Short communication: Molecular markers for epithelial cells across gastrointestinal tissues and fecal RNA in preweaning dairy calves.

The objective of this study was to compare the transcription of gene markers for gastrointestinal (GI) epithelial cells, including fatty acid binding protein 2 (FABP2) and cytokeratin 8 (KRT8), and tight junction complex genes (TJP1, CLDN1, CLDN4) in fecal RNA against several GI tract tissue sections in dairy calves. Eight healthy Jersey calves were euthanized at 5 wk of age, and postmortem samples were collected from rumen, duodenum, jejunum, ileum, large intestine, cecum, and feces for total RNA isolation. Tissues and fecal samples were immediately frozen in liquid nitrogen until RNA isolation. A real-time quantitative PCR analysis was performed using a single standard curve composited of equal amounts of all samples, including cDNA from fecal and GI tract tissues. The mRNA expression of the tight junctions TJP1, CLDN1, and CLDN4 was greater in fecal RNA compared with lower GI tract tissues (i.e., duodenum, jejunum, ileum, large intestine, and cecum). Similar to fecal RNA, rumen tissue had greater expression of tight junctions CLDN1 and CLDN4 than lower GI tract tissues. Similarly, rumen tissue had greater expression of TPJ1 than all lower GI tract tissues except duodenum. The expression of TJP1 and CLDN4 was greater in fecal RNA than in rumen tissue; in contrast, CLDN1 mRNA expression was greater in rumen tissue than in the fecal RNA. The expression of FABP2 was greater in duodenum in comparison to all tissue except ileum. The mRNA expression of FABP2 in fecal samples was similar to jejunum and ileum. The expression of KRT8 in fecal samples was similar to duodenum, large intestine, and cecum. The fecal RNA had a greater expression of KRT8 in comparison to jejunum and ileum. The rumen tissue had the lowest mRNA expression of KRT8. The expression levels of FABP2, KRT8, and tight junction genes observed in fecal transcripts suggest that a considerable amount of RNA derived from GI tract epithelial cells can be detected in fecal RNA, which is in agreement with previous data in neonatal dairy calves and other biological models including humans, rodents, and primates. The greater expression of tight junctions in fecal RNA in comparison to sections of the low GI remains to be understood, and due to the importance of tight junctions in GI physiology, further clarification of this effect is warranted. The similarities in mRNA expression of FABP2 and KRT8 between fecal RNA and intestinal sections add up to the accumulating evidence that fecal RNA can be used to investigate molecular alterations in the GI tract of neonatal dairy calves. Further research in this area should include high-throughput transcriptomic analysis via RNA-seq to uncover novel molecular markers for specific sections of the GI tract of neonates.

Effects of peripartal yeast culture supplementation on lactation performance, blood biomarkers, rumen fermentation, and rumen bacteria species in dairy cows.

Feeding yeast culture fermentation products has been associated with improved feed intake and milk yield in transition dairy cows. These improvements in performance have been further described in terms of rumen characteristics, metabolic profile, and immune response. The objective of this study was to evaluate the effects of a commercial yeast culture product (YC; Culture Classic HD, Phibro Animal Health) on performance, blood biomarkers, rumen fermentation, and rumen bacterial population in dairy cows from -30 to 50 d in milk (DIM). Forty Holstein dairy cows were enrolled in a randomized complete block design from -30 to 50 DIM and blocked according to expected calving day, parity, previous milk yield, and genetic merit. At -30 DIM, cows were assigned to either a basal diet plus 114 g/d of ground corn (control; n = 20) or a basal diet plus 100 g/d of ground corn and 14 g/d of YC (n = 20), fed as a top-dress. Cows received the same close-up diet from 30 d prepartum until calving [1.39 Mcal/kg of dry matter (DM) and 12.3% crude protein (CP)] and lactation diet from calving to 50 DIM (1.60 Mcal/kg of DM and 15.6% CP). Blood samples and rumen fluid were collected at various time points from -30 to 50 d relative to calving. Cows fed YC compared with control showed a trend for increased energy-corrected milk (+3.2 kg/d). Lower somatic cell counts were observed in YC cows than in control. We detected a treatment × time interaction in nonesterified fatty acids (NEFA) that could be attributed to a trend for greater NEFA in YC cows than control at 7 DIM, followed by lower NEFA in YC cows than control at 14 and 30 DIM. In the rumen, YC contributed to mild changes in rumen fermentation, mainly increasing postpartal valerate while decreasing prepartal isovalerate. This was accompanied by alterations in rumen microbiota, including a greater abundance of cellulolytic (Fibrobacter succinogenes) and lactate-utilizing bacteria (Megasphaera elsdenii). These results describe the potential benefits of supplementing yeast culture during the late pregnancy through early lactation, at least in terms of rumen environment and performance.

Effects of yeast culture supplementation on lactation performance and rumen fermentation profile and microbial abundance in mid-lactation Holstein dairy cows.

