Animal, feed and rumen fermentation attributes associated with methane emissions from sheep fed brassica crops.

Methane emissions from ruminants enhance global warming and lead to a loss of feed energy. The emissions are low when fed brassica crops, but the factors contributing to low emissions are unknown. A meta-analysis was conducted with individual animal data collected from seven experiments. In these experiments, methane emissions were measured using respiration chambers. Animal characteristics, feed chemical composition and rumen fermentation parameters were included for the analysis using multiple regression models. Feed intake level, animal live weight and age were animal factors that were weakly and negatively related to methane yield (g/dry matter intake). The duration in which sheep were fed brassica crops was a significant contributor in the model, suggesting that the effect on emissions diminishes with time. Among a range of feed chemical composition characters, acid detergent fibre and hot-water-soluble carbohydrate contributed significantly to the model, suggesting that both structural and soluble carbohydrates affect methane formation in the rumen. There was no significant correlation between the concentration of sulphate in brassicas and emissions, but nitrate was moderately and negatively correlated with methane yield (r = -.53). Short-chain fatty acid profiles in the rumen of animals fed brassicas were different from those fed pasture, but these parameters only moderately correlated to methane emissions (r = .42). Feeding forage rape resulted in low rumen pH. The pH before morning feeding was strongly correlated to methane yield (r = .90). Rumen pH, together with microbial communities mediated by pH, might lead to low emissions. Bacteria known to produce hydrogen were relatively less abundant in the rumen contents of brassica-fed animals than pasture-fed animals. In conclusion, animal and feed factors, rumen fermentation and microbial communities all affect methane emissions to some extent. The interactions of these factors with each other thus contribute to methane emissions from brassica-fed sheep.

Roles of vitamin D and its receptor in the proliferation and apoptosis of luteinised granulosa cells in the goat.

The objective of this study was to investigate the dose-dependent effect of 1α,25-(OH)2VD3 (Vit D3) on invitro proliferation of goat luteinised granulosa cells (LGCs) and to determine the underlying mechanisms of its action by overexpressing and silencing vitamin D receptor (VDR) in LGCs. Results showed that VDR was prominently localised in GCs and theca cells (TCs) and its expression increased with follicle diameter, but was lower in atretic follicles than in healthy follicles. The proliferation rate of LGCs was significantly higher in the Vit D3-treated groups than in the control group, with the highest proliferation rate observed in the 10nM group; this was accompanied by changes in the expression of cell cycle-related genes. These data indicate that Vit D3 affects LGC proliferation in a dose-dependent manner. Contrary to the VDR knockdown effects, its overexpression upregulated and downregulated cell cycle- and apoptosis-related genes respectively; moreover, supplementation with 10nM of Vit D3 significantly enhanced these effects. These results suggest that changes in VDR expression patterns in LGCs may be associated with follicular development by regulation of cell proliferation and apoptosis. These findings will enhance the understanding of the roles of Vit D3 and VDR in goat ovarian follicular development.

YAP1 regulates PPARG and RXR alpha expression to affect the proliferation and differentiation of ovine preadipocyte.

Adipose tissue development is regulated by a serial of developmental signaling pathways. The Hippo pathway is a novel signaling cascade closely associated with adipogenesis. While most of Hippo pathway components had been verified that have a vital role in preadipocytes proliferation and differentiation, little is known about the function of Yes-associated protein 1 (YAP1) in mammalian adipose tissue development. Therefore, we investigated the role of YAP1 in ovine adipose tissue development by in vitro and in vivo experiments. We observed that the adipocyte size in subcutaneous adipose tissue increased with development. YAP1 expression increased during adipose tissue development, while decreased during the differentiation of ovine preadipocytes in vitro. YAP1 knockdown notably promoted lipid accumulation and suppressed ovine preadipocyte proliferation. In addition, we observed that YAP1 deficiency significantly upregulated peroxisome proliferator-activated receptor gamma (PPARG) and retinoid X receptor alpha (RXR alpha) expression. By contrast, overexpression of YAP1 led to the suppression of preadipocyte differentiation, lipid droplets formation, and PPARG expression. In brief, our findings demonstrated that YAP1 regulates the proliferation and differentiation of ovine preadipocyte via altering PPARG and RXR alpha expression.

Characterization of RUNX1T1, an Adipogenesis Regulator in Ovine Preadipocyte Differentiation.

