Rumen-protected methionine supplementation during the transition period under artificially induced heat stress: impacts on cow-calf performance.

Dairy cows experiencing heat stress (HS) during the pre-calving portion of the transition period give birth to smaller calves and produce less milk and milk protein. Supplementation of rumen-protected methionine (RPM) has been shown to modulate protein, energy, and placenta metabolism, making it a potential candidate to ameliorate HS effects. We investigated the effects of supplementing RPM to transition cows under HS induced by electric heat blanket (EHB) on cow-calf performance. Six weeks before expected calving, 53 Holstein cows were housed in a tie-stall barn and fed a control diet (CON, 2.2% Met of MP) or a CON diet supplemented with Smartamine®M (MET, 2.6% Met of MP, Adisseo Inc., France). Four weeks pre-calving, all MET and half CON cows were fitted with an EHB. The other half of the CON cows were considered thermoneutral (TN), resulting in 3 treatments: CONTN (n = 19), CONHS (n = 17), and METHS (n = 17). Respiratory rate (RR), skin temperature (ST), and rectal temperature (RT) were measured thrice weekly and core body temperatures recorded bi-weekly. Post-calving body weights (BW) and BCS were recorded weekly, and DMI was calculated and averaged weekly. Milk yield was recorded daily and milk components were analyzed every third DIM. Biweekly AA and weekly nonesterified fatty acids (NEFA), ß-hydroxybutyrate (BHB), insulin, and glucose were measured from plasma. Calf birth weight and 24 h growth, thermoregulation, and hematology profile were measured and apparent efficiency of absorption (AEA) of immunoglobulins was calculated. Data were analyzed using the MIXED procedure of SAS with 2 preplanned orthogonal contrasts: CONTN vs. the average of CONHS and METHS (C1) and CONHS vs. METHS (C2). Relative to TN, EHB cows had increased RT during the post-calving weeks and increased RR and ST during the entire transition period. Body weight, BCS, DMI, and milk yield were not impacted by the EHB or RPM. However, protein % and SNF were lower in CONHS, relative to METHS cows. At calving, METHS dams had higher glucose concentrations, relative to CONHS, and during the post-calving weeks, the EHB cows had lower NEFA concentrations than TN cows. Calf birthweight and AEA were reduced by HS, while RR was increased by HS. Calf withers height tended to be shorter and RT were lower in CONHS, compared with MTHS heifers. Overall, RPM supplementation to transition cows reverts the negative impact of HS on blood glucose concentration at calving and milk protein % in the dams and increases wither height while decreasing RT in the calf.

Feeding rumen-protected methionine during the peripartum period improved milk fat content and reduced the culling rate of Holstein cows in a commercial herd.

Researchers have reported the benefits of feeding rumen-protected methionine (RPM) during the peripartum on the health parameters of dairy cows. Rumen-protected methionine has reportedly improved milk yield, milk components and liver health, but the literature is scarce on its effects in commercial herds. Therefore, we aimed to determine the effects of feeding RPMet (Smartamine M®, Adisseo Inc., Antony, France) prepartum (8 g per cow per day) and postpartum (15 g per cow per day) on performance, metabolic profile, and culling rate of Holstein cows in a commercial herd. One-hundred and 66 (n = 166) Holstein cows, 58 nulliparous and 108 parous, were randomly assigned to 1 of 2 dietary treatments, consisting of TMR top-dressed with RPMet (2.35 and 2.24% Met of MP for close-up and fresh cows, respectively) or without (control, CON, (2.03 and 1.89% Met of MP for close-up and fresh cows, respectively), fed from 21 ± 6 d prepartum until 16 ± 5 d postpartum. From 17 d in milk (DIM) until dry-off, all cows received RPMet. Daily milk yield was recorded, and milk samples were collected in the first and second weeks after calving to determine their composition. Blood samples were collected before the morning feeding on -14, -7, +1, +7, and +14 d relative to calving. Mortality and morbidity were recorded during the first 60 DIM. Cows supplemented with RPMet had greater milk yield during the first 16 DIM (31.76 vs. 30.37 kg/d; SEM = 1.04, respectively), and had greater milk fat content (4.45 vs. 4.10%; SEM = 0.11, respectively), but not milk total protein (3.47 vs. 3.39%; SEM = 0.04, respectively) and casein contents (2.74 vs. 2.66%; SEM = 0.04, respectively) than CON cows. Cows in RPMet had increased plasma Met concentrations than cows in CON (24.9 vs. 21.0 µmol/L; SEM = 1.2, respectively). Although morbidity was similar between treatments, the culling rate from calving until 60 DIM was lower for RPMet cows than for CON cows (2.4 vs. 12.1%; SEM = 0.02). In conclusion, cows receiving RPMet have greater milk yield, improved milk fat content, and a lower culling rate at 60 DIM than CON cows.

Balancing dairy cattle diets for rumen nitrogen and methionine or all essential amino acids relative to metabolizable energy.

