Estrus synch Buffalo.pdf

The Pharma Innovation Journal 2019; 8(2): 54-62 ISSN (E): 2277- 7695 ISSN (P): 2349-8242 NAAS Rating: 5.03 TPI 2019; 8(2): 54-62 © 2019 TPI www.thepharmajournal.com Received: 01-12-2018 Accepted: 05-01-2019 GN Purohit Department of Veterinary Gynecology and Obstetrics, College of Veterinary and Animal Sciences, Bikaner, Rajasthan University of Veterinary and Animal Sciences, Bikaner, Rajasthan, India Pankaj Thanvi Department of Veterinary Anatomy, College of Veterinary and Animal Sciences, Bikaner, Rajasthan University of Veterinary and Animal Sciences, Bikaner, Rajasthan, India Munesh Pushp Veterinary University Training and Research Centre, Kumher, Bharatpur, Rajasthan, India Mitesh Gaur Department of Veterinary Gynecology and Obstetrics, College of Veterinary and Animal Sciences, Navania, Vallabhnagar, Rajasthan University of Veterinary and Animal Sciences, Bikaner, Rajasthan, India Chandra Shekher Saraswat Department of Veterinary Gynecology and Obstetrics, College of Veterinary and Animal Sciences, Navania, Vallabhnagar, Rajasthan University of Veterinary and Animal Sciences, Bikaner, Rajasthan, India Estrus synchronization in buffaloes: Prospects, approaches and limitations GN Purohit, Pankaj Thanvi, Munesh Pushp, Mitesh Gaur, Chandra Shekher Saraswat, Atul Shanker Arora, Surya Prakash Pannu and Trilok Gocher Abstract Estrus synchronization and timed inseminations can overcome two distinct problems of buffalo breeding: poor overt estrus expression and seasonality of breeding. Evidence is accumulating that, similar to cattle, the synchronization of estrus in buffalo is dependent on the presence of dominant follicle (DF) and corpus luteum (CL) on the ovary. Approaches for estrus synchronization govern either the progression of DF to ovulation following luteolysis (prostaglandins PG) or regression of a DF, followed by growth of a new DF that progresses to ovulation (progestins and GnRH). The use of prostaglandins for estrus synchronization has led to estrus rates (ER) of 60-80% and conception rates (CR) of 12.5%-60% in buffalo heifers and ER of 70-100% and CR of 40-80% in adult buffaloes. Such an approach has the disadvantage of poor response in postpartum anestrus buffaloes and buffalo heifers with absence of CL and follicular growth. Moreover, the effects are suboptimal during the non-breeding season and during nutritional inadequacies. Progestagen treatments are more useful in buffalo heifers and adult buffaloes during the non-breeding season when combined with estradiol, eCG, prostaglandins and GnRH/hCG. A slightly longer duration (12-14 d) of progesterone therapy is suggested during the non-breeding season along with estradiol at the time of progesterone implant insertion, as this effectively regresses large follicles and/or initiates a new follicular growth. Such therapies effectively produce additional pregnancies during the unfavourable season, which is clearly advantageous. The ovsynch is the most commonly used GnRH based estrus synchronization protocol used in buffaloes with conception rates of up to 60% during the breeding season however, during the non-breeding season and in buffalo heifers the conception rates are suboptimal (11-20%). The nutritional status of buffaloes appears important before initiating estrus synchronization. In conclusion, estrus and ovulation can be effectively synchronized in buffaloes using ovsynch protocols during the breeding season; however during the non-breeding season progestagen based protocols in combination with estradiol, eCG, PG and GnRH are a better option for timing insemination and planning calvings. Keywords: Buffalo, estrus synchronization, PG, progestagens, ovsynch Atul Shanker Arora Directorate of Extension Education, Rajasthan University of Veterinary and Animal Sciences, Bikaner, Rajasthan, India Surya Prakash Pannu Department of Veterinary Gynecology and Obstetrics, College of Veterinary and Animal Sciences, Bikaner, Rajasthan University of Veterinary and Animal Sciences, Bikaner, Rajasthan, India Trilok Gocher Department of Veterinary Gynecology and Obstetrics, College of Veterinary and Animal Sciences, Bikaner, Rajasthan University of Veterinary and Animal Sciences, Bikaner, Rajasthan, India Correspondence GN Purohit Department of Veterinary Gynecology and Obstetrics, College of Veterinary and Animal Sciences, Bikaner, Rajasthan University of Veterinary and Animal Sciences, Bikaner, Rajasthan, India Introduction The success of estrus synchronization and timed inseminations in dairy and beef cattle (Bridges and Lake, 2011; Colazo and Mapletoft, 2014) [15, 30] has led to increased adoption of these approaches in the buffalo, a species with inherent problems of poor estrus expression and seasonality of reproduction. It is almost around 45 years since the first report on estrus synchronization appeared in buffalo (Baruselli et al., 2013) [10] yet there are still considerable limitations. The wide scale adoption of this technique can become popular in a very few buffalo rearing countries on account of smaller number of buffaloes per herd, poor awareness of farmers and poor responses of most estrus synchronization regimens especially during the hot summer months. Although buffaloes have been shown to have 2-3 follicular growth waves during estrous cycle, with 2 waves being common (Baruselli et al., 2013) [10], yet the number of follicles recruited per follicular wave is lower in buffalo compared to cattle (Gimenes et al., 2009; Campanile et al., 2010) [46, 21]. Thus, the results of estrus synchronization in buffaloes are expected to be low. Similar to cattle, approaches for estrus synchronization in the buffalo have utilized prostaglandins, progestins and GnRH alone or in combination (De Renis and Lopez-Gatius, 2007) [34]. These pharmaceutical agents regulate corpus luteum (CL) regression, follicle growth/regression and ovulation. Studies have shown that the dominant follicle (DF) and CL on the ovary are pivotal to estrus synchronization protocols. The mechanism of estrus synchronization involves either the progression of DF to ovulation following luteolysis (prostaglanidns), or the regression of DF, followed by growth of a new DF that progresses to ovulation (progestins and GnRH based protocols). ~ 54 ~ The Pharma Innovation Journal Markandeya and Bharkad, 2002) [7, 69] and animals return to estrus within 50-96 hr of administration. Thus, for prostaglandins to be effective animals should be cyclic. The administration of PG causes luteolysis and this permits the dominant follicle (DF) of the follicular growth wave to grow to a stage that allows estrus and ovulation (Day and Geary, 2005) [33]. An obvious increase in the size of the DF (from 0.97 ± 0.07 cm to 1.4 ± 0.09 cm) was recorded in Egyptian buffaloes on Day 2 of PG injection, with concomitant decrease in plasma progesterone and estrus in 3-4 days post injection (Noseir et al., 