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ThesisItem Open Access Effect of gonadotropin releasing hormone and prostaglandin for improving reproductive efficiency in goats(Department of Animal Reproduction, Gynaecology and Obstetrics, College of Veterinary and Animal Sciences, Mannuthy, 2008) Julliet; KAU; Joseph, MathewWith the objective of studying the effect of GnRH and prostaglandin for improving reproductive efficiency in goats the study was carried out at University Sheep and Goat Farm, Mannuthy using 42 cycling goats. Based on the behavioural and physiological changes associated with oestrum the goats were divided into two groups viz., Group I and Group II. Group I animals were those that exhibited pronounced oestrus signs and were divided into two subgroups namely Group IA and Group IB. Group II animals were those that exhibited weak oestrus signs and were divided into three subgroups namely Group IIA, IIB and IIC. Group IA animals were administered 0.0042 mg Buserelin (1 ml Receptal) a potent GnRH analogue on day 0, and Group IB served as the Control. Blood was collected prior to GnRH administration and breeding from all does. The mean duration of oestrum in Group IA and IB was 19.33 ± 0.45 and 33 ± 0.58 h respectively. The conception rate in Group IA and IB was 50 per cent and 66.66 per cent respectively. The serum P4 level on day 0 in does in Group IA and IB was 0.43 ± 0.05 ng/ml and 0.40 ± 0.05 ng/ml respectively. Group IIA and Group IIB does were treated as per the CO-Synch protocol (i/m inj. of 0.0042 mg of Buserelin (1 ml Receptal) on day 0, 125 µg cloprostenol (0.5 ml clostenol) on day 7; 0.0042 mg of Buserelin and mating on day 9) and prostaglandin protocol respectively (two intramuscular injections of 125 µg cloprostenol (0.5 ml clostenol) 11 days apart followed by mating at 72 and 96 h), while Group IIC served as the control. The oestrus response, oestrus onset interval, duration of oestrum and conception rate in Group IIA was 90.9 per cent, 47.6 ± 0.45 h, 24.5 ± 0.63 h and 40 per cent respectively. The oestrus intensity score of induced oestrus ranged from 0 to 13. The serum P4 level in pregnant and non pregnant does was not significantly different on days 0, 7 and 9 (P>0.05). The oestrus response, oestrus onset interval, duration of oestrum and conception rate in Group IIB was 81.8 per cent, 54 ± 1.006 h, 39.77 ± 1.54 h and 66.66 per cent respectively. The oestrus intensity scores in induced oestrus ranged from 0 to 13. The serum progesterone level in does that became pregnant and those that were non pregnant were not significantly different on day 0, 11, and at 72 and 96 h. In Group II C the duration of oestrum and pregnancy rates was 40 ± 0.91 h and 33.33 per cent respectively. Pregnancy diagnosis was done at three months of gestation by abdominal palpation and the accuracy of the method was 90.9 per cent. Mean gestation length was 146.03 ± 0.76 days. Litter size at birth in Group IA, IB, IIA, IIB and IIC was 2, 2, 2, 1.83 and 2 respectively. Average birth weight of kids was 2.35 ± 0.164 kg and the mean birthweight of male and female kid was 2.42 ± 0.98 kg and 2.28 ± 0.36 kg respectively. Thus from the present study, it can be concluded that :- 1. Administration of GnRH on the day of oestrum in animals exhibiting pronounced oestrus signs failed to improve conception rate when compared to the control. 2. In animals exhibiting weak oestrus signs both CO-Synch and double prostaglandin protocols resulted in higher conception rate when compared to control group. 3. The double prostaglandin protocol was found to be more efficient in improving conception rate in animals exhibiting weak oestrus signs.ThesisItem Open Access Comparative efficacy of bromocriptine, cabergoline and thyroxine in inducine oestrus in bitches(Department of Animal Reproduction, Gynaecology and Obstetrics, College of Veterinary and Animal Sciences, Mannuthy, 2008) Ajitkumar, G; KAU; Sreekumaran, TDetailed survey on breeding, feeding and management of 1721 dogs belonging to 817 owners was conducted in different parts of Kerala State. Among the dogs surveyed, 518 (30.10%) were male and 1203 (69.90%) were female. Breed-wise details of dogs maintained by individual owners revealed German shepherd dog (20.74%) as the most popular breed of dog followed by Labrador retriever (18.13%), Dachshund (11.39%), Rottweiler (9.12%) and Spitz (5.52%). The percentage of