The continuous trend for a narrowing margin between feed cost and milk prices across dairy farms in the United States highlights the need to improve and maintain feed efficiency. Yeast culture products are alternative supplements that have been evaluated in terms of milk performance and feed efficiency; however, less is known about their potential effects on altering rumen microbial populations and consequently rumen fermentation. Therefore, the objective of this study was to evaluate the effect of yeast culture supplementation on lactation performance, rumen fermentation profile, and abundance of major species of ruminal bacteria in lactating dairy cows. Forty mid-lactation Holstein dairy cows (121 ± 43 days in milk; mean ± standard deviation; 32 multiparous and 8 primiparous) were used in a randomized complete block design with a 7-d adaptation period followed by a 60-d treatment period. Cows were blocked by parity, days in milk, and previous lactation milk yield and assigned to a basal total mixed ration (TMR; 1.6 Mcal/kg of dry matter, 14.6% crude protein, 21.5% starch, and 38.4% neutral detergent fiber) plus 114 g/d of ground corn (CON; n = 20) or basal TMR plus 100 g/d of ground corn and 14 g/d of yeast culture (YC; n = 20; Culture Classic HD, Cellerate Yeast Solutions, Phibro Animal Health Corp.). Treatments were top-dressed over the TMR once a day. Cows were individually fed 1 × /d throughout the trial. Blood and rumen fluid samples were collected in a subset of cows (n = 10/treatment) at 0, 30, and 60 d of the treatment period. Rumen fluid sampled via esophageal tubing was analyzed for ammonia-N, volatile fatty acids (VFA), and ruminal bacteria populations via quantitative PCR amplification of 16S ribosomal DNA genes. Milk yield was not affected by treatment effects. Energy balance was lower in YC cows than CON, which was partially explain by the trend for lower dry matter intake as % body weight in YC cows than CON. Cows fed YC had greater overall ruminal pH and greater total VFA (mM) at 60 d of treatment period. There was a contrasting greater molar proportion of isovalerate and lower acetate proportion in YC-fed cows compared with CON cows. Although the ruminal abundance of specific fiber-digesting bacteria, including Eubacterium ruminantium and Ruminococcus flavefaciens, was increased in YC cows, others such as Fibrobacter succinogenes were decreased. The abundance of amylolytic bacteria such as Ruminobacter amylophilus and Succinimonas amylolytica were decreased in YC cows than CON. Our results indicate that the yeast culture supplementation seems to promote some specific fiber-digesting bacteria while decreasing amylolytic bacteria, which might have partially promoted more neutral rumen pH, greater total VFA, and isovalerate.

Consumer knowledge and perceptions of milk fat in Denmark, the United Kingdom, and the United States.

This study examined the relationship between consumers' country of origin: Denmark (DK), the United Kingdom (UK), and the United States (US) and their knowledge and perceptions of milk fat. Adult participants answered a web-based survey, and data were collected online from December 2018 to April 2019, in DK, the UK, and the US. A total of 694 participants completed the online survey. Most respondents were between 18 and 39 years old, female, highly educated, and employed. Most UK respondents consumed milk daily (73%), whereas in DK (56%) and the US (46%) consumption was significantly lower. Whole milk and semi-skim milk were consumed less by respondents in DK (20 and 36%, respectively) compared with the UK (50 and 49%, respectively) and the US (47 and 50%, respectively). Danish respondents (53%) consumed more skim milk than respondents from the UK (16%) and the US (19%). Concern about milk fat was higher in DK (60%) than in the UK (34%) and the US (31%). More respondents considered milk fat to be "healthy" or "very healthy" in the UK (31 and 10%, respectively) and the US (37 and 19%, respectively), than in DK (23 and 6%, respectively). Nutritional benefit was the most important reason for perceiving milk fat as healthy in the 3 countries. Awareness of milk saturated fat was higher among respondents from the UK (53%) than from DK (44%) and the US (38%). Results suggest that consumers in DK are different in their perceptions of milk fat, but consumers in the UK and the US share common characteristics.

Thiamine ameliorates inflammation of the ruminal epithelium of Saanen goats suffering from subacute ruminal acidosis.

This study aimed to examine the role of thiamine in the local inflammation of ruminal epithelium caused by high-concentrate diets. Eighteen mid-lactating (148 ± 3 d in milk; milk yield = 0.71 ± 0.0300 kg/d) Saanen goats (body weight = 36.5 ± 1.99 kg; body condition score = 2.73 ± 0.16, where 0 = emaciated and 5 = obese) in parity 1 or 2 were selected. The goats were randomly divided into 3 groups (n = 6/group): (1) control diet (concentrate:forage 30:70), (2) high-concentrate diet (HC; concentrate:forage 70:30), and (3) high-concentrate diet with 200 mg of thiamine/kg of dry matter intake (THC; concentrate:forage 70:30). Goats remained on experimental diets for 8 wk. On the last day of 8 wk, ruminal and blood samples were collected to determine ruminal parameters, endotoxin lipopolysaccharide, and blood inflammatory cytokines. Goats were slaughtered to collect ruminal tissue to determine gene and protein expression of toll-like receptor 4 (TLR4) signaling pathways. Thiamine supplementation increased ruminal pH (6.03 vs. 5.42) compared with the HC group. Propionate (21.08 vs. 31.61 mM), butyrate (12.08 vs. 19.39 mM), lactate (0.52 vs. 0.71 mM), and free lipopolysaccharide (42.16 vs. 55.87 × 103 endotoxin units/mL) concentrations in ruminal fluid were lower in THC goats compared with HC goats. Similar to plasma interleukin 1ß (IL-1ß) concentration (209.31 vs. 257.23 pg/mL), blood CD8+ percentage (27.57 vs. 34.07%) also decreased in response to thiamine. Compared with HC goats, THC goats had lower ruminal epithelium activity of the enzymes myeloperoxidase and matrix metalloproteinase (MMP) 2 and 9. In contrast to HC, THC had downregulated mRNA expression of nuclear factor-κB (NFKB), TLR4, IL1B, MMP2, and MMP9 in ruminal epithelium. Thiamine supplementation led to lower relative protein expression of IL-1ß, NF-κB unit p65, and phosphorylated NF-κB unit p65 in ruminal epithelium. Taken together, these results suggest that thiamine supplementation mitigates HC-induced local inflammation and ruminal epithelial disruption.

Altered rumen fermentation patterns in lactating dairy cows supplemented with phytochemicals improve milk production and efficiency.

