| It’s common to feed lactating ruminants with high concentrate diet to meet the energy requirement for high milk production. However, feeding excessive amounts of high proportions of fermentable concentrate and forage with low physically effective fiber usually cause metabolic disorder especially subacute ruminal acidosis (SARA). SARA is a well-recognized digestive disorder in most dairy herds. Additionally, the clinical signs of SARA include the depression of food intake, diarrhea, laminitis, and inflammatory response, indicating that ruminants suffered SARA may live in a certain stress. However, till now, the data on the activity of the stress axis (Hypothalamus-pituitary-adrenal gland, HPA) is not available when ruminants experienced SARA. Therefore, the purpose of this study was to investigate the changes of HPA axis exhibiting as cortisol concentration in circulating system and key factors mRNA and protein expression in adrenal gland when dairy goats suffered SARA fed diets enriched with high proportion of concentrate (60%). Moreover, in order to elucidate the mechanism behind the depression of fat content in milk, the alternations of lipid metabolic parameters and hepatic genes expression involved in lipids metabolism were compared between SARA and control goats.1Effect of SARA on cortisol secretion and HPA axis in non-lactation goats12rumen-cannulated goats during non-lacatation stage were randomly allocated to a control (concentrate:forage4:6) or a high-grain (concentrate:forage6:4) diet in a randomized study. The whole experiment lasted for21d. Samples including serum, rumen fluid, liver tissues and adrenal gland tissues were collected for biochemical analysis. The value of pH in rumen fluid was immediately determined by a portable pH meter. The levels of cortisol in blood as well as in rumen fluid were detected by radioimmunoassay (RIA) method. Serum concentration of total cholesterol (TCH) was measured by commercial kits with biochemical methods. The expression of genes involved in cortisol synthesis in HPA axis and glucocorticoids receptor (GR) in different tissues was detected with real-time PCR. Protein content of3β-hydroxysteroid dehydrogenase (3β-HSD) and17a-hydroxylase cytochrome P450(P45017a) in adrenal gland were measured by western blot with their specific antibodies.The results showed that SARA was successfully induced in goats by feeding a high-grain (concentrate:forage6:4) diet for three weeks, with ruminal pH remaining below5.8for an average of4hours per day. However, goats in the control group received a control diet (concentrate:forage4:6) showed rumen pH above6.0during the whole experimental time. RIA results showed that there was no significant difference in cortisol concentration in peripheral vein system between SARA and control goats (P>0.05). However, SARA goats had significantly higher level of cortisol in hepatic portal vein as well as in hepatic vein (P<0.05). Diet significantly affected the concentration of TCH in blood, and Teh level was significantly decreased in serum of SARA goats compared to control counterparts (P<0.01). Real-time PCR results showed that corticotropin releasing hormone (CRH) mRNA expression in the hypothalamus and proopiomelanocortin (POMC) mRNA expression in pituitary were significantly decreased in SARA goats compared to control (P<0.05), whereas corticotropin releasing hormone receptor (CRHR) mRNA transcription in pituitary had a tendency to increase (.P=0.07) in SARA goats. In adrenal gland, genes expression of3β-HSD (P<0.05) and P45017a (P<0.01) were markedly up-regulated in SARA goats, and paralleled an increase of3β-HSD and cholesterol side-chain cleavage (P450scc) protein content in adrenal gland by58.6%and39.4%in SARA goats, respectively, but did not reach the statistical significance (P>0.05). However, melanocortin type2receptor (MC2R), steroidogenic acute regulatory protein (StAR) and11β-hydroxylase cytochrome P450(P45011β) mRNA expression in adrenal gland was significantly down-regulated in SARA goats compared with control (P<0.05). In SARA goats, glucocorticoid receptor (GR) mRNA expression was significantly down-regulated in the liver (P<0.01), and had a tendency to decrease in the hypothalamus (P=0.07), yet significantly increased in the rumen epithelium (P<0.01). No significant changes in GR gene expression were observed in the hippocampus or pituitary (P>0.05). These results suggested that SARA goats experienced a certain stress status, exhibiting the activation of HPA axis. In SARA goats, GR gene expression in central and peripheral tissues might lead to changes of material metabolism and nutrition redistribution in target organ, especially in the liver and rumen epithelium of goats. 