2014) [79]. The return to estrus would depend upon the status (Size and dominance) of dominant follicle and CL (at the time of PG administration). Most buffaloes with a CL and a DF below 1.0 cm would respond to PG however, buffaloes with a CL and a DF above 1.0 cm respond poorly (Brito et al., 2002) [16]. Since buffaloes are known to have a preponderance of two wave cycle (63.3%) (Baruseli et al 2013) and a lower number of follicles are recruited per follicular wave (Campanile et al 2010; Baruselli et al., 2013) [10, 21, 19], the probability of buffalo returning to estrus following PG administration is lower compared to cows. Palpation of a CL in buffaloes appears difficult due to poor projection on the ovarian surface (Sharifuddin and Jainudeen, 1983) [96]. However, estrus rates after a single PG administration following palpation of a CL or without palpation of CL in different studies (Table 1) appear to be high. A smaller number of animals (>25) were utilized in most of these studies, conducted during the breeding season. Besides many variables like, day’s postpartum (Usmani, 2001) [109], nutritional status (Hussein and Abdel-Raheem, 2013) [52] and others that regulate effects of prostaglandins, an important determinant appears to be the season of administration. Administration of a single PG to buffalo heifers resulted in estrus rates of 60% during the low breeding season with poor subsequent conception rates (22.8 25.6%) (Chohan et al., 1993) [27], whereas, during the breeding season estrus rates were 86.6% and conception rates varied from 47.8 to 53% (Table 1). Results of estrus synchronization using PG in pre-pubertal buffalo heifers and postpartum anestrus buffaloes may be suboptimal (Honparkhe et al., 2008) [49]. None of the 12 postpartum acyclic buffaloes treated with PG responded to the treatment. Eight of the 12 Murrah buffalo heifers (32 months age) managed at an organized farm responded well to the PG injections with estrus and ovulation within 48 h of PG injection (Singh and Madan, 2000) [49] however, this might not be applicable to farmers buffaloes at similar age (due to diverse nutritional and managemental conditions) (Brito et al., 2002; Misra et al., 2003) [16, 71]. Thus, the nutritional status and ongoing follicular and luteal activity appears important when selecting a diverse group of buffaloes comprising of buffalo heifers and postpartum anestrus buffaloes. The intravulvo-submucosal route of administration of PG has been suggested for estrus induction/synchronization in buffaloes (Totewad et al., 2009; Lucy et al., 2010) [107, 64] to reduce the cost but estrus synchronization with such a treatment was suboptimal and delayed compared to the intramuscular use. Administration of 2 PG 11-12 days apart is suggested for cyclic animals in well managed buffalo herds during the breeding season with satisfactory conception rates (Table 1) employing natural service or timed artificial insemination. Estrus synchronization protocols in swamp buffaloes have been recently reviewed (Yendraliza et al., 2015) [115]. In the present review, the prospects, approaches and limitations of estrus synchronization in river buffaloes are discussed. Prospects and prerequisites The benefits of estrus synchronization in dairy cows have been analysed previously. It was reported that the use of ovsynch reduced intervals to first AI, days open and/or culling of cows for infertility (Tenhagen et al., 2004) [105]. The pregnancy rates can be increased further by accurate estrus detection methods to re-inseminate cows that spontaneously return to estrus, concurrent with routine pregnancy diagnosis and re-synchronization of non-pregnant cows (Galvao et al., 2013) [42]. Estrus synchronization in buffaloes can address two distinct problems of breeding. First, estrus signs are less marked in buffaloes (Roy and Prakash, 2009) [90] which can be efficiently managed by estrus synchronization, followed by timed insemination without estrus detection (Baruselli et al., 2013) [10] and second, anestrus postpartum buffaloes can be effectively impregnated during the hot summer, which would have otherwise continued to be non-pregnant during the nonbreeding season, thus reducing days open and achieving yearly calving (Kumar et al., 2012) [63]. This has been shown in many studies (Malik et al., 2011b; Ghuman et al., 2014) [112, 44] . Using estrus synchronization, 50-75% pregnancy rate can be achieved during the first month of the breeding season without estrus detection (Crudeli and de La Sota, 2011; Baruselli et al., 2013) [10, 31]. Additional pregnancies during non-breeding season would result in calvings during a time of milk deficit improving overall profitability of buffalo farming. Pregnancy rates are, however, dependent upon many factors such as estrus detection, nutritional status, body condition score and postpartum interval. Low pregnancy rates during low breeding season result from high embryonic mortalities (Campanile et al., 2005; Russo et al., 2010) [19, 46]. Animals should be in good body condition before initiating estrus synchronization. It is beneficial to examine animals by transrectal palpation and transrectal ultrasonography to evaluate uterine and ovarian status (De Rensis and Lopez Gatius, 2007) [34] before initiating estrus synchronization. Buffaloes with a DF and a CL are good candidates for inclusion in the synchronization programs. Buffalo heifers should have attained around 60% of their adult body weight and a minimum of 50% of heifers should have normal estrous cycles. Use of high fertility buffalo bulls and high fertility semen are also essential for attaining success with estrus synchronization. Approaches and limitations Similar to cattle, estrus synchronization approaches in the buffalo have used prostaglandins, progestins and GnRH based protocols in combination with other hormones such as estradiol and hCG. The use of a particular approach would depend upon many factors which should be considered prior to use of a particular product. Prostaglandins Use of prostaglandins is one of the oldest approaches for synchronization of estrus in buffaloes (De Rensis and LopezGatius, 2007) [34]. The admsinistration of natural or synthetic prostaglandins to the buffalo results in a marked decrease in progesterone due to luetolysis (Chohan et al., 1992; Progestins Progestin treatments inhibit the maturation of DF due to ~ 55 ~ The Pharma Innovation Journal period is important for the expression of estrus as well as for subsequent normal luteal function (Baruselli et al., 2007) [8]. A longer period of progesterone treatment (12-14 d) has been suggested to mediate these effects (Bhat et al., 2015) [13]. However, a recent report depicted reduction in hypothalamopituitary responsiveness to synchronization treatments including PRID and ovsynch during increasing daylight (Barile et al., 2015) [7]. Progestins have been used as oral