After birth, a newborn calf has to adapt to an extrauterine life characterized by several physiol... more 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
A B S T R A C T Ruminants have a very special niche in the animal kingdom, and are the most impor... more A B S T R A C T Ruminants have a very special niche in the animal kingdom, and are the most important livestock species providing milk, meat, and wool for humans from consumption of highly-fibrous feedstuffs. Cattle, goat and sheep have been widely-used for years as models to study ruminal fermentation and the mechanisms whereby tissues utilize nutrients for milk synthesis, growth, wool accretion, and reproduction. The advent of high-throughput technologies to study an animal's genome, proteome, and metabolome (i.e., " omics " tools) offered ruminant scientists the opportunity to study multiple levels of biological information to better understand the whole animal response to nutrition, environment, physiological state, and their interactions. The omics revolution gave rise to the field of nutrigenomics, i.e. the study of the genome-wide influences of nutrition through alteration in mRNA, protein, and metabolite expression or abundance. This field of research is relatively new in ruminants, and particularly sheep and goats. Dietary compounds affect gene expression directly or indirectly via interactions with transcription factors including ligand-dependent nuclear receptors. New knowledge generated through the application of functional analyses of transcriptomic, proteomic, and metabolomic data sets in goat and sheep is discussed.
Feeding a higher-energy diet by increasing cereal grains at the expense of forage during the last... more 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 non-pregnant, 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.
A B S T R A C T Gene reporter technology (GRT) has opened several new avenues for monitoring biol... more A B S T R A C T 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 0.4 μL/well), and three doses of Green Fluorescent Protein plasmid DNA (i.e., 10, 25, and 50 ng/well). Transfection efficiency and mortality rate were analyzed using CellProfiler software. Transfection efficiency increased until the end of the experiment (20 h 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 0.3 μL/well of LipoLTX plus 25 ng/well of plasmid DNA (ca. 29.5 ± 1.9%) and 0.15 μL/well of LipoLTX plus 25 ng/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 10 nM or 100 nM of synthetic Peroxisome Proliferator-activated Receptor β/σ (PPARβ/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 100 nM of the PPARβ/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.
Mastitis is a major disease in dairy cows resulting in significant economic losses. In vitro work... more Mastitis is a major disease in dairy cows resulting in significant economic losses. In vitro works suggest that ruminants peroxisome proliferator-activated receptor gamma (PPARí µí»¾) 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 PPARí µí»¾ 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 (í µí± = 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 PPARí µí»¾ 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.
Background: Feeding higher-energy prepartum is a common practice in the dairy industry. However, ... more 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.
Supplying trace minerals (TM) in more bioavailable forms such as amino acid complexes could help ... more Supplying trace minerals (TM) in more bioavailable forms such as amino acid complexes 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 amino acid complexes (AAC) during the peripartal period on expression of 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 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 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). Locomotion score was recorded before enrollment and weekly throughout the experiment. Incidence of hoof health problems at 30 DIM was evaluated prior to performing a hoof biopsy in a subset of cows (INO: n = 10; AAC: n = 9). Locomotion score did not differ between treatments in the prepartum or postpartum period. However, incidence of heel horn erosion was lower in AAC cows, while the incidence of sole ulcers did not differ. The 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 incidence of heel erosion in response to AAC. Among the genes associated with oxidative stress, the AAC cows had greater expression of NFE2L2, which is 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, while TLR2, IL1B and TNF were lower compared with INO. Overall, the data indicate that supplementation with metal amino acid complexes during the peripartal period affected the expression of genes involved in the composition, oxidative stress and inflammation status in the corium. The hoof biopsy procedure used should be further perfected and implemented in future lameness research to expand our understanding of the hoof biology in dairy cows.
The hoof digital cushion is a complex structure composed of adipose tissue beneath the dis-tal ph... more The hoof digital cushion is a complex structure composed of adipose tissue beneath the dis-tal 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 upregu-lated 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.
