Effects of sodium salicylate and time postpartum on mammary tissue proliferation, gene transcript profile, and DNA methylation.

Previous studies have demonstrated nonsteroidal antiinflammatory drug treatment in early lactation had a positive impact on whole-lactation milk production in older cows. The objective of this study was to evaluate proliferative, transcriptional, and epigenetic changes in the mammary gland that could explain increased production responses due to nonsteroidal antiinflammatory drug treatment. Sodium salicylate (SAL; 125 g/d) or water (CON) were administered via oral drench to multiparous Holstein cows (n = 8/treatment) once daily for 3 d beginning approximately 24 h after parturition, and mammary tissue was collected on d 1, 4, and 45 postpartum. Day 1 tissue was collected immediately preceding the initial drench, and d 4 tissue was collected 24 h following the final drench. Blood was collected twice weekly and analyzed for plasma glucose, insulin, ß-hydroxybutyrate, free fatty acids, and prolactin. Cows were milked twice daily until d 7 of lactation, and thrice daily for the remainder of the study. Total RNA extracted from tissue was deep-sequenced and analyzed for differential gene expression using DESeq2. We detected no treatment effect on milk yield or plasma metabolites through 45 d of lactation; additionally, no change in mammary epithelial cell proliferation was detected when assessed by Ki67 labeling. Comparison of SAL versus CON revealed that only 16 of 18,286 genes were differentially expressed (false discovery rate <0.1) in mammary tissue collected on d 45, whereas no differentially expressed genes due to treatment were detected on d 1 or 4. Analysis of transcriptional differences over time showed downregulation of pathways related to immune cell recruitment and differentiation, and extensive overlap with pathways related to cholesterol synthesis and liver X receptor signaling. Global DNA methylation of mammary tissue was decreased for CON compared with SAL. Transcriptome analysis emphasized extensive involvement of immune-related signaling pathways in the switch from lactogenesis to galactopoiesis, and changes in methylation with SAL treatment merit future investigation into epigenetic effects on milk production.

Sodium salicylate reduced mRNA abundance of hypoxia-associated genes in MAC-T cells.

Hypoxia is an oxygen deficiency commonly found in growing tissues and is speculated to occur in the rapidly developing mammary gland in peripartum dairy cattle. Low oxygen concentrations can activate hypoxia-inducible factor-1 (HIF-1), which increases transcription of genes involved in angiogenesis (VEGFA) and glucose transport (GLUT1), among other processes. The mRNA stability of these genes is positively regulated by heterogeneous nuclear ribonucleoprotein D (HNRNPD; also known as AUF1). In our previous research, postpartum administration of sodium salicylate (SS) increased whole-lactation milk yield in multiparous cows but tended to reduce milk yield in primiparous cows. Because rapid mammary tissue development likely occurs in cows approaching first lactation, we hypothesized that SS inhibited the activation of HIF-1α and decreased transcription of downstream targets. MAC-T cells were treated with SS (100 µM) or control medium before incubation under either hypoxic (1% O2) or normoxic conditions for 12 h. Additionally, cells were transfected with either HIF1A small interfering RNA (siRNA) or a scrambled siRNA negative control 48 h before hypoxia treatments. HIF1A, GLUT1, VEGFA, and HNRNPD were quantified relative to the internal control gene NENF. Transcript abundance was assessed using a linear mixed model with the fixed effects of SS, hypoxia, siRNA, and all 2- and 3-way interaction terms and the random effect of plate nested within hypoxia. Treatment with SS interacted with hypoxia for GLUT1, as SS reduced GLUT1 when MAC-T cells were cultured in normoxic conditions; however, no effect of SS was found in hypoxia-treated cells. Regardless of oxygen status, SS reduced HNRNPD and tended to decrease VEGFA mRNA relative to untreated cells. Hypoxia increased GLUT1, yet no effect was observed on VEGFA or HNRNPD. Small interfering RNA knocked down HIF1A, but no effect was found on GLUT1, VEGFA, or HNRNPD. In conclusion, SS reduced transcript abundance of genes involved with mammary gland development but generally did not interact with oxygen status.

Associations between body condition score at parturition and microRNA profile in colostrum of dairy cows as evaluated by paired mapping programs.

