Fernanda Batistel

Alterations in Hepatic FGF21, Co-Regulated Genes, and Upstream Metabolic Genes in Response to Nutrition, Ketosis and Inflammation in Peripartal Holstein Cows.

In rodents, fibroblast growth factor 21 (FGF21) has emerged as a key metabolic regulator produced by liver. To gather preliminary data on the potential importance of FGF1, co-regulated genes, and upstream metabolic genes, we examined the hepatic mRNA expression in response to nutrition and inflammation in dairy cows. In experiment 1, induction of ketosis through feed restriction on d 5 postpartum upregulated FGF21, its co-receptor KLB, and PPARA but only elicited a numerical increase in serum FGF21 concentration. In experiment 2, cows in control (CON) or receiving 50 g/d of L-carnitine (C50) from -14 through 21 d had increased FGF21, PPARA, and NFIL3 on d 10 compared with d 2 postpartum. In contrast, compared with CON and C50, 100 g/d L-carnitine (C100) resulted in lower FGF21, KLB, ANGPTL4, and ARNTL expression on d 10. In experiment 3, cows were fed during the dry period either a higher-energy (OVE; 1.62 Mcal/kg DM) or lower-energy (CON; 1.34 Mcal/kg DM) diet and received 0 (OVE:N, CON:N) or 200 μg of LPS (OVE:Y, CON:Y) into the mammary gland at d 7 postpartum. For FGF21 mRNA expression in CON, the LPS challenge (CON:Y) prevented a decrease in expression between d 7 and 14 postpartum such that cows in CON:N had a 4-fold lower expression on d 14 compared with d 7. The inflammatory stimulus induced by LPS in CON:Y resulted in upregulation of PPARA on d 14 to a similar level as cows in OVE:N. In OVE:Y, expression of PPARA was lower than CON:N on d 7 and remained unchanged on d 14. On d 7, LPS led to a 4-fold greater serum FGF21 only in OVE but not in CON cows. In fact, OVE:Y reached the same serum FGF21 concentration as CON:N, suggesting a carryover effect of dietary energy level on signaling mechanisms within liver. Overall, results indicate that nutrition, ketosis, and inflammation during the peripartal period can alter hepatic FGF21, co-regulated genes, and upstream metabolic genes to various extents. The functional outcome of these changes merits further study, and in particular the mechanisms regulating transcription in response to changes in energy balance and feed intake.

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.

Ethyl-cellulose rumen-protected methionine enhances performance during the periparturient period and early lactation in Holstein dairy cows

The onset of lactation in dairy cows is characterized by severe negative energy and protein balance. Increasing Met availability during this time may improve milk production, hepatic lipid metabolism, and immune function. The aim of this study was to evaluate the effect of feeding ethyl-cellulose rumen-protected methionine (RPM; Mepron, Evonik Nutrition and Care GmbH, Hanau-Wolfgang, Germany) on the performance of dairy cows during prepartum and early-lactation periods. Sixty multiparous Holstein cows were used in a block design and assigned to either a control or an ethyl-cellulose RPM diet. Ethyl-cellulose RPM was supplied from -28 to 60 d relative to parturition at a rate of 0.09% and 0.10% of dry matter during the prepartum and postpartum periods, respectively. That rate ensured that the ratio of Lys to Met in metabolizable protein was close to 2.8:1. Cows fed ethyl-cellulose RPM had dry matter intakes (DMI) that were 1.2 kg/d greater during the prepartum period and consequently had overall greater cumulative DMI than cows in the control group. Compared with controls, during the fresh period (1-30 d in milk; DIM) feeding ethyl-cellulose RPM increased DMI by 1.7 kg/d, milk yield by 4.1 kg/d, fat yield by 0.17 kg/d, milk protein yield by 0.20 kg/d, 3.5% fat-corrected milk by 4.3 kg/d, and energy-corrected milk by 4.4 kg/d. Although ethyl-cellulose RPM supplementation increased milk protein content by 0.16 percentage units compared with the control during the fresh period, no differences were observed for milk fat, lactose, and milk urea nitrogen concentration. During the high-producing period (31-60 DIM), cows fed ethyl-cellulose RPM increased DMI and milk yield by 1.45 and 4.4 kg/d, respectively. Ethyl-cellulose RPM also increased fat yield by 0.19 kg/d, milk protein yield by 0.17 kg/d, 3.5% fat-corrected milk by 4.7 kg/d, and energy-corrected milk by 4.8 kg/d compared with controls. Ethyl-cellulose RPM supplementation reduced plasma fatty acids in the fresh period and decreased γ-glutamyl transferase, indicating better liver function. In conclusion, when lysine was adequate, feeding ethyl-cellulose RPM to achieve a ratio close to 2.8:1 in metabolizable protein improved dairy cow performance from parturition through 60 DIM. The greater milk production was, at least in part, driven by the greater voluntary DMI and better liver function.