local non-descript dogs was found to be 14.64 only. The prevalence, nature and magnitude of anoestrus among the female dogs were evaluated based on breeding details. Haematological, hormonal, mineral and cholesterol profile of anoestrous bitches were estimated. Data on breeding revealed that 92.93 per cent of bitches were allowed to breed and the remaining 7.07 per cent were kept as virgin throughout their lifetime. The age at first breeding in majority of the bitches (57.85%) was between 1 to 2 years with an average of 16.30 months. The average number of oestrus periods and the number of breeding per year were 2.14 ± 0.24 and 1.47 ± 0.32 respectively. The average duration of proestrual bleeding was found to be 10.87 ± 1.24 days. Most of the bitches (51.97 %) were bred twice in one oestrus and the period of acceptance varied from 4 to 22 days with an average of 8.94 ± 2.18 days. The incidence of failure of conception, abortion, stillbirth and pseudopregnancy were 18.87, 5.72, 15.56 and 13.69 per cent respectively. The neonatal problems encountered in 13.88 per cent of bitches included agalactia / hypogalactia / mammitis (5.29%), cannibalism (2.65%), puppy fading syndrome (2.09%), puerperal tetany (1.98%) and rejection of young one by dam / poor mothering ability (1.87%). Among the bitches surveyed, 134 (11.14%) were identified as in the stage of anoestrus, 65.67 per cent in primary and 34.33 per cent in secondary anoestrus and the average age was 22.68 and 34.34 months respectively. The average duration of secondary anoestrus was 11.71 months. Analysis of breed-wise prevalence revealed that it was high in breeds such as Dachshund (18.18%), Labrador retriever (16.74%), Great Dane (16.67%), Dalmatian (15.38%) and Doberman pinscher (14.00%) and was lowest (2.44%) among local non-descript bitches. Data on body condition revealed that 42 per cent of the anoestrous bitches were with the ideal body score of five, 46 per cent with body score more than five (obese) and 12 per cent with body score less than five (lean). Analysis of haematological profile of anoestrous bitches revealed normochromic normocytic anaemia. The mean serum progesterone, prolactin and thyroxine levels in anoestrous bitches were of 0.57 ± 0.03 ng/ml, 0.75 ± 0.05 ng/ml and 1.80 ± 0.06 µg/dl respectively. The mean serum calcium and phosphorus levels in anoestrous bitches were 8.94 ± 0.24 and 3.48 ± 0.12 mg per cent respectively. The mean serum iron, copper, cobalt, zinc, manganese and cholesterol levels were found to be within the normal range. Oestrus induction trials were carried out in forty anoestrous bitches and the data obtained were compared with that of control bitches. Out of 10 animals treated in each group, five (50%) in Group I (bromocriptine @ 50 μg/kg. body weight), nine (90%) in Group II (cabergoline @ 5 μg/kg. body weight), eight (80%) in Group III (thyroxine @10 μg/kg. body weight) and seven (70%) in Group IV (thyroxine @ 5 μg/kg. body weight) evinced proestrual bleeding. The mean treatment onset to proestrus in Groups I, II, III and IV was 28 ± 3.39, 13.44 ± 3.12, 24.50 ± 3.18 and 33 ± 2.21 days respectively. The average duration of proestrus in the treatment groups was 9.80 ± 0.86, 10.11 ± 0.68, 11.25 ± 0.88 and 10.71 ± 0.68 days respectively as against 9.70 ± 0.42 days in the control group. The average duration of oestrus in the four treatment groups and the control group was 7.60 ± 0.24, 8 ± 0.29, 8.5 ± 0.63, 7.85 ± 0.46 and 8 ± 0.39 days respectively. Fifty per cent of the animals treated with bromocriptine and 10 per cent of the animals treated with cabergoline exhibited nausea and vomiting as side effects, whereas none of the animals treated with thyroxine exhibited side effects. The mean serum progesterone and prolactin levels during anoestrus, on the first day of proestrus and on the day of first breeding did not differ significantly within and between the treatment groups and the control group. The mean serum thyroxine level during anoestrus and on the first day of proestrus did not differ significantly within and between the treatment and the control groups, whereas on the day of first breeding the mean thyroxine level recorded statistically significant difference between the treatment and control groups, the lowest being 1.56 ± 0.18 μg/dl in Group I and the highest being 2.10 ± 0.13 μg/dl in Group IV. Animals treated with levothyroxine (Group III & IV) exhibited a