supplements, daily injections, auricular implants (norgestomet) and vaginal implants however, use of commercially available vaginal implants (CIDR and PRID) has been most common. Use of indigenously prepared methyl-acetoxy progesterone vaginal sponges was reported in one study with estrus rates of 50-80% and conception rates of 33% in acyclic and 50% in cyclic Nili-Ravi buffaloes (Kausar et al., 2013) [58] sponges. suppression of both FSH and LH which promote the atresia of all follicles present on the ovary in cattle and buffaloes (Baruselli et al., 2007; Suadsong, 2011) [8]. Once the DF undergoes atresia, its suppressive effects on other small follicles and FSH are removed which allows a new follicular wave to begin with formation of a DF which attains ovulatory capacity following withdrawal of progesterone implant. The use of estradiol or GnRH on the day of implant insertion significantly suppresses DF growth and enhances new wave emergence (Suadsong, 2011; Bhat et al., 2015) [104, 13]. Follicular waves emerged in Murrah buffaloes on days 4.22±0.12, 3.12±0.33 and 5.14±0.42 of treatment with estradiol + CIDR, GnRH + CIDR or CIDR alone (Bhat et al., 2015) [13]. Similarly, in another study on Italian buffaloes treated with estradiol and progestagens, a new follicular wave occurred around the 4th day of treatment (Niasari-Naslagi et al., 2007). The diameter of the largest follicle was reduced from about 8mm (Day 0) to 5 mm around the day of wave emergence. Also, the mean number of 4-5 mm follicles increased from 1.58 (Day 0) to 5 around the day of wave emergence (Bhat et al., 2015) [13]. Progesterone based treatments in combination with estradiol, PG, GnRH or hCG and timed inseminations are considered more useful during the non-breeding season in buffaloes due to poor responses of other estrus synchronization treatments such as PG and ovsynch during this time (Carvalho et al., 2013b; Vecchio et al., 2013) [22, 111]. Pre-pubertal buffalo heifers have low blood progesterone concentrations (Ahmed et al., 2010) [10], which peak around puberty (Haldar and Prakash, 2006) [47], thus progesterone treatments should be more suited to anestrus buffalo heifers for estrus synchronization. This was shown in a small trial (n=6) on PRID treated anestrus buffalo heifers, all of which exhibited estrus after PRID removal (Singh et al., 2009) [49] and similar results were shown for anestrus buffaloes treated with progesterone implants in combination with other hormones (Carvalho et al., 2013a; Vikash et al., 2014) [23, 112]. A short period of elevated progesterone concentration during anestrus Oral supplements Oral administration of progestin melengestrol acetate (MGA; 1-2 mg daily for 9-14 days) alone or combined with 500 µg IM estradiol 24-48 hrs before last MGA dose to buffalo heifers has been reported in a few studies with estrus rates of 62.7-100% however, conception rates were only 35% (Shanker et al 1996) [95]. Administration of low levels of oral progestin (MGA) in the absence of a corpus luteum can result in the formation of persistent follicles in cattle (Smith et al., 2011) [102] however; similar occurrence could not be recorded in trials involving small number of buffaloes. Progesterone injections Progesterone injections can be used as an alternative to oral feeding of progestagens, however these injections have to be administered on a daily basis for 5-8 days which limits their practical applicability. The daily administration of progesterone injections (250 mg IM) for 5 days followed by 500 IU eCG or 5 mg estradiol on day 6 to postpartum buffaloes resulted in estrus rates of 40-59% and conception rates of 45.5% (Thakur, 1989; Kumar et al., 2010) [62, 106]. Table 1: Effect of prostaglandins on estrus response and conception rates in buffaloes Type Shot Estrus rate (%) Heifers PG 1 shot 60-68 Heifers Heifers Adult Adults PG 1 shot PG 2 shot PG 2 shot Single injection after CL palpation 60(LBS) 86.6(PBS) 71.4% 88.6(LBS) 76.6-80 Single injection 77.5-100 2 injection 11 d apart 25-95 2 injection 11 days apart TAI 72-96 h after 2nd injection 47.8-63.6 Conception rate (%) Reference Honnapagol and Patil, (1991); Chohan et al. (1993) [27]; Chohan, (1998) 22.8-5.6(LBS) 47.8-53(PBS) Chohan et al. (1993) [27]; Chohan, (1998) 45 Singh and Dabas, (1998) [44] 32.48 Phadnis et al., 1994 [14] 70-80 Ribeiro et al. (1998) [89] Pant and Singh, (1991) [45]; Honappagol and Patil, 41.5-72.9 (1991); Khattab et al. (1996) [20]; Brito et al. (2002) [16] Chohan et al. (1993) [27]; Diaz et al. (1994) [39]; Singh 22.8-83 and Dabas, (1998) [49]; Misra et al. (2003) [71]; Srivastava, (2005) [105]; Hussain and Honappa, (2008) [51] Chohan, (1998); Yuan et al. (2008) [117] Yuan et al. 47.8-63.6 (2010) [118]; Dadarwal et al. (2009) [45] 12.5-62.5 buffaloes (>15), estrus rates were high and conception rates were similar across regimens utilizing norgestomet in combination with eCG and PG or norgestomet alone (Table 2). A larger trial on 100 anestrus buffaloes however, yielded 75% estrus rates (Yadav et al., 1994) [114] only, and in another trial on 96 buffaloes the conception rates were41.4% (Phadnis et al., 1994) [14]. However, another study on Murrah buffaloes evidenced norgestomet to be ineffective in inducing cyclicity in postpartum anestrus buffaloes (Garg et al., 1999) [43]. Ear implants Norgestomet is a synthetic progestagen ear implant that is administered subcutaneously and kept in place for 8-10 days, followed by its removal. An estradiol valerate capsule and/ or estradiol valerate (5 mg IM) is administered simultaneously to synchronize estrus. Buffaloes show estrus within 24-48 hr of withdrawal of the auricular implant. Estrus rates in buffalo heifers were high (70-100%) in trials utilizing a smaller number of animals (n=10) (Patel et al., 2003; Kumar and Mandape, 2004) [61, 81]. Similarly, in experiments on adult ~ 56 ~ The Pharma Innovation Journal Table 2: Estrus and conception rates in adult buffaloes administered norgestomet ear implants alone or with combination with eCG and PG Norgestomet (8-10 days) + 5 mg E2 Norgestomet + 500 IU eCG Norgestomet + PG (9 days) Estrus rates (%) Conception rates (%) References 60-100 30-70 Phadnis et al. (1994) [14]; Chattry et al. (1999); Patel et al. (2003) [81] 86-97.6 44.6-66.7 Luthra et al. (1994) [65]; Malik et al. (2011a) [113] 100 45-66.7 Utage et al. (2010) [110]; Chaudhary et al. (2015) [25] of progestagen treatments in buffalo heifers. A trial in Venezuela buffalo heifers treated with PRID + E2 + PG showed no conception in treated buffalo heifers (Noguera et al., 2013) [78]. Anestrus buffaloes evidence a poor response, especially during the summer season when the implants are used alone (Sekerden et al 2005; Murugavel et al., 2009) [73]. It has thus been suggested to administer 1-2 mg estradiol and PG along with the CIDR/PRID (Caesar et al., 2011; Carvalho et al 2013a) [22]. The combined use of estradiol and