Tannins and other phytochemicals are known to improve RUP in the diet by binding protein and then limiting ruminal degradation, which may improve milk yield and milk protein synthesis. The objective of this study was to evaluate the effects of dietary phytochemicals (tannins and Capsicum species) as rumen modifiers on production parameters and milk efficiency in dairy cows. Twenty-four multiparous Holstein cows (96 ± 16 d in milk; mean ± standard deviation) were used in a replicated 3 × 3 Latin square design balanced to measure carryover effects. Cows were blocked according to days in milk, milk production, and body weight and randomly assigned to 1 of 3 groups (n = 8/group). Each group was assigned to a unique treatment sequence across the 3 periods in the Latin square. The experiment consisted of a 14-d covariate period and three 30-d treatment periods. Cows received a basal diet supplemented with soybean meal pellets (SB) as the control diet, phytochemicals (RUM; Rumiviv, CCPA, Janzé, France) pelleted with soybean meal, or expeller soybean meal (ESBM; SoyPlus, West Central Soy, Ralston, IA). Milk production and dry matter intake during the last 4 d of each period were used for statistical analysis. Blood and rumen fluid samples were collected on d 27 of each period. Rumen fluid was analyzed for ammonia N and volatile fatty acids as well as ruminal bacteria via quantitative PCR amplification of 16S ribosomal DNA genes. Greater milk yield (37.9 vs. 36 kg/d), energy-corrected milk (39.7 vs 37.1 kg/d), and protein yield (1.15 vs. 1.08 kg/d) were observed in RUM compared with SB, but these parameters were similar between RUM and ESBM. Concentrations of total volatile fatty acids (118.1 vs. 101.5 mM) were greater in RUM in comparison to SB and ESBM diets. Cows fed RUM had greater ß-hydroxybutyrate (0.49 vs. 0.42 mmol/L) than SB and ESBM. Selenomonas ruminantium, Succinimonas amylolytica, and Streptococcus bovis in rumen fluid were lower in RUM fed cows in comparison to SB and ESBM. Increased total volatile fatty acids and lower ruminal abundance of bacteria associated with low feed efficiency in RUM cows can partially explain the improvements observed in milk yield and milk efficiency. Overall, these data suggest that feeding a combination of tannin mixture and Capsicum can significantly affect rumen fermentation characteristics via partial manipulation of rumen microbiota, and these effects were reflected in improved milk production and efficiency.

Short communication: Comparative gene expression analysis on the enrichment of polymorphonuclear leukocytes and gastrointestinal epithelial cells in fecal RNA from nondiarrheic neonatal dairy calves.

Increased understanding of the biology of the gastrointestinal tract (GIT) in neonatal dairy calves during their adaptation to an extrauterine environment will decrease health problems such as diarrhea while increasing feed efficiency and average daily gain in preweaned dairy calves. Within this context, a noninvasive method, based on fecal RNA, to study the GIT in neonatal dairy calves through the isolation of RNA from fecal samples for quantitative reverse-transcription PCR analysis can provide valuable information on GIT biological adaptations during the preweaning period. We aimed to evaluate the potential enrichment of RNA from immune cells or GIT epithelial cells during fecal RNA isolation. Eight neonatal Holstein calves less than 3 wk old (14.9 ± 5.5 d of age at sampling ± standard deviation) and a fecal score of 2.0 ± 0.7 (mean ± standard deviation) were used. During a single sampling, fecal and blood samples were taken simultaneously from each calf before the morning feeding. Fecal samples were immediately frozen in liquid nitrogen until RNA isolation, whereas polymorphonuclear leukocytes (PMN) were isolated from blood samples before RNA isolation. An quantitative reverse-transcription PCR analysis was performed using a single standard curve composited of equal amounts of all samples including cDNA from fecal and PMN. The genes myeloperoxidase (MPO) and L-selectin (SELL) were selected for their specific known function in PMN, whereas keratin 8 (KRT8) and aquaporin 3 (AQP3) have been associated with epithelial enterocytes. Our results showed a contrasting gene expression profile between PMN and fecal RNA; whereas greater mRNA expression of SELL was observed in PMN, a greater KRT8 expression was observed in fecal RNA. The mRNA expression of AQP3 tended to be greater in PMN than fecal RNA. Additionally, MPO was not amplified in fecal RNA. Our findings suggest that under nondiarrheic conditions RNA isolated from stool samples of neonatal dairy calves will have a considerable number of GIT epithelial cells, which confirms the reliability of this method under these conditions. However, further research needs to be done to determine if the same effects are observed during diarrhea or throughout the preweaning period of dairy calves.

Technical note: A novel approach to estimate dry matter intake of lactating dairy cows through multiple on-cow accelerometers.

The objective of this study was to evaluate the feasibility of using multiple 3-dimensional accelerometers to estimated individual dry matter intake (DMI) of lactating dairy cows. Twenty-four Holstein cows in late lactation were assigned into 2 groups, a calibration group (n = 12) and a validation group (n = 12). All cows were fitted with 3 sensors that recorded 3-dimensional acceleration (i.e., x, y, and z) at 10-s intervals, 1 on the lateral side of the left hind leg and 2 attached directly to a halter over the nose and jaw area on the left side. Then, 3 accelerations were generated from each accelerometer (e.g., Leg-X, Leg-Y, and Leg-Z). Six new variables were created based on the change in acceleration in the nose and jaw accelerometers between 2 consecutive time points (e.g., LagJaw-X). For both groups (i.e., calibration and validation), cows were continuously video recorded while data on acceleration and intake of total mixed ration were collected for 10 consecutive days. Cows were fed once daily using an individual gate system, and individual refusals were recorded next day before morning feeding. Cows were fed a common lactating cow diet (17.9% crude protein; 1.70 Mcal/kg of dry matter). In the calibration group, individual eating bouts were obtained based on video recordings and merged with the corresponding accelerometer data. Then, a stepwise regression analysis was conducted using the REG procedure of SAS (SAS Institute, Cary, NC) to determine the ranges in acceleration that accounted for the highest variation in DMI (highest R2) in each acceleration variable. All 32,767 potential acceleration combinations were tested in the validation group using the acceleration ranges predetermined in the calibration group. The CORR procedure of SAS was used to test the Pearson correlation coefficient (r) between the type of DMI [i.e., based on accelerations (DMIaccel) or actual DMI (DMIactual)]. The MIXED procedure of SAS was used to perform a repeated-measures analysis with type (DMIaccel vs. DMIactual), day, and their interaction (T × D) in the model. From this analysis, 8 candidate acceleration models were selected based on high r and similarity (P > 0.15) in terms of T and T × D between DMIaccel and DMIactual. A simulated effect on DMIactual was artificially created in the validation group by dividing this group (n = 12) into high and low intake cows (n = 6/group; DMI of 24.1 vs. 18.7 kg/d), and the candidate models were tested to determine whether they were sensitive enough to detect this effect. From these candidate models, AEN (Leg-X + Jaw-Z + LagJaw-Z) showed a weak correlation (r = 0.36) between DMIaccel and DMIactual, but DMIaccel and DMIactual were highly similar (21.2 vs. 21.4 kg/d of DMI). In addition, this was the only model that could detect the simulated effect on DMIactual (22.1 vs. 20.3 kg/d of DMI) in the validation group. The fact that the simulated effect on DMIactual was detected based only on accelerations is highly significant, and models such as AEN could be substantially improved if they were derived from a greater sample size and included different physiological stages in dairy cows.

