Application Of Selenium-Enriched Probiotics In Dairy Cattle Production And Its Mechanism

Selenium (Se) is an essential trace element for animals and humans. Se has many biological functions including enhancing the antioxidative ability, immune function, reproductive performance, and reducing the risk of diabetes, cardiovascular disease, certain types of cancer and resisting to detoxification from some heavy metals. Se is incorporated into selenoproteins as selenocysteine and exerts its biological functions. Expression and activity of selenoenzymes are regulated by Se. Probiotics possess many beneficial effects including improving gastrointestinal flora, resisting to infection of enteropathogenic bacteria, regulating immune function, increasing availability of diets and promoting growth. Precious studies have suggested that organic forms of Se (e.g., Se-enriched yeast, which contains selenomethionine) are less toxic than inorganic forms of Se such as sodium selenite, and the bioavailability of organic Se is relatively high. The preparation of Se-enriched Probiotics was made in our lab and used for nutritional supplementation in diets, which can exert dual effects of organic Se and probiotics at the same time. Invention patent for the preparation was awarded by State Intellectual Property Office of The P.R.C. The present study was conducted to evaluate the effects of supplementary Se-enriched probiotics on transform of Se, antioxidative ability, milk production and somatic cell count (SCC) in dairy cattle, and to explore its mechanism at the cellular and molecular levels, so that to provide a theoretical basis for application of Se-enriched probiotics in dairy cattle industry.Experiment 1. Effect of supplementary selenium-enriched probiotics on absorption and transform of selenium in dairy cattleThirty-six multiparous Holstein cows were divided into four groups, as similar as possible with respect to expected calving date, parity, the previous year's lactation averages, age and body condition score. The control group received a basal diet. The selenium-enriched probiotics (Se-Pro) group received a basal diet plus 108.2 g of Se-Pro/d per cow (mean 5 mg of Se and 320.27x107 cfu/d per cow). The probiotics (Pro) group received a basal diet plus 20.8 g of Pro/d per cow (mean 320.32×107 cfu/d per cow). The sodium selenite (Na2SeO3) group received a basal diet plus 5 ml of Na2SeO3 solution/d per cow (mean 5 mg of Se/d per cow). Experiment period was from d 30 prepartum to 3 mo postpartum. Blood samples were collected for analysis of whole blood selenium (Se) concentrations on d 0,30,60,90 and 120. Milk samples were collected for analysis of milk Se concentrations on d 2,30,60 and 90 of postpartum. Se-Pro group and Na2SeO3 group had significantly higher Se concentrations in whole blood than did control group and Pro group (P<0.05) on d 30,60,90 and 120, respectively. Se-Pro group and Na2SeO3 group had significantly higher Se concentrations in colostrum and milk than did control group and Pro group (P<0.05) on d 2,30,60 and 90 of postpartum, respectively. The Se concentrations of Se-Pro group in whole blood and milk were significantly higher than that of Na2SeO3 group (P<0.05), respectively. The Se concentrations in whole blood, colostrum and milk were not different between Pro group and control group (P>0.05). The results suggest that the effects of Se-Pro are greater than that of Na2SeO3 on increase of Se concentrations in whole blood, colostrum and milk.Experiment 2. Effect of supplementary selenium-enriched probiotics on antioxidative ability of dairy cattleExperiment design is the same as the experiment 1. Experiment period was from d 30 prepartum to 3 mo postpartum. Blood samples were collected for analysis of erythrocyte glutathione peroxidase (RBC GPx1) activity, serum GPx3 activity and plasma Malondialdehyde (MDA) concentrations on d 0,30,60,90, and 120. Se-Pro group and Na2SeO3 group had significantly higher RBC GPx1 activities than did control group and Pro group (P<0.05) on d 90 and 120, respectively, while there were no differences between Pro group and control group (P>0.05). The serum GPx3 activities were not different between the treatment groups and the control group in the whole experiment period (P >0.05). The plasma MDA concentrations in Se-Pro group and Na2SeO3 group were significantly lower than that in control group and Pro group (P<0.05) on d 30,60,90 and 120, respectively. Furthermore, the plasma MDA concentrations in Se-Pro group were significantly lower than that in Na2SeO3 group (P<0.05) on d 30,60,90 and 120, respectively. The results suggest that the effects of Se-Pro are greater than that of Na2SeO3 on increase of antioxidative ability and on reduction of plasma MDA concentrations in dairy cattle. Experiment 3. Effect of supplementary selenium-enriched probiotics on milk production and somatic cell count in dairy cattleExperiment design is the same as the experiment 1. Daily milk yields were determined once monthly after calving. Milk samples were collected for analysis of milk component (fat, protein, and lactose) and somatic cell count (SCC) on d 30,60 and 90 of postpartum. Mean daily milk yield (expressed as 4% fat-corrected milk) for the first 3 mo of lactation was 2.96,1.64 and 0.69 kg-d-'higher in Se-Pro group, Pro group and Na2Se03 group than in control group, respectively. Milk fat percentage and protein percentage were not affected by supplementation of Se-Pro, Pro or Na2SeO3. Milk lactose percentage in Se-Pro group was significantly higher than that in Na2SeO3 group and control group (P< 0.05), but not different when compared with Pro group (P> 0.05). Mean SCC in milk in Se-Pro group was significantly lower than that in control group (P< 0.05), but not different when compared with Na2Se03 group and Pro group (P> 0.05), respectively. Mean SCC from low to high ranking was Se-Pro group< Na2SeO3 group< Pro group< control