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ThesisItem Open Access YIELD AND QUALITY OF TRANSVAGINALLY RETRIEVED OOCYTES IN NORMAL AND REPEAT BREEDING CATTLE(COLLEGE OF VETERINARY AND ANIMAL SCIENCES MANNUTHY, THRISSUR, 2017-12-01) ABHILASH R. S; ABHILASH R. S; Metilda Joseph; Metilda JosephThe study was conducted to assess the efficacy of transvaginal oocyte recovery (TVOR) and to evaluate the quality of oocytes recovered from 12 normal and 12 repeat breeder crossbred cows stationed at the University Livestock Farm and Fodder Research Station, Mannuthy. Both normal and repeat breeder cows further divided into two groups of six animals each based on the TVOR frequency. Transvaginal oocyte recovery was performed at a frequency of once weekly in groups I and III and twice weekly in group II and IV for a period of two months (Group I and II- normal cows, and group III and IV- repeat breeders, six animals each). Frequency of TVOR or reproductive status of animal was not found to influence ovarian biometry. Number of small ( 9 mm) follicles were significantly lower (p ≤ 0.05) in normal and repeat breeder cows subjected to twice weekly TVOR. The number of follicles aspirated, number of oocytes retrieved, culture grade oocytes, matured oocytes, fertilized oocytes, cleaved oocytes per session and oocyte recovery rate (%) were siginicantly higher (p ≤ 0.05) in normal animals and those animals subjected to twice weekly TVOR. However, there was no significant difference between maturation rate, fertilization rate and cleavage rates between normal and repeat breeder animals and animals subjected to once and twice weekly TVOR. Evaluation of oocyte maturation rate using Hoechst 33342 and FDA also revealed that maturation rate was higher in normal breeders. No significant difference in the serum progesterone and blood urea nitrogen level were observed when normal and repeat breeder cows were subjected to TVOR at varying frequencies. A significantly higher follicular fluid progesterone concentration was observed in normal animals when compared to repeat breeders. The present study revealed that oocyte quality in repeat breeders is significantly lower than normal breeders, and TVOR at twice weekly interval was found to be an effective tool for harvesting maximum number of oocytes from crossbred cows.ThesisItem Open Access DIAGNOSIS AND THERAPEUTIC MANAGEMENT OF CANINE PYOMETRA FOR RESTORING BREEDING EFFICIENCY(COLLEGE OF VETERINARY AND ANIMAL SCIENCES, MANNUTHY, THRISSUR, 2018-05-09) UNNIKRISHNAN M. P.; M.O.KurienA study was undertaken to evaluate the efficacy of different therapeutic protocols in the treatment of canine pyometra, based on clinical, haematobiochemical, B-mode ultrasonographic and Doppler ultrasonographic evaluation. Combination treatment using mifepristone @ 2.5mg/kg b. wt. bid orally for five days, along with incremental doses of dinoprost from 10 to 50 µg/kg b. wt. tid; 48 h after initiation of mifepristone, till complete uterine evacuation was superior for therapeutic management of canine pyometra in terms of clinical recovery and future fertility. Haematology recorded anaemia, leucocytosis with neutrophilia and lymphopenia in patients with pyometra, which resolved after treatment. Serum biochemistry revealed marginal rise in blood urea nitrogen, high C-reactive protein and rise in progesterone values, which reduced to normal by day 15 of treatment. Early reduction in progesterone was noticed in cabergoline-based group. B-mode ultrasonography revealed uterine horn distension with anechoic to hypoechoic contents and thickened wall, which reduced to normal by day 15 of treatment. Delay in uterine resolution was noticed in cabergoline-based group. Doppler ultrasonography revealed increased uterine blood flow velocity (peak systolic velocity: 0.69 ± 0.05 to 0.75 ± 0.07; end diastolic velocity: 0.27 ± 0.02 to 0.31 ± 0.01) and decreased resistive index (0.59 ± 0.04 to 0.62 ± 0.06), which returned to normal after treatment. Bacteriological isolates from anterior vagina revealed E. coli (52.63 %), followed by Staphylococcus spp. (37.74 %), Streptococcus spp. (5.66 %) and Pseudomonas (3.77 %). Escherichia coli and Pseudomonas spp. were sensitive to amikacin, whereas Staphylococcus spp was most sensitive to ceftriaxone-tazobactam. Streptococcus spp. was 100 per cent sensitive to cephalosporin. Hyper-salivation was the most