Maternal supply of methionine during late pregnancy is associated with changes in immune function and abundance of microRNA and mRNA in Holstein calf polymorphonuclear leukocytes.

Pregnancy and early life are critical periods during which environmental factors such as nutrition can affect development. Rumen-protected methionine (Met; RPM) supplementation during the prepartum period improves not only performance but immune responses in dairy cows. We investigated the effects of enhanced maternal supply of Met via feeding RPM on whole-blood in vitro lipopolysaccharide (LPS; 0, 0.01, or 5 µg/mL of blood) challenge and targeted microRNA and mRNA abundance in calf blood polymorphonuclear leukocytes (PMNL). Calves (n = 12/maternal diet) born to cows fed RPM at 0.08% of diet dry matter (DM)/d (MET) for the last 21 ± 2 d before calving or fed a control diet with no added Met (CON) were used. The PMNL were isolated at birth (before colostrum feeding) and d 1 (24 h after colostrum intake), 14, 28, and 50 of age. Maternal blood was collected at -10 ± 1.3 d relative to calving. Cows in the MET group had greater DM intake and lower prepartal haptoglobin concentration. In CON cows, haptoglobin was positively correlated with proinflammatory and host-defense mRNA abundance in CON calves. Except for NOS2 and NFE2L2, abundance of CASP8, MPO, ZBP1, and TNF was lower at birth in MET calves. Interleukin 1ß concentration in response to LPS challenge in CON and MET calves was greatest at birth, underscoring the role of this cytokine for lymphocyte activation. Compared with 1 d of age, the interleukin-1ß response to incremental doses of LPS was greater at 14 through 28 d, suggesting that the neonatal calf can mount a robust response to inflammatory stimuli. Greater abundance in CON calves of NOS2, CADM1, and TLR2 coupled with lower SELL from 1 through 50 d of age suggested a chronic activation of the PMNL. There was a marked upregulation over time of MIR125b, MIR146a, MIR155, and MIR9 in both CON and MET calves, suggesting that these microRNA could affect gene transcription associated with differentiation and inflammatory function in PMNL. Regardless of maternal diet, the gradual downregulation of MIR223 (the most abundant microRNA in PMNL) is in line with the progressive increase over time in the proinflammatory signature of the PMNL. Data revealed the potential for maternal supply of Met during late pregnancy through either greater DM intake or Met to elicit some changes in PMNL function during early postnatal life, partly through changes in mRNA expression encompassing cell adhesion and chemotaxis, oxidative stress, Toll-like receptor signaling, and Met metabolism.

Supplementation with rumen-protected methionine or choline during the transition period influences whole-blood immune response in periparturient dairy cows.

Methionine, together with Lys, is the most limiting AA for milk production in dairy cows. Besides its crucial role in milk production, Met and its derivate metabolites (e.g., glutathione, taurine, polyamines) are well-known immunonutrients in nonruminants, helping support and boost immune function and activity. In the present study, the effects of Met or choline, as its precursor, were investigated using an ex vivo whole blood challenge. The study involved 33 multiparous Holstein cows (from a larger cohort with a factorial arrangement of treatments) assigned from d -21 to +30 relative to parturition to a basal control (CON) diet, CON plus rumen-protected Met (MET, Smartamine M, Adisseo NA, Alpharetta, GA) at a rate of 0.08% of dry matter, or CON plus rumen-protected choline (CHOL, ReaShure, Balchem Inc., New Hampton, NY) at 60 g/d. Blood was sampled on d -15, -7, 2, 7, and 20 for ex vivo lipopolysaccharide (LPS) challenge, and on d 1, 4, 14, and 28 relative to parturition for phagocytosis and oxidative burst assays. The MET cows had greater energy-corrected milk production and milk protein content. Overall, IL-6 response to LPS increased around parturition, whereas IL-1ß remained constant, casting doubt on the existence of systemic immunosuppression in the peripartal period. Supplementation with MET dampened the postpartal blood response to LPS (lower IL-1ß), while improving postpartum neutrophil and monocyte phagocytosis capacity and oxidative burst activity. In contrast, CHOL supplementation increased monocyte phagocytosis capacity. Overall, the data revealed a peripartal immune hyper-response, which appeared to have been mitigated by MET supplementation. Both MET and CHOL effectively improved immune function; however, MET affected the immune and antioxidant status before parturition, which might have been beneficial to prepare the cow to respond to metabolic challenges after parturition. These results provide insights on potential differences in the immunomodulatory action of methionine and choline in dairy cows. As such, the effects observed could have implications for ration formulation and dietary strategies.

Maternal supplementation with rumen-protected methionine increases prepartal plasma methionine concentration and alters hepatic mRNA abundance of 1-carbon, methionine, and transsulfuration pathways in neonatal Holstein calves.

