μl using the split method. Milk yield of both morning and evening milking of individual buffalo was recorded in kilogram using a standard weighing balance and values were pooled to express the daily milk yield. Milk yield was recorded at a weekly interval up to 12th week of lactation. The data were analyzed statistically by student t-test as per Snedecor and Cochran (1994). Results and Discussion Average mean concentration (mmol/L) of individual volatile fatty acids viz., acetate, propionate, butyrate, isobutyrate, isovalerate, valerate and TVFA in control and treatment group at 2nd and 12th week of lactation is presented in Table 1. The concentration of individual and total volatile fatty acids was similar in the rumen liquor samples of all the buffaloes at the initiation of the experiment. Monensin supplementation significantly (P<0.05) decreases acetate concentration, increases propionate concentration and decreases the ratio of acetate to propionate (A/P) in the rumen liquor of early lactating buffaloes without altering the TVFA. However, its supplementation in the treatment group had no effect on the concentration of other volatile fatty acids and TVFA after 12 week of lactation. Decrease in the rumen acetate and acetate: propionate ratio and increase in the ruminal propionate concentration without altering the TVFA concentration in present study was similar to the observation of the previous studies by Randall et al. (1978) in steers, Maas et al. (2001) in sheep, Erasmus et al. (2005) and Lamba et 35 Table 1: Average concentration of individual volatile fatty acids (VFA) and total VFA in the rumen liquor of control and monensin supplemented lactating buffaloes at 2nd and 12th of lactation (Mean±SE) Volatile fatty acids (mMol/L) Acetate 2nd week 12th week* Propionate 2nd week 12th week* Isobutyrate 2nd week 12th week Butyrate 2nd week 12th week Isovalerate 2nd week 12th week Valerate 2nd week 12th week Control group Treatment group 52.20±0.98 53.16a ±0.52 51.72±0.48 46.49b ±0.65 12.52±0.29 12.21a ±0.15 12.27±0.24 19.39b ±0.90 0.24±0.10 0.21±0.10 0.23±0.10 0.22±0.10 8.26±0.47 8.40±0.19 8.26±0.10 8.67±0.55 0.44±0.10 0.42±0.10 0.43±0.10 0.41±0.10 0.37±0.10 0.34±0.10 0.35±0.10 0.32±0.10 74.03±1.17 74.13±0.48 73.27±0.53 75.49±0.91 4.18±0.10 4.22±0.10 4.17a ±0.10 2.60b ±0.18 TVFA 2nd week 12th week A:P ratio 2nd week * 12th week ®Means with different superscript in each row differ significantly with other (P<0.05) Table 2: Mean milk yield (kg/d) of control and monensin supplemented buffaloes Week of Lactation 4th 5th 6th 7th 8th 9th 10th 11th 12th Average Control 6.43±0.52 7.20±0.73 7.38±0.47 7.33±0.40 7.42±0.26 a 7.22±0.69 a 7.15±0.07 a 7.30±0.32 a 7.09±0.27 a 7.17±0.10 a Treatment 6.65±0.33 6.82±0.40 6.97±0.47 8.15±0.40 8.10±0.26 b 8.28±0.18 b 8.54±0.66 b 7.96±0.34 b 8.38±0.45 b 7.76±0.24 b Means with different superscript in each row differ significantly with other (P<0.05) Indian J. Vet Sci. Biotech (2018) Vol. 14 No. 2 36 al. (2013) in cows. The increased propionate and decreased acetate concentration in the ruminal fluid of monensin supplemented early lactating buffaloes is due to selective antimicrobial action of monensin in rumen. Monensin sodium selectively inhibits growth of gram-positive organisms and shift the flow of electrons from formate and methane to succinate or propionate as alternate electron sink products in the mixed microbial population (Van Nevel and Demeyer, 1977), thereby reduces acetic acid production and methanogenesis and increases the production of propionate in rumen liquor (Goodrich , 1984). of rumen fermentation, thereby increasing the glucose availability for milk production. The mean milk yield (Kg/d) of control and treatment groups from 4th week to 12th week of lactation were presented in Table 2. There was no significant difference in milk yield between control and monensin supplemented buffaloes from 4th to 7th week of lactation, where as monensin supplemented buffaloes showed significantly (P<0.05) higher milk yield than that of control group from 8th week to 12th week of lactation. The average milk yield (Kg/d) of the entire experimental period was 7.17±0.10 and 7.76±0.24 in control and treatment group respectively. The average milk yield was significantly higher (P<0.05) in treatment group as compared to that of control group. On an average, monensin supplemented early lactating buffaloes yielded 8.236 per cent (0.59kg/d) more milk than the control group during the experimental period. Results of the this experiment are similar to the findings of Lynch et al. (1990), Van Der Werf et al.(1998), Ipharraguerre and Clark. (2003) and Gandra et al. (2010) who have reported increased milk yield in different breeds of dairy cows upon dietary supplementation of monensin. However, Khodamoradi et al. (2013), Lamba et al. (2013) and Van Der Merwe (2001) had observed no significant difference in the milk yield in monensin supplemented cows. McGuffey et al. (2001) reported that the supplementation of ionophores in animal feed resulted in an alteration of ruminal bacterial populations, which altered the final products of fermentation by increasing propionate proportion and reducing acetate and butyrate concentration. Increased milk yield in monensin supplemented buffaloes might therefore be due to increased supply of glucogenic precursor like propionate, resulting from changes in the pattern Erasmus L J, Rabinson P H, Ahmadi A, Hinders R and Gerret J E. (2005). Influence of prepartum and post partum supplementation of yeast culture and monensin or both on ruminal fermentation and performance of multiparous dairy cows. Animal Feed Technol 122, 219-239. Indian J. Vet Sci. Biotech (2018) Vol. 14 No. 2 Based on the results of the present study, it can be concluded that monensin sodium supplementation @200mg/head/day to early lactating buffaloes may be beneficial in improving the production as it shifts the rumen fermentation more towards propionate and increases the blood glucose availability for more milk yield. Conflict of Interest All authors declare no conflict of interest. References: Fourichon C, Seegers H, Bareille N and Beaudeau F. (1999). Effects of disease on milk production in the dairy cow. Prev. Vet. Med 41, 1-35. Gandra J R, Renno F P, Freitas J E, Santos M V, Silva L F P. and Araujo A P C. (2010). Productive performance and milk protein fraction composition of dairy cows supplemented with sodium monensin. R. Bras. Zootec., 39(8):1810-1817. Goodrich R D, Garrett J E, Gast D R, Kirick M A, Larson D A and Meiske J C. (1984). Influence of monensin on the performance of cattle. Journal of Animal Science 58(6), 1484-1498. Ipharraguerre I R and Clark J H. (2003). Usefulness of ionophores for lactating dairy cows: a review. Animal Feed Science and Technology., 106:39– 57. Khodamoradi S H, Fatahnia F, Taherpour K, Pirani V, Rashidi L and Azarfar A. (2013). Effect of monensin and vitamin e on milk production and composition of lactating dairy cows. J.Anim.Phys.Anim.Nutr., 97:666-674. Lamba J S, Grewal R S Ahuja C S, Malhotra P and Tyagi, N., (2013). Effect of monensin on the milk production, milk composition, rumen metabolism and blood biochemical profile in crossbred cows. Indian J. Anim. Nutr 30(1), 38-42. Lynch G A, Hunt M E and McCutcheon S N. (1990). A note on the effect of monensin sodium administered by intraruminal controlled-release devices on productivity of dairy cows at pasture. Anim.Prod., 51:418. 37 Maas J A, Wilson G F, Mccutcheon S N, Lynch G A, Burnham D L and France J. (2001). The effect of season and monensin sodium on the digestive characteristics of autumn and spring pasture fed to sheep. J. Anim. Sci 79, 1052–1058. McGuffey R K, Richardson L F and Wilkinson J I D. (2001). Ionophores for Dairy Cattle: Current Status and Future Outlook. J. Dairy Sci., 84: E194-E203. Paul S S, Mandal A B and Pathak N N. (2002). Feeding standards for lactating riverine buffaloes under tropical condition. Journal of dairy research (UK), 69, 173-180. Randall W, Van Maanen, Joseph H, Herbein A, Dare McGilliard and Jerry Young W. (1978). Effects of monensin on in vivo rumen propionate production and blood glucose kinetics in cattle. J. Nutr 108, 1002-1007. Schelling, G.T. (1984) Monensin : Mode of action in the rumen. J. Anim Sc. 58:1518-1527 Snedecor G W and Cochran. (1994): Statistical Methods. 8thedn. Oxford and IBH. Pub.Co., Calcutta. Van Der Merwe B J, Dugmore T J and Walsh K P. (2001). The effect of monensin on milk production, milk urea nitrogen and body condition score of grazing dairy cows. S.Afr.J.Anim.Sci., 31(1):4955. Van Der Werf J H J, Jonker L J and Oldenbroek J K. (1998). Effect of Monensin on Milk Production by Holstein and Jersey Cows. J.Dairy Sci., 81(2):427–43. Van Nevel C J, Demeyer D I. (1977). Effect of monensin on rumen metabolism in vitro. Appl. Environ. Microb 34, 25-30. ❏ Indian J. Vet Sci. Biotech (2018) Vol. 14 No. 2 38 The Indian Journal of Veterinary Sciences & Biotechnology (2018) Volume 14, Issue 2, 39-43 ISSN (Print) : 2394-0247 : ISSN (Print and online) : 2395-1176, abbreviated as IJVSBT 10.21887/ijvsbt.14.2.9 Ultrasonographic Fetometry and Fetal Structures during Early Pregnancy in Surti Buffaloes Mitesh Gaur1 and G.N. Purohit2* 1 Department of Gynecology and Obstetrics, College of Veterinary and Animal Sciences, Navania, Vallabhnagar, RAJUVAS, Udaipur, 2Department of Gynecology and Obstetrics, RAJUVAS, Bikaner, Rajasthan Publication Info Article history: Received : 05-09-2018 Accepted : 09-09-2018 Published : 17-10-2018 Key Words: Amniotic vesicle, crown-rump length, fetal hear t beat, ultrasonography. *Corresponding author: gnpobs@gmail.com Abstract Ultrasonographic evaluations of early pregnant Surti buffaloes revealed that the uterine fluid was visible on Day 18 of pregnancy, yet this was not a sure sign of pregnancy. The amniotic vesicle could be detected by Day 26 and was a sure sign of pregnancy. The embryo appeared on Day 26 but with clarity on Day 30. Fetal heart beats were visible from Day 26 and increased significantly (p<0.05) from Day 42 compared to Day 30, 34 and 38 of gestation and remained elevated till Day 62 of gestation. The crown rump length (CRL) of the embryo / fetus could be measured using inbuilt callipers starting from Day 30 till Day 58. This work is licensed under the Creative Commons Attribution International License (http:// creativecommons.org/licenses /by/4.0/P), which permits unrestricted use, distribution and reproduction in any medium, provided the original work is properly cited. Copyright @: 2018 by authors and SVSBT. Introduction Transrectal ultrasonography has gained popularity for evaluation of follicular dynamics (Baruselli et al., 1997; Awasthi et al., 2006), early pregnancy, fetal deaths and fetal growth (Karen et al., 2007; Ali and Fahmy, 2008; Russo et al., 2009). Real time B-mode ultrasound was used to detect and monitor the early conceptus, its growth and its anatomical features in Murrah (Pawshe et al., 1994), Egyptian (Karen et al., 2007), Nili Ravi (Naseer et al., 2012), Bangladeshi (Rabidas and Gofur, 2017) and Mediterranean Indian J. Vet Sci. Biotech (2018) Vol. 14 No. 2 (Russo et al., 2010) buffaloes. Sonographic fetometry was performed in a few studies (Pawshe et al., 1994; Ali and Fahmy, 2008). Similar evaluations in Surti breed of buffaloes are unavailable. The present study evaluated the early appearance of embryo/fetus and its annexes during pregnancy. Materials and Methods Surti buffaloes belonging to the elite herd of Network Project on Buffalo Improvement at Livestock Research Station, Vallabhnagar, district Udaipur (RAHUVAS) were included in this study. 39 Buffaloes inseminated during a spontaneous estrus (n=9) detected using a teaser (Purohit and Rao, 2018) and early pregnant buffaloes of the herd (n=19) were examined every 3-4 days by transrectal ultrasonography (Exago, ECMNoveko International Inc., Angoulème, France) equipped with a 5.0-7.5 MHz linear-array transducer for early appearance of fetus and its annexes starting from Day 18 of insemination till Day 90. Only 5 of the inseminated buffaloes were found pregnant thus total 24 buffaloes were examined in the present study. The first appearance of uterine fluid, amniotic vesicle, embryo and its heart beat and crown rump lengths were measured/recorded and the images were saved. The data obtained were analyzed as per statistical procedures described previously (Snedecor and Cochran, 1994). formation was recorded. Anechoic amniotic vesicle formation was observed in fluid filled uterine chamber and could be easily identified (Fig. 2). Results and Discussion Fig. 2 : Sonograph depicting the presence of amniotic vesicle on Day 26 as confirmatory diagnosis of early pregnancy in a Surti buffalo. Uterine fluids On Day 18 post-AI, a very little amount of anechoic fluid was seen in the lumen of uterine horn ipsi-lateral to CL (Fig.1) indicating sign of probable pregnancy. At this stage the echoic uterine wall along with anechoic fluid could be easily identified. In none of the animals examined at this stage the embryo proper could be visualized. The appearance of fluid in the uterine lumen was not a sure sign of pregnancy as 4 animals that evidenced fluid on Day 18 were found to be non-pregnant at later days of evaluation. Similar findings were previously reported by Pawshe et al., (1994). The amount and size of anechoic fluid increased with advancement of gestation. The confirmatory diagnosis of early pregnancy was established with the finding of the amniotic vesicle as all buffaloes with evidence of amniotic vesicle were found confirmed pregnant. There is variation in reports on first appearance of uterine fluid from Day 19 (Pawhse et al., 1994) to Day 21-22 (Herera et al., 2007; Ferreira et al., 2012; Sharma et al., 2012), however, these workers also mentioned that the appearance of uterine fluid was not a definitive sign of pregnancy in buffaloes at this time and for definitive diagnosis of pregnancy examination at later stage seem mandatory. Similar to the present findings, embryo and embryonic membranes were observed on Day 26 or 27 in previous studies on buffaloes (Groza et al., 2012; Sharma et al., 2012). In a few recordings during the present study the embryo was not visible, although the amniotic vesicle was clearly visible. This could be due to the different angle of placement of the probe during examination. Fig. 1 : Sonograph showing the presence of little amount of anechoic fluid in the lumen of uterine horn on Day 18 post AI in a Surti buffalo. Embryo Amniotic vesicle On day 26, the amount of anechoic fluid in the uterine lumen increased and compartment Indian J. Vet Sci. Biotech (2018) Vol. 14 No. 2 The embryo proper could although be visualized on Day 26 of gestation as a small echogenic spot within the amniotic vesicle (Fig. 2) however, it was clearly visible as an echoic structure on Day 30 of pregnancy surrounded by 40 anechoic amniotic fluid (Fig. 3). Starting the same day the crown rump length of a crescent shaped embryo could also be measured. The amniotic membrane surrounding the embryo could clearly be differentiated on Day 38 of gestation and on later days of scanning. Ferreira et al. (2011) assessed buffalo embryo on day 25.18±1.91 post-breeding whereas Pawshe et al. (1994, 2011) observed embryo on day 19.0±1.69 of gestation in buffaloes and in cattle on day 22.5±0.7 of gestation which is earlier than present study. This difference observed may either be due to species variation, expertise of operator. (2013) reported that embryo and fetal heart beats are first visible from Days 25 to 29 based on findings of different studies. Fig. 4 : Sonograph showing the embryonic heart beats as a flickering on the screen by day 26 of pregnancy in a Surti buffalo. Fig. 3 : Sonograph showing the presence of embryo as an echoic structure on Day 30 of pregnancy surrounded by anechoic amniotic fluid in a Surti buffalo. Fetal heart beat The fetal heart could be visualized by Day 26 of gestation. It appeared as a flickering structure on the screen and the beats could be measured from Day 34 (Fig. 4) onwards. Fetal heart beats could be visualized and recorded till Day 90 of gestation. Fetal heart beats increased significantly (P<0.05) from Day 42 compared to Day 34 and 38 of gestation and remained elevated till Day 62 of gestation. Thereafter, from Day 66 the