Mining key circRNA-associated-ceRNA networks for milk fat metabolism in cows with varying milk fat percentages.

BACKGROUND: Cow milk fat is an essential indicator for evaluating and measuring milk quality and cow performance. Growing research has identified the molecular functions of circular RNAs (circRNAs) necessary for mammary gland development and lactation in mammals. METHOD: The present study analyzed circRNA expression profiling data in mammary epithelial cells (MECs) from cows with highly variable milk fat percentage (MFP) using differential expression analysis and weighted gene co-expression network analysis (WGCNA). RESULTS: A total of 309 differentially expressed circRNAs (DE-circRNAs) were identified in the high and low MFP groups. WGCNA analysis revealed that the pink module was significantly associated with MFP (r = - 0.85, P = 0.007). Parental genes of circRNAs in this module were enriched mainly in lipid metabolism-related signaling pathways, such as focal adhesion, ECM-receptor interaction, adherens junction and AMPK. Finally, six DE-circRNAs were screened from the pink module: circ_0010571, circ_0007797, circ_0002746, circ_0003052, circ_0004319, and circ_0012840. Among them, circ_0002746, circ_0003052, circ_0004319, and circ_0012840 had circular structures and were highly expressed in mammary tissues. Subcellular localization revealed that these four DE-circRNAs may play a regulatory role in the mammary glands of dairy cows, mainly as competitive endogenous RNAs (ceRNAs). Seven hub target genes (GNB1, GNG2, PLCB1, PLCG1, ATP6V0C, NDUFS4, and PIGH) were obtained by constructing the regulatory network of their ceRNAs and then analyzed by CytoHubba and MCODE plugins in Cytoscape. Functional enrichment analysis revealed that these genes are crucial and most probable ceRNA regulators in milk fat metabolism. CONCLUSIONS: Our study identified several vital circRNAs and ceRNAs affecting milk fat synthesis, providing new research ideas and a theoretical basis for cow lactation, milk quality, and breed improvement.

Identification and characterization of whole blood gene expression and splicing quantitative trait loci during early to mid-lactation of dairy cattle.

BACKGROUND: Characterization of regulatory variants (e.g., gene expression quantitative trait loci, eQTL; gene splicing QTL, sQTL) is crucial for biologically interpreting molecular mechanisms underlying loci associated with complex traits. However, regulatory variants in dairy cattle, particularly in specific biological contexts (e.g., distinct lactation stages), remain largely unknown. In this study, we explored regulatory variants in whole blood samples collected during early to mid-lactation (22-150 days after calving) of 101 Holstein cows and analyzed them to decipher the regulatory mechanisms underlying complex traits in dairy cattle. RESULTS: We identified 14,303 genes and 227,705 intron clusters expressed in the white blood cells of 101 cattle. The average heritability of gene expression and intron excision ratio explained by cis-SNPs is 0.28 ± 0.13 and 0.25 ± 0.13, respectively. We identified 23,485 SNP-gene expression pairs and 18,166 SNP-intron cluster pairs in dairy cattle during early to mid-lactation. Compared with the 2,380,457 cis-eQTLs reported to be present in blood in the Cattle Genotype-Tissue Expression atlas (CattleGTEx), only 6,114 cis-eQTLs (P < 0.05) were detected in the present study. By conducting colocalization analysis between cis-e/sQTL and the results of genome-wide association studies (GWAS) from four traits, we identified a cis-e/sQTL (rs109421300) of the DGAT1 gene that might be a key marker in early to mid-lactation for milk yield, fat yield, protein yield, and somatic cell score (PP4 > 0.6). Finally, transcriptome-wide association studies (TWAS) revealed certain genes (e.g., FAM83H and TBC1D17) whose expression in white blood cells was significantly (P < 0.05) associated with complex traits. CONCLUSIONS: This study investigated the genetic regulation of gene expression and alternative splicing in dairy cows during early to mid-lactation and provided new insights into the regulatory mechanisms underlying complex traits of economic importance.

Incidence of uterine infections, major bacteria and antimicrobial resistance in postpartum dairy cows in southern Ethiopia.

