Hongyang Wei

Soybean oil and linseed oil supplementation affect profiles of ruminal microorganisms in dairy cows.

The objective of this study was to evaluate changes in ruminal microorganisms and fermentation parameters due to dietary supplementation of soybean and linseed oil alone or in combination. Four dietary treatments were tested in a Latin square designed experiment using four primiparous rumen-cannulated dairy cows. Treatments were control (C, 60 : 40 forage to concentrate) or C with 4% soybean oil (S), 4% linseed oil (L) or 2% soybean oil plus 2% linseed oil (SL) in a 4 × 4 Latin square with four periods of 21 days. Forage and concentrate mixtures were fed at 0800 and 2000 h daily. Ruminal fluid was collected every 2 h over a 12-h period on day 19 of each experimental period and pH was measured immediately. Samples were prepared for analyses of concentrations of volatile fatty acids (VFA) by GLC and ammonia. Counts of total and individual bacterial groups (cellulolytic, proteolytic, amylolytic bacteria and total viable bacteria) were performed using the roll-tube technique, and protozoa counts were measured via microscopy in ruminal fluid collected at 0, 4 and 8 h after the morning feeding. Content of ruminal digesta was obtained via the rumen cannula before the morning feeding and used immediately for DNA extraction and quantity of specific bacterial species was obtained using real- time PCR. Ruminal pH did not differ but total VFA (110 v. 105 mmol/l) were lower (P < 0.05) with oil supplementation compared with C. Concentration of ruminal NH3-N (4.4 v. 5.6 mmol/l) was greater (P < 0.05) due to oil compared with C. Compared with C, oil supplementation resulted in lower (P < 0.05) cellulolytic bacteria (3.25 × 108 v. 4.66 × 108 colony-forming units (CFU)/ml) and protozoa (9.04 × 104 v. 12.92 × 104 cell/ml) colony counts. Proteolytic bacteria (7.01 × 108 v. 6.08 × 108 CFU/ml) counts, however, were greater in response to oil compared with C (P < 0.05). Among oil treatments, the amount of Butyrivibrio fibrisolvens, Fibrobacter succinogenes and Ruminococcus flavefaciens in ruminal fluid was substantially lower (P < 0.05) when L was included. Compared to C, the amount of Ruminococcus albus decreased by an average of 40% regardless of oil level or type. Overall, the results indicate that some ruminal microorganisms, except proteolytic bacteria, are highly susceptible to dietary unsaturated fatty acids supplementation, particularly when linolenic acid rich oils were fed. Dietary oil effects on ruminal fermentation parameters seemed associated with the profile of ruminal microorganisms.

In vitro culture and characterization of a mammary epithelial cell line from Chinese Holstein dairy cow.

Background The objective of this study was to establish a culture system and elucidate the unique characteristics of a bovine mammary epithelial cell line in vitro. Methodology Mammary tissue from a three year old lactating dairy cow (ca. 100 d relative to parturition) was used as a source of the epithelial cell line, which was cultured in collagen-coated tissue culture dishes. Fibroblasts and epithelial cells successively grew and extended from the culturing mammary tissue at the third day. Pure epithelial cells were obtained by passages culture. Principal Findings The strong positive immunostaining to cytokeratin 18 suggested that the resulting cell line exhibited the specific character of epithelial cells. Epithelial cells cultured in the presence of 10% FBS, supraphysiologic concentrations of insulin, and hydrocortisone maintained a normal diploid chromosome modal number of 2n = 60. Furthermore, they were capable of synthesizing β-casein (CSN2), acetyl-CoA carboxylase-α (ACACA) and butyrophilin (BTN1A1). An important finding was that frozen preservation in a mixture of 90% FBS and 10% DMSO did not influence the growth characteristics, chromosome number, or protein secretion of the isolated epithelial cell line. Conclusions The obtained mammary epithelial cell line had normal morphology, growth characteristics, cytogenetic and secretory characteristics, thus, it might represent an useful tool for studying the function of Chinese Holstein dairy cows mammary epithelial cell (CMECs).