Improving the ability of diet formulation models to more accurately predict AA supply while appropriately describing requirements for lactating dairy cattle provides an opportunity to improve animal productivity, reduce feed costs, and reduce N intake. The goal of this study was to evaluate the sensitivity of a new version of the Cornell Net Carbohydrate and Protein System (CNCPS) to formulate diets for rumen N, Met, and all essential AA (EAA). Sixty-four high-producing dairy cattle were randomly assigned to 1 of the 4 following diets in a 14-wk longitudinal study: (1) limited metabolizable protein (MP), Met, and rumen N (Base), (2) adequate Met but limited MP and rumen N (Base + M), (3) adequate Met and rumen N, but limited MP (Base + MU), and (4) adequate MP, rumen N, and balanced for all EAA (Positive). All diets were balanced to exceed requirements for ME relative to maintenance and production, assuming a nonpregnant, 650-kg animal producing 40 kg of milk at 3.05% true protein and 4.0% fat. Dietary MP was 97.2, 97.5, 102.3, and 114.1 g/kg of dry matter intake for the Base, Base + M, Base + MU, and Positive diets, respectively. Differences were observed for dry matter intake and milk yield (24.1 to 24.7 and 39.4 to 41.1 kg/d, among treatments). Energy corrected milk, fat, and true protein yield were greater (2.9, 0.13, and 0.08 kg/d, respectively) in cows fed the Positive compared with the Base diet. Using the updated CNCPS, cattle fed the Base, Base + M, and Base + MU diets were predicted to have a negative MP balance (-231, -310, and -142 g/d, respectively), whereas cattle fed the Positive diet consumed 33 g of MP/d excess to ME supply. Bacterial growth was predicted to be depressed by 16 and 17% relative to adequate N supply for the Base and Base + M diets, respectively, which corresponded with the measured lower apparent total-tract NDF degradation. The study demonstrates that improvements in lactation performances can be achieved when rumen N and Met are properly supplied and further improved when EAA supply are balanced relative to requirements. Formulation using the revised CNCPS provided predictions for these diets, which were sensitive to changes in rumen N, Met, all EAA, and by extension MP supply.

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.

Methionine supply during the peripartum period and early lactation alter immunometabolic gene expression in cytological smear and endometrial tissue of holstein cows.

The objective of the present study was to evaluate the effect of feeding rumen-protected methionine (RPM) during the peripartal period and early lactation on mRNA gene expression profiles of uterine cytological smear and endometrial samples of Holstein cows (n = 20). Treatments consisted of a supplementation with RPM [MET; n = 11; RPM at a rate of 0.08 % of DM: Lys:Met = 2.8:1, (Smartamine® M Adisseo, Alpharetta, GA, USA)] and no supplementation (CON; n = 9; Lys:Met = 3.5:1). Uterine cytology smears and endometrial samples were collected at 15, 30, and 73 days in milk (DIM) and analyzed for expression of genes related with metabolism, inflammation, and methionine metabolism. Regarding the cytological smear samples, RPM supplementation tended to increase mRNA expression of methionine adenosyltransferase 1 alpha (MAT1A) and increased the mRNA expression of fibroblast growth factor 7 (FGF7), with an effect of time for the latter. On the other hand, RPM decreased mRNA expression for glucose transporter 4 (GLUT4), interleukin 1 beta (IL-1ß), interleukin 6 (IL-6), interleukin 8 (IL-8), prostaglandin E synthase 3 (PTGES3), translocator protein 18 kDa (TSPO), mucin 1 (MUC1) and superoxide dismutase (SOD1) in cytological smear samples. There was an effect of time for all variables except MAT1A, with decreasing expression over time. There was a TRT × TIME interaction for GLUT4 mRNA expression, with higher GLUT4 mRNA expression for cows fed CON than for cows fed RPM at time 15 and a tendency to higher expression for cows fed CON on time 30 when compared with cows fed RPM. For uterine tissue samples, feeding RPM increased the mRNA expression of lecithin-cholesterol acyltransferase (LCAT), S-adenosyl-l-homocysteine hydrolase (SAAH), FGF7, GLUT4, and apolipoproteins 3 (APOL3), with an effect of time for APOL3 where its expression increased over time. There was a tendency for cows fed RPM to have decreased IL1ß mRNA expression. In conclusion, feeding RPM during transition period and early lactation is beneficial for uterine immune response and metabolism in early lactation as indicated by the favorable expressions of genes affecting the uterine immunometabolism during such a challenging period.

Effects of diet fermentability and supplementation of 2-hydroxy-4-(methylthio)-butanoic acid and isoacids on milk fat depression: 2. Ruminal fermentation, fatty acid, and bacterial community structure.