Phosphorylation of nuclear factor erythroid 2-like 2 (NFE2L2) in mammary tissue of Holstein cows during the periparturient period is associated with mRNA abundance of antioxidant gene networks.

Changes in the production of reactive oxygen species in the mammary gland of dairy cows during the periparturient period could lead to oxidative stress and potentially impair mammary function. Phosphorylation of the transcription factor nuclear factor erythroid 2-like 2 (NFE2L2), also known as nuclear factor-E2-related factor 2, controls mRNA abundance of genes encoding antioxidant proteins and enzymes. The hypothesis was that NFE2L2 phosphorylation status and target gene mRNA abundance in the mammary gland of dairy cows is altered around parturition. Total NFE2L2 protein, phosphorylated protein (p-NFE2L2), and ratio of p-NFE2L2 to NFE2L2 along with mRNA abundance of 24 genes related to the NFE2L2 signaling pathway, apoptosis, and cell proliferation were measured in mammary tissue samples from Holstein cows at -30, 1, 15, and 30 d relative to parturition. Although total NFE2L2 protein abundance did not differ, p-NFE2L2 and p-NFE2L2-to-NFE2L2 ratio were greater after parturition. The upregulation of DNA damage inducible transcript 3 (DDIT3) postpartum indicated a localized oxidative stress state. Among genes evaluated, thioredoxin (TXN), glutathione peroxidase 1 (GPX1), and glutathione S-transferase mu 1 (GSTM1) had the highest (37.1, 15.1, and 4.8% of total mRNA measured, respectively) abundance. The mRNA abundance of various target genes with detoxifying enzymatic functions and free radical scavenging activities [glutamate-cysteine ligase catalytic subunit (GCLC); glutathione reductase (GSR); ferrochelatase (FECH); TXN; thioredoxin reductase 1 (TXNRD1); and NAD(P)H quinone dehydrogenase 1 (NQO1)] were consistently upregulated (linear effect of time) as parturition approached and lactation began. Among the transcription regulators, NFE2L2 had the highest mRNA abundance (7.3% of total mRNA measured). Abundance of NFE2L2 and other transcription factors [nuclear factor kappa B subunit 1 (NFKB1), retinoid X receptor α (RXRA), and mitogen-activated protein kinase 14 (MAPK14)] were upregulated (linear effect of time) from -30 d to 30 d relative to parturition. Overall, NFE2L2 phosphorylation and downstream signaling leading to postpartal upregulation of genes associated with oxidative stress and inflammation in the mammary gland seem to be key components of normal cellular function to maintain proper redox homeostasis. However, if the longitudinal increases in mRNA and protein abundance of these antioxidant mechanisms are a reflection of cellular oxidative stress, then the likelihood of protein and DNA damage would be greater and might be one factor compromising cell viability and potentially lactation persistency. The actual cues coordinating these molecular responses remain to be determined.

Prepartal standing behavior as a parameter for early detection of postpartal subclinical ketosis associated with inflammation and liver function biomarkers in peripartal dairy cows.

A degree of negative energy balance is commonly experienced by cows during early lactation. This physiological state, if pronounced or prolonged, leads to partial oxidation of nonesterified fatty acids as an energy source and, consequently, increasing blood ß-hydroxybutyrate (BHB) concentrations and potentially development of ketosis in postpartal dairy cows. Twenty-four multiparous Holstein cows received a common prepartal and postpartal diet. Cows were fitted with an accelerometer mounted laterally on the distal left hind leg using vet wrap from -30 to 15 d relative to parturition. A retrospective analysis was performed using the postpartal BHB data at 8 time points from 0 to 15 d in milk measured with the Precision Xtra (Abbott Diabetes Care, Alameda, CA). Cows with an average blood BHB <1.4 mmol/L were designated nonketotic (NONKET; n = 12), and those with ≥1.4 mmol/L were designated ketotic (KET; n = 12). A total of 8 samples per cow were used for this analysis. Subsequent analyses of behavioral patterns and blood biomarkers were performed using this group effect. On average, blood BHB reached subclinical levels (1.4 ± 0.3 mmol/L; mean ± standard error of the mean) at 3 d postpartum for all cows in this study. Behavioral patterns were obtained from accelerometer data, and correlation analysis was performed between these behaviors such as standing and lying time from -30 to 3 d relative to parturition and blood BHB concentration at 3 d postpartum. The strongest correlation was obtained between standing time at 3 d before calving and blood BHB at 3 d postpartum. Dry matter intake was greater (ca. 3 kg/d) in NONKET cows than in KET cows. An interaction of group × time for milk yield resulted in an overall increase of 5.7 kg/d in NONKET cows in comparison with KET. The blood concentrations of biomarkers for liver function (γ-glutamyltransferase and glutamic-oxaloacetic transaminase), inflammation (IL-6), and metabolism (nonesterified fatty acids) were increased at various time points in KET cows in comparison with NONKET during the transition period. Overall, lower bilirubin in NONKET cows than in KET further confirmed an impaired liver function in the latter group of cows. Our findings revealed the potential for establishing correlations between prepartal behavioral patterns derived from accelerometer data and postpartal subclinical ketosis, and further confirming the latter by physiological alterations in biomarkers related to inflammation and liver function. Our data also indicate that cows with a predisposition to postpartal subclinical or clinical ketosis will remain standing for fewer hours during the days leading to parturition, which decreased DMI, and this condition was further reflected in lower milk yield.

