Physiological Conditions Leading to Maternal Subclinical Ketosis in Holstein Dairy Cows Can Impair the Offspring's Postnatal Growth and Gut Microbiome Development.

Maternal metabolic disruptions, such as ketosis, can have adverse effects on fetal development and influence postnatal factors. Twelve Holstein calves were randomly enrolled in this study at birth and monitored until 8 weeks of age. The study was conducted from fall 2018 until spring 2019. After completing the data collection period, calves were classified according to their respective dams ketotic condition after parturition. This classification was based on dam blood ß-hydroxybutyrate < 1.4 mmol/L nonketotic (NONKET; n = 6 calves) or ≥1.4 mmol/L subclinical-ketotic (SK; n = 6 calves). SK calves had greater birth body weight (p = 0.05) but exhibited a slower growth rate compared to NONKET calves from 1 to 8 weeks (p = 0.02). At birth, SK calves had lower (p < 0.01) levels of non-esterified fatty acids and bilirubin compared to NONKET calves. Analysis of feces alpha diversity indicates that by 3 weeks, NONKET calves had greater diversity, richness, and evenness. Butyricicoccus pullicaecorum and Gallibacterium anatis were more abundant in SK calves (p < 0.05) at 3 weeks. In contrast, NONKET calves had a greater (p < 0.05) abundance of Sharpae azabuensis at 3 weeks. These findings suggest that subclinical ketosis in cows can impact the in-utero development, postnatal growth, and maturing gut microbiome of their offspring.

Yeast Culture Supplementation Effects on Systemic and Polymorphonuclear Leukocytes' mRNA Biomarkers of Inflammation and Liver Function in Peripartal Dairy Cows.

This study evaluated the effects of feeding a commercial yeast culture on blood biomarkers and polymorphonuclear leukocyte (PMNL) gene expression in dairy cows during the transition period until 50 d postpartum. Forty Holstein dairy cows were used in a randomized complete block design from -30 to 50 d. At -30 d, cows were assigned to a basal diet plus 114 g/d of top-dressed ground corn (control; n = 20) or 100 g/d of ground corn and 14 g/d of a yeast culture product (YC; n = 20). Blood samples were collected at various time points from -30 to 30 DIM to evaluate blood biomarkers and PMNL gene expression related to inflammation, liver function, and immune response. Liver function biomarkers, gamma-glutamyl transferase (GGT) and albumin were greater and lower, respectively, in YC cows in comparison to control. However, these biomarkers remained within physiological levels, indicating an active inflammatory process. Genes in PMNL expression related to inflammation (NFKB1, TNFA, TRAF6), anti-inflammation (IL10), and cell membrane receptors (SELL) were upregulated in the YC group in comparison to control. These results suggest that YC could stimulate a more active inflammatory response with signs of a resolution of inflammation in transition cows.

Early Life Fecal Microbiota Transplantation in Neonatal Dairy Calves Promotes Growth Performance and Alleviates Inflammation and Oxidative Stress during Weaning.

This study aimed to evaluate the effects of early life fecal microbiota transplantation (FMT) on the health and performance of neonatal dairy calves. The donor was selected based on health and production records and fecal material testing negative for infectious pathogens. Sixteen healthy newborn Holstein calves were randomized to either a baseline nutritional program (CON) or 1×/d inoculations with 25 g of fecal donor material (FMT) mixed in the milk replacer (n = 8/TRT) from 8 to 12 days of age. Blood and fecal samples were collected weekly, and calves were weaned at 7 weeks of age. A TRT × Week interaction was observed in haptoglobin, which was reflected in a positive quadratic effect in FMT calves but not in CON. A trend for a TRT × Week interaction was observed in the liver function biomarker paraoxonase, which resulted in greater paraoxonase in FMT calves than CON at three weeks of age. Fecal microbial community analysis revealed a significant increase in the alpha-diversity between week 1 and week 5 for the FMT calves. These results suggest that early life FMT in neonatal calves has positive effects in mediating the inflammatory response and gut microbial maturation.

An Exploration of the Effects of an Early Postpartum Intravenous Infusion with Carnosic Acid on Physiological Responses of Transition Dairy Cows.