BACKGROUND: Uterine infections, primarily caused by bacterial pathogens, pose a significant problem for dairy farmers worldwide, leading to poor reproductive performance and economic losses. However, the bacteria responsible for uterine infections have not been adequately studied, nor has the antibiotic susceptibility of the causative bacteria been frequently tested in Ethiopia. This study aims to estimate the cumulative incidence of uterine infections in postpartum dairy cows, identify bacterial causes and determine antimicrobial susceptibility profile of the isolated bacteria. METHODS: A prospective cohort study was conducted in which 236 cows from 74 dairy farms were monitored biweekly from calving to 90 days postpartum for metritis, endometritis and other disorders. Aseptic uterine swab samples were collected from 40 cows with uterine infections. The samples were cultured, and the isolated bacteria were tested for antimicrobial susceptibility using the disk diffusion method. RESULTS: Out of 236 cows monitored during the postpartum phase, 45 (19.1%) were found to have contracted uterine infection. The cumulative incidence of metritis was 11.4% (n = 27), while the cumulative incidence of endometritis was 7.6% (n = 18). Of the 40 cultured swab samples, 29 (72.5%) had one or more bacteria isolated. The most commonly isolated bacteria were Escherichia coli (45%), coagulase-positive staphylococci (30%), and Klebsiella spp. (22.5%). Other bacterial spp, including Arcanobacterium pyogenes (12.5%), Fusobacterium spp. (12.5%), Enterobacter aerogenes (12.5%), coagulase-negative staphylococci (12.5%), Streptococcus spp. (7.5%), Salmonella spp, (5%) Proteus spp (5%) and Pasteurella spp (2.5%) were also isolated. All of the isolated bacteria demonstrated resistance to at least one of the antimicrobials tested. Multidrug resistance was observed in E. coli, Klebsiella spp., A. pyogenes, and Fusobacterium spp. Gentamicin was found to be the most effective antimicrobial against all bacteria tested, while tetracycline was the least effective of all. CONCLUSION: The study found that a significant proportion of cows in the population were affected by uterine infections and the isolated bacteria developed resistance to several antimicrobials. The study emphasizes the need for responsible use of antimicrobials to prevent the emergence of antimicrobial resistance. It also highlights the importance of raising awareness among dairy farmers to avoid the indiscriminate use of antibiotics and its consequences.

The rumen microbiota contributed to the development of mastitis induced by subclinical ketosis.

BACKGROUND: Mastitis is a serious disease which affects animal husbandry, particularly in cow breeding. The etiology of mastitis is complex and its pathological mechanism is not yet fully understood. Our previous research in clinical investigation has revealed that subclinical ketosis can increase the number of somatic cell counts (SCC) in milk, although the underlying mechanism remains unclear. Recent studies have further confirmed the significant role of mastitis. RESULTS: In this study, we aimed to examine the SCC, rumen microbiota, and metabolites in the milkmen of cows with subclinical ketosis. Additionally, we conducted a rumen microbiota transplant into mice to investigate the potential association between rumen microbiota disturbance and mastitis induced by subclinical ketosis in dairy cows. The study has found that cows with subclinical ketosis have a higher SCC in their milk compared to healthy cows. Additionally, there were significant differences in the rumen microbiota and the level of volatile fatty acid (VFA) between cows with subclinical ketosis and healthy cows. Moreover, transplanting the rumen microbiota from subclinical ketosis and mastitis cows into mice can induce mammary inflammation and liver function damage than transplanting the rumen flora from healthy dairy cows. CONCLUSIONS: In addition to the infection of mammary gland by pathogenic microorganisms, there is also an endogenous therapeutic pathway mediated by rumen microbiota. Targeted rumen microbiota modulation may be an effective way to prevent and control mastitis in dairy cows.

Phenotypic and genotypic assessment of iron acquisition in diverse bovine-associated non-aureus staphylococcal strains.