The experiment was conducted to understand ruminal effects of diet modification during moderate milk fat depression (MFD) and ruminal effects of 2-hydroxy-4-(methylthio)-butanoic acid (HMTBa) and isoacids on alleviating MFD. Five ruminally cannulated cows were used in a 5 × 5 Latin square design with the following 5 dietary treatments (dry matter basis): a high-forage and low-starch control diet with 1.5% safflower oil (HF-C); a low-forage and high-starch control diet with 1.5% safflower oil (LF-C); the LF-C diet supplemented with HMTBa (0.11%; 28 g/d; LF-HMTBa); the LF-C diet supplemented with isoacids [(IA) 0.24%; 60 g/d; LF-IA]; and the LF-C diet supplemented with HMTBa and IA (LF-COMB). The experiment consisted of 5 periods with 21 d per period (14-d diet adaptation and 7-d sampling). Ruminal samples were collected to determine fermentation characteristics (0, 1, 3, and 6 h after feeding), long-chain fatty acid (FA) profile (6 h after feeding), and bacterial community structure by analyzing 16S gene amplicon sequences (3 h after feeding). Data were analyzed using the MIXED procedure of SAS (SAS Institute Inc., Cary, NC) in a Latin square design. Preplanned comparisons between HF-C and LF-C were conducted, and the main effects of HMTBa and IA and their interaction within the LF diets were examined. The LF-C diet decreased ruminal pH and the ratio of acetate to propionate, with no major changes detected in ruminal FA profile compared with HF-C. The α-diversity for LF-C was lower compared with HF-C, and ß-diversity also differed between LF-C and HF-C. The relative abundance of bacterial phyla and genera associated indirectly with fiber degradation was influenced by LF-C versus HF-C. As the main effect of HMTBa within the LF diets, HMTBa increased the ratio of acetate to propionate and butyrate molar proportion. Ruminal saturated FA were increased and unsaturated FA concentration were decreased by HMTBa, with minimal changes detected in ruminal bacterial diversity and community. As the main effect of IA, IA supplementation increased ruminal concentration of all branched-chain volatile FA and valerate and increased the percentage of trans-10 C18 isomers in total FA. In addition, α-diversity and the number of functional features were increased for IA. Changes in the abundances of bacterial phyla and genera were minimal for IA. Interactions between HMTBa and IA were observed for ruminal variables and some bacterial taxa abundances. In conclusion, increasing diet fermentability (LF-C vs. HF-C) influenced rumen fermentation and bacterial community structure without major changes in FA profile. Supplementation of HMTBa increased biohydrogenation capacity, and supplemental IA increased bacterial diversity, possibly alleviating MFD. The combination of HMTBa and IA had no associative effects in the rumen and need further studies to understand the interactive mechanism.

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.

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.

Methionine and choline supply during the peripartal period alter polymorphonuclear leukocyte immune response and immunometabolic gene expression in Holstein cows.

Polymorphonuclear leukocytes (PMNL) are the first responders upon pathogen invasion and hence play an important role in inflammatory and immune responses. Rumen-protected methionine (MET) and choline (CHOL) during the peripartal period affect the immune response and inflammatory status in dairy cows to different extents. We aimed to examine the effect of MET and CHOL supply on expression of genes regulating key PMNL functions and associations with whole-blood immune challenge. Thirty multiparous Holstein cows from a larger cohort randomly assigned from -21 to 30 d relative to parturition to a basal control (CON) diet, CON plus MET at a rate of 0.08% of dry matter, or CON plus CHOL at 60 g/d were used. Blood was sampled at -10, 7, and 30 d relative to parturition for inflammatory biomarker analyses and PMNL isolation. Neutrophil and monocyte phagocytosis and oxidative burst in vitro were assessed in whole blood at 1, 7, and 28 d. Although neutrophil and monocyte phagocytosis did not differ, oxidative burst in neutrophils and monocytes was greater in MET-supplemented cows relative to CON cows. Compared with CON, PMNL adhesion and migration-related genes (ITGAM, ITGB2, ITGA4) were downregulated in response to MET and CHOL. Expression of CADM1 and SELL was also lower in MET-supplemented cows compared with CON cows but not in CHOL cows. In contrast, compared with CON cows, the expression of ICAM1 was lower in CHOL but not MET cows. Similar to adhesion and migration-related genes, cows receiving MET- or CHOL-supplemented diets had lower expression of inflammation-related genes (IL1ß, IL10RA, NFKB1, STAT3, TLR2). However, expression of IRAK1 and TLR4 was lower in MET- but not CHOL-supplemented cows. Plasma taurine concentration was greater in MET cows compared with CHOL and CON cows, suggesting a better redox status in plasma. In agreement with plasma taurine, oxidative stress-related genes (CBS, CTH, GPX1, GSS, SOD2) in PMNL were lower in response to MET and to CHOL supply. Overall, immunometabolic gene expression profile and blood biomarker analyses suggest an overall better redox status in PMNL during the transition period in response to MET and CHOL supply. These adaptations in PMNL might be beneficial for mounting a better bactericidal response upon challenge.

Maternal supply of methionine during late pregnancy is associated with changes in immune function and abundance of microRNA and mRNA in Holstein calf polymorphonuclear leukocytes.

Pregnancy and early life are critical periods during which environmental factors such as nutrition can affect development. Rumen-protected methionine (Met; RPM) supplementation during the prepartum period improves not only performance but immune responses in dairy cows. We investigated the effects of enhanced maternal supply of Met via feeding RPM on whole-blood in vitro lipopolysaccharide (LPS; 0, 0.01, or 5 µg/mL of blood) challenge and targeted microRNA and mRNA abundance in calf blood polymorphonuclear leukocytes (PMNL). Calves (n = 12/maternal diet) born to cows fed RPM at 0.08% of diet dry matter (DM)/d (MET) for the last 21 ± 2 d before calving or fed a control diet with no added Met (CON) were used. The PMNL were isolated at birth (before colostrum feeding) and d 1 (24 h after colostrum intake), 14, 28, and 50 of age. Maternal blood was collected at -10 ± 1.3 d relative to calving. Cows in the MET group had greater DM intake and lower prepartal haptoglobin concentration. In CON cows, haptoglobin was positively correlated with proinflammatory and host-defense mRNA abundance in CON calves. Except for NOS2 and NFE2L2, abundance of CASP8, MPO, ZBP1, and TNF was lower at birth in MET calves. Interleukin 1ß concentration in response to LPS challenge in CON and MET calves was greatest at birth, underscoring the role of this cytokine for lymphocyte activation. Compared with 1 d of age, the interleukin-1ß response to incremental doses of LPS was greater at 14 through 28 d, suggesting that the neonatal calf can mount a robust response to inflammatory stimuli. Greater abundance in CON calves of NOS2, CADM1, and TLR2 coupled with lower SELL from 1 through 50 d of age suggested a chronic activation of the PMNL. There was a marked upregulation over time of MIR125b, MIR146a, MIR155, and MIR9 in both CON and MET calves, suggesting that these microRNA could affect gene transcription associated with differentiation and inflammatory function in PMNL. Regardless of maternal diet, the gradual downregulation of MIR223 (the most abundant microRNA in PMNL) is in line with the progressive increase over time in the proinflammatory signature of the PMNL. Data revealed the potential for maternal supply of Met during late pregnancy through either greater DM intake or Met to elicit some changes in PMNL function during early postnatal life, partly through changes in mRNA expression encompassing cell adhesion and chemotaxis, oxidative stress, Toll-like receptor signaling, and Met metabolism.