Runt-related transcription factor 1 translocation partner 1 (RUNX1T1), a potential novel regulator of adipogenesis, exists in two splice variants: a long (RUNX1T1-L) and a short (RUNX1T1-S) isoform. However, there is no data showing the existence of RUNX1T1 in ovine subcutaneous fat at different stages of developmental and its role on ovine adipogenesis. Therefore, the objectives of this study were to evaluate the presence of RUNX1T1 in subcutaneous fat of five-day-old to 24-month-old sheep and to investigate the role of RUNX1T1 in ovine adipogenesis. In this study, we detected a 1829 bp cDNA fragment of RUNX1T1 which contains a 1815 bp coding sequence that encodes 602-amino acid and 14 bp of 5' untranslated region, respectively. The amino acid sequence of RUNX1T1 has 31.18⁻94.21% homology with other species' protein sequences. During fat development, the RUNX1T1 protein expression was higher in subcutaneous fat of 24-month-old Hu sheep. In addition, the expression of RUNX1T1-L mRNA decreased first, then subsequently increased during ovine preadipocyte differentiation. Knockdown of RUNX1T1-L in ovine preadipocytes promoted preadipocyte differentiation and lipid accumulation. Taken together, our data suggests that RUNX1T1 is an important functional molecule in adipogenesis. Moreover, it showed for the first time that RUNX1T1-L was negatively correlated with the ovine preadipocyte differentiation.

PPP2R2A promotes testosterone secretion in Hu sheep Leydig cells via activation of the AKT/mTOR signaling pathway.

The serine-threonine protein phosphatase 2A (PP2A) is a heterotrimeric enzyme complex that plays a vital role in regulating male reproductive activities. However, as an essential member of the PP2A family, the physiological functions of PP2A regulatory subunit B55α (PPP2R2A) in testis remain inconclusive. Hu sheep are noted for their reproductive precocity and fertility, and are ideal models for the study of male reproductive physiology. Here, we analyzed the expression patterns of PPP2R2A in the male Hu sheep reproductive tract at different developmental stages and further investigated its role in testosterone secretion and its underlying mechanisms. In this study, we found that there were temporal and spatial differences in PPP2R2A protein expression in the testis and epididymis, especially the expression abundance in the testis at 8 months old (8M) was higher than that at 3 months old (3M). Interestingly, we observed that PPP2R2A interference reduced the testosterone levels in the cell culture medium, which is accompanied by a reduction in Leydig cell proliferation and an elevation in Leydig cell apoptosis. The level of reactive oxygen species in cells increased significantly, while the mitochondrial membrane potential (ΔΨm) decreased significantly after PPP2R2A deletion. Meanwhile, the mitochondrial mitotic protein DNM1L was significantly upregulated, while the mitochondrial fusion proteins MFN1/2 and OPA1 were significantly downregulated after PPP2R2A interference. Furthermore, PPP2R2A interference suppressed the AKT/mTOR signaling pathway. Taken together, our data indicated that PPP2R2A enhanced testosterone secretion, promoted cell proliferation, and inhibited cell apoptosis in vitro, all of which were associated with the AKT/mTOR signaling pathway.

Supplementation of graded levels of rumen-protected choline to a pelleted total mixed ration did not improve the growth and slaughter performance of fattening lambs.

Choline is an essential nutrient in ruminant diets, which contributes to the fundamental biological functions of the animal. However, choline is easily degraded in the rumen before it can be absorbed. Rumen-protected choline (RPC) supplementation might support the fast growth of ruminants. This study aimed to investigate the effects of supplementing graded levels of RPC in a pelleted total mixed ration for fattening lambs. Sixty three-month-old male Small Tail Han and northeast fine wool sheep hybrid lambs with a liveweight of 15.3 ± 1.8 kg (mean ± SD) were fed designated diets and randomly assigned into five treatment groups (n = 12 per group). The five treatments were the rate of RPC supplementation at 0, 1.25, 2.50, 3.75, and 5.00 g (equivalent to 0, 0.31, 0.63, 0.94, and 1.25 g of choline chloride, respectively)/kg basal diet and the RPC-supplemented feed was offered for 112 days after 12 days of adaptation. Average daily gain, dry matter intake, and nutrient digestibility were similar across treatments. The rumen pH was quadratically significant among treatments, with the lowest and highest pH observed from the 2.5 and 5 g/kg RPC supplement groups, respectively (P = 0.02). After feeding, the ruminal ammonia concentrations among treatments were different (P < 0.05), with the highest value observed from the 5 g/kg RPC supplement group. Microbial crude protein level was different, with the highest value recorded from the 0 g/kg RPC supplement group (P = 0.028). A linear effect (P < 0.05) was observed from short-chain fatty acid values among treatments before and after feeding. Serum albumin (P = 0.003) and albumin/globulin ratio (P = 0.002) had a quadratic effect, with the highest value found in the 0 g/kg RPC supplement group. Abdominal fat was higher in RPC-supplemented groups (P < 0.05) compared to the control group. Drip loss was 65% higher in RPC-supplemented groups compared to the control group (P = 0.012). Overall, the study results showed an effect of RPC on ruminal parameters, but the supplementation of low-level RPC did not improve the growth and slaughter performance of fattening lambs.

