Characterization of rumen microbiome and metabolome from oro-esophageal tubing and rumen cannula in Holstein dairy cows.

Less invasive rumen sampling methods, such as oro-esophageal tubing, became widely popular for exploring the rumen microbiome and metabolome. However, it remains unclear if such methods represent well the rumen contents from the rumen cannula technique. Herein, we characterized the microbiome and metabolome in the rumen content collected by an oro-esophageal tube and by rumen cannula in ten multiparous lactating Holstein cows. The 16S rRNA gene was amplified and sequenced using the Illumina MiSeq platform. Untargeted metabolome was characterized using gas chromatography of a time-of-flight mass spectrometer. Bacteroidetes, Firmicutes, and Proteobacteria were the top three most abundant phyla representing ~ 90% of all samples. Although the pH of oro-esophageal samples was greater than rumen cannula, we found no difference in alpha and beta-diversity among their microbiomes. The overall metabolome of oro-esophageal samples was slightly different from rumen cannula samples yet more closely related to the rumen cannula content as a whole, including its fluid and particulate fractions. Enrichment pathway analysis revealed a few differences between sampling methods, such as when evaluating unsaturated fatty acid pathways in the rumen. The results of the current study suggest that oro-esophageal sampling can be a proxy to screen the 16S rRNA rumen microbiome compared to the rumen cannula technique. The variation introduced by the 16S rRNA methodology may be mitigated by oro-esophageal sampling and the possibility of increasing experimental units for a more consistent representation of the overall microbial population. Studies should consider an under or over-representation of metabolites and specific metabolic pathways depending on the sampling method.

Rumen-protected methionine supplementation alters lipid profile of preimplantation embryo and endometrial tissue of Holstein cows.

Our objective is to evaluate the effects of feeding rumen-protected Met (RPM) throughout the transition period and early lactation on the lipid profile of the preimplantation embryos and the endometrial tissue of Holstein cows. Treatments consisted of feeding a total mixed ration with top-dressed RPM (Smartamine® M, Adisseo, Alpharetta, GA, United States; MET; n = 11; RPM at a rate of 0.08% of DM: Lys:Met = 2.8:1) or not (CON; n = 9, Lys:Met = 3.5:1). Endometrial biopsies were performed at 15, 30, and 73 days in milk (DIM). Prior to the endometrial biopsy at 73 DIM, preimplantation embryos were harvested via flushing. Endometrial lipid profiles were analyzed using multiple reaction monitoring-profiling and lipid profiles of embryos were acquired using matrix assisted laser desorption/ionization mass spectrometry. Relative intensities levels were used for principal component analysis. Embryos from cows in MET had greater concentration of polyunsaturated lipids than embryos from cows in CON. The endometrial tissue samples from cows in MET had lesser concentrations of unsaturated and monounsaturated lipids at 15 DIM, and greater concentration of saturated, unsaturated (specifically diacylglycerol), and monounsaturated (primarily ceramides) lipids at 30 DIM than the endometrial tissue samples from cows in CON. In conclusion, feeding RPM during the transition period and early lactation altered specific lipid classes and lipid unsaturation level of preimplantation embryos and endometrial tissue.

Effects of Autolyzed Yeast Supplementation in a High-Starch Diet on Rumen Health, Apparent Digestibility, and Production Variables of Lactating Holstein Cows.

Fifteen multiparous rumen-cannulated Holstein cows were assigned to one of five treatments in a replicated 5 × 5 Latin square design. The treatments were low-starch (LS) (22.8 ± 1% of dry matter; DM) without autolyzed yeast (AY; LS0, control), high-starch (HS) (31.2 ± 4% of DM) without AY (HS0), and HS with either 15 g (HS15), 30 g (HS30), or 45 g (HS45) of AY supplementation. Cows in HS0 had increased (p < 0.03) dry matter intake (DMI; 24.9 kg/d) and energy-corrected milk (ECM; 34.4 kg/d) compared to cows in LS0 (19.9 and 31.3 kg/d, respectively). There was a tendency for a quadratic treatment effect for feed efficiency (ECM/DMI, p = 0.07) and crude protein (CP) apparent digestibility (AD) (p = 0.09). Cows in HS45 tended (p = 0.09) to have increased DMI (25.6 kg/d) compared to cows in HS0 (24.9 kg/d). Cows in HS0 had greater (p < 0.04) milk protein nitrogen (N; 166 g/d) and microbial N production (161 g/d) than those in LS0 (140 and 138 g/d, respectively). In conclusion, the addition of AY tended to improve DMI, feed efficiency, and CP AD when cows were fed the HS diet.

Increased supply of methionine during a heat-stress challenge in lactating holstein cows alters mammary tissue mTOR signaling and its response to lipopolysaccharide.

