The Application Of Nerve Growth Factor-beta In Promoting Ovulation Of Mongolian Mares

The importance of breeding soundness examination(BSE),especially evaluates the estrus and ovulation timing of mares for breeders in breeding seasons can never be underestimated,particularly when it comes to increasing fertilization rates.Ovulation process is regulated by endocrine system and nervous system.The effects of hormones in endocrine system on mare’s ovulation have been fully studied,so was the clinical application of hormones on accurately predicting ovulation.However,specific to the local equine breeds in China,the detailed ultrasonic application of BSE of hormone levels and the pre-ovulation follicle diameter threshold were rarely systematically described.Nerve growth factor-Beta(NGF-β)is a part of the neurotrophic factor family.In recent studies,NGF-β has been found to act not only on nervous system,but also on reproductive system.It was expressed on granulosa cell and oocyte in different animals,such as buffaloes,chicken,and sheep.NGF-β is a major component of alpaca seminal plasma and induces ovulation in female alpacas and camelids,but there is still very little research on equine;Therefore,based on previous research,the research was significantly conducted to pilot explore the application of NGF-β in promoting ovulation of mares.In vivo experiments were carried out on Mongolian mares by using BSE and NGF-β promoting ovulation application(Ⅰ+Ⅱ).Breeding soundness examination(BSE)of 94 Mongolian mares was conducted,and the average 35.8 mm value of 56 mares of the dominant follicles,48 to 72 hours before ovulation was calculated.Forty-two young mares with normal BSE results and 2 hCG-tolerant mares were selected as the main subjects of NGF-β promoting ovulation application(Ⅰ+Ⅱ).In the pilot study(Ⅰ),when the mare dominant follicles reached 35 mm,Pre-examination of different administration routes(intramuscular injection,intravenous injection,and intrauterine infusion)and dosage of NGF-β was performed in groups and provided the best dosing regimen for the NGF-β injection application experiment(Ⅱ).In the experiment of NGF-β injection application(Ⅱ),when 12 follicles(n=3/group)of the mare reached35 mm,they were intramuscularly administered with stallion crude seminal plasma,0.5 mg NGF-β,1.0 mg deslorelin and equivalent amount of 0.9% saline to promote ovulation during four estrus cycle.To study their ovulation,the other two hCGtolerant mares were injected with 2000 IU hCG,0.5 mg NGF-β,1.0 mg deslorelin and equivalent amount of 0.9% saline.The results were judged by rectal ultrasound monitoring of follicular ovulation.In vitro experiments were the determination of the follicle stimulating hormone(FSH),luteinizing hormone(LH)and NGF-β in Mongolian mare’s serum,the SDSPAGE test of stallion crude seminal plasma protein,NGF-β of mare fallopian tube TrkA immunohistochemistry test and NGF-β promoted Mongolian horse oocyte maturation test in vitro which using the primary Mongolian horse cumulus-oocyte complex(COC)cultured in vitro,after adding 50 ng/mL NGF-β assess the maturation of oocytes.In vitro experiments explored the endocrine hormone law and normal ovulation index of Mongolian mares,whether NGF-β was present in the oviduct and seminal plasma of horses,and whether it promoted the maturation of Mongolian oocytes.The screening experiment of NGF-β doses and administration route(Ⅰ)that in the1.0 mg and 0.5 mg doses administration groups,except the intrauterine infusion group,the rest of the experimental group had 100% ovulation rate(P<0.01).The optimum dosage regimen of Ovulation Induction Experiment with NGF-β Injection(II)should be intramuscular injection with 0.5 mg NGF-β.It turns out that when the dominant follicle of a Mongolian mare reached 35 mm,ovulation induction was effective by whether intramuscular or intravenous injection with the same dosage of NGF-β,which is not significantly different.NGF-β(0.5 mg)had a similar ovulation promoting effect to general analogues of GnRH agonist(e.g.deslorelin).NGF-β,with ovulation induction potential,is effective in promoting the ovulation of hCG-tolerant mares.Stallion crude seminal plasma and intrauterine infusion had poor effect.The study discovered that NGF-β exists in seminal plasma of Mongolian horses.NGF-βand its receptor TrkA were expressed on oviducts of mares during estrus and diestrus,and the expression during oestrus was higher than that during dioestrus.Since only one equine oocyte matured in culture,this part could not be concluded.Breeding soundness examination(BSE)is significant to clinical application of practitioner.Describe basic clinical and endocrine parameters and normal ovulation of Mongolian horses,which can be a contribution to the general knowledge of equine reproductive biology.The research explored whether NGF-β can be an ovulationinduction agent,and serve as important references to the development of veterinary drugs,diagnosis and treatment,especially for the treatment of hCG-tolerant mares and will contribute to follow-up horse breeding research.