Ratio of lysine to methionine alters expression of genes involved in milk protein transcription and translation and mTOR phosphorylation in bovine mammary cells

This study was conducted to determine the optimum ratio of lysine and methionine (Lys:Met) to enhance milk protein concentration in vitro, focusing on the regulation of genes related to the JAK2-STAT5 and the mammalian target of rapamycin (mTOR) signaling pathways. A preliminary dose response study revealed that casein concentration peaked (2.5-2.7 ppm) at a supplemental Lys concentration of 1.2 mM and Met at 0.5 mM. At the peak casein concentration cell proliferation rate also was higher. Furthermore, the expression of CSN1S1, CSN1S2, CSN2, CSN3, LALBA, JAK2, STAT5, and MTOR was upregulated with both Lys and Met compared with the control. A subsequent experiment was conducted as a 5 × 3 factorial design with supplemental Lys plus Met at different ratios. When the supplemental concentration of Lys was 1.2 mM and Met was 0.4 mM (∼3:1), the concentration of casein peaked. Therefore, we measured gene expression, mTOR protein expression, and phosphorylated mTOR (p-mTOR) in cultures incubated with 3:1 Lys:Met (Lys&Met). Expression of CSN1S1 and LALBA were the most highly expressed genes (P < 0.01). The upregulation of CSN2, CSN3, CSN1S2 isoforms (P < 0.01) and JAK2, ELF5, mTOR (P < 0.05) was also observed. Total mTOR protein expression was greater (P < 0.05) with Lys alone and also Lys&Met. However, Lys&Met resulted in the greatest (P < 0.05) p-mTOR. Results suggest that peak concentration of casein at a supplemental 3:1 Lys:Met is driven in part via upregulation of the mRNA expression of components of the JAK-STAT and mTOR pathways.

Effect of incremental levels of fish oil supplementation on specific bacterial populations in bovine ruminal fluid

The objective of this study was to determine the effects of incremental replacement of dietary linoleic acid by >20-carbon polyunsaturated fatty acids (PUFA) on changes in population of ruminal micro-organisms associated with fibre digestion and biohydrogenation using real-time PCR of bacterial 16S rRNA sequences. Four beef steers with ruminal cannulas were randomly assigned to control (CK, 65:35 forage to concentrate), CK with 3% sunflower oil plus 1% fish oil (S3F1), 2.5% sunflower oil plus 1.5% fish oil (S2.5F1.5) or 2% sunflower oil plus 2% fish oil (S2F2) in a 4 × 4 Latin square design with 21-day periods. Ruminal fluid was collected on day 15 of each period. Compared with CK, oil addition led to lower ruminal acetate and butyrate but greater propionate concentration. DNA copy number of Anaerovibrio lipolytica in ruminal fluid was greater with oil (average 5.38 vs. 3.62 × 10(5) DNA copy number), particularly with S2F2 relative to CK. Fibrobacter succinogenes and Butyrivibrio fibrisolvens DNA copy number decreased by 74% (1.06 vs. 4.01 × 10(5)) and 39% (5.16 vs. 8.42 × 10(7)) in response to S2F2 compared with CK. DNA copy numbers of Ruminococcus flavefaciens and Ruminococcus albus were not affected by incremental fish oil. Results suggest that greater availability of PUFA with >20 carbons (i.e. eicosapentaenoic acid and docosahexaenoic acid) promoted changes in bacterial populations that are relevant for fibre digestion and biohydrogenation.

Influence of linolenic acid content on the oxidation of milk fat.

Increasing the content of alpha-linolenic acid in milk fat might be desirable to meet consumer concerns about dietary healthfulness. However, the rich content of polyunsaturated fatty acids (PUFAs) will influence the oxidative stability of milk fat. This experiment was carried out to determine the effects of infusion with different amounts of high-linolenic perilla fatty acid (HLPFA) emulsion into the duodenum of dairy cows on milk fatty acid profile and the susceptibility of milk fat to oxidation. In a crossover design, 4 multiparous Holstein cows were infused duodenally with increasing amounts (0, 40, 80, 120, or 160 g/day) of free fatty acids from HLPFA emulsion or with carrier alone. Continuous infusions (20 to 22 h/day) were for 7 days at each amount. Infusions were homogenates of HLPFA with 15 g/day of xanthan gum, 5 g/day sodium alginate, and 25 g/day Tween 80; controls received carrier only. The concentration of n-3 PUFAs, especially alpha-linolenic acid, in milk fat increased linearly as HLPFA infusion increased, but the saturated fatty acids decreased linearly. The milk production and the activity of superoxide dismutase, glutathione peroxidase, and catalase in milk tended to decrease quadratically. The milk fat percentage, however, tended to increase. The concentration of malondialdehyde increased quadratically in milk fat. Results suggest that infusion with HLPFA emulsion at varying amounts enhanced the content of n-3 PUFAs in milk fat over the length of experiment but decreased the oxidative stability of milk fat.