fetal heart beats returned to values that were nonsignificantly higher compared to heart beats on Day 34. The fetal heart beats could not be visualized beyond Day 90 of gestation as the fetus was located more cranially in abdominal cavity beyond the reach of transducer. Pawshe et al. (1994) observed fetal heart beats of 203.8±9.0 on day 29.6±1.57 and the heart beats decreased to 150 beats per minute on day 62. The variations in fetal heart beat may be due to breed or individual difference. Pawshe and Purohit Indian J. Vet Sci. Biotech (2018) Vol. 14 No. 2 Fig. 5 : Sonograph demonstrating the first measurement of CRL on Day 30 of pregnancy in a Surti buffalo. Crown rump length (CRL) of the fetus The fetus was seen as elongated hyperechoic structure surrounded by hypo-echoic fluid in the lumen of uterus. The crown rump length (CRL) of the embryo and/or fetus could be measured using inbuilt callipers starting from Day 30 till Day 58 of gestation. The CRL was 11.41±1.56 mm on Day 30 (Fig. 5) and this increased sequentially for the days of gestation evaluated. Compared to CRL length on Days 30 and Day 34 the increase in CRL was significant (p<0.05) on Days 38 and Day 42. There was further gradual and significant (P<0.01) increase in the CRL on Days 46 and 50, and again on Day 54 and Day 58 compared to previous days records to attain a CRL of 46.34±2.73 mm on 41 Table 1: Crown rump length (Mean ±SE) of embryo/fetus during different days of gestation in Surti buffaloes Days of Gestation (days) 26 30 34 38 42 46 50 54 58 CRL (mm) 11.41±1.56a 11.5±0.84a 13.57±1.10a 17.2±0.73b 18.01±0.86b 28.30±0.96c 29.15±1.72c 41.27±2.16d 46.34±2.73d Mean values with different superscripts (a, b, c, d, e) differ significantly (P<0.05) Day 58 (Table 1). After day 60, image of complete fetus could not be scanned due to large size of fetus that exceeded the scanning range of transducer used. Pawshe et al. (1994) recorded increase in CRL of the embryo from 4.2 ± 0.89 to 53.6 ± 2.11 mm from Day 19 to Day 62 in buffaloes. Groza et al. (2012) recorded CRL on Day 40 as 50 mm which increased to 120 and 150 mm on Days 60 and 80, respectively, in buffaloes. They recorded much greater CRL on Day 60 than that of present finding on Day 58 (120 vs. 46.34 mm), which may be due to position of the fetus, expertise of operator, ultrasound instrument and breed. Acknowledgement The help extended by the Dr. Dinesh Jhamb, Assistant Professor, CVAS, Navania is thankfully acknowledged. Conflict of Interest The authors have no conflicts of interest. References: Ali, A. and Fahmy, S. (2008). Ultrasonographic fetometry and determination of fetal sex in buffaloes (Bubalus bubalis). Anim. Reprod. Sci., 106: 90-99. Awasthi, M.K., Khare, A., Kavani, F.S.,Siddiquee, G.M., Panchal, M.T. and Shah, R.R. (2006). Is one-wave follicular growth during the estrous cycle a usual phenomenon in water buffaloes Indian J. Vet Sci. Biotech (2018) Vol. 14 No. 2 (Bubalus bubalis)? Anim. Reprod. Sci., 92: 241253. Baruselli, P.S., Mucciolo, R.G., Visintin, J.A., Viana, W.G., Arruda, R.P., Madureira, E.H., Oliveira, C.A. and Molero-Filho, J.R. (1997). Ovarian follicular dynamics during the estrous cycle in buffalo (Bubalus bubalis). Theriogenology, 47: 1531-1547. Ferreira, J.C.P., Martin, I. and Irikura, C.R. (2011). Ultrasonographic monitoring of early pregnancy development in Murrah buffalo heifers (Bubalus bubalis). Livestock Sci., 138: 174-179. Groza, I., Tomai, G. and Cenariu, M. (2012). Ultrasonography, a modern tool for pregnancy diagnosis and identification of fetal structures in domestic buffaloes. Bull UASVM Vet. Med., 69: 1-9. Herrera, P., Campo, E. and Denis, R. (2007). Relationship between the size of fetal structures and time of pregnancy in river buffaloes. Rev. Salud. Anim., 29: 28-31. Karen, A., Darwish, B. and Raman, A.D. (2007). Accuracy of ultrasonography and pregnancy associated glycoprotein test for pregnancy diagnosis in buffaloes. Theriogenology, 68:11501155. Naseer, Z., Ahmed, M., Khan, M.I.R., Ahmed, E., Tahir, M.Z. and Singh, J. (2012). Effect of GnRH and estradiol benzoate on follicular wave emergence, estrus, ovulation and pregnancy rate in CIDR treated Nili Ravi buffaloes. J. Anim. Plant Sci., 22: 142-146. Pawshe, C.H., Patil, S.R., and Ingawale, M.V. (2011). Ultrasonography a diagnostic tool for detection of early pregnancy and study of embryonic development in buffalo and cattle. Intas Polivet, 12: 1-4. Pawshe, C.H. and Purohit, G.N. (2013). Approaches for diagnosis of pregnancy in female buffaloes. In: Bubaline Theriogenology, Purohit GN (Ed) www.ivis.org A5708.0613. Pawshe, C.H., Apparao, K.B.C. and Totey, S.M. (1994). Ultrasonographic imaging to monitor early pregnancy and embryonic development in buffalo (Bubalus bubalis). Theriogenology, 41: 697-709. Purohit, G.N. and Rao, T.K.S. (2018). Estrus detection in buffaloes. In: Bubaline Theriogenology , Purohit G.N. (Ed.). International Veterinar y Information Service, Ithaca NY (www.ivis.org), Last updated: 12-Mar-2018; A5704.0318 42 Rabidas, S.K. and Gofur, R.M. (2017). Synchronization of estrus using Ovsynch protocol and fixed timed artificial insemination in indigenous dairy buffaloes: An effective buffalo breeding program in Bangladesh. Asian J .Biol., 2: 1-8. M.J.C. and Campanile, G. (2010). Corpus luteum function and pregnancy outcome in buffaloes during the transitionperiod from breeding to nonbreeding season. Reprod. Domest. Anim., 45: 988-991. Russo, M., Luise, L., Rossi, P., Grasso, F.L., Cocchia, N. and Catone, G. (2009). Ultrasonographic appearance of early embryonic mortality in buffalo (Bubalus bubalis). Italian J. Anim. Sci., 8(sup 2): 138-140. Sharma, R.K., Singh, J.K. and Khanna, S. (2012). Fetal age determination in Murrah buffaloes from day 22 through 60 with ultrasonography. Indian J. Anim. Sci., 82: 374-376. Russo, M., Vecchio, D., Neglia, G., Pacelli, C., Prandi, A., Gasparrini, G., Zicarelli, L., D-Occhio, Snedecor, G.W. and Cochran, W.G. (1994). Statistical methods. 8th edn. Oxford & IBH Publishing Co., Calcutta, India. ❏ Indian J. Vet Sci. Biotech (2018) Vol. 14 No. 2 43 The Indian Journal of Veterinary Sciences & Biotechnology (2018) Volume 14, Issue 2, 44-48 ISSN (Print) : 2394-0247 : ISSN (Print and online) : 2395-1176, abbreviated as IJVSBT 10.21887/ijvsbt.14.2.10 MTNR-1A Gene Polymorphism in Two Tropical Sheep breeds of South India C.R. Vibha, Nagaraja Ramakrishnappa, G.S. Naveen Kumar, R. Jayashree, B.M. Ravindranath Department of Animal Genetics and Breeding, Veterinary College, Bengaluru Publication Info Article history: Received : 25-08-2018 Accepted : 15-09-2018 Published : 17-10-2018 Key Words: Ovine MTNR1A, PCR-RFLP, RsaI-RFLP, Mandya sheep, NARI -Suwarna sheep. *Corresponding author: gsnaveenkumar@yahoo.com This work is licensed under the Creative Commons Attribution International License (http:// creativecommons.org/licenses /by/4.0/P), which permits unrestricted use, distribution and reproduction in any medium, provided the original work is properly cited. Abstract The present study was carried out to evaluate the genetic polymorphism in Ovine MTNR1A of two tropical breeds ie., highly prolific NARI Suwarna sheep and Mandya sheep from southern peninsular region of India with respect to differential fertility. Fifty animals from each breed were randomly selected and subjected to RsaI-RFLP analysis of exon-2 of Ovine MTNR1A. The study revealed monomorphic patterns in all the animals for both the breeds. PCR amplicon of 824 bp fragments with four cut sites at 53 bp, 320 bp, 343 bp and 754 bp, resulted in 411 bp, 267 bp, 70 bp, 53 bp and 23 bp. Only presence of CC genotypes at rs406779174 SNP of Ovine MTNR1A in both the breeds supports non-seasonality in breeding of these animals and no association of SNP with increased fecundity of NARI Suwarna sheep over Mandya sheep. Copyright @: 2018 by authors and SVSBT. Introduction Reproductive efficiency in sheep often varies among the breeds, which is largely influenced by genetic and environmental factors. Seasonal variation in reproductive activity is controlled by photoperiod in sheep (Thiery et al., 2002). The non-seasonal sheep breeds exhibit reproductive responses to improved nutrition irrespective of photoperiod, whereas the seasonal breeds respond to nutrition only during breeding season (Hotzel et al., 2003). The endocrine signal, circadian rhythm of melatonin secretion is responsible for mediating the effects of photoperiod on the hypothalamo pituitary axis and plays an important role in regulation of Indian J. Vet Sci. Biotech (2018) Vol. 14 No. 2 seasonal reproduction (Gall et al., 2002). Reproductive effects of melatonin are mediated via ligand specific guanine nucleotide-binding protein (G-protein) coupled receptors located in the hypophyseal pars tuberalis (Ebisawa et al., 1994). Two melatonin receptor subtypes: Mel1a (or MTNR1A) and Mel1b (or MTNR1B) have been characterized in mammals (Reppert et al., 1994). Melatonin receptor 1A gene (MTNR1A) is located on chromosome 26 of the sheep genome. It consists of two exons and an intronic sequence of about 8 kb in length (Reppert et al., 1994). Exon 1 codes for the first transmembrane domain and the first intracellular loop while exon 2 codes 44 for the remaining part of the receptor, which has been extensively reported in the literature (Barrett et al., 1997). The MTNR1A has been identified as a candidate gene which plays a key role in the photoperiodic control of seasonality mediated by the circadian changes in melatonin concentrations (Carcangiu et al., 2011 and Pelletier et al., 2000). Mel1a has been reported to be present in small ruminants (Migaud et al., 2002). Several studies have reported relationships of MTNR1A with seasonal reproduction activity (Pelletier et al., 2000 and Carcangiu et al., 2011). Since Mandya and NARI Suwarna are two tropical sheep breeds from southern peninsular region of India with varied reproductive efficiency producing single and multiple lambs per lambing, respectively, the present study was carried out to elucidate the polymorphism of MTNR1A in Mandya and NARI Suwarna sheep and its role in differential fertility. Materials and methods Fifty unrelated Mandya sheep were randomly selected from the flock maintained at Livestock Information and Research Center (Mandya sheep), Nagamangala and farmer’s flock from the breeding tract and fifty unrelated NARI Suwarna sheep were randomly selected from the flock maintained at regional campus, KVAFSU, Bengaluru. In the beginning about 9 ml of venous blood was collected through jugular vein puncture in vacutainer tubes containing EDTA (1.5 mg/ml). Blood samples were stored at 4 °C till further processing. Genomic DNA was isolated by following Miller’s high salt method (Miller et al., 1988). The quality and quantification of the DNA was ascertained by electrophoretic analysis using 0.8% agarose gel. A standard procedure of PCR was adopted to amplify the exon-2 segment of the ovine MTNR1A gene from the genomic DNA template. The primer sequences previously published by Reppert et al. (1994) were utilized. The sequence of the forward primer was 5’ TGT GTT TGT GGT GAG CCT GG 3’ and that of the reverse primer was 5’ ATG GAG AGG GTT TGC GTT TA 3’. The PCR was carried out in 25 μl reaction mixture containing 1 μl each of forward and reverse primers of 10 pM/ μl, 1 μl of genomic DNA of 100 ng/ μl, 12.5 μl of 2X Red PCR Master Mix and 9.5 μl of nuclease free water. The PCR cyclic Indian J. Vet Sci. Biotech (2018) Vol. 14 No. 2 conditions were: initial denaturation at 94 °C for 5 min followed by 35 cycles of denaturation (94 °C for 1 min), annealing (61 °C for 45 sec) and extension (72 °C for 2 min) and final extension at 72 °C for 8 min. The PCR amplification was confirmed on 1.5% agarose gel. The restriction enzyme digestion was carried out in 15 µl reaction mixture containing autoclaved triple distilled water 8.5 µl, 10 x Assay buffer for RE 1.0 µl, RsaI (10 U/ µl ) 0.5 µl and PCR product 5.0 µl. The reaction mixture was incubated at 37 °C for 10 minutes. The restriction enzyme digested PCR products were electrophoresed on 3% percent agarose gel. The restriction pattern resolved by agarose gel electrophoresis was photographed and analyzed using Gel Documentation System (Bio Rad Molecular imager Gel Doc XR+, USA). Results and discussion Melatonin exerts circadian rhythms and reproduction changes in seasonally reproducing mammals through binding to high-affinity, Gprotein coupled receptors. In the present study PCR–RFLP technique was employed to verify the polymorphism in exon-2 of Ovine MTNR1A gene in two tropical sheep breeds with varied fertility. PCR-RFLP analysis of the MTNR1A exon-2 resulted in a similar banding pattern in all the samples studied from both Mandya and NARI Suwarna sheep breeds indicating absence of polymorphism at rs406779174 SNP in the exon-2 of MTNR1A gene sequence (Fig.1). Polymorphism at rs406779174 SNP is attributable to the presence or absence of second RsaI cleavage site in the 824 bp PCR amplicon. The presence of nucleotide C at 322 bp position of the amplified product creates restriction site, resulting in five DNA fragments of 411bp, 267 bp, 70 bp 56 bp and 23 bp and is considered as allele ‘R’, whereas the presence of nucleotide T at 322 bp position of the amplified product resulting in absence of RE cut site resulting in 411 bp, 290 bp, 70 bp and 56 bp DNA fragments and is considered as allele ‘r’ (Meena et al., 2013). In the present study electrophoretic analysis revealed clear bands of 411 bp and 267 bp and faint bands of 70 bp and 56 bp indicating presence of only one allele in homozygous state in all the animals (Fig.2). 45 Figure 1: RE mapping and Nucleotide sequence of PCR amplified exon-2sequence of MTNR1A gene in Mandya and NARI Suwarna sheep 1 2 3 4 411bp 267bp 5 6 7 8 500bp 300bp 100bp Figure 2: RsaI RFLP of exon-2 of MTNR1A gene in NARI Suwarna and Mandy sheep exhibiting monomorphic pattern. Indian J. Vet Sci. Biotech (2018) Vol. 14 No. 2 46 It is evident from the literature that rs406779174 SNP of Ovine MTNR1A exon-2 resulted in all the possible genotypes viz., RR (CC), Rr (CT) and rr (TT) in several sheep breeds. In the sheep breeds of temperate regions the frequency of allele R was low to moderate and ranged from 0.04 in Suffolk breed (Chu et al., 2006; Ioannis et al., 2016)) to 0.58 in Sarda sheep breed (Mura et al., 2014). India has extraordinary variety of climatic regions, ranging from tropical in the south to temperate and alpine in the Himalayan north. The frequency of allele R for hilly temperate breeds Sandya and Nilgiri was 0.46 and 0.56 respectively and for Malpura and Patanwadi breeds from dry aired regions of Rajasthan was 0.93 and 0.72 respectively (Saxena et al., 2015). In the present study lack of polymorphism at RsaI/MTNR1A gene and presence of only R type of allele in Mandya sheep may be attributable to its higher acclimatization to tropical climate. Similarly, no evidence for polymorphism of RsaI/MTNR1A gene observed in NARI Suwarna sheep in spite of it being developed from Garole sheep with moderate frequency of both the alleles. The probable reason for absence of polymorphism in NARI Suwarna sheep may be attributable to the fact the breed is Deccani sheep with only introgression of Fec-B gene from Garole. (Nimbkar et al., 2009). Further, the absence of polymorphism in both highly prolific NARI Suwarna sheep as well as Mandya sheep indicates no association of SNP with increased fecundity of NARI Suwarna sheep. In conclusion the present study revealed the absence RsaI/MTNR1A polymorphism of exon2 sequence in both Mandya and NARI Suwarna sheep. Only one genotype was observed in all the animals studied. However, the present observation needs to be revalidated involving a larger sample size and more number of south Indian tropical sheep breeds. References: Barrett, P., Conway, S., Jockers, R., Strosberg, A.D., Guardiola-lemaitre, B., Delagrange, P. and Morgan, P.J. (1997). Cloning and functional analysis of a polymorphic variant of the ovine Mel 1a melatonin receptor. Biochem. Biophys.Acta.,1356: 299-307. Carcangiu, V., Luridiana, S., Vacca, G.M., Daga, C. and Mura, M.C. (2011). A polymorphism at the Indian J. Vet Sci. Biotech (2018) Vol. 14 No. 2 melatonin receptor 1A (MTNR1Aa) gene in Sarda ewes affects fertility after AI in the spring. Reprod. Fertil. Dev., 23: 376–380. Chu, M.X., Cheng, D.X., Liu, W.Z., Fang, L. and Ye, S.C. (2006). Association between melatonin receptor 1A gene and expression of reproductive seasonality in sheep. Asian-Austral. J. Anim. Sci., 19: 1079–1084. Ebisawa, T., Karne, S., Lerner, M.R. and Reppert, S.M. (1994). Expression cloning of a high-affinity melatonin receptor from Xenopusdermal melanophores. Proc Natl Acad Sci., 91: 61336137. Gall, C.V., Stehle, G.H. and Weaver, D.R. (2002). Mammalian melatonin receptors: molecular biology and signal transduction. Cel. Tiss. Res., 309: 151–162. Hotzel, M.J., Walkden-Brown, S.W., Fisher, J.S. and Martin, G.B. (2003). Determinants of the annual pattern of reproduction in mature male Merino and Suffolk sheep: responses to a nutritional stimulus in the breeding and non-breeding seasons. Reprod. Fertil. Dev., 15: 1–9. Ioannis, A., Giantsis, George, P., Laliotis, Olympia, S. and Melpomeni, A. (2016). Polymorphism of the melatonin receptor 1A (MNTR1A) gene and association with easonality of reproductive activity in a local Greek sheep breed. J of Biol ResThessaloniki, 23: 9. Meena, A.S., Kumar, R., Kumari, R., Jyotsana, B., Prince, Lii. and Kumar, S. (2013). Genetic polymorphism of melatonin receptor 1A (MTNR1A) gene in Indian sheep breeds. Indian J. of Ani. Sci., 83: 1284–1287. Migaud, M., Gavet, S. and Pelletier, J. (2002). Partial cloning and polymorphism of the melatonin 1a (Mel1a) receptor gene in two breeds of goat with different reproductive seasonality. Reproduction., 124: 59-64. Miller,S.A., Dykes, D.D. and Polesky, H.F. (1988). A simple salting out procedure for extracting DNA from human nucleated cells. Nucleic Acids Res., 16(3): 1215 Mura, M.C., Luridiana, S., Bodano, S., Daga, C., Cosso, G. and Diaz, M.L. (2014). Influence of melatonin receptor 1A gene polymorphism on seasonal reproduction in Sarda ewes with different body condition scores and ages. Anim. Reprod. Sci., 149: 173-177. 