Studies on gene expression for Heat Shock Protein 70 and identification of sperm membrane proteins in relation to semen quality and fertility in buffalo bulls
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Date
2011-01-11
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Abstract
The present study was conducted to investigate Heat Shock Protein 70 and
sperm membrane proteins in relation to semen quality and fertility in buffalo bulls. Frozen
semen from 20 buffalo bulls was collected, sperm motion traits were analyzed through
Computer Assisted Semen Analysis (CASA), live sperm count and HOS reactive sperms
were assessed. The total RNA was harvested from Percoll washed sperm with Trizol
reagent. The first strand cDNA was synthesized and HSP70 was quantified using a Real
time PCR. To predict fertility, bulls were ranked on the basis of semen evaluation traits.
Fertility Associated Antigens (FAA) were investigated in fresh semen using Reprotest
(Lateral Flow Cassettes). Sperm membrane proteins were harvested from frozen semen
using protein extraction buffer and SDS-PAGE was carried out by loading 100μg proteins
on 10% SDS polyacrylamide gels. The gel was stained with Coomassiae stain and the
protein bands were imaged using Syngene Gel Doc (Synoptic Ltd) and band analysis was
carried out using Gene Snap Image Acquisition Software. HSP70 was also investigated in
beef bull sperm membrane proteins through western blotting technique.
Buffalo bulls were divided into 3 groups on the basis of conception rates as
good fertility (63.30%), average fertility (37.72) and poor fertility (17.22%). The average
conception rate in good fertility bulls was significantly (p<0.05) higher than the average and
poor fertility bulls. The average individual motility in good fertility buffalo bulls (49.73%)
was significantly (p<0.05) higher than the poor fertility bulls (36.29%). The expression for
HSP70 in good, average and poor fertility buffalo bull sperm were 0.501, 0.413 and 0.331
Log10RQ, respectively, the differences were non significant (p>0.05) between the groups.
Buffalo bulls (16/20, 80%) were FAA positive which indicate good fertility. However, only
45% (9/20) buffalo bulls were of good fertility on the basis of fertility trial. Non significant
differences were observed in the semen traits between the FAA positive and negative bulls.
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The 130 and 43 kDa proteins were not found in poor fertility buffalo bull sperm. The spot
for HSP70 could not be detected in immunoblot of frozen beef bull sperm.
It was concluded that CASA based individual motility and expression for
HSP70 in buffalo bull sperm could be used for the differentiation of good fertility buffalo
bulls from poor fertility. On the basis of presence of FAA, good fertility bulls could not be
identified. The 130 and 43 kDa proteins were not found in poor fertility buffalo bull sperm
indicating a role in conception. The spot for HSP70 could not be detected in immunoblot of
frozen sperm indicating cryopreservation mediated cleavage or denaturation of HSP70 in
beef bull sperm.
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Keywords
Buffalo bulls, Conception rate, Fertility Associated Antigens (FAA), Heat Shock Protein 70 (HSP70), Sperm membrane proteins