Effects of nutrient restriction and arginine treatment on oxidative stress in the ovarian tissue of ewes during the luteal phase.

The aim of this study was to determine whether nutrient restriction and arginine treatment affect energy metabolism changes and oxidative stress through the mitochondrial pathway in the ovarian tissue of ewes during the luteal phase. On days 6-15 of the estrous cycle, 24 multiparous Hu sheep (BW = 43.56 ±â€¯1.53 kg) were randomly assigned to three groups: control group (CG; n = 6), restriction group (RG; n = 9), and l-arginine group (AG; n = 9) administered Arg treatment (or vehicle) three times per day. The ewes were slaughtered at the end of treatment, and blood samples and ovaries were collected for analysis. In this study, the expression levels of antioxidase enzymes (SOD2, CAT and GPX1) and mitochondrial biogenesis-related genes (ESRRA and TFAM), as well as antioxidase activity and mitochondrial function were examined in ovarian tissue. Nutrient restriction resulted in activation of ESRRA and TFAM and an increase in relative mtDNA copy number, whereas arginine treatment led to a pronounced recovery of ovarian tissue. In addition, we observed increased AMPK phosphorylation at Thr172 and SIRT3 levels in nutrient restricted ewes, and these effects decreased with arginine treatment. In conclusion, the present results indicated that short-term nutritional restriction led to changes in energy metabolism and oxidative stress. These changes disrupted the redox balance, thus leading to apoptosis through the mitochondria-dependent apoptosis pathway. Arginine treatment altered gene expression in ovarian tissue and increased the resistance to oxidative stress and the anti-apoptosis capacity. The results presented here suggest a potential method to increase agricultural productivity and economic benefits in the sheep industry by using dietary supplementation with arginine to decrease temporary undernutrition of ewes.

Effects of NRF1 on steroidogenesis and apoptosis in goat luteinized granulosa cells.

During goat follicular development, abnormal expression of nuclear respiratory factor 1 (NRF1) in granulosa cells may drive follicular atresia with unknown regulatory mechanisms. In this study, we investigated the effects of NRF1 on steroidogenesis and cell apoptosis by overexpressing or silencing it in goat luteinized granulosa cells (LGCs). Results showed that knockdown of NRF1 expression significantly inhibited the expression of STAR and CYP19A1, which are involved in sex steroid hormones synthesis, and led to lower estrogen levels. Knockdown of NRF1 resulted in an increased percentage of apoptosis, probably due to the release of cytochrome c from mitochondria, accompanied by upregulating mRNA and protein levels of apoptosis-related markers BAX, caspase 3 and caspase 9. These data indicate that NRF1 might be related with steroidogenesis and cell apoptosis. Furthermore, NRF1 silence reduced mitochondrial transcription factor A (TFAM) transcription activity, mtDNA copy number and ATP level. Simultaneously, knockdown of NRF1 suppressed the transcription and translation levels of SOD, GPx and CAT, decreased glutathione level and increased 8-OHdG level. However, the overexpression of NRF1 in LGCs or gain of TFAM in NRF1 silenced LGCs increased the expression of genes involved in mitochondrial function and biogenesis, and elevated the antioxidant stress system and steroids synthesis. Taken together, aberrant expression of NRF1 could induce mitochondrial dysfunction and disturb the cellular redox balance, which lead to disturbance of steroid hormone synthesis, and trigger LGC apoptosis through the mitochondria-dependent pathway. These findings will be helpful for understanding the role of NRF1 in goat ovarian follicular development and atresia.

Effects of diet and arginine treatment during the luteal phase on ovarian NO/PGC-1α signaling in ewes.

The aim of this study was to determine whether arginine (Arg) supplementation of malnourished ewes affects the expression of key NO/PGC-1α signaling pathway genes in the ovary. On Day 6-15 of the estrous cycle, 24 multiparous Hu sheep (BW = 43.56 ± 1.53 kg) were randomly assigned to three groups: control group (CG; n = 6), restriction group (RG; n = 9) and l-arginine group (AG; n = 9), and administered Arg treatment (or vehicle) three times per day. The ewes were slaughtered at the end of treatment, and blood samples and ovaries were collected for analysis. The results of our analyses showed that both short-term feed-restriction and/or supplementation with L-Arg-HCl affected the number of different size follicles observed in the ovary, and the relative day of estrus behavior initiation of ewes. Specifically, the relative day of estrus behavior initiation was significantly advanced in AG compared with that in RG ewes (P < 0.05). Both the number of ≤2 mm-ovarian follicles (P < 0.05) and the total number of ovarian follicles (P < 0.05) were significantly increased in the RG and AG compared with that in the CG ewes. RG ewes exhibited a higher proportion of ≤2 mm (P < 0.05), but a lower proportion of >5 mm follicles than did CG ewes (P < 0.05). The mean number of corpus lutea ≥5 mm was significantly increased in AG as compared to that in either CG or RG ewes. Furthermore, the expression of eNOS, nNOS, iNOS, PDE5A, PDE9A, PRKG2, and PPARGC1A varied significantly among the treatment groups (P < 0.05). GUCY1A3 mRNA levels were significantly increased in RG and AG as compared to those in CG ewes (P < 0.05), whereas conversely, GUCY1B3 mRNA levels were significantly decreased in CG and RG as compared to those in AG ewes (P < 0.05). P53 mRNA levels were found to vary significantly among the three experimental treatment groups (P < 0.05), and similarly, the relative expression levels of P53 were greater in AG and RG than in CG ewes (P < 0.05). The levels of eNOS protein were significantly higher in RG than in either CG or AG ewes (P < 0.05). The relative expression levels of PGC-1α were significantly higher in RG (P < 0.05) and significantly lower in AG ewes (P < 0.05) than in CG ewes. In conclusion, the results of the present study indicate that feed-restriction negatively affects follicular development, and that Arg-supplementation may modulate the expression of key NO/PGC-1α signaling pathway genes in the ovary and thereby accelerate ovulatory processes and the estrous rate. Elucidation of mechanisms underlying these effects of Arg on gene expression in the ewe ovary requires further investigation.

