PhD

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 Folltropin- V and Prosolvin, starting on the
day i 1 of the cycie. Of the i 6 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 fiushings from] 3
cows, non-surgically on day 7. A total of 56 embryos (mean 3.50 ± 0.822) were
recovered in the first treatment; from 13 Bushings. In one cow, though, there was 80
per cent Bushing, no embryos could be recovered. While 22 embryos (mean 2.75 ±
0.861) were recovered in the second treatment from 8 flushings, only 7 (mean j.4 ±
0.4) embryos were recovered in the third treatment, from 5 Bushings.
A total of 72 transferable (60 morulae and 12 blastocysts) were slected fur 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 (l0 morulae and 2 blastocysts), the initial cooling was at a
rate of l°C/min from room temperature to _QC and 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 0.3°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 1 M 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
mg/100 rnl; 14.17 KA to 22.3 KA and 119.02 to 129.00 KA (mean 128.075 ± 9.019,
18.675 ± 0.667 and 122.67 ± 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

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ill 11 cows which showed subsequent heat after the transfer were 0.357 ± 2.140, 3.053
± 0.420 and 2.572 ± 0.627 ng/rnl and that of the animals which failed to show oestrum
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were 0.157 ± 0.166, 3.793 ± 0.406 and 3.867 ± 0.362 ng/ml respectively.
While significant difference was seen between the freezing media I and II and Il
and III respectively on the morphology of embryo after the freezing and thawing, no
significant differences were seen between the media I and Ill, 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 media,
freezing protocols and the cryoprotectant removal procedure after the culture on the
morphology of the embryos.