γ-Linolenic Acid Prevents Lipid Metabolism Disorder in Palmitic Acid-Treated Alpha Mouse Liver-12 Cells by Balancing Autophagy and Apoptosis via the LKB1-AMPK-mTOR Pathway.

Excessive fat deposition is the main character in nonalcoholic fatty liver disease (NAFLD), while γ-linolenic acid (GLA) is a polyunsaturated fatty acid that can reduce lipid deposition. This study investigated the effect and regulatory mechanism of GLA (100 µM) on lipid metabolism in alpha mouse liver 12 (AML-12) cells treated by 400 µM palmitic acid (PA). GLA reduced lipid content and increased fatty acid ß oxidation, as indicated by decreasing triglyceride and cholesterol contents and increasing mRNA and protein expressions of CPT1α and PPARα. GLA relieved oxidative stress caused by PA, upregulated mRNA levels of superoxide dismutase and glutathione peroxidase, and decreased reactive oxygen species content. GLA reduced apoptosis, as indicated by decreases in the BAX/BCL2 expression level and apoptosis percentage. GLA activated autophagy, autophagosome-lysosome fusion, and LKB1-AMPK-mTOR signaling and upregulated mRNA and protein expressions of Beclin-1, autophagy-related 5, and liver kinase B1 (LKB1). These effects of GLA on lipid metabolism disorders of PA-treated hepatocytes were reversed by autophagy inhibitor 3MA and AMPK inhibitor compound C, confirming our conclusions. Overall, GLA can protect AML-12 cells from lipid metabolism disorder caused by PA via balancing autophagy and apoptosis mediated by the LKB1-AMPK-mTOR pathway. Consequently, GLA, as a dietary supplement, can help to prevent and treat NAFLD by regulating lipid metabolism and autophagy.

Effects of dietary betaine supplementation on biochemical parameters of blood and testicular oxidative stress in Hu sheep.

Betaine, a highly valuable feed additive, has been observed to alter the distribution of protein and fat in the bodies of ruminants and to exhibit strong antioxidant properties. However, the effects of dietary betaine supplementation on the biochemical parameters of blood and on testicular oxidative stress remain unknown. This study aimed to investigate the effects of dietary betaine supplementation on lipid metabolism, immunity, and testicular oxidative status in Hu sheep. Experimental sheep (n=3, three sheep per group) were fed betaine-containing diets, a basal diet supplemented with 0 g/day (control group), 1 g/day (B1), and 3 g/day betaine (B2). There were no differences in the serum concentrations of triglycerides and cholesterol in Hu sheep receiving diets supplemented with betaine. The ratio of basophils significantly increased in the B1 and B2 groups. ELISA (enzyme-linked immunosorbent assay) results showed that testicular superoxide dismutase (SOD) and glutathione peroxidase (GSH-Px) activity were significantly higher, whereas malondialdehyde (MDA) content significantly decreased, after feeding betaine-supplemented diets. qPCR results showed that the mRNA expression levels of CAT, SOD2, and GSH-Px were significantly upregulated in both the B1 and B2 groups compared to those in the control group. Furthermore, the expression of proliferating cell nuclear antigen (PCNA) was significantly lower in the testes of betaine-treated Hu sheep than in the control group. Moreover, LKB1 (liver kinase B1) expression significantly increased, and mRNA expression of AMPK (AMP-activated serine/threonine protein kinase) significantly decreased in the B1 group. The relative gene expression of mTOR (mechanistic target of rapamycin) was significantly higher in the B2 group than in the control group. RAPTOR expression significantly increased in the B1 group. Western blot revealed that the ratio of P-mTOR and mTOR significantly increased after feeding betaine-supplemented diets. In conclusion, betaine supplementation improved serum lipid metabolism, immune response, and increased the testicular antioxidant capacity of Hu sheep, which might be regulated via mTOR signaling pathway.

Pelleting of a Total Mixed Ration Affects Growth Performance of Fattening Lambs.

