Effect Of Short-term Nutritional Supplementation On Folliculogenesis And Its Mechanism Of Regulation In Sheep During The Luteal Phase

This study was to evaluate the effects of short-term nutritional supplementation on follicular development and its mechanism of regulation in sheep.The plasma and follicular fluid of energy metabolites,hormones concentrations,plasma lipid metabolism and expression of genes related to granulose cells were detected to investigate the mechanisms of effects of short-term nutritional supplementation on folliculogenesis,and to analyze the hypothalamic-pituitary-ovarian axis transcriptome to reveal the molecular regulation mechanism of short-term nutritional supplementation on folliculogenesis during luteal phase in Sheep.The purpose of this study is for references of nutrition regulations on reproductive performances of Sheep.The study contains the following 4 parts:1 The certain of the optimal short-term nutritional supplementation schedule in Sheep during luteal phase60 Dorset×Han Crossbred ewes（9-10 months age）of 40 kg approximately were divided randomly into 4 groups（15 per group）,All ewes were fed on basal diet（DE 11.72MJ/d;DP 79.71 g/d）initially,the basal diet were offered to control group ewes throughout the experiment and treatment group ewes were fed experimental diet（DE18.75 MJ/d;DP108.44 g/d）during supplementation phase then regain fed the basal diet after end of supplementation.The ewes were treated into synchronized oestrus（CIDR pessary inserted for 12 d,injection of PG 0.1mg for twice）.The day of pessary insertion was named day 0.The experimental diet was fed to the treatment group since day 2,day 6,day 8 until day 11,thus supplement for 10 d,6 d,4 d respectively.Laparoscopy examination was taken on day20 after implantation of pessary in order to determine the optimal schedule of supplementation for ovulation according to the number of corpus luteum of estrual sheep.The weight changes and corpus luteum were recorded before and after supplementation.There was no significant difference in the body weight increase among treatments（P>0.05）.Compared with the control group,the ovulation rates were increased by 75%,25%,0%by supplementation for 10 d,6 d,4 d respectively.In conclusion,short-term nutritional supplementation for 10 d before luteolysis can be used as the follow-up optimal feeding scheme.2 Effects of short-term nutritional supplementation on the follicular improvement and the plasma correlation index in Sheep during luteal phase60 Dorset×Han Crossbred ewes of 40 kg approximately were divided randomly into 2groups（30 per group）:the control group and treatment group.Feeding experiments were conducted according to the 10 d short-term supplementation and the oestrus synchronization was also same as before.Plasma gonadotrophins,metabolic hormones,energy metabolism substrates of control and treatment group were measured on day 1,2,4,6,8,10 during supplementation.The follicles and follicular fluid of small,medium and large ovarian follicles（≤3.0 mm,3.0-5.0 mm,≥5.0 mm respectively）from the ovary of6 ewes were calculated respectively after end of nutritional supplementation.Plasma gonadotrophins,metabolic hormones and expression of genes were measured on differential follicles.The results showed that compared with control group,supplementation decreased the number of small follicles（<3.0 mm）while siginificantly increased the number of medium follicles（3.0-5.0 mm）and large follicles（≥5.0 mm）siginificantly.The levels of glucose and insulin were significantly increased in medium follicles and large follicles.The concentration of leptin significantly increased in large follicles.Supplementation ewes had higher intrafollicular FSH level in small follicles than control group（P<0.05）.In large follicles,the concentration of LH was significantly increased.The concentration of E₂ was higher in large follicles than small and medium follicles.The levels of glucose,insulin and leptin were marked increases in the plasma during the supplementation phase.There was no significant difference between the control group and the treatment group concerning the level of gonadotrophins in their plasma,the level of FSH and LH were increased and the concentration of E₂ decreased during the whole supplementation phase.The levels of total cholesterol,free fatty acids and urea protein were significantly decreases,however high-density lipoprotein cholesterol in plasma significantly increased.In conclusion,Short-term supplementation could promoted the positive balance of energy in body and make follicular cells take in more glucose and use them effectively to regulate the follicular development.3 Effects of short-term nutritional supplementation on the gene expression of follicular granular cells in Sheep during luteal phaseThe granulose cells of small,medium,large follicles（≤3.0 mm,3.0-5.0 mm,≥5.0mm respectively）from each group were collected randomly as in experiment 3 to detect the expression of genes by qRT-PCR.The results showed that Short-term supplementation significantly improve the genes relative expression of FSHR1 mRNA and FSHR3 mRNA in small and medium follicles,however suppressed the relative expression of FSHR2 and FSHR4 mRNA relatively.The FSHR1 and FSHR3 are the predominant forms of the FSHR in the short-term nutritional supplementation regulating follicular development during luteal phase.In medium and large follicles,short-term nutritional supplementation significantly enhanced the expression of OB-R and IGF-1 mRNA,resulting the significant increases of LHR,ESR1 mRNA expression than control group in large follicles to make influence on follicular development.It is suggested that short-term nutritional supplementation regulates secretion of reproductive hormones to effect follicle development through paracrine or autocrine factors.Compared to control group ewes,expression of CYP17A1mRNA in all kinds of follicles is significantly higher in supplementation group,and the expression of STAR and CYP19A1 mRNA in large follicles significantly improved.4 Transcriptome analysis of the hypothalamus-pituitary-ovary in Sheep by short-term nutritional supplementation during luteal phase3 ewes per group were slaughtered on day 13（early estrus）,14（mid-estrus）,15（metestrus）of the estrous cycle and their Hypothalamus（B）,Pituitary（P）,Ovary（O）were collected respectively to take RNA-seq analysis.By Illumina Hiseq 4000 System,the DEGs in 18 hypothalamus,pituitary,ovary during early estrus,mid-estrus,metestrus in control and supplementation groups were detected to explore the signal pathway and relevant candidate genes regulating follicular development by hypothalamus-pituitary-ovary through which the short-term supplementation take effects in time.The results showed that,18 RNA libraries were constructed and bioinformatics analysis was performed.Solexa sequencing provided varying reads from hypothalamus,hypophysis and ovary libraries ranging 33149868⁵7138286 reads.There were29560138⁵2705150 reads mapped to the reference genome of sheep which account to89.17%⁹4.53%of genes in total reads.A total of 54339 transcripts were obtained including 19207 genes expressed.Among these genes,12464 genes more than 2000bp which accounted for 64.89%of the conding transcripts that provided sufficient data for subsequent genes analysis.During the follicular phase,short-term nutritional supplementation regulated more follicles to develop into dominant follicle and maturation on hypothalamus-pituitary-ovary by the calcium signaling pathway,MAPK signaling pathway,Insulin secretion,Progesterone-mediated oocyte maturationThrough hypothalamus-pituitary-ovary axis,analysis of DEGs in three tissues indicated enrichment in some reproduction-related genes such as AGRP、LEPR、PRL、RERG、AKR1D1、IGF1、NLRP14、PRR11、EGR1,and all DEGs could be candidate genes for reproduction who participate in regulation of follicles development by shor-term nutritonal supplementation.