Alterations in skeletal muscle abundance of protein turnover, stress, and antioxidant proteins during the periparturient period in dairy cows fed ethyl-cellulose rumen-protected methionine.

Skeletal muscle turnover helps support the physiological needs of dairy cows during the transition into lactation. We evaluated effects of feeding ethyl-cellulose rumen-protected methionine (RPM) during the periparturient period on abundance of proteins associated with transport AA and glucose, protein turnover, metabolism, and antioxidant pathways in skeletal muscle. Sixty multiparous Holstein cows were used in a block design and assigned to a control or RPM diet from -28 to 60 d in milk. The RPM was fed at a rate of 0.09% or 0.10% of dry matter intake (DMI) during the prepartal and postpartal periods to achieve a target Lys:Met ratio in the metabolizable protein of ∼2.8:1. Muscle biopsies from the hind leg of 10 clinically healthy cows per diet collected at -21, 1, and 21 d relative to calving were used for western blotting of 38 target proteins. Statistical analysis was performed using the PROC MIXED statement of SAS version 9.4 (SAS Institute Inc.) with cow as random effect, whereas diet, time, and diet × time were the fixed effects. Diet × time tended to affect prepartum DMI, with RPM cows consuming 15.2 kg/d and controls 14.6 kg/d. However, diet had no effect on postpartum DMI (17.2 and 17.1 ± 0.4 kg/d for control and RPM, respectively). Milk yield during the first 30 d in milk was also not affected by diet (38.1 and 37.5 ± 1.9 kg/d for control and RPM, respectively). Diet or time did not affect the abundance of several AA transporters or the insulin-induced glucose transporter (SLC2A4). Among evaluated proteins, feeding RPM led to lower overall abundance of proteins associated with protein synthesis (phosphorylated EEF2, phosphorylated RPS6KB1), mTOR activation (RRAGA), proteasome degradation (UBA1), cellular stress responses (HSP70, phosphorylated MAPK3, phosphorylated EIF2A, ERK1/2), antioxidant response (GPX3), and de novo synthesis of phospholipids (PEMT). Regardless of diet, there was an increase in the abundance of the active form of the master regulator of protein synthesis phosphorylated MTOR and the growth-factor-induced serine/threonine kinase phosphorylated AKT1 and PIK3C3, whereas the abundance of a negative regulator of translation (phosphorylated EEF2K) decreased over time. Compared with d 1 after calving and regardless of diet, the abundance of proteins associated with endoplasmic reticulum stress (XBP1 spliced), cell growth and survival (phosphorylated MAPK3), inflammation (transcription factor p65), antioxidant responses (KEAP1), and circadian regulation (CLOCK, PER2) of oxidative metabolism was upregulated at d 21 relative to parturition. These responses coupled with the upregulation of transporters for Lys, Arg, and His (SLC7A1) and glutamate/aspartate (SLC1A3) over time were suggestive of dynamic adaptations in cellular functions. Overall, management approaches that could take advantage of this physiological plasticity may help cows make a smoother transition into lactation.

Alterations in one­carbon metabolism and protein synthesis signals due to methionine supplementation and lipopolysaccharide challenge in Holstein fetal liver explants.

One­carbon metabolism (OCM) fueled by methionine (Met), choline, and folic acid is key for embryo development and fetal growth. We investigated effects of lipopolysaccharide (LPS) to induce inflammation in fetal liver tissue with or without Met on components of OCM and protein synthesis activity. Fetal liver harvested at slaughter from six multiparous pregnant Holstein dairy cows (37 ± 6 kg milk/d, 100 ± 3 d gestation) were incubated (0.2 ± 0.02 g) for 4 h at 37 °C with each of the following: ideal profile of amino acids (control; Lysine:Met 2.9:1), control plus LPS (1 µg/mL), increased Met supply (Met, Lys:Met 2.5:1), and Met+LPS. Data were analyzed as a 2 × 2 factorial (PROC MIXED, SAS 9.4). Ratios of mechanistic target of rapamycin (p-mTOR:mTOR) and eukaryotic elongation factor 2 (p-eEF2:eEF2) protein were lowest (P < 0.0 5) with LPS and highest with Met. Tissue amino acid concentrations were lowest (P < 0.0 5) with Met regardless of LPS suggesting enhanced use via mTOR. The marked increase (P = 0.02) in phosphorylation of S6 ribosomal protein (p-RPS6) with LPS suggested a pro-inflammatory response that was partly alleviated with Met+LPS. No effect (P = 0.4 5) on methionine adenosyl transferase 1 A (MAT1A) protein abundance was detected. Activity of betaine-homocysteine S-methyltransferase (BHMT) was greatest with Met, but Met+LPS dampened this effect (P = 0.0 5). Overall, fetal liver responds to inflammatory challenges and Met supply. The latter can stimulate protein synthesis via mTOR and alter some OCM reactions while having a modest anti-inflammatory effect.