progestagen vaginal implant at the beginning of the regime induces a new follicular wave due to atresia of all follicles present (Baruseli et al., 2007). Alternatively, PG plus eCG or GnRH or both (eCG and GnRH) at withdrawal of implant have been suggested during off season use (Caeser et al 2011; Frares et al 2013) [41]. The administration of eCG at the time of withdrawal of progestagen implants significantly increased the size of the DF rapidly before ovulation (12 mm) from Day 9 to Day 12 (Malik et al., 2011a) [113] and the estrus and ovulation rates (Caesar et al., 2011). The ovulation rate can further be increased by the administration of GnRH two days after the administration of eCG and PG (Carvalho et al 2013c) [23]. Timed insemination at 60-72 h after withdrawal of CIDR/PRID, and repeated at 24 h, has been suggested (Carvalho et al 2013a) [24]. Experiments using GnRH (administered 2 days after implant withdrawal and eCG administration at withdrawal) suggest fixed time insemination 24 hr after the GnRH administration (Carvalho et al 2013b) [22] during the non-breeding season. Timed insemination in 119 buffaloes 64-68 hr after withdrawal of norgestomet ear implants and administration of PG+ eCG before withdrawal during summer resulted in conception rates of 49.6% (Carvalho et al., 2013a) [24] and similar conception rates were recorded in other trials on timed insemination in adult buffaloes following estrus synchronization with norgestomet in combination with eCG, PG and GnRH (Baruselli et al., 2007; Diaz et al., 1994) [8, 39] Vaginal progesterone implants Two commercially available progesterone vaginal implants (CIDR and PRID) developed for cattle have been widely used in buffaloes. The implants are applied for 8-10 days (Ghuman et al., 2009; Murugavel et al., 2009) [45, 73]. Estrus and ovulation occur within 48-96 hrs after withdrawal of progestagen implant in treated buffaloes (Bhosrekar et al., 1994) [82]. In experiments utilizing lesser number (n=20) of buffaloes the estrus response to progesterone vaginal implants was depicted to be high (Table 3) however, the estrus response was lower in trials using a higher number of buffaloes (<50). The estrus response and subsequent conception rates are dependent upon the reproductive status, days postpartum, ovarian follicular status, season of the year and the simultaneous administration of eCG, PG and GnRH (De Rensis and Lopez-Gatius, 2007) [34] . The mean diameter of the largest follicle was significantly smaller in nulliparous Italian buffalo heifers compared to mixed parity buffaloes during the first 4 days after PRID removal (Presicce et al., 2004) [83], suggesting lower response Table 3: Estrus rates and conception rates in buffaloes administered CIDR/PRID intravaginal progesterone implants in different studies No of animals Physiological state of buffalo Estrus rates (%) Conception rates (%) >20 Acyclic/sub estrus 80-100 56-80 21-50 21-50 Anestrus Postpartum 50-80 46- 85.7 25.3 40.7 36.0 36.5-46.4 >50 Anestrus >50 Postpartum/ Acyclic 83-100 71.4-100 NBS-32.5-82 BS-5493 - >50 Lactating/ Acyclic - References De Santis et al. (2003) [36]; Naseer et al. (2011) [74]; Yotov et al. (2012) [116]; Kajaysri et al. (2015) [55] Singh et al. (1988) [44]; Singh, (2003) [45] Sathiamoorthy et al. (2007) [93]; Caesar et al. (2011) Rao and Rao, (1983); Bhoserkar et al. (1994); Ghuman et al. (2009) [45] Neglia et al., 2003 [76] Carvalho et al. (2013a) [23]; Noguera et al. (2013) [78]; Urdaneta et al. (2013) [108] follicle present at the time of administration of first GnRH may regress or persist throughout to ovulate subsequently (Day and Geary, 2005) [33]. The success of this protocol depends on the presence of a DF at the time of first GnRH (De Rensis and Lopez-Gatius, 2007) [34] and this can be determined by ultrasound examination of the ovaries (De Rensis et al., 2005) [35]. In a study on Mediterranean buffaloes the largest follicle present at the time of first GnRH administration regressed in 46.6% of nulliparous and 78.5% of pluriparous animals (Presicce et al., 2005) [85]. In another study on postpartum anestrus Murrah buffaloes, all animals (n=15) ovulated one largest follicle in response to first GnRH treatment (Malik et al., 2011b) [112]. The ovulation of the largest follicle at the time of first GnRH administration is probably dependent on the size of the dominant follicle (De Rensis et al., 2005) [35]. When the size of the largest follicle was >8mm at the time of first GnRH injection, 87.5% of GnRH based Protocols Approaches utilizing administration of GnRH alone, followed by PG to postpartum buffaloes have not become popular, although they can stimulate estrus and ovulation (Khasatiya et al., 2005) [59]. Pursley et al. (1995) [85] were the first to demonstrate that the administration of GnRH, followed by administration of PG 7 days later results in more precise control of ovarian follicle growth and estrus and the ovulation is assured by a second injection of GnRH 2 days after the PG. This has led to a wider use of GnRH in estrus synchronization protocols in cattle and such a protocol is popularly known as ovsynch protocol. By far the ovsynch protocol is the most widely used regime in buffaloes. This ovulation synchronization protocol employs the IM administration of GnRH, PG and GnRH on days 0, 7 and 9. The administration of GnRH promotes ovulation of a DF and this initiates a new wave of follicular growth. The largest ~ 57 ~ The Pharma Innovation Journal heifers and 100% of adult buffaloes ovulated (Derar et al., 2012) [112]. The ovulation of the DF is also dependent on the stage of the DF. In buffaloes, Day 6 and Day 10 of the estrous cycle are considered the growth phase and regression phase of the first wave DF and the proportion of buffaloes that ovulate in response to GnRH during the growth and regression phase was 75.0 and 16.67%, respectively (Dharani et al., 2010) [38]. Similarly, Campanile et al. (2008) [20] recorded ovulation in 62% of the buffaloes when GnRH was administered on Day 5 of the estrous cycle. Moreover, the DF of the first anovulatory wave reached the maximum size earlier and remained in static phase for a greater number of days, indicating an early loss of LH receptors in buffaloes compared to cows (Sateshkumar et al., 2011) [87]. The ovulation of a DF initiates a new follicular wave and formation of a new DF.A new follicular wave started one day after the first GnRH dose in Egyptian buffaloes and this wave resulted in the development of a new DF with a diameter of 1.03+0.07 cm on day 7 after treatment (Day of PG administration) (Noseir et al., 2014) [79]. In another study, the interval from first GnRH administration to the onset of new follicular wave was 48 to 54 h and a new DF developed from this wave (Ali et al., 2008). The administration of PG 7 days later causes luteolysis and the new DF or the continually growing unovulated DF reaches