Improved uterine immune mediators in Holstein cows supplemented with rumen-protected methionine and discovery of neutrophil extracellular traps (NET).

During the transition from prepartum to early lactation, dairy cows often experience negative energy balance (NEB) that may result in reproductive stress and decreased fertility. The objective of this study was to observe the effects of rumen-protected methionine (RPM) on plasma amino acid concentrations, uterine cytology, immunohistochemistry (IHC) of glutathione peroxidase 1 (GPX) and superoxide dismutase 1 (SOD), and to confirm neutrophil extracellular trap (NET) formation. Multiparous Holstein cows (n = 20) were randomly assigned to two treatments starting at 21 d before calving until 73 days in milk (DIM). Treatments were: CON (n = 9, no supplementation, TMR with a Lys:Met = 3.5:1) and MET (n = 11, TMR + Smartamine® M with a Lys:Met = 2.8:1). Uterine endometrial biopsies, uterine cytology, and blood samples from the coccygeal artery or vein were collected at 15, 30, and 73 DIM. Blood plasma samples were analyzed for amino acids and metabolites. Uterine biopsies were analyzed for NET formation, neutrophil numbers, as well as GPX and SOD by IHC. Additionally, uterine cytology was analyzed for polymorphonuclear neutrophil (PMN) to epithelial cell percentage. Cows in CON had lower methionine plasma concentrations (18.05 ±â€¯2.0 µM) than cows in MET (30.39 ±â€¯1.6 µM). Cows in CON had greater cystine plasma concentrations (3.62 ±â€¯0.3 µM) than cows in MET (2.8 ±â€¯0.3 µM). No treatment differences were observed for SOD or GPX in the endometrium. Cows in CON tended to have a high score for positively immunolabeled GPX cells at 15 DIM than cows in MET. No treatment differences were observed for the percentage of PMN in uterine cytology, number of neutrophils, or extent of NET formation in the endometrium. A treatment by time interaction was observed for PMN percentage and the number of neutrophils: cows in MET tended to have greater PMN percentages than cows in CON at 15 DIM which decreased for subsequent days and cows in MET had greater neutrophil numbers in the endometrium at 30 DIM than cows in CON. In conclusion, dietary supplementation of RPM altered plasma amino acid concentrations and increased neutrophil infiltration in the postpartum period, suggesting improved uterine immunity.

Effects of rumen-protected methionine and choline supplementation on steroidogenic potential of the first postpartum dominant follicle and expression of immune mediators in Holstein cows.

Multiparous Holstein cows were assigned in a randomized complete block design into four treatments from 21 d before calving to 30 d in milk (DIM). Treatments were: MET [n = 19, fed the basal diet + rumen-protected methionine at a rate of 0.08% (w/w) of the dry matter, Smartamine® M], CHO (n = 17, fed the basal diet + choline 60 g/d, Reashure®), MIX (n = 21, fed the basal diet + Smartamine® M at a rate of 0.08% (w/w) of the dry matter and 60 g/d Reashure®), and CON (n = 20, no supplementation, fed the close-up and fresh cow diets). Follicular development was monitored via ultrasound every 2 d starting at 7 DIM until ovulation (n = 37) or aspiration (n = 40) of the first postpartum dominant follicle (DF). Follicular fluid from 40 cows was aspirated and cells were retrieved immediately by centrifugation. Gene expression of TLR4, TNF, IL1-ß, IL8, IL6, LHCGR, STAR, 3ß-HSD, P450scc, CYP19A1, IRS1, IGF, MAT1A, and SAHH, was measured in the follicular cells of the first DF. Cows in CON had higher TNF, TLR4, and IL1-ß mRNA expression (11.70 ± 4.6, 21.29 ± 10.4, 6.28 ± 1.4, respectively) than CHO (2.77 ± 0.9, 2.16 ± 0.9, 2.29 ± 0.7, respectively), and MIX (2.23 ± 0.7, 1.46 ± 0.6, 2.92 ± 0.8, respectively). Cows in CON had higher IL1-ß expression (6.27 ± 1.4) than cows in MET (3.28 ± 0.6). Expression of IL8 mRNA was lower for cows in CHO (0.98 ± 0.3) than cows in CON (4.90 ± 0.7), MET (6.10 ± 1.7), or MIX (5.05 ± 1.8). Treatments did not affect mRNA expression of LHCGR, STAR, P450scc, CYP19A, SAHH, MAT1A, or IL6 however, 3ß-HSD expression was higher for cows in MET (1.46 ± 0.3) and MIX (1.25 ± 0.3) than CON (0.17 ± 0.04) and CHO (0.26 ± 0.1). Supplementation of methionine, choline, and both methionine and choline during the transition period did not affect days to first ovulation or number of cows that ovulated the first follicular wave. Plasma and follicular fluid estradiol and progesterone concentrations were not different among treatments. Methionine concentrations in the follicular fluid of the first postpartum DF was higher for cows in MET (18.2 ± 0.1 µM) than cows in CON (11.1 ± 0.9 µM). In conclusion, supplementing choline and methionine during the transition period changed mRNA expression in follicular cells and dietary methionine supplementation increased plasma and follicular fluid concentrations of methionine of the first postpartum DF in Holstein cows.

