Improved uterine immune mediators in Holstein cows supplemented with rumen-protected methionine and discovery of neutrophil extracellular traps (NET).

During the transition from prepartum to early lactation, dairy cows often experience negative energy balance (NEB) that may result in reproductive stress and decreased fertility. The objective of this study was to observe the effects of rumen-protected methionine (RPM) on plasma amino acid concentrations, uterine cytology, immunohistochemistry (IHC) of glutathione peroxidase 1 (GPX) and superoxide dismutase 1 (SOD), and to confirm neutrophil extracellular trap (NET) formation. Multiparous Holstein cows (n = 20) were randomly assigned to two treatments starting at 21 d before calving until 73 days in milk (DIM). Treatments were: CON (n = 9, no supplementation, TMR with a Lys:Met = 3.5:1) and MET (n = 11, TMR + Smartamine® M with a Lys:Met = 2.8:1). Uterine endometrial biopsies, uterine cytology, and blood samples from the coccygeal artery or vein were collected at 15, 30, and 73 DIM. Blood plasma samples were analyzed for amino acids and metabolites. Uterine biopsies were analyzed for NET formation, neutrophil numbers, as well as GPX and SOD by IHC. Additionally, uterine cytology was analyzed for polymorphonuclear neutrophil (PMN) to epithelial cell percentage. Cows in CON had lower methionine plasma concentrations (18.05 ±â€¯2.0 µM) than cows in MET (30.39 ±â€¯1.6 µM). Cows in CON had greater cystine plasma concentrations (3.62 ±â€¯0.3 µM) than cows in MET (2.8 ±â€¯0.3 µM). No treatment differences were observed for SOD or GPX in the endometrium. Cows in CON tended to have a high score for positively immunolabeled GPX cells at 15 DIM than cows in MET. No treatment differences were observed for the percentage of PMN in uterine cytology, number of neutrophils, or extent of NET formation in the endometrium. A treatment by time interaction was observed for PMN percentage and the number of neutrophils: cows in MET tended to have greater PMN percentages than cows in CON at 15 DIM which decreased for subsequent days and cows in MET had greater neutrophil numbers in the endometrium at 30 DIM than cows in CON. In conclusion, dietary supplementation of RPM altered plasma amino acid concentrations and increased neutrophil infiltration in the postpartum period, suggesting improved uterine immunity.

Effects of rumen-protected methionine and choline supplementation on steroidogenic potential of the first postpartum dominant follicle and expression of immune mediators in Holstein cows.

Multiparous Holstein cows were assigned in a randomized complete block design into four treatments from 21 d before calving to 30 d in milk (DIM). Treatments were: MET [n = 19, fed the basal diet + rumen-protected methionine at a rate of 0.08% (w/w) of the dry matter, Smartamine® M], CHO (n = 17, fed the basal diet + choline 60 g/d, Reashure®), MIX (n = 21, fed the basal diet + Smartamine® M at a rate of 0.08% (w/w) of the dry matter and 60 g/d Reashure®), and CON (n = 20, no supplementation, fed the close-up and fresh cow diets). Follicular development was monitored via ultrasound every 2 d starting at 7 DIM until ovulation (n = 37) or aspiration (n = 40) of the first postpartum dominant follicle (DF). Follicular fluid from 40 cows was aspirated and cells were retrieved immediately by centrifugation. Gene expression of TLR4, TNF, IL1-ß, IL8, IL6, LHCGR, STAR, 3ß-HSD, P450scc, CYP19A1, IRS1, IGF, MAT1A, and SAHH, was measured in the follicular cells of the first DF. Cows in CON had higher TNF, TLR4, and IL1-ß mRNA expression (11.70 ± 4.6, 21.29 ± 10.4, 6.28 ± 1.4, respectively) than CHO (2.77 ± 0.9, 2.16 ± 0.9, 2.29 ± 0.7, respectively), and MIX (2.23 ± 0.7, 1.46 ± 0.6, 2.92 ± 0.8, respectively). Cows in CON had higher IL1-ß expression (6.27 ± 1.4) than cows in MET (3.28 ± 0.6). Expression of IL8 mRNA was lower for cows in CHO (0.98 ± 0.3) than cows in CON (4.90 ± 0.7), MET (6.10 ± 1.7), or MIX (5.05 ± 1.8). Treatments did not affect mRNA expression of LHCGR, STAR, P450scc, CYP19A, SAHH, MAT1A, or IL6 however, 3ß-HSD expression was higher for cows in MET (1.46 ± 0.3) and MIX (1.25 ± 0.3) than CON (0.17 ± 0.04) and CHO (0.26 ± 0.1). Supplementation of methionine, choline, and both methionine and choline during the transition period did not affect days to first ovulation or number of cows that ovulated the first follicular wave. Plasma and follicular fluid estradiol and progesterone concentrations were not different among treatments. Methionine concentrations in the follicular fluid of the first postpartum DF was higher for cows in MET (18.2 ± 0.1 µM) than cows in CON (11.1 ± 0.9 µM). In conclusion, supplementing choline and methionine during the transition period changed mRNA expression in follicular cells and dietary methionine supplementation increased plasma and follicular fluid concentrations of methionine of the first postpartum DF in Holstein cows.