The first objective was to investigate the effects of feeding rumen-protected methionine (RPM) during a heat stress (HS) challenge on abundance and phosphorylation of mechanistic target of rapamycin (mTOR)-related signaling proteins in mammary gland. The second objective was to investigate how HS and RPM may modulate the response of mammary gland explants to an inflammatory challenge using lipopolysaccharide (LPS). Thirty-two multiparous, lactating Holstein cows (184 ± 59 DIM) were randomly assigned to 1 of 2 environmental treatment groups, and 1 of 2 dietary treatments [TMR with RPM (Smartamine M; Adisseo Inc.; 0.105% DM as top dress) or TMR without RPM (CON)] in a crossover design. There were two periods with two phases per period. In phase 1 (9 d), all cows were in thermoneutral conditions (TN) and fed ad libitum. During phase 2 (9 d), group 1 (n = 16) cows were exposed to HS using electric heat blankets, whereas group 2 cows (n = 16) remained in TN but were pair-fed to HS counterparts to control for DMI decreases associated with HS. After a washout period (14 d), the study was repeated (period 2). Environmental treatments were inverted in period 2 (sequence), whereas dietary treatments remained the same. Mammary tissue was harvested via biopsy at the end of both periods. Tissue was used for protein abundance analysis and also for incubation with 0 or 3 µg/mL of LPS for 2 h and subsequently used for mRNA abundance. Data were analyzed using PROC MIXED in SAS. Analysis of protein abundance data included the effects of diet, environment and their interaction, and period and sequence to account for the crossover design. The explant data model also included the effect of LPS and its interaction with environment and diet. Abundance of phosphorylated mTOR and ratio of phosphorylated eukaryotic translation elongation factor 2 (p-EEF2) to total EEF2 in non-challenged tissue was greater with RPM supplementation (P = 0.04 for both) and in both cases tended to be greater with HS (P = 0.08 for both). Regardless of RPM supplementation, incubation with LPS upregulated mRNA abundance of IL8, IL6, IL1B, CXCL2, TNF, NFKB1, and TLR2 (P < 0.05). An environment × LPS interaction was observed for NFKB1 (P = 0.03); abundance was greater in LPS-treated explants from non-HS compared with HS cows. Abundance of CXCL2, NFKB1, NOS2, NOS1, and SOD2 was lower with HS (P < 0.05). Although LPS did not alter mRNA abundance of the antioxidant transcription factor NFE2L2 (P = 0.59), explants from HS cows had lower abundance of NFE2L2 (P < 0.001) and CUL3 (P = 0.04). Overall, RPM supplementation may alter mTOR activation in mammary tissue. Additionally, although HS reduced explant immune and antioxidant responses, RPM did not attenuate the inflammatory response induced by LPS in vitro.

Heat stress (HS) is an environmental issue worldwide and occurs when animals experience a heat load that exceeds their thermoregulatory capacity. Milk protein synthesis and overall production often decrease when cows are exposed to HS conditions, in part due to lower feed intake and a limit in the mammary supply of amino acids. Increasing post-ruminal supply of methionine to late-lactation cows upregulated abundance of p-mTOR in mammary tissue, providing a link with the greater milk protein production. Exposure of cows to a HS challenge also increased abundance of p-mTOR, but did not alter milk protein suggesting this response might have been associated with synthesis of other proteins. Further work at a translational level is needed to understand potential mechanisms whereby methionine may modulate mammary metabolism during periods of HS.

Insulin signaling and antioxidant proteins in adipose tissue explants from dairy cows challenged with hydrogen peroxide are altered by supplementation of arginine or arginine plus methionine.

Arginine (Arg) and methionine (Met) can elicit anti-inflammatory and antioxidant effects in animals. Unlike Met, however, it is unknown if the supply of Arg can impact key aspects of adipose tissue (AT) function in dairy cows. Since Met and Arg metabolism are linked through the synthesis of polyamines, it is also possible that they have a complementary effect on aspects of AT function during a stress challenge. In this experiment, subcutaneous AT was harvested from four lactating multiparous Holstein cows (~27.0 kg milk per day, body condition score 3.38 ± 0.23) and used for incubations (4 h) with the following: control medium with an "ideal" profile of essential amino acids (IPAA; CTR; Lys:Met 2.9:1), IPAA plus 100 µM H2O2 (HP), H2O2 plus greater Arg supply (HPARG; Lys:Arg 1:1), or H2O2 plus greater Arg and methionine (Met) supply (HPARGMET; Lys:Met 2.5:1 and Lys:Arg 1:1). Western blotting was used to measure abundance of 18 protein targets associated with insulin and AA signaling, nutrient transport, inflammation, and antioxidant response. Reverse transcription polymerase chain reaction (RT-PCR) was used to assess effects on genes associated with Arg metabolism. Among the protein targets measured, although abundance of phosphorylated (p) AKT serine/threonine kinase (P = 0.05) and p-mechanistic target of rapamycin (P = 0.04) were lowest in HP explants, this effect was attenuated in HPARG and especially HPARGMET compared with CTR. Compared with HP, incubation with HPARG led to upregulation of the AA transporter solute carrier family 1 member 3 (L-glutamate transporter; P = 0.03), the reactive oxygen species detoxification-related enzyme glutathione S-transferase mu 1 (GSTM1; P = 0.03), and fatty acid synthase (P = 0.05). Those effects were accompanied by greater abundance of solute carrier family 2 member 4 (insulin-induced glucose transporter) in explants incubated with HPARG and also HPARGMET (P = 0.04). In addition, compared with other treatments, the peak response in abundance of the intracellular energy sensor 5'-prime-AMP-activated protein kinase was detected with HPARGMET (P = 0.003). There was no effect of Arg or Arg plus Met on the mRNA abundance of genes associated with Arg metabolism (ARG1, NOS2, AMD1, SMS, and SRM). Overall, supplementation of Arg alone or with Met partially alleviated the negative effects induced by H2O2. More systematic studies need to be conducted to explore the function of Arg supply with or without Met on AT function.