MicroRNA (miRNA) are abundant in milk, and likely have regulatory activity involving lactation and immunity. The objective of this study was to determine the miRNA profile in colostrum of overconditioned cows compared with cows of more moderate body condition score (BCS) at calving. Multiparous cows with either high (≥4.0 on a scale of 1 to 5; n = 7) or moderate BCS (2.75 to 3.50; n = 9) in the week before parturition were selected from a commercial dairy herd. Blood and colostrum were sampled within 24 h after calving. Blood serum was analyzed for free fatty acid (FFA) concentration. MicroRNA was isolated from colostrum samples after removing milk fat and cells. MicroRNA were sequenced, and reads were mapped to the bovine genome and to the existing database of miRNA at miRBase.org. Two programs, Oasis 2.0 and miRDeep2, were employed in parallel for read alignment, and analysis of miRNA count data was performed using DESeq2. Identification of differentially expressed miRNA from DESeq2 was not affected by the differences in miRNA detected by the 2 mapping programs. Most abundant miRNA included miR-30a, miR-148a, miR-181a, let-7f, miR-26a, miR-21, miR-22, and miR-92a. Large-scale shifts in miRNA profile were not observed; however, colostrum of cows with high BCS contained less miR-486, which has been linked with altered glucose metabolism. Colostrum from cows with elevated serum FFA contained less miR-885, which may be connected to hepatic function during the transition period. Potential functions of abundant miRNA suggest involvement in development and maintenance of cellular function in the mammary gland, with the additional possibility of influencing neonatal tissue and immune system development.

Effects of fat supplementation to diets high in nonforage fiber on production responses of midlactation dairy cows.

The effects of dietary nonforage fiber sources on production responses of lactating dairy cattle have been well described, but interactions with other components of the diet have been less thoroughly explored. We investigated the effects of adding 2 commonly fed fat sources to a ration featuring high levels of nonforage fiber supplied by a corn milling by-product. Midlactation Holstein cows were blocked by parity, stratified by days in milk, and randomly assigned to 1 of 6 pens (12 cows/pen). Pens were randomly assigned to treatment sequences in a 3 × 3 Latin square design, where the treatments consisted of prilled saturated fat (SAT; Energy Booster 100, Milk Specialties Co., Dundee, IL), calcium salts of long-chain fatty acids (UNS; Megalac, Church and Dwight Co. Inc., Princeton, NJ), or no added dietary fat (control), with fat sources included to provide 1.2% added fat (dry matter basis). Treatment periods were 21 d; milk and feed samples were collected and milk yield and feed intake were recorded for the last 4 d of each period. Results were analyzed with mixed models with pen as the experimental unit, and orthogonal contrasts were employed to evaluate the overall effect of added fat and the effect of fat source. Dry matter intake and milk yield tended to increase with added fat. Protein content decreased with fat supplementation, to a greater degree for UNS than for SAT, but protein yield was not affected. Fat content, fat yield, and energy-corrected milk yield were not affected by treatment. Conversion of feed to milk tended to increase for UNS compared with SAT. Fat supplementation to diets high in nonforage fiber had effects that were similar to those reported for more traditional lactation diets, except for the dry matter intake response.

Effects of early postpartum sodium salicylate treatment on long-term milk, intake, and blood parameters of dairy cows.

Previous research has shown that cows who receive treatment with nonsteroidal anti-inflammatory drugs after calving may have increased milk yield beginning near peak lactation, resulting in greater 305-d milk production. It has not been demonstrated whether this response is associated with greater feed intake following the first 3 wk of lactation. Dry matter intake (DMI) and milk yield were measured daily for 56 cows over the first 120 d in milk. Cows in their second parity and greater were blocked by parity and alternately enrolled 12 to 36 h after calving into 1 of 2 treatments: either 3 daily drenches of water or 3 daily drenches of a similar volume of water containing 125 g of sodium salicylate (SAL) beginning 12 to 36 h after calving. Cows were housed in individual stalls to monitor DMI. Blood samples were collected before calving and on the last day of treatment, as well as at 7, 11, 14, 18, 21, 35, 49, 63, 77, 91, 105, and 120 d in milk. The SAL treatment did not affect estimated 305-d milk, fat, or protein yields (from monthly test days), daily milk yield or components, energy-corrected milk, fat-corrected milk, or DMI; however, an interaction between parity and treatment was observed for DMI, where second-parity SAL cows had decreased intake with no differences observed in older cows. This resulted in a parity by treatment interaction for the ratio of energy-corrected milk to DMI. Similarly, no main effects of treatment were observed for plasma glucose, ß-hydroxybutyrate (BHB), or fatty acid concentrations, but we noted interactions between treatment and parity for glucose, BHB, and insulin. Older cows had greater plasma glucose and insulin concentrations and decreased plasma BHB following SAL but no differences were observed in second parity animals. Alterations in glucose and insulin resulted in a tendency for a treatment by time interaction for the revised quantitative insulin sensitivity check index. Feeding behavior was also altered following SAL administration, resulting in fewer but longer meals, as well as a tendency for greater meal weight. A tendency for a treatment by week interaction for inter-meal interval was observed, as well as a parity by treatment interaction for meal weight. Despite the lack of a milk yield response, SAL had a prolonged programming effect on feeding behavior and blood variables over the first 120 DIM, with responses largely dependent on parity.