the ovulatory size and the animal evidences estrus. The second GnRH treatment ensures ovulation of the ovulatory follicle. The ovulation rate to first GnRH appears important and can be used as a preliminary screening of buffaloes to be inseminated, reducing timed insemination costs (Neglia et al., 2013) [76], as pregnancy rates were significantly higher (81%) in buffaloes that ovulated after first GnRH injection compared to 22.3% in those that did not ovulate with first GnRH treatment (Neglia et al., 2013) [76]. The ovsynch protocol has been used in buffaloes in a large number of countries with estrus rates varying from 41.6 to 91.9% and conception rates from 11.11 to 68.8% (Table 4). Similar pregnancy rates were recorded in studies using a combination of CIDR and ovsynch or norgestomet and ovsynch (Table 4). Although the DF at first GnRH did not ovulate or regress in 50% of buffaloes treated with CIDR in combination with ovsynch treatment, yet the addition of a progestagen implant at the time of first GnRH injection improved conception rates in acyclic postpartum buffaloes (Ali et al., 2008; Alyas et al., 2013) [6]. Estrus synchronization in buffalo heifers using ovsynch has yielded lower conception rates (18.8-40%) compared to those in adult buffaloes (Irikura et al., 2003; Presicce et al., 2005) [85, 53] . Many previous reports (Baruselli et al., 2002; Warriach et al., 2008; Jabeen et al., 2013) [9, 113, 54] have shown that estrus rates and conception rates in buffaloes with the ovsynch protocols are higher during the breeding season (Estrus rates 87.5% versus 36.3% and conception rates 40% versus 11.1%) (Warriach et al., 2008; Jabeen et al., 2013) [113, 54] . Table 4: Estrus rates and conception rates in buffaloes with ovsynch treatments in different reports Ovsynch Estrus Rates (%) Conception Rates (%) 46.3-91.9% 28.0 - 66.6 Ovsynch + CIDR Norgestomet + Ovsynch PG + Ovsynch 58.3-86 18.18 – 63.6 Reference Berber et al. (2001) [11]; Francillo et al. (2005) [40]; Ali and Fahmy, (2007) [37]; Sathiamoorthy et al. (2007) [93]; Ravikumar et al. (2009) [88]; Derar et al. (2012) [112]; Hoque et al. (2014) [50] Campanile et al. (2005) [19]; Ravikumar et al. (2008) [87]; Kalwar et al. (2015) [56] - 71.4 Malik et al. (2010) [111] - 36.4 Ovsynch + TAI - 18 - 59 CIDR + Ovsynch + TAI 100 66.67 Camelo et al. (2002) Hoque et al. (2014) [50] ; Karen and Darwish, (2010) [57]; Akhtar et al. (2013) [2]; Neglia et al. (2013) [76] [18] Alyas et al. (2013) [6] A variant to the ovsynch protocol is the Heat synch protocol in which the second GnRH is replaced with estradiol. Heat synch protocols resulted in estrus induction rates of 100% and conception rates of 50% (Ali et al., 2012). Such a treatment is considered to result in better estrus expression in buffaloes, however, the conception rates were low (Akhtar et al., 2013) [2] , especially in heavily lactating buffaloes (Berber et al., 2005) [12]. Although the protocol has been suggested for subestrus buffaloes for timed inseminations (Mohan et al., 2009; Carvalho et al., 2012b) [24, 72], yet due to potential dangers of milk suppression with estradiol the same should be discouraged or at least used cautiously. Timed inseminations following ovsynch treatments are suggested 16 to 24 h after the second GnRH with variable conception rates (18 to 60%) (Malik et al., 2011b; Derar et al., 2012) [112]. Pre synchronization with PG 12 days before ovsynch treatment resulted in slight improvement in pregnancy rates with timed inseminations (Hoque et al., 2014) [50] . Recently, another protocol estra-doublesynch with PG on Day 0, GnRH on Day 2, PG on Day 9 and estradiol benzoate on Day 10 and timed insemination 48 and 60 h after estradiol benzoate was suggested for cyclic and anestrus Murrah buffaloes (Mirmahmoudi et al., 2014) [70], with pregnancy rates of 62-64%. References 1. Ahmed WM, Bashandy MM, Ibrahim AK, Shalaby SIA, El-Moez SIA et al. Investigation on delayed puberty in Egyptian buffalo-heifers with emphasis on climatepathological changes and treatment using GnRH (Receptal). Global Vet. 2010; 4:78-85. 2. Akhtar MS, Ullah S, Farooq AA, Mazhar M, Murtaza S, et al. Pregnancy rate in lactating buffaloes treated with or without estradiol after estrus synchronization protocols at timed AI. Buffalo Bull. 2013; 32:366-369. 3. Ali A, Fahmy S. Ovarian dynamics and milk progesterone concentrations in cycling and non-cycling buffalo cows (Bubalus bubalis) during ovsynch program. Theriogenology. 2007; 68:23-28. 4. Ali A, Fahmy S, Abdel-Khalek AKh, Solouma GM. Effect of controlled internal drug release supplement on the response of cyclic buffalo cows to gonadotrophin releasing hormone and prostaglandin F2 treatment. J Agric Vet Sci Qassim Univ. 2008; 1:81-87. 5. Ali R, Shukla SP, Nema SP. Hormonal induction of ovarian cyclicity and conception rate in postpartum anestrus buffaloes. Indian J Field Vet. 2012; 7:44-46. 6. Alyas M, Razzaque WAA, Ali R, Rao MM, Kumar S et al. Supplementation of progesterone in ovsynch to ~ 58 ~ The Pharma Innovation Journal 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 2340. 20. Campanile G, Vecchio D, Neglia G, Di Palo R, Prandi A, D’Occhio MJ. Progesterone and pregnancy ststus of buffaloes treated with a GnRH agonist. Livestock Sci. 2008; 115:242-248. 21. Campanile G, Baruselli PS, Neglia G, Vecchio D, Gasparrini B et al. Ovarian function in the buffalo and implications for embryo development and assisted reproduction. Anim Reprod Sci. 2010; 121:1-11. 22. Carvalho NAT de, Soares JG, Reis EL, Vannucci FS, Sales JNS et al. Use of different progestagens for ovulation synchronization and TAI in buffaloes during the non-breeding season. Buffalo Bull. 2013a; 32:527531. 23. Carvalho NAT de, Soares JG, Souza DC de, Maio JRG, Sales JNS et al. Use of intravaginal progesterone devices during eight or nine days in the ovulation synchronization protocol for TAI in buffaloes during the non-breeding season. Buffalo Bull. 2013b; 32:532-536. 24. Carvalho NAT, Soares JG, Porto Filho RM, Gimenes LU, Souza DC et al. Equine chorionic gondaotropin improves the efficacy of a timed artificial insemination protocol in buffalo during the non-breeding season. Theriogenology. 2013c; 79:423-428. 25. Chaudhary JK, Khasatiya CT, Parmar SC, Patel RV, Tyagi KK. Estrus induction and fertility response in postpartum silent estrus Surti buffaloes treated with norgestomet ear implants alone and in combination with PGF2α. Sch J Agric Vet Sci. 2015; 2(3A):156-165. 26. Chhatry U, Pandit RK, Agarwal RG. Fertility following progesterone induced estrus in Murrah buffaloes. Indian J Anim Sci. 1999; 69:672-675. 27. Chohan KR. Estrus synchronization with low doses of PGF2α and subsequent fertility in subestrus buffalo. Theriogenology. 1998; 50:1101-1108. 28. Chohan KR, Chaudhary RA, Khan NU, Chaudhary MA, Ahmed M. Oestrus behavior and fertility in normal cycling and oestrus synchronized buffaloes. Indian J Dairy Sci. 1992; 45:588-590. 