Nutrient restriction during late gestation reduces milk yield and mammary blood flow in lactating primiparous beef females.

Fall-calving primiparous beef females [body weight (BW): 451 ±â€…28 (SD) kg; body condition score (BCS): 5.4 ±â€…0.7] were individually-fed 100% (control; CON; n = 13) or 70% (nutrient restricted; NR; n = 13) of estimated metabolizable energy and metabolizable protein requirements from day 160 of gestation to calving. Post-calving, all dams were individually-fed tall fescue hay supplemented to meet estimated nutrient requirements for maintenance, growth, and lactation until day 149 of lactation. Four-hour milk yields were collected on days 21, 42, 63, 84, 105, and 147 of lactation, and milk nutrient composition was determined. Doppler ultrasonography of both pudendoepigastric arterial trunks was conducted every 21 d from days 24 to 108 of lactation. Total mammary blood flow was calculated, and hemodynamics from both sides were averaged. Data were analyzed as repeated measures with nutritional plane, day of lactation, their interaction, calving date, and calf sex (if P < 0.25) as fixed effects. We previously reported that post-calving, NR dams weighed 64 kg less and were 2.0 BCS lower than CON, but calf birth weight was not affected. Milk weight and volume were 15% less (P = 0.04) for NR dams than CON. Milk protein concentration was lower (P = 0.008) for NR dams than CON, but triglyceride and lactose concentrations were not affected (P ≥ 0.20) by nutritional plane. Milk urea N concentration of NR dams tended to be greater (P = 0.07) on day 42 but was lower (P = 0.01) on day 147 of lactation than CON. Total milk protein, triglyceride, and lactose yields were less (P ≤ 0.05) for NR dams than CON. Total milk urea N yield was less (P ≤ 0.03) for NR dams than CON on days 21, 63, and 147 of lactation. Maternal heart rate was greater (P = 0.008), but pudendoepigastric arterial trunk peak systolic velocity, resistance index, and cross-sectional area were less (P ≤ 0.04) and pulsatility index tended to be less (P = 0.06) for NR dams than CON. Mammary blood flow was 19% less (P = 0.004) for NR dams than CON, but mammary blood flow relative to milk weight or dam BW was not affected (P ≥ 0.14) by nutritional plane. Most milk yield, milk nutrient composition, and mammary blood flow variables were affected (P ≤ 0.04) by day of lactation. In summary, first-parity beef females that were nutrient restricted during late gestation and then fed to meet estimated nutrient requirements during lactation had decreased milk nutrient yield and a similar reduction in mammary blood flow.

Mammary development in preparation for lactation is largely complete at the time of calving, and final prepartum mammary growth and differentiation are occurring for the first time in heifers. Nutrient requirements increase substantially during late gestation, resulting in competition for nutrient use among maternal growth, fetal growth, and mammary growth in primiparous beef females. Undernutrition during late gestation can occur due to poor forage nutrient availability or drought, potentially impacting mammary gland development and subsequent milk production. We report that first-parity beef females that were nutrient restricted during late gestation and then fed to meet estimated nutrient requirements during lactation had 15% lower milk yield, reduced milk protein concentration, and less total milk protein, triglycerides, lactose, and urea N available for their calves than controls. Additionally, previously nutrient restricted dams had 19% less total mammary blood flow, and the major arteries supplying the mammary gland were smaller.

Systemic and local responses of cytokines and tissue histology following intramammary lipopolysaccharide challenge in dairy cows.