An important mechanism of nutritional "programming" induced by supplementation with methyl donors during pregnancy is the alteration of mRNA abundance in the offspring. We investigated the effects of rumen-protected Met (RPM) on abundance of 17 genes in the 1-carbon, Met, and transsulfuration pathways in calf liver from cows fed the same basal diet without (control, CON) or with RPM at 0.08% of diet dry matter/d (MET) from -21 through +30 d around calving. Biopsies (n = 8 calves per diet) were harvested on d 4, 14, 28, and 50 of age. Cows fed RPM had greater plasma concentration of Met (17.8 vs. 28.2 µM) at -10 d from calving. However, no difference was present in colostrum yield and free AA concentrations. Greater abundance on d 4 and 14 of betaine-homocysteine S-methyltransferase 2 (BHMT2), adenosylhomocysteinase (AHCY; also known as SAHH), and cystathionine-ß-synthase (CBS) in MET calves indicated alterations in Met, choline, and homocysteine metabolism. Those data agree with the greater abundance of methionine adenosyltransferase 1A (MAT1A) in MET calves. Along with CBS, the greater abundance of glutamate-cysteine ligase (GCLC) and glutathione reductase (GSR) on d 4 in MET calves indicated a short-term postnatal alteration in the use of homocysteine for taurine and glutathione synthesis (both are potent intracellular antioxidants). The striking 7-fold upregulation at d 50 versus 4 of cysteine sulfinic acid decarboxylase (CSAD), catalyzing the last step of taurine synthesis, in MET and CON calves underscores an important role of taurine during postnatal calf growth. The unique role of taurine in the young calf is further supported by the upregulation of CBS, GCLC, and GSR at d 50 versus 14 and 28 in MET and CON. Although betaine-homocysteine S-methyltransferase (BHMT) activity did not differ in MET and CON, it increased ∼50% at d 14 and 28 versus 4. A significant positive correlation (r = 0.79) was present between BHMT abundance and BHMT activity regardless of treatment. The gradual upregulation over time of BHMT2 and SAHH coupled with the gradual upregulation of MAT1A and the DNA (cytosine-5-)-methyltransferases (DNMT1, DNMT3A, DNMT3B) in MET and CON calves was indicative of adaptations potentially driven by differences in intake of milk replacer and starter feed as calves grew. In that context, the ∼2.5-fold increase in abundance of DNMT3B at d 50 versus 4 in MET and CON indicate that DNA methylation might be an important component of the physiologic adaptations of calf liver. The data indicate that calves from MET-supplemented cows underwent alterations in Met, choline, and homocysteine metabolism partly to synthesize taurine and glutathione, which would be advantageous for controlling metabolic-related stress. Whether the effects in MET calves were directly related to increased Met supply in utero remains to be determined.

Maternal rumen-protected methionine supplementation and its effect on blood and liver biomarkers of energy metabolism, inflammation, and oxidative stress in neonatal Holstein calves.

In nonruminants, nutrition during pregnancy can program offspring development, metabolism, and health in later life. Rumen-protected Met (RPM) supplementation during the prepartum period improves liver function and immune response in dairy cows. Our aim was to investigate the effects of RPM during late pregnancy on blood biomarkers (23 targets) and the liver transcriptome (24 genes) in neonatal calves from cows fed RPM at 0.08% of diet dry matter/d (MET) for the last 21 d before calving or controls (CON). Blood (n=12 calves per diet) was collected at birth before receiving colostrum (baseline), 24 h after receiving colostrum, 14, 28, and 50 d (post-weaning) of age. Liver was sampled (n=8 calves per diet) via biopsy on d 4, 14, 28, and 50 of age. Growth and health were not affected by maternal diet. The MET calves had greater overall plasma insulin concentration and lower glucose and ratios of glucose-to-insulin and fatty acids-to-insulin, indicating greater systemic insulin sensitivity. Lower concentration of reactive oxygen metabolites at 14 d of age along with a tendency for lower overall concentration of ceruloplasmin in MET calves indicated a lesser degree of stress. Greater expression on d 4 of fructose-bisphosphatase 1 (FBP1), phosphoenolpyruvate carboxykinase 1 (PCK1), and the facilitated bidirectional glucose transporter SLC2A2 in MET calves indicated alterations in gluconeogenesis and glucose uptake and release. The data agree with the greater expression of the glucocorticoid receptor (GR). Greater expression on d 4 of the insulin receptor (INSR) and insulin-responsive serine/threonine-protein kinase (AKT2) in MET calves indicated alterations in insulin signaling. In that context, the similar expression of sterol regulatory element-binding transcription factor 1 (SREBF1) in CON and MET during the preweaning period followed by the marked upregulation regardless of diet after weaning (d 50) support the idea of changes in hepatic insulin sensitivity during early postnatal life. Expression of carnitine palmitoyltransferase 1A (CPT1A) was overall greater and acyl-CoA oxidase 1 (ACOX1) was lower in MET calves, indicating alterations in fatty acid oxidation. Except forkhead box O1 (FOXO1), all genes changed in expression over time. Transcriptome results indicated that calves from MET-supplemented cows underwent a faster maturation of gluconeogenesis and fatty acid oxidation in the liver, which would be advantageous for adapting to the metabolic demands of extrauterine life.

