C. Rekkas

INDI_SHEEP TRADI_CHEESE is a research project under the SEEERA.NET PLUS Call for Joint European Research Projects of the Seventh Framework Programme. The project falls into the topic AgroFood and particularly into the subtopic “Preservation of indigenous species and traditional food products”. Cheese production represents each geographical area, a fact well recognized via established special labels of origin. Consumers recognize traditional cheeses for their unique quality characteristics, while they ask for certification of their hygiene. The objective of this project is to support the local producers in making safe and certified traditional cheeses from indigenoussheep-breeds milk. This can be achieved (1) through ensuring the supply of milk from indigenous sheep breeds and (2) through ensuring the hygiene and underscoring the qualitative superiority of traditional cheeses. In order to achieve the project objectives, the work plan has been structured as following: there are 4 Rese...

Substrates used in in vitro embryo production (IVP) mimic the synthesis of the fluids where maturation and fertilization of oocytes and early embryo development occur in vivo, namely the pre-ovulatory follicular fluid and the oviductal fluid. Oxidative processes are considered significant moderators of IVP outcome. In order to decide the proper modifications of IVP substrates, which would mimic the total antioxidant capacity (TAC) of the respective biological fluids in vivo, we assessed TAC of these fluids. Pre-ovulatory follicular- (FO: ovine, FB: bovine) and oviductal- (OO: ovine, OB: bovine) fluid samples were collected from eight Holstein cows and 10 Karagouniki ewes in oestrus. We assessed TAC according to the sample ability to inhibit rat hepatic microsomal membrane lipid peroxidation (LP). LP was assayed spectrophotometrically, by the determination of the 2-thiobarbituric acid reactive material. Statistical analysis was performed by t-test; p < 0.05 was considered signific...

This study investigates the effect of superovulation on ovarian steroid concentrations, plasminogen activator activity (PAA) and glycosidases activity in the uterine luminal fluid of ewes, since the latter constitutes the environment in which embryos grow during early pregnancy. At the end of a 12-day progestagen treatment, 13 adult Chios ewes received superovulation treatment (SOV; Folltropin-200 mg, n=6; or Intergonan-1000 IU, n=7); 7 ewes served as controls (C). The embryos and a sample of each uterine horn flushing (UHF) were collected on day 5 (day of intrauterine artificial insemination = day 0), at slaughter. Embryo quality was evaluated stereoscopically. PAA, α-mannosidase activity and β-N-acetyloglucosaminidase (β-NAGASE) activity were determined spectrophotometrically. Progesterone and estradiol-17β concentrations were determined using radioimmunoassay. No significant differences were noticed between the two superovulation treatments, so data were pooled. In UHF of SOV ewe...

Plasminogen activators/Plasmin system plays pivotal role in regulating reproductive functions of mammals. Here, we examined the effects of modification of in vitro fertilization medium (IVF medium) with the addition of tissue-type plasminogen activator (t-PA), on bovine embryo development and quality, assessed by quantification of expression of various genes related to metabolism, oxidation, implantation and apoptosis. In addition, plasminogen activator activity (PAA) and plasminogen activator inhibition (PAI) were measured in the spent media. After conventional IVM, 2016 cumulus-oocyte complexes (COCs) were divided into four groups with modified composition of the IVF medium containing t-PA and/or its inhibitor epsilon-aminocaproic acid (control, t-PA, t-PA+ε-ACA, ε-ACA). Presumptive zygotes were cultured for 8 days in synthetic oviductal fluid (SOF) medium; gene expression studies were carried out on morulae and blastocysts. t-PA alone significantly suppressed cleavage and blastoc...

Possible hormonal aberrations precluding conception or maintenance of pregnancy in dairy ewes subjected to ovulation synchronisation were investigated in this study. The pituitary response to exogenous gonadotrophin-releasing hormone (GnRH) was tested at different luteal stages in 36 ewes. Oestruses were synchronised by using progestagen-impregnated sponges and the animals were randomly allotted into one of three treatment groups (A, B and C; n = 12 for each). Treatments commenced on Days 4, 9 and 14 of the new cycle (oestrus was defined as Day 0). Ewes were given two GnRH injections, 5 days before and 36 h after a prostaglandin F2+/- (PGF2+/-) injection, and the animals were inseminated 12-14 h after the second GnRH injection (modified OVSYNCH). For luteinising hormone (LH) determination blood samples were withdrawn from six ewes of each group at the time of GnRH administration, and 30, 90, 180, 270 and 360 min later. Progesterone was assayed in samples taken every other day starti...