Grain challenge affects systemic and hepatic molecular biomarkers of inflammation, stress, and metabolic responses to a greater extent in Holstein than Jersey cows.

Long-term feeding of high-grain diets to dairy cows often results in systemic inflammation characterized by alterations in acute-phase proteins and other biomarkers, both in plasma and immune-responsive tissues like the liver. The molecular and systemic changes that characterize an acute grain feeding challenge remain unclear. The current study involved 6 Holstein and 6 Jersey cows in a replicated 2 × 2 Latin square. Periods (10 d) were divided into 4 stages (S): S1, d 1 to 3, served as baseline with total mixed ration (TMR) ad libitum; S2, d 4, served as restricted feeding, with cows offered 50% of the average daily intake observed in S1; S3, d 5, a grain challenge was performed, in which cows were fed a TMR ad libitum without (CON) or with an additional pellet wheat-barley (1:1; HIG) at 20% of dry matter intake top-dressed onto the TMR; S4, d 6 to 10, served as recovery during which cows were allowed ad libitum access to the TMR. Among the 28 biomarkers analyzed in blood 12 h after grain challenge on d 5, the concentrations of fatty acids and bilirubin increased in HIG Holstein but not Jersey cows. In Holsteins, feeding HIG also increased total protein and albumin while decreasing ceruloplasmin, myeloperoxidase, and alkaline phosphatase concentrations. At the molecular level, hepatic genes associated with inflammation (IL1B, IL6, TNF, TLR4, MYD88, and NFKB1) were upregulated in Holstein cows fed HIG versus CON. Despite such response, expression of the acute-phase proteins SAA and HP in Holsteins fed HIG compared with CON was markedly downregulated. In Holsteins fed HIG versus CON, the marked downregulation of SCD, ELOVL6, and MTTP along with upregulated CPT1A, ACOX1, and APOA5 indicated alterations in fatty acid and lipoprotein metabolism during grain challenge. Genes related to ketogenesis (HMGCS2 and ACAT1) were upregulated in Jerseys, and gluconeogenic genes (PDK4 and PCK1) were upregulated in Holstein cows fed HIG, suggesting alterations in ketone body and glucose production. Expression of phosphorylated p70S6K1, RPS6, and 4EBP1 proteins, as well as total mechanistic target of rapamycin (mTOR) protein, decreased in Holsteins fed HIG, whereas phosphorylated mTOR and 4EBP1 proteins increased in Jerseys fed HIG. From a metabolic and inflammatory biomarker standpoint, data indicate that Jersey cows better tolerated the acute grain challenge. Alterations in mTOR signaling proteins in both Jerseys and Holsteins fed HIG suggest a potential role for exogenous AA in the hepatic adaptations to grain challenge. It remains to be determined if these acute responses to a grain challenge can elicit long-term liver dysfunction, which could negatively affect welfare of the cow.

Reticulo-rumen mass, epithelium gene expression, and systemic biomarkers of metabolism and inflammation in Holstein dairy cows fed a high-energy diet.

Feeding a higher-energy diet by increasing cereal grains at the expense of forage during the last 3 to 4 wk prepartum is a traditional approach to help the rumen "adapt" to the traditional diets fed at the onset of lactation. Increasing grain/concentrate in the diet changes ruminal fermentation and in sheep and goats elicits marked changes in mRNA expression of immune-related genes in ruminal epithelium. Whether such changes at the epithelial and systemic levels occur in dairy cows when the dietary energy content increases at a fixed level of concentrate is unknown. Fourteen nonpregnant, nonlactating Holstein cows were fed a control lower-energy (CON, 1.30 Mcal/kg of dry matter) diet to meet 100% of estimated nutrient requirements for 3 wk, after which half of the cows were assigned to a higher-energy diet (OVE, 1.60 Mcal/kg of dry matter) and half of the cows continued on CON for 6 wk. Levels of forage and concentrate for CON and OVE were 80 and 79% and 20 and 21%, respectively. Plasma samples were collected 1 d before slaughter to examine biomarkers of metabolism, liver function, inflammation, and oxidative stress. The reticulo-rumen mass was recorded at slaughter, and samples of epithelium were harvested from all cows. The expression of 29 genes associated with tight junctions, immune function, and nutrient transport (volatile fatty acids, urea, and trace minerals) was examined. Overfeeding energy led to consistently greater dry matter intake over time, and lowered plasma concentrations of haptoglobin, paraoxonase, bilirubin, fatty acids, and myeloperoxidase (secreted by neutrophils). In contrast, OVE resulted in greater hydroxybutyrate and cholesterol concentrations. A greater reticulo-rumen mass in cows fed OVE did not alter genes associated with tight junctions (CDLN1, CDNL4, OCLN, TJP1), immune function (IL1B, IL10, NFKB1, TLR2, TLR4, TNF), oxidative stress (SOD1, SOD2), or most nutrient transporters. However, feeding OVE upregulated the acute-phase protein SAA3 by 3.5-fold and downregulated a volatile fatty acid transporter (SLC16A1) and a Fe and Cu transporter (SLC11A2). The lack of effect on mRNA expression along with lower plasma concentrations of inflammation biomarkers indicates that long-term intake of a higher-energy diet ad libitum was not detrimental to ruminal epithelium integrity. In that context, a protective function of SAA3 could be envisioned with a role in opsonizing gram-negative bacteria that produce endotoxins. The long-term control of volatile fatty acid absorption and trace minerals from the rumen in cows overfed energy does not seem to be controlled at the gene transcription level. The relevance of these findings to the nutritional management of pregnant dry cows merits further research.