Total Replacement of Soybean Meal with Sundried Soymilk Residue in the Total Mixed Ration has a Negative Impact on Intake, Digestibility, and Milk Production in Dairy Goats.

This study aimed to evaluate whether total replacement of soybean meal (SBM) with sundried soymilk residue (SSR) in a total mixed ration (TMR) affects intake, digestibility, milk production, and blood metabolites in dairy goats. A total of 12 healthy Saanen dairy goats (40.12 ± 5.80 kg of BW) in midlactation (31.23 ± 10.12 days) were used in a randomized complete design (n = 4 goats/group). Dietary treatments were based on a TMR as follows: control TMR without SSR (CON) or SBM-based TMR with 50% or 100% of SSR replacing SBM (SSR-50 and SSR-100, respectively). All goats had ad libitum access to feed and clean water throughout the experiment. The dry matter (DM) intake decreased (p < 0.05) with the increasing replacement ratio of SBM and was lowest in the SSR-100 group. Similarly, organic matter (OM) digestibility was lowest (p < 0.05) in the SSR-100 group. However, the digestibility of DM, CP, NDF, and ADF did not change (p > 0.05) by dietary treatments. Compared with CON, the milk yield decreased significantly (p < 0.05) with increasing replacement ratio of SBM. In contrast, milk composition such as total solids, solids-not-fat, milk fat, lactose, protein, and pH were not influenced (p > 0.05) by feeding dietary SSR. Compared with other treatments, blood glucose concentration was lower (p < 0.05) in the SSR-100 group. In contrast, packed cell volume, glucose, and plasma urea nitrogen concentrations did not differ (p > 0.05). The results indicated that SSR could replace SBM in a TMR at less than 50%. Thus, the present study provides support for further investigation to enhance the utilization of soybean waste as an alternative protein source in the TMR for dairy goats and potentially other ruminants.

Abundance of proteins and genes associated with nutrient signaling, protein turnover, and transport of amino acids and glucose in fetuses from lactating Holstein cows.

Availability of nutrients in maternal circulation and abundance of nutrient transporters, metabolic enzymes, and nutrient-responsive proteins in fetal tissues coordinate growth. To begin characterizing these mechanisms, we evaluated the abundance of nutrient signaling genes and proteins in bovine fetal tissues. Liver, entire intestine, and semitendinosus muscle were harvested from fetuses (4 female, 2 male) collected at slaughter from 6 clinically-healthy multiparous Holstein dairy cows (167 ± 7 days in milk, 37 ± 6 kg milk/d, 100 ± 3 d gestation). Data were analyzed using PROC MIXED in SAS 9.4. Among proteins measured, abundance of the amino acid (AA) utilization and insulin signaling proteins p-AKT and p-mTOR was greater (P < 0.01) in liver and intestine. The abundance of p-EEF2 (translation elongation) and SLC2A4 (glucose uptake) was greater (P < 0.05) in liver relative to intestine and muscle suggesting this organ has a greater capacity for anabolic processes. In contrast, among mTOR signaling genes, the abundance of IRS1 was greatest (P < 0.01) in muscle and lowest in the intestine, whereas, abundance of AKT1 and mTOR was greater (P < 0.01) in intestine and muscle than liver. Abundance of the protein degradation-related genes UBA1, UBE2G1, and TRIM63 was greater (P < 0.01) in muscle than intestine and liver. Among nutrient transporters, abundance of glucose transporters SLC5A1 and SLC2A2 was greatest (P < 0.01) in the intestine than liver and muscle. Several AA transporters had greater (P < 0.01) abundance in the intestine or liver compared with muscle. Overall, these molecular analyses highlighted important biological differences on various aspects of metabolism in fetal tissues.

Body condition prepartum and its association with term placentome nutrient transporters, one­carbon metabolism pathway activity, and intermediate metabolites in Holstein cows.

