Effects Of LHRH-A3 On Concentrations Of Reproductive Hormones And Metabolite In Kazak Ewes And Hu Ewes

Although GnRH(Gonadotropin-releasing hormone)has been widely used in fixed time insemination in sheep,but effects of GnRH on pregnancy rate of ewes after insemination is unclear.This study investigated the effects of GnRH agonist LHRH-A3 injected at insemination on concentrations of reproductive hormones,metabolite and pregnancy in Kazak ewes and Hu ewes.A intravaginal sponge impregnated with flurogestone acetate was left in the vagina for 12 days followed with an injection of 330 IU eCG i.m.at the sponge removal and transcervically inseminated 55 h after sponge removal.Kazak ewes were assigned randomly to the treatment group(TY group,n=380,25 μg of LHRH-A3 i.m.at the first insemination)and control group(CY group,n=281,1mL saline i.m.at the first insemination).Hu ewes were assigned randomly to the treatment group(TB group,n=180,25 μg of LHRH-A3 i.m.at the first insemination)and control group(CY group,n=153,1mL saline i.m.at the first insemination).The second transcervical insemination was performed 70 h after the removal of intravaginal devices.Blood samples were collected 5 days after the first insemination of Kazak ewes and Hu ewes for reproductive hormones concentrations and metabolism analysis.The pregnancy rate was measured 45 days after the first insemination.The results showed that the pregnancy rate of Kazak ewes treated with GnRH(46.6%)was significantly lower than the control group(74.4%)(P<0.05),while there was no significant difference in the pregnancy rate of Hu ewes treated with GnRH(85.6%vs 87.6%).The hormone analysis results showed that TY group had significantly higher concentrations of PGF2α,E2,LH compared to CY group,while OXT concentration was significantly lower(P<0.05),and P4 concentration did not differ significantly.In contrast,the concentrations of PGF2α,E2,and LH in the TB group were significantly lower than CB group,while the concentrations of OXT and P4 did not differ significantly.The results of the metabolomics analysis indicated that a total of 839 metabolites in positive ion mode and 683 metabolites in negative ion mode were identified.The PCA plots in the both positive and negative modes showed significant variation between Kazak ewes,while the variation between Hu ewes was low.In combination with OPLS-DA(Orthogonal Partial Least Squares-Discriminant Analysis)and the P value of univariate statistical analysis T test to screen the significantly different metabolites between different groups.The differential metabolites of CB group and CY group were mainly enriched in the biosynthesis of unsaturated fat acids、linoleic acid metabolis、retrograde endocannabinoid signaling.Notably,the arachidonic acid of Kazak ewes was down-regulated in biosynthesis of unsaturated fat acids,linoleic acid metabolism,retrograde endocannabinoid signaling.The differential metabolites of the CY and TY groups were mainly enriched in the cAMP signaling pathway and the degradation of aromatic compounds.In conclusion,effects of GnRH administration at insemination on pregnancy rate of ewes was breed dependent,GnRH administration at insemination significantly changes the concentration of four reproductive hormones,affects metabolism and reduces pregnancy rate of kazak ewes.Acetylcholine,epinephrine,and 3-Hydroxybutanoic were landmark differential metabolites.