Differences in liver functionality indexes in peripartal dairy cows fed rumen-protected methionine or choline are associated with performance, oxidative stress status, and plasma amino acid profiles.

The liver functionality index (LFI) represents an assessment of transition cow metabolic health by measuring changes in biomarkers associated with liver plasma protein synthesis (albumin), lipoprotein synthesis (cholesterol), and heme catabolism (bilirubin). The present analysis was conducted to determine the role of peripartal rumen-protected Met or choline (CHOL) supplementation on LFI groupings, and to assess relationships with performance, inflammation, oxidative stress status, and plasma AA profiles. A cohort of 40 multiparous Holstein cows that were part of a randomized complete block design with 2 × 2 factorial arrangement of Met (Smartamine M, Adisseo NA, Alpharetta, GA) and CHOL (ReaShure, Balchem Inc., New Hampton, NY) level (with or without) were used. From -21 d to calving, cows received the same close-up diet and were assigned randomly to each treatment. From calving to 30 d, cows were on the same postpartal diet and continued to receive the same treatments until 30 d. Addition of Met was adjusted daily at 0.08% dry matter of diet and CHOL was fed at 60 g/cow per day. Liver (-10, 7, 20, and 30 d) and blood (-10, 4, 8, 20, and 30 d) samples were harvested for biomarker analyses. Cows were ranked retrospectively and assigned to low (LLFI, LFI <0) and high (HLFI, LFI >0) LFI groups regardless of Met or CHOL supplementation. Compared with cows in LLFI, close-up and lactation DMI, milk yield, milk fat yield, and milk protein yield were greater in HLFI cows. As expected, cows in LLFI had lower plasma cholesterol and albumin but greater bilirubin concentrations around parturition. Plasma haptoglobin concentration was also lower in HLFI cows, but plasma paraoxonase and hepatic total and reduced hepatic glutathione concentrations were greater. Although higher concentrations of His, Met, and Trp, as well as a tendency for greater Ile, were observed in HLFI cows, overall essential AA concentrations did not differ with LFI status. In contrast, overall concentrations of nonessential AA were greater in HLFI cows due to greater circulating concentrations of Ala, Asn, Gln, Pro, and Ser. Similarly, overall concentrations of total AA and total sulfur-containing compounds were greater in cows with HLFI. Feeding Met compared with CHOL led to a tendency for more cows classified as HLFI. Overall, results support the broader application of the LFI in the management of transition cows. In that context, the fact that precalving concentrations of compounds such as reduced glutathione, total sulfur-containing compounds, Met, Tau, and homocysteine differed between HLFI and LLFI independent of Met or CHOL feeding also underscores their potential for monitoring cows that might be at a greater risk of developing health problems after calving. Further studies on the applicability of these biomarkers to monitor transition success appears warranted.

Supplementation with rumen-protected methionine or choline during the transition period influences whole-blood immune response in periparturient dairy cows.

Methionine, together with Lys, is the most limiting AA for milk production in dairy cows. Besides its crucial role in milk production, Met and its derivate metabolites (e.g., glutathione, taurine, polyamines) are well-known immunonutrients in nonruminants, helping support and boost immune function and activity. In the present study, the effects of Met or choline, as its precursor, were investigated using an ex vivo whole blood challenge. The study involved 33 multiparous Holstein cows (from a larger cohort with a factorial arrangement of treatments) assigned from d -21 to +30 relative to parturition to a basal control (CON) diet, CON plus rumen-protected Met (MET, Smartamine M, Adisseo NA, Alpharetta, GA) at a rate of 0.08% of dry matter, or CON plus rumen-protected choline (CHOL, ReaShure, Balchem Inc., New Hampton, NY) at 60 g/d. Blood was sampled on d -15, -7, 2, 7, and 20 for ex vivo lipopolysaccharide (LPS) challenge, and on d 1, 4, 14, and 28 relative to parturition for phagocytosis and oxidative burst assays. The MET cows had greater energy-corrected milk production and milk protein content. Overall, IL-6 response to LPS increased around parturition, whereas IL-1ß remained constant, casting doubt on the existence of systemic immunosuppression in the peripartal period. Supplementation with MET dampened the postpartal blood response to LPS (lower IL-1ß), while improving postpartum neutrophil and monocyte phagocytosis capacity and oxidative burst activity. In contrast, CHOL supplementation increased monocyte phagocytosis capacity. Overall, the data revealed a peripartal immune hyper-response, which appeared to have been mitigated by MET supplementation. Both MET and CHOL effectively improved immune function; however, MET affected the immune and antioxidant status before parturition, which might have been beneficial to prepare the cow to respond to metabolic challenges after parturition. These results provide insights on potential differences in the immunomodulatory action of methionine and choline in dairy cows. As such, the effects observed could have implications for ration formulation and dietary strategies.

