STUDIES IN BULL SEMINAL PLASMA PROTEOME AND REDUCTION OF SPERM DOSES ON CRYO-SURVIVABILITY AND IN VITRO SPERM FUNCTIONS
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Date
2020
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Publisher
ICAR-NDRI, KARNAL
Abstract
The present study was conducted for the assessment of in vitro sperm functions and
seminal plasma proteome of bulls with differential fertility, method(s) for immobilization and
revival of bull sperm and cryo-survivability of low sperm number per dose in Murrah bulls. A
total no. of Murrah (n=8) and Sahiwal bulls (n=8) were divided into high-fertile (HF) and lowfertile
(LF) groups based on conception rate of 43-58 % and 23-34%, respectively. In post-thaw,
PM, live and AI in HF bulls was found to be highly significant (p≤0.05) as compared to LF
Murrah bulls, however: VAP, STR, TM, PM, moribund and dead sperm, B-pattern, AR-pattern
were differed significantly (p≤0.05) in HF and LF bulls. For sperm kinetics and sperm function,
VCL, ALH, LIN, RM, live sperm, F-pattern, LAI and MMP were significantly (p≤0.05) higher in
HF group as compared to LF Murrah bulls. Furthermore, a significant (p≤0.05) higher difference
was also found in LAR, DAR, CMA3 deficiency and lipid peroxidation in LF as compared to HF
Murrah bulls. For Sahiwal bulls, sperm kinetics and sperm function, which includes post-thaw,
PM, HOST, VCL and ALH differ significantly (p≤0.05) in HF as compared to LF. Further, postthaw
sperm live, AI, TM, PM and RM, live sperm, LAI and JC-1 was highly significant (p≤0.05)
in HF as compared to LF. However, significant (p≤0.05) differences in STR, LIN, and SM,
moribund and dead sperm, LAR, DAR and CMA3 were found in LF as compared to HF. In the
case of sperm immobilization, the PM of sperm was highly significant (p≤0.05) at zero, 30, 60,
90,120, 150 and 180 mins in different groups of Ouabain viz. I (Control) II, III, IV, V and VI
respectively and above time interval different groups of Amiloride viz. Groups; I (Control), II, III,
IV and V respectively in HF Murrah bulls. In revival with Nigericin, PM, live, HOST and AI of
sperm were found to be highly significant (p≤0.05) at 185 mins in different groups of Ouabain
viz. I (control), II, III, IV, V and VI respectively, however PM, live, HOST and AI of sperm was
highly significant (p≤0.05) in different groups of Amiloride viz. Groups; I (Control), II, III, IV
and V respectively. In case of low dose sperm Murrah bulls, the post-thaw PM, live, HOST, AI,
VAP and VSL were significantly (P≤0.05) high in 10, 15 and 20 million (M) sperm doses as
compared to 5M sperm doses in HF Murrah bulls. Further, TM differed significantly (P≤0.05) 5,
10, 15 and 20M sperm doses in HF bulls. The RM was significantly (P≤0.05) higher in 15 and 20
M sperm doses as compared to 5 and 10 M sperm doses in HF bulls. The post-thaw live sperm, Fpattern,
LAI and MMP was significantly (P≤0.05) high in 10, 15 and 20M sperm doses as
compared to 5 M sperm doses in HF bulls. The post-thaw AR, LAR, DAI and LPO of sperm
were significantly (P≤0.05) high in 5, 10 and 15M sperm doses as compared to 20 M sperm doses
in HF bulls. For seminal plasma proteome studies: 8 Murrah bulls (4HF + 4LF) and 8 Sahiwal
bulls (4HF + 4LF) were included and undergone with Tandem Mass Tag. The total no. of
proteins in Murrah bulls was found to be 1545. In conclusion, both HF Murrah and Sahiwal bulls
exhibited better post-thaw in vitro sperm functional attributes in comparison to their respective
LF bulls. Differential sperm motility inhibition potential of Ouabain and Amiloride with the
revival of sperm functionality with Nigericin treatment was found to be significant. In low dose
sperm /straw (20, 15, 10 and 5 M); high sperm doses (20, 15 and 10 M) performed better during
in vitro sperm function tests than the low sperm dose (<10 M). The total no. of SP protein group
and peptides in Murrah bulls identified was 934 and 9868. Up and down-regulated proteins were
30 and 6.