Evaluation of a commercially available radioimmunoassay and enzyme immunoassay for the analysis of progesterone and estradiol and the comparison of two extraction efficiency methods.

The measurement of progesterone (P4) and estradiol (E2) is essential for monitoring reproductive cycles and can aid in diagnosing the cause of poor reproductive performance in dairy cattle. Readily available, reproducible, accurate, non-radioactive assays are needed for the assessment of P4 and E2 in bovine serum. The gold standard for hormone assessment, radioimmunoassay (RIA), was compared with enzyme-linked immunoassay (EIA). Serum collected from various points in the estrous cycle was extracted with radiolabeled P4 (ie, 3H-P4; HE) and without 3H-P4 (CE) before being used in the assay. For the assessment of P4, there is a great degree of correlation between the RIA and EIA (adjusted R-square = 0.95; Pearson correlation coefficient (PCC) = 0.98, P < 0.001). A difference between the RIA and EIA methods was not detected for E2 concentrations (P = 0.16), but the correlation between techniques was poor (adjusted R-squared = 0.73; PCC = 0.87, P = 0.002). There was no difference in the serum extraction efficiency as measured with 3H-P4 as opposed to without (P = 0.94). The two methods for the measurement of serum extraction efficiency were highly correlated (adjusted R-square = 0.83; PCC = 0.92, P < 0.001). The concordance correlation coefficient (CCC) showed an excellent agreement between RIA and EIA for P4 determination (0.89) and between HE and CE methods (0.90). Although the 95% limits of agreement of the Bland-Altman plots encompassed 89% (8/9) and 92% (12/13) of the differences between methods for P4 quantification and extraction respectively, the CCC indicated an excellent agreement among them. The CCC between RIA and EIA for E2 quantification was 0.68 which corresponds with a fair agreement; however, the 95% limits of agreement of the Bland-Altman plot encompassed 100% (9/9) of differences between methods. The EIA and CE methods are comparable alternatives to the RIA and HE methods, respectively and can be used to quantify P4 and E2 for bovine serum.

Effects of rumen-protected methionine and choline supplementation on the preimplantation embryo in Holstein cows.

Our objective was to determine the effects of supplementing methionine and choline during the prepartum and postpartum periods on preimplantation embryos of Holstein cows. Multiparous cows were assigned in a randomized complete-block design into four treatments from 21 days before calving to 30 days in milk (DIM). Treatments (TRT) were MET (n = 9, fed the basal diet + rumen-protected methionine at a rate of 0.08% [w:w] of the dry matter [DM], Smartamine M), CHO (n = 8, fed the basal diet + choline 60 g/d, Reashure), MIX (n = 11, fed the basal diet + Smartamine M and 60 g/d Reashure), and CON (n = 8, no supplementation, fed the close-up and fresh cow diets). Cows were randomly reassigned to two new groups (GRP) to receive the following diets from 31 to 72 DIM; control (CNT, n = 16, fed a basal diet) and SMT (n = 20, fed the basal diet + 0.08% [w:w] of the dry matter intake as methionine). An progesterone intravaginal insert (CIDR) device was inserted in all cows after follicular aspiration (60 DIM) and superovulation began at Day 61.5 using FSH in eight decreasing doses at 12-hour intervals over a 4-day period. On Days 63 and 64, all cows received two injections of PGF2α, and CIDR was removed on Day 65. Twenty-four hours after CIDR removal, ovulation was induced with GnRH. Cows received artificial insemination at 12 hours and 24 hours after GnRH. Embryos were flushed 6.5 days after artificial insemination. Global methylation of the embryos was assessed by immunofluorescent labeling of 5-methylcytosine, whereas lipid content was assessed by staining with Nile red. Nuclear staining was used to count the total number of cells per embryo. There was no difference between TRT, GRP, or their interaction (P > 0.05) for embryo recovery, embryos recovered, embryo quality, embryo stage, or cells per embryo. Methylation of the DNA had a TRT by GRP interaction (P = 0.01). Embryos from cows in CON-CNT had greater (P = 0.04) methylation (0.87 ± 0.09 arbitrary units [AU]) than embryos from cows in MET-CNT (0.44 ± 0.07 AU). The cytoplasmic lipid content was not affected (P > 0.05) by TRT or their interaction, but lipid content was greater (P = 0.04) for SMT (7.02 ± 1.03 AU) than that in CNT (3.61 ± 1.20 AU). In conclusion, cows in MET-CNT had embryos with lower methylation, and SMT cows had a higher lipid content than CNT. Methionine supplementation seems to impact the preimplantation embryo in a way that enhances its capacity for survival because there is strong evidence that endogenous lipid reserves serve as an energy substrate.