47 Nimbkar, C., Ghalsasi, P.M., Nimbkar, B.V., Ghalsasi, P.P., Gupta, V.S., Pardeshi, V.C., Maddox, J.F., Van Der Werf, J.H, Gray, G.D., Walkden-Brown, S.W. (2009). Biological and economic consequences of introgression of the Fec-B gene into Deccani sheep. Pelletier, J., Bodin, L., Hanocq, E., Malpaux, B., Teyssier, J., Thimonier, J. and Cheminea, U. P. (2000). Association between expression of reproductive seasonality and alleles of the gene Mel1a receptor in the ewe. Biol. Reprod., 62: 1096–1101. Reppert, S.M., Weaver, D.R. and Ebisawa, T. (1994). Cloning and characterization of a mammalian Indian J. Vet Sci. Biotech (2018) Vol. 14 No. 2 melatonin receptor that mediates reproductive and circadian responses. Neuron, 13: 1177-1185. Saxena, V.K., Jha, B.P., Meena, A.M. and Naqvi, S.M.K. (2015). Characterization of MTNR1A gene in terms of genetic variability in a panel of subtemperate and subtropical Indian sheep breeds. Journal of Genetics, 94(4): 715-721. Thiery, J.C., Cheminea, U.P., Hernandez, X., Migaud, M. and Malpaux, B. (2002). Neuroendocrine interactions and seasonality. Domest. Anim. Endocrinol., 23: 87-100. ❏ 48 The Indian Journal of Veterinary Sciences & Biotechnology (2018) Volume 14, Issue 2, 49-51 ISSN (Print) : 2394-0247 : ISSN (Print and online) : 2395-1176, abbreviated as IJVSBT 10.21887/ijvsbt.14.2.11 Comparative evaluation of Egg Quality Parameters of Kadaknath and Vanaraja Chicken in Intensive Farming System S. S. Pathak*, U. Tamuli1, S. Khargharia2, G. Bordoloi3, L. S. Khuman4, P. Chabukdahara5, A. Gogoi6, P. Rajbongshi7, K. Saikia8, K. Boruah9s Department of Veterinary Extension Education,2 Veterinary Pharmacology & Toxicology,3 Veterinary Parasitology, 5 Veterinary Physiology & Biochemistry,6 Veterinary Animal Genetics & Breeding, 7 Livestock Farm Complex, 8,9 Livestock Production Management, Lakhimpur College of Veterinary Science, AAU, Joyhing, Lakhimpur-787051, India 1,4 Publication Info Article history: Received : 09-07-2018 Accepted : 29-08-2018 Published : 17-10-2018 Key Words: Kadaknath, Vanaraja, chickens, egg quality, deep litter *Corresponding author: siddharthapathak83@gmail.com Abstract In the present study , a total of 60 eggs were assessed for egg quality parameters of Kadaknath and Vanaraja chicken reared under deep litter farming system . All chickens were provided uniform diet of Chick (0-8 weeks), Grower (9-20 weeks) and Layer (above 20 weeks) ration. Except for the shape index, the internal egg quality parameters were significantly (P<0.05) higher in Vanaraja eggs as compared to Kadaknath. Results revealed that the quality of egg of Vanaraja chicken was found to be better as compared to Kadaknath chicken. This work is licensed under the Creative Commons Attribution International License (http:// creativecommons.org/licenses /by/4.0/P), which permits unrestricted use, distribution and reproduction in any medium, provided the original work is properly cited. Copyright @: 2018 by authors and SVSBT. Introduction Next to the cow milk egg is the only animal origin balanced food which is consumed and relished by majority of the population of the world (Baishya et al., 2008). In India, Per capita availability of eggs is only 69 while Indian Council of Medical Research recommendation is 182 (Amandeep Singh, 2018). The Kadaknath breed, also known as Kalamashi in Hindi, is known for its black-coloured meat. It is native of Jhabua and Dhar districts in the western region Indian J. Vet Sci. Biotech (2018) Vol. 14 No. 2 of Madhya Pradesh and in adjoining areas of Gujarat and Rajasthan states (Valavan et al., 2016). The meat is known for its medicinal characteristics, fetching a higher demand among the consumers. Vanaraja, a dual purpose variety developed by Directorate of Poultry research, Hyderabad, India is highly popular among the rural people of Assam as one of the income generating source especially for the rural women. The success of poultry farming largely depends on the total number of good quality eggs 49 produced especially in layers and dual purpose birds (Niranjan et al., 2008). The evaluation of external and internal quality of the egg is helpful to the consumers. Hence an attempt was made to compare the egg quality of Kadaknath and Vanaraja reared in intensive system of management. Materials and Methods The present study was conducted on the Poultry farm maintained at the Department of Livestock Farm Complex, Lakhimpur College of Veterinary Science, Assam Agricultural University. Thirty chickens each of Kadaknath and Vanaraja were included in the present study. The experimental chickens were fed uniform diet of Chick (0-8 weeks), Grower (9-20 weeks) and Layer (above 20 weeks) ration as per BIS (1992) throughout the rearing period. The external egg quality parameters observed were egg weights and shape index. The egg quality parameters like shell colour, shell thickness, egg weight, shape index, albumen index, yolk index, yolk weight and albumen weight were estimated as per method described by Singh and Panda ( 1987). Haugh unit score, a measure of internal quality of egg was also computed (Kondaiah et al., 1983). The values obtained were subjected to statistical analysis (Snedecor and Cochran, 1994). Results and Discussion In the present investigation results indicated that most of the studied egg quality parameters viz Egg weight, shell thickness, albumen index, Haugh unit, Yolk Index, Yolk weight and Albumen weight are found to be significantly (Pd”0.05) higher in Vanaraja, while, data for shape index is found significantly higher in Kadaknath (Table 1). The smaller egg size/ weight of the Kadaknath eggs is in agreement with the findings of Mohan et al., (2008), who reported that the average egg weight at 21 weeks was 32.50 ± 0.70 g. The higher egg weight in vanaraja chicken might be due to utilization of exotic germplasm for the development of these rural varieties (Sharma et al., 2006). A significantly (Pd”0.05) higher shape index in Kadaknath chicken eggs is in agreement with the reports of Parmar et al., (2006) and Valavan et al., (2016). The higher shape index Indian J. Vet Sci. Biotech (2018) Vol. 14 No. 2 Table 1 Egg quality parameters of Kadaknath and Vanaraja chicken reared in deep litter system of management Parameters Egg Weight (g) Shape index % Shell Thickness (mm) Albumen Index Haugh Unit Yolk Index Yolk weight (g) Albumen weight(g) Kadaknath eggs 36.37a ± 0.59 76.819b ± 0.417 0.318a ± 0.005 0.079a ± 0.035 71.910a ± 0.472 0.352a ± 0.261 13.722a ± 0.161 21.823a ± 0.241 Vanaraja eggs 42.30b ± 0.87 69.000a ± 0.494 0.382b ±0.010 0.128b ± 0.006 74.703b ± 0.779 0.591b ± 0.004 16.750b ± 0.209 25.200b ± 0.467 Means having same superscript do not differ significantly (Pd”0.05) value of indigenous chicken might be due to broad and short size and shape of the eggs (Pathak et al., 2015). The higher shell thickness in the birds developed for backyard poultry keeping was a better indication for their suitability in rural backyard farming (Sinha et al., 2017). The indices for Kadaknath chicken is in line with the reports of Valavan et al. (2016), this might be attributed to the difference in egg weight which has a positive correlation with the albumen index (ªekeroìlu and Altunta, 2009 and Sinha et al., 2017 ) and the influence of genetic group on the yolk index values (Rajkumar et al., 2009 and Momoh et al., 2010). Higher egg weight in Vanaraja may be due to higher yolk and albumen weight . Sinha et al. (2017) reported that the average albumen and yolk weight increased gradually with the increase in egg weight. The significantly (Pd”0.05) higher Haugh unit values for vanaraja eggs might be attributed to improved quality of the albumen and increase in height of the albumen. Sinha et al. (2017) revealed that the albumen height subsequently increases with the increase in egg weight. The Haugh unit for Kadaknath eggs recorded were comparable to the findings of Valavan et al. (2016) and Parmar et al. (2006). A wide variation in Haugh unit values for Kadaknath birds, ranging from 62.58 to 90.00 for eggs collected under field conditions was recorded by Parmar et al., (2006). Acknowledgments The authors are highly thankful to Assam 50 Agricultural University, Jorhat, Assam for providing the facilities. The author is also thankful to the I/C HOD, Department of Livestock Farm Complex, Lakhimpur College of Veterinary Science, Assam Agricultural University, Joyhing, Lakhimpur-787051, India. Conflict of Interest All authors declare no conflict of interest. References: Amandeep, Singh. (2018). Current livestock Production and statistics of India. http:// www.vetextension.com/current-livestock-animalhusbandry-statistics-india/ Baishya, D., Dutta, K. K., Mahanta, J. D. and Borpujari, R. N. (2008). Studies on Certain Qualities Of Different Sources Of Chicken Eggs. Tamil Nadu J. Vety., & Anim. Sci. 4 (4): 139-141. BIS.1992. Bureau of Indian Standard of poultry feed. Manik Bhawan, Bahadur Shah Zafer Marg, New Delhi. Kondaiah, N., Panda, B. and Singhal, R. A. (1983). Internal egg quality measure for quail eggs; Indian J. Anim. Sci. 53: 1261-64. Mohan, J., Sastry, K. V. H., Moudgal, R. P. and Tyagi J. S. (2008). Performance profile of Kadaknath desi hens under normal rearing system. Int. J. Poult. Sci. 43:379–381. Momoh, O. M., Ani, A.O. and Ugwuowo, L. C. (2010). Part-period egg production and egg quality characteristics of two ecotypes of Nigerian local chicken and their F1 crosses. Int. J. Poult. Sci., 9 (8): 744-748. Niranjan, M., Sharma, R. P., Rajkumar, U., Chatterjee, R. N., Reddy, B. L. N. and Bhattacharya, T. K. (2008). Egg quality traits in chicken varieties developed for backyard poultry farming in India. Livest. Res. for Rural Dev. 20 (12). Indian J. Vet Sci. Biotech (2018) Vol. 14 No. 2 Parmar, S. N. S., Thakur, M. S., Tomar, S. S., and Pillai, P. V. A. (2006). Evaluation of egg quality traits in indigenous Kadaknath breed of poultry. Livest. Res. for Rural Dev.18 (9). Pathak, S.S., Barua, N. and Kalita, N. (2015). Comparison of Egg Quality Characteristics of Indigenous and Broiler Parent Line crossed with Indigenous chicken. Indian J. Vet Sci. Biotech. 11(3): 12-16. Rajkumar, U., Sharma, R.P., Rajaravindira, K. S., Niranjan, M., Reddy, B.L.N., Bhattacharya, T.K. and Chaterjee, R.N. (2009). Effect of genotype and age on egg quality traits in Naked neck chicken in Tropical climate from India. Int. J. of Poult. Sci. 8(12): 1151-1155. Sekeroglu, A. and Altunta, E. (2009). Effects of egg weight on egg quality characteristics. J. Sci. Food Agric. 8(3): 379-383. Sharma, R. P., Chatterjee, R. N. and Niranjan, M. (2006). Poultry production under backyard system: Improvement approaches. In: National symposium on conservation and improvement of animal genetic resources under low input system; Challenges and strategies, NBAGR, Karnal. PP 72-77 Singh, R. P. and Panda, B. (1987). Effect of seasons on physical quality and component yields of eggs. Indian J. Anim. Sci. 57 : 50-55. Sinha, B., Mandal, K. G. and Kumar, R. (2017). Effect of Egg Weight on Egg Quality Traits of Laying Hens. Int. J. Pure App. Biosci., 5(3): 293-300. Snedecor, G. W. and Cochran W. C. (1994). Statistical methods; 8th edition. Iowa state University Press, Ames, Iowa. Valavan, Ezhil. S. A., Omprakash, V. and Bharatidhasan, A. (2016). Production Performance of Kadaknath in an organized Poultry Farm. Int. J. Appl. Pure Sci. Agric., 02 (11):125-128. ❏ 51 The Indian Journal of Veterinary Sciences & Biotechnology (2018) Volume 14, Issue 2, 52-57 ISSN (Print) : 2394-0247 : ISSN (Print and online) : 2395-1176, abbreviated as IJVSBT 10.21887/ijvsbt.14.2.12 Pathological Studies on Acute Marek’s Disease in Native and Crossbred Chickens of Organized Poultry Farm Kinnari R. Makwana*, B. P. Joshi, D. J. Ghodasara, C. J. Dave, Monika P. Patel Department of Veterinary Pathology, College of Veterinary Science & Animal Husbandry, AAU, Anand, Gujarat, India Publication Info Article history: Received : 22-09-2018 Accepted : 15-10-2018 Published : 17-10-2018 Key Words: Marek’s disease, Poultry, Visceral lymphomas. *Corresponding author: kinnarimakwana94@gmail.com This work is licensed under the Creative Commons Attribution International License (http:// creativecommons.org/licenses /by/4.0/P), which permits unrestricted use, distribution and reproduction in any medium, provided the original work is properly cited. Abstract The present study was aimed to detect the occurrence of Marek’s disease in a native and cross-bred chickens based on gross and microscopic lesions. Seven different native as well as crossbred strains of poultry flocks were evaluated the mortality pattern based on farm as well as autopsy records and for the gross and microscopic examination of the birds suspected for Marek’s disease. Visceral lymphomas like lesions were recorded in liver, spleen, proventriculus, heart, kidney and gonads. Fragments of the affected organs were collected and placed in 10% formalin for routine histopathology. Under microscopic examination, pleomorphic population of lymphocytes and lymphoblasts were observed. The incidence of MD observed was from 12 to 61 weeks of age with peak mortality from 12 to 31 weeks of age during summer in different strains. The strain wise mortality due to MD ranged between 13.42 (random bred controls) to 36.57% (IWN×Native, F1) with an average of 26.80 %, and it was higher in female than male birds. Copyright @: 2018 by authors and SVSBT. Introduction Marek’s Disease (MD) is a lymphoproliferative and neuropathic disease of domestic chickens and less commonly of turkeys and quails, caused by a highly contagious, cell-associated, oncogenic herpes virus (Schat and Nair, 2008). Marek’s Disease Virus (MDV) strains that induce disease in chicken are classified as serotype-1 under alpha-herpesvirus. The disease is usually characterized by mononuclear cellular infiltration in peripheral nerves and various other organs and tissues including iris and skin. Marek’s disease virus spreads rapidly throughout flocks by direct Indian J. Vet Sci. Biotech (2018) Vol. 14 No. 2 and indirect contact with infected chicken, premises, litter, dust and chopped feathers. Most important is the airborne route of infection (Josipovic, 1990). This study was aimed to describe the epidemiology of Marek’s disease based on gross and microscopic lesions, i.e. visceral lymphoma, in seven different native and crossbred chicken strains maintained at University farm in Anand