Rumen-protected methionine during heat stress alters mTOR, insulin signaling, and 1-carbon metabolism protein abundance in liver, and whole-blood transsulfuration pathway genes in Holstein cows.

We investigated effects of rumen-protected Met (RPM) during a heat stress (HS) challenge on (1) hepatic abundance of mTOR, insulin, and antioxidant signaling proteins, (2) enzymes in 1-carbon metabolism, and (3) innate immunity. Holstein cows (n = 32; mean ± standard deviation, 184 ± 59 d in milk) were randomly assigned to 1 of 2 environmental groups, and 1 of 2 diets [total mixed ration (TMR) with RPM (Smartamine M; 0.105% dry matter as top-dress) or TMR without (CON); n = 16/diet] in a split-plot crossover design. There were 2 periods with 2 phases. During phase 1 (9 d), all cows were in thermoneutral conditions (TN; temperature-humidity index = 60 ± 3) and fed ad libitum. During phase 2 (9 d), half the cows (n = 8/diet) were exposed to HS using electric heat blankets. The other half (n = 8/diet) remained in TN, but was pair-fed to HS counterparts. After a 14-d washout and 7-d adaptation period, the study was repeated (period 2) and environmental treatments were inverted relative to phase 2, but dietary treatments were the same. Blood was collected on d 6 of each phase 2 to measure immune function and isolate whole-blood RNA. Liver biopsies were performed at the end of each period for cystathione ß-synthase (CBS) and methionine adenosyltransferase activity, glutathione concentration, and protein abundance. Data were analyzed using PROC MIXED in SAS. Abundance of CUL3, inhibitor of antioxidant responses, tended to be downregulated by HS suggesting increased oxidative stress. Heat-shock protein 70 abundance was upregulated by HS. Phosphorylated mTOR abundance was greater overall with RPM, suggesting an increase in pathway activity. An environment × diet (E × D) effect was observed for protein kinase B (AKT), whereas there was a tendency for an interaction for phosphorylated AKT. Abundance of AKT was upregulated in CON cows during HS versus TN, this was not observed in RPM cows. For phosphorylated AKT, tissue from HS cows fed CON had greater abundance compared with all other treatments. The same effect was observed for EIF2A (translation initiation) and SLC2A4 (insulin-induced glucose uptake). An E × D effect was observed for INSR due to upregulation in CON cows during HS versus TN cows fed CON or RPM. There was an E × D effect for CBS, with lower activity in RPM versus CON cows during HS. The CON cows tended to have greater CBS during HS versus TN. An E × D effect was observed for methionine adenosyltransferase, with lower activity in RPM versus CON during HS. Although activity increased in CON during HS versus TN, RPM cows tended to have greater activity during TN. Neutrophil and monocyte oxidative burst and monocyte phagocytosis decreased with HS. An (E × D) effect was observed for whole-blood mRNA abundance of CBS, SOD1 and CSAD; RPM led to upregulation during TN versus HS. Regardless of diet, CDO1, CTH, and SOD1 decreased with HS. Although HS increased hepatic HSP70 and seemed to alter antioxidant signaling, feeding RPM may help cows maintain homeostasis in mTOR, insulin signaling, and 1-carbon metabolism. Feeding RPM also may help maintain whole-blood antioxidant response during HS, which is an important aspect of innate immune function.

Effects of rumen-protected methionine on lactation performance and physiological variables during a heat stress challenge in lactating Holstein cows.

Milk yield, content, and composition are altered by heat stress. Thirty-two multiparous, lactating Holstein cows [balanced by days in milk (mean ± standard deviation; 184 ± 59); body surface area (5.84 ± 0.34 m2)] were randomly assigned to 1 of 2 dietary treatments [total mixed ration with rumen-protected Met (RPM; Smartamine M; Adisseo Inc., Antony, France; 1.05 g of RPM/kg of dry matter intake) or total mixed ration without RPM (CON)], and within each dietary treatment group cows were randomly assigned to 1 of 2 environmental treatment groups in a split-plot crossover design. The study was divided into 2 periods with 2 phases per period. In phase 1 (9 d), all cows were in thermoneutral conditions and fed ad libitum. In phase 2 (9 d), group 1 (n = 16) was exposed to a heat stress challenge (HSC) using electric heat blankets. Group 2 (n = 16) remained in thermoneutral conditions but was pair-fed (PFTN) to HSC counterparts. After a 21-d washout period, the study was repeated (period 2) and the environmental treatments were inverted relative to treatments from phase 2 of period 1, whereas dietary treatments (RPM or CON) remained the same for each cow. Cows were milked 3× per day and samples were taken on d 1, 5, and 9 of each phase. Vaginal temperature was measured every 10 min, rectal temperature and skin temperature were measured 3× per day, and respiration rate and heart rate were recorded once per day. Cow activity was measured using an accelerometer. Paired difference values were calculated for each cow for each period based on the difference between phase 1 baseline means and phase 2 values for each variable. Cows in HSC had a greater increase in vaginal temperature and respiration rate (+0.2°C and +13.7 breaths/min, respectively) compared with cows in PFTN (0.0°C and -1.6 breaths/min, respectively). Cows in PFTN had a greater decrease in dry matter intake and milk yield (-3.9 and -2.6 kg/d, respectively) compared with cows in HSC (-3.2 and -0.9 kg/d, respectively). Cows in CON had a greater decrease in milk protein concentration for PFTN (-0.10 percentage units) and HSC (-0.06 percentage units) compared with cows in RPM for PFTN (0.00 percentage units) and HSC (-0.02 percentage units). Cows in CON for HSC had greater decrease in milk fat concentration compared with cows in RPM for HSC (-0.10 and +0.12 percentage units, respectively). In conclusion, HSC altered physiological and production parameters of cows. Additionally, RPM helped maintain milk protein and fat concentration during HSC, whereas dry matter intake, milk yield, and feed efficiencies were not affected by RPM.