In practice, the etiologic treatment of the repeat-breeder cow is nearly infeasible. In this study, we tested the hypothesis that a combined treatment would benefit the conception rate of repeat-breeder cows. The components of this regimen target ovulation defects, late progesterone (P4) rise, and premature luteolysis. In a 5-year period, 402 repeat-breeder cows were divided in five groups, and treatment regimens consisted of the following: gonadotropin-releasing hormone (GnRH; Group 1, n=115, 0.012mg buserelin im 4 to 6h before artificial insemination); P4 (Group 2, n=51, 100mg P4 intravaginally, on Days 5 to 7); meloxicam (Group 3, n=31, 0.5mgkg(-1), 24h(-1) melomicronxicam sc, on Days 16 to 18); GnRH+P4+meloxicam (Group 4, n=98); and no treatment (Group 5, control, n=107). Artificial insemination was conducted only after overt estrus; thereafter, the duration of the estrous cycle was assessed in all cows that were detected to return to heat. The conception and pregnancy rate was compared among groups. The proportion of cows that returned to estrus after artificial insemination did not differ among groups; the duration of estrous cycle was the shortest in Group 1 and the longest in Group 4. In Group 4, pregnancy rate was higher (P<0.05) than that of Groups 1 and 5 (35.71% vs. 20.00% and 17.76% for Groups 4, 1, and 5, respectively), but though numerically higher, it did not differ statistically from that of Groups 2 (27.45%) and 3 (22.58%). Our results imply that a multifaceted protocol has to be applied for the successful treatment of the repeat-breeder cow.

A novel method for oestrus-ovulation synchronization in sheep followed by fixed time insemination is presented herewith. Mature dry ewes (n = 28) of Karagouniko breed being at an unknown stage of the oestrous cycle, were used during the middle of breeding season. The treatment protocol consisted of an initial administration of a GnRH analogue followed 5 days later by a prostaglandin F2alpha injection. Thirty-six hours later a second GnRH injection was administered to synchronize ovulation, and laparoscopic intrauterine insemination was performed 12-14 h later. Three days after insemination, fertile rams were introduced into the flock twice daily and oestrus-mating detection was carried out. For progesterone (P(4)) determination, blood samples were collected on alternate days, starting 2 days before the first GnRH injection and continuing for 17 days after insemination. An additional sample was taken on the day of insemination. Pregnancy diagnosis was carried out by trans-abdominal ultrasonography. Fourteen ewes (50%) conceived at insemination and maintained pregnancy; from the remainder 14 ewes 10 became pregnant at natural service, while four, although they mated at least two to three times, failed to conceive. In response to the first GnRH, P(4) concentration increased at higher levels in ewes that conceived at AI compared with those that failed to conceive (47.54 and 22.44%, respectively; p < 0.05). Significant differences (p < 0.05) in mean P(4) concentration between pregnant and non-pregnant animals were detected 1 day before AI (0.17 +/- 0.06 and 0.26 +/- 0.14 ng/ml, respectively) on the day of AI (0.15 +/- 0.04 and 0.24 +/- 0.08 ng/ml, respectively) as well as 9 and 11 days thereafter (0.48 +/- 0.12 and 0.38 +/- 0.12 ng/ml; 0.68 +/- 0.14 and 0.50 +/- 0.18 ng/ml, respectively). These results indicate that using the proposed protocol, an acceptable conception rate can be achieved which could be further improved by modifying the time intervals between interventions.

Survival and growth of early mammalian embryos before implantation largely depend on uterine environment. Glucosidases play a significant role as initiators of the adhesive phase of implantation and may be involved in early embryonic development by altering membrane permeability allowing the entry of important metabolites. This study investigated the effect of eCG used for estrus synchronization or superovulation on glucosidase activity in the genital tract of ewes, before implantation. At the end of a 12-day progestagen treatment, 13 adult Chios ewes received either 500 IU (ES, n = 6) or 1000 IU (SOV, n = 7) eCG (Intergonan); 7 ewes served as controls. On day 5 (day of intrauterine artificial insemination = day 0), the embryos, a sample of
each uterine horn flushing (UHF) and samples of caruncular endometrium (CE) and intercaruncular endometrium (ICE) were collected, after slaughter. Embryo quality was evaluated stereoscopically.
a-mannosidase (a-man) and b-N-acetylo-glucosaminidase (b-NAGASE) activities were determined spectrophotometrically. Compared to controls: a-man activity was higher (p < 0.01) in UHF, CE and ICE of SOV ewes and in UHF and ICE of ES ewes; b-
NAGASE activity was lower (p < 0.01) in UHF and ICE of SOV ewes. Positive linear relationships were noticed between a-man activity in UHF and the number of embryos or high quality embryos collected (p < 0.001). In conclusion, eCG used for estrus synchronization or
superovulation affected glucosidase activity in the genital tract of ewes, before implantation.