Feeding pelleted total mixed rations (TMR) instead of traditional loose concentrate plus forage to fattening lambs is an emerging practice. This study aimed to determine the effects of feeding pelleted TMR to fattening lambs on feed intake behaviour, growth performance, feed digestion, rumen fermentation characteristics, rumen microbial community, serum parameters, slaughter performance, meat quality, and the economic outcome. Two physical forms (pelleted vs. un-pelleted) of TMR composed of the same ingredients with the same particle sizes were compared in three animal experiments. Feed intake and average daily gain were higher when the TMR was pelleted, but apparent total tract digestibility of nutrients (organic matter, crude protein, neutral detergent fibre, acid detergent fibre, and ether extract) and serum parameters were not affected and apparent total tract dry matter digestibility was slightly lower. Feeding pelleted TMR increased total short-chain fatty acid concentration and decreased rumen pH. Rumen microbial community was not affected by the physical form of the TMR at phylum level but changed slightly at genus level. Liveweight at slaughter and hot carcass weight were higher for lambs fed the pelleted compared to the un-pelleted TMR, while dressing percentage and meat quality were not affected. In conclusion, feeding pelleted TMR improves growth performance of fattening lambs mainly due to an increase in feed intake. Feeding pelleted TMR is a feasible strategy for intensive lamb fattening operations.

Dietary Supplementation of Yeast Culture Into Pelleted Total Mixed Rations Improves the Growth Performance of Fattening Lambs.

There is a growing interest in the use of yeast (Saccharomyces cerevisiae) culture (YC) for the enhancement of growth performance and general animal health. Grain-based pelleted total mixed rations (TMR) are emerging in intensive sheep farming systems, but it is uncertain if the process of pelleting results in YC becoming ineffective. This study aimed to examine the effects of YC supplemented to pelleted TMR at two proportions of corn in the diet on animal performance, feed digestion, blood parameters, rumen fermentation, and microbial community in fattening lambs. A 2 × 2 factorial design was adopted with two experimental factors and two levels in each factor, resulting in four treatments: (1) low proportion of corn in the diet (LC; 350 g corn/kg diet) without YC, (2) LC with YC (5 g/kg diet), (3) high proportion of corn in the diet (HC; 600 g corn/kg diet) without YC, and (4) HC with YC. Fifty-six 3-month-old male F2 hybrids of thin-tailed sheep and Northeast fine-wool sheep with a liveweight of 19.9 ± 2.7 kg were randomly assigned to the four treatment groups with an equal number of animals in each group. The results showed that live yeast cells could not survive during pelleting, and thus, any biological effects of the YC were the result of feeding dead yeast and the metabolites of yeast fermentation rather than live yeast cells. The supplementation of YC resulted in 31.1 g/day more average daily gain regardless of the proportion of corn in the diet with unchanged feed intake during the 56-day growth measurement period. The digestibility of neutral detergent fibre and acid detergent fibre was increased, but the digestibility of dry matter, organic matter, and crude protein was not affected by YC. The supplementation of YC altered the rumen bacterial population and species, but the most abundant phyla Bacteroidetes, Firmicutes, and Proteobacteria remained unchanged. This study indicates that YC products can be supplemented to pelleted TMR for improved lamb growth performance, although live yeast cells are inactive after pelleting. The improved performance could be attributed to improved fibre digestibility.

Effects of the Supplementation of Lysophospholipids through Pelleted Total Mixed Rations on Blood Biochemical Parameters and Milk Production and Composition of Mid-Lactation Dairy Cows.

Lysophospholipids (LPL), a new feed additive, were supplemented to a pelleted total mixed ration (TMR) of dairy cows to examine its effects on feed intake, production, and composition of milk and plasma biochemical parameters. Two dietary treatments included diets supplemented without (control diet; CON) or with LPL at a dose of 0.5 g/kg of pelleted TMR. Twelve multiparous, mid-lactation, Holstein cows (Bodyweight 730 ± 9.3 kg; 100 ± 6.0 days in milk) were randomly assigned to one of the two dietary treatments with a 42-day measurement period after a 14-day adaptation period. Feed and water were provided ad libitum. Feed intake and milk yields were recorded daily, blood samples were collected fortnightly, and milk samples weekly. The results showed that the supplementation of LPL did not change feed dry matter intake, milk yields, and milk composition. However, it increased total protein and globulin and the activity of alkaline phosphatase and decreased total cholesterol in plasma. This study suggests that LPL may have beneficent effects in animal health but might be not a feasible feed additive to increase production for dairy cows fed a pelleted TMR.

Estradiol-17ß regulates proliferation and apoptosis of sheep endometrial epithelial cells by regulating the relative abundance of YAP1.