During bovine mastitis, immune responses include the release of cytokines and the recruitment of leukocytes, resulting in profound structural and functional changes in the mammary gland. Our aims were to delineate systemic and local cytokine responses and to quantify histological changes in the mammary tissue of lactating cows after acute intramammary lipopolysaccharide (LPS) challenge. Ten multiparous dairy cows were paired to either treatment (TRT) or control (CON) groups. For TRT cows, one side of the udder was randomly assigned to receive treatment with LPS (50 µg in 10 mL of saline, TL) into both the front and rear quarters; the contralateral quarters received saline (10 mL). Udder-halves of CON cows were similarly assigned randomly to receive either saline (10 mL, CS) or no infusion (untreated). Temporal changes in the concentrations of 15 cytokines in the blood (0, 3, 6, 12, and 24 h relative to the LPS infusion) and in mammary tissue (0, 3, and 12 h) were determined, as were concomitant changes in mammary histology. The cytokines IL-6, IL-10, MCP-1, and MIP-1ß showed a systemic response as their concentrations were significantly different in the plasma of TRT cows as compared with CON cows after LPS challenge. The cytokines IL-1α, IL-1ß, IL-6, IL-8, IL-17A, IL-36RA, IP-10, MCP-1, MIP-1α, MIP-1ß, TNF-α, and VEGF-A showed a local response in TL glands, and 8 cytokines, IL-1ß, IL-6, IL-10, IL-17A, IL-36RA, IP-10, MIP-1ß, and VEGF-A showed systemic changes in the nonchallenged mammary glands adjacent to LPS-infused glands. Endotoxin challenge evoked changes in the histology of mammary tissue that included a 5.2- and 7.2-fold increases in the number of neutrophils in alveolar lumens at 3 h and 12 h, respectively. In summary, LPS challenge induced specific local and systemic responses in cytokine induction and elicited neutrophil infiltration in bovine mammary tissue.

Effects of copper, zinc, and manganese source and inclusion during late gestation on beef cow-calf performance, mineral transfer, and metabolism.

To determine effects of Cu, Zn, and Mn source and inclusion during late gestation, multiparous beef cows [n = 48; 649 ±â€…80 kg body weight (BW); 5.3 ±â€…0.5 body condition score (BCS)] were individually-fed hay and supplement to meet or exceed all nutrient recommendations except Cu, Zn, and Mn. From 91.2 ±â€…6.2 d pre-calving to 11.0 ±â€…3.2 d post-calving, cows received: no additional Cu, Zn, or Mn (control, CON), sulfate-based Cu, Zn, and Mn (inorganic, ITM) or metal methionine hydroxy analogue chelates (MMHAC) of Cu, Zn, and Mn at 133% recommendations, or a combination of inorganic and chelated Cu, Zn, and Mn (reduce and replace, RR) to meet 100% of recommendations. Data were analyzed with treatment and breeding group (and calf sex if P < 0.25 for offspring measures) as fixed effects, animal as experimental unit, and sampling time as a repeated effect for serum, plasma, and milk measures over time. Post-calving cow liver Cu was greater (P ≤ 0.07) in MMHAC compared with all other treatments. Calves born to RR had greater (P ≤ 0.05) liver Cu than ITM and CON, and MMHAC had greater (P = 0.06) liver Cu than CON. Liver Mn was less (P ≤ 0.08) for RR calves than all other treatments. Calf plasma Zn was maintained (P ≥ 0.15) from 0 to 48 h of age in ITM and MMHAC but decreased (P ≤ 0.03) in CON and RR. Gestational cow BW, BCS, and metabolites were not affected (P ≥ 0.13) by treatment, but gestational serum thiobarbituric acid reactive substances (TBARS) were greater (P = 0.01) for CON than MMHAC. Treatment did not affect (P ≥ 0.13) calf birth size, vigor, placental size and minerals, or transfer of passive immunity. Neonatal calf serum Ca was greater (P ≤ 0.05) for MMHAC than all other treatments; other calf serum chemistry and plasma cortisol were not affected (P ≥ 0.12). Pre-suckling colostrum yield, and lactose concentration and content, were greater (P ≤ 0.06) for MMHAC compared with ITM and RR. Colostral triglyceride and protein concentrations were greater (P ≤ 0.08) for RR than MMHAC and CON. Cow lactational BW and BCS, milk yield and composition, and pre-weaning calf BW and metabolism were not affected (P ≥ 0.13) by treatment. Lactational serum TBARS were greater (P = 0.04) for RR than CON at day 35 and greater (P ≤ 0.09) for MMHAC at day 60 than all other treatments. Source and inclusion of Cu, Zn, and Mn altered maternal and neonatal calf mineral status, but calf size and vigor at birth, passive transfer, and pre-weaning growth were not affected in this study.

Technical note: colorimetric methods for accurate determination of nutrient composition in beef cow colostrum and milk.