slight increase in the mean level of serum thyroxine on the first day of bleeding as well as on the day of first breeding, but the values were found to be within the normal limits. The haemoglobin level and total erythrocyte count were found to be increased on the first day of proestrus and on the day of first breeding in animals of Group III and IV. The mean serum phosphorus level on the first day of proestrus and on the day of first breeding in the treatment groups were found to continue at low levels as in the anoestrous stage. The mean serum copper level on the first day of proestrus exhibited statistically significant difference between the treatment and control groups. The mean serum level of calcium, iron, cobalt, zinc and manganese during anoestrus, on the first day of proestrus and on the day of first breeding did not reveal statistically significant difference between groups. The overall conception rate in the treatment groups were 40, 70, 50 and 40 per cent respectively as against 70 per cent in the control group. The average gestation length and litter size did not differ significantly between the treatment and control groups.ThesisItem Open Access Detection of serum relaxin as a diagnostic tool for early pregnancy diagnosis in bitches(Department of Animal Reproduction, Gynaecology and Obstetrics, College of Veterinary and Animal Sciences, Mannuthy, 2007) Deepthi, L; KAU; Sreekumaran, TWith the object of fioding a suitable and reliable method of early pregnancy diagnosis in bitches, the study was undertaken to investigate the efficacy of trans abdominal palpation, ultrasound scanning and relaxin detection was conducted. The study consisted of 45 apparently healthy bitches whieh were brought to the clinics for finding the optimal breeding time. Out of this, ten animals were selected at random for pregnancy diagnosis and were subjected to different methods of pregnancy diagnosis at different gestational age-16 to 20 days, 21 to 24 days and 25 to 30 days post breeding. Blood samples were collected for the estimation of haemoglobin, packed eell volume and erythrocyte sedimentation rate at the day of breeding and also at the above gestation periods. Body weights were reeorded at the day of breeding and also at different gestation periods. In the present study, it was found that abdominal palpation was difficult m diagnosing pregnancy between 16 to 20 days of gestation. When palpation was done in between 21 to 24 and 25 to 30 days post breeding, the accuracy obtained was 50% and 70% respeetively. This study suggests that trans abdominal palpation was not useful in diagnosing early pregnancy. By ultrasound scanning, the percentage accuracy at 16 to 20 days was 50%, which improved to 80 percent and 100 percent at 21-24 and 25-30 days post breeding respeetively. Foetal heartbeat could be observed in all the positive cases from 24 days of gestation. Pseudo-pregnancy, pyometra and abortion could be easily identified by this method. The earliest positive result obtained for serum relaxin detection was obtained at 20" day post breeding and the percentage accuracy was 50% at this period, as against 100% at 21-30 days of gestation. In the present study, it was found that serum relaxin test was not influenced by pseudo-pregnancy and uterine pathological conditions like pyometra. There was significant variation in haemogram (P <0.01) at the day of breeding and at different gestational age. Haemoglobin concentration at 16-20, 21-24 and 25-30 days of gestation were 10.88+0.31, 10.24+0.22, 8.77+0.28g/dl, which was lower than the value 11.56+0.27 obtained prior to breeding. The packed cell volume values were 34.66+0.9, 30.77+0.94, 28.22+1.02 and 26±0.94 percent at day 0, 16-20, 21-24, 25-30 days post breeding. There was significant variation in the values before and after conception. There was significant variation in erythrocyte sedimentation rate between day zero and at different gestational age. The values obtained varied significantly and recorded as 4.6±0.33, 14.3±1.09, 17.8±1.28 and 21.76±1.47mm/hr at day 0, 16-20, 21-24 and 25-30 days of gestation respectively. The body weight of all the ten animals varied significantly (P<0.01). It was observed that the body weight had shown a steady and progressive increase as the pregnancy advanced. The study revealed that abdominal palpation was not very useful in diagnosing early pregnancy. By ultrasound scanning, uterus