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.

Supplementing Zn, Mn, and Cu from amino acid complexes and Co from cobalt glucoheptonate during the peripartal period benefits postpartal cow performance and blood neutrophil function.

The physiologic and metabolic stresses that dairy cows experience during the transition into early lactation can promote oxidative stress, inflammation, and immune dysfunction. Optimal supply of micronutrients such as trace minerals (e.g., Zn, Mn, Cu, and Co) via more bioavailable forms (e.g., AA complexes) might minimize these negative effects. Multiparous Holstein cows were enrolled at 60 d before dry-off (~110 d before calving) and remained on experiment until 30 d in milk (DIM). Cows were offered a common diet supplemented entirely with inorganic trace minerals (INO) from -110 to -30 d before calving. From -30 to calving cows received a common prepartal [1.5 Mcal/kg of dry matter (DM), 15% crude protein] diet, and from calving to 30 DIM a common postpartal (1.76 Mcal/kg of DM, 18% crude protein) diet. Both diets were partially supplemented with an INO mix of Zn, Mn, and Cu to supply 35, 45, and 6 mg/kg, respectively, of the total diet DM. Cows were assigned to treatments in a randomized complete block design to receive an oral bolus with a mix of INO (n=21) or organic AA complexes (AAC; n=16) of Zn, Mn, Cu, and Co to achieve supplemental levels of 75, 65, 11, and 1mg/kg, respectively, in the total diet DM. Inorganic trace minerals were provided in sulfate form and AAC were supplied via Availa Zn, Availa Mn, Availa Cu, and COPRO (Zinpro Corp., Eden Prairie, MN). Liver tissue was harvested on -30, -15, 10, and 30 d, and blood samples for biomarker analyses were obtained more frequently from -30 to 30 DIM. Short-term changes in blood ketones were measured via Precision Xtra (Abbott Diabetes Care, Alameda, CA) every other day from 1 to 15 d postpartum. Prepartal DM intake was lower in AAC cows. In contrast, a tendency for a diet by time (D × T) interaction resulted in greater postpartal DM intake of approximately 2 kg/d in cows fed AAC. Milk and milk protein yield had a D × T interaction because AAC cows produced approximately 3.3 kg/d more milk and 0.14 kg/d more protein during the first 30 DIM. Although blood glucose, fatty acids, and liver triacylglycerol were not affected by diet, the Precision Xtra ketones (1.44 vs. 2.18 mmol/L) and γ-glutamyltransferase (liver function biomarker) were lower in AAC than INO. Furthermore, feeding AAC increased (D × T) polymorphonuclear neutrophilic lymphocyte phagocytosis, antioxidant capacity postpartum, and overall concentration of liver tissue Co and Cu. Overall, the positive response in milk yield and milk protein in AAC cows might be partly explained by the beneficial effects of AAC on postpartal DM intake driven at least in part by better liver and immune function as a result of improved antioxidant status.

Corium molecular biomarkers reveal a beneficial effect on hoof transcriptomics in peripartal dairy cows supplemented with zinc, manganese, and copper from amino acid complexes and cobalt from cobalt glucoheptonate.

Supplying trace minerals in more bioavailable forms such as amino acid complexes (AAC) could help ameliorate the incidence of hoof disorders in peripartal dairy cows. The aim of this study was to evaluate the effects of supplementing metal AAC during the peripartal period on expression of 28 genes in corium tissue related to claw composition, oxidative stress, inflammation, chemotaxis, and transcriptional regulation. Forty-four multiparous Holstein cows received a common diet from -30 to 30 d relative to parturition and were assigned to receive an oral bolus containing either inorganic trace minerals (INO) or AAC (i.e., organic) Zn, Mn, Cu, and Co to achieve supplemental levels of 75, 65, 11, and 1 ppm, respectively, in the total diet dry matter. Inorganic trace minerals were provided in sulfate form, and AAC were supplied via Availa Zn, Availa Mn, Availa Cu, and COPRO (Zinpro Corp., Eden Prairie, MN). Locomotion score was recorded before enrollment and weekly throughout the experiment. Incidence of hoof health problems at 30 d in milk was evaluated before a hoof biopsy in a subset of cows (INO=10; AAC=9). Locomotion score did not differ between treatments in the prepartum or postpartum period. The incidence of heel horn erosion was lower in AAC cows, but the incidence of sole ulcers did not differ. Downregulation of KRT5, CTH, CALML5, and CYBB, and upregulation of BTD in AAC cows indicated a decrease in the need for activation of cellular pathways to regenerate corium tissue and increase biotin availability in the sole claw. These molecular changes in the sole could have been triggered by the lower incidence of heel erosion in response to AAC. Among the genes associated with oxidative stress, the AAC cows had greater expression of NFE2L2, a transcription factor that regulates the antioxidant response, and the antioxidant enzyme SOD1. Among genes associated with inflammation, AAC cows had greater expression of TLR4, and lower expression of TLR2, IL1B, and TNF compared with INO cows. Supplementation with metal AAC during the peripartal period affected the expression of genes involved in composition, oxidative stress, and inflammation status in the corium. The hoof biopsy procedure used in the present study should be further perfected and implemented in future lameness research to expand our understanding of hoof biology in dairy cows.

Hepatic purinergic signaling gene network expression and its relationship with inflammation and oxidative stress biomarkers in blood from peripartal dairy cattle.

