Dengpan Bu

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.

Factors Affecting the Lactoferrin Concentration in Bovine Milk

Lactoferrin (LF) concentrations in the milk with different levels of the somatic cell count score were examined using an ELISA to determine whether milk LF concentration is influenced by parity of the cow, stage of lactation, and the somatic cell count. The study animals were 198 Chinese Holstein cows randomly chosen from more than 1,600 cows in 4 dairy farms in the Beijing area. The cows had shown no sign of mastitis for 2 mo. Daily milk production was recorded, and milk samples were taken from individual cow samples. The LF concentration varied between 31.78 and 485.63 microg/mL in milk from normal animals. Lactoferrin was significantly associated with stage of lactation (r = 0.557) and daily milk production (r = -0.472). Nevertheless, there was no significant relationship with parity. Moreover, milk LF concentration tended to be correlated with the somatic cell count score (r = 0.375). This finding suggests that milk LF may be helpful as an indicator for intramammary infection in dairy cows.

MicroRNA expression patterns in the bovine mammary gland are affected by stage of lactation.

The objective of this work was to determine the expression pattern of microRNA (miR) associated with cellular proliferation, lipid metabolism, and innate immunity in dairy cow mammary gland tissue at different stages of lactation. The expression of miR-10a, miR-15b, miR-16, miR-21, miR-31, miR-33b, miR-145, miR-146b, miR-155, miR-181a, miR-205, miR-221, and miR-223 was studied by real-time reverse-transcription PCR in tissue (n=7/stage) harvested via repeated biopsies during the dry period (-30 d prepartum), the fresh period (7 d postpartum), and early lactation (30 d postpartum). Except for miR-31, all miR studied increased in expression between the dry and fresh periods. Among those upregulated, the expression of miR-221 increased further at early lactation, suggesting a role in the control of endothelial cell proliferation or angiogenesis, whereas the expression of miR-223 decreased at early lactation but to a level that was greater than in the dry period, suggesting it could play a role in the mammary response to pathogens soon after parturition. The expression of miR-31, a hormonally regulated miR that inhibits cyclin gene expression, was greater at early lactation compared with the dry period. From a metabolic standpoint, the consistent upregulation of miR-33b during early lactation compared with the dry period suggests that this miR may exert some control over lipogenesis in mammary tissue. Overall, results indicate that expression of miR associated with transcriptional regulation of genes across diverse biological functions is altered by stage of lactation. The specific roles of these miR during lactation will require further research.

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).

Proteomic Analysis of Cow, Yak, Buffalo, Goat and Camel Milk Whey Proteins: Quantitative Differential Expression Patterns

To aid in unraveling diverse genetic and biological unknowns, a proteomic approach was used to analyze the whey proteome in cow, yak, buffalo, goat, and camel milk based on the isobaric tag for relative and absolute quantification (iTRAQ) techniques. This analysis is the first to produce proteomic data for the milk from the above-mentioned animal species: 211 proteins have been identified and 113 proteins have been categorized according to molecular function, cellular components, and biological processes based on gene ontology annotation. The results of principal component analysis showed significant differences in proteomic patterns among goat, camel, cow, buffalo, and yak milk. Furthermore, 177 differentially expressed proteins were submitted to advanced hierarchical clustering. The resulting clustering pattern included three major sample clusters: (1) cow, buffalo, and yak milk; (2) goat, cow, buffalo, and yak milk; and (3) camel milk. Certain proteins were chosen as characterization traits for a given species: whey acidic protein and quinone oxidoreductase for camel milk, biglycan for goat milk, uncharacterized protein (Accession Number: F1MK50 ) for yak milk, clusterin for buffalo milk, and primary amine oxidase for cow milk. These results help reveal the quantitative milk whey proteome pattern for analyzed species. This provides information for evaluating adulteration of specific specie milk and may provide potential directions for application of specific milk protein production based on physiological differences among animal species.

Transfer efficiency of melamine from feed to milk in lactating dairy cows fed with different doses of melamine.

This study was conducted to investigate the transfer efficiency of melamine (1,3,5-triazine-2,4,6-triamine) from feed to milk of lactating cows fed with different doses of melamine. Twenty-four China Holstein dairy cows were divided into 2 blocks according to milk yield (block 1 and block 2 for low- and high-producing cows). Cows of block 1 or block 2 each were randomly assigned to 1 of 4 treatments in a randomized complete block design and each treatment had 6 cows. The cows of treatments 1 to 4 were dosed with melamine at 0 (control), 90 (treatment 1), 270 (treatment 2), and 450 (treatment 3) mg/d per cow, respectively. The trial lasted 19 d. During the first 13 d, cows were fed melamine at the respective treatment levels, and the last 6 d was the clearance period after melamine was withdrawn. The results indicated that the levels of melamine used did not affect milk yield or composition. The mean milk melamine concentration increased during the initial 3 d after melamine feeding in all the melamine-supplemented groups, and then fluctuated slightly over the remaining 10 d of melamine feeding. No melamine was detected in the milk of any groups on d 4 of the clearance period. Milk melamine concentration measured between 3 to 13 d was significantly affected by melamine feeding doses, but was not influenced by milk yield. The transfer efficiency of melamine from feed to milk was not affected by melamine doses (0.95, 0.70, and 0.66% for treatments 1, 2, and 3, respectively), but was linearly related with milk yield (0.56% for block 1 and 0.95% for block 2, R(2)=0.80). The milk melamine concentration was linearly related with melamine intake (R(2)=0.84). The present study demonstrated that when the daily intake of melamine exceeds 312.7mg/cow, the milk should not be used to produce infant formula powder.