The objective of the present study was to evaluate the effects of an antioxidant and anti-inflammatory compound found in rosemary plants (Salvia rosmarinus) named carnosic acid during the transition period of dairy cows. From day 1 to 3 after calving, 16 multiparous Holstein cows received a daily intravenous infusion of either 500 mL of saline (NaCl 0.9%; Saline; n = 8) or carnosic acid at a rate of 0.3 mg/kg of BW supplied in 500 mL of saline (CA; n = 8). Blood samples were taken at -7, 2, 5, 7, 14, and 21 d relative to parturition, then analyzed for metabolites related to energy metabolism, muscle mass catabolism, liver function, inflammation, and oxidative stress. CA infusion tended to improve milk performance; however, DMI was unaffected by treatment. At 2 d relative to parturition, CA cows had lower blood concentrations of haptoglobin, paraoxonase, FRAP, and NO2- than saline cows. After treatment infusions, haptoglobin remained lower in CA cows than saline at 5 d relative to parturition. Our results demonstrate that carnosic acid promoted positive responses on inflammation and oxidative stress biomarkers and may promote beneficial effects on lactation performance in peripartal dairy cows.

Gut health, stress, and immunity in neonatal dairy calves: the host side of host-pathogen interactions.

The cumulative evidence that perinatal events have long-lasting ripple effects through the life of livestock animals should impact future nutritional and management recommendations at the farm level. The implications of fetal programming due to malnutrition, including neonatal survival and lower birth weights, have been characterized, particularly during early and mid-gestation, when placental and early fetal stages are being developed. The accelerated fetal growth during late pregnancy has been known for some time, while the impact of maternal stressors during this time on fetal development and by extent its postnatal repercussions on health and performance are still being defined. Maternal stressors during late pregnancy cannot only influence colostrogenesis but also compromise adequate intestinal development in the fetus, thus, that further limits the newborn's ability to absorb nutrients, bioactive compounds, and immunity (i.e., immunoglobulins, cytokines, and immune cells) from colostrum. These negative effects set the newborn calf to a challenging start in life by compromising passive immunity and intestinal maturation needed to establish a mature postnatal mucosal immune system while needing to digest and absorb nutrients in milk or milk replacer. Besides the dense-nutrient content and immunity in colostrum, it contains bioactive compounds such as growth factors, hormones, and cholesterol as well as molecular signals or instructions [e.g., microRNAs (miRNAs) and long non-coding RNAs (lncRNAs)] transferred from mother to offspring with the aim to influence postnatal gut maturation. The recent change in paradigm regarding prenatal materno-fetal microbiota inoculation and likely the presence of microbiota in the developing fetus intestine needs to be addressed in future research in ruminants. There still much to know on what prenatal or postnatal factors may predispose neonates to become susceptible to enteropathogens (e.g., enterotoxigenic Escherichia coli), causing diarrhea. From the host-side of this host-pathogen interaction, molecular data such as fecal RNA could, over time, help fill those gaps in knowledge. In addition, merging this novel fecal RNA approach with more established microbiome techniques can provide a more holistic picture of an enteropathogenesis and potentially uncover control points that can be addressed through management or nutrition at the farm level to minimize preweaning morbidity and mortality.

Quantitative determination of histone methylation via fluorescence resonance energy transfer (FRET) technology in immortalized bovine mammary alveolar epithelial cells supplemented with methionine.

Methionine (Met) is an essential precursor of S-adenosylmethionine (SAM), which is the primary methyl donor required for biological processes such as DNA and histone methylation, which alter gene expression. In dairy cows, dietary Met has been observed to exert transcriptional alterations with beneficial effects on milk biosynthesis; however, the extent of these effects via SAM remains unknown. Therefore, we evaluated the effect of Met supply on histone methylation in lysine residues K9 and K27 in the histone tail H3 via a fluorescence resonance energy transfer (FRET) system in immortalized bovine mammary alveolar epithelial cells (MACT) incubated varying concentration of Met. The histone methylation data was complemented with global DNA methylation, cellular protein synthesis, and RT-qPCR analysis of genes related to Met cycle, DNA and histone methylation, AA transporters, and protein synthesis. The histone methylation data was performed on MACT cells seeded at 30,000 cells/well in 96-well plates 24 h prior to transfection. The transfections of FRET gene reporter plasmids H3K9 and H3K27 was performed with 0.3 µL/well of Lipofectamine® 3000 and 50 ng of plasmid DNA per well. At 24 h post-transfection, cells were treated with 0, 125, 250, and 500 µM of Met, and quantification of histone methylation was performed at 0, 12, and 24 h post-treatment as well as cell viability at 24 h using CellProfiler software. An inverted microscope for live imagining (EVOS® FL Auto) equipped with a motorized scanning stage, and an environment-controlled chamber at 37˚C and 5.0% of CO2 was used to take 4 pictures/well at 4x magnification. A more defined response on histone methylation was observed in H3K9 than H3K27 to Met supply, where maximal histone methylation in H3K9 was observed with 125 µM of Met. This greater histone methylation in H3K9 at 125 µM was accompanied by greater cellular protein concentration. The linear increase in Met supply causes a linear decrease in global DNA methylation, while linearly upregulating genes related to the Met cycle (i.e., MAT1A, PEMT, SAHH, and MTR). The histone methylation data suggest that, to some extent, methyl-donors such as Met may affect the methylation sites, H3K9 and H3K27, and consequently causing a different epigenetic alteration. In the context of the dairy cow, further refinement to this FRET assay to study histone methylation could lead to establishing novel potential mechanisms of how dietary methyl donors may control the structural conformation of the bovine genome and, by extension, gene expression.