The liver plays a central role in allowing dairy cattle to make a successful transition into lactation. In liver, as in other tissues, extracellular nucleotides and nucleosides trigger cellular responses through adenosine and ATP receptors. Adenosine triphosphate and certain nucleotides serve as signals that can heighten purinergic receptor activation in several pathologic processes. We evaluated the mRNA expression of genes associated with the purinergic signaling network in liver tissue during the peripartal period. Seven multiparous Holstein cows were dried off at d -50 relative to expected parturition and fed a controlled-energy diet (net energy for lactation=1.24 Mcal/kg of DM) for ad libitum intake during the entire dry period. After calving, all cows were fed a common lactation diet (net energy for lactation=1.65 Mcal/kg of DM) until 30 DIM. Biopsies of liver were harvested at d -10, 7, and 21 for mRNA expression of 9 purinergic receptors, 7 ATP and adenosine transport channels, and 10 enzymes associated with ATP hydrolysis. Blood collected at d -21, -10, 7, 14, and 21 was used to measure concentrations of inflammation and oxidative stress biomarkers. The expression of type 1 purinergic receptors (ADORA2A and ADORA3), several nucleoside hydrolases [ectonucleoside triphosphate diphosphohydrolase 7 (ENTPD7), ectonucleotide pyrophosphatase/phosphodiesterase 2 (ENPP2), ENPP3, and adenosine deaminase (ADA)], and a type 2 purinergic receptor (P2RX7) was downregulated after calving. In contrast, the expression of type 2 purinergic receptors (P2RX4 and PR2Y11), an ATP release channel (gap junction hemichannel GJB1), and an adenosine uptake protein (SLC29A1) followed the opposite response, increasing after calving and remaining elevated through 21 d. Haptoglobin, ceruloplasmin, and reactive oxygen metabolite concentrations increased gradually from d -21 d through at least d 7. The opposite response was observed for albumin, paraoxonase, α-tocopherol, and nitric oxide, which decreased gradually to a nadir at 7 and 14 d. Our results suggest that alterations after calving of the expression of hepatic purinergic signaling genes could be functionally important because in nonruminants, they play roles in bile formation, glucose metabolism, cholesterol uptake, inflammation, and steatosis. The correlation analysis provided evidence of a link between purinergic signaling genes and biomarkers of inflammation and oxidative stress.

Smartamine M and MetaSmart supplementation during the peripartal period alter hepatic expression of gene networks in 1-carbon metabolism, inflammation, oxidative stress, and the growth hormone-insulin-like growth factor 1 axis pathways.

Peripartal cows likely require greater amounts of Met not only at the tissue and cell level for methylation reactions but also for milk protein synthesis after calving. 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 Inc., Antony, France; n=12), or CON plus Smartamine M (SM; Adisseo Inc.; n=13). The Met supplements were adjusted daily and top-dressed over the total mixed ration at a rate of 0.19 or 0.07% (dry matter) of feed for MS or SM. Liver tissue was collected on -10, 7, and 21 d for transcriptome profiling of genes associated with Met and glutathione metabolism as well as components of the inflammation, oxidative stress, growth hormone/insulin-like growth factor-1 axis, and DNA methylation pathways. Data were analyzed using PROC MIXED of SAS (SAS Institute Inc., Cary, NC) with the preplanned contrasts CON versus SM + MS and SM versus MS. The S-adenosylhomocysteine hydrolase (SAHH) gene was the most abundant among all genes evaluated, with overall greater expression in Met-supplemented cows than CON, and in SM than MS. Expression of Met adenosyltransferase 1A (MAT1A) was greater in Met-supplemented cows than CON by 21 d postpartum. A greater overall expression of 5-methyltetrahydrofolate-homocysteine methyltransferase (MTR) occurred in Met-supplemented cows than CON. In contrast, the expression of glutathione synthase (GSS); glutamate-cysteine ligase, catalytic subunit (GCLC); and superoxide dismutase 1, cytosolic (SOD1) was lower in Met-supplemented cows than CON. A greater overall expression of nuclear factor of kappa light polypeptide gene enhancer in B-cells 1 (NFKB1) and greater upregulation of haptoglobin (HP) on d 7 occurred in Met-supplemented cows than CON. Expression of DNA cytosine-5-methyltransferase 3 alpha (DNMT3A) was greater but expression of DNMT1 was lower in Met-supplemented cows than CON. The response observed in SAHH reflects its importance to Met supplementation during the peripartum period. Despite greater HP expression after calving, the lower expression of glutathione (GSS and GCLC) metabolism genes and SOD1 due to Met reflect a lower oxidative stress and mild inflammatory status. The extent to which changes in expression of DNMT3A and DNMT1 result in epigenetic effects partly responsible for the previously observed enhanced performance in Met-supplemented cows remains to be examined. Increasing the supply of Met as SM or MS can affect expression of genes in the Met cycle to various extents and, hence, the supply of methyl donors such as S-adenosylmethionine and antioxidants such as glutathione. These compounds likely are in high demand during the peripartum period.

Biomarkers of inflammation, metabolism, and oxidative stress in blood, liver, and milk reveal a better immunometabolic status in peripartal cows supplemented with Smartamine M or MetaSmart.

The peripartal dairy cow experiences a state of reduced liver function coupled with increased inflammation and oxidative stress. This study evaluated the effect of supplementing basal diets with rumen-protected Met in the form of MetaSmart (MS) or Smartamine M (SM) (both from Adisseo Inc., Antony, France) during the peripartal period on blood and hepatic biomarkers of liver function, inflammation, and oxidative stress. Thirty-seven multiparous Holstein cows were fed the same basal diet from -50 to -21 d relative to expected calving [1.24 Mcal/kg of dry matter (DM); no Met supplementation]. From -21 d to calving, the cows received diets (1.54 Mcal/kg of DM) with no added Met (control, CON; n=13), CON plus MS (n=11), or CON plus SM (n=13). From calving through 30 d in milk (DIM), the cows received the same postpartal diet (1.75 Mcal/kg of DM; CON), or CON plus MS or CON plus SM. Liver and blood samples were harvested at various time points from -21 to 21 d relative to calving. Preplanned contrasts of CON versus SM + MS during prepartum (-21 and -10 d before calving) and postpartum (7, 14, and 21 d after calving) responses were evaluated. Cows fed MS or SM compared with CON had lower overall concentrations of plasma ceruloplasmin and serum amyloid A (SAA). Compared with CON, Met-supplemented cows had greater overall plasma oxygen radical absorbance capacity. Liver concentrations of glutathione and carnitine also were greater overall with Met supplementation. Milk choline and liver phosphatidylcholine were lower overall in cows fed Met compared with controls. Liver tissue choline concentrations did not differ. Data indicate that supplemental Met enhanced de novo glutathione and carnitine synthesis in liver and, thus, increased antioxidant and ß-oxidation capacity. The greater decrease of IL-6 after calving coupled with lower ceruloplasmin and SAA in Met-supplemented cows indicated a reduction in proinflammatory signaling within liver. The lower hepatic phosphatidylcholine in Met-supplemented cows might have been associated with greater assembly or export of very low density lipoproteins. Overall, biomarker analyses in blood and tissue indicate that the beneficial effect of feeding SM and MS on postpartal cow performance is due in part to a better immunometabolic status.