Identification of Bovine Casein Phosphorylation Using Titanium Dioxide Enrichment in Combination with Nano Electrospray Ionization Tandem Mass Spectrometry

Abstract Protein phosphorylation is an important post-translational modification that regulates milk protein structure and function. The objective of this study was to analyze the presence of phosphorylated casein. Bovine milk proteins were first separated by SDS polyacrylamide gel electrophoresis. After in gels digestion and extraction, phosphorylated peptides were enriched by titanium dioxide and identified by ultra performance liquid chromatography coupled with nano electrospray ionization tandem mass spectrometry. This method ensured the identification of 20 phosphorylated peptides, including 7 phosphorylated forms of α s1 -casein, 8 α s2 -casein, and 5 β-casein. Eight phosphorylated sites derived from 3 α s1 -caseins, 3 α s2 -caseins, and 2 β-caseins were also identified, and localized on residues Ser 61 , Ser 63 and Ser 130 in α s1 -casein; Thr 145 , Ser 146 and Ser 158 in α s2 -casein; and Ser 50 and Thr 56 in β-casein. These findings provide valuable information for investigating casein phosphorylation of the bovine milk.

Development and Application of a PCR Approach for Detection of Bovis, Sheep, Pig, and Chicken Derived Materials in Feedstuff

A PCR method for detection of bovis, sheep, pig, and chicken derived materials in feedstuff was established, and the existing method was improved according to the research on general primer and species-specific primers. First, general primer designed according to 16S rRNA gene sequence of bovis, sheep, pig, chicken, fish, and horse mtDNA was used for primary detection of animal derived materials in feedstuff. Species-specific primers designed according to conserved sequence of mtDNA of bovis, sheep, pig, and chicken were used for amplification of a 271, 274, 149, and 266 bp fragment, respectively. Further confirmation of the detection result was then carried out. PCR method for detection of animal derived materials in unknown feedstuff was developed by using general primer, relevant PCR system, and PCR condition. Also a PCR method for detection of each species (bovines, sheep, pig, and chicken) was designed by using our species- specific primers. High sensitivity and specificity of our method were confirmed with a minimum detection level of 0.1%. Method for detection of animal derived materials in this research is not only cheap and easy for operation but also precise and reliable results can be obtained. It could be one of the effective methods for the detection of animal derived materials in feedstuff.

A fluorescence-based Coomassie Blue protocol for two-dimensional gel-based proteomics.

A sensitive and convenient “visible SYPRO” staining protocol was developed for visualizing proteins after SDS-PAGE. Gels were sensitized with SYPRO Ruby and then stained with the Coomassie Brilliant Blue G-250 protocol (Blue Silver). This combined protocol had similar or better linearity than staining with only SYPRO Ruby or Blue Silver, respectively. In addition, this method was more sensitive than that of Blue Silver, simpler than that of SYPRO Ruby, and compatible with subsequent mass spectrometry analysis.

The Effect of Dietary Vegetable Oilseeds Supplement on Fatty Acid Profiles in Milk Fat from Lactating Dairy Cows

To determine the effect of dietary supplementation with veetab1e oilseeds on the composition of bovine milk fatty acids (FAs), 40 Holstein dairy cows were used with a complete randomized design. At the beginning of the experiment, the cows were 150±25 day in milk (DIM). Total duration of the experiment was six weeks. Measurements were made during the last three weeks. Cows in four treatments were fed with a basal diet (CT) or basal diet supplemented with either whole full fat soybean (WFS), full fat expanded soybean (FPS) or whole full fat soybean with whole cottonseed and full fat expanded soybean (MIX). The composition of the milk fat was analyzed by gas chromatography. Relative to control, the conjugated linoleic acid (CLA) concentration in milk fat from cows on FPS was significantly increased by 83.88% (P＜0.05). The proportions of C 12:0 were decreased by 35.7, 35.51, and 38.65% in milk fat from cows on WFS, MIX, and FPS compared with cows on CT. Similar decreases in C 14:0 were 23.83, 24.85, and 31.48% in WFS, MIX, and FPS treatments, respectively. Feeding vegetable oilseeds increased the proportion of healthy FAs (mainly CLA), whereas decreased the concentration of C 12:0 and C 14:0. Therefore, milk and dairy products would have higher nutritive and therapeutic value.