and received for post-mortem in Pathology department over a period of one year. Materials and Methods The present study was carried out for a period of one year, i.e. from February 2017 to 52 January 2018, on seven different native as well as crossbred strains, viz., Anand Specific White Leghorn (ASWLH), Bantomized White Leghorn (BWLH), Rhode Island Red (RIR), Native, RC (Random breed Control), F1 (IWN×Native) (First generation cross), TC (F1×RIR) (Terminal Cross) and Indian White Native (IWN) of poultry flocks maintained at Poultry Research Station, AAU, Anand and received for post-mortem diagnosis and suspected for Marek’s disease based on gross pathological lesions, i.e. visceral lymphoma. The study comprised the epidemiological information in relation to mortality due to MD with respect to strain, age, sex, season and month wise susceptibility, gross and histopathological examination of different organs. Epidemiological Study The information pertaining to the strength of different native and crossbred strains, and age and sex wise as well as month wise mortality due to Marek’s disease was collected based on records available at Poultry Research Station of the College. The necropsy incidence of Marek’s disease from February-2017 to January-2018 was worked out based on necropsy records available at Department of Pathology. Gross Lesions and Histopathology Regular autopsies were conducted on the birds of different strains. Detailed post-mortem examination was carried out and gross pathological lesions observed were recorded giving special attention to visceral lymphoma. During autopsy, the tissue pieces of organs showing gross lesions of Marek’s disease like liver, spleen, heart, proventriculus, kidney, gonads and sciatic nerve were collected for histopathological examination. Tissues were preserved in 10% formalin for further processing. The trimmed tissues were processed in automatic tissue processor and sections of 5 to 6 micron thickness were cut with automatic section cutting machine (Leica, Germany). The sections were stained with haematoxylin and eosin stain and histopathological lesions were observed using light microscope. Results and Discussion Epidemiological Studies between mortality summer different 12 to 61 weeks of age with peak between 12 to 31 weeks of age during months of March to June 2017 in strains. The strain wise mortality due to MD ranged between 13.42 to 36.57 % with an average of 26.80 %. Out of total 4,498 birds of different strains, 1203 birds were died due to Marek’s disease. Among the total population of different strains, the highest mortality was observed in IWN×Native (F1) (36.57%) followed by Native (32.09%), BWLH (30.08%), F1×RIR (TC) (25.29%), ASWLH (25.18%), RIR (23.64%) and RC (13.42%) [Fig. 1]. Among the birds died due to MD, the mortality pattern was of the order of Native (24.94%) followed by BWLH (18.20%), RIR (18.04%), ASWLH and F1 (14.38%), RC (6.40%), and TC (3.66%). The age wise mortality pattern revealed that the mortality due to MD started from 12 weeks onwards and lasted till 61 weeks of age. The majority of birds (94.74%) belonged to all the strains died between the ages of 12 to 31 weeks of age, and overall age wise mortality ranged between 0.16 to 51.45 % among different age groups of all strains. The highest mortality due to MD was observed in the age group of 17-21 weeks (51.45%) followed by 22-26 weeks (21.94%), 12-16 weeks (15.54%), 27-31 weeks (5.81%), 32-36 weeks (2.41%), 42-46 weeks (1.25%), 37-41 weeks (0.74%), 47-56 weeks (0.33%) and 58-62 weeks (0.16%) of age [Fig. 2]. The month wise mortality pattern showed that majority of birds (95.50%) died during months of February to June 2017. The incidence decreased from July onwards till January in all the strains and amounted to 4.5% only. The highest mortality due to MD was observed during March (39.23%) followed by April (32.75%), May (10.88%), February (8.81%), June (3.82%), July (1.99%), September (1.16%), November (0.50%), August, October (0.33% each) and December and January (0.08% each). The month wise mortality incidence reflected the season wise incidence as 86.69 % during summer months, 9.47 % during winter months and 3.82 % during monsoon months. The incidence was high during hot months of summer during March to June. The present study recorded incidence of MD Indian J. Vet Sci. Biotech (2018) Vol. 14 No. 2 53 Where the population of both male and female birds was reared together, the sex wise mortality in male and female birds was 22.97 and 26.23 % in ASWLH, 24.00 and 32.38% in BWLH and 15.58 and 17.50% in native strain, respectively. lesions in the ovary were whitish nodular growths and typical cauliflower-like appearance due to tumorous masses. The testes showed marked asymmetry following unilateral or bilateral enlargement. The mortality due to MD has been reported in birds above 12 weeks of age or during peak production among layer flocks (Zhuang et al., 2015; Balasubramaniam et al., 2017). Present study also recorded incidence of MD between 12 to 61 weeks of age with peak mortality between 12 to 31 weeks of age during summer months (March to June) in all the strains. The significance of higher mortality during summer months in the present study could not be explained as the age of the flocks above 12 weeks coincided with months of February onwards till June. Gross lesions in visceral organs suggestive of MD, i.e. presence of greyish white tumorous nodules or diffuse enlargement of the organs like liver, spleen, kidneys and gonads (ovary and testes) were reported from time to time by various workers (Kamaldeep et al., 2007; AbdEllatieff et al., 2018). Significant gross lesions of numerous lymphomas of various size and greyishwhite in colour on the surface of the visceral organs and thickening of proventricular wall suggestive of Marek’s disease were observed in number of birds affected with MD. There were no gross lesions in peripheral nerves especially in brachial and sciatic nerves in any of the strains like enlargement and oedema with loss of striations. Clinical Signs Chickens with MD lymphoma initially exhibited non-specific clinical signs such as emaciation and anorexia almost in all the affected flocks which progressed to depression, weight loss, diarrhoea and paleness of comb and wattles. At later stage, there was marked reduction in egg production and skin around feather follicles was raised and roughened with enlargement of feather tips. The most severely affected birds died due to starvation and dehydration, with presence of visceral lymphomas in various organs including liver, spleen, proventriculus, heart, kidneys, ovary and testes. Gross Pathological findings The gross pathological lesions were predominantly found in various visceral organs. The gross lesions in liver and spleen varied from few large size greyish white nodules to many multiple small size nodules with little or no normal tissue visible [Fig.3, 4, 5]. The lesions in the heart were nodular growths with diffuse enlargement and single small greyish nodules commonly noticed on the ventricular muscles leading to distortion of the shape. Proventriculus revealed diffuse thickening of the wall with white nodular areas through the serosal surface and focal ulceration and inter-papillary haemorrhages on the mucosal surface [Fig. 6]. In kidneys bilateral enlargement due to lymphoma formation with nodular growths of greyish white colour involving few lobes of the kidney was observed. The gross Indian J. Vet Sci. Biotech (2018) Vol. 14 No. 2 Histopathological findings Microscopic changes were most consistently observed in tissue sections of liver, spleen and proventriculus representative of all the strains. The lesions in the hepatic parenchyma and spleen were characterized by proliferation of pleomorphic lymphoid cells, i.e. lymphoblasts and small to large lymphocytes with perivascular lymphoid proliferation in some of the sections [Fig. 7, 8]. In proventriculus, there was extensive infiltration of lymphoid cells which has replaced and distorted the mucosal and glandular architecture [Fig. 9]. Kidney sections showed extensive infiltration and proliferation of pleomorphic lymphocytes in the interstitial connective tissue accompanied by partial to complete obliteration of renal tubules of cortex and medulla. In ovary there was infiltration and proliferation of pleomorphic lymphoid cells with focal infiltration of lymphoid cells and plasma cells in the stromal tissue, whereas in testes pleomorphic lymphoid cells proliferation was observed. In the heart lesions were characterized by focal aggregation of pleomorphic lymphoid cells among the muscle fibres resulting in separation of muscle fibres noticed in the interstitial spaces [Fig. 10]. Focal to diffuse infiltration of pleomorphic 54 Fig.1. Graph showing strain wise incidence Fig.2.Graph showing age wise incidence of MD in different pure as well as cross-bred strains Fig.3.Liver showing diffuse enlargement with large sized greyish-white masses on its surface Fig.4. Spleen showing diffuse enlargement with greyish-white nodular growth Fig.5. Mottling of spleen with diffuse enlargement Fig.6.Proventriculus: focal ulceration, small to medium sized nodular growth and inter papillary haemorrhages Indian J. Vet Sci. Biotech (2018) Vol. 14 No. 2 55 Fig.7. Liver section showing extensive pleomorphic perivascular lymphocytic proliferation replacing the normal parenchyma (H & E × 120) Fig.8. Liver section showing neoplastic lymphoid cell proliferation in the hepatic parenchyma (H & E × 1000) Fig.9. Proventriculus section showing diffuse infiltration of pleomorphic lymphoid cells in glandular parenchyma (H & E × 480) Fig.10. Heart showing pleomorphic lymphoid cells proliferation in the pericardium (H & E × 480) cells in liver, spleen, heart and lungs leading to disruption of the architecture of these organs and infiltration of pleomorphic cells in mucosa, submucosa, muscularis and serosal layers of proventriculus, intestines and inter tubular spaces of renal parenchyma are the characteristic microscopic lesions commonly observed in Marek’s disease (Sharma et al., 2017). During the present study most of the gross and microscopic lesions of lymphoma were observed in visceral organs suggestive of acute visceral form of marek’s disease in native and crossbred strains reared in confinement. As the gross and microscopic lesions suggestive of MD were not evident in sciatic nerve and brain, the present outbreak of Marek’s disease was acute form of Marek’s disease without involvement of classical or neural form. Indian J. Vet Sci. Biotech (2018) Vol. 14 No. 2 Conclusion The epidemio-pathological study of Marek’s disease occurrence in seven native and crossbred strains of chicken revealed varying pattern of mortality based on farm as well as autopsy records with clear visceral lymphomas like lesions in vital organs like liver, spleen, proventriculus, heart, kidney and gonads. The incidence of MD varied between 12 to 61 weeks of age with peak mortality between 12 to 31 weeks of age during summer months. Highest mortality was seen in March (39.23%), in age group of 17-21 weeks (51.45%), in IWN×Native, (F1) strain (36.57 %), and in female birds. Acknowledgement We thank the Principal Scientist and Head, 56 Poultry Research station and Dept. of Veterinaty Pathology for providing materials and confirmatory diagnosis and authorities of the University for the facilities provided. Conflict of Interest Authors have no conflict of interest. References: Abd-Ellatieff, H.A., Abou Rawash, A.A., Ellakany, H.F., Goda, W.M., Suzuki, T. and Yanai, T. (2018). Molecular characterization and phylogenetic analysis of a virulent Marek’s disease virus field strain in broiler chickens in Japan. Avian Path., 47(1): 47-57. Balasubramaniam, A., Saravanajayam, M. and Arulmozhi, A. (2017). Recurrent incidence of Marek’s disease in native breed chickens. J. Anim. Res., 7(4): 789-791. Josipovic, D. (1990). Marek’s disease: Still a problem in poultry. L’aviculture en Mediterranee, 185-194. Kamaldeep, P.C., Sharma, C. and Narang, G. (2007). Occurrence of Marek’s disease in vaccinated poultry flocks of Haryana (India). Intl. J. Poult. Sci., 6(5): 372-377. Schat, K.A. and Nair, V. (2008). Marek’s disease. Dis. Poultry, 12: 452-514. Sharma, D., Gupta, K. and Singh, A. (2017). Studies on prevalence and pathology of mixed infections of reticuloendotheliosis and Marek’s disease under field conditions. Indian J. Vet. Path., 41(2): 146150. Zhuang, X., Zou, H., Shi, H., Shao, H., Ye, J., Miao, J. and Qin, A. (2015). Outbreak of Marek’s disease in a vaccinated broiler breeding flock during its peak egg-laying period in China. BMC Vet. Res., 11(1): 157. ❏ Indian J. Vet Sci. Biotech (2018) Vol. 14 No. 2 57 The Indian Journal of Veterinary Sciences & Biotechnology (2018) Volume 14, Issue 2, 58-59 ISSN (Print) : 2394-0247 : ISSN (Print and online) : 2395-1176, abbreviated as IJVSBT 10.21887/ijvsbt.14.2.13 In vitro efficacy of cypermethrin against Hyalomma anatolicum anatolicum L. Prasad, R.K. Bagherwal, A.K. Jayraw*1, N. Rajput2, N. Yadav, M.Shakya1 and P.Thakur 1 Department of Veterinary Parasitology 2Department of Veterinary Pharmacology & Toxicology, Department of Veterinary Medicine College of Veterinary Science and Animal Husbandry Nanaji Deshmukh Veterinary Science University, Jabalpur Mhow – 453 446 (M.P.) India Publication Info Article history: Received : 11-07-2018 Accepted : 18-08-2018 Published : 17-10-2018 Key Words: Hyalomma anatolicum anatolicum, cypermethrin, in vitro efficacy *Corresponding author: jayrawanant@yahoo.co.in Abstract The present investigation was undertaken to evaluate in vitro efficacy of cypermethrin against H. a.anatolicum. The engorged H. a.anatolicum females dropped on ground were collected from dairy farms of nearby villages of Mhow and were maintained in the laboratory till hatching of the eggs and larvae were used for further study. Larvae were placed in filter paper packets impregnated with 100, 200, 400, and 800 ppm concentrations of cypermethrin for 24 hours which revealed the per cent mortality 89.84, 93.70, 100 and 100%, while the corrected per cent mortality and efficacy was found 89.30, 93.36, 100 and 100% . This work is licensed under the Creative Commons Attribution International License (http:// creativecommons.org/licenses /by/4.0/P), which permits unrestricted use, distribution and reproduction in any medium, provided the original work is properly cited. Copyright @: 2018 by authors and SVSBT. Introduction Hyalomma anatolicum anatolicum has gained noticeable importance because of its role as the major vector of Theileria annulata in cattle. Use of acaricides is the most common method of tick control adopted by the cattle owners in India and these acaricides are applied on infested animals at frequent intervals. Indiscriminate use with incorrect concentrations of acaricides might be Indian J. Vet Sci. Biotech (2018) Vol. 14 No. 2 the cause of development of resistance against acaricides. Commonly used Cypermethrin (synthetic pyrethroid) is commercially available acaricide for tick control in India. Apart from its application against agriculturally important pests, it is also extensively used for the control of mosquitoes. The indiscriminate use of acaricides with incorrect concentrations of acaricides results in development of resistance against the acaricides. Therefore the present study was 58 conducted to assess the in vitro efficacy of cypermethrin against H. a. anatolicum. Materials and Methods The engorged H. a.anatolicum females dropped on ground were collected from dairy farms of nearby villages of Mhow. Each fully engorged female tick was placed in a test tube and these test tubes were transferred in a desiccator having 10% potassium hydroxide solution at the base and the desiccators were placed in incubator at 28 ± 1°C and 85 ± 5% relative humidity and maintained till hatching of the eggs and released larvae were used to assess the efficacy of cypermethrin as per the method described by Shyma et al. (2012) with minor modifications. Pieces of Whatsman filter paper No. 11 ( 5 x 10 cm in diameter )were used. The filter papers were impregnated with 100, 200, 400 and 800 ppm concentrations of analytical grade cypermethrin (Sigma-Aldrich). Sevenday-old hungry larvae were placed in openended packets which