| The present paper comparatively studied the follicular fluid metabolites and hormones concentrations form different-sized follicles at the same reproductive and nutritional physiology, investigated the effects of short-term different nutritional intake on folliculogenesis, plasma and follicular fluid metabolites and hormones concentrations and sterol-regulatory genes expression of follicular and luteal cells during luteal phase in Hu sheep. The objectives of this study were to reveal the mechanism in which short-term different nutritional intake influenced folliculogenesis, and to provide certain references for scientifically rearing barren ewe in large-scale sheep farms and further studying precise signal pathway by which nutrition affected ovine reproductive performance. This study contained the following5parts:Experiment1. A comparative study of follicular fluid metabolites and hormones concentrations from different-sized follicles in Hu sheep11multiparous Hu sheep of proven fertility were used. After estrus synchronization, all ewes were slaughtered and follicular fluid was collected according to different-sized follicles on day12of estrous cycle. The results showed that there were no significant effects of follicle size on plasma serum ammonia, NEFA, urea, insulin and progesterone concentrations, however, compared with follicles<2.5mm in diameter, the follicular fluid glucose and estradiol concentrations in follicles>2.5mm in diameter were increased (P< 0.05) and the testosterone and glucagon concentrations and LDH activity were decreased (P<0.05). The follicular fluid concentration of estradiol was positively correlated with glucose (P<0.05) and progesterone (P=0.051) and negatively correlated with LDH (P<0.01), glucagon (P<0.05) and testosterone (P<0.01). In conclusion, follicle development was co-regulated by intrafollicular metabolites and hormones.Experiment2. Effect of different levels of short-term feed intake on folliculogenesis in Hu sheep during the luteal phase28multiparous Hu sheep (3-4years) weighed approximately40kg were selected and randomly assigned to3groups:the control group (n=6) received a maintenance diet (M) while the supplemented group (n=11) and restricted group (n=11) received1.5xM and0.5×M, respectively, on days6-12of their estrous cycle. After estrus synchronization,6ewes per group were slaughtered on day12of the estrous cycle. The ovarian follicles>1.0mm were dissected free and the number and volume of follicles1.0-2.0mm,2.0-2.5mm,2.5-3.5mm and>3.5mm per ewe were calculated, respectively. The remaining ewes were detected for estrous behavior. During the experimental period, body condition and body weight were measured four times:at pessary insertion, at pessary removal, at the start of different feeds and on the day they were killed. The results showed that there were no significant differences in the body weight and body condition score among times or treatments, however, compared with restriction, supplementation shortened the estrus length (P<0.05), decreased the number of follicles2.5-3.5mm (P<0.05), increased the number of follicles>3.5mm (P<0.05) and augmented the volume of follicles>2.5mm (P<0.05). In conclusion, short-term dietary restriction for6days before Iuteolysis inhibits folliculogenesis.Experiment3. Effect of different levels of short-term feed intake on plasma physiochemical indexes in Hu sheep during the luteal phaseAfter estrus synchronization, all ewes were slaughtered on day12of the estrous cycle and live and organ weights were determined. Blood samples were taken during experiment period. The results showed that as dietary intake decreased, plasma urea, total cholesterol, LDL-C, NEFA, FSH, estradiol, progesterone concentrations and progesterone:estradiol ratio and insulin:glucagon ratio increased (P<0.05), and plasma glucose, triglyceride, insulin and glucagon concentrations decreased (P<0.05). There was no significant effect of treatment on plasma uric acid, plasma ammonia and high density lipoprotein and spartate transaminase, alanine transaminase activities (P>0.05), however, there were significant effect of day on plasma uric acid, ammonia, low density lipoprotein concentrations and aspartate transaminase activity (P<0.05). Plasma leucine, valine, isoleucine, tyrosine, serine, ethanol amine, essential amino acid (EAA), essential amino acid derivatives (EAAD), amino acid metabolites (AAM) and total amino acids (TAA) concentrations in R group ewes were lower, while plasma taurine and citrulline concentrations and ammonia/energy metabolites (AEM):TAA ratio than those in S group ewes during experiment period. Restricted group ewes exhibited greater spleen weight and spleen weight per live weight, and less liver and small intestine weights, liver and stomach weights per live weight than supplemented group ewes (P<0.05). In conclusion, different nutritional requirement and metabolic feature were present in different physiological periods during ovine luteal phase and the mechanism by which dietary restriction inhibited folliculogenesis may involve responses to increased the capacity of lipolysis and protein degradation, decreased the capacity of lipid and protein synthesis, changed amino acids metabolism and varied levels of metabolic and reproductive hormones.Experiment4. Effect of different levels of short-term feed intake on follicular fluid microenvironment in Hu sheep during the luteal phase6ewes per group were slaughtered on day12of the estrous cycle and the follicular fluid of follicles1.0-2.0mm,2.0-2.5mm,2.5-3.5mm and>3.5mm per ewe was collected, respectively. Compared with supplementation, restriction increased follicular fluid estradiol, phosphorylethanolamine, urea (P<0.05) and8-hydroxylysine (P=0.085) concentrations, decreased ornithine concentration, EAA:EAAD ratio (P<0.05) and AEM:TAA ratio (P=0.075). Restricted ewes had higher intrafollicular insulin concentration than C group ewes (P<0.05), but it was similar to that of supplemented ewes. There were significant follicle size effect on intrafollicular glucose, estradiol, testosterone, insulin, glucagon and lactate dehydrogenase activity (P<0.05), however, only in restricted ewes were intrafollicular lactate dehydrogenase and testosterone concentrations in follicles>2.5mm not different from those in follicles<2.5mm. In conclusion, the mechanism by which short-term dietary restriction inhibits folliculogenesis may involve responses to intrafollicular influenced amino acids metabolism, increased estradiol, testosterone and lactate dehydrogenase levels in late-stage follicles.Experiment5. Effect of different levels of short-term feed intake on sterol-regulatory gene expression of follicular and luteal cells in Hu sheep during the luteal phase6ewes per group were slaughtered on day12of the estrous cycle and the follicular and luteal cells of follicles1.0-2.0mm,2.0-2.5mm,2.5-3.5mm and>3.5mm per ewe was collected, respectively. The results showed that short-term different nutritional intake affected the genes expression of ESR1, CYP17A1and CYP19A1in follicular cell (P<0.05). Compared to S group ewes, R and C group ewes decreased the CYP17A1mRNA expression (P<0.05) and R group ewes increased CYP19A1mRNA expression (P<0.05) and decreased ESR1mRNA expression (P<0.05) in follicles>2.5mm. There is follicle size effect on VLDLR, ESR2, FSHR (P<0.05), CYP17A1and CYP19A1(P<0.01) mRNA expression, however, only in S group ewes, were follicles>2.5mm less ESR2mRNA expression than follicles≤2.5mm (P<0.05). In conclusion, the mechanism in which short-term restriction inhibited folliculogenesis during luteal phase is that restriction influenced CYP17A1, CYP19A1and ESR1mRNA expression in late-stage follicles. |
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