Although the role of iron in bacterial infections has been well described for Staphylococcus (S.) aureus, iron acquisition in (bovine-associated) non-aureus staphylococci and mammaliicocci (NASM) remains insufficiently mapped. This study aimed at elucidating differences between four diverse bovine NASM field strains from two species, namely S. chromogenes and S. equorum, in regards to iron uptake (with ferritin and lactoferrin as an iron source) and siderophore production (staphyloferrin A and staphyloferrin B) by investigating the relationship between the genetic basis of iron acquisition through whole genome sequencing (WGS) with their observed phenotypic behavior. The four field strains were isolated in a previous study from composite cow milk (CCM) and bulk tank milk (BTM) in a Flemish dairy herd. Additionally, two well-studied S. chromogenes isolates originating from a persistent intramammary infection and from a teat apex were included for comparative purpose in all assays. Significant differences between species and strains were identified. In our phenotypical iron acquisition assay, while lactoferrin had no effect on growth recovery for all strains in iron deficient media, we found that ferritin served as an effective source for growth recovery in iron-deficient media for S. chromogenes CCM and BTM strains. This finding was further corroborated by analyzing potential ferritin iron acquisition genes using whole-genome sequencing data, which showed that all S. chromogenes strains contained hits for all three proposed ferritin reductive pathway genes. Furthermore, a qualitative assay indicated siderophore production by all strains, except for S. equorum. This lack of siderophore production in S. equorum was supported by a quantitative assay, which revealed significantly lower or negligible siderophore amounts compared to S. aureus and S. chromogenes. The WGS analysis showed that all tested strains, except for S. equorum, possessed complete staphyloferrin A (SA)-synthesis and export operons, which likely explains the phenotypic absence of siderophore production in S. equorum strains. While analyzing the staphyloferrin A and staphyloferrin B operon landscapes for all strains, we noticed some differences in the proteins responsible for iron acquisition between different species. However, within strains of the same species, the siderophore-related proteins remained conserved. Our findings contribute valuable insights into the genetic elements associated with bovine NASM pathogenesis.

Mycobiome of the external ear canal of healthy cows.

Malassezia yeasts belong to the normal skin microbiota of a wide range of warm-blooded animals. However, their significance in cattle is still poorly understood. In the present study, the mycobiota of the external ear canal of 20 healthy dairy Holstein cows was assessed by cytology, culture, PCR, and next-generation sequencing. The presence of Malassezia was detected in 15 cows by cytology and PCR. The metagenomic analysis revealed that Ascomycota was the predominant phylum but M. pachydermatis the main species. The Malassezia phylotype 131 was detected in low abundance. Nor M. nana nor M. equina were detected in the samples.

The mycobiota of the external ear canal of healthy cows was assessed by cytology, culture, PCR, and NGS. The presence of Malassezia was detected by cytology and PCR. Ascomycota was the main phylum and M. pachydermatis the main species. The Malassezia phylotype 131 was also detected in the samples.

Oxylipins biosynthesis and the regulation of bovine postpartum inflammation.

Uncontrolled or dysregulated inflammation has adverse effects on the reproduction, production and health of animals, and is a major pathological cause of increased incidence and severity of infectious and metabolic diseases. To achieve successful transition from a non-lactation pregnant state to a non-pregnant lactation state, drastic metabolic and endocrine alteration have taken place in dairy cows during the periparturient period. These physiological changes, coupled with decreased dry matter intake near calving and sudden change of diet composition after calving, have the potential to disrupt the delicate balance between pro- and anti-inflammation, resulting in a disordered or excessive inflammatory response. In addition to cytokines and other immunoregulatory factors, most oxylipins formed from polyunsaturated fatty acids (PUFAs) via enzymatic and nonenzymatic oxygenation pathways have pro- or anti-inflammatory properties and play a pivotal role in the onset, development and resolution of inflammation. However, little attention has been paid to the possibility that oxylipins could function as endogenous immunomodulating agents. This review will provide a detailed overview of the main oxylipins derived from different PUFAs and discuss the regulatory role that oxylipins play in the postpartum inflammatory response in dairy cows. Based on the current research, much remains to be illuminated in this emerging field. Understanding the role that oxylipins play in the control of postpartum inflammation and inflammatory-based disease may improve our ability to prevent transition disorders via Management, pharmacological, genetic selection and dietary intervention strategies.

Nasal carriage of Staphylococcus aureus in healthy dairy cows in Algeria: antibiotic resistance, enterotoxin genes and biofilm formation.