were transferred in desiccators. After 24 hours incubation in BOD incubator, live and dead larvae were counted and Corrected mortality (%) was calculated by using Abbortt’s formula. Results and Discussion In the present study, the per cent mortality was observed as 89.84, 93.70, 100 and 100% while the corrected per cent mortality and efficacy was recorded as 89.30, 93.36, 100 and 100% at 100, 200, 400, and 800 ppm concentration of cypermethrin, respectively (Table 1).The data revealed that per cent mortality increased with increasing the concentration of cyprtmethrin and 100% mortality was observed at 400 ppm. The efficacy of 89.30% recorded at the recommended concentration is in line with the findings of Arunachalam et al. (2007) and Sajid et al. (2009). At the same time Sangwan et al. Table 1: Efficacy of cypermethrin against H. a.anatolicum Cypermethrin (ppm) Control 100 200 400 800 Average no. of larvae Treated 135 128 143 137 126 Dead 07 115 134 137 126 Mortality (%) Corrected mortality (%) 05.18 89.84 93.70 100 100 89.30 93.36 100 100 Indian J. Vet Sci. Biotech (2018) Vol. 14 No. 2 (1993) and Singh et al. (2015) reported resistance against cypermethrin. Continuous and indiscriminate use with incorrect concentrations of acaricides results in development of resistance against these compounds, which is attributable to the reduced efficacy of cypermethrin in the present investigation. Acknowledgement Authors are thankful to the Honorable Vice Chancellor, NDVSU, Jabalpur and Dean, College of Veterinary Science and Animal Husbandry, Mhow for providing the necessary facilities to carry out the research work. Conflict of Interest All authors hereby declare that there is no conflict of interest. References: Arunachalam, K., Raman, M., Narendrababu, R. and Karunanidhi, K. (2007).Comprative evaluation of acaricides in natural tick infestation of mecheri sheep in an organized farm.Indian J. Small Ruminants, 13: 216-221. Sajid, M.S., Iqbal, Z., Khan, M.N., and Muhammad, G. (2009). In vitro and in vivo efficacies of ivermectin and cypermethrin against the cattle Hyalommaanatolicumanatolicum tick (Acari:Ixodidae). Parasitology Research, 105: 1133-1138. Sangwan, A.K., Chhabra, M.B. and Singh, S. (1993). Acaricide resistance status of common livestock ticks in Haryana. Indian Vet. J., 70: 20-24. Shyma, K.P., Kumar, S., Sharma, K.A., Ray, D.D., and Ghosh, S. (2012). Acaricide resistance status in Indian isolates of Hyalommaanatolicum . Experimental and Applied Acarology, 58: 471481. Singh N.K., Jyoti, Haque, M., Singh, S.S., Rath, and Ghosh, S. (2014) A comparative study on cypermethrin resistance in Rhipicephalus (Boophilus) microplus and Hyalommaanatolicum from Punjab (India). Ticks and Tick Borne Diseases, 5: 90-94. Singh, N.K., Gehlot, I.S., Jyoti.,Bhat, S.A., Singh, H., and Singh, V. (2015). Detection of acaricidal resistance inHyalommaanatolicumanatolicum from Banaskantha district, Gujarat.J. Parasit. Dis.39: 563–566. ❏ 59 The Indian Journal of Veterinary Sciences & Biotechnology (2018) Volume 14, Issue 2, 60-63 ISSN (Print) : 2394-0247 : ISSN (Print and online) : 2395-1176, abbreviated as IJVSBT 10.21887/ijvsbt.14.2.14 Effect of Garlic (Allium sativum) Extract on Recovery and Conception Rate in Infectious Repeat Breeder Crossbred Cows Anavil Bhardwaz1, S.P. Nema1, S.S. Mahour1, Daljeet Chabbra2, N. Rajput3 and K. Sudarshan1* Dept of 1Veterinary Gynaecology & Obstetrics, 2Veterinary Microbiology, 3Pharmacology, College of Veterinary Science and AH, NDVSU, Mhow (MP), India Publication Info Article history: Received : 26-07-2018 Accepted : 16-08-2018 Published : 17-10-2018 Key Words: Repeat breeder, uterine flushing, Garlic extract, bacterial count, PMNs%, Whiteside test, Conception rate *Corresponding author: drsudarshandogra@yahoo.com Abstract The study was undertaken to evaluate the therapeutic efficacy of Garlic (Allium sativum) extract in 20 infectious repeat breeder crossbred cows. Animals were divided into two groups of ten in each. The animals in first group were treated intrauterine with 30 ml 15% Garlic extract w/v dissolved in saline at 24 hrs interval for 7 days, while the animals in control group were infused with 30 ml saline for 7 days. Bacterial count in uterine flushing declined significantly (p<0.05) after treatment in Garlic group. A significant increase (p< 0.05) in total cellular count and PMNs % was found in Garlic group than in control group. An overall recovery rate of 80 vs 20 % and conception rate of 60 vs 10 % was found in Garlic treated cows over control placebo group proving its efficacy in infectious repeat breeders. This work is licensed under the Creative Commons Attribution International License (http:// creativecommons.org/licenses /by/4.0/P), which permits unrestricted use, distribution and reproduction in any medium, provided the original work is properly cited. Copyright @: 2018 by authors and SVSBT. Introduction One calf a year by cow is essential for economic viability in dairying. Repeat breeding, comprise a heterogeneous group of subfertile cows with a tune of 7.31 to 23.88 % among bovines. Though, antibiotics are quite effective in treating endometritis, but use of herbal medicines as antimicrobial and/or immunomodulator is becoming popular due to toxicity and side effects of allopathic medicines. Extract of Garlic (Kumar, 2013 and Singh, 2016) has been suggested as alternative therapy for repeat breeding cattle and Indian J. Vet Sci. Biotech (2018) Vol. 14 No. 2 buffaloes. The present study was carried out to evaluate the efficacy of Garlic extract I/Ut infusion in repeat breeding crossbred cows. Materials and Methods The study was conducted on clinical cases of endometritis in crossbred cows belonging to farmers brought to the AI centre of Department of Veterinary Gynaecology and Obstetrics of the College of Veterinary Science and A.H. Mhow and nearby Government AI centres during period from December 2015 to April 2017. All the cows 60 (> 90 days postpartum) were screened as per history, gynaecological examination, nature of estrual cervico-vaginal mucus and positive to whiteside test (WST) as per method of Popov (1969) to identify them as infectious repeat breeding crossbred cows. Which were divided into two groups with ten animals in each, T1 (treatment group and T2 was kept as negative control). Fresh cloves of Garlic (Allium Sativum) were crushed in sterilized pestle-mortar and was first filtered through muslin cloth twice and fihnally through whatman filter paper. Alcoholic extract of Garlic filtrate was prepared using soxhlet extraction method. The residue obtained after evaporation of alcoholic extract was dissolved in saline to obtain 15 % w/v for intrauterine infusion. Animals of T1 group were infused intrauterine with 30 ml of the above extract at 24 hrs interval for 7 days, whereas animals of control group were infused 30 ml saline for 7 days as placebo. The estrual cervico-vaginal mucus was examined for pH before and after treatment at subsequent estrus using pH paper strips. All animals (T1 and T2) were flushed before start of the therapy and after 24 hours of last administration of the Garlic extract (T1) and saline and 8-12 hours after they showed first signs of heat. Total bacterial count in uterine flushing was done as per Bauer et al. (1966). Total cellular count in the uterine flushing was determined by haemocytometeric technique (Jain, 1986). Polymorphonuclear (PMNs %) cell count in the uterine flushing was made in smears prepared from the cell suspension received by centrifugation (3000 rpm for five minutes) on clean grease free glass slides, and stained with Giemsa stain (Kasimanickam et al., 2004). Statistical analysis was carried out by using completely randomized design as per Snedecore and Cochran (1980). Results and Discussion The observations on the nature of the CVM revealed that during infection purulent discharge was observed in 30% cows and muco-purulent discharge in 60% cows, whereas the CVM of 10% cows was clear. Any alteration in the colour of estrual mucus will indicate genital infections (Bhat et al., 2015). Following intrauterine infusion of Garlic extract, the discharge at subsequent Indian J. Vet Sci. Biotech (2018) Vol. 14 No. 2 estrus was observed as clear in 70% cows in comparison to untreated control group (10 %). The present findings are in agreement with the reports of Rahi (2011), Kumar (2013) and Singh (2016) following treatment with Garlic extract in crossbred cows. In the present study, the higher values of pH in repeat breeder cows are in close proximity to the values reported earlier (Modi et al., 2011; Sudarshan Kumar et al., 2015 and Bhardwaz et al ., 2018). In repeat breeding cows with endometritis due to infection, the metabolites of bacteria and inflammatory exudates may alter the pH of estrual cervical mucus to alkaline side resulting in failure of conception due to death of spermatozoa (Ravikumar et al., 2007; Bhardwaz et al., 2018). Once the infection is eliminated, the pH of cervical mucus returns towards the normal neutral side. Whiteside test (WST) was performed on the estrual cervico-vaginal mucus of control and treated animals, before and after treatment. Most of the cows became negative to Whiteside test following treatment with Garlic (80.00%) as compared to only 20.00 % cows in control group which reveals that the Garlic was most effective treatment for endometritis. The present findings corroborated with earlier study (Singh, 2016). A significant decline (p<0.05) in bacterial count (x104/ml) was observed from pre-treatment to post-treatment estrus in uterine flushing of Garlic group (309.59 ± 2.53 to 1.17 ± 0.02) as compared to control group (328.62±0.17 to 296.96±0.22). Xiang (2009), Singh (2016) and Bhardwaz et al. (2018) reported significant drop in bacterial load in uterine flushings following treatment with ciprofloxacin. Reduction in bacterial load in control group may be due to natural uterine defense mechanisms. Besides this, uterine flushings might have also reduced bacterial load. A significant increase (p<0.05) in the total cellular count (104/ml) and (PMNs) % values was observed from pre-treatment to posttreatment in uterine flushing of Garlic group (0.461±0.01 to 3.50±0.05) and (27.91±0.14 to 43.05±0.1), respectively. A non- significant change was found in TCC in control cows after normal saline infusion. The non-significant increase in cellular count in control group might be due to natural uterine defense mechanism. 61 Table 1: Effect of Garlic (Allium sativum) extract on CVM, pH, Bacterial count, Total cellular count and PMN (%) in infectious Repeat Breeder Crossbred Cows Garlic (n = 10) Parameters/Observations CVM appearance Whiteside test Control ( n = 10) Pre-treatment estrus Post-treatment estrus Pre-treatment estrus Post-treatment estrus Purulent 30.00 (3) 10.00 (1) 60.00 (6) 50.00 (5) Muco-purulent 60.00 (6) 20.00 (2) 40.00 (4) 40.00 (4) Clear 10.00 (1) 70.00 (7) 0.00 10.00 (1) Positive 100.00 (10) 20.00 (2) 100.00 (10) 80.00 (8) Negative 0.00 80.00 (8) 0.00 20.00 (2) A 7.49±0.01 B 8.36±0.02 X 8.31±0.02Y CVM –pH (Mean ± SE) 8.11±0.01 Bacterial count (104/ml) 309.59± 2.53A 1.17±0.02B 328.62±0.17 296.96±0.22 Total cellular count (TCC) 0.461±0.01A 3.50±0.05B 0.433±0.02X 0.581±0.01Y PMNs (%) 27.91±0.14A 43.05±0.12B 27.84±0.12 25.57±0.27 Means bearing different superscripts within the row for a group differ significantly (p<0.05). Table 2: Recovery and conception rate in different groups of infectious repeat breeding crossbred cows after treatment Groups No. of Conception rates (%) cows Recovery rate (%) 1st AI 2nd AI 3rd AI Overall Garlic 10 80.00 (8) 40.00 (4) 20.00 (2) -- 60.00 (6) Control 10 20.00 (2) -- 10.00 (1) -- 10.00 (1) The present findings are also in agreement with the finding of Davis and Kuttan (1999). Singh (2016) reported increase in TCC after treatment with Garlic. The herbal extract may act as attractant substance for PMNs and thus activates infiltration of PMNs into the uterine lumen. Conception rate and Recovery rate After treatment with Garlic extract, 80 % recovery rate and 60 % conception rate was recorded in the present study as compared with control group (20 and 10 %). Recovery rate was found to be significant (p<0.05). This could be due to the significant reduction of bacterial count in Garlic extract-treated animal, as Garlic extract is well known for its antimicrobial property against Gram positive and Gram negative bacteria (Chung et al., 2003). The treatment with Garlic Indian J. Vet Sci. Biotech (2018) Vol. 14 No. 2 extract has been shown to stimulate the release of cytokines such as IL-2, IFN-α, IFNγ and increase the natural killer activity and enhances phagocytic activity of peritoneal macrophages (Kyo et al., 1998). Acknowledgement Authors are thankful to Vice Chancellor, NDVSU, Jabalpur and Dean, College of Veterinary Science and AH, Mhow for providing facilities to undertake this study. Conflict of Interest All authors declare no conflict of interest. References: Bauer, A.W., Kirby, W.M.M., Sherris, J.C. and Truck, M. (1966). Antibiotic susceptibility testing by a 62 standardized single disk method. Am. J. Clin. Path., 45(4): 493-496. Bhardwaz, A., Nema, S.P., Sudarshan, K,. Chabbra Daljeet, Shukla, S. and Madhwani, R. (2018). Effect of ciprofloxacin on recovery and conception rate in infectious repeat breeder crossbred cows. Indian J. Vet. Sci. & Biotech., 14(1): 71-74. Bhat, F.A., Bhattacharya, H.K..Fazili, M.R., Hussain, S.A. and Khan, M.Z. (2015). Studies on oestrual cervical mucus of repeat breeding cows with special reference to ovularory disturbance and genitial infection. Theriogenology, 5(2): 113-123. Chung, K.S., Kang, S.Y. and Kim, J.Y. (2003). The antibacterial activity of garlic juice against pathogenic bacteria and lactic acid bacteria. Korean J. Microbiol. Biotech., 31(1): 32-35. Davis, L. and Kuttan, G. (1999). Effect of Withania somnifera on cell mediated immune response in mice. J. Exptl Clin. Cancer Res., 21(4): 585590. Jain, N.C. (1986). Schalm’s Veterinary Haematology. 4th edn. Lea and Febiger, Philadelphia, pp 526527. Kasimanickam, R., Duffielda, T.F., Fosterb, R.A., Gartleya, C.J., Lesliea, K.E., Walton, J.S. and Johnsona, W.H. (2004). Endometrial cytology and ultrasonography for the detection of subclinical endometritis in postpar tum dair y cows. Theriogenology, 62: 9-23. Kumar, A. (2013). Evaluation of immunomodulatory and therapeutic efficacy of turmeric (Curcuma longo) neem (Azadirachta indica) and garlic (Allium sativum) on endometritis in repeat breeding crossbred cows. M.V.Sc. Thesis, G.B. Pant Univ. of Agric and Technol., Pantnagar, India. Kyo, E., Uda, N., Suzuki, A., Kakimoto, M., Ushigina, M., Kasuga, S. and Itakura, Y. (1998). Immunomodulation and antitumour activities of aged garlic extract. Phytomedicine, 5(4): 259267. Indian J. Vet Sci. Biotech (2018) Vol. 14 No. 2 Modi, L.C., Suthar, B.N., Nakhashi, H.C., Sharma, V.K. and Panchasara, H.H. (2011). Physical characteristics of oestrual cervical mucus and conception rate in repeat breeder Kankrej cattle. Intl. J. Agril. Sci.