Background: Main objectives were to determine to what extent Smartamine M (SM) supplementation to... more Background: Main objectives were to determine to what extent Smartamine M (SM) supplementation to a prepartal higher-energy diet could alter neutrophil (PMN) and liver tissue immunometabolic biomarkers, and whether those responses were comparable to those in cows fed a prepartal lower-energy diet (CON). Results: Twenty-eight multiparous Holstein cows were fed CON (NE L = 1.24 Mcal/kg DM) during d −50 to d −22 relative to calving. From d −21 to calving, cows were randomly assigned to a higher-energy diet (OVE, n = 9; NE L = 1.54 Mcal/kg DM), OVE plus SM (OVE + SM, n = 10; SM = 0.07 % of DM) or remained on CON (n = 9). All cows received the same basal lactation diet (NE L = 1.75 Mcal/kg DM). Supplementation of SM (OVE + SM) continued until 30 d postpartum. Liver biopsies were harvested at d −10, 7, and 21 relative to parturition. Blood PMN isolated at −10, 3, and 21 d relative to calving was used to evaluate gene expression. As expected, OVE increased liver lipid content postpartum; however, cows fed OVE + SM or CON had lower concentrations than OVE. Compared with OVE, cows in CON and OVE + SM had greater DMI postpartum and milk production. Furthermore, cows fed OVE + SM had the greatest milk protein and fat percentage and lowest milk SCC despite having intermediate PMN phagocytic capacity. Adaptations in PMN gene expression in OVE + SM cows associated with the lower SCC were gradual increases from −10 to 21 d in genes that facilitate migration into inflammatory sites (SELL, ITGAM), enzymes essential for reducing reactive oxygen metabolites (SOD1, SOD2), and a transcription factor(s) required for controlling PMN development (RXRA). The greater expression of TLR4 on d 3, key for activation of innate immunity due to inflammation, in OVE compared with CON cows suggests a more pronounced inflammatory state. Feeding OVE + SM dampened the upregulation of TLR4, despite the fact that these cows had similar expression of the pro-inflammatory genes NFKB1 and TNF as OVE. Cows in CON had lower overall expression of these inflammation-related genes and GSR, which generates reduced glutathione, an important cellular antioxidant.
The demand for high quality milk is increasing worldwide. The efficiency of milk synthesis can be... more 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 post-transcriptional 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 bovine, 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 post-transcriptional 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.
After birth, a newborn calf has to adapt to an extrauterine life characterized by several physiol... more 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
A B S T R A C T Ruminants have a very special niche in the animal kingdom, and are the most impor... more A B S T R A C T Ruminants have a very special niche in the animal kingdom, and are the most important livestock species providing milk, meat, and wool for humans from consumption of highly-fibrous feedstuffs. Cattle, goat and sheep have been widely-used for years as models to study ruminal fermentation and the mechanisms whereby tissues utilize nutrients for milk synthesis, growth, wool accretion, and reproduction. The advent of high-throughput technologies to study an animal's genome, proteome, and metabolome (i.e., " omics " tools) offered ruminant scientists the opportunity to study multiple levels of biological information to better understand the whole animal response to nutrition, environment, physiological state, and their interactions. The omics revolution gave rise to the field of nutrigenomics, i.e. the study of the genome-wide influences of nutrition through alteration in mRNA, protein, and metabolite expression or abundance. This field of research is relatively new in ruminants, and particularly sheep and goats. Dietary compounds affect gene expression directly or indirectly via interactions with transcription factors including ligand-dependent nuclear receptors. New knowledge generated through the application of functional analyses of transcriptomic, proteomic, and metabolomic data sets in goat and sheep is discussed.
Feeding a higher-energy diet by increasing cereal grains at the expense of forage during the last... more 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 non-pregnant, 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.
A B S T R A C T Gene reporter technology (GRT) has opened several new avenues for monitoring biol... more A B S T R A C T 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 0.4 μL/well), and three doses of Green Fluorescent Protein plasmid DNA (i.e., 10, 25, and 50 ng/well). Transfection efficiency and mortality rate were analyzed using CellProfiler software. Transfection efficiency increased until the end of the experiment (20 h 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 0.3 μL/well of LipoLTX plus 25 ng/well of plasmid DNA (ca. 29.5 ± 1.9%) and 0.15 μL/well of LipoLTX plus 25 ng/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 10 nM or 100 nM of synthetic Peroxisome Proliferator-activated Receptor β/σ (PPARβ/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 100 nM of the PPARβ/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.
Mastitis is a major disease in dairy cows resulting in significant economic losses. In vitro work... more Mastitis is a major disease in dairy cows resulting in significant economic losses. In vitro works suggest that ruminants peroxisome proliferator-activated receptor gamma (PPARí µí»¾) 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 PPARí µí»¾ 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 (í µí± = 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 PPARí µí»¾ 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.
Background: Feeding higher-energy prepartum is a common practice in the dairy industry. However, ... more 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.
Supplying trace minerals (TM) in more bioavailable forms such as amino acid complexes could help ... more Supplying trace minerals (TM) in more bioavailable forms such as amino acid complexes 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 amino acid complexes (AAC) during the peripartal period on expression of 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 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 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). Locomotion score was recorded before enrollment and weekly throughout the experiment. Incidence of hoof health problems at 30 DIM was evaluated prior to performing a hoof biopsy in a subset of cows (INO: n = 10; AAC: n = 9). Locomotion score did not differ between treatments in the prepartum or postpartum period. However, incidence of heel horn erosion was lower in AAC cows, while the incidence of sole ulcers did not differ. The 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 incidence of heel erosion in response to AAC. Among the genes associated with oxidative stress, the AAC cows had greater expression of NFE2L2, which is 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, while TLR2, IL1B and TNF were lower compared with INO. Overall, the data indicate that supplementation with metal amino acid complexes during the peripartal period affected the expression of genes involved in the composition, oxidative stress and inflammation status in the corium. The hoof biopsy procedure used should be further perfected and implemented in future lameness research to expand our understanding of the hoof biology in dairy cows.