as well as foetus could be visualized after 23 days of gestation. Serum relaxin detection could be used as an early tool for pregnancy diagnosis in bitches from 20 days post breeding. Results of the present study suggest that the relaxin test was accurate in diagnosing early pregnancy and its advantage being that it could be conducted and interpreted easily by a dog breeder or a dog owner. It could be concluded that detection of serum relaxin is a quick, simple and accurate tool for diagnosing early pregnancy under field conditionsThesisItem Open Access Comparison of different methods of pregnancy diagnosis in bitches(Department of Animal Reproduction, College of Veterinary and Animal Sciences, Mannuthy, 2005) Asha Merina, Kuriakose; KAU; Joseph MathewWith the object of comparing different methods of pregnancy diagnosis and to determine the early reliable method, a study was undertaken to investigate the efficacy of transabdominal palpation, ultrasound scanning and haematological profile during pregnancy at various stages of gestation in bitches. Sixty six apparently normal healthy bitches were included in the study. The bitches subjected to pregnancy diagnosis were grouped based on gestational period as 20 to 30, 31 to 40 and 41 to 65 days post breeding. The data obtained were compiled and tabulated.ThesisItem Open Access Early pregnancy diagnosis using ultrasonography in goats(Department of Animal Reproduction, Gynaecology and Obstetrics, College of Veterinary and Animal Sciences, Mannuthy, 2009) Sreejith, J R; KAU; Athman, K VThe objective of the study was to evaluate the efficacy of B-mode ultrasonography in early pregnancy diagnosis in goats and to identify the optimum stage of gestation for early pregnancy diagnosis using of transrectal and transabdominal ultrasonography. Thirty apparently healthy does with the history of breeding were selected for the study and these goats were randomly divided into three groups consisting of ten animals each. Group I consisted of ten goats which were scanned between third and fourth week (15- 28 days) post-breeding. Ten goats scanned between fifth and sixth week (29- 42 days) post-breeding, were included in group II and group III consisted of ten goats which were scanned between seventh and eighth week (43- 56 days) post-breeding. These animals were subjected to B-mode real-time ultrasound scanning transrectally (7.5 MHz probe)) and transabdominally (3.5 MHz probe). The accuracy of transrectal scanning in group I, II and III was 90, 100 and 100 per cent respectively and the accuracy for corresponding weeks was 50, 100 and 100 per cent respectively for transabdominal scanning. The embryonic vesicle was detected earliest on day 19 of gestation by transrectal scanning and on day 26 by transabdominal scanning. The embryo was first observed on day 22 and day 28 by transrectal and transabdominal scanning respectively. The foetal heartbeat which was an indication of foetal viability was detected earliest by transrectal scanning on day 24 of gestation. But by transabdominal ultrasonography, it could be detected only on day 34 of gestation. Placentomes and foetal skeleton were observed on day 42 and 54 of gestation respectively using both methods of scanning. The mean diameter of gestational sac recorded was 5.1 mm on day 19 and 27 mm on day 36 of gestation by transrectal scanning. The mean diameter of gestational sac recorded was 15.7 mm on day 26 and 34.4 mm on day 36 of gestation by transabdominal scanning. The mean crown-rump length ranged from 7.2 mm on day 22 to 34.4 mm on day 43 of gestation using transrectal scanning. By transabdominal scanning, the mean crown-rump length recorded was 16.7 mm on day 28 and 32.7 mm on day 43 of gestation. The diameter of the placentomes recorded by transrectal and transabdominal scanning on day 42 of gestation was 8.4 mm and 8.5 mm respectively. All the foetal measures by transrectal and transabdominal scanning were highly correlated (r > 0.9) with gestational age. The overall accuracy for the prediction of foetal numbers by transrectal and transabdominal scanning was 80 and 50 per cent respectively. The accuracy for prediction of foetal number using transrectal scanning in pregnant animals of group I and III was 71.43 per cent. The accuracy for the prediction was 100 per cent in pregnant animals of group II. By transrectal scanning, the accuracy for the prediction of