In nonruminants, oxygen-derived free-radicals such as hydrogen peroxide produced during stressful events impair insulin responsiveness including glucose uptake, protein synthesis, and fatty acid metabolism. Arginine and methionine supply induce anti-inflammatory and antioxidant responses during stressful conditions. We studied the acute effect of arginine supplementation alone or combined with methionine on protein abundance in adipose tissue explants from lactating Holstein cows challenged with hydrogen peroxide. Hydrogen peroxide reduced protein abundance of key insulin and amino acid signaling proteins. Most pronounced and positive effects were detected with arginine alone, restoring abundance of key target proteins including those involved in glucose, amino acid, and glutathione metabolism. Potential benefits of enhanced post-ruminal arginine supply during stressful periods such as the transition into lactation merit further study.

Immune and metabolic effects of rumen-protected methionine during a heat stress challenge in lactating Holstein cows.

Multiparous, lactating Holstein cows (n = 32) were randomly assigned to one of two dietary treatments [TMR with rumen-protected Met (RPM) or TMR without RPM (CON)], and within each dietary treatment group cows were randomly assigned to one of two environmental treatment groups in a split-plot crossover design. In phase 1 (9 d), all cows were fed ad libitum and in thermoneutral conditions (TN). In phase 2 (9 d), group 1 (n = 16) was exposed to a heat stress (HS) challenge (HSC). Group 2 cows (n = 16) were pair-fed (PFTN) to HSC counterparts and remained in TN. 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, while dietary treatments remained the same for each cow. During phase 1, cows in RPM had greater plasma Met concentration compared with cows in CON (59 and 30 µM, respectively; P < 0.001). Cows in PFTN had a greater decrease (P < 0.05) in plasma insulin than cows in HSC at 4 h (-2.7 µIU/mL vs. -0.7 µIU/mL) and 8 h (-7.7 µIU/mL vs. -0.4 µIU/mL) during phase 2. Compared with cows in PFTN, cows in HSC had an increase (P < 0.05) in plasma serum amyloid A (-59 µg/mL vs. +58 µg/mL), serum haptoglobin (-3 µg/mL vs. +33 µg/mL), plasma lipopolysaccharide binding protein (-0.27 and +0.11 µg/mL), and plasma interleukin-1ß (-1.9 and +3.9 pg/mL) during phase 2. In conclusion, HSC elicited immunometabolic alterations; however, there were limited effects of RPM on cows in HSC.

Branched-Chain Amino Acid Supplementation Alters the Abundance of Mechanistic Target of Rapamycin and Insulin Signaling Proteins in Subcutaneous Adipose Explants from Lactating Holstein Cows.

The objective of this study was to investigate changes in protein abundance of mTOR and insulin signaling pathway components along with amino acid (AA) transporters in bovine s.c. adipose (SAT) explants in response to increased supply of Leu, Ile, or Val. Explants of SAT from four lactating Holstein cows were incubated with high-glucose serum-free DMEM, to which the 10 essential AAs were added to create the following treatments: ideal mix of essential AA (IPAA; Lys:Met 2.9:1; Lys:Thr 1.8:1; Lys:His 2.38:1; Lys:Val 1.23:1; Lys:Ile 1.45:1; Lys:Leu 0.85:1; Lys:Arg 2.08:1) or IPAA supplemented with Ile, Val, or Leu to achieve a Lys:Ile of 1.29:1 (incIle), Lys:Val 1.12:1 (incVal), or Lys:Leu (incLeu) 0.78:1 for 4 h. Compared with IPAA, incLeu or incIle led to greater activation of protein kinase B (AKT; p-AKT/total AKT) and mTOR (p-mTOR/total mTOR). Total EAA in media averaged 7.8 ± 0.06 mmol/L across treatments. Incubation with incLeu, incIle, or incVal led to greater protein abundance of solute carrier family 38 member 1 (SLC38A1), a Gln transporter, and the BCAA catabolism enzyme branched-chain α-keto acid dehydrogenase kinase (BCKDK) compared with IPAA. Activation of eukaryotic elongation factor 2 (eEF2; p-eEF2/total eEF2) was also greater in response to incLeu, incIle, or incVal. Furthermore, compared with incLeu or incIle, incVal supplementation led to greater abundance of SLC38A1 and BCKDK. BCKDK is a rate-limiting enzyme regulating BCAA catabolism via inactivation and phosphorylation of the BCKD complex. Overall, data suggested that enhanced individual supplementation of BCAA activates mTOR and insulin signaling in SAT. Increased AA transport into tissue and lower BCAA catabolism could be part of the mechanism driving these responses. The potential practical applications for enhancing post-ruminal supply of BCAA via feeding in rumen-protected form support in vivo studies to ascertain the role of these AAs on adipose tissue biology.

Endometrial glycogen metabolism on days 1 and 11 of the reproductive cycle in dairy cows.