BACKGROUND: Staphylococcus aureus can colonize and infect a variety of animal species. In dairy herds, it is one of the leading causes of mastitis cases. The objective of this study was to characterize the S. aureus isolates recovered from nasal swabs of 249 healthy cows and 21 breeders of 21 dairy farms located in two provinces of Algeria (Tizi Ouzou and Bouira). METHODS: The detection of enterotoxin genes was investigated by multiplex PCRs. Resistance of recovered isolates to 8 antimicrobial agents was determined by disc-diffusion method. The slime production and biofilm formation of S. aureus isolates were assessed using congo-red agar (CRA) and microtiter-plate assay. Molecular characterization of selected isolates was carried out by spa-typing and Multi-Locus-Sequence-Typing (MLST). RESULTS: S. aureus was detected in 30/249 (12%) and 6/13 (28.6%) of nasal swabs in cows and breeders, respectively, and a total of 72 isolates were recovered from positive samples (59 isolates from cows and 13 from breeders). Twenty-six of these isolates (36.1%) harbored genes encoding for staphylococcal enterotoxins, including 17/59 (28.8%) isolates from cows and 9/13 (69.2%) from breeders. Moreover, 49.1% and 92.3% of isolates from cows and breeders, respectively, showed penicillin resistance. All isolates were considered as methicillin-susceptible (MSSA). Forty-five (76.3%) of the isolates from cows were slime producers and 52 (88.1%) of them had the ability to form biofilm in microtiter plates. Evidence of a possible zoonotic transmission was observed in two farms, since S. aureus isolates recovered in these farms from cows and breeders belonged to the same clonal lineage (CC15-ST15-t084 or CC30-ST34-t2228). CONCLUSIONS: Although healthy cows in this study did not harbor methicillin-resistant S. aureus isolates, the nares of healthy cows could be a reservoir of enterotoxigenic and biofilm producing isolates which could have implications in human and animal health.

Assessing lameness prevalence and associated risk factors in crossbred dairy cows across diverse management environments.

BACKGROUND: A thorough understanding of lameness prevalence is essential for evaluating the impact of this condition on the dairy industry and assessing the effectiveness of preventive strategies designed to minimize its occurrence. Therefore, this cross-sectional study aimed to ascertain the prevalence of lameness and identify potential risk factors associated with lameness in Holstein Friesian crossbred cows across both commercial and smallholder dairy production systems in Bengaluru Rural District of Karnataka, India. METHODS: The research encompassed six commercial dairy farms and 139 smallholder dairy farms, involving a total of 617 Holstein Friesian crossbred cattle. On-site surveys were conducted at the farms, employing a meticulously designed questionnaire. Lameness in dairy cattle was assessed subjectively using a locomotion scoring system. Both bivariate and binary logistic regression models were employed for risk assessment, while principal components analysis (PCA) was conducted to address the high dimensionality of the data and capture the underlying structure of the explanatory variables. RESULTS: The overall lameness prevalence of 21.9% in commercial dairy farms and 4.6% in smallholder dairy farms. Various factors such as age, body weight, parity, body condition score (BCS), floor type, hock and knee injuries, animal hygiene, provision of hoof trimming, and the presence of hoof lesions were found to be significantly associated with lameness. Binary logistic regression analysis indicated that the odds of lameness in crossbred cows increased with higher parity, decreased BCS, presence of hard flooring, poor animal hygiene, and the existence of hoof lesions. These factors were identified as potential risk factors for lameness in dairy cows. Principal component analysis unveiled five components explaining 71.32% of the total variance in commercial farms and 61.21% in smallholder dairy farms. The extracted components demonstrated higher loadings of housing and management factors (such as hoof trimming and provision of footbath) and animal-level factors (including parity, age, and BCS) in relation to lameness in dairy cows. CONCLUSIONS: The findings suggest that principal component analysis effectively reduces the dimensionality of risk factors. Addressing these identified risk factors for lameness is crucial for the strategic management of lameness in dairy cows. Future research in India should investigate the effectiveness of management interventions targeted at the identified risk factors in preventing lameness in dairy cattle across diverse environments.

Lipopolysaccharide-binding protein in follicular fluid is associated with the follicular inflammatory status and granulosa cell steroidogenesis in dairy cows.

Metabolic stress and subsequent hepatic dysfunction in high-producing dairy cows are associated with inflammatory diseases and declining fertility. Lipopolysaccharide (LPS)-binding protein (LBP) is produced by hepatocytes and controls the immune response, suggesting that it is involved in the pathophysiology of inflammation-related attenuation of reproductive functions during metabolic stress. This study investigated the effect of LBP on the inflammatory status, oocyte quality, and steroidogenesis in the follicular microenvironment of dairy cows. Using bovine ovaries obtained from a slaughterhouse, follicular fluid and granulosa cells were collected from large follicles to evaluate the follicular status of metabolism, inflammation, and steroidogenesis. Cumulus-oocyte complexes were aspirated from small follicles and subjected to in vitro embryo production. The results showed that follicular fluid LBP concentrations were significantly higher in cows with fatty livers and hepatitis than in those with healthy livers. Follicular fluid LBP and LPS concentrations were negatively correlated, whereas LPS concentration showed a positive correlation with the concentrations of non-esterified fatty acids (NEFA) and ß-hydroxybutyric acid in follicular fluid. The blastulation rate of oocytes after in vitro fertilization was impaired in cows in which coexisting large follicles had high NEFA levels. Follicular fluid NEFA concentration was negatively correlated with granulosa cell expression of the estradiol (E2) synthesis-related gene (CYP19A1). Follicular fluid LBP concentration was positively correlated with follicular fluid E2 concentration and granulosa cell CYP19A1 expression. In conclusion, follicular fluid LBP may be associated with favorable conditions in the follicular microenvironment, including low LPS levels and high E2 production by granulosa cells.