Aluminosilicate clay improves production responses and reduces inflammation during an aflatoxin challenge in lactating Holstein cows.

Mitigation strategies are vital in minimizing the health and economic risks associated with dairy cattle exposure to aflatoxin (AF). The objective of this study was to determine the effects of a commercially available aluminosilicate clay in a lactation diet on production responses, blood chemistry, and liver inflammatory markers of multiparous lactating Holstein cows during an AF challenge. Sixteen multiparous lactating Holstein cows [body weight (mean ± SD) = 758 ± 76 kg; days in milk = 157 ± 43 d] were assigned to 1 of 4 treatments in a replicated 4 × 4 Latin square design with 21-d periods: no adsorbent and no AF challenge (CON), no adsorbent and an AF challenge (POS), 113 g of aluminosilicate clay top-dressed on the total mixed ration (adsorbent; FloMatrix, PMI Nutritional Additives, Arden Hills, MN) with an AF challenge (F4), or 227 g of adsorbent with an AF challenge (F8). The challenge consisted of 100 µg of AFB1/kg of dietary dry matter intake administered orally. For each period, milk was sampled 3× daily from d 14 to 21; blood, feces, and urine were sampled on d 14, 18, and 21; and liver samples were taken on d 18. Liver tissue was assessed for gene expression and histological hepatocyte inflammation. Statistical analysis was preformed using the MIXED and GLIMMIX procedures of SAS (SAS Institute Inc., Cary, NC). Fat-corrected milk (POS = 37.2, F4 = 39.2, and F8 = 38.9 kg/d) increased as concentration of adsorbent in the diet increased. There was a decrease in milk AFM1 concentration at d 18 as concentration of adsorbent in the diet increased (POS = 0.33, F4 = 0.32, and F8 = 0.27 µg/kg). There was a decrease in AFM1 concentration in urine (POS = 2.10, F4 = 1.89, and F8 = 1.78 µg/kg) and AFB1 concentration in feces (POS = 4.68, F4 = 3.44, and F8 = 3.17 µg/kg) as concentration of adsorbent in the diet increased. Cows in CON had greater concentrations of serum cholesterol (202 mg/dL) and plasma superoxide dismutase (2.8 U/mL) compared with cows in POS (196 mg/dL and 2.6 U/mL, respectively). Plasma glutamate dehydrogenase increased as concentration of adsorbent in the diet increased (POS = 37.8, F4 = 39.3, and F8 = 39.1 U/L). The expression of NFKB1 was greater in the liver of cows in POS (0.78) compared with cows in CON (0.70). The expression of MTOR was greater in the liver of cows in CON (1.19) compared with cows in POS (0.96). When compared with cows in CON, cows in POS had greater odds ratio for hepatocyte inflammation (odds ratio = 5.14). In conclusion, the adsorbent used in this study had a positive effect on milk production and hepatocyte inflammation and reduced AF transfer.

Methionine and choline supply alter transmethylation, transsulfuration, and cytidine 5'-diphosphocholine pathways to different extents in isolated primary liver cells from dairy cows.

