Development Of High Efficiency Horse And Donkey Reproduction Technology And Their Applications

It is of decisive significance to realize the high-efficiency reproduction on the horse and donkey production and animal protection.In the horse,we evaluated the effect of melatonin and calcium ion inhibitor BAPTA-AM on horse semen in vitro cryopreservation,and drugs effects on mare ovulation and estrus.In the donkey,we also investigated the donkey cryopreservation semen extender.The factors that influence the quality of semen,maternal physiological characteristics and exogenous drugs on donkeys ovulation,synchronization estrus.Our study found that:In the horse,melatonin and BAPTA-AM protect stallions sperm from cryopreservation injuries in a dose-dependent manner.The addition of 1 mM melatonin in the freezing extender could increase intracellular ATP,MMP and DNA integrity while decrease BimEL,ROS and MDA levels after thawing compared with the control.The extender supplemented 1 mM melatonin could prolong the survival period of 4 ℃ cooled stallion sperm and improve the fertility of frozen sperm.In comparison with the control group,sperm frozen in the extender containing 50μM BAPTA-AM showed increased intracellular ATP,MMP,ROS abundance and MDA content after thawing.The addition of BAPTA-AM to the extender did not affect the pregnancy rate of mares inseminated by sperm preserved at 4 ℃ 48 h.After 7 days of ovulation,mares muscle injection of PG,PGF2α,PG+PGF2a or PGF2a+PG for two consecutive days could shorten the ovulation cycle to 18±2d,17±2d,14±2d,15±2d respectively.Repeated use of hCG over 4 times extended the interval time between injection and ovulation.An average of 1.6 embryos can be recovered per 3.3 follicles after GnRH analogue treated cycle.In the donkey,the total sperm number of adult jack has a significant relation with body height,body length,rump length,cannon circumference.There is no correlation between climbing,cleft lip number,erection time,mating time and the total amount of sperm,a negative correlation between semen volume and density.Meanwhile,semen volume,sperm motility has a correlation with seasonal variation of year and age.The activity of 4 ℃ cooled sperm which diluted by extender SM3 for 96h was higher than other SM groups,and the AI pregnancy rate of SM3 treated 600 million sperm that cryopreservation for 96h was no difference with the fresh semen.SM3 containing 5%dimethylformamide would be beneficial for donkey sperm freezing.Meanwhile,the viability of semen which thawed in 50 ℃ 12～16s was better than in 35 ℃,36s.Moreover,there was no statistical fertility rate difference between the fresh and frozen semen.In breeding seasons,the life length of jennies CL was 14.0±4.8d,and an average of two follicular waves pre estrus cycle in DEZHOU donkey.The first follicle recruitment occurred after 5.2～1.9 days of ovulation,lasting about 8.0 ± 4.0 days until the corpus luteum was ablated,however the follicles won’t developed to be the dominant follicles.The next ovulation dominant follicles which usually appeared at 16.5 ± 1.0 day of after ovulation,and ovulated 6.2 ±1.3 days later,so the interovulatory interval was 20.0 ± 5.2 days.After analyzing the hormonal changes,we found that blood E2 levels began to increase gradually,and reached the crest value at 3 days before ovulation,the low concentration of E2 lasted in the diestrus(7-17d)and 4-6d of ovulation.The highest FSH value was shown in the interval interovulatory,while FSH concentration of later estrus declined slightly than that of the estrus.The concentration of P4 gradually increased after ovulation and reached a peak at 7-9d,then gradually decreased.The LH concentration increased gradually after the appearance of dominant follicles and peaked 2 days before ovulation.The development of follicles was controled by the hormones.Body temperature,vaginal resistance value cannot effectively identify the donkey estrus state.The interval of jenny ovulation of intramuscular injected 5,10mg PGF2a,and 0.4mg PG at 5～8d after ovulation was 22.58±5.72d,18.11±1.92d and 19.13±3.38d,meanwhile after injection 9～11d,the jenny’s ovulation rate was 36.5%,88.8%and 63.5%respectively.The average ovulation diameter is 38.8 ± 5.1mm in DEZHOU donkey.The ovulation rate of GnRH treated jennies with follicle size over 30mm that ovulated within 48h or between 25 and 48h following injection was significantly higher than jennies in the hCG and control groups.Furturemore,the number of ovulated follicle did not shown significantly difference among the groups.Jennies,which injected GnRH on 0 day,plug progesterone suppository 2 days later,im.PG on 7th day,im.PG and withdrawal device on the 9th day can make 85.69%of them in estrus at 10～14 days.