Embryos need glucose or fructose to remain viable; however, it is not well understood how secretion of these carbohydrates is regulated. This study was conducted to evaluate endometrial glycogen and associated enzymes on Days 1 and 11 of the estrous cycle (Day 0 = behavioral estrus) in cattle. Diastase-liable periodic acid-Schiff (PAS) staining of luminal epithelia decreased 81 % between Days 1 and 11. Similarly, glycogen content of glandular epithelia was 66 % less on Day 11 than Day 1. There was dense PAS staining in the lumen of some glands, and this staining was removed when there was pretreatment with diastase. Based on western blot results, there was no difference in glycogen metabolizing enzymes between Days 1 and 11. Results from conducting immunohistochemistry procedures indicated hexokinase 1 was more abundant in the epithelial cells than stroma, but immunostaining was not different between Day 1 and 11. In contrast, phospho-glycogen synthase was undetectable on Day 1 but was present in glandular epithelia on Day 11. Glycogen synthase was localized to the epithelia, and was in larger abundance on Day 1. The abundance of glycogen phosphorylase was greater in the epithelium than stroma and on Day 11 than 1. Furthermore, glucose-6-phosphatase 3 was more abundant in the epithelium on both Days 1 and 11. In conclusion, in the uterus of cattle glycogen is stored in a reproductive cycle-dependent manner. Glucose released from endometrial glycogen stores could potentially be utilized by the endometrium or secreted into the uterine lumen.

Methionine and Arginine Supply Alters Abundance of Amino Acid, Insulin Signaling, and Glutathione Metabolism-Related Proteins in Bovine Subcutaneous Adipose Explants Challenged with N-Acetyl-d-sphingosine.

The objective was to perform a proof-of-principle study to evaluate the effects of methionine (Met) and arginine (Arg) supply on protein abundance of amino acid, insulin signaling, and glutathione metabolism-related proteins in subcutaneous adipose tissue (SAT) explants under ceramide (Ce) challenge. SAT from four lactating Holstein cows was incubated with one of the following media: ideal profile of amino acid as the control (IPAA; Lys:Met 2.9:1, Lys:Arg 2:1), increased Met (incMet; Lys:Met 2.5:1), increased Arg (incArg; Lys:Arg 1:1), or incMet plus incArg (Lys:Met 2.5:1 Lys:Arg 1:1) with or without 100 µM exogenous cell-permeable Ce (N-Acetyl-d-sphingosine). Ceramide stimulation downregulated the overall abundance of phosphorylated (p) protein kinase B (AKT), p-mechanistic target of rapamycin (mTOR), and p-eukaryotic elongation factor 2 (eEF2). Without Ce stimulation, increased Met, Arg, or Met + Arg resulted in lower p-mTOR. Compared with control SAT stimulated with Ce, increased Met, Arg, or Met + Arg resulted in greater activation of mTOR (p-mTOR/total mTOR) and AKT (p-AKT/total AKT), with a more pronounced response due to Arg. The greatest protein abundance of glutathione S-transferase Mu 1 (GSTM1) was detected in response to increased Met supply during Ce stimulation. Ceramide stimulation decreased the overall protein abundance of the Na-coupled neutral amino acid transporter SLC38A1 and branched-chain alpha-ketoacid dehydrogenase kinase (BCKDK). However, compared with controls, increased Met or Arg supply attenuated the downregulation of BCKDK induced by Ce. Circulating ceramides might affect amino acid, insulin signaling, and glutathione metabolism in dairy cow adipose tissue. Further in vivo studies are needed to confirm the role of rumen-protected amino acids in regulating bovine adipose function.

Steroidogenic, Metabolic, and Immunological Markers in Dairy Cows Diagnosed With Cystic Ovarian Follicles at Early and Mid-Late Lactation.

The etiology of cystic ovarian follicles (COF) remains a conundrum with steroidogenic, immunological, and metabolic dysfunctions linked to its development. Studies suggest that COF development may occur as a result of disruption of the insulin signaling pathway and the severity of a negative energy balance in dairy cows, but mid to late lactation cows diagnosed with COF are unlikely to have issues with energy metabolism. Herein, we characterized the mRNA expression of steroidogenic (LHCGR, StAR, CYP11A1, 3ß-HSD, CYP19A), immunological (IL-1ß, IL-6, IL-8, TLR-4, TNF), and metabolic markers (IGF-1, IRS1) in follicular fluid; and plasma and follicular fluid levels of E2, IL-1ß, glucose, and NEFA in early and mid-late lactation COF cows. Lactating dairy cows were diagnosed as having COF (n = 11, follicle >20 mm persistent for 7 days, absence of corpus luteum, and flaccid uterus) while 11 herdmates cycling with a dominant follicle were classified as the control cows. Cows diagnosed with COF were classified as early lactation (COF-E, n = 5) cows, <35 days in milk (DIM); or mid-late lactation (COF-M/L, n = 6), ≥118 DIM cows. Results revealed that mRNA expression StAR was greater (P < 0.01) in COF-E cows than COF-M/L cows and the control cows. The mRNA expression CYP19A1 was lower (P < 0.01) in COF-E cows and COF-M/L cows than in the control cows. The mRNA expression IL-6 and IRS-1 tended to be greater and lower, respectively, in COF-M/L cows compared to the control cows. The mRNA expression IGF-1 was greater (P < 0.01) in COF-E and COF-M/L cows than in the control cows. The plasma and follicular fluid concentration of NEFA was greater (P < 0.05) in COF-E cows than in COF-M/L and the control cows. Cows with COF-E had disturbances in steroidogenic and metabolic markers, while cows with COF-M/L had steroidogenic, immunological, and metabolic dysregulations, suggesting that COF pathogenesis may vary between early and mid-late lactation dairy cows.