Ethyl-cellulose rumen-protected methionine alleviates inflammation and oxidative stress and improves neutrophil function during the periparturient period and early lactation in Holstein dairy cows

The periparturient period is the most critical phase in the productive cycle of dairy cows and is characterized by impairment of the immune system. Our objective was to evaluate the effect of feeding ethyl-cellulose rumen-protected methionine (RPM) starting at d -28 from expected parturition through 60 d in milk on biomarkers of inflammation, oxidative stress, and liver function as well as leukocyte function. Sixty multiparous Holstein cows were used in a block design and assigned to either a control or the control plus ethyl-cellulose RPM (Mepron, Evonik Nutrition & Care GmbH). Mepron was supplied from -28 to 60 d in milk at a rate of 0.09% and 0.10% dry matter during the prepartum and postpartum period. That rate ensured that the ratio of Lys to Met in the metabolizable protein was close to 2.8:1. Blood samples from 15 clinically healthy cows per treatment were collected at d -30, -14, 1, 7, 21, 30, and 60 and analyzed for biomarkers of liver function, inflammation, and oxidative stress. Neutrophil and monocyte function in whole blood was measured in vitro at -14, 1, 7, 21, and 30 d in milk. The statistical model included the random effect of block and fixed effect of treatment, time, and its interaction. Compared with control, ethyl-cellulose RPM increased plasma cholesterol and paraoxonase after parturition. Among the inflammation biomarkers measured, ethyl-cellulose RPM led to greater albumin (negative acute-phase protein) and lower haptoglobin than control cows. Although concentration of IL-1β was not affected by treatments, greater IL-6 concentration was detected in response to ethyl-cellulose RPM. Cows supplemented with ethyl-cellulose RPM had greater plasma concentration of ferric-reducing antioxidant power, β-carotene, tocopherol, and total and reduced glutathione, whereas reactive oxygen metabolites were lower compared with control cows. Compared with control, ethyl-cellulose RPM enhanced neutrophil phagocytosis and oxidative burst. Overall, the results indicate that ethyl-cellulose RPM supply to obtain a Lys-to-Met ratio of 2.8:1 in the metabolizable protein during the periparturient period and early lactation is an effective approach to help mitigate oxidative stress and inflammation as well as enhance liver and neutrophil function in dairy cows.

Far-off and close-up dry matter intake modulate indicators of immunometabolic adaptations to lactation in subcutaneous adipose tissue of pasture-based transition dairy cows

The common practice of increasing dietary energy density during the close-up dry period (last ∼3 wk prepartum) has been recently associated with a higher incidence of metabolic disorders after calving. Despite these reports, over-feeding of metabolizable energy (ME) during the far-off, nonlactating period is a common management policy aimed at achieving optimum calving body condition score (BCS) in pasture-based systems, as cows are generally thinner than total mixed ration cows at the end of lactation. Our hypothesis was that both far-off and close-up overfeeding influence the peripartum adipose tissue changes associated with energy balance and inflammatory state. Sixty mid-lactation, grazing dairy cows of mixed age and breed were randomly allocated to 1 of 2 groups that were managed through late lactation to achieve a low and high BCS (approximately 4.25 and 5.0 on a 10-point scale) at dry-off. The low BCS cows were then overfed ME to ensure that they achieved the same BCS as the higher BCS group by calving. Within each rate of BCS gain treatment, cows were offered 65, 90, or 120% of their pre-calving ME requirements for 3 wk pre-calving in a 2 × 3 factorial arrangement of treatments (i.e., 10 cows/treatment). Subcutaneous adipose tissue was collected via biopsy at -1, 1, and 4 wk relative to parturition. Quantitative PCR was used to measure mRNA and microRNA expression of targets related to adipogenesis and inflammation. Cows overfed in the far-off period had increased expression of miR-143 and miR-378 prepartum (-1 wk) indicating greater adipogenesis, consistent with their rapid gain in BCS following dry-off. Furthermore, the lower postpartum expression of IL6, TNF, TLR4, TLR9, and miR-145, and a higher abundance of miR-99a indicated lower body fat mobilization in early lactation in the same group. In the close-up period, feeding either 65 or 120% of ME requirements caused changes in FASN, IL1B, IL6R, TLR9, and the microRNA miR-143, miR-155, and miR-378. Their respective expression patterns indicate a tentative negative-feedback mechanism in metabolically compromised, feed-restricted cows, and a possible immune-related stimulation of lipolysis in apparently static adipocytes in overfed cows. Data from cows fed 90% of ME requirements indicate the existence of a balance between lipolytic (inflammatory-related) and anti-lipolytic signals, to prime the mobilization machinery in light of imminent lactation. Overall, results indicate that far-off dry cow nutrition influences peripartum adipose tissue metabolism, with neither strategy negatively affecting the physiological adaptation to lactation. Furthermore, to ensure a favorable transition, cows should be subjected to a small feed restriction in the close-up period, irrespective of far-off nutritional management.