2Effect of SARA on cortisol and lipid metabolism in lactating goats1) Short-term experiment:Seven goats during mid-lactating peroid fitted with permanent rumen cannulas were employed in a2×2latin square experiment design with different concentrate to forage ratio of6:4and4:6, respectively. Each term of experiment lasted for two weeks. Rumen fluid were collected for detecting pH by a portable pH meter. The levels of cortisol in blood and rumen fluid were detected by RIA. The expression of GR mRNA in liver was detected with real-time PCR.The results showed that SARA was induced successfully by feeding the diet with concentrate to forage ration of6:4. pH of the rumen fluid in SARA goats was below5.8after feed delivery, lasting for about6hours. The concentration of cortisol in blood of SARA goats was higher than that in the control group (P=0.05) prior to feeding, however, there was no statistical significance between two groups at6h after feeding (P>0.05). The level of cortisol in rumen fluid was significantly higher than control during2,4and6h after feeding, while no significance was observed between two groups after10h feed delivery (P>0.05). Real-time PCR results showed that GR mRNA expression was significantly down-regulated in liver of SARA goats compared to control (P<0.05). Our results indicated that SARA goats experienced stress response with a significantly higher glucocorticoid concentrate in circulating system and in rumen fluid, which accompanied with a significant down-regulation of GR gene expression in liver, indicating the alterations in substance metabolism and nutrients redistribution in liver.2) Long-term experiment:Eight Saanen dairy goats during mid-lactating stage were cannulated in rumen and were randomly allocated to the control group (Control group, n=4) and high concentrate group (Treated group, n=4) with F:C=6:4and F:C=4:6diets, respectively. Rumen fluid and milk sample were collected for every two weeks for analyzing rumen pH or the contents of fat, protein and lactose in milk. The level of cortisol in blood, rumen fluid and milk were detected by radioimmunoassay method. Glutamic-oxalacetic transaminease (GOT), globulin, triglycerol (TG), nonesterified fatty acid (NEFA) and other biochemical parameters were detected by automatic blood analyzer with commercial kits. The expression of genes related to lipid metabolism and GR in liver was detected by real-time PCR. Protein content of GR and long-chain acyl-CoA synthetase1(ACSL1) in liver were measured using western blot with their specific antibodies.Results:During2th-3th week, pH in rumen fluid of treated goats was below5.8after feeding, lasting for about4hours per day. When the experiment time prolonged from4th to9th week, ruminal pH was recovered to>6.0in treated goats, but still significantly lower than control group (P<0.05). Compared to control goats, the contents of milk fat and milk protein were significantly decreased in treated goats from6th to9th week (P<0.05), and milk fat contents was greatly depressed from2th-3th week (P<0.01). The levels of GOT and NEFA in blood of treated goats had a tendency to increase (P<0.1), and glucose concentration was significantly higher in treated goats (P<0.05), whereas the contents of TG and CHO in liver were significantly lower (P<0.05) compared to control. The concentration of cortisol in rumen fluid in treated group have a tendency to increase (P<0.1) prior to feed delivery, and was significantly higher at2h after feeding (P<0.01). The level of cortisol in rumen fluid and blood were significantly increased at8h after feeding in treated goats than in control group (P<0.05). Milk cortisol concentration was not significantly changed between two groups before4th week diet treatment (P>0.05), while the concentration was increased at8th week and reached the statistically significance in treated goats (P<0.05). Real-time PCR results showed that the expression of Acetyl coenzyme A acyltransferase2(ACAA2) and3-hydroxyacyl Co A dehydrogenase (HADH) genes in liver were significantly up-regulated in treated goats (P<0.05), and ACSL1mRNA expression was up-regulated (P<0.1) and paralleled a significant increase in protein contents in liver of treated goats (P<0.01). Compared to the control group, both GR mRNA expression and protein were markedly down-regulated (P<0.05and P<0.01, respectively) in liver of treated goats receiving high concentrate diets. These results suggested that lactating goats fed high concentrate diets for a long time increased milk yield but significantly depressed milk quality with lower fat and protein contents. The level of cortisol in body fluid was elevated, indicating the activation of stress response during long-term feeding high concentrate challenge. The significant increase of NEFA in blood indicated the enhanced catabolism of fatty acids in liver. The increases in sress response and lipids catabolism in liver might result in the nutrient partitioning, which consequently lead to the decrease of nutrients precursor to mammary gland for fat synthesis. |
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