Our objectives were to develop colorimetric methods to accurately measure nutrient concentrations of beef cow colostrum and milk, to determine if the yield of colostrum from a single rear quarter is representative of complete collection of colostrum in beef cows, and to compare data from our developed colorimetric methods with Fourier transform infrared spectroscopy (FTIR) analysis to determine the accuracy of FTIR for beef cow colostrum and milk. In Exp. 1, colostral weight and volume of the most full rear quarter were compared with complete collection of colostrum from post-calving, unsuckled beef heifers. Both volume and weight had r2 = 0.85 (P < 0.001) between single-quarter and 4 quarter yields. In Exp. 2, colostrum (n = 35) and milk at d 35 (n = 42) and d 60 (n = 38) of lactation were collected from multiparous, fall-calving, crossbred beef cows. Subsamples were submitted for FTIR analysis and frozen for colorimetric analysis. Colorimetric analyses were developed for lactose, triglycerides (measure of fat), protein, and urea N. To validate method accuracy, spike recoveries were determined for lactose (96.8% average) and milk protein (100.1% average), triglyceride concentration was compared with fat concentration determined by the Mojonnier method (r2 ≥ 0.91, P < 0.001), and colostral or milk urea N was compared with serum urea N from the same sampling day (r2 ≥ 0.72, P < 0.001). Coefficients of determination between colorimetric methods and FTIR were determined for colostrum, d 35 milk, and d 60 milk. Colostral lactose concentration from FTIR was positively associated (r2 = 0.24, P = 0.01) with colorimetric analysis, but there was no relationship (r2 ≤ 0.09, P ≥ 0.14) between methods for colostral fat, protein, or urea N. Milk nutrient composition was positively associated for all nutrients measured at d 35 (r2 = 0.28 to 0.58, P < 0.001), and coefficients of determination strengthened for all nutrients measured at d 60 (r2 = 0.38 to 0.82, P < 0.001). In conclusion, colostrum yield of a single rear quarter can be used to indicate complete collection of colostrum for beef cows, and colorimetric methods developed have adequate accuracy for beef cow colostral and milk nutrient analysis. Based on our analyses, nutrient composition of beef cow colostrum was not accurately analyzed by FTIR. Accuracy of FTIR for beef cow milk varies with component and may be affected by the day of lactation.

The purpose was to develop laboratory methods to measure lactose, fat, protein, and urea nitrogen in beef cow colostrum (first milk) and milk and to validate single-quarter colostrum yield as a predictor of total colostrum yield. Additionally, new methods were compared with Fourier transform infrared spectroscopy (FTIR), which is the primary method of dairy cow milk nutrient analysis. New laboratory methods were determined to be accurate for beef cow colostrum and milk analysis, and single-quarter colostrum yield was successful in predicting total colostrum yield. Overall, our data suggest that beef cow colostrum cannot be accurately analyzed by FTIR instruments, and accuracy of FTIR for beef cow milk analysis varies with nutrients and may be affected by the day of lactation.

Evidence for a Role of Prolactin in Mediating Effects of Photoperiod during the Dry Period.

Photoperiod manipulation during the lactation cycle alters milk yield, with long days (LDPP) increasing yield in lactation and short days (SDPP) in the dry period improving subsequent yield. Circulating prolactin (PRL) is directly related to day length, with LDPP increasing and SDPP decreasing PRL, respectively. Two blocks of 24 multiparous Holstein cows were used during two consecutive years to test the hypothesis that the mammary response to SDPP is the result of decreased concentrations of PRL in the circulation relative to LDPP. Cows were randomly assigned to one of three treatment groups during the dry period: SDPP, LDPP, or SDPP+PRL. Cows were returned to ambient photoperiod at calving and milk yield and DMI recorded for 120 d and 42 d, respectively. Mammary biopsies were obtained to determine rates of [³H]-thymidine incorporation into DNA in vitro. Treatment of SDPP cows with PRL caused a rapid increase in systemic PRL that reached concentrations similar to cows under LDPP. The periparturient PRL surge was similar for LDPP and SDPP+PRL cows, but those groups had greater surge concentrations versus SDPP. Cows exposed to SDPP produced more milk than LDPP cows, and there was a trend for SDPP+PRL cows to produce more milk than LDPP cows. Milk production was inversely related to the periparturient PRL surge. There was a trend for a treatment effect on mammary cell proliferation with greater proliferation in mammary tissue of SDPP cows relative to LDPP or SDPP+PRL on day -20 relative to parturition. Replacement of PRL to cows on SDPP when dry resulted in milk yield intermediate to cows on SDPP or LDPP, supporting the concept of a link between dry period PRL and yield.