singletons, twins and triplets by transrectal scanning was 100, 83.33 and 50 per cent respectively. By transabdominal ultrasonography, it was not possible to predict foetal number accurately in pregnant animals group I. The accuracy for the prediction of foetal number in group II by transabdominal scanning was 66.66 per cent while it was 85.71 per cent in group III. . The accuracy for the prediction of singletons, twins and triplets by ultrasonography was 50, 58.33 and 25 per cent respectively. In conclusion, transrectal scanning was accurate for pregnancy diagnosis from fourth week of gestation and transabdominal from fifth week of gestation and that real-time ultrasound scanning by both transrectal and transabdominal approaches was found to be reliable, safe and accurate for the diagnosis of pregnancy in goats from fifth week of gestation.ThesisItem Open Access Preservability of bovine preantral follicles in situ(Department of Animal Reproduction, Gynaecology and Obstetrics, College of Veterinary and Animal Sciences, Mannuthy, 2008) Harinarayanan, P M; KAU; Vijayakumaran, VThe study was designed and conducted with the objectives of assessing: (1) efficiency of two different temperatures, media and duration of storage for short term preservation of bovine preantral follicles in situ and (2) efficiency of conventional vitrification (CV) and solid surface vitrification (SSV), for long term cryopreservation of bovine preantral follicles in situ. Ovaries of six freshly slaughtered adult crossbred cows were collected and transported to the laboratory within one hour in normal saline at room temperature. The ovarian cortex from both ovaries of each animal was separated, divided further into fifteen pieces. One hour after the slaughter a cortical piece was selected at random from each animal and fixed in Bouin’s fluid for histological processing later on (control). Twelve cortical pieces from each animal were randomly allotted for short term preservation, of which, six pieces each were placed in vials containing normal saline and remaining in Dulbecco’s phosphate buffered saline (DPBS). Three vials from normal saline group and DPBS group were stored at room temperature (25ºC) and the remaining three vials from each group were stored at refrigeration temperature (3-5ºC). One cortical piece was taken out, from each media, from both the storage temperatures, at four, eight and twelve hours of storage and fixed for histological analysis. The remaining two cortical pieces were further divided into smaller fragments of approximately 1mm3 size. The fragments from one piece were vitrified using conventional vitrification (CV) and the rest by solid surface vitrification (SSV) and stored in liquid nitrogen for ten days. The fragments were warmed and fixed after the storage period. The quality of preantral follicles in the fixed ovarian tissues was evaluated based on morphology in histological sections. The preantral follicles were counted, classified as primordial, primary and secondary. Storage at refrigeration temperature preserved the preantral follicles very much better than the storage at room temperature. The amount of normal preantral follicles reduced progressively with passage of time. Primordial follicles were better adapted to preservation than primary and secondary follicles. The preantral follicles were best preserved at refrigeration temperature up to four hours of storage. The morphologically normal preantral follicles in normal saline (55.17 %) and DPBS (56.17 %) at this temperature and storage period did not show significant difference from that of control (59 %). The primordial follicles were preserved in both normal saline (61.42 %) and DPBS (61.15 %) at refrigeration temperature up to four hours, at levels similar to control (62.63 %). However, only DPBS at refrigeration temperature could ideally preserve primary (51.13 % vs. 53.06 % in control) and secondary follicles (42.10 % vs. 48.83 % in control). The number of morphologically normal preantral follicles in situ was significantly reduced after both CV (25.5 %) and SSV (37.17 %). But, SSV was significantly better than CV for preserving the quality of preantral follicles. According to this study DPBS at refrigeration temperature is ideal for preserving all the three classes of bovine preantral follicles in situ up to four hours of storage. Though vitrification of bovine ovarian tissue could not preserve