common side effect of prostaglandin treatment, exhibited in 31.25 per cent of dogs. Time taken for complete uterine evacuation varied between 5.88 ± 0.40 and 8.25 ± 0.73 days, with more in cabergoline-based group. Fertility studies revealed better cyclicity and conception in 75 and 66.67 per cent, respectively of mifepristone-dinoprost combination treated dogs, whereas overall cyclicity and conception was 68.75 and 54.55 per cent, respectively. Overall recurrence of 28.13 per cent was recorded after medical treatment with minimum recurrence (12.5 %) in mifepristone-dinoprost treated dogs.ThesisItem Open Access CHARACTERIZATION OF MALABARI BUCK SEMINAL PLASMA PROTEINS IN RELATION TO SEMEN FREEZABILITY AND FERTILITY(COLLEGE OF VETERINARY AND ANIMAL SCIENCESMANNUTHY, THRISSUR, 2016-12-30) AMBILI JOHN; Hiron M. HarshanThe study was undertaken to characterize the protein profile of Malabari buck seminal plasma for identifying protein markers of freezability and fertility and to compare the in vivo fertility of Malabari buck semen with low and high semen freezability. Adult Malabari bucks with post thaw sperm progressive motility of more than 35 per cent were classified as having high semen freezability while those with less than 30 per cent were classified as having low semen freezability (four each) were utilized for the study. Tris - egg yolk based extender was used for cryopreservation of the ejaculates (n = 48; 24 each from bucks having high and low semen freezability). Significantly higher values were noted in the pre-freeze acrosome integrity and membrane cholesterol levels and post-thaw motility, viability, acrosome integrity, hypo osmotic sperm swelling response (HOS) and membrane cholesterol levels of high semen freezability bucks when compared with low semen freezability bucks. Whereas the seminal plasma ALP and LDH were found to be significantly lower in high semen freezability bucks compared with low semen freezability bucks at post thaw stage. The total protein content and average number of protein bands in seminal plasma of high and low semen freezability bucks did not differ significantly (75.82 ± 4.62 mg/ml vs 67.07 ± 6.07 mg/ml; 20.83 ± 0.53 vs 22.09 ± 0.49). The 24 kDa, 79 kDa, 84 kDa and 134 kDa proteins were found to have significantly higher occurence in seminal plasma of bucks with high semen freezability whereas the occurence of 15 kDa, 19.9 kDa, 21.5 kDa, 70kDa and 89 kDa proteins were found significantly higher in seminal plasma of bucks with low semen freezability. On analysis of 2D gels, an average of 67.67 ± 4.06 and 75.33 ± 4.28 spots (pI 3-10) were detected in semen of high and low semen freezability bucks, respectively and the difference was not significant. On western blot analysis, osteopontin, a protein fertility marker, was found to be present in seminal plasma of all the bucks studied. For fertility studies 134 does were inseminated with semen preserved by chilling and 91 with cryopreserved semen. The conception rate (CR) with cryopreserved semen was significantly higher in bucks with high semen freezability than with low semen freezability. In the semen preserved by chilling, no significant difference was noticed between low and high semen freezability animals in the spermatozoa progressive motility, viability, acrosome integrity, HOS response or seminal plasma ALP and LDH levels after dilution or after 24 h of storage at refrigeration. But when chilled semen was used, the CR was significantly higher for bucks with low semen freezability than for bucks with high semen freezability.ThesisItem Open Access AUGMENTING REPRODUCTIVE EFFICIENCY IN POST PARTURIENT DOES WITH INTRAVAGINAL PROGESTERONE SPONGES, PROSTAGLANDIN F2α AND GONADOTROPIN RELEASING HORMONE(COLLEGE OF VETERINARY AND ANIMAL SCIENCES MANNUTHY, THRISSUR, 2016-12-30) UPASANA RATNAKARAN; K.N. Aravinda GhoshThesisItem Open Access CLINICAL, HISTOPATHOLOGICAL AND IMMUNOHISTOCHEMICAL CHANGES IN VAGINAL HYPERPLASIA IN DOGS(COLLEGE OF VETERINARY AND ANIMAL SCIENCES MANNUTHY, THRISSUR, 2019-08-16) ANILA BABU; Bibin BechaThe study was undertaken with the objectives of the objectives of clinico gynaecological examination, haematological studies, exfoliative vaginal cytology, ultrasonographic assessment of ovarian status, histopathological studies and immunohistochemical localisation of oestrogen receptor-α in normal oestrus dogs (Group I) and in