MORPHOLOGY AND VIABILITY OF BOVINE EMBRYOS FROZEN IN MEDIA CONTAINING BSA AND PROP ANEDIOL By K. RAMACHANDRAN 'r THESIS Submitted in partial fulfilment of the requirement for the degree of Boctorof 'hilosophy Faculty of Veterinary and Animal Sciences Kerala Agricultural University COLLEGE Department of Animal Reproduction OF VETERINARY AND ANIMAL SCIENCES MANNUTHY, THRISSUR -680651 KERALA 2000 'l-
CERTIFICATE Certified that the thesis, entitled "MORPHOLOGY AND VIA.BILTTY OF BOVINE EMBRYOS FROZEN IN MEDIA. CONTAINING BSA AND PROPANEDIOL" is a record of research work done independently by Dr. K. Ramachandran, under my guidance and supervision and that it has not previously formed the basis for the award of any degree, fellowship or associateship to him. Thiruvananthapuram \b. \0-2000 Si Dr. S.p. resan Nair (Chairman, Adviso Committee) Associate Pro ssor & Head Department of Animal Husbandry College of Agriculture, Vellayani Thiruvananthapuram
ABSTKACT The observation of the study was to compare the effect of Bovine Serum Albumin (BSA) and i,2 Propanediol on the morphology and viability of bovine embryos frozen under two freezing and thawing protocols. A total of sixteen crossbred cows, kept under identical conditions, maintained in the Network Project on Embryo Transfer attached to the Department of Animal Reproduction, College of Veterinary and Animal Sciences, Thrissur. The animals were superovulated by FoHtropin-V and Prosolvin, starting on the day 11 of the cycle. Of the 16 cows superovulated i 3 showed good response. While two cows did not show any response, there were multiple follicles in both the ovaries without any evidence of ovulation in the third animal. A total of 85 (76 transferable and 9 non transferable) embryos were recovered from a total of 24 Bushings from i 3 cows, non-surgically on day 7. A total of 56 embryos (mean 3.50 :i: 0.822) were recovered in the first treatment;. from i 3 Bushings. In one cow, though, there was 80 per cent Bushing, no embryos could be recovered. While 22 embryos (mean 2.75 :i: 0.861) were recovered in the second treatment from 8 Bushings, only 7 (mean i.4 :i: 0.4) embryos were recovered in the third treatment, from 5 Bushings. A total of 72 transferable (60 morulae and i 2 blastocysts) were sleeted for the freezing trials. The embryos were divided into three groups with 24 (20 morulae and 4
blastocysts) embryos and assigned to three media. The first medium was FBS with 10 per cent glycerol, second PBS containing 10 per cent glycerol and 1 per cent BSA; the third medium was with a composition of 10% glycerol and 20% 1,2 propanediol in PBS. Two freezing protocols were used for freezing of the embryos. In the first protocol, with 12 embryos (10 morulae and 2 blastocysts), the initial cooling was at a rate of I C/min from room temperature to _OCand then at a rate of 0.3 C/min to -35 C, while in the second protocol the initial rate of cooling was at 5 C/min to -7 C and then at O.3 C/min to -30 C before transferring to liquid nitrogen. Thawing was carried out at 37 C for 20 sec after 30 days of preservation. Cryoprotectants were removed by two methods, a four step-wise using decreasing concentrations of cryoprotectants in the first method and one step using 1M sucrose phosphate buffered saline in the second. Thirty four embryos (26 morulae and 8 blastocysts) found normal after freezing and thawing were subjected to culture for 24 h in PBS enriched with 4 per cent BSA at 37 C and 5 per cent CO2 tension. Sixteen embryos (13 morulae and 3 blastocysts) were transferred to 15 recipient cows. While one cow was confirmed pregnant on examination 60 days after transfer, eleven cows returned to heat subsequently, two cows came to oestrus on days 34 and 35 respectively, after the transfer. The third showed oestrum on 45th day of transfer. The glucose, acid. phosphatase and alkaline phosphatase values showed a normal range of 86.2 to 195.2 mgjloo ml; 14.17 KA to 22.3 KA and 119.02 to.129.00 KA (mean 128.075 :f: 9.019, 18.675 :f: 0.667 and 122.67 :f: 0.788) respectively in the luminal fluid of the recipient animals. The average serum progesterone levels on day 0, 14 and 28 days after oestrus
11 in 11 cows which showed subsequent heat after the transfer were 0.357 :f: 2.140, 3.053 :f: 0.420 and 2.572 :f: 0.627 ng/ml and that of the animals which failed to show oestrum were 0.157:f: 0.166,3.793:f: 0.406 and 3.867:f: 0.362 ng/ml respectively. While significant difference was seen between the freezing media I and II and II and III respectively on the morphology of embryo after the freezing and thawing, no significant differences were seen between the media I and III, between the freezing protocols and cryoprotectant removal procedures on the morphology of embryos frozen. No significant differences were noticed on the effect of the freezing medi~ freezing protocols and the cryoprotectant removal procedure after the culture on the morphology of the embryos.