| Estrus is one of the important reproductive traits of female rabbits,which has a certain periodicity.The estrus cycle of female rabbits is divided into Diestrus,Early estrus,Estrus,Late estrus four periods,the conception rate of female rabbits can reach the highest only when the female rabbits are bred in the estrus period,so as to maximize the fertility of the female rabbits,but it takes more time for most female rabbits to reach the peak estrus period.Long and short estrus duration,which results in female rabbits not being able to fully develop their productivity.Therefore,to further explore the regulation mechanism of female rabbit estrus can help us shorten the production cycle of female rabbit and improve production efficiency.In this study,New Zealand rabbits were selected as the research object.By comparing the daily behavior,reproductive hormone changes and follicular development of female rabbits during estrus,the behavioral and physiological changes of female rabbits during estrus were investigated.In addition,transcriptome sequencing was used to screen differential genes in interestrus and oestrus,and to verify the function of differential genes,in order to understand the molecular regulation mechanism of female estrus.The test results are as follows:1.The behavior of female rabbits in the whole estrus cycle was recorded through installation monitoring,and the behavior changes in each estrus stage were compared.It was found that the duration and frequency of grooming behavior of female rabbits in estrus were significantly higher than those in diestrus and early estrus（P<0.05）,and the duration of feeding behavior was significantly lower than that in early estrus and diestrus（P<0.05）;The frequency of resting behavior in the estrus was significantly lower than that of the other three stages;the frequency of drinking water behavior decreased significantly after estrus（P<0.05）;the frequency of motor behavior and biting behavior increased significantly in the estrus（P<0.05）;wait-and-see behavior.There was no significant difference between the different stages of standing and standing behavior.The changes of reproductive hormones in different estrus stages were compared,and it was found that FSH increased first and then decreased in the whole estrus cycle of New Zealand rabbits,it was significantly higher in the early estrus than in the later estrus（P<0.05）;the change trend of LH concentration was similar to that of FSH,estrus was significantly higher than diestrus and late estrus（P<0.05）;The concentration of P4 was the lowest at the estrus,and increased significantly at the late estrus（P<0.05）;the concentration of E2 gradually increased from the beginning of estrus and reached the highest at the late estrus,and the concentration of early estrus was significantly lower than the other three stages（P<0.05）.The number of follicles at all levels in the four estrus stages was observed and counted,and it was found that there was no significant difference in the number of primordial follicles in the four estrus stages;the number of primary follicles in the estrus stage was the largest,which was significantly higher than that in the other three estrus stages（P<0.05）;The number of secondary follicles in estrus was significantly higher than that in interestrus and early estrus（P<0.05）.To sum up,during the estrus period,the female rabbit’s feed intake and water intake decreased,and the amount of exercise increased,and the concentration of reproductive hormones changed regularly throughout the estrus cycle,which could regulate follicle development through synergistic or inhibitory effects,thereby achieving The role of regulating the estrus cycle of female rabbits.2.Using RNA-seq technology to analyze the ovarian tissue of female rabbits in diestrus（group D）and estrus（group E）,using |log2FoldChange|>1＆P-value<0.05 as the screening criteria,a total of 967 There were 623 up-regulated genes and 344 down-regulated genes.GO annotations to cell surface receptor signaling pathway,cell development process and other items.Through KEGG signaling pathway analysis,the Estrogen signaling pathway,Steroid hormone biosynthesis,Aldosterone synthesis and secretion and other pathways were enriched,including CYP19A1 and CYP1B1 and other genes related to reproductive hormone synthesis.Six genes（ESR1,IGF1R,CYP19A1,CYP1B1,BMPR1B,HOXA9）were randomLy selected from the screened differential genes for fluorescence quantitative verification.It was found that the quantitative results were consistent with the transcriptome sequencing results,indicating that the sequencing results were reliable.3.According to transcriptomic sequencing data,the key gene CYP19A1 was cloned.Through bioinformatics prediction,it was found that the protein encoded by CYP19A1 was a stable hydrophilic protein,with α helix and random curl as the main secondary structure,and curved helix as the tertiary structure,containing a domain,belonging to the P450 family,with high homology with other species.It’s a conserved gene.In addition,rabbit ovarian granulosa cells were isolated and identified,and the overexpression and interference of CYP19A1 were carried out.It was found that overexpression of CYP19A1 could significantly up-regulate the expressions of IGF1R,CYP1B1,and CYP1A1（P<0.01）,and significantly down-regulate the expressions of BMP6 and CYP17A1.amount（P<0.01）,the interference result was opposite to the overexpression result.The effect of CYP19A1 on the function of ovarian granulosa cells was further verified,and it was found that overexpression of CYP19A1 would promote the proliferation of ovarian granulosa cells and inhibit cell apoptosis,and the interference results were opposite.Therefore,CYP19A1 may affect the estrus cycle of female rabbits by regulating the expression of genes related to the synthesis of reproductive hormones,and can promote the proliferation of ovarian granulosa cells,which is of great significance to follicular development. |
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