A rapid blood test to monitor immunity shift during pregnancy and potential application for animal health management.

The immune health of a farm animal can have significant impact on its overall health, welfare and productivity. One of the most vulnerable physiological states for both humans and animals is pregnancy. Many systemic changes correlate with the gravid state, including shifts in the immune system that may impact the ability to respond optimally to pathogen challenge. Because of this, it would be beneficial to be able to monitor the immune health of the pregnant animals closely. Recently, we developed a new nanoparticle-enabled rapid blood test that can detect ongoing immune responses from both laboratory and farm animals. Here, we report that this novel test reveals highly repeatable and acute changes associated with pregnancy and peri-parturition period in laboratory mice and in cattle. We hypothesize that the test score change reflects changes in the immune status of the gravid females related to the humoral immune response. The test is easy to conduct, of low cost, with results obtained in less than 20 min. This rapid test could be potentially used as an onsite test in local farms and small clinics for animal health management.

Hepatic metabolomics and transcriptomics to study susceptibility to ketosis in response to prepartal nutritional management.

BACKGROUND: Ketosis in dairy cows is associated with body fat mobilization during the peripartal period. Sub-clinical and clinical ketosis arise more frequently in cows that are overfed energy during the entire dry (last 50 to 45 days prior to parturition) or close-up period (last ~ 28 days prepartum). METHODS: A retrospective analysis was performed on 12 cows from a larger cohort that were fed a higher-energy diet [1.54 Mcal/kg of dry matter (DM); 35.9% of DM corn silage and 13% of DM ground corn] during the close-up dry period, of which 6 did not develop clinical ketosis (OVE, 0.83 mmol/L plasma hydroxybutyrate; BHB) and 6 were diagnosed with clinical ketosis (KET, 1.4 mmol/L BHB) during the first week postpartum. A whole-transcriptome bovine microarray (Agilent Technologies) and metabolomics (GC-MS, LC-MS; Metabolon® Inc.) were used to perform transcript and metabolite profiling of liver tissue harvested at - 10 days relative to parturition which allowed to establish potential associations between prepartal transcriptome/metabolome profiles and susceptibility to clinical ketosis postpartum. RESULTS: Cows in KET had greater (P = 0.01) overall body weight between - 2 and 1 week around parturition, but similar body condition score than OVE. Although dry matter intake (DMI) did not differ prepartum, KET cows had lower (P < 0.01) DMI and similar milk yield as OVE cows during the first week postpartum. Transcriptome analysis revealed a total of 3065 differentially expressed genes (DEG; P ≤ 0.05) in KET. Metabolomics identified 15 out of 313 total biochemical compounds significantly affected (P ≤ 0.10) in KET. Among those, greater concentrations (P ≤ 0.06, + 2.3-fold) of glycochenodeoxycholate in KET cows also have been detected in humans developing non-alcoholic fatty liver disease. Bioinformatics analysis using the Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway database and the DEG revealed that, among the top 20 most-impacted metabolic pathway categories in KET, 65% were overall downregulated. Those included 'Metabolism of cofactors and vitamins', 'Biosynthesis of other secondary metabolites', 'Lipid', 'Carbohydrate', and 'Glycan biosynthesis and metabolism'. The lower relative concentration of glucose-6-phosphate and marked downregulation of fructose-1,6-bisphosphatase 2 and pyruvate dehydrogenase kinase 4 support a strong impairment in gluconeogenesis in prepartal liver of cows developing KET postpartum. Among the top 20 most-impacted non-metabolic pathways, 85% were downregulated. Pathways such as 'mTOR signalling' and 'Insulin signalling' were among those. 'Ribosome', 'Nucleotide excision repair', and 'Adherens junctions' were the only upregulated pathways in cows with KET. CONCLUSIONS: The combined data analyses revealed more extensive alterations of the prepartal liver transcriptome than metabolome in cows overfed energy and developing ketosis postpartum. The causative link between these tissue-level adaptations and onset of clinical ketosis needs to be studied further.