Insufficient supply of Met and choline (Chol) around parturition could compromise hepatic metabolism and milk protein synthesis in dairy cows. Mechanistic responses associated with supply of Met or Chol in primary liver cells enriched with hepatocytes (PHEP) from cows have not been thoroughly ascertained. Objectives were to isolate and culture PHEP to examine abundance of genes and proteins related to transmethylation, transsulfuration, and cytidine 5'-diphosphocholine (CDP-choline) pathways in response to Met or Chol. The PHEP were isolated from liver biopsies of Holstein cows (160 d in lactation). More than 90% of isolated cells stained positively for the hepatocyte marker cytokeratin 18. Cytochrome P450 (CYP1A1) mRNA abundance was only detectable in the PHEP and liver tissue compared with mammary tissue. Furthermore, in response to exogenous Met (80 µM vs. control) PHEP secreted greater amounts of albumin and urea. Subsequently, PHEP were cultured with Met (40 µM) or Chol (80 mg/dL) for 24 h. Compared with control or Chol, mRNA and protein abundance of methionine adenosyltransferase 1A (MAT1A) and phosphatidylethanolamine methyltransferase (PEMT) were greater in PHEP treated with Met. The mRNA abundance of S-adenosylhomocysteine hydrolase (SAHH), betaine-homocysteine methyltransferase (BHMT), and sarcosine dehydrogenase (SARDH) was greater in Met-treated PHEP compared with control or Chol. Compared with control, greater expression of 5-methyltetrahydrofolate-homocysteine methyltransferase (MTR), betaine aldehyde dehydrogenase (BADH), and choline dehydrogenase (CHDH) was observed in cells supplemented with Met and Chol. However, Chol led to the greatest mRNA abundance of CHDH. Abundance of choline kinase α (CHKA), choline kinase ß (CHKB), phosphate cytidylyltransferase 1 α (PCYT1A), and choline/ethanolamine phosphotransferase 1 (CEPT1) in the CDP-choline pathway was greater in PHEP treated with Chol compared with control or Met. In the transsulfuration pathway, mRNA and protein abundance of cystathionine ß-synthase (CBS) was greater in PHEP treated with Met compared with control or Chol. Similarly, abundance of cysteine sulfinic acid decarboxylase (CSAD), glutamate-cysteine ligase, catalytic subunit (GCLC), and glutathione reductase (GSR) was greater in response to Met compared with control or Chol. Overall, these findings suggest that transmethylation and transsulfuration in dairy cow primary liver cells are more responsive to Met supply, whereas the CDP-choline pathway is more responsive to Chol supply. The relevance of these data in vivo merit further study.

Injectable trace minerals (selenium, copper, zinc, and manganese) alleviate inflammation and oxidative stress during an aflatoxin challenge in lactating multiparous Holstein cows.

Trace minerals are vital in the antioxidant response during oxidative stress; however, limited research is available on the effects of trace mineral supplementation during an aflatoxin (AF) challenge. The objective of the study was to determine the effects of 2 subcutaneous injections of 15 mg/mL of Cu, 5 mg/mL of Se, 60 mg/mL of Zn, and 10 mg/mL of Mn (Multimin 90, Multimin North America, Fort Collins, CO) given at 1 mL/90.7 kg of average body weight in response to an AF challenge. Fifty-eight Holstein cows [body weight (mean ± SD) = 734 ± 6 0kg; days in milk = 191 ± 93] were assigned to 1 of 3 treatments in a randomized complete block design. The experimental period (63 d) was divided into an adaptation phase (d 1-56) and a measurement phase (d 57-63). From d 57 to 59, cows received an AF challenge that consisted of 100 µg of aflatoxin B1/kg of dietary dry matter intake (DMI) administered orally via balling gun. Treatments were saline injection and no AF challenge (NEG), saline injection and AF challenge (POS), and trace mineral injection and AF challenge (MM). Injections were performed subcutaneously on d 1 and 29. Milk was sampled 3 times daily from d 56 to 63, blood was sampled on d 0, 56, 60, and 63, and liver samples were taken on d 0 and 60. Two treatment orthogonal contrasts [CONT1 (NEG vs. POS) and CONT2 (POS vs. MM)] were made. Cows in NEG had lower AF excretion in milk and greater 3.5% fat-corrected milk (32.1 ± 1.37 kg/d) compared with cows in POS (28.6 ± 1.43 kg/d). Feed efficiencies (3.5% fat-corrected milk/DMI, energy-corrected milk/DMI, and milk/DMI) were greater for cows in NEG (1.42 ± 0.07, 1.46 ± 0.07, and 1.45 ± 0.07, respectively) than cows in POS (1.16 ± 0.08, 1.18 ± 0.08, and 1.22 ± 0.07, respectively). Cows in POS had greater milk urea nitrogen and blood urea nitrogen than cows in MM. Liver concentrations of Se and Fe were greater for cows in MM compared with cows in POS. Cows in MM tended to have greater plasma glutathione peroxidase activity compared with cows in POS. An upregulation of liver GPX1 was observed for cows in POS compared with cows in MM. In conclusion, subcutaneous injection of trace minerals maintained an adequate antioxidant response when an AF challenge was present.