group. The results suggest that (i) there is a trend for Se-Pro to increase milk yield, (ii) milk fat percentage and protein percentage are not affected by Se-Pro supplementation, whereas milk lactose percentage is significantly increased by Se-Pro supplementation and (iii) the effects of Se-Pro are greater than that of Na2SeO3 and Pro on reduction of milk SCC.Experiment 4. Development of the method for isolation and primary culture of bovine hepatocytesIn the present study, collagenase perfusion method was used to isolate the bovine hepatocytes of caudate lobe obtained from a neonatal calf serum factory. Total cell count and survival rate of freshly isolated hepatocytes were determined by trypan blue exclusion. Hepatocytes were seeded onto a cattle tail collagen coated 6-well microplate at a density of 1×106cells/well. Morphological assessment of hepatocyte monolayers and determination of LDH activity in the culture supernatant were performed. The results showed that the cell yield was 3.558±0.228×108 hepatocytes per caudate lobe. The survival rate of freshly isolated hepatocytes was 84.46%±3.56%. The optimal growth state of hepatocyte monolayers was observed during the 24-72 h culture period, which was applicable in studies relating to hepatocytes metabolism, toxicosis, and gene expression.Experiment 5. Development of the method for determination of GPx1, GPx4, and D1 mRNA levels in bovine hepatocytes by real-time PCRAccording to the published mRNA sequences of gene ofβ-actin, GPxl, GPx4, and D1, four pairs of specific primers were designed by biological software and synthesized. Oligo dTs were used as primer and first strand cDNAs were synthesised from total RNA, which was isolated from bovine hepatocytes. Using the synthesised cDNAs as template, four target fragments were amplified by polymerase chain reaction (PCR), respectively. The PCR products were electrophoresed on 2% agarose gels and extracted. The extracted PCR products were cloned into pMD18-T Vector and sequenced. The copies of extracted plasmids were measured and serially diluted by 10-folds. The diluted plasmids were used as standard templates and real-time PCRs were performed. In addition, the contents and annealing temperatures of four pairs of specific primers were optimized, respectively. The results showed that one target band was amplified for each specific primer pair and consistent with the fact. The optimal contents and annealing temperatures of four primer pairs were 10μmol·L-1 and 62℃, respectively. Amplification efficiencies of four primer pairs for gene ofβ-actin, GPx1, GPx4, and D1 were 91.99%,96.04%,96.51%, and 91.61%, respectively. The melting curve analysis showed only one peak for each PCR product. These results indicate that the method for determination GPx1, GPx4 and D1 mRNA levels in bovine hepatocytes by real-time PCR is successfully developed.Experiment 6. Regulation of cellular glutathione peroxidase by different forms and concentrations of selenium in primary cultured bovine hepatocytesPrimary cultured bovine hepatocyte monolayers derived from neonatal Holstein male calves (aged 1-2 d) were incubated for 24 h with 0 (control),0.5,1,1.5,2,3,4, or 5μmol·L-1 Se as DL-selenomethionine (Se-Met), sodium selenite (Na2SeO3), or Kappa-selenocarrageenan (Se-Car). Compared to controls, significantly lower lactic dehydrogenase (LDH) release was observed at 0.5-5μmol·L-1 Se-Met,0.5-1μmol·L-1 Na2SeO3 and 0.5μmol·L-1 Se-Car, but significantly higher LDH release was observed at 2-5μmol·L-1 Na2SeO3 and 3-5μmol·L-1 Se-Car in a dose-dependent manner. Intracellular reduced glutathione (GSH) contents in all Se-treated hepatocytes were significantly lower than controls. Significant increases of GPxl mRNA level were obtained in all Se-treated hepatocytes, with maximal effects at 3μmol·L-1 Se-Met,1.5μmol·L-1 Na2SeO3 and 2μmol·L-1 Se-Car, respectively. Furthermore,3μmol·L-1 Se from Se-Met resulted in the peak GPx1 mRNA level in all groups treated with Se. After reaching a maximal increase, higher Se supplementation led to reduction of GPxl mRNA level. GPx1 activity showed similar patterns with less magnitude. We conclude that (i) regulation of GPx1 mRNA level and activity by different Se forms are different in primary cultured bovine hepatocytes, (ⅱ) the optimal concentrations of different Se forms are various, which support the full expression of GPx1 in bovine hepatocytes and (ⅲ) the effects of Se-Met are best.Experiment 7. Effects of different forms and concentrations of selenium on mRNA expression of phospholipid hydroperoxide glutathione peroxidase and typeⅠiodothyronine deiodinase in primary cultured bovine hepatocytesPrimary cultured bovine hepatocyte monolayers derived from neonatal Holstein male calves (aged 1-2 d) were incubated with 0 (control),0.5,1,1.5,2,3,4, or 5μmol·L-1 Se as DL-selenomethionine (Se-Met), sodium selenite (Na2SeO3), or Kappa-selenocarrageenan (Se-Car) for 24 h. Compared to controls, significantly lower GPx4 mRNA was observed in all Se-treated hepatocytes, whereas significantly higher D1 mRNA was observed in all Se-treated hepatocytes. The maximal increases of D1 mRNA in the hepatocytes treated with different Se forms were observed at 1.5μmol·L-1 Se-Met,1μmol·L-1 Na2SeO3 and 1μmol·L-1 Se-Car, respectively. Furthermore,1.5μmol·L-1 Se from Se-Met resulted in the peak D1 mRNA level in all Se-treated hepatocytes. After reaching a maximal increase, higher concentration of Se as Se-Met, Na2SeO3, or Se-Car led to reduction of D1 mRNA in a dose-dependent manner. The data suggest that (ⅰ) Se concentration required for full expression of GPx4 mRNA in bovine hepatocytes is relatively low (speculated<0.5μmol/L), (ⅱ) regulation of D1 mRNA by different Se forms is different in bovine hepatocytes, (ⅲ) the optimal concentrations of different Se forms are various, which support the full expression of D1 in bovine hepatocytes and (iiii) the effects of Se-Met are best.