Regulation of gene expression in milk secretory pathways during secretory activation in Holstein cows.

Increased transcription of genes involved in lactose synthesis is a key feature of secretory activation of the mammary gland. We determined which genes are transcriptionally regulated during the transition from colostrum to milk in cows and whether more frequent milking of one udder half would increase milk yields and alter gene expression. We enrolled 12 Holstein cows immediately after calving, harvested colostrum, then randomly assigned one udder half to 12 h (2X) or 6 h (4X) milking intervals for 48 h after first milking. After 48 h, all quarters were milked twice daily until d7, when final quarter milk yields were collected. Yields and composition of colostrum and milk were recorded for each 12 h interval. After each milking, a strip sample of hind milk was collected for isolation of RNA from milk fat and quantification of selected transcripts via qPCR. Milk, milk fat, total protein, and lactose yields increased significantly over the initial 48 h and at d7 after calving. Quarters on 4X treatment produced more milk than the contralateral 2X quarters. Genes upregulated concomitantly with milk yields encoded α-lactalbumin (LALBA), ß-1,4-galactosyltransferase (B4GALT1), fatty acid synthase (FASN), ß casein (CSN2), and folate receptor α (FOLR1). Downregulated genes encoded monosaccharide transporters (SLC2A3, SLC2A8, SLC35A2) and enzymes involved in galactose synthesis (HK1, PGM1, GALE). Three genes were initially downregulated but later upregulated at d7 (LPIN1, SLC2A1, UGP2). Notably, milking frequency had no effect on gene expression. Sequential upregulation first of genes encoding the former enzymes, then of UGP2, may be necessary for copious milk production. However, the local effects of milking frequency on milk production during lactogenesis do not appear to be controlled by transcription of these genes related to lactose synthesis.

Graduate Student Literature Review: Systemic mediators of inflammation during mastitis and the search for mechanisms underlying impaired lactation.

The negative effect of mastitis on lactation is well established, yet the mechanisms causing reduced milk production in the afflicted dairy cow are not. As one of the major inflammatory diseases in the dairy industry, mastitis has rightly received considerable research interest for decades. However, the focus on distinct, pathologic effects in mastitic glands has largely overlooked systemic effects on noninflamed mammary glands. This is particularly evident in the severe, acute response to the potent inflammatory mediator, lipopolysaccharide (LPS). Whereas secretory cell death, impaired tight junctions, and migration of leukocytes are locally restricted to an inflamed, LPS-challenged gland, changes in milk yield and milk components may be detectable in all mammary glands. Further, these differences extend to the mammary transcriptome. Notably, few transcriptomic studies have been designed to test for effects of systemic mediators of inflammation on gene expression. Relevant changes in the noninflamed mammary gland, identified through biochemical analyses and transcriptional studies, warrant further research. Current evidence suggests proinflammatory cytokines play a role in regulating lactose synthesis, but additional candidates and mechanisms continue to be identified. Ultimately, understanding how systemic mediators of inflammation affect mammary function may lead to the development of interventions that enable more efficient milk production without sacrificing the benefits of inflammation.

Hypogalactia in mammary quarters adjacent to lipopolysaccharide-infused quarters is associated with transcriptional changes in immune genes.

Infusion of lipopolysaccharides (LPS) into a mammary gland can provoke inflammatory responses and impair lactation in both the infused gland and neighboring glands. To gain insight into the mechanisms controlling the spatiotemporal response to localized mastitis in lactating dairy cows, we performed RNA sequencing on mammary tissue from quarters infused with LPS, neighboring quarters in the same animals, and control quarters from untreated animals at 3 and 12 h postinfusion. Differences in gene expression were annotated to Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways. Comparing mammary transcriptomes from all 3 treatments revealed 3,088 and 1,644 differentially expressed (DE) genes at 3 and 12 h, respectively. Of these genes, >95% were DE only in LPS-infused quarters and represented classical responses to LPS: inflammation, apoptosis, tissue remodeling, and altered cell signaling and metabolism. Although relatively few genes were DE in neighboring quarters (56 at 3 h; 74 at 12 h), these represented several common pathways. At 3 h, tumor necrosis factor (TNF), nuclear factor-κB, and nucleotide-binding and oligomerization domain (NOD)-like receptor signaling pathways were identified by the upregulation of anti-inflammatory (NFKBIA, TNFAIP3) and cell adhesion molecule (VCAM1, ICAM1) genes in neighboring glands. Additionally, at 12 h, several genes linked to 1-carbon and serine metabolism were upregulated. Some responses were also regulated over time. The proinflammatory response in LPS-infused glands diminished between 3 and 12 h, indicating tight control over transcription to re-establish homeostasis. In contrast, 2 glucocorticoid-responsive genes, FKBP5 and ZBTB16, were among the top DE genes upregulated in neighboring quarters at both time points, indicating potential regulation by glucocorticoids. We conclude that a transient, systemic immune response was sufficient to disrupt lactation in neighboring glands. This response may be mediated directly by proinflammatory factors from the LPS-infused gland or indirectly by secondary factors released in response to systemic inflammatory signals.