2,4-Thiazolidinedione in Well-Fed Lactating Dairy Goats: II. Response to Intra-Mammary Infection.

In a prior experiment, treatment of goats with the putative PPARγ agonist 2,4-thiazolidinedione (2,4-TZD) ameliorated the response to intramammary infection without evidence of PPARγ activation. The lack of PPARγ activation was possibly due to deficiency of vitamin A and/or a poor body condition of the animals. Therefore, the present study hypothesized that activation of PPARγ by 2,4-TZD in goats supplemented with adequate amounts of vitamin A can improve the response to sub-clinical mastitis. Lactating goats receiving a diet that met National Research Council requirements, including vitamin A, were injected with 8 mg/kg BW of 2,4-TZD (n = 6) or saline (n = 6; control (CTR)) daily. Two weeks into treatment, all goats received Streptococcus uberis (IMI) in the right mammary gland. Blood biomarkers of metabolism, inflammation, and oxidative status plus leukocytes phagocytosis were measured. Mammary epithelial cells (MEC) and macrophages were isolated from milk and liver tissue collected for gene expression analysis. Milk fat was maintained by treatment with 2,4-TZD, but decreased in CTR, after IMI. Haptoglobin was increased after IMI only in 2,4-TZD without any effect on negative acute phase proteins, indicating an improved liver function. 2,4-TZD vs. CTR had a greater amount of globulin. The expression of inflammation-related genes was increased by IMI in both macrophages and MEC. Except for decreasing expression of SCD1 in MEC, 2,4-TZD did not affect the expression of measured genes. Results confirmed the successful induction of sub-clinical mastitis but did not confirm the positive effect of 2,4-TZD on the response to IMI in well-fed goats.

2,4-Thiazolidinedione in Well-Fed Lactating Dairy Goats: I. Effect on Adiposity and Milk Fat Synthesis.

Background: In a prior experiment, treatment of goats with the putative PPARγ agonist 2,4-thiazolidinedione (2,4-TZD) did not affect milk fat or expression of milk-fat related genes. The lack of response was possibly due to deficiency of vitamin A and/or a poor body condition of the animals. In the present experiment, we tested the hypothesis that PPARγ activation affects milk fat synthesis in goats with a good body condition and receiving adequate levels of vitamin A. Methods: Lactating goats receiving a diet that met NRC requirements, including vitamin A, were injected with 8 mg/kg BW of 2,4-TZD (n = 6) or saline (n = 6; CTR) daily for 26 days. Blood metabolic profiling and milk yield and components were measured including fatty acid profile. Expression of genes related to glucose and lipid metabolism was measured in adipose tissue and in mammary epithelial cells (MEC). Size of adipocytes was assessed by histological analysis. Results: NEFA, BHBA, and fatty acids available in plasma decreased while glucose increased in 2,4-TZD vs. CTR. Size of cells and expression of insulin signaling and glucose metabolism-related genes were larger in 2,4-TZD vs. CTR in adipose tissue. In MEC, expression of SCD1 and desaturation of stearate was lower in 2,4-TZD vs. CTR. Conclusions: Overall data revealed a lack of PPARγ activation by 2,4-TZD and no effect on milk fat synthesis despite a strong anti-lipolysis effect on adipose tissue.

Short communication: Characterization of gene expression profiles related to yak milk protein synthesis during the lactation cycle.

This research assessed the gene expression patterns related to the synthesis of milk in yak, which is characterized by high fat and protein content but low yield. The yak (Bos grunniens) is one of the most crucial domestic animals in Tibetan life; however, the genetic and molecular factors underlying yak milk protein synthesis remain understudied. Yak mammary biopsies harvested during late-pregnancy (d -15) through the end of subsequent lactation (d 1, 15, 30, 60, 180, and 240) were used to evaluate gene expression via real-time quantitative PCR. The expression pattern of 41 genes encompassing multiple pathways integral to milk protein synthesis including insulin, mammalian target of rapamycin (mTOR), 5' AMP-activated protein kinase, Jak2-Stat5 signaling, and the expression of glucose and AA transporters was evaluated. Our results confirmed that most upregulated genes increased from d -15 and peaked at d 30 or 60 and then remained relatively highly expressed. Specifically, there was an increased expression of mTOR-related amino acid transporters (SLC1A5, SLC7A5, and SLC36A1), glucose transporters (SLC2A1, SLC2A3, and SLC2A8), Jak2-Stat5 pathway (ELF5), and insulin signaling pathway components (IRS1, PDPK1, and AKT1). For activation of proteins synthesis, MTOR was significantly increased only at d 1. Among inhibitors of mTOR signaling, TSC1 and PRKAA2 were significantly upregulated during lactation. The RPL23 was downregulated among ribosomal components. In conclusion, a critical role for AA and glucose transporters and insulin signaling through mTOR for regulation of yak milk protein synthesis was revealed in this study of the yak mammary gland.