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.

Effects of Arginine Concentration on the In Vitro Expression of Casein and mTOR Pathway Related Genes in Mammary Epithelial Cells from Dairy Cattle

Arginine (Arg) is a conditionally-essential amino acid that is taken up by bovine mammary gland in excess of its output in milk. In this study we evaluated the effects of Arg concentration on the expression of casein and signaling pathway-related genes in mammary epithelial cells. The treatments (applied for 24 h) were designed to be devoid of Arg 0X (control; 0.00 mg/L), resemble the profile of Arg in casein (Arg 1X; 278.00 mg/L), be deficient [Arg 0.25X (69.50 mg/L) and Arg 0.5X (139.00 mg/L)], or be in excess of the amount in casein [Arg 2X (556.00 mg/L), Arg 4X (1,112 mg/L), and Arg 8X (2,224 mg/L)]. The expression of CSN1S, CSN3 and mTOR in the experimental groups was higher than those of the control group (P<0.05). Except for Arg 0.25X and Arg 8X (P>0.05), the expression of CSN1S2, CSN2 and JAK2 in other experimental groups was higher (P<0.05) than those in the control group. Except for Arg 8X (P>0.05), the expression of STAT5 in the other experimental groups was higher than those of the control (P<0.05). It also was observed that except for Arg 0.5X, the S6K expression was higher in other experimental groups than the control (P<0.05). In contrast, except for Arg 0.25X the other experimental groups resulted in lower 4EBP1 expression than the control (P<0.05). Among groups, the expression of CSN1S1, CSN1S2, CSN2, CSN3, JAK2, STAT5, mTOR and S6K gene was highest with Arg 2X (P<0.05); the reverse was true for 4EBP1 gene, with the lowest expression in this group (P<0.05). Taken together, Arg appears to play an important role in the transcriptional regulation of casein genes and mTOR-related genes in bovine mammary epithelial cells.

Effects of rumen-protected γ-aminobutyric acid on performance and nutrient digestibility in heat-stressed dairy cows

This experiment was conducted to investigate the effects of rumen-protected γ-aminobutyric acid (GABA) on performance and nutrient digestibility in heat-stressed dairy cows. Sixty Holstein dairy cows (141±15 d in milk, 35.9±4.3kg of milk/d, and parity 2.0±1.1) were randomly assigned to 1 of 4 treatments according to a completely randomized block design. Treatments consisted of 0 (control), 40, 80, or 120mg of true GABA/kg of dry matter (DM). The trial lasted 10wk. The average temperature-humidity indices at 0700, 1400, and 2200h were 78.4, 80.2, and 78.7, respectively. Rectal temperatures decreased linearly at 0700, 1400, and 2200h with increasing GABA concentration. Supplementation of GABA had no effect on respiration rates at any time point. Dry matter intake, energy-corrected milk, 4% fat-corrected milk, and milk fat yield tended to increase linearly with increasing GABA concentration. Supplementation of GABA affected, in a quadratic manner, milk protein and lactose concentrations, and milk protein yield, and the peak values were reached at a dose of 40mg of GABA/kg. Milk urea nitrogen concentration responded quadratically. Total solids content increased linearly with increasing GABA concentration. Supplementation of GABA had no effect on milk yield, lactose production, total solids, milk fat concentration, somatic cell score, or feed efficiency. Apparent total-tract digestibilities of DM, organic matter, crude protein, neutral detergent fiber, and acid detergent fiber were similar among treatments. These results indicate that rumen-protected GABA supplementation to dairy cows can alleviate heat stress by reducing rectal temperature, increase DM intake and milk production, and improve milk composition. The appropriate supplemental GABA level for heat-stressed dairy cows is 40mg/kg of DM.

Identification and characterization of microRNA sequences from bovine mammary epithelial cells.

The bovine mammary gland is composed of various cell types including bovine mammary epithelial cells (BMEC). The use of BMEC to uncover the microRNA (miRNA) profile would allow us to obtain a more specific profile of miRNA sequences that could be associated with lactation and avoid interference from other cell types. The objective of this study was to characterize the miRNA sequences expressed in isolated BMEC. The miRNA were identified by Solexa sequencing technology (Illumina Inc., San Diego, CA). Furthermore, novel miRNA were uncovered by stem-loop reverse transcription-PCR and sequencing of PCR products. To detect tissue specificity, expression of novel miRNA sequences was measured by stem-loop RT-PCR and sequencing of PCR products in mammary gland, liver, adipose, ileum, spleen and kidney tissue from 3 lactating Holstein cows (50±10 d postpartum). After bioinformatics analysis, 12,323,451 reads were obtained by Solexa sequencing, of which 11,979,706 were clean reads, matching the bovine genome. Among clean reads, 9,428,122 belonged to miRNA sequences. Further analysis revealed that the miRNA bta-mir-184 had the most abundant expression, and 388 loci possessed the typical stem-loop structures matching known miRNA hairpins. In total, 38 loci with novel hairpins were identified as novel miRNA and were numbered from bta-U1 to bta-U38. One novel miRNA (bta-U21) was specific to mammary gland. Seven novel miRNA, including bta-U21, had tissue-restricted distribution. Uncovering the specific roles of these novel miRNA during lactation appears warranted.