preantral follicles at ideal levels, SSV was found to be far superior to CV in preserving bovine preantral follicles in situ.ThesisItem Open Access Reproductive performance of cross bred heifers under special livestock breeding programme of Kerala(Department of Animal Reproduction, Gynaecology and Obstetrics, College of Veterinary and Animal Sciences, Mannuthy, 2007) Sathyaraj N; KAU; Aravinda Ghosh, K NThe study was conducted to assess the influence of better feeding and management of calves selected under Special Livestock Breeding Programme (SLBP) implemented by the Department of Animal Husbandry of Kerala. Twenty two calves which were covered under SLBP and 11 calves which were not covered under SLBP were selected at random to form group I and II respectively. All the animals in both groups belonged to the farmers below poverty line (BPL) of Anthikad, Villadom and Ollukkara Villages of Thrissur District. Group I animals were fed with good quality compounded cattle feed supplied to farmers at 50 per cent subsidized rate from Department of Animal Husbandry and provide extension support, adequate health and insurance cover. These animals were closely monitored at monthly intervals and were dewormed at regular intervals. Group II animals were maintained by poor farmers under field condition and their feeding and management were fully dependent on the interest and capability of the farmers. The body weight of all animals in group I and II were recorded at 6th, 12th and 18th month of age and at puberty and sexual maturity. The mean body weight in animals belonging to group I at 6th, 12th and at 18th month of age and at puberty and maturity were 68.32 ± 0.88, 116.59 ± 0.94 and 178.36 ± 1.36 , 165.5 ± 0.08 and 174.55 ± 1.7 kg and in group II were 69.36 ± 1.0, 83.26 ± 0.84 and 102.16 ± 0.29, 155.26 ± 0.29 and 165.24 ± 0.2 kg respectively. The daily weight gain of animals belonging to group I and II from 6th to 12th month of age were 268.22 g per day and 77.2 g per day respectively and that from 12th to 18th month of age were 343.16 and 105 g per day respectively. Statistical analysis revealed that group I animals had higher level of significance (P<0.01) compared group II animals. It was found that all the animals in group I exhibited puberty before 21 months of age while only 2 (18.2 per cent) exhibited puberty in control group by 24th month of age. The overall age at puberty in group I experimental animals were 448.68 ± 16.20 days whereas in group II animals were 645 days. Similarly, all the experimental animals in group I reached maturity by 24th month of age while only 2 (18.2%) reached maturity in control group. The overall age at maturity in group I experimented animals was 515.09 ± 15.06 days whereas in group II animals it was 686 days. There is higher level of significance in age at puberty and maturity between these two groups. A total of 14 (63.6%) in group I whereas only 2 (18.2%) in group II conceived by 24th month of age. The overall age at conception in group I experimental animals was 619 ± 22.66 days whereas in group II control animals it was 716 days. The number of AI per conception in group I animals was 1.86 whereas in group II was 2.5. The heifers covered under SLBP had reached puberty and maturity at an early age and obtained a higher conception rate when compared to control group. Haematological parameters such as haemoglobin, packed cell volume, total leukocyte counts, and total erythrocyte counts were estimated in all the animals at 6th, 12th and 18th month of age and at puberty and maturity. It was found that all the haematological parameters except leukocytes counts were significantly higher in group I animals from 12th month of age to maturity compared to group II animals. The blood biochemical constituents like calcium, copper, iron, cobalt, zinc and manganese were estimated by Perkin Elmer Atomic Absorption Spectrophotometry and phosphorus by colourimetry. The serum phosphorus, iron, cobalt and copper were found to be significantly higher in group I whereas there was no significant difference in serum calcium, zinc and manganese levels between the two groups. It is concluded that calves enrolled under SLBP implemented by AH Department of the State attained puberty and maturity at an early age and yielded a satisfactory conception rate under field conditions.ThesisItem Open Access Cryopreservability of caprine oocytes and embryos