Hepatic betaine-homocysteine methyltransferase and methionine synthase activity and intermediates of the methionine cycle are altered by choline supply during negative energy balance in Holstein cows.

Although choline requirements are unknown, enhanced postruminal supply may decrease liver triacylglycerol (TAG) storage and increase flux through the methionine cycle, helping cows during a negative energy balance (NEB). The objective was to investigate effects of postruminal choline supply during NEB on hepatic activity of betaine-homocysteine methyltransferase (BHMT), methionine synthase (MTR), methionine adenosyltransferase, transcription of enzymes, and metabolite concentrations in the methionine cycle. Ten primiparous rumen-cannulated Holstein cows (158 ± 24 d postpartum) were used in a replicated 5 × 5 Latin square design with 4-d treatment periods and 10 d of recovery (14 d/period). Treatments were unrestricted intake with abomasal infusion of water (A0), restricted intake (R; 60% of net energy for lactation requirements to induce NEB) with abomasal infusion of water (R0) or R plus abomasal infusion of 6.25, 12.5, or 25 g/d of choline ion. Liver tissue was collected on d 5 after the infusions ended, blood on d 1 to 5, and milk on d 1 to 4. Statistical contrasts were A0 versus R0 (CONT1) and tests of linear (L), quadratic (Q), and cubic (C) effects of choline dose. Plasma choline increased with R (CONT1) and choline (L). Although R decreased milk yield (CONT1), choline increased milk yield and liver phosphatidylcholine (PC), but decreased TAG (L). No differences were observed in plasma PC or very-low-density lipoprotein concentrations with R or choline. Activity and mRNA abundance of BHMT were greater with R (CONT1) and increased with choline (L). Although activity of MTR was lower with R (CONT1), it tended to increase with choline (L). No effect of R was detected for activity of methionine adenosyltransferase, but it changed cubically across dose of choline. Those responses were associated with linear increases in the concentrations of liver tissue (+13%) and plasma methionine concentrations. The mRNA abundance of CPT1A, SLC22A5, APOA5, and APOB, genes associated with fatty acid oxidation and lipoprotein metabolism, was upregulated by choline (Q). Overall, enhanced supply of choline during NEB increases hepatic activity of BHMT and MTR to regenerate methionine and PC, partly to help clear TAG. The relevance of these effects during the periparturient period merits further research.

Placentome Nutrient Transporters and Mammalian Target of Rapamycin Signaling Proteins Are Altered by the Methionine Supply during Late Gestation in Dairy Cows and Are Associated with Newborn Birth Weight.

Background: To our knowledge, most research demonstrating a link between maternal nutrition and both fetal growth and offspring development after birth has been performed with nonruminants. Whether such relationships exist in large ruminants is largely unknown.Objective: We aimed to investigate whether increasing the methionine supply during late pregnancy would alter uteroplacental tissue nutrient transporters and mammalian target of rapamycin (mTOR) and their relation with newborn body weight.Methods: Multiparous Holstein cows were used in a randomized complete block design experiment. During the last 28 d of pregnancy, cows were fed a control diet or the control diet plus ethylcellulose rumen-protected methionine (0.9 g/kg dry matter intake) (Mepron; Evonik Nutrition & Care GmbH) to achieve a 2.8:1 ratio of lysine to methionine in the metabolizable protein reaching the small intestine. We collected placentome samples at parturition and used them to assess mRNA and protein expression and the phosphorylation status of mTOR pathway proteins.Results: Newborn body weight was greater in the methionine group than in the control group (44.1 kg and 41.8 kg, respectively; P ≤ 0.05). Increasing the methionine supply also resulted in greater feed intake (15.8 kg/d and 14.6 kg/d), plasma methionine (11.9 µM and 15.3 µM), and plasma insulin (1.16 µg/L and 0.81 µg/L) in cows during late pregnancy. As a result, mRNA expression of genes involved in neutral amino acid transport [solute carrier (SLC) family members SLC3A2, SLC7A5, SLC38A1, and SLC38A10], glucose transport [SLC2A1, SLC2A3, and SLC2A4], and the mTOR pathway [mechanistic target of rapamycin and ribosomal protein S6 kinase B1] were upregulated (P ≤ 0.07) in methionine-supplemented cows. Among 6 proteins in the mTOR pathway, increasing the methionine supply led to greater (P ≤ 0.09) protein expression of α serine-threonine kinase (AKT), phosphorylated (p)-AKT, p-eukaryotic elongation factor 2, and the p-mTOR:mTOR ratio.Conclusion: Supplemental methionine during late gestation increases feed intake and newborn body weight in dairy cows, and this effect may be mediated by alterations in the uteroplacental transport of nondispensable and dispensable amino acids and glucose at least in part through changes in gene transcription and mTOR signaling.

Polymorphisms of the Toll-Like Receptor 2 of Goats (Capra hircus) may be Associated with Somatic Cell Count in Milk.

