Cytokines Screening And Its Effect On Milk Fat And Protein Synthesis In Bovine Mammary Epithelial Cells

In China, milk fat and protein content, two important dairy quality indicators, arecurrently lower than developed countries. Recently, dairy safety standards havedecreased from2.95to2.8gram of milk protein content per100-gram milk;developed countries have a minimum standard of3.0gram per100-grams milk. Thecomparative poor quality of milk in China is directly related to the substandard dietgiven to dairy cows. Low milk protein content has become the bottleneck for healthydevelopment of the dairy industry. Milk fat and protein synthesis are influenced bymany factors including, nutritional content of diet, overall health of the cow, hormonelevels, and roughage. China has a shortage of arable land resulting in limited space forgrowth and production of high-quality forage. Crop straw is the common source ofroughage, in dairy farming, and has a low nutritional value. This study will comparethe effect of two different types of roughage by evaluating the subsequent relationshipbetween cytokines in the blood and quality of milk. Understanding the relationshipbetween milk fat and protein synthesis and roughage consumption is imperative toimprove milk conditions in China. Regulating the feed given to dairy cows couldpotentially eliminate the current practices of adulterating milk powder with foreignand harmful additives.Two different diets were fed to Chinese Holstein cows: group A were fed cornstraw (CS), while group B ate alfalfa hay and whole plant corn silage mixed roughage(MF). Analyze the quality of milk, and venous blood was collected from10cow pergroup. Blood serum samples were evaluated using Enzyme-Linked Immuno-SorbentAssay (ELISA) and the concentration of the following factors were determined: serumleptin (LEP), adiponectin (ADPN), prolactin inhibitory factor (PIF), prolactin releasing factor (PRF), interferon gamma （IFNγ）, insulin-like growth factor-1（IGF-1）, transforming growth factor beta1(TGFβ1), corticotropin releasinghormone （CRH） and interleukin2,6,10,12. Radioimmunosorbent assay was used todetermine the concentrations, in blood serum, of the following factors: somatostatin(SS), tumor necrosis factor alpha (TNFα), and growth hormone releasing hormone(GHRH). Finally, serum concentration of prolactin (PRL), cortisol, growth hormone(GH) and insulin were determined using chemiluminescent. There was a significantdifference (p <0.05) for the rate of the milk protein between the two groups.Spearman correlation test was run to determine the relationship between serumconcentrations of hormones and cytokines and the rate of milk protein, and therelationship between TGFβ1, IGF-1, GH and PRL, IFNγ and the rate of milk proteinwere correlative and the serum concentrations of hormones and cytokines have asignificant difference (p <0.05) between the two groups.Bovine mammary epithelial cells (BMECs) were analyzed to establish a lactationmodel. Mammary tissues were explant cultured and BMECs were purified todetermine the relationship between serum cytokine concentration and the resultingmilk fat and protein synthesis. BMECs were cultured for eight hours in serum-freeDMEM/F12. The cultured BMECs were transferred and re-cultured for twenty-fourhours in growth medium supplemented with varying concentrations of the followingfactors: GH (0,2.5,5,7.5,10ng/mL), PRL (0,0.5,2,4,8μg/mL), TGFβ1(0,2.5,5,10,20ng/mL) and IFNγ (0,5,10,20,40ng/mL). Real-time-PCR (qPCR) was used to detectrelative expression levels of β-casein (CSN2) and acetyl coenzyme A carboxylasealpha (ACACA). The results showed that GH and PRL promoted expression of CSN2mRNA. IFNγ and TGFβ1inhibited expression of CSN2mRNA in BMEC. GH had nosignificant effect on ACACA mRNA expression. However, GH and PRL increasedACACA expression, TGFβ1decreased ACACA expression, and IFNγ can eitherincrease or decrease ACACA expression.After establishing a bovine mammary epithelial cells lactation model, this studyexplored the effect of methionine, lysine, or in combination with TGFβ1and IFNγ on milk fat and protein synthesis. Again, BMECs were cultured for8hours in serum-freeDMEM/F12. Cells were then grown for24hours under the following conditions:growth medium (control), growth medium+methionine (30μg/mL), growth medium+lysine (60μg/mL), growth medium+methionine (30μg/mL)+TGF β1(5ng/mL),growth medium+methionine (30μg/mL)+IFNγ(5ng/mL), growth medium+lysine(60μg/mL)+TGF β1(5ng/mL), growth medium+lysine (60μg/mL)+IFNγ (5ng/mL).Total RNA was then extracted from the BMECs and analyzed using qPCR for mRNAexpression of CSN2and ACACA. The results showed that methionine and lysinepromoted CSN2and ACACA mRNA expression in BMEC. When methionine andlysine were present at the appropriate concentrations, IFNγ and TGFβ1did notinhibit milk protein synthesis in BMECs.The results showed that TGFβ1, IGF-1, GH, PRL and IFNγ are associated withthe rate of milk protein and there is a significant difference (p <0.05) directly relatedto nutritional content of dairy cow feed. This study has shown that GH and PRLpromote protein synthesis and that IFNγ and TGFβ1inhibit protein synthesis inBMEC. This study has also determined that methionine and lysine promote milk fatand protein synthesis in BMEC; when combined at the appropriate concentrationsmethionine and lysine will lower the inhibitory effect of IFNγ and TGFβ1on milksynthesis. This study has provided a theoretical basis and an experimental foundationfor safe and effective improvements for dairy quality in China.