dogs affected with vaginal hyperplasia (Group II). The overall occurrence of vaginal hyperplasia was found to be 1.02 per cent. Occurrence was more in Labrador retriever breed (48.94%), in dogs of one to two years age group (43.62%), nulliparous dogs (61.70%) and was observed during proestrus (35.11%) and oestrus (60.64%) stages of ovarian cycle in intact dogs. Presence of varying degrees of vaginal mass (86.17%), followed by vaginal discharge (25.53%) were major symptoms reported. Physiological parameters like rectal temperature, heart rate, respiratory rate and colour of mucous membranes of these dogs were within reference ranges. Clinical examination of prolapsed vaginal tissue of hyperplastic dogs revealed 83.33 per cent as Type II and 16.67 per cent as Type III vaginal hyperplasia with 50 per cent dry and pale prolapsed vaginal tissue. The mean anuclear cell index in group I and II dogs were 63.17±6.11 and 63.33±6.24, respectively. Mean recorded follicular diameter (mm) of left and right ovaries were 3.90±0.40 and 4.60±0.50 among Group I dogs, whereas in Group II it was found to be 4.70±0.50 and 4.20±0.20, respectively. Mean recorded follicular number of left and right ovaries are 3.67±0.49 and 3.50±0.42 among Group I dogs, whereas in Group II it was found to be 2.68±0.55 and 3.66±0.55 and they differed non-significantly between the groups. There was no ovarian abnormalities could be observed in Group I and Group II dogs during presented day of examination. The oestradiol levels in Group I and II were 74.11±10.52 and 82.95±11.56 pg/mL, respectively and they differed non-significantly. On histopathological analysis, oestrogenic response of the vaginal epithelium was found to be more evident in Group II than Group I dogs. The mean thickness of stratum germinativum, stratum granulosum and stratum corneum of Group I dogs were measured as 31.42±2.73, 13.93±0.73 and 32.86±6.32µm, respectively. The respective values for Group II dogs were obtained as 27.11±1.45, 13.69±1.03 and 29.77±4.10µm and no significant difference was observed between groups. The proportional score of ER-α in the vaginal epithelium was found to be significantly high (p<0.05) in Group II (3.00±0.25) than Group I dogs (1.66±0.21) and that in the stroma was found to be same (2.50 ± 0.22) in both groups. It was concluded that ER-α expression was more in vaginal epithelium of vaginal hyperplasia affected dogs.ThesisItem Open Access PREDICTION OF WHELPING DATE IN DOGS BASED ON VAGINAL EXFOLIATIVE CYTOLOGY, VAGINOSCOPY AND PERI-OVULATORY SERUM PROGESTERONE ASSAY(COLLEGE OF VETERINARY AND ANIMAL SCIENCES, POOKODE WAYANAD, 2019-08-20) NIKITA NITIN, PATIL; Chacko, LeebaThe study was designed to assess the efficacy of vaginal exfoliative cytology (VEC), vaginoscopy and peri-ovulatory serum progesterone assay in predicting the whelping date in dogs. VEC and vaginoscopy were performed in 43 animals with the history of proestrual bleeding on all days till the onset cytological dioestrus (D1). Serum progesterone (P4) level was estimated on alternate days till the value reached four ng/mL or higher and two breeding were recommended. Eighteen whelped dogs were selected randomly for the current study. On VEC, mean ± SE cornification index peak was 71.63 ± 2.55. On vaginoscopy, mean ± SE period of shrinkage with angulation and period of declining shrinkage with rounding out of mucosa was 5.22 ± 0.31 and 2.89 ± 0.26 days, respectively. Mean serum P4 values in ng/mL on the day of LH surge and ovulation were 2.53 ± 0.10 and 4.99 ± 0.14, respectively. Whelping was predicted on 57th, 61st, 65th and 63rd day from D1, day of maximum angulation (S5), day of LH surge and day of ovulation, respectively. When calculated from D1, 22.22 per cent animals delivered on 57th day. The accuracy of prediction of whelping date within ±1 and ±2 days was 83.33 and 100 per cent, respectively. When calculated from S5, 22.22 per cent animals delivered on 61st day. The accuracy of prediction of whelping date within ±1, ±2, ±3 and ±4 days was 44.44, 77.78, 94.44 and 100 per cent, respectively. When calculated from the day of LH surge, 33.34 animals delivered on 65th day. The accuracy of prediction of whelping date within ±1, ±2 and ±3 days was 66.67, 83.34 and 100 per cent, respectively. When calculated from the day of ovulation, 27.78 per cent dogs delivered on 63rd day. The accuracy of prediction of