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

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

Supplemental methionine, choline, or taurine alter in vitro gene network expression of polymorphonuclear leukocytes from neonatal Holstein calves

Isolated PMNL from neonatal calves were used to evaluate the effect of Met, choline, and taurine supplementation on mRNA expression of genes related to the Met cycle and innate immunity. Five neonatal Holstein calves (3 wk old) were used for PMNL isolation and in vitro culture. The selected genes were related to the 1-carbon and Met cycles, cell signaling and cytokine mediators, inflammation, antimicrobial and killing mechanism associated genes, immune mediators, adhesion, and pathogen recognition. The results indicated that supplementation of Met, choline, and taurine increased homocysteine synthesis through upregulation of SAHH. Furthermore, the lower expression of CXCR1, IL10, IL6, IRAK1, NFKB1, NR3C1, SELL, TLR4, and TNFA indicated that all treatments mitigated the inflammatory activation of blood PMNL. As indicated by the modulation of GCLC and GPX1, choline and taurine supplementation also affected the antioxidant system. However, data indicate that oversupplementation could alter the inflammatory and oxidative status, suggesting the existence of cytotoxicity thresholds. Overall, multiple biological processes in calf PMNL related to inflammatory response and cytoprotection against oxidative stress were affected by Met, choline, and taurine supplementation. These data underscore an important role of these compounds in pre-weaning calf nutritional management.

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

BackgroundImmune 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.ResultsTwenty 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.ConclusionsData 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.

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 phosphorylation status of serine/threonine kinase 1, mammalian target of rapamycin signaling proteins, and growth rate in Holstein heifer calves in response to maternal supply of methionine

The study investigated whether methionine supply during late pregnancy is associated with liver mammalian target of rapamycin (MTOR) pathway phosphorylation, plasma biomarkers, and growth in heifer calves born to cows fed a control diet (CON) or the control diet plus ethylcellulose rumen-protected methionine (MET; 0.09% of dry matter intake) for the last 28 d prepartum. Calves were fed and managed similarly during the first 56 d of age. Plasma was harvested at birth and 2, 7, 21, 42, and 50 d of age and was used for biomarker profiling. Liver biopsies were harvested at 4, 14, 28, and 50 d of age and used for protein expression. Body weight, hip height, hip width, wither height, body length, rectal temperature, fecal score, and respiratory score were measured weekly. Starter intake was measured daily, and average daily gain was calculated during the first 8 wk of age. During the first 7 wk of age, compared with calves in the CON group, calves in the MET group had greater body weight, hip height, wither height, and average daily gain despite similar daily starter intake. Concentration of methionine in plasma was lower at birth but increased markedly at 2 and 7 d of age in MET calves. Plasma insulin, glucose, free fatty acids, and hydroxybutyrate did not differ. A greater ratio of phosphorylated α-serine/threonine kinase (AKT):total AKT protein expression was detected in MET calves, namely due to differences at 4 d of age. The phosphorylated MTOR:total MTOR ratio also was greater in MET calves due to differences at 28 and 50 d (8 d postweaning). The decrease in phosphorylated MTOR:total MTOR between 14 and 28 d in CON calves agreed with the increase in phosphorylated eukaryotic translation initiation factor 4E binding protein 1 (EIF4EBP1):total EIF4EBP1 ratio during the same time frame. The overall expression of phosphorylated ribosomal protein S6 kinase B1 (RPS6KB1):total RPS6KB1 and phosphorylated eukaryotic translation elongation factor 2 (EEF2):total EEF2 was lower in MET calves. Regardless of methionine supply prepartum, there was an 11-fold temporal decrease from 4 to 50 d in phosphorylated AKT:total AKT. Similarly, regardless of methionine supply, there were overall decreases in phosphorylation ratios of AKT, MTOR, RPS6KB1, and eukaryotic translation initiation factor 2A (EIF2A) over time. Data provide evidence of a positive effect of methionine supply during the last month of pregnancy on rates of growth during the first 7 wk of age. Phosphorylation status of some components of the MTOR pathway in neonatal calf liver also was associated with greater maternal supply of methionine. Thus, the data suggest that molecular mechanisms in the liver might be programmed by supply of methionine during late pregnancy. The exact mechanisms coordinating the observed responses remain to be determined.