by conventional straw and open pulled straw vitrification(Department of Animal Reproduction, Gynaecology and Obstetrics, College of Veterinary and Animal Sciences, Mannuthy, 2006) Ratheesh Babu, M; KAU; Vijayakumaran, VObjective of the study was to compare the cryopreservability of caprine oocytes and embryos by Conventional Straw (CS) and Open Pulled Straw (OPS) vitrification. Oocytes retrieved by slicing the abattoir origin ovaries of Malabari goats/ its cross bred, having at least one complete layer of cumulus cells were randomly assigned for CS and OPS vitrification procedure. After thawing and cryoprotectant removal oocytes were examined again for morphological viability and membrane integrity by FDA staining. The total recovery of good quality oocytes from 73 ovaries and its average yield per ovary were 206 and 2.82 respectively. Out of this 85 and 54 oocytes respectively were subjected to CS and OPS vitrification. The percentage recovery of morphologically viable oocytes after thawing was higher for OPS than CS vitrification (70.59 vs. 42.11). The higher recovery rate of oocytes for OPS over CS vitrification indicated the superiority of the technique in maintaining the morphological viability of oocytes. Only 3.92 per cent of the OPS vitrified thawed oocytes showed ZP damage, which was lower than that after CS vitrification (5.25). Higher cumulus damage observed after CS than OPS vitrification (36.8 vs. 11.7 %) indicated the higher efficiency of OPS in reducing the osmotic damage of cumulus cells. A higher percentage of oocytes (52.9) were recorded as FDA viable after OPS than CS vitrification (31.58). The vitrification efficiency of caprine embryos were studied using embryos obtained from fresh and repeatedly superovulated adult Malabari goats. Superovulation was achieved with 133 mg pFSH after synchronization using 1.5 mg norgestomet and 10 mg of prostaglandin. Thirty four transferable quality embryos were equally divided and subjected to vitrification by CS and OPS methods. After a storage period of minimum 10 days the vitrified embryos were examined for morphological characteristics and membrane integrity by FDA staining. The response to synchronization was hundred per cent in fresh and repeatedly superovulated groups. The average number of anovulatory follicles observed in fresh and repeatedly superovulated goat was 7.17 ± 2.0 and 5.17 ± 1.08 respectively. Average number of ovulation in fresh goat (18.5 ± 2.19) was higher than that of repeatedly superovulated goat (11 ± 2.74). Significantly higher (P<0.05) average recovery of embryos was noted in fresh animal (9.33 ± 3.4) than in repeatedly superovulated animal (0.67 ± 0.49). Adhesion of reproductive tract due to earlier surgical handling led to lower recovery of embryos from repeatedly superovulated goats. Embryo recovery rate after OPS was higher than CS vitrification (100 % vs. 82.35 %). Similarly the recovery rate of morphologically viable embryos was also higher for OPS (88.24 %) than CS vitrification (50 %). After CS vitrification, 14.28 per cent of the vitrified embryos showed ZP damage while none of the embryos subjected to OPS vitrification showed similar damage. After FDA staining a higher percentage (76.47) of OPS vitrified embryos retained membrane integrity of blastomeres than CS vitrified embryos (28.57). Present study indicated that OPS is a far superior technique than CS vitrification for cryopreservation of caprine oocytes and embryos. Presence of morphologically viable non-fluorescein oocytes and embryos with FDA staining inferred that viability assessment of oocyte and embryo based on morphological characteristics alone is not enough, but it should be coupled with other non-invasive technique such as FDA staining. The study further revealed that the embryo yielding efficiency of fresh animal was significantly higher than repeatedly superovulated animals.ThesisItem Open Access Effect of ovum retrieval methods and cumulus-oocyte complex morphology on in vitro maturation of bovine oocytes(Department of Animal Reproduction, College of Veterinary and Animal Sciences, Mannuthy, 2005) Magnus Paul, K; KAU; Sreekumaran, TThis study was designed to evaluate the effect of different retrieval methods like aspiration, slicing and puncture on yield of different grades of oocytes and their in vitro maturation potential. The effect of cumulus oocyte complex morphology on in vitro