Growth and wear characteristics of individual claws in young dairy calves.

Data are limited on how cow hooves develop early in life and how this will affect susceptibility to lameness and claw disorders later in life. The aim of this study was to characterize individual claw growth and wear in dairy calves. A total of 90 male Holstein calves <1 wk old were enrolled in this completely randomized design experiment and monitored until wk 20 of age. Evaluation of hoof development was conducted by visual inspection, and individual claw measurements were taken with a graduated ruler. Claws were numbered from 1 to 8 to maintain consistent data collection, where front claws were numbered 1 to 4 (left to right) and rear claws were 5 to 8. Lateral claws were numbered 1, 4, 5, and 8, and medial claws were 2, 3, 6, and 7. Measurements included length from the coronary band to the end of the hoof wall and from the coronary band to the groove line, resulting in the variables claw length (CL) and groove length (GL), respectively. Hoof development was evaluated at wk 0, 5, 10, 15, and 20. All evaluations were performed by the same person throughout the study to eliminate variation between observers. Hoof growth and wear were derived from CL and GL measurements and analyzed from wk 0 to 20. Claw length at wk 0 was different across claw positions, and maximal claw length was observed in claws in positions 6 and 7 (i.e., rear medial claws). A similar growth pattern in claw length was observed when evaluated from wk 0 to 20, where a greater claw length was observed in claws 6 and 7 compared with other claws. In contrast to claw length, claw wear at wk 20 was lower in claws 6 and 7 compared with other claws. Compared with rear claws, front claws had greater wear at wk 20, and compared with medial claws, lateral claws had greater wear at wk 20. Factors associated with claw length differences at wk 0 remain to be elucidated, but the fact that this uneven claw length difference was maintained 20 wk after birth is puzzling. The complementary claw length to wear described greater wear in claws 1 and 4 (i.e., front lateral) in young calves, which is translated into lower claw length in the same claws by wk 20 after birth. Future research in postnatal hoof growth should strive to understand the potential biological significance of this effect on lameness resistance and longevity of dairy cows.

Purinergic signaling gene network expression in bovine polymorphonuclear neutrophils during the peripartal period.

An effective immune response relies on efficient activation of polymorphonuclear neutrophilic leukocytes (PMNL). The PMNL release cellular ATP in response to inflammatory mediators. Although extracellular ATP is rapidly degraded to adenosine, both compounds can readily bind to either the purinergic receptor P1 (adenosine) or P2 (ATP). The P1 and P2 receptors are members of the G-protein-coupled receptor family. The peripartal period is characterized by marked changes in metabolic and inflammatory status that are functionally related with immune responses in the cow. We evaluated the mRNA expression of genes associated with purinergic signaling in PMNL during the peripartal period. Seven multiparous Holstein cows were dried off at d -50 relative to expected parturition and fed a controlled-energy diet (net energy for lactation=1.24 Mcal/kg of dry matter) for ad libitum intake during the entire dry period. After calving, all cows were fed a common lactation diet (net energy for lactation=1.65 Mcal/kg of dry matter) until 30 d in milk. Blood PMNL collected at -10, 3, and 21 d in milk were used to study the expression of 22 genes associated with adhesion to endothelium, chemoattractant binding at the plasma membrane, and purinergic signaling. Other blood samples around calving were used to analyze concentrations of insulin, metabolites, and whole-blood phagocytosis. The expression of purinergic receptor P2Y, G-protein coupled, 2 (P2RY2) increased on d 3 and then decreased on d 21. This response suggested that ATP could play a role in the amplification of chemotactic signals. In contrast, the expression of genes encoding cell adhesion [selectin L (SELL) and selectin P ligand (SELPLG)], chemoattractant receptors [complement component 5a receptor 1 (C5AR1), IL-8 receptor α (CXCR1), IL-8 receptor ß (CXCR2), and platelet-activating factor receptor (PTAFR)], and adenosine receptors [adenosine A1 receptor (ADORA1) and adenosine A3 receptor (ADORA3)] decreased between -10 and 3 d. The decrease coincided with a marked increase in blood nonesterified fatty acids and hydroxybutyrate concentrations, and a decrease in glucose and insulin concentrations. The increase in metabolites also was associated with greater expression of leukotriene B4 receptor (LTB4R) on d 3 and 21 compared with d -10, which is involved in inflammatory prostaglandin synthesis. Most chemoattractant receptors increased by 21 d, but cell adhesion genes and blood leukocyte phagocytosis was lower. The expression of adenosine A2a receptor (ADORA2A), which is associated with immunosuppression of PMNL and that of adenosine uptake channels [solute carrier family 29 (nucleoside transporters), member 1 (SLC29A1) and member 2 (SLC29A2)] and the nucleotidase adenosine deaminase (ADA) was greater at 3 and 21 d compared with -10d. The reduction in key immune responses, such as cell adhesion and chemotaxis, by bovine PMNL could partly be a function of changes in mRNA expression of genes associated with purinergic signaling.