Maternal supplementation with rumen-protected methionine increases prepartal plasma methionine concentration and alters hepatic mRNA abundance of 1-carbon, methionine, and transsulfuration pathways in neonatal Holstein calves.

An important mechanism of nutritional "programming" induced by supplementation with methyl donors during pregnancy is the alteration of mRNA abundance in the offspring. We investigated the effects of rumen-protected Met (RPM) on abundance of 17 genes in the 1-carbon, Met, and transsulfuration pathways in calf liver from cows fed the same basal diet without (control, CON) or with RPM at 0.08% of diet dry matter/d (MET) from -21 through +30 d around calving. Biopsies (n = 8 calves per diet) were harvested on d 4, 14, 28, and 50 of age. Cows fed RPM had greater plasma concentration of Met (17.8 vs. 28.2 µM) at -10 d from calving. However, no difference was present in colostrum yield and free AA concentrations. Greater abundance on d 4 and 14 of betaine-homocysteine S-methyltransferase 2 (BHMT2), adenosylhomocysteinase (AHCY; also known as SAHH), and cystathionine-ß-synthase (CBS) in MET calves indicated alterations in Met, choline, and homocysteine metabolism. Those data agree with the greater abundance of methionine adenosyltransferase 1A (MAT1A) in MET calves. Along with CBS, the greater abundance of glutamate-cysteine ligase (GCLC) and glutathione reductase (GSR) on d 4 in MET calves indicated a short-term postnatal alteration in the use of homocysteine for taurine and glutathione synthesis (both are potent intracellular antioxidants). The striking 7-fold upregulation at d 50 versus 4 of cysteine sulfinic acid decarboxylase (CSAD), catalyzing the last step of taurine synthesis, in MET and CON calves underscores an important role of taurine during postnatal calf growth. The unique role of taurine in the young calf is further supported by the upregulation of CBS, GCLC, and GSR at d 50 versus 14 and 28 in MET and CON. Although betaine-homocysteine S-methyltransferase (BHMT) activity did not differ in MET and CON, it increased ∼50% at d 14 and 28 versus 4. A significant positive correlation (r = 0.79) was present between BHMT abundance and BHMT activity regardless of treatment. The gradual upregulation over time of BHMT2 and SAHH coupled with the gradual upregulation of MAT1A and the DNA (cytosine-5-)-methyltransferases (DNMT1, DNMT3A, DNMT3B) in MET and CON calves was indicative of adaptations potentially driven by differences in intake of milk replacer and starter feed as calves grew. In that context, the ∼2.5-fold increase in abundance of DNMT3B at d 50 versus 4 in MET and CON indicate that DNA methylation might be an important component of the physiologic adaptations of calf liver. The data indicate that calves from MET-supplemented cows underwent alterations in Met, choline, and homocysteine metabolism partly to synthesize taurine and glutathione, which would be advantageous for controlling metabolic-related stress. Whether the effects in MET calves were directly related to increased Met supply in utero remains to be determined.

Rumen-protected methionine compared with rumen-protected choline improves immunometabolic status in dairy cows during the peripartal period.

The immunometabolic status of peripartal cows is altered due to changes in liver function, inflammation, and oxidative stress. Nutritional management during this physiological state can affect the biological components of immunometabolism. The objectives of this study were to measure concentrations of biomarkers in plasma, liver tissue, and milk, and also polymorphonuclear leukocyte function to assess the immunometabolic status of cows supplemented with rumen-protected methionine (Met) or choline (CHOL). Forty-eight multiparous Holstein cows were used in a randomized complete block design with 2×2 factorial arrangement of Met (Smartamine M, Adisseo NA, Alpharetta, GA) and CHOL (ReaShure, Balchem Inc., New Hampton, NY) level (with or without). Treatments (12 cows each) were control (CON), no Met or CHOL; CON and Met (SMA); CON and CHOL (REA); and CON and Met and CHOL (MIX). From -50 to -21d before expected calving, all cows received the same diet [1.40Mcal of net energy for lactation (NEL)/kg of DM] with no Met or CHOL. From -21d to calving, cows received the same close-up diet (1.52Mcal of NEL/kg of DM) and were assigned randomly to each treatment. From calving to 30d, cows were on the same postpartal diet (1.71Mcal of NEL/kg of DM) and continued to receive the same treatments until 30d. The Met supplementation was adjusted daily at 0.08% DM of diet, and CHOL was supplemented at 60g/cow per day. Liver (-10, 7, 21, and 30d) and blood (-10, 4, 8, 20, and 30d) samples were harvested for biomarker analyses. Neutrophil and monocyte phagocytosis and oxidative burst were assessed at d 1, 4, 14, and 28d. The Met-supplemented cows tended to have greater plasma paraoxonase. Greater plasma albumin and IL-6 as well as a tendency for lower haptoglobin were detected in Met- but not CHOL-supplemented cows. Similarly, cows fed Met compared with CHOL had greater concentrations of total and reduced glutathione (a potent intracellular antioxidant) in liver tissue. Upon a pathogen challenge in vitro, blood polymorphonuclear leukocyte phagocytosis capacity and oxidative burst activity were greater in Met-supplemented cows. Overall, liver and blood biomarker analyses revealed favorable changes in liver function, inflammation status, and immune response in Met-supplemented cows.

