The Effects Of IGF-I And GSH On Quality Of Ovine Straw Frozen Semen In Different Seasons

Sheep is an important economic animal in our country, providing agricultural products for human need.Although artificial insemination, IVF(in vitro fertilization) and other techniques can be used to improve the reproductive efficiency of sheep reproductive rate and reduce feeding costs especially in non-breeding season too poor to use. To find a suitable frozen dilution for physiological characteristics of ram sperm in whole season is an urgent matter.In order to improve the quality of ram frozen semen in all seasons, seven Texel rams’ semen collected in breeding(November) and non-breeding(June) season, different individuals’ sperm in the same season were mixed together.Supplied different concentration of IGF-I or GSH in frozen semen, made it to straw frozen semen by one step dilution method. After thawing semen, 37 ℃ warm bath semen 0,1, 2 hours,tested sperm acrosome membrane integrity, sperm plasma membrane integrity, semen DNA integrity, by SYBR-14/PI fluorescent staining, hypotonic swelling test, acridine orange staining methods.tested cleavage rate and blastocyst rate after IVF and in vitro propagation.All experiments above were made to explore how various IGF-I and GSH effected on spernm, and provided a theoretical basis for improving the utilization efficiency of semen in whole season.The results were as follows :1. The effects of different seasons(breeding season and non-breeding season) on the quality of thawed semen: The sperm acrosome membrane integrity, the sperm plasma membrane integrity, the cleavage rate and the blastocyst rate of semen in breeding season were higher than those semen in non-breeding season.2. The effects of different concentration of IGF-I(0, 50, 100, 150, 250 ng/m L) on frozen semen in breeding season:(1) 50, 100, 150 ng/m L IGF-I supplied in the initial thaw frozen semen could improved sperm acrosome membrane integrity significantly(P<0.05). High concentration of IGF-I group(150, 250ng/m L) in the 2 h post-thaw semen had a lower sperm acrosome membrane integrity than control group(P<0.05).(2) Incubated for 1h at 37 ℃, 100 ng/m L IGF-I group had a higher sperm plasma membrane integrity than other groups(P<0.05), and incubated for 2 h at 37 ℃, high concentration of IGF-I group(150, 250 ng/m L) had a lower sperm plasma membrane integrity than other groups.(3) Incubated for 1 h at 39 ℃, 100 ng/m L IGF-I added in semen did a better affection on the semen DNA integrity(P<0.05).(4) supplied IGF-I did nothing to cleavage rate and blastocyst rate.3. The effects of different concentration of IGF-I(0, 50, 100, 150, 250 ng/m L) on frozen semen in non-breeding season:(1)Incubated for 0h at 37 ℃,50, 100, 150 ng/m L IGF-I groups had higher sperm acrosome membrane integrity than other groups(P<0.05). Incubated for 1h at 37 ℃,50, 100, 150, 250ng/m L IGF-I groups had higher sperm acrosome membrane integrity than other groups(P<0.05).(2)Incubated for 0 h and 1 h at 37 ℃, 100, 150 ng/m L IGF-I groups had higher sperm plasma membrane integrity than other groups(P<0.05).(3) Incubated for 1 h and 2 h at 37 ℃, 250 ng/m L IGF-I supplied group had a decreased DNA integrity than any other groups(P<0.05).(4) 100, 150 ng/m L IGF-I supplied in the semen improved the cleavage rate significantly(P<0.05).4. The effects of different concentration of GSH(0, 1, 2, 2.5, 3 m M) on frozen semen in breeding season:(1) Incubated for 0 h and 1h at 37 ℃, 1 m M and 2.5 m M GSH supplied groups had a significant improvement on the perm acrosome membrane integrity than other groups(P<0.05).(2) 1 m M, 2 m M GSH supplied groups in incubated for 0 h, 2 m M, 2.5 m M groups in incubated for 1 h at 37 ℃ improved sperm plasma membrane integrity(P<0.05).(3) 1 and 2 m M GSH added in the initial thaw frozen semen and 1m M GSH group in the 1, 2 h post-thaw semen perm improved semen DNA integrity(P<0.05).(4)Supplied GSH did no significant effects to cleavage rate and blastocyst rate(P<0.05).5. The effects of different concentration of GSH(0, 1, 2, 2.5, 3 m M) on frozen semen in non-breeding season:(1) 1 m M GSH did some positive effect on the sperm acrosome membrane integrity at 0 h,1 h post-thaw seme(P<0.05).(2) The sperm plasma membrane integrity at 1 h post-thaw seme of 1 m M GSH group was higher than other group(P<0.05).(3) GSH supplied did no significant effect on the DNA integrity of frozen semen(P>0.05).(4) 1 m M and 2 m M GSH supplied groups had a significant improvement on cleavage rate and blastocyst rate(P<0.05).All in all, 100 ng/m L IGF-I or 1 m M GSH supplied in frozen dilution could improve the sperm quality in breeding season and non-breeding season in some extent, but a high concentration like 250 ng/ml IGF-I or 3 m M GSH may reduce the sperm quality.