Toll-like receptor 2 (TLR2) plays an important role in recognition by the innate immune system of Gram-positive bacteria. As Gram-positive bacteria cause mastitis, we examined variations in the region of the TLR2 gene that codes for the extracellular domain. Samples of forty goats from a single dairy herd were collected, half with low SCC (≤200,000 cells/mL), and half with higher SCC. Two synonymous single nucleotide polymorphisms (SNPs) were identified: 840G > A and 1083A > G. One nonsynonymous SNP 739G > A was identified. This coded for valine or isoleucine, which have similar physiochemical properties, and was not in a region coding for a known functional domain. Surprisingly, the least square mean SCC of the heterozygous goats (146,220) was significantly lower than the SCC of homozygous GG goats (537,700; p = 0.004), although these two groups were similar in days in milk (p = 0.984), and there was no significant difference by breed (p = 0.941). Because factors other than mastitis can affect SCC and our sample sizes were limited, additional studies are needed to corroborate an association between TLR2 genotype and SCC or mastitis in goats.

Effects of rumen-protected methionine and choline supplementation on vaginal discharge and uterine cytology of Holstein cows.

Methionine is one of the most limiting amino acids in dairy diets and low feed intake around the time of calving could lead to decreased synthesis of phosphatidylcholine. An alternative pathway for phosphatidylcholine is to have choline as a precursor. The objective of this study was to determine the effects of feeding rumen-protected methionine and choline pre - and postpartum on reproduction of Holstein cows. Seventy-two Holstein cows were randomly assigned to four treatments from 21 days before calving to 30 days in milk (DIM): supplementation with rumen-protected methionine (MET; n = 20, received 0.08% of the dry matter (DM) of the diet/d as methionine, Smartamine M® to a Lys:Met = 2.9:1), rumen-protected choline (CHO; n = 17, received 60 g/d choline, Reassure), both rumen protected methionine and choline (MIX; n = 19, received 0.08% of the DM of the diet/d as methionine to a Lys:Met = 2.9:1 and 60 g/d choline), or no supplementation to serve as control (CON; n = 16, fed total mixed ration with a Lys:Met = 3.5:1). Cows were evaluated at 4, 7, 10, 13, 15, 17, and 30 d after calving for the presence of secretion using the Metricheck® device. On 15, 30, and 72 d after calving, the uterine endometrium of all cows was sampled using a cytological brush and streaked onto slides for analysis of the presence of polymorphonuclear neutrophils (PMN). We hypothesized that cows supplemented with methionine would have lower metricheck smell scores and lower rates of PMN than non-supplemented cows. On d 30, a treatment difference was detected using the metricheck score and smell (P < 0.04), with treatment MIX (score = 0.38) having a lower score than CHO (score = 2.11). Supplementing cows with rumen-protected methionine may have a beneficial effect on cows' uterine health.

The effect of tail paint formulation and heifer behavior on estrus detection.

Although heifers can have better conception rates than cows, they are still subject to poor estrus detection and economic losses from reduced reproductive efficiency. Tail paint has been successful in identifying estrus, but behaviors such a licking or rubbing have been believed to remove the paint and lead to false-positives. To investigate tail paint utilization and potential relationships among behaviors, eighteen Holstein heifers were randomly assigned to one of three treatments: a control tail chalk (CON), tail chalk with proprietary ingredient (CHALK+); and a spray formulation (SPRAY). Experimental design was a replicated 3 × 3 Latin square. Visual observations were performed in 30 min segments every 2 h from 6 AM to 6 PM. Ovaries were examined via ultrasound imaging on d 0, 7, and 9 of each period. The presence of follicles or a corpus luteum (CL) was recorded with their respective sizes. Heifers receiving SPRAY had a lower number of licks received per day and less tail paint removed regardless of day or follicle size when compared with CON or CHALK+. Rump lick received, chin rest received, anogenital sniff received, mount received, and both initiated and received behaviors for attempt to mount occurred more in heifers with large follicles regardless of day. Producers looking for heifers to breed should focus on those receiving rump lick, chin resting, anogenital sniff, mount, and attempt to mount. The use and combination of these estrus detection tools can improve reproductive efficiency in dairy operations.

Induction of Subacute Ruminal Acidosis Affects the Ruminal Microbiome and Epithelium.

Subacute ruminal acidosis (SARA) negatively impacts the dairy industry by decreasing dry matter intake, milk production, profitability, and increasing culling rate and death loss. Six ruminally cannulated, lactating Holstein cows were used in a replicated incomplete Latin square design to determine the effects of SARA induction on the ruminal microbiome and epithelium. Experimental periods were 10 days with days 1-3 for ad libitum intake of control diet, followed by 50% feed restriction on day 4, and ad libitum access on day 5 to the basal diet or the basal diet with an additional 10% of a 50:50 wheat/barley pellet. Based on subsequent ruminal pH, cows were grouped (SARA grouping; SG) as Non-SARA or SARA based on time <5.6 pH (0 and 3.4 h, respectively). Ruminal samples were collected on days 1 and 6 of each period prior to feeding and separated into liquid and solid fractions. Microbial DNA was extracted for bacterial analysis using 16S rRNA gene paired-end sequencing on the MiSeq Illumina platform and quantitative PCR (qPCR). Ruminal epithelium biopsies were taken on days 1 and 6 before feeding. Quantitative RT-PCR was used to determine gene expression in rumen epithelium. Bray-Curtis similarity indicated samples within the liquid fraction separated by day and coincided with an increased relative abundance of genera Prevotella, Ruminococcus, Streptococcus, and Lactobacillus on day 6 (P < 0.06). Although Firmicutes was the predominant phyla in the solid fraction, a SG × day interaction (P < 0.01) indicated a decrease on day 6 for SARA cows. In contrast, phylum Bacteroidetes increased on day 6 (P < 0.01) for SARA cows driven by greater genera Prevotella and YRC22 (P < 0.01). Streptococcus bovis and Succinivibrio dextrinosolvens populations tended to increase on day 6 but were not affected by SG. In ruminal epithelium, CLDN1 and CLDN4 expression increased on day 6 (P < 0.03) 24 h after SARA induction and a tendency for a SG × day interaction (P < 0.10) was observed for CLDN4. Overall, results indicate more rapid adaptation to an induced bout of SARA in the solid fraction ruminal microbiome compared with ruminal epithelium.