Equine chorionic gonadotropin treatment and timed artificial insemination for dairy cow production under heat stress.

This study investigated the effects of timed artificial insemination (TAI) and equine chorionic gonadotropin (eCG) administration on lactating dairy cows under heat-stress conditions (average temperature-humidity index: 80). Timed artificial insemination was performed on the cows with (n = 57) or without (control, n = 41) supplementation with 500 IU of eCG at the day of PGF2α treatment using the CIDR-Ovsynch protocol. GnRH was administered, and a progesterone device (CIDR) was inserted on Day -10 of the treatment protocol. The CIDR was removed on Day -3, and the cows were treated with PGF2α. Two days later, a 2nd GnRH injection was administered. Subsequently, AI was performed on Day 0 (16-20 h after the 2nd GnRH injection), and pregnancy was diagnosed on Days 32 and 60. Plasma progesterone (P4) concentrations were measured after AI. Results showed that the eCG group had a higher pregnancy per AI (P/AI) than the control group (43.9 vs. 12.2%, P = 0.002), which was also accompanied by elevated P4 levels. Four cows in the eCG group had multiple calves, representing 7.0 and 16.0% of the group and pregnant cows, respectively. In conclusion, 500 IU of eCG combined with CIDR-Ovsynch in lactating dairy cows under severe heat stress conditions successfully improved fertility. However, the protocol may have a slight risk of multiple births.

Prevalence of growth hormone receptor gene polymorphisms and their association with milk production and fertility-related traits of cross-bred dairy cows in Sri Lanka.

This study investigated the association of selected growth hormone receptor (GHR) gene SNPs with selected fertility and milk production-related phenotypes of cross-bred dairy cows (n = 153) reared on three National Livestock Development Board farms in Sri Lanka. Selected cows were genetically screened for SNPs in the exon 08 (n = 153) and 5' upstream (n = 118) regions of the GHR gene using the target sequencing method. The relationships between different genotypes and fertility traits (average calving interval, average number of services per conception, and age at first calving) and milk production-related traits (average total lactation yield, average lactation length, and average milk yield) were analyzed using the General Linear Model in SPSS. Among the identified Four GHR SNPs, rs1099014416 was significantly associated with average calving interval and age at first calving. Cows with GG genotype exhibited younger age at first calving (918.51 ± 113.42 days) and longer calving intervals (543.41 ± 43.29 days) compared to cows with GT (1275.18 ± 38.31, 515.09 ± 24.49 days) and TT (1212.89 ± 88.22, 364.52 ± 54.01 days) genotypes. Other SNPs did not show associations with the studied traits. SNP rs109014416 has the potential to be used as a genetic marker for fertility-related traits in the selection of cross-bred dairy cows in Sri Lanka which should be validated with a larger population.

Estimation of genetic parameters and single-step genome-wide association studies for eating time and rumination time in Holstein dairy cows.

