| Estrus synchronization is an essential breeding method for livestock of large-scale intensive farms.The principle of concurrent oestrus technique is to change the oestrus cycle of female animals by using some exogenous hormone methods,so that they can be oestrous and ovulate in a certain period of time.In order to accelerate the rapid breeding of excellent livestock,improve production efficiency and increase economic benefits,large-scale intensive breeding farms often carry out synchronization estrus treatment on mating females in every breeding year,and females need to receive repeated estrus synchronization treatment in their breeding years.Thus,previous studies have shown that repeated hormone stimulation is easy to cause side effects,resulting in the decline of animal reproductive performance.However,the specific molecular mechanism is still unclear.This research used two different gonadotrophins for two estrus synchronization treatments,PMSG or FSH.Ninety-six dairy goats were divided in to 4 groups at random,each group is 24 does,respectively.The does of 1-PMSG group or 3-PMSG group were treated with once or trice PMSG mediated estrus synchronization treatment;the does of 1-FSH group or 3-FSH group were treated with once or trice FSH mediated estrus synchronization treatment.This experiment has researched the effect of repeated PMSG or FSH estrus synchronization treatment on the reproductive performance,growth and development,lactation performance,blood hormone and biochemical index concentration,ovarian morphology,m RNA and protein expression of ovarian tissue.In the present study,the promising genes that affect reproductive performance were screened out and verified at the cellular level,to explore the specific molecular mechanism of repeated estrus treatment influence reproductive performance of dairy goats,and the following results were obtained:1.Effect of repeated estrus synchronization treatment on the production performance of dairy goats.The results showed that the estrus rate,pregnancy rate and litter size were significantly reduced by repeated PMSG mediated estrus synchronization treatment(P < 0.05).Repeated FSH mediated estrus synchronization treatment could reduce estrus rate and litter size(P < 0.05).Repeated PMSG or FSH mediated estrus synchronization treatment had no significant effect on the body condition of kids which were delivered by the experiment does,nor did it affect lactation performance,growth and development,and blood biochemical index concentration of dairy goats.2.Effect of repeated estrus synchronization treatment on the hormone concentration of dairy goatsThe results showed that repeated PMSG mediated estrus synchronization treatment could significantly increase the blood estrodial concentration at 36,48,72 hours after CIDR withdrawal,and also influence the progesterone concentration during the CIDR insetion period(P < 0.05).Repeated PMSG mediated estrus synchronization treatment could significantly increase blood PMSG antibody concentration at 24,48,72 hours after CIDR withdrawal(P < 0.05).Repeated FSH mediated estrus synchronization treatment significantly increased the concentration of FSH antibody at the time of CIDR insertion of the third estrus synchronization treatment(P < 0.05),but had no effect on the concentration of PGF2? and anti-PGF2?.The contents of FSH,LH,Gn RH,MT and anti-P4 were not affected by repeated estrus synchronization treatment.Repeated PMSG or FSH mediated estrus synchronization treatment had no significant effect on the concentration of hormone and hormone antibody of pregnancy goats during the pregnancy period.3.Effect of repeated estrus synchronization treatment on ovarian morphology of dairy goatTo collect the unilateral ovaries of does,we selected 12 does in early estrus from the 1-PMSG group,3-PMSG,1-FSH group,and 3-FSH group,each group included 3 does.At the process of operation,the ovarian size and the follicle development situation in the ovary surface were measured.The left ovary tissue was collected and longitudinal cut into two parts: one part was used for the extraction of total RNA and total protein,another part was used to make paraffin section for HE staining.Repeated PMSG or FSH mediated estrus synchronization treatment did not affect the size of left and right ovaries of dairy goats,as well as the diameter and number of follicles in ovarian cortex.HE staining results showed that there was no significant difference between 1-PMSG group and 3-PMSG group,1-FSH group and 3-FSH group in the morphology and proportion of primordial follicles,primary follicles,secondary follicles and antral follicles.4.Effect of repeated estrus synchronization treatment on m RNA and protein expression of dairy goat ovariesThe bioinformatics analysis results of RNA sequencing indicated that repeated PMSG mediated estrus synchronization treatment changed 114 gene expressions of ovarian tissue.Compared with 1-FSH group and 3-FSH group,319 genes were significantly changed.The candidate genes were verified by RT-q PCR,WB,and immunohistochemistry.Results showed that compared with the 1-PMSG group,GCG m RNA and protein expression in the 3-PMSG group were significantly up-regulated.Compared with the 1-FSH group,the expression of GH,PTGIS,DRD1 m RNA and protein in the 3-FSH group were significantly increased(P < 0.05),the expression of ESR1 and DHB1 m RNA and protein were significantly decreased(P < 0.05).There were four common differential genes AQP3,SPIB,KIF28 B and Novel00378 in 1-PSMG group,3-PMSG group,1-FSH group,and 3-FSH group.Compared with the 1-PMSG group and 1-FSH group,the expression of AQP3 m RNA and protein of goat ovary were both up-regulated in the 3-PMSG and 3-FSH group(P < 0.05).5.Study on the function of AQP3 gene in ovarian granulosa cells and oocytes of dairy goatsOverexpression of AQP3 decreased the expression of apoptosis-related gene BCL-2,and increased the expression of Caspase 3 and Caspase 9 in goat ovarian granulosa cells(P < 0.01),and the secretion of progesterone and estrogen of ovarian granulosa cells were significantly decreased 12,24 and 36 h after serum-free culture(P < 0.05).Overexpression of AQP3 in goat oocytes significantly reduced the maturation rate,cleavage rate after parthenogenetic activation and cleavage rate after in vitro fertilization(P < 0.05).In conclusion,repeated PMSG or FSH mediated estrus synchronization reduced the reproductive performance of dairy goat,increased the concentration of PMSG and FSH antibody,lead to the change of the m RNA and protein expression of severial genes in goat ovarian tissue.The expression of AQP3 m RNA and protein of ovarian tissue in the 3-PMSG group and 3-FSH group were both increased.Furthermore,overexpression of AQP3 lead to the apoptosis of ovarian granulosa cells,and decreased the concentration of steroid hormone in ovarian granulosa cells.Overexpression of AQP3 significantly damaged the growth and reproductive performance of oocytes.AQP3 is a key gene that reduced the reproductive performance caused by repeated estrus synchronization of dairy goats.This study explains the specific molecular mechanism of repeated estrus synchronization treatment to reduce the reproductive performance of dairy goats,provides the theoretical basis for optimizing the application of estrus synchronization,and lay the foundation for the rapid development of dairy goat breeding technology. |
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