whelping date within ±1, ±2 and ±3 days was 66.67, 83.34 and 100 per cent, respectively. Significant correlation was found between serum P4 value on the day of LH surge (R2 = 0.867) and gestation length. However, no significant correlation was found between serum P4 value on the day of ovulation (R2 = 0.461) and gestation length. Two gestational equations were also derived from the observations. In conclusion, VEC was the most accurate method for prediction of whelping date. Whereas serum P4 values were found to be more accurate than vaginoscopy.ThesisItem Open Access EVALUATION OF SEMEN QUALITY ON LIQUID STORAGE OF SPECIFIC FRACTIONS OF LARGE WHITE YORKSHIRE BOAR SEMEN(COLLEGE OF VETERINARY AND ANIMAL SCIENCES, POOKODE WAYANAD, 2019-08-09) MALATI NAIK; Harshan, Hiron M.The present research was carried out to assess the quality of Large White Yorkshire (LWY) boar semen in specific fractions within an ejaculate and to assess the keeping quality of fractions of LWY boar semen stored in liquid state at 15C. Two fractions of the semen were taken for the study, initial 10 mL of sperm rich fraction (SRF) formed F1/Group I, rest of the sperm rich fraction formed F2/group II and mixture of half of F1 and F2 each formed group III. A total of 30 ejaculates were used for the study as fractions from four adult healthy LWY boars (16 in Phase I and 14 in Phase II). In phase I, differences in fresh semen characteristics of F1 and F2 were evaluated with respect to pH, concentration, seminal plasma protein content and SDS PAGE profile were analysed. Significant differences were observed between fractions with respect to pH and sperm concentration. The sperm progressive motility and total protein content of semen did not differ significantly between F1 and F2 fraction. An average number of 13.5 ± 0.51 bands were observed in F1, 12.92 ± 0.44 bands in F2 and 12.78 ± 0.23 bands in the sperm free fraction. The proteins observed varied in the molecular weight from 18 - 244 kDa, 8 – 242 kDa and 9 – 248 kDa in F1, F2 and sperm free fraction, respectively. In Phase II of study, semen fractions were analyzed for their keeping quality at 15C after being extended in BTS as group I, group II and group III. Semen was extended with BTS to a final concentration of 50 × 106 sperms/mL and gradually cooled to 22°C, held at same temperature for three hours. The semen was subsequently cooled to 15°C and stored for 72h and evaluated at 4, 24, 48 and 72 h of storage. The pH of semen was significantly (p < 0.01) higher in group II at all time point intervals. The progressive motility of semen was significantly (p < 0.05) higher in group I at 24h, 48 h and 72 h of storage. The sperm viability was significantly higher (p < 0.05) in group I at 72 h of storage alone. The sperm abnormality, acrosome integrity and rapid HOS response was significantly higher in group I at 24 h, 48 h and 72 h of storage alone. Sperm plasma membrane integrity showed a trend of being higher in group I at 72 h of liquid storage (p < 0.1). The cholesterol values at 0h and 72h was significantly higher (p < 0.01) in group I than other groups. The study revealed that initial 10 mL of SRF had showed better preservability at 15C than rest of SRF.ThesisItem Open Access STORAGE QUALITY OF LIQUID SEMEN OF LARGE WHITE YORKSHIRE BOAR SUPPLEMENTED WITH CHOLESTEROL LOADED CYCLODEXTRIN(COLLEGE OF VETERINARY AND ANIMAL SCIENCES, POOKODE WAYANAD, 2019-09-04) T., SHYLESH; Harshan, Hiron M.The present study was carried out to assess the effect of supplementation of cholesterol loaded cyclodextrin on quality of liquid semen of Large White Yorkshire (LWY) boar stored at 15C. A total of 12 ejaculates were used for the study from four adult healthy LWY boars maintained at Centre for pig production and research, Mannuthy. The fresh semen characteristics were evaluated immediately after collection and semen was extended with Beltsville thaw solution to a final concentration of 120 × 106 sperms/mL. The extended semen was split into five groups and processed as Group I (without any supplementation); Group II (supplemented with cholesterol loaded cyclodextrin (CLC) at the rate of 0.75 mg/120 million sperms); Group III (supplemented with CLC at the rate of 1.5 mg/120 million sperms); Group IV (supplemented with CLC at the rate of 3 mg/120 million sperms) and Group V (supplemented with methyl- cyclodextrin at the rate of 1.5 