| It has been proved that long-term high concentrate feeding can lead to the damage of body health and the decrease of milk quality in lactating goats,and the addition of buffering agents in high concentrate diet can improve the body health status and milk quality.This study explored the effects of the lactating goats health,as well as the formation,transformation and distribution of lactating goat milk fat or lactose precursors under the high concentrate feeding mode with buffering agents in order to clarify its protective effect on the body and the mechanism of milk fat and lactose synthesis.The research mainly consists of the following four aspects:1 The effect of buffering agents on the synthesis of milk fat/lactose and the metabolism of their precursors in lactating goats fed with high concentrate dietsTwelve similarly weighted Saanen dairy goats in the mid of lactating period were randomly divided into 2 groups of six,the high concentrate group(high concentrate:roughage ratio 60:40)and the buffering agents group(high concentrate:roughage ratio 60:40+buffering agents).Then various experiments were carried out for a period of 20 weeks.Amount of milk yield was recorded every day.Content of milk fat or lactose was determined every week.The pH value of rumen fluid was measured every two weeks.On the 20th week,rumen fluid and blood samples were collected and then the lipopolysaccharide(LPS)content,volatile fatty acid(VFA),blood related biochemical indexes,hormones and the content of milk fat or lactose precursors were determined.Results:1)At the early stage of feeding,there were no significant differences in the milk yield amount,milk fat content and milk lactose content between the two groups.From the 3rd week on,the milk yield amount and lactose content in the buffering agents group remained higher than those of the high concentrate group.From the 6th week on,the milk fat content in the buffering agents group also remained higher until the end of the experiment.2)The rumen pH in the high concentrate group decreased(average 5.8±0.08)after long-term high concentrate feeding,which results in subacute ruminal acidosis(SARA),while in the buffering agents group,the rumen pH was always at a higher level(average 6.0 ± 0.10)and the LPS content in the rumen juice also significantly decreased(P<0.01).3)The concentration and proportion of VFA in rumen fluid and blood changed in both groups.The content of propionic acid and butyric acid in rumen was found significantly higher in the buffering agents group(P<0.05).The content of butyric acid in blood was also significantly higher than that in the buffering agents group(P<0.05).4)AST,ALT and AKP activity significant lower in the buffering agents group than that of the high concentrate group(P<0.05).The level of lactic acid and TNF-α in the buffering agents group was found significantly lower(P<0.05).The content of insulin(INS),non-esterified fatty acid(NEFA)and glucose(GLU)in buffering agents group was higher than that of the high concentrate group,and the content of INS and NEFA was significantly higher than that of the high concentrate group(P<0.05).Conclusion:Adding buffering agents to the high concentrate feed of lactating goat can improve the milk quality and milk yield,alleviate the decrease of rumen pH,change the fermentation state of rumen and reduce the production of abnormal metabolite and the change of metabolic flow direction of precursors.Therefore,the improvement of body health,milk quality and milk yield.The increase of lactose and milk fat may be related to the increase of NEFA and other precursors in blood.2 The effect of buffering agents on distribution and redistribution of glycolipid precursor in liver of lactating goats fed with high concentrate dietsThe metabolic changes of liver glycolipid precursors in lactating goats fed with high concentrate were investigated.The redistributive mechanism of milk fat or lactose precursor in liver was analyzed in combination with liver metabonomics.The signal mechanism of INS-mediated PI3K/AKT signaling pathway was further revealed.Blood(portal vein and hepatic vein)and liver tissue were collected at the 20th week.The following experiments were conducted:1)Histological observation of the morphological changes in the liver of lactating goats.2)The content of the milk fat or lactose precursors in the blood(portal vein and hepatic vein)were determined.3)LC/MS-QTOF technique was used to determine the difference in liver metabolites in both groups of lactating goats.4)Analyze and determine the expression of genes and related protein involved in fatty acid metabolism,gluconeogenesis and pentose phosphate pathway in the liver of lactating goats.5)The levels of INS receptor and its downstream phosphorylation level of PI3K/AKT pathway key proteins in liver were analyzed.Results:1)Hemorrhage in the central vein of the liver and high edema of the liver cells in the high concentrate group;However,in the buffering agents group,the central venous structure of the liver was clear,and the structure of the liver cells was dense and complete.2)Compared with the high concentrate group,the net output of NEFA and GLU in the liver of lactating goats was significantly increased in the buffering agents group(P<0.05).3)Liver metabolomics analysis:a total of 268 differential metabolites were screened under the Pos model,of which 118 were upregulated and 150 were down-regulated.A total of 264 differential metabolites were screened under the Neg model,of which 153 were upregulated and 111 were down-regulated.Through KEGG pathway analysis,it was found that metabolic pathways related to fatty acid metabolism,amino acid metabolism and pentose phosphate pathway were activated in the buffering agents group.4)The key enzymes ACC and SCD-1 of fatty acid synthesis and the upstream transcription factor SREBP-1c in the buffering agents group were significantly up-regulated(P<0.05),while the key enzyme CPT-1 of fatty acid catabolism and the upstream transcription factor PPARa were