Intramammary lipopolysaccharide infusion induces local and systemic effects on milk components in lactating bovine mammary glands.

Each quarter of the bovine mammary gland is an anatomically and functionally distinct gland. However, mastitis in one quarter may affect function of adjacent, uninfected glands. To investigate the mechanisms and potential mediators of these effects, we quantified early responses of the mammary gland to intramammary lipopolysaccharide (LPS) challenge, distinguishing between local and systemic effects. Ten multiparous cows over 70 d in milk were blocked into pairs by breed, cow-level somatic cell count (SCC), and milk yield. Within block, one cow was assigned to LPS treatment (T) such that both the front and the rear quarter of a randomly selected udder half received an infusion of 50 µg of LPS in 10 mL of saline (T-L); the contralateral quarters received only 10 mL of saline (T-S). Similarly, each paired control cow (C) received either 10 mL of saline (C-S) or no infusion (C-N) into udder halves. Cows were quarter milked twice daily, with foremilk samples (∼30 mL, front quarters) taken at -24, 0, 3, 6, 12, and 24 h relative to infusions. At 24 h, average milk yield in T-L and T-S quarters fell to 23 and 32% of pre-infusion levels, respectively. For T cows, systemic effects were observed by 3 h post-infusion as rectal temperature was elevated and foremilk fat concentration was reduced in both T-L and T-S. However, SCC and concentrations of l-lactate and total protein in foremilk indicated a local response to LPS: protein was transiently higher at 3 h, whereas SCC and lactate were higher at 6 h in T-L compared with T-S. Lactose concentration showed a local effect at 6 h, being lower in T-L than in T-S, and then a systemic effect at 12 h, being lower in both T-L and T-S than C quarters. Concomitant with changes in milk, systemic effects were also observed in blood. Plasma antioxidant potential and glucose concentration were lower in T cows than in C cows at 6 or 12 h, respectively, although neither variable remained different at 24 h. In summary, unilateral LPS infusion induced distinct, time-dependent effects on each milk component. Depending on the component, effects were local, systemic, or both, suggesting involvement of multiple different mediators that collectively result in systemic inhibition of milk production.

The impact of direct-fed microbials and enzymes on the health and performance of dairy cows with emphasis on colostrum quality and serum immunoglobulin concentrations in calves.

Thirty-six cows were blocked by calving date and randomly assigned to one of three treatments. Cows were on treatments 3 weeks prepartum through 8 weeks post-partum. Treatments were as follows: (i) no direct-fed microbial (DFM) or cellulase and amylase enzymes (C), (ii) 45.4 g/day of DFM (D) or (iii) 45.4 g/day of DFM and 18.2 g/day of enzyme (DE). Total mixed ration fed and refused were measured daily to determine dry matter intake (DMI). Blood samples were taken three times weekly and analysed for ß-hydroxybutyrate, glucose and non-esterified fatty acids. Body weight (BW) was measured weekly. Colostrum was weighed and analysed for IgA and IgG concentration. Calves were fed 4 L of colostrum within 2 hr of birth. Calf blood samples were taken at 0 and 24 hr for analysis of IgA and IgG concentrations and apparent efficiency of absorption. Milk yield was measured daily and samples collected weekly. Initial BW was different among treatments with D being lesser than C or DE treatments. Body weight, weight gain, efficiency of gain, DMI and blood parameters were unaffected. Treatment did not affect colostrum yield. Ash percentage of colostrum tended to increase with D and DE, while IgA and total solids yield decreased with D. Colostrum fat yield was decreased in D and DE. Treatments did not impact BW, serum IgA and IgG concentrations or apparent efficiency of absorption of calves. Post-partum BW, DMI, blood parameters, milk production and composition were unaffected by treatment. However, cows on D gained more BW and tended to have greater efficiency of gain compared to those on DE, but were similar to C. Somatic cell scores were greatest for D. Results indicate that DFM and enzyme supplementation did not improve health and performance of dairy cattle during the pre- and post-partum periods under conditions of this study.