Hepatic global DNA and peroxisome proliferator-activated receptor alpha promoter methylation are altered in peripartal dairy cows fed rumen-protected methionine.

The availability of Met in metabolizable protein (MP) of a wide range of diets for dairy cows is low. During late pregnancy and early lactation, in particular, suboptimal Met in MP limits its use for mammary and liver metabolism and also for the synthesis of S-adenosylmethionine, which is essential for many biological processes, including DNA methylation. The latter is an epigenetic modification involved in the regulation of gene expression, hence, tissue function. Thirty-nine Holstein cows were fed throughout the peripartal period (-21 d to 30 d in milk) a basal control (CON) diet (n=14) with no Met supplementation, CON plus MetaSmart (MS; Adisseo NA, Alpharetta, GA; n=12), or CON plus Smartamine M (SM; Adisseo NA; n=13). The total mixed ration dry matter for the close-up and lactation diets was measured weekly, then the Met supplements were adjusted daily and top-dressed over the total mixed ration at a rate of 0.19 (MS) or 0.07% (SM) on a dry matter basis. Liver tissue was collected on -10, 7, and 21 d for global DNA and peroxisome proliferator-activated receptor alpha (PPARα) promoter region-specific methylation. Several PPARα target and putative target genes associated with carnitine synthesis and uptake, fatty acid metabolism, hepatokines, and carbohydrate metabolism were also studied. Data were analyzed using PROC MIXED of SAS (SAS Institute Inc., Cary, NC) with the preplanned contrast CON versus SM + MS. Global hepatic DNA methylation on d 21 postpartum was lower in Met-supplemented cows than CON. However, of 2 primers used encompassing 4 to 12 CpG sites in the promoter region of bovine PPARA, greater methylation occurred in the region encompassing -1,538 to -1,418 from the transcription start site in cows supplemented with Met. Overall expression of PPARA was greater in Met-supplemented cows than CON. Concomitantly, PPARA-target genes, such as ANGPTL4, FGF21, and PCK1, were also upregulated overall by Met supplementation. The upregulation of PPARα target genes indicates that supplemental Met, likely through the synthesis of S-adenosylmethionine, activated PPARA-regulated signaling pathways. Upregulation of hepatic PPARA has been associated with improved lipid metabolism and immune function, both of which were reported in companion publications from this study. In turn, those positive effects resulted in improved postpartal health and performance. Further research is needed to study more closely the mechanistic connections between global DNA and promoter region-specific PPARA methylation with PPARA expression and functional outcomes in liver.

Monitoramento da variabilidade genética de pacu, Piaractus mesopotamicus, do programa de aumento de estoque do rio Paranapanema / Monitoring of the genetic variability of pacu, Piaractus mesopotamicus, of the stock enhancement program of the Paranapanema River

Avaliou-se a variabilidade genética dos estoques de reprodutores e dos peixes jovens de Piaractus mesopotamicus de três pisciculturas do estado do Paraná, utilizadas no programa de aumento de estoque de peixes no rio Paranapanema. Foi utilizado o marcador RAPD para avaliar as amostras do estoque de reprodutores e dos peixes jovens das pisciculturas de Palotina, Cambará e Andirá. A porcentagem de fragmentos polimórficos e o índice de diversidade genética de Shannon dos estoques de reprodutores variaram de 75,0 por cento a 71,4 por cento e de 0,434 a 0,376, respectivamente. Os peixes jovens das pisciculturas apresentaram valores mais elevados para ambos os parâmetros, com exceção da piscicultura de Palotina, na qual o índice de diversidade genética de Shannon foi semelhante. Os estoques de reprodutores apresentaram alta variabilidade genética, e esta foi mantida nos peixes jovens.

The genetic variability of broodstocks and juveniles of Piaractus mesopotamicus raised in three hatchery stations in Parana state, that were used in the fish stock enhancement program of the Paranapanema River, was estimated. The RAPD marker was used to evaluate samples taken from broodstocks and juveniles in the hatchery stations of Palotina, Cambará, and Andirá cities. The percentage of polymorphic fragments and the Shannon genetic diversity index of broodstocks ranged from 75.0 percent to 71.4 percent and from 0.434 to 0.376, respectively. The juveniles of the hatchery stations presented higher values for both parameters, except in the hatchery station of Palotina in which the Shannon genetic diversity index was similar. The broodstocks presented high genetic variability, and this was maintained in the juveniles.