MOHAMMED, K., ISMAIL, S., REZK, Y., TAWFIK, M. (2000). FACTORS AFFECTING VIABILITY OF DEEP FROZEN BUFFALO-EMBRYOS. Egyptian Journal of Agricultural Research, 78(2), 877-892. doi: 10.21608/ejar.2000.322513
KAMEL M. E. MOHAMMED; SAYED T. ISMAIL; YAHUA REZK; MAHMOUD S. TAWFIK. "FACTORS AFFECTING VIABILITY OF DEEP FROZEN BUFFALO-EMBRYOS". Egyptian Journal of Agricultural Research, 78, 2, 2000, 877-892. doi: 10.21608/ejar.2000.322513
MOHAMMED, K., ISMAIL, S., REZK, Y., TAWFIK, M. (2000). 'FACTORS AFFECTING VIABILITY OF DEEP FROZEN BUFFALO-EMBRYOS', Egyptian Journal of Agricultural Research, 78(2), pp. 877-892. doi: 10.21608/ejar.2000.322513
MOHAMMED, K., ISMAIL, S., REZK, Y., TAWFIK, M. FACTORS AFFECTING VIABILITY OF DEEP FROZEN BUFFALO-EMBRYOS. Egyptian Journal of Agricultural Research, 2000; 78(2): 877-892. doi: 10.21608/ejar.2000.322513
FACTORS AFFECTING VIABILITY OF DEEP FROZEN BUFFALO-EMBRYOS
1Animal Reproduction Research Institute, Agricultural Research Centre, Ministry of Agriculture, Dokki, Giza, Egypt
2Faculty of Vetrinary Medicine, Cairo University, Giza, Egypt
Abstract
Fitly buffalo embryos were recovered non-surgically on day 5-6 of the estrous cycle. The collected embryos were exposed to an increasing concentration of glycerol in phosphate buffer saline (PBS). either by a three step addition or six step addition. In the final concentration (1.5M glycerol), each embryo was aspirated into 0.25 ml straw. Two freezing procedures were used Procedure (A): conventional freezing (slow cooling) in which the freezing rate was 2°C/min. from room temperature to -70C and cooled at 0.30C/min to -350C, then. to -800C at cooling rate 100C/min before being plunged into liquid nitrogen (LN). Procedure (6): Rapid cooling or vitrification, the embryos were directly plunged into LN alter addition of cryoproteclant solution. Two techniques of thawing were applied on embryos frozen, either by rapid or slow methods. The first technique included plunging the straws into a water bath at 37°0 for 30 seconds, while the second one included holding the straws in air at room temperature for 2 minutes. Cryoprotectant was removed and embryos were washed. The viability was assessed using morphological evaluation and by staining the embryos with fluorescein diacetate (FDA). Embryos were examined under a reflected•ligh fluorescence microscope, and according to the fluorescence observed the embryos were classified as bright and non-bright (fluorescence was not detected). Twenty-one of them were frozen by rapid cooling method (vitrification). 10 of them were frozen after adding glycerol in 3 steps. On staining, the post-thawed embryos by FDA stain for evaluation 6 (60%) and 4 (40%) embryos were bright and non-bright, respectively. When embryos were frozen after adding glycerol in 6 stops (mr11), the respective values were 9 (81.8%) and 2 (18.2%). Differences among the 2 regimens of glycerol addition (3 and 6 steps) were significant (P<0.01). On the other side. 22 embryos were frozen by slow cooling. On adding glycerol in steps. 7 (77.8%) and 2 (22.2%), embryos were bright and non-bright, respectively. Six steps Glycerol additions revealed that 10 (76.9%) and (23.1%) embryos were bright and non-bright, respectively. No significant differences were noted between the two regimens of glycerol addition. Irrespective of the regimen of glycerol addition, the comparison between rapid and slow methods of freezing illustrated that, on using rapid freezing, 15 (71.4%) and 6 (28.6%). embryos were bright and non-bright. respectively. The respective values on using slow method were 17 (77.3%) and 5 (22.7%). Differences between the two methods of freezing were non-significant. Across both methods of freezing, 14 blastocysts (67.5%) and 18 morulae (66.7%) were bright, the differences between the 2 stages were highly significant (P<0.01). Seven out 22 embryos (31.8%) developed damaged ZP on using slow method of freezing, while, only 4 out of 21 (19.1%) embryous showed similar lesions on using rapid freezing, the difference between them was significant (P<0.05). Embryos thawed in water bath resulted in recovery rate of 82.8%, while, those thawed in air at room temperature resulted in 90.5% recovery rate. The difference between the methods of thawing was non-significant.
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