The aims of this study were to estimate genetic parameters and to identify genomic regions associated with eating time (ET) and rumination time (RUT) in Holstein dairy cows. Genetic correlations among ET, RUT, and milk yield traits were also estimated. The data were collected from 2019 to 2022 in 6 dairy herds located in the Walloon Region of Belgium. The dataset consisted of daily ET and RUT records on 284 Holstein cows, from which 41 cows had records only for the first parity (P1), 101 cows had records from both the first and second parities, and 142 cows had records only for the second parity (P2). The number of daily ET and RUT records in the P1 and P2 cows were 18,569 (on 142 cows) and 34,464 (on 243 cows), respectively. Data on 28,994 SNPs located on 29 Bos taurus autosomes (BTA) of 747 animals (435 males) were used. Random regression test-day models were used to estimate genetic parameters through the Bayesian Gibbs sampling method. The SNP solutions were estimated using a single-step genomic best linear unbiased prediction approach. The proportion of genetic variance explained by each 20-SNP sliding window (with an average size of 1.52 Mb) was calculated, and regions accounting for at least 1.0% of the total additive genetic variance were used to search for candidate genes. Mean (standard deviation; SD) averaged daily ET and RUT were 327.0 (85.66) and 559.4 (77.69) min/d for cows in P1 and 316.0 (82.24) and 574.2 (75.42) min/d for cows in P2, respectively. Mean (standard deviation; SD) heritability estimates for daily ET and RUT were 0.42 (0.09) and 0.45 (0.06) for cows in P1 and 0.45 (0.04) and 0.43 (0.02) for cows in P2, respectively. Mean (SD) daily genetic correlations between daily ET and RUT were 0.27 (0.07) for P1 and 0.34 (0.08) for P2. Genome-wide association analyses identified 6 genomic regions distributed over 5 chromosomes (BTA1, BTA4, BTA11, 2 regions of BTA14, and BTA17) associated with ET or RUT. The findings of this study increase our preliminary understanding of the genetic background of feeding behavior in dairy cows; however, larger datasets are needed to determine whether ET and RUT might have the potential to be used in selection programs.

Resilience indicator traits in 3 dairy cattle breeds in Baden-Württemberg.

In recent years, research in animal breeding has increasingly focused on the topic of resilience, which is expected to continue in the future due to the need for high-yielding, healthy, and robust animals. In this context, an established approach is the calculation of resilience indicator traits with time series analyses. Examples are the variance and autocorrelation of daily milk yield in dairy cows. We applied this methodology to the German dairy cow population. Data from the 3 breeds (German Holstein, German Fleckvieh, and German Brown Swiss) were obtained, which included 13,949 lactations from 36 farms from the state Baden-Württemberg in Germany working with automatic milking systems. Using the milk yield data, the daily absolute milk yields, deviations between observed and expected daily milk yields, and relative proportions of daily milk yields in relation to lactation performance were calculated. We used the variance and autocorrelation of these data as phenotypes in our statistical analyses. We estimated a heritability of 0.047 for autocorrelation and heritabilities between 0.026 and 0.183 for variance-based indicator traits. Furthermore, significant breed differences could be observed, with a tendency of better resilience in Brown Swiss. The breed differences can be due to both genetic and environmental factors. A high value of a variance-based indicator trait indicates a low resilience. Performance traits were positively correlated with variance-based indicator traits calculated from absolute daily milk yields, but they were negatively correlated with variance-based indicators calculated from relative daily milk yields. Thus, they can be considered as different traits. Although variance-based indicators based on absolute daily milk yields were affected by the performance level, variance-based indicators based on relative daily milk yields were corrected for the performance level and also showed higher heritabilities. Thus, they seem to be more suitable for practical use. Further studies need to be conducted to calculate the correlations between resilience indicators, functional traits, and health traits.

Fatty acids promote M1 polarization of monocyte-derived macrophages in healthy or ketotic dairy cows and a bovine macrophage cell line via impairing mTOR-mediated autophagy.

