Supplementing rumen-protected methionine to lactating multiparous dairy cows did not improve reproductive performance.
ABSTRACT: There is evidence that supplementing methionine has positive effects on uterine environment, oocyte quality and embryo development in cattle. Thus, the objective of this study was to evaluate reproductive traits of cows supplemented with rumen-protected methionine (RPM) during early to mid-lactation in comparison with an untreated control group (CON). An additional focus was on the effect of puerperal diseases on reproductive performance parameters in RPM-supplemented group MET and in CON. A total of 1,709 multiparous Holstein-Friesian cows were enrolled in this field trial conducted on a commercial dairy farm in Slovakia. Cows were allocated at approximately 12 days post-partum (dpp) to either CON or MET, the latter supplemented with 25.0 g-27.2 g RPM per cow per day incorporated into the total mixed ration (TMR) until leaving the study pen at approximately 140 dpp. The amount of RPM was calculated based on individual feed ingredients analysis and adjusted during the study period when TMR changed. Cows were monitored during the post-partum period by vaginal examination (day 5 pp), measuring of beta-hydroxybutyrate in blood (3, 5, and 8 dpp) and by vaginal examination, uterine cytology and measuring of back fat thickness by ultrasound (all at 31 ± 3 dpp). Compared with CON, cows supplemented with RPM did not show better reproduction performance parameters (first service submission rate, days to first service, conception risk, days open 140). Results from binary logistic regression model for the risk of conception showed that metritis had a significant effect, but the supplementation of methionine had not. Results of Cox regression analysis for the odds of conception within 140 dpp revealed only metritis and clinical endometritis as significant factors. In conclusion, supplementation of RPM had no beneficial effect on reproductive performance in this study farm compared with an untreated control group.
Project description:The objective of this study was to evaluate the progression of the uterine microbiota from calving until establishment of metritis. Uterine swabs (n = 72) collected at 0, 2, and 6 ± 2 days postpartum (dpp) from 12 metritic and 12 healthy cows were used for metagenomic sequencing of the 16S rRNA gene on the Illumina MiSeq platform. A heat map showed that uterine microbiota was established at calving. The microbiota changed rapidly from 0 to 6 ± 2 dpp, with a decrease in the abundance of Proteobacteria and an increase in the abundance of Bacteroidetes and Fusobacteria, which were dominant in metritic cows. Uterine microbiota composition was shared; however, metritic and healthy cows could be discriminated using relative abundance of bacterial genera at 0, 2, and 6 ± 2 dpp. Bacteroides was the main genus associated with metritis because it was the only genus that showed significantly greater abundance in cows with metritis. As the abundance of Bacteroides organisms increased, the uterine discharge score, a measure of uterine health, worsened. Fusobacterium was also an important genus associated with metritis because Fusobacterium abundance increased as Bacteroides abundance increased and the uterine discharge score worsened as the abundance increased. The correlation with uterine discharge score and the correlation with Bacteroides or Fusobacterium showed that other bacteria, such as Helcoccocus, Filifactor, and Porphyromonas, were also associated with metritis. There were also bacteria associated with uterine health, such as "Candidatus Blochmannia," Escherichia, Sneathia, and Pedobacter.
Project description:This experiment was conducted to evaluate the impact of yeast and lactic acid bacteria (LAB) on mastitis and milk microbiota composition of dairy cows. Thirty lactating Holstein cows with similar parity, days in milk were randomly assigned to five treatments, including: (1) Health cows with milk SCC?<?500,000 cells/mL, no clinical signs of mastitis were found, fed basal total mixed ration (TMR) without supplementation (H); (2) Mastitis cows with milk SCC?>?500,000 cells/mL, fed basal TMR without supplementation (M); (3) Mastitis cows fed basal TMR supplemented with 8 g day-1 yeast (M?+?Y); (4) Mastitis cows fed basal TMR supplemented with 8 g day-1 LAB (M?+?L); (5) Mastitis cows (milk SCC?>?500,000 cells/mL) fed basal TMR supplemented with 4 g day-1 yeast and 4 g day-1 LAB (M?+?Y?+?L). Blood and milk sample were collected at day 0, day 20 and day 40. The results showed efficacy of probiotic: On day 20 and day 40, milk SCC in H, M?+?Y, M?+?L, M?+?Y?+?L was significantly lower than that of M (P?<?0.05). Milk concentration of TNF-?, IL-6 and IL-1? in M?+?Y?+?L were significantly reduced compared with that of M on day 40 (P?<?0.05). Milk Myeloperoxidase (MPO) and N-Acetyl-?-D-Glucosaminidase (NAG) activity of M?+?Y, M?+?L, M?+?L?+?Y were lower than that of M on day 40 (P?<?0.05). At genus level, Staphylococcus, Chryseobacterium and Lactococcus were dominant. Supplementation of LAB decreased abundance of Enterococcus and Streptococcus, identified as mastitis-causing pathogen. The results suggested the potential of LAB to prevent mastitis by relieving mammary gland inflammation and regulating milk microorganisms.