Linking Peripartal Dynamics of Ruminal Microbiota to Dietary Changes and Production Parameters.

During the peripartal period, proper acclimatization of rumen microorganisms to variations in nutritional management can facilitate the transition into lactation. This study characterized the temporal shifts in the composition and functional properties of ruminal microbiota during the periparturient period in dairy cows subjected to a typical two-tiered feeding management approach. Ruminal digesta samples from eight multiparous fistulated Holstein cows were collected on days -14, -7, 10, 20, and 28 relative to parturition. High-throughput Illumina sequencing of the V4 region of the bacterial 16S rRNA gene revealed distinct clustering patterns between pre- and postpartal ruminal microbiota. During the prepartal period, when the voluntary dry matter intake was lower, we observed strikingly lower inter-animal variations in the composition of the ruminal microbiota. Genera Ruminococcus and Butyrivibrio, which are considered major fibrolytic rumen dwellers, were overrepresented in the prepartal rumen ecosystem. In contrast, increased postpartal voluntary DMI was associated with enrichment of bacterial genera mainly consisting of proteolytic, amylolytic, and lactate-producer species (including Prevotella, Streptococcus, and Lactobacillus). These, together with the postpartal enrichment of energy metabolism pathways, suggested a degree of acclimatization of the ruminal microbiota to harvest energy from the carbohydrate-dense lactation diet. In addition, correlations between ruminal microbiota and parameters such as milk yield and milk composition underscored the metabolic contribution of this microbial community to the cow's performance and production.

Acute mammary and liver transcriptome responses after an intramammary Escherichia coli lipopolysaccharide challenge in postpartal dairy cows.

The study investigated the effect of an intramammary lipopolysaccharide (LPS) challenge on the bovine mammary and liver transcriptome and its consequences on metabolic biomarkers and liver tissue composition. At 7 days of lactation, 7 cows served as controls (CTR) and 7 cows (LPS) received an intramammary Escherichia coli LPS challenge. The mammary and liver tissues for transcriptomic profiling were biopsied at 2.5 h from challenge. Liver composition was evaluated at 2.5 h and 7 days after challenge, and blood biomarkers were analyzed at 2, 3, 7 and 14 days from challenge. In mammary tissue, the LPS challenge resulted in 189 differentially expressed genes (DEG), with 20 down-regulated and 169 up-regulated. In liver tissue, there were 107 DEG in LPS compared with CTR with 42 down-regulated and 65 up-regulated. In mammary, bioinformatics analysis highlighted that LPS led to activation of NOD-like receptor signaling, Toll-like receptor signaling, RIG-I-like receptor signaling and apoptosis pathways. In liver, LPS resulted in an overall inhibition of fatty acid elongation in mitochondria and activation of the p53 signaling pathway. The LPS challenge induced changes in liver lipid composition, a systemic inflammation (rise of blood ceruloplasmin and bilirubin), and an increase in body fat mobilization. The data suggest that cells within the inflamed mammary gland respond by activating mechanisms of pathogen recognition. However, in the liver the response likely depends on mediators originating from the udder that affect liver functionality and specifically fatty acid metabolism (ß-oxidation, ketogenesis, and lipoprotein synthesis).

Postpartal subclinical endometritis alters transcriptome profiles in liver and adipose tissue of dairy cows.

Transcriptome alterations in liver and adipose tissue of cows with subclinical endometritis (SCE) at 29 d postpartum were evaluated. Bioinformatics analysis was performed using the Dynamic Impact Approach by means of KEGG and DAVID databases. Milk production, blood metabolites (non-esterified fatty acids, magnesium), and disease biomarkers (albumin, aspartate aminotransferase) did not differ greatly between healthy and SCE cows. In liver tissue of cows with SCE, alterations in gene expression revealed an activation of complement and coagulation cascade, steroid hormone biosynthesis, apoptosis, inflammation, oxidative stress, MAPK signaling, and the formation of fibrinogen complex. Bioinformatics analysis also revealed an inhibition of vitamin B3 and B6 metabolism with SCE. In adipose, the most activated pathways by SCE were nicotinate and nicotinamide metabolism, long-chain fatty acid transport, oxidative phosphorylation, inflammation, T cell and B cell receptor signaling, and mTOR signaling. Results indicate that SCE in dairy cattle during early lactation induces molecular alterations in liver and adipose tissue indicative of immune activation and cellular stress.