Effect of PGC-1α overexpression or silencing on mitochondrial apoptosis of goat luteinized granulosa cells.

During goat follicular development, abnormal expression of peroxisome proliferator- activated receptor gamma coactivator-1 alpha (PGC-1α) in granulosa cells (GCs) may contribute to follicular atresia with unknown regulatory mechanisms. In this study, we investigate the effect of ectopic expression or interference of PGC-1α on cell apoptosis of goat first passage granulosa cells (FGCs) in vitro. The results indicate that PGC-1α silencing by short hairpin RNA (shRNA) in goat FGCs significantly reduced mitochondrial DNA (mtDNA) copy number (P < 0.05), changed mitochondria ultrastructure, and induced cell apoptosis (P < 0.05). The transcription and translation levels of the apoptosis-related genes BCL-2-associated X protein (BAX), caspase 3, and caspase 9 were significantly up-regulated (P < 0.05, respectively). Moreover, the ratio of BAX/B-cell lymphoma 2 (BCL-2) was reduced (P < 0.05), and the release of cytochrome c (cyt c) and lactate dehydrogenase (LDH) was significantly enhanced (P < 0.05, respectively) in PGC-1α interference goat FGCs. Furthermore, the expression of anti-oxidative related genes superoxide dismutase 2 (SOD2), glutathione peroxidase (GPx) and catalase (CAT) was down-regulated (P < 0.05, respectively) and the activity of glutathione/glutathione disulfide (GSH/GSSG) was inhibited (P < 0.05). While enforced expression of PGC-1α increased the levels of genes involved in the regulation of mitochondrial function and biogenesis, and enhanced the anti-oxidative and anti-apoptosis capacity. Taken together, our results reveal that lack of PGC-1α may lead to mitochondrial dysfunction and disrupt the cellular redox balance, thus resulting in goat GCs apoptosis through the mitochondria-dependent apoptotic pathway.

Determination of energy and protein requirement for maintenance and growth and evaluation for the effects of gender upon nutrient requirement in Dorper × Hu Crossbred Lambs.

This study aimed to determine energy and protein requirement of Dorper × Hu crossbred lambs and further to evaluate the effect of gender upon nutrient requirement parameters. Forty-two female lambs (18.60 ± 1.57 kg) and 42 male lambs (18.30 ± 1.28 kg) were used. In comparative slaughter trial, 30 of animals from each gender group were randomly selected and assigned to ad libitum (AL), low restriction (LR) and high restriction (HR) group, and then were slaughtered when lambs under AL treatment reached target BW of 20, 28, and 35 kg, to determine body energy and nitrogen retained. In digestibility trial, remaining 12 female (18.01 ± 1.66 kg) and 12 male lambs (18.43 ± 1.17 kg) were randomly assigned to three feeding treatments in accordance with the design of comparative slaughter trial, to evaluate dietary energetic values at different feed intake levels. The combined data indicated that metabolizable energy (ME) requirement for maintenance (MEm; 400.61 ± 20.31 vs. 427.24 ± 18.70 kJ kg(-1) of shrunk BW(0.75); SBW(0.75)), partial efficiency of ME utilization for maintenance (k m; 0.64 ± 0.02 vs. 0.65 ± 0.03), partial efficiency of ME utilization for growth (k g ; 0.42 ± 0.03 vs. 0.44 ± 0.02), and net protein (NP) requirement for maintenance (NPm; 1.83 ± 0.17 vs. 1.99 ± 0.28 g kg(-1) of SBW(0.75)) did not differ (P > 0.05) due to gender; although not statistically different, the mean value of Net energy (NE) requirement for maintenance (NEm) for male lambs (260.62 ± 13.21 kJ kg(-1) of SBW(0.75)) were 5 % greater than that (274.16 ± 11.99 kJ kg(-1) of SBW(0.75)) of female lambs. Additionally, rams have greater amounts of NP requirement for growth (NPg, 15.94 to 44.32 g d(-1)) than those of ewes (13.07 to 32.95 g d(-1)) at the similar condition of BW and ADG. In conclusion, we suggested that our results of energy and protein requirement for growth ranged between the NRC recommendation for early and later maturating growing sheep, and the effect of gender upon energy requirement parameters was similar in tendency but was less evidently than those frequently recommended previously.