& Vet. Med., 5(4): 416-423. Popov, Y.N. (1969). Diagnosis of occult endometritis in cow (using Whiteside test in cervical mucus). Veterinariya Moscow, 4: 85-87. Rahi, S. (2011). Immunotherapeutic effect of ashwagandha and garlic on endometritic in repeat breeding crossbred cows. M.V.Sc. thesis, G.B. Pant Univ. of Agril and Technol., Pantnagar, India, pp 68. Ravikumar, B.P., Devaraj, M. and Jayakumar, K. (2007). Certain biochemical studies on the uterine flushings of normal and endometritis cows. Indian J. Anim. Reprod., 28(2): 101-103. Singh, G. (1991). Studies on incidence of various reproductive disorders in bovines with special reference to mycotic infections in repeat breeding animals. M.V.Sc. thesis, Punjab Agricultural University, Ludhiana, India. Singh, S. (2016). Phytotherapeutic measures for endometritis in crossbred cows. M.V.Sc &A.H thesis. Nanaji Deshmukh Veterinary Science University, Jabalpur, India. Snedecor, G.W. and Cochran, W.G. (1980). Statistical Methods, 14th edn. Oxford and IBH Publishing House, New Delhi, India. Sudarshan Kumar, Bhardwaz, A. and Srivastava, A.K. (2015). Whiteside test-a field test on the cervical mucus of cows for diagnosis of endometritis. IntasPolivet, 16(2): 207-213. Xiang, C. H. (2009). Observation on the curative effect of integrated traditional Chinese and western medicine on endomertitis sterility in cows and analysis on its function mechanisms. J. Anhui Agric. & Sci., 25: 83. ❏ 63 The Indian Journal of Veterinary Sciences & Biotechnology (2018) Volume 14, Issue 2, 64-65 ISSN (Print) : 2394-0247 : ISSN (Print and online) : 2395-1176, abbreviated as IJVSBT 10.21887/ijvsbt.14.2.15 Osteodystrophia Fibrosa in a Goat - A case report Tejaswini K.G 1,Usha Narayana Pillai1, Madhavan Unny N1*and Sudheesh S Nair2 Department of Veterinary Clinical Medicine, Ethics and Jurisprudence1, Department of Veterinary Surgery and Radiology, College of Veterinary and Animal Sciences, Mannuthy, Thrissur, Kerala Publication Info Article history: Received : 09-07-2018 Accepted : 29-07-2018 Published : 17-10-2018 Key Words: Osteodystrophia, Fibrosa, Goat *Corresponding author: madhavanunny@gmail.com Osteodystrophia fibrosa is a condition associated with fibrous replacement of the resorbed bony matrix. In India, it is commonly reported in horses, and is most often associated with bran feeding, leading to higher availability of phosphorus and imbalance in the calcium phosphorus ratio. Low calcium or high phosphorus diet can result in secondary nutritional hyperparathyroidism leading to osteodystrophia fibrosa (Ozmen et al., 2017). Bone resorption of calcium occurs associated with low calcium level in circulation. Deformities of the bone manifests associated with the severity of the condition. This work is licensed under the Creative Commons Attribution International License (http:// creativecommons.org/licenses /by/4.0/P), which permits unrestricted use, distribution and reproduction in any medium, provided the original work is properly cited. Copyright @: 2018 by authors and SVSBT. Case history and Clinical observations A nine month old male kid weighing 12 kg was presented to the University Veterinary Hospital, Mannuthy with a complaint of facial swelling for the past two months, reduced feed intake and loss of body condition. The animal was fed exclusively with concentrate feed consisting of bran, soyabean meal and coconut cake. On examination, the general appearance of animal was found abnormal puffy face and prominent eyeballs along with respiratory distress.The animal appeared dull and depressed, dehydrated with poor body condition. The gait was abnormal characterized by lameness and swollen joints. On clinical examination, mucous Indian J. Vet Sci. Biotech (2018) Vol. 14 No. 2 membranes were normal in colour, body o temperature was102.2 F. On oral examination, symmetrical enlargement of face and jaws which were soft in texture and difficulty in opening the mouth was observed. The tongue was protruded outwards with frothy salivation. Routine laboratory examination of dung sample and blood smear were negative. Serum biochemistry revealed elevated (1420 IU/L) alkaline phosphatase (ALP), serum calcium and phosphorus were 7.6 mg/dl and 2.5 mg/dl respectively, serum protein, albumin and globulin were recorded 5.9 g/dl , 3.4 g/dl and 2.5 g/dl respectively. Radiograph of lateral and dorso-ventral view of skull indicated radiolucence of skull bones especially mandible 64 and maxilla (Fig. 1 and 2). Reduced bone density characterized by osteolytic changes of alveolar sockets with irregular alignment of all teeth roots were noted. Facial bones showed excessive demineralization and osseous changes which were more prominent towards mandibular symphysis. Based on history, clinical signs and radiograph, the condition was diagnosed as osteodystrophia fibrosa. The prognosis of this case was grave due to irregular arrangement of teeth, which would gradually loosens itself and falls off over time making it difficult for prehension and mastication. The owner was advised to provide a balanced goat feed avoiding bran containing feed stuff and oral calcium and vitamin D supplements was also advised. Osteodystrophia fibrosa is a metabolic condition associated with excessive bone resorption, proliferation of fibrous connective tissue and insufficient mineralization of the bone tissue and thus the name osteodystrophia fibrosa (Bandarra et al., 2011). Enlargements or deformities of facial bones (mandible and maxilla) are characteristic alterations observed in goats affected by fibrous osteodystrophy (Thompson, 2007). Report of the occurrence of the condition in southern India is scarce (Manju and George, 2011). It is a major disorder of horses and is also referred as Miller’s disease, Bran disease or Big head disease due to high phosphorus content in feed. Improvement in skeletal deformities may result with modifications in feed to reduce phosphorus level and improving the calcium content by supplemenatation over a period of time. It is sporadically reported in goats, pigs, reptiles, guinea pigs, dogs and cats and rare in sheep and cattle. References: Bandarra, P.M., Pavar ini, S.P., Santos, A.S., Antoniassi, N.A.B., Cruz, C.E. and Driemeier, D. (2011). Nutritional fibrous osteodystrophy in goats. Pesqui. Vet. Bras.31: 875-878. Manju, S and George, V. (2011). Osteodystrophia fibrosa in a goat- a case report. J. Indian Vet. Assoc. 9: 53 Fig. 1 : Radiolucence of skull bones – lateral view Ozmen, O., Sahinduran, S., Aydogan, A., Sevgisunar, N. S. and Haligur, M. (2017). Clinical and pathological studies on nutritional fibrous osteodystrophy in goats.Ankara. Univ. Vet. Fak. Derg. 64: 55-59. Thompson K. (2007). Jubb, Kennedy and Palmer´s Pathology of Domestic Animals.5th Ed.Elsevier, Philadelphia.p 82-83. ❏ Fig. 2 : Radiolucence of skull bones – dorso-ventral view Indian J. Vet Sci. Biotech (2018) Vol. 14 No. 2 65 The Indian Journal of Veterinary Sciences & Biotechnology (2018) Volume 14, Issue 2, 66-68 ISSN (Print) : 2394-0247 : ISSN (Print and online) : 2395-1176, abbreviated as IJVSBT 10.21887/ijvsbt.14.2.16 Late-Term Abortion and Retention of Placenta in Donkey (Equus acinus): A Report of Two Cases S.C. Parmar*, J.A. Patel, D.V. Chaudhari, K.K. Hadiya and A.J. Dhami Department of Animal Reproduction, Gynaecology and Obstetrics College of Veterinary Science and Animal Husbandry Anand Agricultural University, Anand – 388001, Gujarat Publication Info Article history: Received : 26-07-2018 Accepted : 09-08-2018 Published : 17-10-2018 Key Words: Retention, Placenta, LateTerm, Donkey. *Corresponding author: ajdhami@aau.in This work is licensed under the Creative Commons Attribution International License (http:// creativecommons.org/licenses /by/4.0/P), which permits unrestricted use, distribution and reproduction in any medium, provided the original work is properly cited. Copyright @: 2018 by authors and SVSBT. The incidence of equine abortion is higher than that experienced by cattle. Abortion in equine may be infectious or non-infectious. Infectious agents such as bacteria, viruses or fungi may attack the fetus or its membranes, resulting in fetal death and its expulsion. Non-infectious agents such as development of twins, torsion of umbilical cord or congenital anomalies may also result in death of the fetus and abortion (Kocabiyik et al., 2005). Many causes of late-term abortion including infectious agents and placental abnormalities have been reported. Sometimes thorough post-abortion and post-delivery examination of the fetus, placenta and umbilical cord may reveal other causes that are not identified with laboratory procedures. Retention of placenta is probably less common in equine because of simple, diffuse type of placentation and the strong uterine contractions aiding the separation of the villi from their maternal crypts and expelling the detached membranes (Roberts, 2004). Fetal membranes were defined as retained if they were not expelled completely within three hours after delivery in equine and has been regarded as a threat to the uterine health and general health of the equine. The reported sequelae of retained fetal membranes range from none to metritis, laminitis, septicemia and death (Provencher et al., 1988). The present paper reports a successfully managed case of abortion at late-term gestation due to twisting of umbilical cord and retained placenta in a donkey. History and Clinical Observations A 9 years old non-descript female donkey (Equus acinus) was presented to Veterinary Clinical Complex of the College of Veterinary Science and A.H. Anand with the complaint of eight month pregnancy with reddish-brown colored discharge from the vulva. Animal was unable to stand and was lying in lateral recumbency. Per-rectal examination revealed presence of dead fetus in the uterus, while pervaginal examination revealed dead fetus in dorsocranial presentation with the head turned laterally Indian J. Vet Sci. Biotech (2018) Vol. 14 No. 2 to the right. The front legs of the fetus were folded backward at the carpus. An another case of six years old non-descript female donkey (Equus acinus) was presented to same Clinic with a history of normal foaling, but the placenta was not expelled till 12 hours after foaling and also a little stump of the placenta hanging out from the vulva. Since foaling, the animal was restlessness and showing abdominal straining. Per-rectal examination was done to rule out the possibility of another fetus in the uterus. Per-vaginal examination revealed the tip 66 of the allantochorion extended in the non-gravid horn. Treatment and Discussion In the first case, the fetal head was repositioned manually and the legs were manipulated into normal position for presentation through the pelvic canal. This dead fetus was delivered along with placenta (Fig. 1) per-vaginally with mild and gentle traction together with straining and abdominal contractions by the animal. Gross examination revealed twisting of the umbilical cord with its edema and discoloration of cord tissue. The present case report is in accordance with Mizushima (2005), who reported late term abortion associated with umbilical cord torsion in the mare and stated that it is an indicative of vascular obstruction preceding fetal death and abortion. In the present case, immediate postpartum examination revealed substantial findings to support the diagnosis of twisting of umbilical cord resulted into fetal death and abortion. Fig. 2: Placenta hanging out from the vulva Fig. 3: Placenta of donkey - Velvety reddish tissue (Outer view) Fig. 1: Aborted fetus with placenta- twisting of the umbilical cord with its edema The second case of retained fetal membranes following normal foaling was treated by applying gentle traction on the part of the placenta hanging out from the vulva (Fig. 2) and the whole of the placenta was successfully pulled out of the uterus (Fig. 3). Gross examination revealed an Indian J. Vet Sci. Biotech (2018) Vol. 14 No. 2 apparently normal placenta without any inflammatory changes (Fig. 4). The chorion appeared as a normal velvety reddish tissue due to the villi. All the placentas showed avillous areas at the cervical pole in correspondence to endometrial folds. Manual removal is the most commonly described treatment for retained placenta in equine (Provencher et al., 1988) and it may result in separation of microvilli from the large portion of the fetal membranes (Azawi and 67 (Mifex, @ 0.5 ml/kg b.wt., Novartis) 200 ml and flunixin meglumine (Megludyne, @ 1 mg/kg b.wt., Virbac) 10 ml intravenously on the first day. Both the cases made uneventful recovery without any complications. Acknowledgements We thank the Dean of the College and authorities of Anand Agricultural University, Anand for the facilities provided for this work. Conflict of Interest Authors declare that they have no conflict of interest. References: Azawi, O.I. and Taha, M.B. (2008). Retention of fetal membranes in an Arabian mare: a case report. Iraqi J. Vet. Sci., 22(1): 57-58. Fig. 4: Placenta of donkey- avillous area at the cervical pole (Inner View) Taha, 2008). According to Vandeplassche et al. (1971), the allantochorion situated in the nongravid horn of the uterus is the portion of the membranes most likely to be retained causing retained fetal membranes in equines. However, the forceful traction should be avoided in cases of retained placenta as it may produce haemorrhage and fur ther complicate the condition. Therapeutically, both the cases were treated with Ceftiofur sodium 1 gm (Xyrofure, @ 2.2 mg/ kg b.wt., Intas Pharma) intramuscularly for five days postpartum. Additionally, the supportive therapy in first case of abortion included 5% dextrose saline 1 litre, calcium borogluconate Kocabiyik, A.L., Sonmez, G., Ulgen, M., Ozakin, C., Kocakaya, E. and Alasonyalilar, A. (2005). Abortion due to Streptococcus equi subspecies zooepidemicus in a mare. Turk. J. Vet. Anim. Sci., 29: 937-940. Mizushima, C. (2005). Late-term abortion associated with umbilical cord torsion in the mare: Case report. J. Equine Vet. Sci., 25(4): 162-163. Provencher, R., Threlfall, W.R., Murdick, P.W. and Keith Wearly, W. (1988). Retained fetal membranes in the mare: A retrospective study. Can. Vet. J., 29: 903-910. Roberts, S.J. (2004). Veterinary Obstetrics and Genital Diseases. 2nd Edn, CBS Publishers Pvt Ltd, New Delhi, India. p. 325-327. Vandeplassche, M., Spincemaille, and R. Bouters, R. (1971). Aetiology, pathogenesis and treatment of retained placenta in the mare. Equine Vet J., 3: 144-147. ❏ Indian J. Vet Sci. Biotech (2018) Vol. 14 No. 2 68 The Indian Journal of Veterinary Sciences & Biotechnology (2018) Volume 14, Issue 2, 69-70 ISSN (Print) : 2394-0247 : ISSN (Print and online) : 2395-1176, abbreviated as IJVSBT 10.21887/ijvsbt.14.2.17 Enamel Hypoplasia In Canine Distemper Recovered Pup : A Case Report V.Vijayanand*, M. Shiju Simon, A. Methai, S.Kavitha and T.Sathiamoorthy Veterinary University Peripheral Hospital, (TANUVAS) Madhavaram Milk Colony, Chennai - 600 051 Publication Info Article history: Received : 07-08-2018 Accepted : 19-08-2018 Published : 17-10-2018 Key Words: Enamel, Hypoplasia, Canine Distemper. *Corresponding author: drvjanand@gmail.com Canine Distemper (CD) is a severe life threatening disease with a worldwide distribution caused by a Morbillivirus of the family Paramyxoviridae similar to the human Measles virus, transmitted between susceptible hosts and the dog still remains the most important reservoir for the infection (Leisewitz et al., 2001). The disease affects mainly domestic dogs but has become a serious problem in a wide range of hosts, threatening captive and freeranging wildlife populations including several marine mammals such as seals, dolphins and whales (Visser et al., 1993). Dogs show signs of severe tooth enamel damage or enamel hypoplasia during the course of the disease particularly in young puppies whose teeth have not fully developed. This work is licensed under the Creative Commons Attribution International License (http:// creativecommons.org/licenses /by/4.0/P), which permits unrestricted use, distribution and reproduction in any medium, provided the original work is properly cited. Copyright @: 2018 by authors and SVSBT. Case History and Observations A seven month old, male, non descript pup was brought to Veterinary University Peripheral Hospital, Madhavaram Milk Colony, Chennai with the primary complaint of non-eruption of permanent teeth following the fall of milk teeth. Examination of oral cavity revealed absence of teeth (incisors, canine, molar and premolars) in both the jaws and the entire dental pad appeared smooth. The dog appeared lean with the tongue protruded (about 2 inches) out of oral cavity on the right side. History revealed that about two months earlier the dog had a recovery from an exposure to Canine Distemper virus. Radiograph Indian J. Vet Sci. Biotech (2018) Vol. 14 No. 2 of the skull revealed absence of incisors and canine tooth while the premoloars and molars in both the jaws appeared less in number and irregular (Fig.1). Haematological and serum biochemical values were within normal range and are as follows: haemoglobin 10.8 g/dl, packed cell volume 31.5 %, red blood cell 5.15 X106/ cmm, white blood cells 13,600 /cmm, differential count – N 75%, L 18%, M 6 %, E 1 % B nil, blood urea nitrogen 8.64 mg/dl, creatinine 0.96 mg/dl, alanine aminotransferase 36 IU/dl, total protein 5.6 g/dl, Albumin 2.7 g/dl, calcium 12.39 mg/dl, phosphorus 7.09 mg/dl, blood parasites – Negative and no abnormalities were detected in the blood picture. 