Project description:Bacterial contamination of the uterus following calving is ubiquitous in the dairy cow, 40% of cows develop postpartum uterine infection, including metritis. While predisposing factors like twinning and dystocia are associated with metritis, it is unclear why some cows remain healthy following calving and others develop uterine infection, negatively impacting animal health, milk production and economic return. Here, we profiled peripheral blood mononuclear cells of cows before calving and during postpartum metritis. We hypothesized that peripheral blood mononuclear cell function and proportions would be altered during the prepartum period in cows that develop postpartum metritis. Using flow cytometry we observed reduced proportions of peripheral CD3+/CD4+, CD4+/CD62L+, FOXP3+ and CD21+ populations from -10 to 40?days relative to calving associated with metritis, while the proportion of peripheral CD3+/CD4+ lymphocytes were specifically reduced in the prepartum period before the onset of metritis. Peripheral blood mononuclear cells from cows with metritis had a perturbed capacity to secrete IL-1? or IFN? in response to in vitro stimulus; cells collected during the prepartum period from cows that would go on to develop metritis failed to increase IL-1? secretion in response to stimulation, while IFN? secretion was altered at calving and postpartum in cows with metritis compared to healthy herd mates. No effect of metritis was observed in the capacity of cows to mount a humoral immune response to antigen administered on the day of calving. The studies discussed here suggest that while minor changes to the prepartum immune system are observed in cows that develop metritis, changes observed in the postpartum period are more prevalent and likely a consequences of disease and not causative. Future studies to modulate the prepartum immune system may help to limit postpartum metritis.
Project description:Milk fat depression (MFD) syndrome represents a significant drawback to the dairy industry. The aim of this study was to unravel the ruminal metabolome-microbiome interaction in response to diet-induced MFD in dairy cows. Twelve healthy second parity Holstein dairy cows (days in milk (DIM) = 119 ± 14) were randomly assigned into control (CON, n = 6) group and treatment (TR, n = 6) group. Cows in TR group received a high-starch total mixed ration (TMR) designed to induce an MFD syndrome. Decreased milk fat yield and concentration in TR cows displayed the successful development of MFD syndrome. TR diet increased the relative abundance of Prevotella and decreased the relative abundance of unclassified Lachnospiraceae, Oribacterium, unclassified Veillonellaceae and Pseudobutyrivibrio in ruminal fluid. Metabolomics analysis revealed that the ruminal fluid content of glucose, amino acids and amines were significantly increased in TR cows compared with CON cows. Correlation analysis revealed that the concentration of amines and amino acids were highly correlated with the abundance of Oribacterium, Pseudobutyrivibrio, RC9_gut_group, unclassified BS11_gut_group and Selenomonas. In general, these findings revealed that TR diet reduced the rumination time and altered rumen fermentation type, which led to changes in the composition of ruminal microbiota and metabolites, and caused MFD.
Project description:Post-partum metritis is among the most prevalent disease in dairy cows affecting animal welfare and inflicting considerable economic loses. While post-partum contamination of the uterus is rife in dairy cows, only a fraction of these animals will develop metritis. Our main objective was to compare the bacterial communities and the inflammatory response in the endometrium of healthy and metritic dairy cows. Holstein-Friesian cows (n?=?35) were sampled immediately following clinical classification as healthy (n?=?21), suffering from metritis (n?=?13) or septic metritis (n?=?1), based on veterinary examination at 5-10 days post-partum. Polymorphonuclear cells (PMN) percentage in endometrial cytology was significantly higher in cows with metritis. Full-thickness uterine biopsy analysis revealed that the luminal epithelium in inter-caruncle areas was preserved in healthy cows, but in metritis it was compromised, with marked PMN infiltration particularly in the apical endometrium. Gram staining revealed that bacterial load and spatial distribution was associated with disease severity. 16S-rDNA bacterial community analysis revealed unique endometrial bacterial community composition in metritic cows, as compared to more diverse communities among healthy cows. The most abundant phyla in healthy cows were Proteobacteria (31.8?±?9.3%), Firmicutes (27.9?±?8.4%) and Bacteroidetes (19.7?±?7.2%), while Bacteroidetes (60.3?±?10.3%), Fusobacteria (13.4?±?5.9%) and Firmicutes (10.5?±?3.3%) were most abundant in the endometrial mucosa of metritic cows. Relative abundance of Bacteroidetes (19.7?±?7.2% vs. 60.3?±?10.3%), Fusobacteria (7.5?±?5.2% vs. 13.4?±?5.9%) and Proteobacteria (31.8?±?9.3% vs. 7.3?±?5.6%) phyla differed significantly between healthy and metritic cows. In summary, endometrial PMN abundance, spatial distribution and bacterial communities differed between healthy and metritic dairy cows at early post-partum.