Blood progesterone (P4) and oestradiol-17b (E2) concentrations were studied in Lesvos ewes during superovulation. The ewes received intravaginal FGA (20 mg) sponges for 12 days, 200 mg p-FSH, in either 8 (SOV1, n = 18) or 6 (SOV2, n = 18) decreasing doses and
200 IU eCG at sponge removal (SR). Five ewes received no FSH (control-C). All ewes received 8 lg GnRH 24 h after SR followed by estrus detection and natural mating. Embryos were collected 7 days after estrus, after slaughter (SL). Blood samples were collected at
sponge insertion (SI), before the onset of FSH treatment (0 h), every 12 h until 132 h, at 180 h and before SL. Serum P4 and E2 concentrations were determined using a radioimmunoassay. Estrus started earlier and was longer in SOV1 or SOV2 compared to C ewes (p < 0.05). At SI and at 0 h, P4 or E2 showed no significant difference among the three groups. The higher (p < 0.05) E2 concentrations in both SOV groups compared to C, 12 to 24 h after SR, as well as the higher (p < 0.05) P4 concentrations after the end of estrus (132 h, 180 h, SL), indicate the success of superovulation. Between  the two SOV groups no significant differences were observed in P4 or E2, in most of the time studied. Still, estrus onset and duration of estrus showed less variation in SOV2 compared to SOV1 group, and SOV2
tended to have higher E2 concentration than SOV1 group during clinical estrus, indicating the better result of SOV2 treatment. (Financed by SEE-ERA.NET Plus, ERA83.)

Reduced viability of embryos after cryopreservation has been associated with lipid peroxidation due to increased levels of free radicals. Thus the addition of antioxidants in the cryoprotectant solutions might be beneficial to embryo survival. Antioxidant caffeic acid, that has been shown to reduce the levels of lipid peroxidation markers in rat erythrocytes, was tested for its ability to improve the cryotolerence of sheep embryos under the two major methods used for embryo cryopreservation. Embryos were collected from 32 superovulated Lesvos ewes, on day 7 after the onset of oestrus, soon after slaughter, by uterine horns flushing and were evaluated under stereoscope. One hundred and thirteen freezable embryos (grades 1, 2), in the morula or in the blastocyst stage, were cryopreserved either by slow freezing (seeding at -6.5°C, 0.3°C/min to -35°C) or by vitrification. Unless differently specified, all chemicals were purchased from Sigma-Aldrich Co. (St Luis, MO, USA). Ethylene glycol, in a final concentration of 1.5M in ECM [Embryo Culture Medium = PBS+20%FCS (Biochrom AG, Berlin, Germany)], was used as cryoprotectant in slow freezing. A final concentration of 25% glycerol and 25% ethylene glycol in ECM was used for vitrification. In half of the cases in each method, 20μM of antioxidant caffeic acid was added in all the cryoprotectant solutions. After thawing / warming, the embryos were cultured in vitro, in SOF, for 72 hours and evaluated for development and hatching. Plasminogen activator activity (PAA), which has been linked to embryo development or degeneration, was determined spectrophotometrically in the media used during the removal of cryoprotectants and in vitro culture. Data was analysed using chi square test, t-test and regression analysis. Overall, 56.0% of the thawed / warmed embryos developed during in vitro culture. At the end of in vitro culture, 42.0% of all the incubated embryos were undergoing or had completed hatching; 42.3% after slow freezing and 41.7% after vitrification. Increased hatching ratio was observed in the embryos cryopreserved in the presence of caffeic acid (52.0% vs. 32.0%, P<0.05); this was apparent in both cryopreservation methods and the difference approached significance after slow freezing (53.8% vs. 30.8%, P<0.10) but not after vitrification (50.0% vs. 33.3%, P<0.20). At the end of in vitro culture, 47.0% of the embryos were degenerating; no statistically significant effect of cryopreservation method or the presence/absence of caffeic acid was observed. PAA in the culture medium at the end of in vitro culture was negatively associated with the ratio of degenerated embryos (R2=0.465, P<0.05). In conclusion, addition of antioxidant caffeic acid seems to improve cryotolerence of sheep embryos and its effect seems to be more prominent when slow freezing is applied.