69 convalescent period after infection develop and function normally (Greene and Appel, 1990). Fig. 1 : Radiograph showing irregular and less number of premolars and molars in both the jaws (incisors and canine tooth – absent) Treatment and Discussion In the present case radiograph of the skull revealed absence of incisors and canine tooth while the premolars and molars in both the jaws appeared less in number and irregular. Hence not much could be done about the dentition the owner was advised to feed on liquid or semiliquid diet until the eruption of the premolars and molars tooth. Canine Distemper Virus (CDV) infection during early developmental stages, before the eruption of permanent dentition, can infect tooth buds and ameloblasts causing clear enamel hypoplasia (Bittegeko et al., 1995 and Hale, 2005). Dogs naturally infected with distemper virus while the adult teeth are developing often have defects in the enamel when the adult teeth emerge ranging from focal depressions in the enamel to segmental lack of enamel formation, while dental structures developing in the Indian J. Vet Sci. Biotech (2018) Vol. 14 No. 2 Diffuse enamel hypoplasia is usually the result of systemic disease with pyrexia or direct infection of the actively enamel producing ameloblasts by microorganisms. Canine Distemper Virus (CDV) infection, due to the epitheliotropic nature of the virus, produces diffuse enamel hypoplasia with direct infection and destruction of the ameloblasts in addition to the effects of fever (Mannerfelt and Lindgren,2009). References: Bittegeko, S.B., Arnbjerg, J., Nkya, R., Tevik, A. (1995). Multiple dental developmental abnormalities following canine distemper infection. J. Am. Anim. Hosp. Assoc., 31 : 42–45. Greene, C.E. and Appel, M.(1990). Canine Distemper In: Infectious diseases of the dog and cat., Greene, C.E. (ed.), W.B. Saunders, Philadelphia, pp. 226 – 241. Hale, F.A. (2005). Juvenile veterinary dentistry.Vet. Clin.Nr. Am. Sm. Anim. Pract., 35 : 789 – 817. Leisewitz, A.L., Carter, A. Van Vuuren, M and Van Blerk, L. (2001). Canine distemper infections, with special reference to South Africa, with a review of the literature. S. Af. Vet. Assoc.,72 : 127 – 136. Mannerfelt, T. and Lindgren, P. (2009). Enamel defects in standard Poodle dogs in Sweden. J. Vet. Dent.,26 : 213 – 215. Visser, I.K., Van Bressem, M.F., Barrett, T. and Osterhaus, A.D. (1993). Morbillivirus infections in aquatic mammals. Vet. Res.,24 : 169 – 178. ❏ 70 The Indian Journal of Veterinary Sciences & Biotechnology (2018) Volume 14, Issue 2, 71-73 ISSN (Print) : 2394-0247 : ISSN (Print and online) : 2395-1176, abbreviated as IJVSBT 10.21887/ijvsbt.14.2.18 Therapeutic Management of Renal Dysfunction Associated with Trypanosomiasis in a Horse - A Case Report T.M. Vidhyalakshmi1, S.K. Raval2, G.M. Akshatha3, P.V. Parikh4 and J.M. Kathri5 1,2,3 Department of Vet. Medicine, 4,5 Department of Vet. Surgery & Radiology College of Veterinary Science & A.H, AAU, Anand, 388001 Gujarat, India Publication Info Article history: Received : 08-09-2018 Accepted : 23-09-2018 Published : 17-10-2018 Key Words: Renal Dysfunction, Trypanosomiasis, Horse *Corresponding author: skraval23@rediffmail.com Trypanosomiasis, an arthropod borne blood protozoan disease commonly known as Surra is caused by Trypanosoma evansi. Several species of hematophagous flies, including Tabanids and Stomoxys are implicated in transferring infection from host to host, acting as mechanical vectors. Trypanosomiasis is diagnosed based on the clinical evidences augmented with some parasitological methods. The “Office international des epizootics” categorized the disease under ‘B’ disease of significance (OIE, 2004). Surra in India is generally considered as a disease prevalent mostly in animals of Northern India (Soodan et al., 1995). The present case deals with the diagnosis of renal dysfunction associated with trypanosomiasis and its’ successful therapeutic management in a mare. This work is licensed under the Creative Commons Attribution International License (http:// creativecommons.org/licenses /by/4.0/P), which permits unrestricted use, distribution and reproduction in any medium, provided the original work is properly cited. Copyright @: 2018 by authors and SVSBT. Case History and Clinical Observations A seven year old mare with a history of reduced feed intake was presented to Teaching Veterinary Clinical Complex, Anand. Clinical manifestations included fever, depression and edema on brisket and ventral region of the body. Clinical examination revealed pyrexia (103.3°F), tachycardia (60 beats/min) and pale to icteric mucous membrane. There was a history of prophylactic therapy against trypanosomes two months back. Blood samples were taken from jugular vein Indian J. Vet Sci. Biotech (2018) Vol. 14 No. 2 in EDTA vial for specific diagnosis and haematological parameters, whereas 9 ml blood was withdrawn into serum clotting accelerator vial for serum separation and biochemical analysis. Haemato-biochemical investigation showed low Hb, RBC, platelet count, albumin, A/ G ratio and high ESR, globulin, total protein, SGPT, BUN and creatinine (5.5 mg/dl). Total leukocyte count was within normal range. Urine analysis revealed acidic pH (5.5), glucosuria (+++), proteinuria (+++) and pyuria (>7 leukocytes/ field). Blood smear examination on Giemsa staining revealed the presence of trypomastigotes 71 (Fig.1). The findings indicated the renal dysfunction/damage associated with Tryanosomiasis in absence of elevated TLC. Fig. 1 : Trypanomastigotes found on Giemsa stained blood smear Treatment and Discussion The mare was treated first with Isometamidium hydrochloride (Nyzome) @ 0.5 mg/kg intramuscularly, followed by intravenous administration of Ketoprofen and Oxytetracycline @ 2.2 mg/kg and 10 mg/kg, respectively for the 1st day. Thereafter, 6 liters of 0.9% NaCl in combination with 5% Dextrose along with Bcomplex Vitamins was also administered at 12 hrs interval on the same day. Oxytetracycline was continued for next 2 days, after which Doxycycline was used @ 10 mg/kg twice in a day orally for next 5 days. Fluids in combination with B-complex vitamins and Furosemide, a loop acting diuretic @ 2 mg/kg twice a day, were continued intravenously for 2 weeks. Dexamethasone 40 mg was also administered intravenously and continued for next 3 days, whereas Prednisolone was used at a tapering dosage for next 10 days. It was used first @ 10 mg/kg intravenously for 5 days and later @ 0.75 mg/kg and 0.5 mg/kg orally for next 3 and 2 days, respectively. The mare was also treated with Silymarin, a hepatoprotectant @ 210 mg thrice a day and haematinics (Ferritas) @ 1 ml/50 kg intramuscularly for the first 5 days. After 2 weeks of intensive therapy, the mare regained appetite and showed relief from edema. After the treatment period, the blood samples were again analyzed for haemato-biochemical parameters (Table 1). An improvement in haemato-biochemical parameters was observed and we couldn’t find out any trypomastigotes on microscopic examination of Giemsa stained blood smear. In case of hemoprotozoan diseases, glomerulonephritis may occur due to the deposition of antigen-antibody or immune complexes on subendothelial and subepithelial surface of the glomerular basement membrane, which may lead to renal dysfunction. Damage to the glomerular basement membrane results in albuminuria, which may lead to hypoalbuminemia and animal may then exhibit signs related to hypoalbuminemia like peripheral edema (Kahn and Scott, 2005) as seen in this case. In the present case, oxytetracycline was given as antitrypanosome (Ekanem and Adeniran, 2003) in order to compete the suspected resistance of protozoa against trypanocidal drugs. Doxycycline, Table 1: Haemato-biochemical parameters of horse before and after therapy of renal failure G=granulocytes; L=lymphocytes; M-monocytes; ND=Not done. Indian J. Vet Sci. Biotech (2018) Vol. 14 No. 2 72 being a safer drug than oxytetracycline in cases of renal dysfunction was selected in order to counteract any secondary bacterial infection (Davis et al., 2006). Prednisolone, corticosteroid, was used in tapering dose for 10 days in the present case as it is effective in cases of immunemediated diseases (Lewis et al., 2007). The diagnostic and therapeutic approaches adopted were fruitful and the animal made uneventful recovery within 15 days. Acknowledgement We thank the University, Dean of Veterinary faculty and Professor & Head of TVCC of the College for the facilities and cooperation provided. Conflict of Interest Authors have no conflict of interest. References: Davis, J.L., Salmon, J.H. and Papich, M.G. (2006). Pharmacokinetics and tissue distribution of doxycycline after oral administration of single and multiple doses in horses. Am. J. Vet. Res., 67(2): 3106. Ekanem, J.T. and Adeniran, I.S. (2003). Oxytetracycline in the possible management of African trypanosomosis. BIOKEMISTRI, 15(2): 67-75. Kahn, C.M. and Scott Line (2005). Merck’s Veterinary Manual. 9th edition. Merck & Co., USA, pp 654, 1272. Lewis, S.S., Valberg, S.J. and Nielsen, I.L. (2007). Suspected immune-mediated myositis in horses. J. Vet. Intern. Med., 21: 496. OIE (2004). Surra (Trypanosomaevansi). In: “Manual of standards for diagnostic tests and vaccines”. Office international des epizootics. 5th edition. WHO for Animal Health, Paris, pp. 891-900. Soodan, J.S., Singh, K.B., Juyal, P.D. and Khakhaira, S.S. (1995). Incidence of Trypanosoma evansi in equines in Punjab State. J. Vet. Parasitol., 9: 133134. ❏ Indian J. Vet Sci. Biotech (2018) Vol. 14 No. 2 73 The Indian Journal of Veterinary Sciences & Biotechnology (2018) Volume 14, Issue 2, 74-75 ISSN (Print) : 2394-0247 : ISSN (Print and online) : 2395-1176, abbreviated as IJVSBT 10.21887/ijvsbt.14.2.19 Cerebral Babesiosis in a Gir Bullock and its Successful Therapeutic Management J.B. Solanki1; B.J. Thakre*, N. Kumar, D.C. Patel and Y.G. Patel Department of Parasitology, Veterinary College, Navsari Agricultural University, Navsari. Department of Veterinary Clinical Complex, Veterinary College, JAU, Junagadh-362 001 Publication Info Article history: Received : 09-07-2018 Accepted : 19-07-2018 Published : 17-10-2018 Key Words: Cerebral, Babesiosis, Gir Bullock. *Corresponding author: drbhupendrakumarthakre@gmail.com Among haemoprotozoan diseases of the animals, babesiosis is a tick-transmitted disease caused by protozoans of the genus Babesia and it is characterized by haemolytic anemia and fever, with occasional hemoglobinuria and death (Ristic, 1981). Among six species causing bovine babesiosis, B. bigemina and B. bovis are the most important in tropical and subtropical regions (Radostits et al., 2008). The one host tick Boophilus microplus is responsible for transmission of the disease in India, both by trans-stadial and transovarian routes (Taylor et al., 2007; Lefevre et al., 2010). We came across an unusual and interesting observation about the Babesia associated with the cerebral form of the disease, the “cerebral babesiosis” in a Gir bullock, hence reporting in this communication. This work is licensed under the Creative Commons Attribution International License (http:// creativecommons.org/licenses /by/4.0/P), which permits unrestricted use, distribution and reproduction in any medium, provided the original work is properly cited. Copyright @: 2018 by authors and SVSBT. Case History and Observations A 9 years old Gir bullock having approximately 270 kg weight was found ill with fever, anorexia and circling movement since last 7 days at a Gaushala in Dharampur, Gujarat. Upon inquiry it was revealed that the animal was treated with systemic antibiotics, antipyretics and appetizers by a local veterinarian, without fruitful results. Clinically the animal had high temperature (105o F), ruminal hypotonocity (1/3 minutes), anorexia, aggressiveness, grinding of teeth, and cessation of defaecation, icterus, anaemia, paleness of conjunctival mucous membrane, hyper-excitability and convulsions. Haematological analysis revealed reduced haemoglobin (11.0 g/ Indian J. Vet Sci. Biotech (2018) Vol. 14 No. 2 dl), PCV (40%), TLC (4.0x103/μl) and altered differential leucocytes counts (Neutrophils 22%, Lymphocytes 69%, Monocytes 4% and Eosinophils 5%). Thin blood smears prepared with peripheral blood withdrawn from the ear tip, stained with Giemsa stain revealed intraerythrocytic pyriform shape of Babesia spp. (Fig.1). The organisms were characteristically pea shaped and lied in pairs forming an acute angle in the red blood corpuscale. The molecular detection revealed Babesia bigemina organisms (Fig. 2) using 100 bp ladder standard (Ludwig Biotecnologia, Porto Alegre, Brazil). Clinical and laboratory findings of the case were suggestive of cerebral babesiosis. 74 babesiosis either with B. bigemina or B. Bovis (Zaugg, 2009; Maharana et al., 2018). Central nervous system signs are caused by brain anoxia resulting from severe anaemia (Zaugg, 2009). The clinical features observed could be due to destruction of large number of erythrocytes by blood parasites. The sudden onset of high fever (105oF) is due to non-specific toxic substances produced during the metabolism of Babesia. The haematological observations were suggestive of milder form of anaemia, severe leucopenia, lymphocytosis and moderate eosinophilia. Fig. 1: Intra-erythrocytic pyriform shape Babesia Treatment with 40 ml Berenil (Diminazene aceturate) Vet 7% RTU intramuscularly was found very effective along with other mentioned supportive therapy. Blood report after 3 days showed magic improvement in blood parameters with absence of piroplasms. Similar effect of diminazene aceturate (3-5 mg/ kg) has also been reported earlier for babesiosis (Cebra and Cebra, 2002). Conflict of Interest All authors declare no conflict of interest. References: Cebra, C., and Cebra, M. (2002). Diseases of the hematologic, immunologic, and lymphatic systems (Multisystem Diseases). In: Pugh, D.G. (Eds): Sheep and Goat Medicine. Saunders, An Imprint of Elsevier. Philadelphia, Pennsylvania. Fig. 2: Molecular detection of Babesia bigemina using PCR 100 bp ladder Treatment and Discussion The animal was treated with 2 lit of Ringer’s lactate, 2 lit of normal saline, 100 ml Steclin (Oxytetracyclin 50 mg/ml) and 15 ml NeuroxinM intravenously (Methylcobalamin 500 mcg, Pyridoxin 50 mg & Nicotinamide 50 mg/ml), and 15 ml of Melonex (Meloxicam 5mg/ml) intramuscularly for four days. 40 ml Berenil Vet 7% RTU was given intramuscularly on first day of treatment, along with Bol. Rumipro for five days. The animal made an uneventful recovery with improved haematology in next three days. In the literature, more or less similar findings were reported in cattle suffering from cerebral Indian J. Vet Sci. Biotech (2018) Vol. 14 No. 2 Lefevre, P.S., Blancou, J., Chermette, R. and Uilenberg, G. (2010). Infectious and Parasitic Diseases of Livestock. Lavoisier Tec & Doc, France. Maharana, B.R., Patel, B.R., Patel, J. And Hirani, N.D. (2018). Parasitiological and molecular based detection of cerebral babesiosis in Kankej bullock and its successful therapeutic management. Asian J. Anim. Vet. Advances, 13(2): 122-127. Radostits, O.M., Gay, C.C., Hinchcliff, K. and Constable, P.D. (2008). A Textbook of the Diseases of Cattle, Horses, Sheep, Pigs, and Goats, 10th ed. Saunders, Elsevier, Philadelphia, USA. Ristic, M. (1981). Babesiosis of Domestic Animals and Man. CRC Press, Boca Raton, Florida, USA. pp: 264. Taylor, M.A., Coop, R.L. and Wall, R. L. (2007). Veterinary Parasitology. 