Peroxisome proliferator-activated receptor ß/δ does not regulate glucose uptake and lactose synthesis in bovine mammary epithelial cells cultivated in vitro.

The hypothesis of the study was that inhibition of PPARß/δ increases glucose uptake and lactose synthesis in bovine mammary epithelial cells by reducing the expression of the glucose transporter mRNA destabiliser calreticulin. Three experiments were conducted to test the hypothesis using immortalised bovine mammary alveolar (MACT) and primary bovine mammary (PBMC) cells. In Experiment 1, the most effective dose to inhibit PPARß/δ activity among two synthetic antagonists (GSK-3787 and PT-s58) was assessed using a gene reporter assay. In Experiment 2, the effect on glucose uptake and lactose synthesis was evaluated by measuring glucose and lactose in the media and expression of related key genes upon modulation of PPARß/δ using GSK-3787, the synthetic PPARß/δ agonist GW-501516, or a combination of the two in cells cultivated in plastic. In Experiment 3, the same treatments were applied to cells cultivated in Matrigel and glucose and lactose in media were measured. In Experiment 1 it was determined that a significant inhibition of PPARß/δ in the presence or absence of fetal bovine serum was achieved with ≥ 1000 nm GSK-3787 but no significant inhibition was observed with PT-s58. In Experiment 2, inhibition of PPARß/δ had no effect on glucose uptake and lactose synthesis but they were both increased by GW-501516 in PBMC. The mRNA abundance of PPARß/δ target gene pyruvate dehydrogenase kinase 4 was increased but transcription of calreticulin was decreased (only in MACT cells) by GW-501516. Treatment with GSK-3787 did not affect the transcription of measured genes. No effects on glucose uptake or lactose synthesis were detected by modulation of PPARß/δ activity on cells cultivated in Matrigel. The above data do not provide support for the original hypothesis and suggest that PPARß/δ does not play a major role in glucose uptake and lactose synthesis in bovine mammary epithelial cells.

Transcriptional changes detected in fecal RNA of neonatal dairy calves undergoing a mild diarrhea are associated with inflammatory biomarkers.

After birth, a newborn calf has to adapt to an extrauterine life characterized by several physiological changes. In particular, maturation of the gastrointestinal tract in a new environment loaded with potential pathogens, which can predispose neonatal calves to develop diarrhea, and is a major cause of morbidity and mortality during the first 4 wks of life. We aimed to investigate the inflammatory adaptations at a transcriptomic level in the gastrointestinal (GI) tract to a mild diarrhea in neonatal dairy calves using RNA isolated from fresh fecal samples. Eight newborn Jersey male calves were used from birth to 5 wks of age and housed in individual pens. After birth, calves received 1.9 L of colostrum from their respective dams. Calves had ad-libitum access to water and starter grain (22% CP) and were fed twice daily a total of 5.6 L pasteurized whole milk. Starter intake, body weight (BW), fecal score, withers height (WH), and rectal temperature (RT) were recorded throughout the experiment. Blood samples were collected weekly for metabolic and inflammatory profiling from wk 0 to wk 5. Fresh fecal samples were collected weekly and immediately flash frozen until RNA was extracted using a Trizol-based method, and subsequently, an RT-qPCR analysis was performed. Orthogonal contrasts were used to evaluate linear or quadratic effects over time. Starter intake, BW, and WH increased over time. Fecal score was greatest (2.6 ± 0.3) during wk 2. The concentrations of IL-6, ceruloplasmin, and haptoglobin had a positive quadratic effect with maximal concentrations during wk 2, which corresponded to the maximal fecal score observed during the same time. The concentration of serum amyloid A decreased over time. The mRNA expression of the proinflammatory related genes TLR4, TNFA, IL8, and IL1B had a positive quadratic effect of time. A time effect was observed for the cell membrane sodium-dependent glucose transporter SLC5A1, for the major carbohydrate facilitated transporter SLC2A2, and water transport function AQP3, where SLC5A1 and AQP3 had a negative quadratic effect over time. Our data support the use of the fecal RNA as a noninvasive tool to investigate intestinal transcriptomic profiling of dairy calves experiencing diarrhea, which would be advantageous for future research including nutritional effects and health conditions.

2,4-Thiazolidinedione Treatment Improves the Innate Immune Response in Dairy Goats with Induced Subclinical Mastitis.