Yes-associated protein 1 (YAP1) transcription regulator of the Hippo protein kinase pathway, serves as a key regulator of tissue growth and organ size by regulating cell proliferation and apoptosis. Effects of YAP1 on proliferation and apoptosis of sheep endometrial epithelial cells (EEC) as a result of estradiol-17ß (E2) treatment, however, remain unclear. In the present study, the abundance of YAP1 protein in the uterine horn was greater than that in the uterine body or cervix. The YAP1 protein was primarily localized in the endometrial luminal and glandular epithelial cells of the uterine horn of ewes on day 2 of the estrous cycle. Compared with control samples, there was a lesser abundance of YAP1 mRNA transcript that was associated with a lesser proliferation and greater apoptosis of EEC. There were also lesser concentrations of epidermal growth factor and insulin-like growth factor 1 in the spent culture medium when there was a lesser abundance of YAP1 mRNA in EEC compared with those in the control group. When there was a greater abundance of YAP1 mRNA transcript, there were greater concentrations of epidermal growth factor and insulin-like growth factor 1 in the spent media. Furthermore, with estradiol-17ß treatment the abundance of YAP1 mRNA transcript was similar to that of the control samples. Taken together, estradiol-17ß may function as an essential regulator of EEC proliferation and apoptosis by modulation of concentrations of YAP1 protein in the sheep uterus. These results indicate there are molecular mechanisms of estradiol-17ß and YAP1 in EEC proliferation and apoptosis of ewes.

Dietary Supplementation of Lysophospholipids Affects Feed Digestion in Lambs.

Five experiments were conducted to examine effects of lysophospholipids (LPL) on live weight gain, nutrient digestibility, ruminal fermentation parameters, serum biochemical parameters and rumen bacterial community profile in fattening lambs. Two dietary treatments (pelleted complete feed supplemented without (control diet; CON) or with 0.05% LPL on dry matter basis) were tested in these experiments. Feed and water were provided ad libitum to lambs. The results showed that average daily gain (ADG) tended to increase or was not affected by LPL supplementation. Compared with CON, the supplementation of LPL resulted in an increase in dry matter, crude protein and organic matter digestibilities, and a decrease in neutral detergent fiber and acid detergent fiber digestibilities. Ruminal pH values did not change with LPL supplementation, but the concentrations of ammonia and total short chain fatty acids (SCFAs) were increased. The molar proportion of major individual SCFAs and the ratio of acetate to propionate were not affected by LPL supplementation. While the activity of lipase was decreased with LPL supplementation, all other serum biochemical parameters did not change. Rumen bacterial community was altered by LPL supplementation with the relative abundance of fibrolytic bacteria in the total bacterial population, such as Prevotella, decreased. In conclusion, LPL supplementation can alter feed digestion, but may not result in consistent positive responses in animal growth performance.

Effects of l-arginine on endometrial estrogen receptor α/ß and progesterone receptor expression in nutrient-restricted sheep.

This study aimed to determine the effects of l-arginine (L-Arg) supplementation on steroid hormone receptors in non-pregnant ovine endometrium. All experimental ewes were randomly assigned to either a control group (n = 6), a nutrient-restricted group (n = 6), or an L-Arg supplemented nutrient-restricted group (n = 6). The effects of L-Arg on estrogen receptor α/ß (ERα/ß) and progesterone receptor (PGR) expression in the ovine endometrium were assessed. Our results showed that levels of ERß and PGR expression were significantly increased by nutrient restriction, but L-Arg counteracted the effect of nutrient restriction on ERß and PGR expression (p < 0.05). Also, expression of endometrial ERα was substantially increased (p < 0.05) by L-Arg supplementation. Furthermore, ERα/ß and PGR were mainly detected in the endometrial luminal epithelium and glandular epithelium. Therefore, we isolated and identified endometrial epithelial cells (EECs) from sheep. Different concentrations of L-Arg were added to investigate the effects on ERα/ß and PGR in EECs. The expression levels of endothelial nitric oxide synthase, ERß, and PGR were significantly increased in response to low-concentration (200 µmol) L-Arg supplementation, which subsequently decreased with a high concentration (800 µmol) (p < 0.05). Otherwise, ERα expression was remarkably increased at both L-Arg concentrations in EECs (p < 0.05). Overall, the results indicated that L-Arg performed crucial roles in the regulation of ovine steroid hormone receptor expression in the endometrium. The results of this study provide a theoretical basis and technical means for the normal function of endometrium in response to low nutrient levels.

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.