We investigated linkages among BCS prior to calving and placentome concentrations of metabolites, proteins in one­carbon metabolism (OCM) and protein synthesis, and nutrient transport. Multiparous Holstein cows retrospectively divided by prepartal BCS at -4 weeks relative to parturition into high BCS (HBCS = 3.58 ± 0.23; n = 9) or normal BCS (NBCS = 3.02 ± 0.17; n = 13) were used. BCS was assessed using a 5-point scale (1 = thin, 5 = fat). Four placentomes per cow were collected at delivery and frozen in liquid N. Western blotting was used for protein abundance. Cystathionine-ß-synthase (CBS) and betaine-homocysteine-S-methyltransferase (BHMT) activity were measured via 14C assays. Amino acids (AA) and metabolites in OCM were measured by liquid chromatography mass spectrometry (LC-MS). Compared with NBCS cows, the cellular stress sensor p-eIF2α was more than 2-fold greater (P = 0.04) in HBCS. Abundance of the AA-catabolism enzyme branched-chain α-ketoacid dehydrogenase complex was lower (P = 0.05) in HBCS cows. Although BHMT activity did not differ, greater concentration of betaine (P = 0.01) and lower (P = 0.05) concentration of dimethylglycine in HBCS cows suggested reduced flux through the methionine cycle. Despite a lack of difference in CBS activity, lower concentrations of cystathionine (P = 0.03) and hypotaurine (P = 0.04) along with lower cysteine and the tendency for lower total GSH (P = 0.10) in HBCS cows suggested a decrease in transsulfuration. Overall, associations between OCM in placentomes and BCS at calving exist. Identifying mechanisms responsible for these effects merits further research.

Intermediate metabolites and molecular correlates of one­carbon and nutrient metabolism differ in tissues from Holstein fetuses.

Methionine and folate cycles along with transsulfuration comprise the one­carbon metabolism (OCM) pathway. Amino acids and other nutrients feed into OCM, which is central to cellular function. mRNA abundance, proteins (Western blotting), and metabolites (GC-MC) associated with OCM were used to characterize these mechanisms in fetal tissues. Liver, whole intestine, and semitendinosus muscle were harvested from fetuses in 6 multiparous Holstein cows (37 kg milk/d, 100 d gestation). Data were analyzed using PROC MIXED (SAS 9.4). Protein abundance of BHMT was greatest (P < 0.01) in liver suggesting active remethylation of homocysteine to methionine. This idea was supported by the greater (P < 0.05) mRNA of CBS, BHMT, MTR, SHMT1, and MAT1A (encoding OCM enzymes) in liver. The antioxidant protein GPX3 had greatest (P < 0.05) abundance in liver, whereas the glutathione-transferase GSTM1 was 5-fold greater (P < 0.05) in intestine than liver and muscle. Greatest concentrations of glycine, serine, and taurine along with lower cysteine underscored the relevance of OCM in fetal liver. Phosphoethanolamine concentration was greatest (4-fold, P < 0.05) in intestine and along with the greatest (P < 0.05) mRNA of SLC44A1 (choline transporter), CHKA, and CEPT1 underscored the importance of the CDP-choline pathway. Greatest (P < 0.05) mRNA of PPARA, CPT1A, and HMGCS2 along with lower PCK1 in liver highlighted a potential reliance on fatty acid oxidation. In contrast, greater (P < 0.05) concentration of myo-inositol in muscle and intestine suggested both tissues rely on glucose as main source of energy. Future research should address how environmental inputs such as maternal nutrition alter these pathways in fetal tissues and their phenotypic outcomes.

Alterations in Skeletal Muscle mRNA Abundance in Response to Ethyl-Cellulose Rumen-Protected Methionine during the Periparturient Period in Dairy Cows.

This study aimed to evaluate the effect of feeding ethyl cellulose rumen-protected methionine (RPM) on skeletal muscle mRNA abundance during the periparturient period. Sixty multiparous Holstein cows were used in a block design and assigned to either a control or RPM diet. The RPM was supplied from −28 to 60 days in milk (DIM) at a rate of 0.09% (prepartum) or 0.10% (postpartum) of dry matter (DM), ensuring a Lys:Met in the metabolizable protein of ~2.8:1. Muscle biopsies were collected at −21, 1, and 21 DIM. Thirty-five target genes associated with nutrient metabolism and biochemical pathways were measured via RT-qPCR. The mRNA abundance of genes associated with amino acid (AA) transport (SLC7A8, SLC43A2), carnitine transport (SLC22A5), insulin signaling (IRS1), and antioxidant response (NFE2L2) had diet × time effect (p < 0.05) due to greater abundance in RPM versus CON cows, especially at 1 and 21 DIM. Members of the AA transport (SLC7A8, SLC25A29, SCL38A9), fatty acid ß-oxidation (ACADVL), vitamin transport (SLC5A6, SLC19A2), mTOR pathway (AKT1 and mTOR), antioxidant response (KEAP1, CUL3), CDP-Choline pathway and arginine metabolism had overall greater abundance (p < 0.05) in RPM versus CON cows. Overall, data indicate that RPM can alter nutrient metabolism in the skeletal muscle around parturition partly through alterations in mRNA abundance.