Potential regulatory role of microRNAs in the development of bovine gastrointestinal tract during early life.

This study aimed to investigate the potential regulatory role of miRNAs in the development of gastrointestinal tract (GIT) during the early life of dairy calves. Rumen and small intestinal (mid-jejunum and ileum) tissue samples were collected from newborn (30 min after birth; n = 3), 7-day-old (n = 6), 21-day-old (n = 6), and 42-day-old (n = 6) dairy calves. The miRNA profiling was performed using Illumina RNA-sequencing and the temporal and regional differentially expressed miRNAs were further validated using qRT-PCR. Analysis of 16S rRNA gene copy numbers was used to quantify total bacteria, Bifidobacterium and Lactobacillus species. The expression of miR-143 was abundant in all three gut regions, at all time points and it targets genes involved primarily in the proliferation of connective tissue cells and muscle cells, suggesting a role in regulating rapid tissue development during the early life of calves. The expression of miR-146, miR-191, miR-33, miR-7, miR-99/100, miR-486, miR-145, miR-196 and miR-211 displayed significant temporal differences (FDR <0.05), while miR-192/215, miR-194, miR-196, miR-205 and miR-31 revealed significant regional differences (FDR <0.05). The expression levels of miR-15/16, miR-29 and miR-196 were positively correlated with the copy numbers of 16S rRNA gene of Bifidobacterium or Lactobacillus species or both (P<0.05). Functional analysis using Ingenuity Pathway Analysis identified the above mentioned differentially expressed miRNAs as potential regulators of gut tissue cell proliferation and differentiation. The bacterial density-associated miRNAs were identified as modulators of the development of lymphoid tissues (miR-196), maturation of dendritic cells (miR-29) and development of immune cells (miR-15/16). The present study revealed temporal and regional changes in miRNA expression and a correlation between miRNA expression and microbial population in the GIT during the early life, which provides further evidence for another mechanism by which host-microbial interactions play a role in regulating gut development.

Milk yield responses to changes in milking frequency during early lactation are associated with coordinated and persistent changes in mammary gene expression.

BACKGROUND: The lactating mammary gland responds to changes in milking frequency by modulating milk production. This response is locally regulated and, in dairy cows, the udder is particularly sensitive during early lactation. Relative to cows milked twice-daily throughout lactation, those milked four-times-daily for just the first 3 weeks of lactation produce more milk throughout that lactation. We hypothesized that the milk yield response would be associated with increased mammary cell turnover and changes in gene expression during frequent milking and persisting thereafter. Cows were assigned to unilateral frequent milking (UFM; left udder halves milked twice-daily; right udder halves milked four-times daily) on days 1 to 21 of lactation, followed by twice-daily milking for the remainder of lactation. Relative to udder halves milked twice-daily, those milked four-times produced more milk during UFM; the difference in milk yield declined acutely upon cessation of UFM after day 21, but remained significantly elevated thereafter. We obtained mammary biopsies from both udder halves on days 21, 23, and 40 of lactation. RESULTS: Mammary cell proliferation and apoptosis were not affected by milking frequency. We identified 75 genes that were differentially expressed between paired udder halves on day 21 but exhibited a reversal of differential expression on day 23. Among those genes, we identified four clusters characterized by similar temporal patterns of differential expression. Two clusters (11 genes) were positively correlated with changes in milk yield and were differentially expressed on day 21 of lactation only, indicating involvement in the initial milk yield response. Two other clusters (64 genes) were negatively correlated with changes in milk yield. Twenty-nine of the 75 genes were also differentially expressed on day 40 of lactation. CONCLUSIONS: Changes in milking frequency during early lactation did not alter mammary cell population dynamics, but were associated with coordinated changes in mammary expression of at least 75 genes. Twenty-nine of those genes were differentially expressed 19 days after cessation of treatment, implicating them in the persistent milk yield response. We conclude that we have identified a novel transcriptional signature that may mediate the adaptive response to changes in milking frequency.

Onset of lactation in the bovine mammary gland: gene expression profiling indicates a strong inhibition of gene expression in cell proliferation.