29. Chohan KR, Iqbal J, Asghar AA. Influence of season on fertility of estrus synchronized buffaloes. Buffalo J. 1993; 9:65-67. 30. Colazo MG, Mapletoft RJ. A review of current timed-AI (TAI) protocols for beef and dairy cattle. Can Vet J. 2014; 55:772-780. 31. Crudeli GA, de la Sota RL. Artificial insemination at fixed time in buffaloes. In: Artificial insemination in farm animals (ed) Manafi M. In Tech Open, 2011, 15-26. 32. Dadarwal D, Ghuman SPS, Honparkhe M, Jagir Singh, 2009. Synchronization of ovulation and subsequent fertility in buffaloes following PGF2αC-PGF2α protocol, with or without GnRH. Indian J Anim Sci. 2011; 79:861865. 33. Day ML, Geary TW. Handbook of estrus synchronization. Western Assoc Experiment Station Directors WRP. 2005; 014:2-39. 34. De Rensis F, Lopez-Gatius F. Protocols for synchronizing estrus and ovulation in buffalo (Bubalus bubalis): A review. Theriogenology. 2007; 67:209-216. 35. De Rensis F, Ronci G, Guarneri P, Nguyen BX, Presicce GA et al. Conception rate after fixed time insemination following ovsynch protocol with and without progesterone supplementation in cyclic and non-cyclic Mediterranean Italian buffaloes (Bubalus bubalis). improve fertility in postpartum anestrus buffaloes. International J Adv Res. 2013; 1:79-82. Barile VL, Terzano GM, Pacelli C, Todini L, Malfatti A, et al. LH peak and ovulation after two different estrus synchronization treatments in buffalo cows in the day light lengthening period. Theriogenology. 2015; 84:286293. Baruselli PS, Carvalho NAT, Gimenes LU, Crepaldi GA. Fixed time insemination in buffalo. Italian J Anim Sci. 2007; 6(2):107-118. Baruselli PS, Carvalho NAT, Henriquez CHP, Amaral R, Nichi M. Synchronization of ovulation for timed artificial insemination during the off breeding season in the buffalo. Proc 1st Buffalo Symp of Americas, Belem, Brazil, 2002, 418-420. Baruselli PS, Soares JG, Gimenes LU, Monteiro BM, Olazarri MJ et al. Control of buffalo dynamics for artificial insemination, superovulation and in vitro embryo production. Buffalo Bull. 2013; 32(1):160-176. Berber RCA, Baruselli PS, Madureira EH. Evaluation of utilization of subsequent estrus after synchronization of ovulation with ovsynch protocols (GnRH vs LH) in buffaloes (Bubalus bubalis). Revista Brasileira de Reproducao Animal. 2001; 25:379-381. Berber RCA, Silva MA, Cruz GD, Takayama NS, Baruselli PS. Effect of estrus synchronization protocols, lactation yield and body condition score on conception rate in buffaloes. Indian Vet J. 2005; 82:45-47. Bhat GR, Dhaliwal GS, Ghuman S and Honparkhe M, 2015. Comparative efficacy of E-17β and GnRH administration on day 0 of a controlled internal drug release (CIDR) based protocol on synchrony of wave emergence, ovulation and conception rates in Murrah buffaloes (Bubalus bubalis). Iranian J Vet Res. 2005; 16: 53-58. Bhosrekar MR, Phadnis YP, Gokhale SB, Mangurkar BR. Behavioural responses and conception rates in oestrus synchronised buffaloes. Proceedings, 4th World Buffalo Congress, São Paulo, Brazil. 1994; 3:468-470. Bridges GA, Lake SL. Comparison of the CIDR select and 5 day Select synch + CIDR protocols that included limited estrus detection and timed insemination in beef heifers. The Professional Anim Scientist. 2011; 27:141146. Brito LF, Satrapa R, Marson EP, Kastelic JP. Efficacy of PGF (2alpha) to synchronize estrus in water buffalo cows (Bubalus bubalis) is dependent upon plasma progesterone concentration, corpus luteum size and ovarian follicular status before treatment. Anim Reprod Sci. 2002; 73(12):23-35. Caesar NK, Shukla SN, Shrivastava OP, Agrawal RG, Agrawal S. Fertility response to modified progesterone based synchronization protocol in true anoestrus buffaloes (Bubalus bubalis). India J Anim Sci. 2011; 81:683-686. Camelo ASA, Ribeiro HFL, Silva AOA, Souza JS, Vale WG. Pregnancy rates in suckled female buffaloes submitted to estrus and ovulation synchronization with artificial insemination in fixed time. Proc 1 st Buffalo Symp Americas, Belem Para, Brazil, 2002, 482-485. Campanile G, Neglia G, Gasparrini B, Galeiro G, Prandi A et al. Embryonic mortality in buffaloes synchronized and mated by AI during the seasonal decline in reproductive function. Theriogenology. 2005; 63:2334~ 59 ~ The Pharma Innovation Journal Theriogenology. 2005; 63:1824-1831. 36. De Santis G, Senatore EM, Presicce GA. Follicle turnover during synchronization treatments in Mediterranean Italian buffaloes. Reprod Fertil Dev. 2003; 16:129. 37. Derar R, Hussein HA, Fahmy S, El-Sherry TM, Megahed G. Ovarian response and progesterone profile during the ovsynch protocol in buffalo heifers and postpartum buffalo cows. Buffalo Bull. 2012; 31:136-147. 38. Dharani S, Kathiresan D, Devanathan TG, Balachandran C, Sateshkumar. Ovulatory response of the first follicular wave growing and regressing phase follicle for GnRH administration in cyclic buffaloes. Buffalo Bull. 2010; 29:199-205. 39. Diaz JS, Fritsch M, Rodrigues JL. Pre-fixed artificial insemination in water buffaloes with synchronized oestrus using prostaglandin F2α. Book- Proceedings, 4th World Buffalo Congress, Sao Paulo, Brazil. 1994; 3:588590. 40. Francillo M, Natale A, Sorgente F, Salizillo F, Neglia G. Synchronization of estrus and artificial insemination in buffalo heifers by using two different protocols. Bubalus bubalis. 2005; 11:18-20. 41. Frares LF, Weiss RR, Kozicki LE, Santangelo RP, Abreu RA de et al. Estrus synchronization and fixed time artificial insemination (FTAI) in dairy buffaloes during seasonal anestrus. Brazilian Arch Biol Technol. 2013; 56:575-580. 42. Galvao KN, Federico P, De Vries A, Schuemann GM, 2013. Economic comparison of reproductive programs for dairy herds using estrus detection, ovsynch or a combination of both. J Dairy Sci. 2013; 96:2681-2693. 43. Garg SL, Chander S, Bugalia NS, Rao AR. Plasma thyroid hormone profiles in postpartum anestrus buffaloes treated with synchromate B. Buffalo Bull. 1999; 18:67-69. 44. Ghuman S, Honparkhe M, Singh J. Comparison of ovsynch and progesterone-based protocol for induction of synchronized ovulation and conception rate in subestrous buffalo during low-breeding season. Iranian J Vet Res. 2014; 15:375-378. 45. Ghuman SPS, Singh J, Honparkhe M, Dadarwal D. Induction of ovulatory estrus using ovsynch protocol and subsequent fertility in true anestrus buffalo heifers. Indian J Anim Reprod. 2009; 30:1-3. 46. Gimenes LU, Neto PF, Arango JSP, Ayres H, Baruselli PS. Follicular dynamics of Bos indicus, Bos taurus and Bubalus bubalis heifers treated with norgestomet ear implant associated or not to injectable progesterone. Anim Reprod. 2009; 6:256. 47. Haldar A, Prakash BS. Growth hormone releasing factor (GRF) induced growth hormone advances puberty in female buffaloes. Anim Reprod Sci. 2006; 92:254-267. 48. Honnappagol S, Patil RV. Oestrus synchronization and fertility in buffalo heifer using carboprost tromethamine. Indian J Anim Reprod. 