Better postpartal performance in dairy cows supplemented with rumen-protected methionine compared with choline during the peripartal period.

The onset of lactation in dairy cows is characterized by high output of methylated compounds in milk when sources of methyl group are in short supply. Methionine and choline (CHOL) are key methyl donors and their availability during this time may be limiting for milk production, hepatic lipid metabolism, and immune function. Supplementing rumen-protected Met and CHOL may improve overall performance and health of transition cows. The objective of this study was to evaluate the effect of supplemental rumen-protected Met and CHOL on performance and health of transition cows. Eighty-one multiparous Holstein cows were used in a randomized, complete, unbalanced block design with 2×2 factorial arrangement of Met (Smartamine M, Adisseo NA, Alpharetta, GA) and CHOL (ReaShure, Balchem Inc., New Hampton, NY) inclusion (with or without). Treatments (20 to 21 cows each) were control (CON), CON+Met (SMA), CON+CHOL (REA), and CON+Met+CHOL (MIX). From -50 to -21d before expected calving, all cows received the same diet (1.40Mcal of NEL/kg of DM) with no Met or CHOL. From -21d to calving, cows received the same close-up diet (1.52Mcal of NEL/kg of DM) and were assigned randomly to treatments (CON, SMA, REA, or MIX) supplied as top dresses. From calving to 30 DIM, cows were fed the same postpartal diet (1.71Mcal of NEL/kg of DM) and continued to receive the same treatments through 30 DIM. The Met supplementation was adjusted daily at 0.08% DM of diet and REA was supplemented at 60g/d. Incidence of clinical ketosis and retained placenta tended to be lower in Met-supplemented cows. Supplementation of Met (SMA, MIX) led to greater DMI compared with other treatments (CON, REA) in both close-up (14.3 vs. 13.2kg/d, SEM 0.3) and first 30d postpartum (19.2 vs. 17.2kg/d, SEM 0.6). Cows supplemented with Met (SMA, MIX) had greater yields of milk (44.2 vs. 40.4kg/d, SEM 1.2), ECM (44.6 vs. 40.5kg/d, SEM 1.0), and FCM (44.6 vs. 40.8kg/d, SEM 1.0) compared with other (CON, REA) treatments. Milk fat content did not differ in response to Met or CHOL. However, milk protein content was greater in Met-supplemented (3.32% vs. 3.14%, SEM 0.04%) but not CHOL-supplemented (3.27 vs. 3.19%, SEM 0.04%) cows. Supplemental CHOL led to greater blood glucose and insulin concentrations with lower glucose:insulin ratio. No Met or CHOL effects were detected for blood fatty acids or BHB, but a Met × time effect was observed for fatty acids due to higher concentrations on d 20. Results from the present study indicate that peripartal supplementation of rumen-protected Met but not CHOL has positive effects on cow performance.

Maternal rumen-protected methionine supplementation and its effect on blood and liver biomarkers of energy metabolism, inflammation, and oxidative stress in neonatal Holstein calves.

In nonruminants, nutrition during pregnancy can program offspring development, metabolism, and health in later life. Rumen-protected Met (RPM) supplementation during the prepartum period improves liver function and immune response in dairy cows. Our aim was to investigate the effects of RPM during late pregnancy on blood biomarkers (23 targets) and the liver transcriptome (24 genes) in neonatal calves from cows fed RPM at 0.08% of diet dry matter/d (MET) for the last 21 d before calving or controls (CON). Blood (n=12 calves per diet) was collected at birth before receiving colostrum (baseline), 24 h after receiving colostrum, 14, 28, and 50 d (post-weaning) of age. Liver was sampled (n=8 calves per diet) via biopsy on d 4, 14, 28, and 50 of age. Growth and health were not affected by maternal diet. The MET calves had greater overall plasma insulin concentration and lower glucose and ratios of glucose-to-insulin and fatty acids-to-insulin, indicating greater systemic insulin sensitivity. Lower concentration of reactive oxygen metabolites at 14 d of age along with a tendency for lower overall concentration of ceruloplasmin in MET calves indicated a lesser degree of stress. Greater expression on d 4 of fructose-bisphosphatase 1 (FBP1), phosphoenolpyruvate carboxykinase 1 (PCK1), and the facilitated bidirectional glucose transporter SLC2A2 in MET calves indicated alterations in gluconeogenesis and glucose uptake and release. The data agree with the greater expression of the glucocorticoid receptor (GR). Greater expression on d 4 of the insulin receptor (INSR) and insulin-responsive serine/threonine-protein kinase (AKT2) in MET calves indicated alterations in insulin signaling. In that context, the similar expression of sterol regulatory element-binding transcription factor 1 (SREBF1) in CON and MET during the preweaning period followed by the marked upregulation regardless of diet after weaning (d 50) support the idea of changes in hepatic insulin sensitivity during early postnatal life. Expression of carnitine palmitoyltransferase 1A (CPT1A) was overall greater and acyl-CoA oxidase 1 (ACOX1) was lower in MET calves, indicating alterations in fatty acid oxidation. Except forkhead box O1 (FOXO1), all genes changed in expression over time. Transcriptome results indicated that calves from MET-supplemented cows underwent a faster maturation of gluconeogenesis and fatty acid oxidation in the liver, which would be advantageous for adapting to the metabolic demands of extrauterine life.