The mammary gland undergoes dramatic functional and metabolic changes during the transition from late pregnancy to lactation. To better understand the molecular events underlying these changes, we analyzed expression profiles of approximately 23,000 gene transcripts in bovine mammary tissue about day 5 before parturition and day 10 after parturition. At the cutoff criteria of the signed fold change >or=2 or

Bovine glucose transporter GLUT8: cloning, expression, and developmental regulation in mammary gland.

GLUT8 is a newly identified member of the facilitative glucose transporter family, which characteristically exhibits high-affinity glucose transport activity. The expression of GLUT8 has been shown to depend on gonadotropin secretion in human testes and to be regulated by insulin in the blastocyst. To characterize GLUT8 and investigate its role in normal mammary gland function, we cloned and sequenced the full-length cDNA of bovine GLUT8. The 2073-base-pair cDNA sequence is predicted to encode a protein of 478 amino acids, with a molecular weight of approximately 51 kDa. The deduced amino acid sequence of bovine GLUT8 is 90%, 84%, 84% and 58% identical to human, mouse, rat and chicken GLUT8, and is 26%, 27% and 24% identical to bovine GLUT1, GLUT3 and GLUT4, respectively. Bovine GLUT8 retains the characteristic structural features of GLUT8 proteins previously identified from other species including membrane spanning helices, glucose transporter motifs, an N-linked glycosylation site on loop 9 and a putative dileucine internalization motif. The major in vitro transcription and translation product of bovine GLUT8 cDNA migrated at an apparent molecular weight of 38 kDa similar to the sizes reported for GLUT8 from other mammalian species. In the presence of canine microsomal membranes, the translation product increased to 40 kDa suggesting glycosylation. Transient transfection studies using a FLAG epitope tagged construct in COS-7 cells revealed that bovine GLUT8 is localized to the cytoplasm in non-stimulated conditions. A 2.1-kb GLUT8 mRNA transcript was detected at high levels in bovine testes, at moderate levels in lactating bovine mammary gland, lung, kidney, spleen, intestine and skeletal muscle, and at low levels in bovine liver. GLUT8 mRNA expression in bovine mammary gland increased about 10-fold (P<0.001) during late pregnancy and early lactation, similar to the pattern of change in GLUT1 mRNA and more dramatic than the increase seen in mouse mammary gland. These results suggest that GLUT8 expression may be regulated by lactogenic hormones and that GLUT8 may play a role in glucose uptake in the lactating mammary gland.

Dietary fat regulates mammary stearoyl coA desaturase expression and activity in lactating mice.

Mice in mid lactation (n=6 per group) were fed a control diet (1.19% fat), or diets containing safflower oil (25% w/w) or olive oil (25% w/w) for 7d. Mammary and liver stearoyl CoA desaturase (SCD) mRNA levels and mammary SCD activities were higher in lactating mice fed the control diet than in those fed the oil-supplemented diets. Further, mammary SCD mRNA was directly related to mammary SCD activity. Milk fat composition was influenced by dietary fat composition. The olive oil diet, high in 18:1, led to high levels of this fatty acid in milk and the safflower oil diet, high in 18:2, resulted in a milk fat with high levels of 18:2. These results show that there is regulation of SCD at the transcriptional level, associated with changes in enzyme activity and in milk fat composition.

Hormonal regulation of mammary differentiation and milk secretion.

The endocrine system coordinates development of the mammary gland with reproductive development and the demand of the offspring for milk. Three categories of hormones are involved. The levels of the reproductive hormones, estrogen, progesterone, placental lactogen, prolactin, and oxytocin, change during reproductive development or function and act directly on the mammary gland to bring about developmental changes or coordinate milk delivery to the offspring. Metabolic hormones, whose main role is to regulate metabolic responses to nutrient intake or stress, often have direct effects on the mammary gland as well. The important hormones in this regard are growth hormone, corticosteroids, thyroid hormone, and insulin. A third category of hormones has recently been recognized, mammary hormones. It currently includes growth hormone, prolactin, PTHrP, and leptin. Because a full-term pregnancy in early life is associated with a reduction in breast carcinogenesis, an understanding of the mechanisms by which these hormones bring about secretory differentiation may offer clues to the prevention of breast cancer.