Mastitis is a major disease in dairy cows resulting in significant economic losses. In vitro works suggest that ruminants peroxisome proliferator-activated receptor gamma (PPARγ) can aid in improving the response to mastitis and can control milk fat synthesis. The objectives of the present experiment were to test if treatment with the putative PPARγ agonist 2,4-thiazolidinedione (TZD) improves (1) the response to subclinical mastitis and (2) milk fat production. Lactating goats received daily injections of 8 mg/kg BW of TZD or saline for 3 weeks. After one week of TZD injection, half of the goats in each group received intramammary infusion of Strep. uberis or saline in both halves for a total of 4 groups (n = 6/group). TZD treatment did not affect milk fat but had positive effect on milk somatic cells count, blood nonesterified fatty acids, inflammatory markers, and liver function. TZD significantly increased myeloperoxidase but did not affect leukocytes phagocytosis or insulin. TZD increased adipocytes size and had minor effect on expression of PPARγ target genes in mammary epithelial cells but not in adipose tissue. Overall, TZD ameliorated the response to intramammary infection but the effect on milk fat synthesis and expression of related transcripts was less than expected.

Plasmid transfection in bovine cells: Optimization using a realtime monitoring of green fluorescent protein and effect on gene reporter assay.

Gene reporter technology (GRT) has opened several new avenues for monitoring biological events including the activation of transcription factors, which are central to the study of nutrigenomics. However, this technology relies heavily on the insertion of foreign plasmid DNA into the nuclei of cells (i.e., transfection), which can be very challenging and highly variable among cell types. The objective of this study was to investigate the optimal conditions to generate reliable GRT assay data on bovine immortalized cell lines, Madin Darby Bovine Kidney (MDBK) and bovine mammary epithelial alveolar (MACT) cells. Results are reported for two experiments. In Experiment 1, using 96 well-plate and a robotic inverted fluorescent microscope, we compared transfection efficiency among commercially available transfection reagents (TR) Lipofectamine® 3000 (Lipo3), Lipofectamine® LTX (LipoLTX), and TransIT-X2® (TransX2), three doses of TR (i.e., 0.15, 0.3, and 0.4µL/well), and three doses of Green Fluorescent Protein plasmid DNA (i.e., 10, 25, and 50ng/well). Transfection efficiency and mortality rate were analyzed using CellProfiler software. Transfection efficiency increased until the end of the experiment (20h post-transfection) at which point MACT had greater transfection than MDBK cells (16.3% vs. 2.2%). It is unclear the reason for the low transfection in MDBK cells. Maximal transfection efficiency was obtained with 0.3µL/well of LipoLTX plus 25ng/well of plasmid DNA (ca. 29.5±1.9%) and 0.15µL/well of LipoLTX plus 25ng/well of plasmid DNA (ca. 4.0±0.4%) for MACT and MDBK cells, respectively. The higher amount of TR and DNA was generally associated with higher cell mortality. Using high, medium, and low transfection efficiency conditions determined in Experiment 1, we performed a GRT assay for peroxisome proliferator-activated response element (PPRE) luciferase in MACT and MDBK cells treated with 10nM or 100nM of synthetic Peroxisome Proliferator-activated Receptor ß/σ (PPARß/σ) agonist. The GRT assay was unaffected by poor transfection in MACT cells although the high transfection hampered the possibility of detecting differences between 10 and 100nM of the PPARß/δ agonist. In MDBK cells, low transfection efficiency (<2.0%) failed to detect any differences with GRT assay. The level of transfection was positively associated with a lower coefficient of variation of GRT data. Overall, our data indicates that results of GRT assays are affected by transfection efficiency and a minimum transfection of 2% is required. Thus, factors such as TR type, TR amount, and DNA plasmid amount need to be optimized for a specific cell type before performing GRT assays.

Peripheral leukocyte and endometrium molecular biomarkers of inflammation and oxidative stress are altered in peripartal dairy cows supplemented with Zn, Mn, and Cu from amino acid complexes and Co from Co glucoheptonate.