Hot topic: Early postpartum treatment of commercial dairy cows with nonsteroidal antiinflammatory drugs increases whole-lactation milk yield.

Previous research has shown that postpartum administration of the nonsteroidal antiinflammatory drug (NSAID) sodium salicylate can increase 305-d milk yield in older dairy cattle (parity 3 and greater). However, in this prior work, sodium salicylate was delivered to cows via the drinking water, a method that does not align well with current grouping strategies on commercial dairy farms. The objective of the current study was to replicate these results on a commercial dairy farm with a simplified treatment protocol and to compare sodium salicylate with another NSAID, meloxicam. Dairy cattle in their second lactation and greater (n=51/treatment) were alternately assigned to 1 of 3 treatments at parturition, with treatments lasting for 3d. Experimental treatments began 12 to 36 h after parturition and were (1) 1 placebo bolus on the first day and 3 consecutive daily drenches of sodium salicylate (125 g/cow per day; SAL); (2) 1 bolus of meloxicam (675 mg/cow) and 3 drenches of an equal volume of water (MEL); or (3) 1 placebo bolus and 3 drenches of water (CON). Blood samples were collected on the first day of treatment, immediately following the last day of treatment, and 7d after the last day of treatment; plasma was analyzed for glucose, ß-hydroxybutyrate (BHB), free fatty acids, haptoglobin, and paraoxonase. Milk production, body condition score, reproductive status, and retention in the herd were monitored for 365 d posttreatment, and effects of treatment, parity, days in milk, and interactions were evaluated in mixed effects models. Significance was declared at P<0.05. Whole-lactation milk and protein yields were greater in NSAID-treated cows, although 305-d fat production was not affected. There was a significant interaction of treatment and parity for plasma glucose concentration; MEL increased plasma glucose concentrations compared with CON and SAL in older cows. Sodium salicylate decreased plasma BHB concentration compared with MEL at 7d posttreatment, although no difference was detected immediately following treatment. Haptoglobin concentrations were elevated in SAL cows compared with CON. There was a tendency for CON cows to be removed from the herd more quickly than MEL cows (42 vs. 26% at 365 d posttreatment). Body condition score, concentrations of plasma free fatty acids and paraoxonase, and time to pregnancy were not affected by treatment. These results indicate that NSAID administration in postpartum cows has the potential to be a viable way to improve productivity and potentially longevity in commercial dairies, although further research is necessary to optimize recommendations for producers.

Feed intake is related to changes in plasma nonesterified fatty acid concentration and hepatic acetyl CoA content following feeding in lactating dairy cows.

The relationship between hepatic acetyl CoA (AcCoA) content and dry matter intake (DMI) was evaluated using 28 multiparous Holstein cows; 14 were early postpartum (PP; 12.6 ± 3.8 d in milk) and 14 were late-lactation cows (LL; 269 ± 30 d in milk). Cows were fed once daily, and DMI was determined for the first 4h after feeding. Liver and blood samples were collected before feeding and 4h after feeding. Feed intake over the 4-h period ranged from 3.7 to 9.6 kg of dry matter and was similar for the 2 stages of lactation. Before feeding, hepatic AcCoA content was greater for PP compared with LL cows (34.4 vs. 12.5 nmol/g), and decreased over the 4h after feeding for PP only (28.7 vs. 34.4 nmol/g). The range for change in AcCoA over the 4-h period was wide for both PP (-24.3 to 10.4 nmol/g) and LL (-5.7 to 16.1 nmol/g), and was related negatively to DMI at 4h for both PP (R(2) = 0.55) and LL (R(2) = 0.31). The reduction in plasma NEFA concentration over the 4-h period was greater for PP than LL cows (-681 vs. -47 µEq/L), and was related to DMI at 4h for both PP and LL (both R(2) = 0.38). Greater DMI among cows over the first 4h after feeding might have been from a sharper reduction in supply of AcCoA in the liver for oxidation during meals because of the reduction in plasma NEFA concentration. Consistent with this is that the change in AcCoA was positively related to the reduction in plasma NEFA concentration for PP cows (R(2) = 0.31). However, change in plasma NEFA concentration was not related to change in hepatic AcCoA in LL cows, indicating that the pool of AcCoA in LL cows is not as dependent on NEFA flux to the liver as that of PP cows. Further research is required to determine production and fate of AcCoA within the timeframe of meals and the effects of feeding on energy charge in hepatic tissue.