Project description:Background:Peripheral blood mononuclear cells (PBMCs), commonly referred to as lymphocytes and monocytes, representing cells of the innate and adaptive immune systems. Aims:To find out whether changes in PBMCs' mRNA expression of pattern recognition receptors (PRRs) are associated with puerperal metritis in Holstein cows. Methods?:Peripheral blood mononuclear cells were collected from 20 cows with puerperal metritis and 20 cows without metritis at 10 days postpartum. Expression of toll-like receptors 2 and 4 (TLR2 and TLR4), and cluster of differentiation 14 (CD14) genes were assessed in PBMCs using a quantitative real time-polymerase chain reaction (qRT-PCR) technique. The data was normalized to glyceraldehyde-3-phosphate dehydrogenase (GAPDH) as a reference gene, and 2-??Ct methodology was used for relative quantification. Results:The results of the present study demonstrated that the expression of TLR4 (P=0.04) and CD14 (P=0.008) was significantly greater in cows with puerperal metritis compared to the control group. However, the expression of TLR2 (P=0.06) was not significantly different between cows with puerperal metritis and healthy cows. Conclusion:This study suggests that puerperal metritis significantly increases the expression of TLR4 and CD14 genes in the PBMCs which contributes to the proper stimulation of inflammation and uterine clearance of bacteria soon after calving.
Project description:Metritis is associated with reduced fertility in dairy cows, but the mechanisms are unclear because the disease resolves several weeks before insemination. One hypothesis is that metritis causes persistent changes in granulosa cells during follicle development, which might be evident in the transcriptome of granulosa cells from dominant follicles weeks after parturition. To test this hypothesis, we collected the follicular fluid and granulosa cells from dominant follicles 63 days post partum from cows previously diagnosed with metritis, at least 6 weeks after resolution of the disease and from cows not diagnosed with metritis (control cows). Bacterial lipopolysaccharide was detected in follicular fluid, and concentrations were associated with follicular fluid IL-8 and glucose concentrations. Transcriptome analysis using RNAseq revealed 177 differentially expressed genes in granulosa cells collected from cows that had metritis compared with control cows. The most upregulated genes were ITLN1, NCF2, CLRN3, FSIP2 and ANKRD17, and the most downregulated genes were ACSM1, NR4A2, GHITM, CBARP and NR1I3. Pathway analysis indicated that the differentially expressed genes were involved with immune function, cell-cell communication, cell cycle and cellular metabolism. Predicted upstream regulators of the differentially expressed genes included NF?B, IL-21 and lipopolysaccharide, which are associated with infection and immunity. Our data provide evidence for a persistent effect of metritis on the transcriptome of granulosa cells in ovarian follicles after the resolution of disease.