mg/120 million sperms). The processed semen was gradually cooled to 22°C and held at same temperature for three hours. The semen was subsequently cooled to 15°C and stored for 72 h and evaluated at 4, 24, 48 and 72 h of storage for semen attributes. The progressive motility of semen was significantly (p < 0.01) higher in group III and IV at 4h of storage and in group III at 24h, 48 h and 72 h of storage. The sperm viability was significantly higher (p < 0.01) in group III at 4, 24, 48 and 72 h of storage. The sperm abnormality was significantly (p < 0.01) higher in MBCD treated group at 48 and 72 h of storage. The HOS response was significantly (p < 0.01) higher in group II and III at all stages of preservation. The acrosome integrity was significantly (p < 0.01) higher in group III at 24 and 48 h, whereas both group II and III had significantly (p < 0.01) higher values at 72 h of preservation. The cholesterol content was significantly (p < 0.01) higher in group IV at 72h. The study revealed that cholesterol treatment of the spermatozoa with means of CLC at a rate of 1.5 mg/120 million sperms improved the quality of stored spermatozoa at various stages of storage at 15C till 72 h. It was also found that the supplementation of cholesterol as CLC could increase the amount of sperm cholesterol. Thus the positive effects of CLC on sperm characteristics during storage at 15C could be due to the supplementation of cholesterol to sperm membrane.ThesisItem Open Access POSTPARTUM RESUMPTION OF OVARIAN ACTIVITY AND ITS ASSOCIATION WITH SUBCLINICAL KETOSIS IN CROSSBRED COWS(COLLEGE OF VETERINARY AND ANIMAL SCIENCES, POOKODE WAYANAD, 2019-08-20) MEGHASHAM, KATHANE SONAM; K., PromodThe aim of the study was Postpartum resumption of ovarian activity and its association with subclinical ketosis in crossbred cows. The work was conducted at Instructional Livestock Farm Complex (ILFC), Pookode and Livestock Research Station (LRS), Thiruvazhamkunnu during the period of September 2018 to May 2019. The age, parity, milk yield, peripartum and postpartum complications of the selected animals was recorded. Crossbred cows on day 30 postpartum, with the history of peripartum and postpartum complications were screened by rectal examination at 12 days interval (30, 42 54 66, 78 and 90) for the assessment of uterine involution and ovarian functional status of the animals. Crossbred dairy cows at 30 days postpartum were screened for clinical ketosis by Rothera’s test of urine samples. On day 45 postpartum, the animals without any clinical signs of ketosis was screened again for subclinical ketosis (SCK) using cow side BHB testing reagent strips. Blood samples were collected from animals of both the groups at 12 days interval (30-90 days postpartum). The following biochemical constituents in serum were analysed in both the groups at 12 days interval as per standard procedures using diagnostic kits. Total serum cholesterol, Total lipids, Blood urea nitrogen (BUN), Aspartate aminotransferase (AST), Serum glucose, BHB. Serum progesterone concentrations analysis was done in both the groups to assess the resumption of ovarian cyclicity. Out of 30 animals 40 per cent of animals showed slightly positive, 33.33 per cent of animals showed moderately positive and 8per cent of animals showed highly positive for Rothera’s test. Control animals observed no colour change due to absence of ketone bodies. Out of 30 animals 105 200µmol/L, 300µmol/L, 350µmol/L, 400µmol/L and 500µmol/L of BHB level were present in 13.33, 26.66, 3.33, 53.33 and 6.66 per cent of animals, respectively. In SCK and non SCK cows mean ± SE of glucose, total cholesterol, total lipid, AST, BUN and BHB were significantly (P < 0.01) varied at 12 days interval (30-90 days postpartum). Mean ± SE of glucose, total cholesterol, total lipid, AST, BUN and BHB in early, late and not resumed animals were non-significance at 12 days interval (30-90 days postpartum). Result indicated that ROA, measured by the appearance of the first significant progesterone rise ( > 1 ng/ ml in blood ) in early postpartum period was not associated with SCK .More than 1.2 to 1.4 mmol/L level of the BHB which indicates presence of SCK. BCS was significantly associated with SCK and ROA as odds ratios were higher. Biochemical parameters were associated with the development of SCK, but no association was observed with postpartum ROA.