Effects of rumen-protected methionine and choline supplementation on the preimplantation embryo in Holstein cows.

Our objective was to determine the effects of supplementing methionine and choline during the prepartum and postpartum periods on preimplantation embryos of Holstein cows. Multiparous cows were assigned in a randomized complete-block design into four treatments from 21 days before calving to 30 days in milk (DIM). Treatments (TRT) were MET (n = 9, fed the basal diet + rumen-protected methionine at a rate of 0.08% [w:w] of the dry matter [DM], Smartamine M), CHO (n = 8, fed the basal diet + choline 60 g/d, Reashure), MIX (n = 11, fed the basal diet + Smartamine M and 60 g/d Reashure), and CON (n = 8, no supplementation, fed the close-up and fresh cow diets). Cows were randomly reassigned to two new groups (GRP) to receive the following diets from 31 to 72 DIM; control (CNT, n = 16, fed a basal diet) and SMT (n = 20, fed the basal diet + 0.08% [w:w] of the dry matter intake as methionine). An progesterone intravaginal insert (CIDR) device was inserted in all cows after follicular aspiration (60 DIM) and superovulation began at Day 61.5 using FSH in eight decreasing doses at 12-hour intervals over a 4-day period. On Days 63 and 64, all cows received two injections of PGF2α, and CIDR was removed on Day 65. Twenty-four hours after CIDR removal, ovulation was induced with GnRH. Cows received artificial insemination at 12 hours and 24 hours after GnRH. Embryos were flushed 6.5 days after artificial insemination. Global methylation of the embryos was assessed by immunofluorescent labeling of 5-methylcytosine, whereas lipid content was assessed by staining with Nile red. Nuclear staining was used to count the total number of cells per embryo. There was no difference between TRT, GRP, or their interaction (P > 0.05) for embryo recovery, embryos recovered, embryo quality, embryo stage, or cells per embryo. Methylation of the DNA had a TRT by GRP interaction (P = 0.01). Embryos from cows in CON-CNT had greater (P = 0.04) methylation (0.87 ± 0.09 arbitrary units [AU]) than embryos from cows in MET-CNT (0.44 ± 0.07 AU). The cytoplasmic lipid content was not affected (P > 0.05) by TRT or their interaction, but lipid content was greater (P = 0.04) for SMT (7.02 ± 1.03 AU) than that in CNT (3.61 ± 1.20 AU). In conclusion, cows in MET-CNT had embryos with lower methylation, and SMT cows had a higher lipid content than CNT. Methionine supplementation seems to impact the preimplantation embryo in a way that enhances its capacity for survival because there is strong evidence that endogenous lipid reserves serve as an energy substrate.

Hepatic global DNA and peroxisome proliferator-activated receptor alpha promoter methylation are altered in peripartal dairy cows fed rumen-protected methionine.

The availability of Met in metabolizable protein (MP) of a wide range of diets for dairy cows is low. During late pregnancy and early lactation, in particular, suboptimal Met in MP limits its use for mammary and liver metabolism and also for the synthesis of S-adenosylmethionine, which is essential for many biological processes, including DNA methylation. The latter is an epigenetic modification involved in the regulation of gene expression, hence, tissue function. Thirty-nine Holstein cows were fed throughout the peripartal period (-21 d to 30 d in milk) a basal control (CON) diet (n=14) with no Met supplementation, CON plus MetaSmart (MS; Adisseo NA, Alpharetta, GA; n=12), or CON plus Smartamine M (SM; Adisseo NA; n=13). The total mixed ration dry matter for the close-up and lactation diets was measured weekly, then the Met supplements were adjusted daily and top-dressed over the total mixed ration at a rate of 0.19 (MS) or 0.07% (SM) on a dry matter basis. Liver tissue was collected on -10, 7, and 21 d for global DNA and peroxisome proliferator-activated receptor alpha (PPARα) promoter region-specific methylation. Several PPARα target and putative target genes associated with carnitine synthesis and uptake, fatty acid metabolism, hepatokines, and carbohydrate metabolism were also studied. Data were analyzed using PROC MIXED of SAS (SAS Institute Inc., Cary, NC) with the preplanned contrast CON versus SM + MS. Global hepatic DNA methylation on d 21 postpartum was lower in Met-supplemented cows than CON. However, of 2 primers used encompassing 4 to 12 CpG sites in the promoter region of bovine PPARA, greater methylation occurred in the region encompassing -1,538 to -1,418 from the transcription start site in cows supplemented with Met. Overall expression of PPARA was greater in Met-supplemented cows than CON. Concomitantly, PPARA-target genes, such as ANGPTL4, FGF21, and PCK1, were also upregulated overall by Met supplementation. The upregulation of PPARα target genes indicates that supplemental Met, likely through the synthesis of S-adenosylmethionine, activated PPARA-regulated signaling pathways. Upregulation of hepatic PPARA has been associated with improved lipid metabolism and immune function, both of which were reported in companion publications from this study. In turn, those positive effects resulted in improved postpartal health and performance. Further research is needed to study more closely the mechanistic connections between global DNA and promoter region-specific PPARA methylation with PPARA expression and functional outcomes in liver.