Project description:This study aimed to evaluate bacterial and host factors causing a fever in cows with metritis. For that, we investigated uterine microbiota using a metagenomic sequencing of the 16S rRNA gene (Study 1), and immune response parameters (Study 2) in metritic cows with and without a fever.Bacterial communities were similar between the MNoFever and MFever groups based on distance metrics of relative abundance of bacteria. Metritic cows showed a greater prevalence of Bacteroidetes, and Bacteroides and Porphyromonas were the largest contributors to that difference. A comparison of relative abundance at the species level pointed to Bacteroides pyogenes as a fever-related species which was significantly abundant in the MFever than the MNoFever and Healthy groups; however, absolute abundance of Bacteroides pyogenes determined by droplet digital PCR (ddPCR) was similar between MFever and MNoFever groups, but higher than the Healthy group. The same trend was observed in the total number of bacteria.The activity of polymorphonuclear leukocyte (PMN) and the production of TNF?, PGE2 metabolite, and PGE2 were evaluated in serum, before disease onset, at 0 and 3 DPP. Cows in the MNoFever had decreased proportion of PMN undergoing phagocytosis and oxidative burst compared with the MFever. The low PMN activity in the MNoFever was coupled with the low production of TNF?, but similar PGE2 metabolite and circulating PGE2.Our study is the first to show a similar microbiome between metritic cows with and without a fever, which indicates that the host response may be more important for fever development than the microbiome. Bacteroides pyogenes was identified as an important pathogen for the development of metritis but not fever. The decreased inflammatory response may explain the lack of a febrile response in the MNoFever group.
Project description:The diversity of the uterine bacterial composition in dairy cows is still poorly understood, although the emerging picture has shown to be increasingly complex. Understanding the complexity and ecology of microorganisms in the uterus of postpartum dairy cows is critical for developing strategies to block their action in reproductive disorders, such as metritis/endometritis. Here, we used PCR-Denaturing Gradient Gel Electrophoresis (DGGE) and DNA pyrosequencing to provide a comprehensive description of the uterine bacterial diversity and compare its succession in healthy, metritic and endometritic Holstein dairy cows at three intervals following calving. Samples were collected from 16 dairy cows housed in a dairy farm located in upstate New York. PCR-DGGE revealed a complex profile with extensive differences in the community structure. With few exceptions, clustering analysis grouped samples from cows presenting the same health status. Analysis of >65,000 high-quality 16S rRNA gene sequences showed that the uterine bacterial consortia, regardless of the health status, is mainly composed of members of the phyla Bacteroidetes, Fusobacteria, Firmicutes, Proteobacteria, and Tenericutes. In addition to these co-dominant phyla, sequences from Spirochaetes, Synergistetes, and Actinobacteria appear less frequently. It is possible that some sequences detected in the uterine fluid resulted from the presence of fecal or vaginal contaminants. Overall, the bacterial core community was different in uterine fluid of healthy cows, when compared to cows suffering from postpartum diseases, and the phylogenetic diversity in all the combined samples changed gradually over time. Particularly at the 34-36 days postpartum (DPP), the core community seemed to be specific for each health status. Our finding reveals that the uterine microbiota in dairy cows varies according with health status and DPP. Also, it adds further support to the hypothesis that there is uterine contamination with diverse bacterial groups following calving and emphasizes the role of unidentified microorganisms in this context.
Project description:The aim of this study was to test whether a combination of plant bioactive lipid compounds (also termed 'essential oils') and biotin (PBLC+B) could decrease the mobilization of body reserves and ketosis incidence in postpartum dairy cows. We compared non-supplemented control (CON) cows with cows receiving monensin (MON) as a controlled-release capsule at d -21, and with cows receiving PBLC+B from day (d) -21 before calving until calving (Phase 1) and further until d 37 after calving (Phase 2), followed by PBLC+B discontinuation from d 38 to d 58 (Phase 3). The PBLC+B cows had higher body weight and higher back fat thickness than CON cows and lesser body weight change than MON and CON cows in Phase 3. Body condition score was not different among groups. Milk protein concentration tended to be higher on the first herd test day in PBLC+B vs. CON cows. Milk fat concentration tended to be highest in PBLC+B cows throughout Phases 2 and 3, with significantly higher values in PBLC+B vs. MON cows on the second herd test day. Yields of energy-corrected milk were higher in PBLC+B vs. CON and MON cows in Phase 2 and higher in PBLC+B and MON cows vs. CON cows in Phase 3. The incidence of subclinical ketosis was 83%, 61% and 50% in CON, PBLC+B and MON cows, respectively, with lower mean ?-hydroxybutyrate values in MON than in PBLC+B cows in Phase 1 prepartum. The serum triglyceride concentration was higher in PBLC+B vs. CON cows on d 37. No differences were observed in serum glucose, urea, non-esterified fatty acids, cholesterol and bilirubin concentrations. Aspartate transaminase and ?-glutamyltranspeptidase but not glutamate dehydrogenase activities tended to be highest in MON and lowest in PBLC+B in Phase 2. We conclude that PBLC+B prevent body weight loss after parturition and are associated with similar ketosis incidence and partly higher yields of energy-corrected milk compared to MON supplementation of dairy cows.