Survival and growth of early mammalian embryos before implantation largely depend on uterine environment. This study investigates the effect of eCG used for estrus synchronization or superovulation on ovarian steroid concentrations and plasminogen activator activity (PAA) in the genital tract of ewes, before implantation. At the end of a 12-day progestagen treatment, 13 adult Chios ewes received either 500 IU (ES, n = 6) or 1000 IU (SOV, n = 7) eCG (Intergonan); 7 ewes served as controls. On day 5 (day of intrauterine artificial insemination = day 0), the embryos, a sample of each uterine horn flushing (UHF) and samples of caruncular endometrium (CE) and intercaruncular endometrium (ICE) were collected, after slaughter. Embryo quality was evaluated stereoscopically. Progesterone and estradiol-17b concentrations were determined using radioimmunoassay. PAA was determined spectrophotometrically. Compared to controls: progesterone concentration was higher (p &lt; 0.01) in the UHF of SOV...

This study investigates the effect of superovulation on ovarian steroid concentrations, plasminogen activator activity (PAA) and glycosidases activity in the uterine luminal fluid of ewes, since the latter constitutes the environment in which embryos grow during early pregnancy. At the end of a 12-day progestagen treatment, 13 adult Chios ewes received superovulation treatment (SOV; Folltropin-200 mg, n=6; or Intergonan-1000 IU, n=7); 7 ewes served as controls (C). The embryos and a sample of each uterine horn flushing (UHF) were collected on day 5 (day of intrauterine artificial insemination = day 0), at slaughter. Embryo quality was evaluated stereoscopically. PAA, α-mannosidase activity and β-N-acetyloglucosaminidase (β-NAGASE) activity were determined spectrophotometrically. Progesterone and estradiol-17β concentrations were determined using radioimmunoassay. No significant differences were noticed between the two superovulation treatments, so data were pooled. In UHF of SOV ewe...

INDI_SHEEP TRADI_CHEESE is a research project under the SEE‐ERA.NET PLUS Call for Joint European Research Projects of the Seventh Framework Programme. The project falls into the topic AgroFood, subtopic “Preservation of indigenous species and traditional food products”. The objective of this project is to support the local producers in making safe and certified traditional cheeses from indigenous‐sheep‐breeds milk. Cheese production represents each geographical area, a fact well recognized via established special labels of origin. Consumers recognize traditional cheeses for their unique quality characteristics, while they ask for certification of their hygiene.
The objective of the project could be achieved through the following approaches:
1. Ensuring the supply of milk from indigenous sheep
• Rapid improvement of productivity of the indigenous‐breed population (Lesvos and Pramenka‐Ovcepolian); the superovulatory response and the freezability of semen and embryos will be investigated.
• Application of natural/alternative methods for the control of estrus and increase of fecundity (ram‐effect/light manipulation/flushing), based on the breed’s reproductive profile (Pramenka, Lika and Sjenicka strains).
2. Ensuring the hygiene and underscoring the qualitative superiority of traditional cheeses
• Evaluation of raw milk quality, including physical and chemical analysis and microbiological examination at different stages of lactation and different regions will be performed
• Evaluation of cheese quality, including biochemical analysis (chemical characteristics during ripening), microbiological examination (effect of milk pasteurization on microbial evolution, composition of NSLAB during ripening, evolution of pathogens), and organoleptic evaluation will be performed. The possible effect of stage of lactation, geographical location (climate, pastures) and differences in production process on the quality of milk and cheese will be studied.
Four traditional cheeses will be studied: Ladotyri Mytilinis, light yellow, hard cheese, stored in olive oil, produced in the island of Lesvos, Ovco belo sirenje, white soft cheese, made in the summer in the mountain regions of FYR of Macedonia, Sir iz mišine light yellow cheese produced in Dalmatia (Croatia) during the summer and Sjenicki cheese, soft cheese stored in brine, produced in the southern part of Serbia.
In addition to all the above, a database management system will be developed, focusing on existing populations, threats and different production systems for Lesvos breed.
Milk sampling and testing at the middle and/or the end of the milking period have been completed; the first lots of the cheeses have been prepared, chemical analysis and microbiological examination of cheese samples have also been performed. The work is under way.

INDI_SHEEP TRADI_CHEESE is a research project under the SEE-ERA.NET PLUS Call for Joint European Research Projects of the Seventh Framework Programme. The project falls into the topic AgroFood and particularly into the subtopic “Preservation of indigenous species and traditional food products”. Cheese production represents each geographical area, a fact well recognized via established special labels of origin. Consumers recognize traditional cheeses for their unique quality characteristics, while they ask for certification of their hygiene. The objective of this project is to support the local producers in making safe and certified traditional cheeses from indigenoussheep-breeds milk. This can be achieved (1) through ensuring the supply of milk from indigenous sheep breeds and (2) through ensuring the hygiene and underscoring the qualitative superiority of traditional cheeses. In order to achieve the project objectives, the work plan has been structured as following: there are 4 Res...