BACKGROUND: Immune dysfunction and a higher risk of uterine infections are characteristics of the transition into lactation in dairy cows. The supply of complexed trace minerals, which are more bioavailable, could help overcome the greater needs of these nutrients in tissues around parturition and early lactation. RESULTS: Twenty Holstein cows received an oral bolus with a mix of inorganic trace minerals (INO) or complexed trace minerals (AAC) to achieve 75, 65, 11, and 1 ppm supplemental Zn, Mn, Cu, and Co, respectively, in the total diet dry matter from -30 d through +30 d relative to parturition. Blood for polymorphonuclear leukocyte (PMNL) isolation was collected at -30, -15, +10, and + 30 d relative to parturition, whereas endometrium biopsies were performed at +14 and +30 d. Feeding AAC led to greater PMNL expression of genes related with inflammation response (DDX58), oxidative stress response (MPO), eicosanoid metabolism (PLA2G4A and ALOX5AP), transcription regulation (PPARG), and cellular adhesion (TLN1). The upregulation by AAC in endometrium of genes related with inflammation response (TLR2, TLR4, NFKB1, TNF, IL6, IL1B, IL10, IL8), prostaglandin synthesis (PTGS2, PTGES), and antioxidant responses (NFE2L2, SOD1) indicated a faster remodeling of uterine tissue and potentially greater capacity to control a local bacterial invasion. CONCLUSIONS: Data indicate that trace mineral supplementation from amino acid complexes improves PMNL activity and allows the prompt recovery of uterine tissue during early lactation. As such, the benefits of complexed trace minerals extend beyond an improvement of liver function and productive performance.

Supplemental Smartamine M in higher-energy diets during the prepartal period improves hepatic biomarkers of health and oxidative status in Holstein cows.

BACKGROUND: Feeding higher-energy prepartum is a common practice in the dairy industry. However, recent data underscore how it could reduce performance, deepen negative energy balance, and augment inflammation and oxidative stress in fresh cows. We tested the effectiveness of rumen-protected methionine in preventing the negative effect of feeding a higher-energy prepartum. Multiparous Holstein cows were fed a control lower-energy diet (CON, 1.24 Mcal/kg DM; high-straw) during the whole dry period (~50 d), or were switched to a higher-energy (OVE, 1.54 Mcal/kg DM), or OVE plus Smartamine M (OVE + SM; Adisseo NA) during the last 21 d before calving. Afterwards cows received the same lactation diet (1.75 Mcal/kg DM). Smartamine M was top-dressed on the OVE diet (0.07% of DM) from -21 through 30 d in milk (DIM). Liver samples were obtained via percutaneous biopsy at -10, 7 and 21 DIM. Expression of genes associated with energy and lipid metabolism, hepatokines, methionine cycle, antioxidant capacity and inflammation was measured. RESULTS: Postpartal dry matter intake, milk yield, and energy-corrected milk were higher in CON and OVE + SM compared with OVE. Furthermore, milk protein and fat percentages were greater in OVE + SM compared with CON and OVE. Expression of the gluconeogenic gene PCK1 and the lipid-metabolism transcription regulator PPARA was again greater with CON and OVE + SM compared with OVE. Expression of the lipoprotein synthesis enzyme MTTP was lower in OVE + SM than CON or OVE. Similarly, the hepatokine FGF21, which correlates with severity of negative energy balance, was increased postpartum only in OVE compared to the other two groups. These results indicate greater liver metabolism and functions to support a greater production in OVE + SM. At 7 DIM, the enzyme GSR involved in the synthesis of glutathione tended to be upregulated in OVE than CON-fed cows, suggesting a greater antioxidant demand in overfed cows. Feeding OVE + SM resulted in lower similar expression of GSR compared with CON. Expression of the methionine cycle enzymes SAHH and MTR, both of which help synthesize methionine endogenously, was greater prepartum in OVE + SM compared with both CON and OVE, and at 7 DIM for CON and OVE + SM compared with OVE, suggesting greater Met availability. It is noteworthy that DNMT3A, which utilizes S-adenosylmethionine generated in the methionine cycle, was greater in OVE and OVE + SM indicating higher-energy diets might enhance DNA methylation, thus, Met utilization. CONCLUSIONS: Data indicate that supplemental Smartamine M was able to compensate for the negative effect of prepartal energy-overfeeding by alleviating the demand for intracellular antioxidants, thus, contributing to the increase in production. Moreover Smartamine M improved hepatic lipid and glucose metabolism, leading to greater liver function and better overall health.

Digital Cushion Fatty Acid Composition and Lipid Metabolism Gene Network Expression in Holstein Dairy Cows Fed a High-Energy Diet.