1991; 12:177-179. 49. Honparkhe M, Singh J, Dadarwal D. Estrus induction and fertility rates in response to exogenous hormonal administration in postpartum anestrus and subestrus bovines and buffaloes. J Vet Med Sci. 2008; 70:13271331. 50. Hoque MN, Talukdar AK, Akter M, Shamsuddin M. Evaluation of ovsynch protocols for timed artificial insemination in water buffaloes in Bangladesh. Turkish J ~ 60 ~ Vet Anim Sci. 2014; 38:418-424. 51. Hussain PM, Honnappa TG. Synchronization of estrus in bovines. Indian J Anim Reprod. 2008; 29:58-61. 52. Hussein HA, Abdel-Raheem SM. Effect of feed intake restriction on reproductive performance and pregnancy rate in Egyptian buffalo heifers. Trop Anim Health Prod. 2013; 45:1001-1006. 53. Irikura CR, Ferreira JCP, Martin I, Gimenes LU, Oba E et al. Follicular dynamics in buffalo heifers (Bubalus bubalis) using the GnRH-PG-GnRH protocol. Buffalo J. 2003; 19:323-327. 54. Jabeen S, Anwar M, Andrabi SM, Mohammad A, Murtaza S et al. Determination of ovsynch efficiency for estrus synchronization by plasma LH and P4 in Nili-Ravi buffalo during peak and low breeding seasons. Pak Vet J. 2013; 33:221-224. 55. Kajaysri J, Chumchoung C, Photikanit G. Estrus and ovulation responses in anestrus postpartum swamp buffaloes following synchronization with a controlled internal drug release device and prostaglandin F2α based protocols. Buffalo Bull. 2015; 34(3):357-368. 56. Kalwar Q, Memon AA, Bhutto MB, Kunbhar HK, Hussain A et al. Estrus response and fertility rate in Kundhi buffaloes following estrus synchronization in breeding season. J Adv Vet Anim Res. 2015; 2:362-365. 57. Karen AM, Darwish S. Efficacy of ovsynch protocol in cyclic and acyclic Egyptian buffaloes in summer. Anim Reprod Sci. 2010; 119(1-2):17-23. 58. Kausar R, Khanum SA, Hussain M, Ahmad N, Ahmad L et al. Estrus synchronization and conception rates using locally prepared methylacetoxy-progesterone sponges in cyclic and acyclic Nili-Ravi buffaloes (Bubalus bubalis). Pak Vet J. 2013; 33:433-437. 59. Khasatiya CT, Bharkad VD, Dhami AJ, Hnsu TV, Panchal MT et al. Effect of GnRH and PGF2 alpha treatment on conception rate and blood biochemical and mineral profile of postpartum true anestrus and subestrus Surti buffaloes. Indian J Anim Sci. 2005; 75:1153-1158. 60. Khattab RM, Ibrahim MAR, Mohsen MK, El-Shama’a IS. Improving oestrous expression of Egyptian buffalo using different analogous of prostaglandin. Annals Agric Sci, Moshtohor. 1996; 34(2):549-554. 61. Kumar H, Mandape MK. Fertility management in rural buffaloes by hormonal therapies during the summer season. Buffalo Bull. 2004; 23(2):30-33. 62. Kumar H, Bhooshan N, Patra MK, Yadav MC. Treatment with progestagen and PMSG to prevent prolonged anestrus in buffaloes. Indian J Anim Sci. 2010; 80:623625. 63. Kumar S, Malik RK, Sharma RK, Dutt R, Singh P et al. Effect of ovsynch protocol in different hormonal combinations on follicular dynamics in anestrus Murrah buffaloes. Vet Practitioner. 2012; 13:273-275. 64. Lucy MC, Patel JL, Smith MF, Spencer TE. Reproduction in domestic ruminants. VII Proc Eighth International symposium on reproduction in domestic ruminants, 2010, 624. 65. Luthra RA, Khar SK, Singh KP. Oestrus induction and synchronization in cows and buffaloes with synthetic progestagens. Indian J Anim Sci. 1994; 64:1060-1061. 66. Malik RK, Pardeep Singh, Sharma RK, Singh I, Tuli RK. Estrus and fertility response of postpartum anestrus Murrah buffaloes to Crestar and Ovsynch treatment regimens. Indian J Anim Sci. 2010; 80:982-985. The Pharma Innovation Journal 67. Malik RK, Singh P, Sharma RK, Singh I, Phulia SK et al. Efficacy of norgestomet ear implant for estrus induction on postpartum anestrus Murrah buffaloes (Bubalus bubalis). Indian J Anim Sci. 2011a; 81:687-690. 68. Malik RK, Singh P, Singh LJ, Sharma RK, Phulia SK et al. Ovarian respose and fertility of ovsynch-treated postpartum anestrus Murrah buffaloes. Buffalo Bull. 2011b; 30:272-276. 69. Markandeya NM, Bharkad GP. Effect of cloprostenol on conception rate during spring in suboestrus murrah buffaloes. Indian Vet J. 2002; 79:1205-1206. 70. Mirmahmoudi R, Souri H, Prakash BS. Endocrine changes, timing of ovulation, ovarian follicular growth and efficacy of a novel protocol (Estra-double-synch) for synchronization of ovulation and timed artificial insemination in Murrah buffaloes (Bubalus bubalis). Theriogenology. 2014; 81:237-242. 71. Misra AK, Kasiraj R, Rao MM, Reddy NSR, Pant HC. Estrus response following PGF2α and superovulation treatments and its relationship with fertilization and viable embryo production in water buffalo. Indian J Anim Sci. 2003; 73:245-248. 72. Mohan K, Sarkar M, Prakash BS. Efficiency of heat synch protocol in estrus synchronization, ovulation and conception in dairy buffaloes (Bubalus bubalis). Asian Austr J Anim Sci. 2009; 22:774-780. 73. Murugavel K Antoine D Raju MS, López-Gatius F. The effect of addition of equine chorionic gonadotropin to a progesterone-based estrous synchronization protocol in buffaloes (Bubalus bubalis) under tropical conditions. Theriogenology. 2009; 71:1120-1126. 74. Naseer Z, Ahmad E, Singh J, Ahmad N. Fertility following CIDR based synchronization regimens in anoestrous Nili-Ravi buffaloes. Reprod Domest Anim. 2011; 46:814-817. 75. Neglia G, Gasparrini B, Cimmino R, Zullo G, Albero G, et al. Response of the first GnRH and pregnancy outcome in buffaloes underwent ovsynch and fixed timed artificial insemination. Buffalo Bull. 2013; 32:483. 76. Neglia G, Gasparrini B, Palo R di, Rosa C de, Zicarelli L, et al. Comparison of pregnancy rates with two estrus synchronization protocols in Italian Mediterranean Buffalo cows. Theriogenology. 2003; 60:125-133. 77. Niasari-Naslagi A, Rastegarina A, Howareshi P, Sarhaddi F, Kordnejad C et al. Effect of steroids on ovarian follicular growth in river buffalo. Italian J Anim Sci. 2007; 6:597-599. 78. Noguera SEG, Vale WG, Ribeiro HFL, Rolim Filho ST, Reis AN et al. Fixed-time artificial insemination in cows and buffaloes using an intravaginal releasing progesterone insert. Livestock Res Rural Dev. 2013; 25(10):171. 79. Noseir WMB, El-Bawab IE, Hassan WR, Fadel MS. Ovarian follicular dynamics in buffaloes during different estrus synchronization protocols. Vet Sci. Dev. 2014; 4:25-29. 80. Pant HC, Singh GD. Application of prostaglandin F2 α (PGF2 α) in the treatment of Sub-oestrus in buffaloes. Indian J Anim Reprod. 1991; 12:55-57. 81. Patel DM, Sarvaiya NP, Patel AV, Parmar AP, Dugwekar YG. Induction of estrus and hormonal profile in buffalo treated with norgestomet ear implant. Indian J Anim Reprod. 2003; 24:67-68. 82. Phandis YP, Bhosrekar MR, Mangurkar BR. On farm ~ 61 ~ 83. 84. 85. 86. 87. 88. 89. 90. 91. 92. 93. 94. 95. 96. 97. 98. studies on oestrus synchronization in cows and buffaloes. Indian J Anim Sci. 1994; 64:1151-1154. Presicce GA, Senatore EM, Bella A, Parmeggiani A. Ovarian follicular dynamics and hormonal profiles in heifer and mixed-parity Mediterranean Italian buffaloes (Bubalus bubalis) following an estrus synchronization protocol. Theriogenology. 