PARENT SESSION
EMBRYOGENESIS

Tuesday, August 3, 2004
10:30 AM–12:30 PM
Buchanan Courtyard

(528) BULL INFLUENCE ON APOPTOSIS IN BOVINE PREIMPLANTATION EMBRYOS.

Giritharan, Gnanaratnam ¹, Ramakrishnappa, Nagaraja¹, Balendran , Anusha ¹, Rajamahendran, Rajadurai ¹, ¹ Faculty of Agricultural Sciences, Vancouver, BC, Canada

ABSTRACT- The overall objective of this study was to develop an in-vitro test to predict fertility of bulls in the field. In this study, we investigated the bull effect on apoptosis in in-vitro produced embryos. Frozen semen from three separate ejaculates of six unrelated bulls (B1-B6) was used. Ejaculates from each bull were pooled to minimize variability among ejaculates. Oocytes obtained from slaughterhouse ovaries were matured and incubated with sperm from experimental bulls in a standard in-vitro fertilization and embryo culture procedure, to obtain Morula to blastocyst stage embryos. Cleavage and morula to blastocyst production rates were determined at 48 h and 162 h post insemination, respectively. The number of live, apoptotic and dead cells of morula to blastocyst stage embryos was counted, after staining with annexin V, propidium iodide, and bisbenzamide. Bax gene expression levels in morula to blastocyst stage embryos were determined by semi-quantitative reverse transcription polymerase chain reaction. Cleavage (B1-76.7±4.7, B2-59.2±2.2, B3-73.8±3.4, B4-60.4±4.9, B5-88.1±2.0, B6-76.0±0.7) and morula to blastocyst production rates (B1-32.9±4.3, B2-22.3±0.4, B3-29.2±3.5, B4-26.5±1.5, B5-45.4±0.7, B6-35.0±2.8) were different (P<0.01) among bulls. Percent apoptotic (B1-7.5±0.6, B2-11.3±0.3, B3-13.3±1.3, B4-10.0±0.9, B5-4.1±0.1, B6-5.3±0.3), live (B1-85.8±0.7, B2-79.8±0.9, B3-81.4±1.0, B4-83.8±0.7, B5-90.9±0.4, B6-90.4±0.3) and dead cells (B1-6.7±0.1, B2-8.9±0.6, B3-5.3±0.3, B4-6.2±0.2, B5-5.0±0.4, B6-4.3±0.1) in morula to blastocyst stage embryos were different (P<0.01) among bulls. The Bax gene expression levels (B1-0.98±0.23, B2-0.86±0.07, B3-0.66±0.11, B4-0.80±0.15, B5-0.84±0.14, B6-1.00±0.23) in morula to blastocyst stage embryos were not different among bulls. Percent live cells were positively correlated with cleavage rate (r=0.61) and morula to blastocyst production rate (r=0.73). Percent apoptotic cells were negatively correlated with cleavage rate (r=0.57) and morula to blastocyst production rate (r=0.67).

KEY WORDS: Apoptosis, Bull fertility, Bax, Embryo