The hoof digital cushion is a complex structure composed of adipose tissue beneath the distal phalanx, i.e. axial, middle and abaxial fat pad. The major role of these fat depots is dampening compression of the corium underneath the cushion. The study aimed to determine expression of target genes and fatty acid profiles in the hoof of non-pregnant dry Holstein cows fed low (CON) or high-energy (OVE) diets. The middle fat pad of the hoof digital cushion was collected soon after slaughter. Despite the lack of effect on expression of the transcription regulators SREBF1 and PPARG, the expression of the lipogenic enzymes ACACA, FASN, SCD, and DGAT2 was upregulated with OVE. Along with the upregulation of G6PD and IDH1, important for NADPH synthesis during lipogenesis, and the basal glucose transporter SLC2A1, these data indicated a pro-lipogenic response in the digital cushion with OVE. The expression of the lipid droplet-associated protein PLIN2 was upregulated while expression of lipolytic enzymes (ATGL, ABDH5, and LIPE) only tended to be upregulated with OVE. Therefore, OVE induced lipogenesis, lipid droplet formation, and lipolysis, albeit to different extents. Although concentration of monounsaturated fatty acids (MUFA) did not differ, among the polyunsaturated fatty acids (PUFA), the concentration of 20:5n3 was lower with OVE. Among the saturated fatty acids, 20:0 concentration was greater with OVE. Although data indicated that the hoof digital cushion metabolic transcriptome is responsive to higher-energy diets, this did not translate into marked differences in the fatty acid composition. The decrease in concentration of PUFA, which could contribute to synthesis of inflammatory molecules, in OVE-fed cows indicated that feeding higher-energy diets might be detrimental for the mediation of inflammation in digital cushion. This effect could be further exacerbated by physiologic and endocrine changes during the peripartal period that favor inflammation.

Immunometabolic Status during the Peripartum Period Is Enhanced with Supplemental Zn, Mn, and Cu from Amino Acid Complexes and Co from Co Glucoheptonate.

The peripartum (or transition) period is the most-critical phase in the productive life of lactating dairy cows and optimal supply of trace minerals through more bioavailable forms could minimize the negative effects associated with this phase. Twenty Holstein cows received a common prepartal diet and postpartal diet. Both diets were partially supplemented with an inorganic (INO) mix of Zn, Mn, and Cu to supply 35, 45, and 6 ppm, respectively, of the diet dry matter (DM). Cows were assigned to treatments in a randomized completed block design, receiving an daily oral bolus with INO or organic trace minerals (AAC) Zn, Mn, Cu, and Co to achieve 75, 65, 11, and 1 ppm supplemental, respectively, in the diet DM. Liver tissue and blood samples were collected throughout the experiment. The lower glutamic-oxaloacetic transaminase concentration after 15 days in milk in AAC cows indicate lower hepatic cell damage. The concentration of cholesterol and albumin increased, while IL-6 decreased over time in AAC cows compared with INO indicating a lower degree of inflammation and better liver function. Although the acute-phase protein ceruloplasmin tended to be lower in AAC cows and corresponded with the reduction in the inflammatory status, the tendency for greater serum amyloid A concentration in AAC indicated an inconsistent response on acute-phase proteins. Oxygen radical absorbance capacity increased over time in AAC cows. Furthermore, the concentrations of nitric oxide, nitrite, nitrate, and the ferric reducing ability of plasma decreased with AAC indicating a lower oxidative stress status. The expression of IL10 and ALB in liver tissue was greater overall in AAC cows reinforcing the anti-inflammatory response detected in plasma. The greater overall expression of PCK1 in AAC cows indicated a greater gluconeogenic capacity, and partly explained the greater milk production response over time. Overall, feeding organic trace minerals as complexed with amino acids during the transition period improved liver function and decreased inflammation and oxidative stress.

Biosynthesis of milk fat, protein, and lactose: roles of transcriptional and posttranscriptional regulation.

The demand for high-quality milk is increasing worldwide. The efficiency of milk synthesis can be improved by taking advantage of the accumulated knowledge of the transcriptional and posttranscriptional regulation of genes coding for proteins involved in the synthesis of fat, protein, and lactose in the mammary gland. Research in this area is relatively new, but data accumulated in the last 10 years provide a relatively clear picture. Milk fat synthesis appears to be regulated, at least in bovines, by an interactive network between SREBP1, PPARγ, and LXRα, with a potential role for other transcription factors, such as Spot14, ChREBP, and Sp1. Milk protein synthesis is highly regulated by insulin, amino acids, and amino acid transporters via transcriptional and posttranscriptional routes, with the insulin-mTOR pathway playing a central role. The transcriptional regulation of lactose synthesis is still poorly understood, but it is clear that glucose transporters play an important role. They can also cooperatively interact with amino acid transporters and the mTOR pathway. Recent data indicate the possibility of nutrigenomic interventions to increase milk fat synthesis by feeding long-chain fatty acids and milk protein synthesis by feeding amino acids. We propose a transcriptional network model to account for all available findings. This model encompasses a complex network of proteins that control milk synthesis with a cross talk between milk fat, protein, and lactose regulation, with mTOR functioning as a central hub.