The hoof digital cushion is a complex structure composed of adipose tissue beneath the dis-tal ph... more The hoof digital cushion is a complex structure composed of adipose tissue beneath the dis-tal 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 upregu-lated 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.
Background: Main objectives were to determine to what extent Smartamine M (SM) supplementation to... more Background: Main objectives were to determine to what extent Smartamine M (SM) supplementation to a prepartal higher-energy diet could alter neutrophil (PMN) and liver tissue immunometabolic biomarkers, and whether those responses were comparable to those in cows fed a prepartal lower-energy diet (CON). Results: Twenty-eight multiparous Holstein cows were fed CON (NE L = 1.24 Mcal/kg DM) during d −50 to d −22 relative to calving. From d −21 to calving, cows were randomly assigned to a higher-energy diet (OVE, n = 9; NE L = 1.54 Mcal/kg DM), OVE plus SM (OVE + SM, n = 10; SM = 0.07 % of DM) or remained on CON (n = 9). All cows received the same basal lactation diet (NE L = 1.75 Mcal/kg DM). Supplementation of SM (OVE + SM) continued until 30 d postpartum. Liver biopsies were harvested at d −10, 7, and 21 relative to parturition. Blood PMN isolated at −10, 3, and 21 d relative to calving was used to evaluate gene expression. As expected, OVE increased liver lipid content postpartum; however, cows fed OVE + SM or CON had lower concentrations than OVE. Compared with OVE, cows in CON and OVE + SM had greater DMI postpartum and milk production. Furthermore, cows fed OVE + SM had the greatest milk protein and fat percentage and lowest milk SCC despite having intermediate PMN phagocytic capacity. Adaptations in PMN gene expression in OVE + SM cows associated with the lower SCC were gradual increases from −10 to 21 d in genes that facilitate migration into inflammatory sites (SELL, ITGAM), enzymes essential for reducing reactive oxygen metabolites (SOD1, SOD2), and a transcription factor(s) required for controlling PMN development (RXRA). The greater expression of TLR4 on d 3, key for activation of innate immunity due to inflammation, in OVE compared with CON cows suggests a more pronounced inflammatory state. Feeding OVE + SM dampened the upregulation of TLR4, despite the fact that these cows had similar expression of the pro-inflammatory genes NFKB1 and TNF as OVE. Cows in CON had lower overall expression of these inflammation-related genes and GSR, which generates reduced glutathione, an important cellular antioxidant.
The demand for high quality milk is increasing worldwide. The efficiency of milk synthesis can be... more 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 post-transcriptional 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 bovine, 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 post-transcriptional 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.
Diarrhea is the most common disease during the neonatal stage of dairy calves. Exfoliated gastroi... more Diarrhea is the most common disease during the neonatal stage of dairy calves. Exfoliated gastrointestinal epithelial (EGE) cells isolated from fecal samples of newborn humans have been used to study GI transcriptomics associated with nutrition. Similarly, EGE from neonatal dairy calves can be used to evaluate nutritional effects as well as health conditions such as diarrhea. The aim of this study was to use the RNA isolated from EGE cells to evaluate the transcriptional adaptations to mild diarrhea by assessing the expression of genes related to the inflammatory response and cell membrane transporters. Eight newborn Jersey male calves were used from birth to 5 wk of age and housed in individual pens at the Oregon State University Dairy Center. 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 whole milk. Starter intake, BW, fecal score, withers height (WH), and rectal temperature (RT) were recorded during the experiment. RNA isolated from EGE fresh fecal samples (~200 mg) collected weekly was used for RT-qPCR analysis. Data were analyzed using the PROC MIXED procedure of SAS. Statistical significance and tendencies were declared at P < 0.05 and P ≤ 0.15, respectively. Starter intake, BW, and WH increased (P < 0.01) over time. Fecal score was greatest (2.6 ± 0.3; P < 0.01) during 2 wk. The mRNA expression of inflammation-related genes TLR4 and TNFA increased (P < 0.01) over time and a trend was observed for NFKB1 (P = 0.07), while a quadratic effect over time (P < 0.01) was observed for IL8 and IL1B. In contrast, IFNG and TLR2 expression decreased (P < 0.01) over time. A time effect (P = 0.01) was observed for SLC5A1 expression, a sodium/glucose transporter, because of a decreased during 3 wk of age. The fecal RNA method utilized in this study was able to detect transcriptional changes during a mild diarrhea event in neonatal dairy calves, which underscore the usefulness of such method for future research including nutritional effects and health conditions.
Background: One of the carcass traits that influence meat quality is the intramuscular fat deposi... more Background: One of the carcass traits that influence meat quality is the intramuscular fat deposition which begins its development before birth. Therefore, maternal nutrition can potentially result in alterations of the development not only of the offspring but of adipose tissue including changes in gene expression. Overall, assess the RNA yield from tissue samples can generate important information for the gene expression analysis. Objectives: Evaluate the total RNA yield in fat tissue from offspring of beef cows that were on different planes of nutrition during late gestation.
Uploads
Papers by Johan Osorio