maturation of bovine oocyte was also studied. Slaughterhouse derived bovine ovaries from South Indian breeds like Kangayam, Khillari, Hallikar and crossbred cattle of Kerala were subjected to three retrieval methods to yield different quality grades of oocytes. In this study, a total of one hundred and fifty ovaries were processed. Fifty ovaries were processed under each method of retrieval. Each quality grade of oocyte obtained through three retrieval method were subjected to maturation for 24 h in TCM-199 medium supplemented with LH, FSH, Estrogen, Pyruvate and Foetal calf serum. Culture environment was set as 38.5°C temperature, 5% carbondioxide tension and maximum humidity in standard CO2 incubator. Maturation changes were assessed by cumulus expansion, formation of M II plates and polar body extrusion. Total yield of COCs in slicing was 5.42 ± 0.56, which was significantly higher than aspiration and puncture which yielded 3.7 ± 0.43 and 3.66 ± 0.43 COCs respectively. This could be due to recovery of oocytes from a heterogeneous population of follicles in slicing method. Aspiration, slicing and puncture yielded A class oocytes at the rate of 44.86%, 37.2% and 43.17% respectively, wherein mean yield per ovary in same methods were 1.66 ± 0.25, 2.02 ± 0.25 and 1.58 ± 0.25 respectively. Yield of B class oocytes by aspiration, slicing and puncture were 32.97%, 29.89% and 31.69% respectively. Mean yield of B class oocytes per ovary was 1.22 ± 0.12, 1.62 ± 0.20 and 1.16 ± 0.16 respectively. Proportionate yield of D class oocytes by aspiration, slicing and puncture was 3.7%, 12.10% and 6.01% respectively. Aspiration, slicing and puncture did not differ significantly in yield of A, B and C class oocytes. But D class oocytes were proportionately more in slicing method. This finding could be attributed to the fact that the slicing recovered oocytes from a heterogeneous population of follicles, which included all kinds of growing follicles. When percentage yield of different classes of oocytes in same retrieval method was compared aspiration yielded A and B class oocytes in significantly higher proportion than C and D class oocytes. The same trend was observed in slicing and puncturing. In all the methods, the yield of D class oocytes was least. Cumulus expansion rate of A class oocytes were 83.08 per cent, 69.70 per cent and 70.37 per cent in aspiration, slicing and puncture method respectively. B class oocytes exhibited cumulus expansion rate of 68.29 per cent, 53.0 per cent and 62.07 per cent in aspiration, slicing and puncture method respectively. Retrieval method was found to have no significant effect on cumulus expansion potential of bovine COCs. Oocytes with more than three layers of cumulus cells exhibited better cumulus expansion rate than oocytes with less cumulus cell layers. Nuclear maturation rate of A class oocytes in aspiration, slicing and puncture was 81.08, 78.84 and 80.0% respectively, whereas in B class oocytes under same methods the percentage of nuclear maturation was 57.10, 41.67 and 52.63% respectively. D class oocytes failed to mature in all methods of retrieval except one oocytes in aspiration method. Aspiration, slicing and puncture did not differ significantly in the maturation percentage of oocytes. Polar body extrusion rate of A class oocytes in aspiration, slicing and puncture method was 45.5 per cent, 42.1 per cent and 44.0 per cent respectively. B class oocytes exhibited polar body extrusion rates of 28.6 per cent, 25.0 per cent and 26.3 per cent respectively in aspiration, slicing and puncture method respectively. Oocytes with more than three layers of cumulus cells exhibited better polar body extrusion rate irrespective of their retrieval method. Staining of 18 oocytes with fluorescent dye Hoechst 33342 as a method of cross checking, revealed 66.67 per cent Metaphase II rate. Ten oocytes tested for viability with FDA stain revealed 80 per cent viability after maturation. TCM-199 medium with 5 µg FSH/ml, 0.5 µg LH/ml, 1 µl Estradiol/ml, 0.2 mM pyruvate and 10 per cent serum was found to be ideal medium for maturation of bovine oocytes in vitro.. Oocytes with more than three layers of cumulus cells matured better than oocytes with less number of cumulus cell layers or denuded oocytes. These experiments proved that cumulus oocytes complex morphology have a very significant role in maturation of oocytes rather than retrieval methods.