| The fertility of female rabbits in the half-frequency dense breeding mode decreased sharply after 6 consecutive births,and the phenomenon of infertility appeared,which seriously affected the utilization value of the female rabbits.Oxidative stress is one of the main factors that cause ovarian function decline in female animals.In this studie female rabbits were used as experimental subjects.In this study,the reproductive performance,ovarian tissue structure,serum hormone,antioxidant enzyme content and ovarian function indexes of P1,P2 and P6 female rabbits were compared,and the ovarian function status of different parities of reproductive female rabbits was clarified.Transcriptome sequencing was used to screen the differential genes of ovarian function in female rabbits and verify the effect of SRD5A2 on ovarian granulosa cells.On the other hand,this study constructed an in vitro induced ovarian oxidative stress model of female rabbits through D-gal（Dgalactose）,and to analyze the alleviating effect of antioxidant NAC（N-acetyl-L-cysteine）on it,and to provide theoretical basis for the research and development of delaying ovarian function decline caused by oxidative stress in female rabbits.The main results were as follows:1.The conception rates of P1 to P6 increased firstly and then decreased,with P2 being the highest and P6 the lowest.With P2 as the control,the survival rate and litter weight at 3 weeks of age of P1 rabbits were significantly decreased（P<0.01）,and the average weight at birth was significantly decreased（P<0.05）,but there were no significant differences in other indexes（P>0.05）.The litter size,the number of live birth,live birth rate and average weight at 3 weeks and 5 weeks of age of female P6 rabbits were significantly lower than that of P2（P<0.05）,the live birth rate and average weight at 3 weeks of age of female P6 rabbits were significantly lower than that of P2 rabbits at 5 weeks of age（P<0.01）,and there was no significant difference in survival rate between female P6 and P2 rabbits at 5 weeks of age（P>0.05）.The number of primordia follicles in P2 was significantly lower than that in P1（P<0.05）,and the number of ovarian follicles in P6 was significantly lower than that in P1 and P2（P<0.01）.The contents of E2,P4,GSH and T-AOC in serum and Klotho and TE in ovary of P2 were significantly higher than those of P1 and P6（P<0.05）,and the contents of FSH,ROS,AMH and MDA were significantly lower than those of P6（P<0.05）,and there were no significant differences in AMH and MDA between P2 and P1（P>0.01）.The results showed that ovarian decline occurred after multiple gestation,which was manifested as follicle loss,decreased immunity,antioxidant capacity and ovarian function index.2.P2 and P6 ovaries of female rabbits were collected for RNA-Seq analysis,and a total of 213 up-regulated and 747 down-regulated genes were screened out.Including CYP21A2,BRCA1,PTGFR,SGK1,PIK3R6,SRD5A2 and other differential genes related to reproductive function.Through GO and KEGG functional analysis,it was found that GO annotated functional items related to ovarian development such as REDOX,germ cell regulation,embryogenesis and hormone secretion.KEGG is mainly enriched in PI3K-Akt signaling pathway,HIF-1α signaling pathway and cAMP signaling pathway,which affect ovarian function.qRT-PCR was used to verify the expression of differential genes,and the results were consistent with RNA-Seq prediction trend,indicating the reliability of transcription sequencing.The results of this study showed that there were genetic differences in the ovaries of different parities during the reproductive process of female rabbits.3.The full-length CDS of SRD5A2 gene was 765 bp,encoding 252 amino acids.Rabbit ovarian granulosa cells were isolated and identified by FSHR.By overexpressing and interfering with SRD5A2 gene expression in rabbit granulosa cells,the show that overexpression of SRD5A2 significantly down-regulated HIF1A and up-regulated INHBB,HSD17B1,GDF9 and BMP 15 expression levels（P<0.01）.However,after the expression of SRD5A2 was interfered,these related genes showed opposite results.At the same time,SRD5A2 overexpression significantly promoted the proliferation of granulosa cells and inhibited the apoptosis of granulosa cells（P<0.05）.After interference,the proliferation of granulosa cells was significantly inhibited and apoptosis was promoted（P<0.05）.These results provide basic data for revealing the molecular mechanism of ovarian decline induced by multiple pregnancies in reproductive female rabbits.4.Rabbit ovarian tissue was collected for in vitro culture,different concentrations of D-gal（5 mg/mL,10 mg/mL,15 mg/mL）were used for treatment.It was found that with the increase of Dgal concentration,the structure of follicle was destroyed,the number of granulosa cells decreased,and the content of SOD and GSH in ovary decreased significantly（P<0.01）,and 10 mg/mL was the optimal concentration for model construction.Furthermore,different concentrations of NAC（0.1 mg/mL,0.3 mg/mL,0.5 mg/mL）were added into the model to analyze its alleviating effect on ovarian recession.After HE staining in ovarian tissue sections,0.1 mg/mL NAC effectively alleviated the granulosa cell apoptosis caused by D-gal,and showed no obvious toxic effect in ovary,so it was the best concentration for alleviating.By detecting the content of antioxidant enzyme and gene expression in ovary,it was found that compared with normal ovary,the contents of GSH,T-SOD and CAT in D-gal（10 mg/mL）group were significantly decreased（P<0.01）.The expression levels of Nrf2,GPX,PRDX4,GSR,SOD1 and TAF4B were significantly down-regulated（P<0.01）.After adding NAC（0.1mg/mL）,the content of antioxidant enzymes and the expression of antioxidant genes in D-Gal group were significantly higher than those in D-gal group（P<0.01）.The results showed that NAC could delay ovarian function decline by alleviating d-Gal induced oxidative stress in female rabbits. |
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