Application Of AMH In Predicting Reproductive Potential Of Dongbao Black-headed Goat

In females,Anti-Müllerian Hormone(AMH)is mainly produced in the granular cells of the sinus follicle and subsequently circulates to the follicular fluid or peripheral blood,and its levels can be known by blood Elisa assay.It has been increasingly demonstrated as an important factor in the negative regulation of follicular growth and development and in the evaluation of ovarian reserve in female animals.In clinical practice,AMH is an important indicator of the health of the female ovary and can indicate the number and quality of available follicles in assisted reproduction.In herbivores,AMH levels have been used to predict the response of follicles to FSH during ovulation promotion and thus to select donors with better ovarian reserve,with dairy cows and sheep being the most studied.As a local improved goat group with excellent reproductive performance,the Dongbao black-headed goat has good performance in producing double lambs by the ewes,but the proportion of ewes producing double lambs and above in the group needs to be further improved.This experiment was conducted to investigate whether the AMH level test could be used to select ewes with superior ovarian reserve and to assist in the selection of individuals with high reproductive potential by combining relevant molecular markers.In this study,the peripheral AMH levels of ewes in the Dongbao black-headed goat were measured,and a reference interval of 2.41 to 4.70 ng/m L was obtained for AMH levels in lambing ewes;7 SNPs carried on AMH gene and 12 SNPs located at AMHR2 gene were obtained with the use of second-generation sequencing,and after further screening,the AMHR2 gene was found to have g.26335365 C > T and g.26338571C>T,they were identified as potential available markers for increasing newborn lambs.Subsequently,a PCR-Ava I-RFLP typing method was initially established in Dongbao black-headed goat for the former with good accuracy.The specific results obtained from this experiment were as follows:1.Peripheral AMH levels were first measured in Dongbao black-headed goat involving 115 lambing ewes among the age group of 1-8 years old.The peripheral AMH levels of lambing ewes gradually decreased with the increase of litter size,and the difference between the groups was significant(P < 0.05).And the AMH level of ewes decreased with increasing age,the mean AMH content of 1-3 years old ewes was 3.96ng/m L,which was significantly higher than that of ewes of other age groups(except 4-year-old ewes),while the difference in AMH level between adjacent groups was not significant.Further analysis revealed a moderate negative correlation between age and AMH levels(r=-0.527,P < 0.001).The analysis of ewes for which information on lambing in the current litter was obtained and for which AMH levels had been measured showed that,the higher the AMH level,the better the production performance accordingly.The distribution of AMH levels in the population was tested for normality and a reference range of 2.41-4.70 ng/m L was obtained for peripheral AMH levels in ewes,suggesting that the ewes were selected for assisted breeding according to AMH levels from highest to lowest in the normal reference interval.Initially,The AMH levels of 53 lambs were further followed for the first six months of their lives,the results showed that as the age increased,AMH levels showed a fluctuating downward trend.2.By interpreting the genome DNA sequencing results of 500 Dongbao blackheaded goat,7 SNPs were found on AMH gene.Besides,we got 12 SNPs on AMHR2 gene.Further analysis of the association between the SNPs of the two genes and the average number of lambs born in ewes and the number of lambs born in each litter showed that no molecular markers that could potentially increase lambing numbers exist on AMH gene.In contrast,the g.26335365C>T and g.26338571C>T loci of the AMHR2 gene had consistent typing and consistent corresponding genotypic individuals in the ewe population,and were closely associated with ewe lambing performance,mainly as follows: the number of first lambs born to TT mutant individuals was 0.83 and 0.85 higher than CT and CC types,respectively(P < 0.05);for the number of lambs in the third litter,the number of lambs in TT pure mutant individuals was significantly higher than CT and CC(P < 0.05);for the number of lambs in the fifth litter,CC individuals had 1.92 fewer lambs than pure mutant TT(P < 0.05),and CT individuals had 1.8 fewer lambs than TT,and the difference between them was significant;the number of lambs in the sixth litter in TT individuals was 1.43 higher than CT individuals(P < 0.05),and CC individuals had 1.43 more lambs than CT and TT individuals(P < 0.05).The difference was not significant(P > 0.05)between the CC type and the CT or TT types.As for the trait of average lambing number,TT individuals also performed significantly better than CT and CC individuals(P < 0.05),with TT individuals outperforming CC and CT individuals by 0.72 and 0.74,respectively.Therefore,preferential selection of ewe individuals with TT genotype which carrying the g.26335365 C > T or g.26338571 C > T loci located at AMHR2 gene may improve the lambing performance of ewe population.3.Sequence analysis of the g.26335365C>T polymorphic locus on the AMHR2 gene of Dongbao black-headed goat revealed that the Ava I endonuclease locus was lost after mutation,so a PCR-Ava I-RFLP method was established to genotype the sequenced ewes.The results showed that the genotype identification results were consistent with the sequencing report,which indicated that the newly established typing method could be more direct and effective than sequencing.