3rd ed. Edition Blackwell Publishing Ltd, UK, pp: 874. Zaugg, J.L. (2009). Babesiosis In: Smith, B.P. (Eds): Large Animal Internal Medicine. Mosby, Elsevier, St. Louis, pp: 1157. ❏ 75 The Indian Journal of Veterinary Sciences & Biotechnology (2018) Volume 14, Issue 2, 76-77 ISSN (Print) : 2394-0247 : ISSN (Print and online) : 2395-1176, abbreviated as IJVSBT 10.21887/ijvsbt.14.2.20 Hydrocephalic Monster in a non Descript Buffalo A.D. Patil*, C. K. Lakde, S. K. Sahatpure, Chandini and Kamaljit Teaching Veterinary Clinical Complex, Nagpur Veterinary College, Maharashtra Animal & Fishery Sciences University, Nagpur, Maharashtra, India Publication Info Article history: Received : 15-06-2018 Accepted : 15-07-2018 Published : 17-10-2018 Key Words: Hydrocephalic Monster, Non Descript Buffalo *Corresponding author: rupanil.1@rediffmail.com Excessive accumulation of fluid in arachnoid and /or in ventricular system of cranium lead to large size of cranium refers to hydrocephalus (Arthur et al., 2001). It is one of the rare congenital anomalies seen in cattle and buffalo while occasionally in ewe and doe (Dhaliwal et al., 1998). It may be caused due to abnormal development of fetus with genetic, nutritional and environmental factors (Kalman, 1989). An autosomal recessive gene is responsible for many hereditary cases but intrauterine infections and nutritional factor have not been fully evaluated except relationship of blue tounge virus with hydrocephaly (Upasana et al., 2012). Deficiency of Vitamin A may be a potent cause of hydrophalus in buffalo (Arthur et al., 2001). This work is licensed under the Creative Commons Attribution International License (http:// creativecommons.org/licenses /by/4.0/P), which permits unrestricted use, distribution and reproduction in any medium, provided the original work is properly cited. Copyright @: 2018 by authors and SVSBT. Case history and Clinical observation A ten year old non-descript buffalo was presented to the Obstetrical Ward, Teaching Veterinary Clinical Complex, Nagpur Veterinary College, Nagpur with the history of full term gestation in her fourth parity. It was observed that the buffalo failed to deliver fetus since 12 hrs. The alanto-chorion membrane was ruptured as the case was unsuccessfully handled by local quack. The history of breeding was with natural service by available buffalo breeding bull with no pedigree record. “No any complication was reported during her previous parturitions”. Indian J. Vet Sci. Biotech (2018) Vol. 14 No. 2 Vaginal examination revealed dilated cervix and enlarged abnormal fetal head was palpated. The fetus was in the anterior longitudinal presentation, dorso-sacral position with extended fore limbs. The ample quantity of fluid was palpated in fetal cranium and thus it was confirmed a case of hydrocephalic fetal monster. Treatment and Discussion Epidural anesthesia (2% lignocaine HCl, 7 ml) was administered in first inter-coccygeal space. Inj. CPM @ 10 ml, fluid therapy was instituted immediately after reporting the case. 76 Carboxyl methyl cellulose sodium @ 20 gm per litre of luke warm water was poured in the birth passage for lubrication of birth canal. Fore limbs were tied with cotton rope for application of traction force. Smooth skin area was palpated on cranium of fetal monster and by use of long sharp needle, it was punctured. Maximum fluid was drained by applying palm pressure on the fluid filled area on fetal cranium which resulted in considerable reduction in size of fetal head. Dead female fetus was delivered per vaginum after puncture of cranium with gentle traction. Placenta was also removed immediately. It was confirmed hydrocephalic monster in a nondescript buffalo and monster was removed nonsurgically per vaginum. cord, both fore and hind limbs. Frontal bone was absent with incomplete fusion of skull (Fig. 2). Dilation of ventricular system and sub-arachnoid space was observed due to accumulation of fluid in the cranium that led to hydrocephalus (Noakes, 2009). Cranial bones were found markedly thin with epical cap of the bony skull missing and thinning of frontal, parietal and temporal skull bones (Sharma et al., 2015). Similar findings have also been reported earlier in cases of hydrocephalus (Patil et al., 2008 and Upasana et al., 2012). Etiology of hydrocephalus could not be ascertained but its vaginal delivery was possible through obstetrical maneuvers (Honparkhe et al., 2012). References: Arthur, G.H., Noakes, D.E., Parkinson, T.J. and England, G.C.W. (2001). Veterinary Reproduction and Obstetrics. 8th (Edn.) W B Saunders company, Philadelphia, Pennsylvania. pp 131-132. Dhaliwal, G.S., Prabhakar, S., Vashista, N.K. and Sharma, R.D. (1988). Dystocia in a buffalo due to hydrocephalic foetus – A case report. Livestock Adviser XIII (IX): 40-41. Honparkhe, M., Singh, A. K., Brar, P. S., and Doddagoudar, V. G. (2012) Dystocia due to hydrocephalus in a buffalo. Indian Journal of Animal Reproduction 33 (1): 90-91 Fig. 1 : Hydrocephalus fetus in a non descript buffalo Kalman,T.S. (1989). Congenital malformations in laboratory and farm animals. Academic Press. INC San Diego, California. Noakes, D.E. (2009). Arthur’s Veterinary Reproduction and Obstetrics. 8th Ed. W.B. Saunder’s Comp., Philadelphia. Patil, A.D., Yadav, G.U., Markandeya, N.M. and Moregaonkar, S.D. (2008). A rare case of acephalia with hydrocephalus in a cow calf. Intas Polivet, 9(1):46-47. Fig. 2 : Dissected hydrocephalic fetus The monster was characterized by small oral cavity without forehead, hollow cranium filled with blood tinged fluid resembling to a large size ball (Fig.1), incomplete development of cranium with normal orbits, ears, neck , normal thoracic cavity and vertebral column, intact umbilical Indian J. Vet Sci. Biotech (2018) Vol. 14 No. 2 Sharma, S.K., Joshi, Monika, Khosa ,J.S. and Singh, Diler (2015) An unusual case of dystocia due to hydrocephalic monster in a buffalo. International Journal of Science, Environment and Technology, 4(2):300-4. Upasana, R., Bhar, G.R., Varughese, E.E., Ghuman, S.P.S. and Honparkhe, M. (2012). Per-vaginal delivery of a murrah buffalo calf with congenital high grade hydrocephalus internus. Veterinary Practitioner, 13(2):310-311. ❏ 77 The Indian Journal of Veterinary Sciences & Biotechnology (2018) Volume 14, Issue 2, 78-81 ISSN (Print) : 2394-0247 : ISSN (Print and online) : 2395-1176, abbreviated as IJVSBT 10.21887/ijvsbt.14.2.21 Prevalence of Bunostomum trigonocephalum infection in sheep and goats in Madhya Pradesh Ravi Kumar Khare1, A.K. Dixit3, R. Kumar3, G. Das4, D. Bhinsara1, Subhamoy Ghosh5, Rohit Singh2, D. Chandra2, M. Sankar1 Division of Temperate Animal Husbandry, ICAR- IVRI, Mukteswar1; IVRI, Izatnagar2; College of Veterinary Science and A.H., Rewa3; College of Veterinary Sciences and A.H., Jabalpur4; College of Veterinary Science & AH, CAU, Selesih, Aizawl, Mizoram5. Publication Info Article history: Received : 25-07-2018 Accepted : 20-08-2018 Published : 17-10-2018 Key Words: Bunostomum Trigonocephalum, Sheep, Goat *Corresponding author: drrkkhare17@gmai.com This work is licensed under the Creative Commons Attribution International License (http:// creativecommons.org/licenses /by/4.0/P), which permits unrestricted use, distribution and reproduction in any medium, provided the original work is properly cited. Abstract Bunostomum trigonocephalum is one of the neglected and highly pathogenic nematodes of small ruminants. The accurate prevalence of bunostomosis is under estimated because it is morphologically indistinguishable with another blood sucking nematode, Gaigeria pachyscelis. The prime objective of the present study was to estimate the prevalence of B.trigonocephalum in different region of Madhya Pradesh and reinforce identification and differentiation characters between B.trigonocephalum and G. pachyscelis. A total of 52 intestines were screened from different places of Madhya Pradesh and B. trigonocephalum and G. pachyscelis were identified morphologically. The overall prevalence of B.trigonocephalum was 30.76% in Madhya Pradesh. The highest prevalence (85.71 %) was found in the Baghelkhand region (Rewa) is followed by Mahakoshal region (Jabalpur) (83.33%) and Vindhyanchal plateau (Bhopal) (66.66%). Beside B.trigonocephalum, G. pachyscelis was also prevalent (57.14%) in Rewa. The characteristic morphological differences between B.trigonocephalum and G. pachyscelis were discussed. Copyright @: 2018 by authors and SVSBT. Introduction Gastrointestinal nematode infection in sheep and goats is ubiquitous and severely affects small ruminants production, worldwide. In India, the income from small ruminants is major source to the middle and marginal farmers (Bandyopadhyay, 1999). However, gastrointestinal parasitism led by strongyles has detrimental effects on production by reduction in growth and development, and mortality in growing animals. Among strongyles, Haemonchus contortus, Trichostrongylus colubriformis and Oesophagostomum columbianum are well studied Indian J. Vet Sci. Biotech (2018) Vol. 14 No. 2 and reported from different states of our country (Annual report GIP, 2013, 2014). There are meager reports on prevalence of Bunostomum trigonocephalum. Moreover, prevalence of B. trigonocephalum is restricted in few pockets of India, mainly from central India, north east and Kashmir valley. The severity of Bunostomum infection is mainly due to blood sucking activity of worm as well as frequent changing of biting sites, which will lead to continuous oozing of blood. B. trigonocephalum is highly pathogenic and few hundreds of worm can kill an animal (Soulsby, 1982). Despite of high pathogenicity, 78 few studies were conducted on prevalence of bunostomosis. Therefore, the present study was conducted to estimate the prevalence of B.trigonocephalum in different regions of Madhya Pradesh and reestablish identification characters of B.trigonocephalum compared with G. pachyscelis. rate was 57.14% (N=8). The overall prevalence of B.trigonocephalum was 30.76% in Madhya Pradesh. The earlier coproculture reports on the prevalence of B.trigonocephalum in goats from Mahakoshal region of Madhya Pradesh were 4.75 % (Singh et al., 2015) and 3.12 % in Nimar region (Rajpoot et al., 2017). Materials and methods: The distribution of Bunostomum spp. is worldwide but it is more abundant in warm and moist regions with humid weather prevails (William et al., 1983). In temperate region of Uttarakhand, the overall prevalence was 4-7% (Annual report GIP, 2013, 2014). The north-east, Shillong, Meghalaya, have the prevalence rates of 41.7% (Yadav and Tondon, 1989) and 22.40 % (Bandyopadhyay et al., 2010) in goats. B. trigonocephalum has a high prevalence in Kashmir valley, where sheep and goat were harboured 37.7% and 30.1%, respectively (Tariq et al., 2008, 2010). The rate of prevalence in the Western and high altitude of Tamilnadu was 2.7% (Arunachalam et al., 2013) and in plains of Tamilnadu varies from 2.15% to 4.72% (Varadharajan and Vijayalakshmi, 2015; Rajarajan et al., 2017). Normally, coproculture studies do not reveal real prevalence of strongyles. In the present study, gastrointestinal tracts were screened for actual prevalence of B.trigonocephalum and G. pachyscelis. The prevalence of B.trigonocephalum was very high but the number of parasites per intestines were low to medium. Study area and collection of parasites Adult nematodes were collected from gastrointestinal tract of goats slaughtered at local abattoir at Rewa, Jabalpur, Mhow, Morena, Bhopal districts of Madhya Pradesh. Parasites were washed thoroughly in PBS (pH 7.4) and identified as per the morphological keys (Taylor et al., 2015). 12 intestines from Jabalpur and Mhow each, 14 from Rewa, 08 from Morena and 06 from Bhopal were screened for B.trigonocephalum. Results and discussion The prevalence rate of B.trigonocephalum infection from different parts of Madhya Pradesh is summarized in Table 1. Table 1. Prevalence of B. trigonocephalum from different parts of Madhya Pradesh Prevalence rate (%) 12 14 12 08 06 Number of intestines positives for B. trigonocephalum 10 12* 4 52 16 30.769% S. No. Places of Sampling Total intestines screened 1. 2. 3. 4. 5 Jabalpur Rewa Mhow Morena Bhopal Total 83.33 85.71 66.66 * Eight intestines are positive for G. pachyscelis (57.14%) B.trigonocephalum was mainly present in the posterior parts of duodenum and jejunum. The number of worms varied from 30-72 per intestine. The maximum prevalence of B.trigonocephalum was 83.33% from Mahakoshal region (Jabalpur), followed by 85.71% from Baghelkhand region (Rewa) and 66.66% from Vindhyanchal plateau (Bhopal). B.trigonocephalum was not recovered from Morena and Mhow. Moreover, in Rewa along with B.trigonocephalum, other hook worm G. pachyscelis was also found and the prevalence Indian J. Vet Sci. Biotech (2018) Vol. 14 No. 2 Morphologically, the hook worms are offwhite in colour. The anterior end was bent in a dorsal direction.. Both B.trigonocephalum and G. pachyscelis look more or less same, macroscopically. Microscopically, the buccal capsule of B.trigonocephalum open in anterodorsal direction and bear a large dorsal tooth and two short ventral teeth (Fig.1). The mouth cavity has two sub-ventral cutting plates and pair of small dorsal plates. (Fig.1). The major difference in dorsal cone of G. pachyscelis and B. trigonocephalum was that it reach 2/3rd of the brim of buccal capsule in the B.trigonocephalum while short dorsal cone was found in G.pachyscelis without dorsal tooth (Fig.2). The bursa of male B.trigonocephalum has two lateral lobes and a small asymmetrical dorsal lobe (Fig.3). The right externo-dorsal ray arise 79 Fig. 1 : Anterior end of B.trigonocephalum; arrow showing well developed dorsal cone upto 2/3rd brim of buccal capsule. Fig. 2 : Anterior end of G.pachyscelis; arrow showing short dorsal cone and reaching up arrow head indicate subventral lancet containing several cusps each. from dorsal stem much before than left externodorsal ray, which arise almost near the bifurcation of the dorsal rays. The dorsal ray was divided in to two tridigitate branches in the extremities. The spicules were slender, alate, spirally twisted and united posteriorly (Fig.3). In G.pachyscelis, the dorsal lobe was large and both the lateral lobe joined in ventral side. The spicules of G.pachyscelis were larger than B. trigonocephalum. The spicules diverge at extremities but were united in middle and their tips were recurved (Fig.4.). In Bunostomum the tips of the spicules were not recurved . This seems to be first report of G.pachyscelis from goats in Baghelkhand region (Rewa). The present observations may help in planning chemotherapeutic and control strategies against these parasites in the study area. Acknowledgements The authors are highly thankful to Indian Council of Agricultural Research (ICAR) New Delhi, India for funding through network programme on gastrointestinal parasitism. Project Coordinator of Network Programme on GI Parasitism and Director of Indian Veterinary Research Institute, Izatnagar, India are also acknowledged for providing necessary facilities. Indian J. Vet Sci. Biotech (2018) Vol. 14 No. 2 Fig. 3 : Posterior end of B.trigonocepahlum; arrow showing characteristic spicules united together posteriorly; bent arrow showing showing well developed lateral lobe. Fig. 4 : Posterior end of G.pachyscelis ; arrow showing the characteristic recurved spicules divergent posteriorly & arrow head showing well developed dorsal lobe and bent arrow indicate lateral lobes joined together ventrally. Conflict of Interest All authors declare no conflict of interest. References: Annual Report, Network project on gastrointestinal parasitism (GIP), ICAR-IVRI, 2013. Annual Report, Network project on gastrointestinal parasitism (GIP), ICAR-IVRI, 2014. Arunachalam, K., Harikrishnan, T.J., Anna. T. and Balasubramaniam, G.A. 2013. Prevalence of gastrointestinal helminth in sheep at western and high altitude hilly region of Tamil Nadu. Indian Vet. J. 90(3):115-116. Bandyopadhyay, B., 1999. Gastrointestinal parasitic infections of sheep and goats at Salboni, West Bengal. J. Vet. Parasitol., 13: 79-80. Bandyopadhyay, S., Devi, P., Bera, A., Bandyopadhyay, S. and Bhattacharya, D., 2010. Prevalence of gastrointestinal parasite in goats in Shillong, Meghalaya, India.Webmedcentral.1(9):WMC00777. Rajarajan, S., Palanivel, K.M., Geetha, M. and Rani, N., 2017. Epidemiology of Gastrointestinal Parasitism in Small Ruminants in Pudukkottai District, India. Int. J. Curr. Microbiol. App. Sci.6(10):4924-4930. Rajpoot, J., Shukla, S., Jatav, G.P., Garg, U.K. and Agrawal, V., 2017. 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Wiley-Blackwall, pp 452-456. Varadharajan, A. and Vijayalakshami, 2015. Prevalence and seasonal occurrence of gastrointestinal parasites in small ruminants of coastal areas of Tamil Nadu. Int. J. Scientific and Res. Publications. 5(2):2250-3153. William, J.C., Sheeham, D.S.and Fuselier, R.Ii. 1983. Experimental and natural infection of calves with Bunostomum phlebotomum. Vet.Parasitol. 13: 225-237. Yadav, A.K. and Tandon, V., 1989. Gastrointestinal nematode infection of goat in a subtropical and humid zone of India. Vet.Parasitol. 33(2):135-14. ❏ 81