2004; 61:1343-1355. Presicce GA, Senatore EM, De Santis G, Bella A. Follicle turnover and pregnancy rates following estrus synchronization protocols in Mediterranean Italian buffaloes (Bubalus bubalis). Reprod Domest Anim. 2005; 40:443-447. Pursley JR, Mee MO, Wiltbank MC. Synchronization of ovulation in dairy cows using PGF2 alpha and GnRH. Theriogenology. 1995; 44:915-923. Rao AR, Rao CC. Synchronization of oestrus and fertility in buffaloes with a progesterone releasing intravaginal device. Vet Rec. 1983; 113:623-624. Ravikumar K, Asokan SA. Ovarian status, serum progesterone level and conception rate in ovsynch treated buffaloes. Indian Vet J. 2008; 85:388-392. Ravikumar K, Asokan SA, Veerapandian C. Inclusion of CIDR in ovsynch protocol to improve fertility in postpartum subestrus buffaloes. Indian J Anim Reprod. 2009; 30:29-31. Ribeiro HFL, Vale WG, Sousa J, Silva AOA. The luteolytic effect of small doses of prostaglandin for oestrus synchronization and fertility in buffaloes. BookProceedings of the 4th SIPAR follow-up seminar on animal reproduction and biotechnology for Latin America, Belem-Para-Brazil. 1998; 2:70-75. Roy KS, Prakash BS. Plasma progesterone, estradiol 17 beta and total estrogen profiles in relation to estrous behavior during induced ovulation in Murrah buffalo heifers. J Anim Physiol Anim Nutr. 2009; 93:486-495. Russo M, Vecchio D, Neglia G, Pacelli C, Prandi A et al. Corpus luteum function and pregnancy outcome in buffaloes during the transition period from breeding to non-breeding season. Reprod Domest Anim. 2010; 45:988-991. Sateshkumar S, Palanisamy A, Rangasamy S, Kathiresan D, Kumanan K. Comparative analysis of follicular and luteal dynamics in estrous cycle of buffaloes and crossbred cattle. Buffalo Bull. 2011; 30:148-151. Sathiamoorthy T, Parthasarathy R, Kathirchelvan M. Efficacy of PGF2α, CIDR and ovsynch treatment on estrus induction and fertility in postpartum buffaloes-A field study. Indian J Anim Reprod. 2007; 28:8-11. Şekerden O, Köroğlu M, Saban E. Possibilities of early pregnancy diagnosis by blood and milk progesterone test post insemination in Anatolian buffalo cows of Hatay Province.Tarim Bilimleri Dergisi. 2005; 11:34-39. Shanker U, Singh MP, Upadhyay MP, Pant HC. Treatment of true anoestrus in bovine with progesterone and oestrogen. Indian J Anim Reprod. 1996; 17:1-5. Sharifuddin W, Jainudeen MR. The accuracy of rectal diagnosis of corporalutea in water buffaloes (Bubalus bubalis). Anim Reprod Sci. 1983; 6:185-189. Singh J, Ghuman SPS, Honparkhe M, Singh N. Investigation on dominant follicle development, estrus response, ovulation time and fertility in PRID treated anestrus buffalo heifers. Indian J Anim Sci. 2009; 79:773-777. Singh, Madan ML. Effects of PGF2 alpha and GnRH The Pharma Innovation Journal during different ovarian status at onset of puberty in Murrah buffalo heifers (Bubalus bubalis). Asian Austr J Anim Sci. 2000; 13:1059-1062. 99. Singh C. Response of anoestrus rural buffaloes (Bubalus bubalis) to intravaginal progesterone implants and PGF2alpha injection in summer. J Vet Sci. 2003; 4:137141. 100. Singh G, Dhaliwal GS, Sharma RD, Biswas RK. Treatment of summer anestrous buffalo (Bubalus bubalis) with progesterone releasing intravaginal device plus pregnant mare serum gonadotropin. Theriogenology. 1988; 29:1201-1206. 101. Singh OV, Dabas YPS. Synchronization of estrus in Murrah buffaloes with prostaglandin to augment milk production inlean period. Indian J Anim Reprod. 1998; 19:93-94. 102. Smith MF, Perry GA, Atkins JA, Jinks EM, Pohler KG, et al. Physiological principles underlying synchronization of estrus. Proc Applied Reprod Strategies in Beef CattleNorthwest September, 2011, 21-38. 103. Srivastava SK. Oestrus induction and conception in buffaloes after hormonal treatment during summer. Indian J Anim Sci. 2005; 75:765-768. 104. Suadsong S. Control of estrus and ovulation in cows. Thai J Vet Med. 2011; 41:95-98. 105. Tenhagen BA, Drillich M, Surholt R, Heuwieser W. Comparison oftimed AI after synchronized ovulation to AI at estrus: reproductive and economic considerations. J Dairy Sci. 2004; 87:85-94. 106. Thakur MS. Synchronization of oestrus in post-partum anoestrus buffaloes (Bubalus bubalis) with short-term steroid treatment. Indian J Anim Reprod. 1989; 10:19-21. 107. Totewad GD, Dhoble RL, Sawale AG, Naik PM, Ambore PM. Induction of estrus using cloprostenol by intra vulvo-submucosal route in sub estrus buffalo. Vet World. 2009; 2:381-382. 108. Urdaneta NSM, Montiel CCCh, Berrios MN, Morillo N, Belandria SJ et al. Assessment of the rate of pregnancy in buffaloes crossbred lactation using two protocols CIDRSynch in 6 and 8 days. Buffalo Bull. 2013; 32:375. 109. Usmani RH. Effects of exogenous GnRH and PGF2α on postpartum estrus activity and fertility of buffaloes during low breeding season. Pak Vet J. 2001; 21:95-99. 110. Utage SG, Raghuwanshi DS, Vhora SC, Khan LA, Sahatpure SK. Efficacy of Crestar PGF2α and GnRH combination in treatment of postpartum anestrous buffaloes. Indian J Anim Reprod. 2010; 31(1):28-29. 111. Vecchio D, Rossi P, Neglia G, Longobardi V, Salzano A, et al. Comparison of two synchronization protocols for timed artificial insemination in acyclic Italian Mediterranean buffalo cows out of the breeding season. Buffalo Bull. 2013; 32:479. 112. Vikash, Virmani M, Malik RK, Singh P. Impact of CIDR in combination with different hormones for treatment of anestrus in buffaloes under field conditions of Haryana. Haryana Vet. 2014; 53:28-33. 113. Warriach HM, Channa AA, Nasim A. Effect of estrus synchronization methods on estrus behavior, timing of ovulation and pregnancy rate during the breeding and low breeding seasons in Nili Ravi buffaloes. Anim Reprod Sci. 2008; 107:62-67. 114. Yadav NK, Lohan IS, Singal SP, Dhanda OP, Arora KL. Efficacy of some hormonal and non-hormonal drugs in the induction of oestrus in buffaloes. International J Anim ~ 62 ~ Sci. 1994; 9:215-217. 115. Yendraliza BP, Zefsin BP, Udin ZJ, Arman C. Estrus synchronization in swamp buffaloes. In: Bubaline Theriogenology, Purohit G.N. (Ed.). International Veterinary Information Service, Ithaca NY, 2015. (www. ivis.org), Last updated: 28-Dec-2015; A5706.1215 116. Yotov S, Atanasov A, Ilieva Y. Therapy of ovarian inactivity in postpartum Bulgarian murrah buffaloes by PRID and ovsynch estrus synchronization protocols. Asian Pacific J Reprod. 2012; 1:293-299. 117. Yuan WL, Tsan LB, Shine JY, Yung LC. Improving the fertility of buffalo cows (Bubalus bubalis) in Taiwan with PGF2α treatment. J Taiwan Livestock Res. 2008; 41:51-62. 118. Yuan WL, Tsan LB, Shine JY, Yung LC. Application of artificial insemination for improving the pregnancy rate of water buffalo (Bubalus bubalis) in Taiwan. J Taiwan Livestock Res. 2010; 43:1-9.