Metabolic evaluation of dairy cows submitted to three different strategies to decrease the effects of negative energy balance in early postpartum / Avaliação metabólica de vacas leiteiras submetidas a três estratégias para diminuir os efeitos do balanço energético negativo no pós-parto inicial

In early lactation dairy cattle suffer metabolic alterations caused by negative energy balance, which predisposes to fatty liver and ketosis. The aim of this study was to evaluate the metabolic condition of high yielding dairy cows subjected to three treatments for preventing severe lipomobilization and ketosis in early lactation. Fifty four multiparous Holstein cows yielding >30 L/day were divided into four groups: control (CN= no treatment), glucose precursor (PG= propylene-glycol), hepatic protector (Mp= Mercepton®), and energy supplement with salts of linolenic and linoleic faty acids (Mg-E= Megalac-E®). Treatments were administrated randomly at moment of calving until 8 weeks postpartum. Blood samples were collected on days 1, 7, 14, 21, 28, 35, 42 and 49 postpartum. Body condition score (BCS) was evaluated at the same periods and milk yield was recorded at 2nd, 4th, 5th, 6th, 7th, and 8th weeks of lactation. Concentrations of non-esterified fatty acids (NEFA), albumin, AST, ß-hydroxybutyrate (BHBA), cholesterol, glucose, total protein, urea and triglycerides were analyzed in blood samples. Cut-off points for subclinical ketosis were defined when BHBA >1.4 mmol/L and NEFA >0.7 mmol/L. General occurrence of subclinical ketosis was 24 percent during the period. An ascendant curve of cholesterol and glucose was observed from the 1st to the 8th week of lactation, while any tendency was observed with BHBA and NEFA, although differences among treatments were detected (p<0.05). BCS decreased from a mean of 3.85 at 1st week to 2.53 at 8th week of lactation (p=0.001). Milk yield was higher in the Mg-E group compared with the other treatment groups (p<0.05) Compared with the CN group, the treatments with Mp and PG did not show significant differences in blood biochemistry and milk yield. Cows receiving PG and Mg-E showed higher values of BHBA and NEFA (P<0.05), indicating accentuated lipomobilization. Supplementation with Mg-E also resulted in significant higher concentrations of cholesterol, BHBA, urea, AST and lower values of glycemia. This performance may be explained by the highest milk yield observed with this treatment. Treatments with PG and Mp did not improve milk yield, compared with control cows, but did not show metabolic evidence of ketosis, fat mobilization or fatty liver. These results suggest that treatment with Mg-E improves milk production but induces a higher negative energy balance leading to moderated lipomobilization and ketone bodies production, increasing the risk of fatty liver.

Durante o início da lactação as vacas leiteiras sofrem transtornos metabólicos causados pelo balanço energético negativo, o que predispõe a infiltração gordurosa hepática e cetose. O objetivo deste estudo foi avaliar o status metabólico de vacas leiteiras de alta produção submetidas a três tratamentos para prevenir severa lipomobilização e cetose no início da lactação. Cinquenta e quatro vacas de raça Holandesa multíparas produzindo >30 L/dia foram divididas em quatro grupos: controle (CN= sem tratamento), precursor de glicose (PG= propileno-glicol), protetor hepático (Mp= Mercepton®) e suplementação com sais de ácidos linolênico e linoléico (Mg-E= Megalac-E®). Amostras de sangue foram coletadas nos dias 1, 7, 14, 21, 28, 35, 42 e 49 do pós-parto. A condição corporal foi avaliada nos mesmos períodos e a produção de leite foi registrada nas semanas 2, 4, 5, 6, 7 e 8 de lactação. As concentrações de ácidos graxos não esterificados (AGNE), albumina, AST, ß-hidroxibutirato (BHB), colesterol, glicose, proteína total, uréia e triglicerídeos foram determinadas nas amostras de sangue. Pontos de corte para diagnosticar cetose subclínica foram definidos quando BHB >1,4mmol/L e AGNE >0,7mmol/L. A ocorrência geral de cetose subclínica foi de 24 por cento durante o período. Uma curva ascendente de colesterol e de glicose foi observada desde a 1ª até a 8ª semana de lactação, enquanto que nenhuma tendência foi observada com BHB e AGNE, embora diferenças entre os tratamentos foram detectadas (p<0,05). A condição corporal diminuiu de uma media de 3,85 na 1ª semana até 2,53 na 8ª semana de lactação (p=0,001). A produção de leite foi superior no grupo de Mg-E comparado com os demais tratamentos. Comparado com o grupo CN, os tratamentos de Mp e PG não mostraram diferenças significativas na bioquímica sanguínea nem na produção de leite (p<0,05) As vacas que receberam PG e Mg-E mostraram maiores valores de AGNE, indicando uma acentuada lipomobilização. A suplementação com Mg-E também resultou em maiores concentrações de colesterol, BHB, uréia, AST e menores valores de glicemia. Este resultado pode ser explicado pela maior produção de leite observada com este tratamento. Os tratamentos com PG e Mp não melhoraram a produção de leite, comparados ao grupo CN, mas também não mostraram evidências metabólicas de cetose, alta lipomobilização nem infiltração gordurosa hepática. Os resultados sugerem que o tratamento com Mg-E melhora a produção de leite, mas induz um balance energético negativo maior levando a moderada lipomobilização e produção de corpos cetônicos, aumentando o risco de fígado gorduroso.