significantly down-regulated(P<0.05),The gluconeogenesis key enzymes PEPCK and G6PC were significantly up-regulated(P<0.05),and the key enzyme 6PGDH in the pentose phosphate pathway was also significantly up-regulated(P<0.05).5)The expression of insulin receptor,insulin receptor substrate and PI3K/AKT pathway protein in the liver of lactating goats were significantly up-regulated in the buffering agents group(P<0.05).Conclusion:Adding buffering agents to the high concentrate feed of lactating goat can enhance the hepatic pentose phosphate pathway,fatty acid synthesis and gluconeogenesis.The liver milk fat or lactose precursor were redistributed,the synthesis of precursors increased,and the net output of NEFA and GLU in liver was significantly increased,thus providing more precursors for the synthesis of milk fat or lactose in the mammary gland.Activation of INS-PI3K/AKT signaling pathway is the main mechanism for enhanced fatty acid synthesis in the liver.3 The effect of buffering agents on milk fat or lactose and its precursors uptake and utilization in mammary gland of lactating goat fed with high concentrate dietsTo investigate the effect and mechanism of buffering agents on the health of mammary gland and the intake and utilization of milk fat or lactose precursors in lactating goats fed with high concentrate.Mammary gland tissue was taken at the 20th week and the following experiments were carried out:1)Histological observation of the morphological changes in the mammary gland of lactating goats.2)Oxidative stress,apoptosis protein and other related indexes in mammary gland tissue were determined.3)The expression level of the G protein-coupled receptor 41(GPR41)protein and the milk fat synthesis related enzymes in mammary gland tissue were determined.4)The expression level of glucose-related transporter,lactose synthesis-related enzymes and lactose content in mammary gland tissue were determined.Results:1)In the high concentrate group,there were inflammatory cell infiltration,acinar atrophy and blurred structure in the mammary gland tissue of lactating goats;However,in the buffering agents group,the acinar filling and the clear structure of the mammary gland were clear.2)Compared with the high concentrate group,the content of SOD,T-AOC in the mammary gland significantly increased(P<0.05)and the content of MDA significantly decreased(P<0.05).The expression levels of apoptotic protein Caspase-3 and Bax significantly decreased(P<0.05)and the anti-apoptotic protein Bcl-2 was significantly up-regulated(P<0.05).3)Compared with the high concentrate group,the mammary gland GPR41 in the buffering agents group was significantly up-regulated(P<0.05),and the DE novo fatty acid synthesis enzymes ACC,SCD-1 and triglyceride synthesis enzymes DGAT1 were significantly up-regulated(P<0.05).4)Compared with the high concentrate group,GLUT1 and the key enzyme HK of lactose synthesis in the mammary gland were significantly up-regulated(P<0.05).Conclusion:The results suggested that adding buffering agents to the high concentrate feed of lactating goat can enhance the anti-oxidant stress ability of mammary gland and alleviate the apoptosis injury and improve the health state.In the mammary gland,milk fat or lactose precursor consumption reduced,enhanced utilization,lactose and milk fat synthesis increased.The results suggested that the improvement of mammary gland health and up-regulation of GPR41 expression in the buffering agents group may be related to the addition of sodium butyrate in the buffering agents,but its mechanism needs to be further studied.4 The protective effect of sodium butyrate on LPS-induced oxidative stress and apoptosis injury of MAC-T cellsThe results of our in vivo experiments have shown that the buffering agents can alleviate the mammary gland damage caused by high concentrate feeding,and it may be related to the addition of sodium butyrate in the buffering agents.We will further verify the protective effect of sodium butyrate on LPS-induced mammary gland injury in vitro and reveal its molecular mechanism.The bovine mammary epithelial cells line(MAC-T)were used as the research object.The injury model of mammary gland was established by adding LPS.At the same time,sodium butyrate+LPS treatment group and sodium butyrate+LPS treatment+PI3K inhibitor group were established.The following experiments were performed:1)The model of MAC-T cell injury induced by LPS was established.2)The oxidative stress and anti-oxidative stress related indicators were determined.3)Flow cytometry was used to determine the degree of early and late apoptosis of MAC-T cells.4)Western blot method was used to determine the protein expression level of apoptosis and PI3K/AKT signal pathway.5)The expression level of PI3K protein was determined by immunofluorescence.Results:1)The injury model of MAC-T induced by LPS was 1μg/mL,and the stimulation time was 9h.2)The activity of antioxidant enzymes such as SOD,GSH-Px,CAT,POD,T-AOC significantly increased(P<0.05 or P<0.01)and the level MDA and protein carbonyl was significantly decreased(P<0.05 or P<0.01)in MAC-T injury model cells.3)Sodium butyrate could significantly decrease the expression of apoptotic protein Caspase-3,Caspase-9 and Bax in MAC-T model cells.The key proteins of PI3K/AKT signaling pathway related to apoptosis were significantly increased(P<0.05 or P<0.01).However,PI3K inhibitor(LY294002)could inhibit the expression of PI3K/AKT protein and increase the expression of apoptosis protein(P<0.05).These results suggest that sodium butyrate can enhance the anti-oxidative stress and inhibit cell apoptosis through PI3K/AKT signaling pathway and alleviate the damage of MAC-T cells induced by LPS.In summary,our experiment suggests that the buffering agents can slow down the SARA and decline of milk quality induced by high concentrate in lactating goats.The results provide theoretical basis for restraining the negative effect of high concentrate and rational use of buffers in production. |
PDF Full Text Request