Excessive concentrations of free fatty acids (FFA) are the main factors causing immune dysfunction and inflammation in dairy cows with ketosis. Polarization of macrophages (the process of macrophages freely switching from one phenotype to another) into M1 or M2 phenotypes is an important event during inflammation induced by environmental stimuli. In non-ruminants, mammalian target of rapamycin (mTOR)-mediated autophagy (a major waste degradation process) regulates macrophage polarization. Thus, the objective was to unravel the role of mTOR-mediated autophagy on macrophage polarization in ketotic dairy cows. Four experiments were performed as follows: (1) In vitro differentiated monocyte-derived macrophages from healthy dairy cows or dairy cows with clinical ketosis (CK) were treated with 100 ng/mL lipopolysaccharide (LPS) and 100 ng/mL interferon-γ (IFN-γ) or 10 ng/mL interleukin-4 (IL4) and 10 ng/mL interleukin-10 (IL10) for 24 h; (2) Immortalized bovine macrophages were treated with 0, 0.3, 0.6, 1.2 mM FFA and LPS and IFN-γ or IL4 and IL10 for 24 h; (3) Macrophages were pretreated with 2 µM 4,6-dimorpholino-N-(4-nitrophenyl)-1,3,5-triazin-2-amine (MHY1485) for 30 min before treatment with LPS and IFN-γ or IL4 and IL10; (4) Macrophages were pretreated with 100 nM rapamycin (RAPA) for 2 h before treatment with LPS and IFN-γ or IL4 and IL10. Compared with healthy cows, cows with CK had a greater mean fluorescence intensity (MFI) of CD86+, but lower MFI of CD206+ and lower number of autophagosomes and autolysosomes in macrophages. Exogenous FFA treatment upregulated protein abundance of inducible nitric oxide synthase (iNOS) and mean fluorescence intensity of CD86, whereas it downregulated the protein abundance of arginase 1 (ARG1) and mean fluorescence intensity of CD206. In addition, FFA increased the p-p65/p65 protein abundance and tumor necrosis factor α (TNFA), interleukin-1B (IL1B), and interleukin-6 (IL6) mRNA abundance, but decreased LC3-phosphatidylethanolamine conjugate (LC3-II) protein abundance and autophagosomes and autolysosomes number. Pretreatment with MHY1485 promoted macrophage M1 polarization and inhibited macrophage M2 polarization via decreased mTOR-mediated autophagy. Activation of mTOR-mediated autophagy by pretreatment with RAPA attenuated the upregulation of inflammation in M1 macrophages that was induced by FFA. These data revealed that high concentrations of FFA promote macrophage M1 polarization in ketotic dairy cows via impairing mTOR-mediated autophagy.

Association between body condition profiles, milk production and reproduction performance in Holstein and Normande cows.

Body-condition dynamics are known to affect the different steps of reproduction in cattle (cyclicity, estrus expression, fertilization, embryo development). This has led to a widespread idea that there is an ideal-target optimal body condition, but no clear profile has yet been identified. Here we investigated the relationships between body condition score (BCS) profiles and reproductive performance in dairy cows. Data were from Holstein or Normande herds in 6 French experimental farms. In the Holstein breed, we discriminated 4 BCS profiles based on combining BCS at calving (Low: around 2.6 points, or High: around 3.3 points) with BCS loss after calving (Moderate (M): ≤ 1.0 points, or Severe (S): > 1.0 points). The Low-M profile mostly included multiparous cows with higher milk yield and lower reproductive performance than cows in the 3 other profiles. Low-M cows that experienced abnormal ovarian activity had lower reproductive performance than their profile-mates. Moreover, 67% of Low-M cows kept the same profile at the following lactation. The High-S profile mostly included primiparous cows with lower milk yield and higher reproductive performance than cows in other profiles. In High-S cows, higher milk yields correlated to higher risk of failure to calf on first insemination. Moreover, 38% of High-S cows kept the same profile at the following lactation, and none changed to Low-M. The other 2 BCS profiles (Low-S and High-M) were intermediate in terms of milk yield and reproductive performance. In Normande, we discriminated 3 BCS profiles based on combining BCS at calving (Low: around 2.6 points, or High: around 3.5 points) with BCS loss after calving (Flat (F): flat with no loss, Moderate (M): around 0.5 points, or Severe (S): around 1.0 point). The Low-M and High-S profiles included cows with similar performance, even though High-S-profile cows showed better but not significantly different milk yield and reproduction performance. The High-F profile included cows that were more likely to experience abnormal ovarian activity and fail at first insemination than cows in other profiles. More than 50% of Normande cows with 2 successive lactations kept in the same BCS profile at the next lactation. Even though a low BCS at calving combined with severe BCS loss (more than 1 point) after calving was found to increase reproductive failure, there was no evidence of an optimal BCS profile for reproduction in dairy cows, and reproductive success or failure is multifactorial.

Effects of dietary chromium supplementation on blood biochemical parameters in dairy cows: A multilevel meta-analytical approach.

Chromium (Cr) has been reported to modulate blood biochemistry in dairy cows. However, there is a discrepancy in the literature regarding the effects of dietary Cr supplementation on various blood parameters. This meta-analysis aimed to evaluate the effects of Cr supplementation in dairy cows on blood glucose, insulin, glucagon, nonesterified fatty acid (NEFA), cortisol, and serum total protein (STP) concentrations. Following relevant literature data extraction, a 3-level meta-analytical random effect model was fitted to the data expressed as standardized mean difference (SMD) of outcome measures of control versus Cr-supplemented cows (i.e., difference in mean between control and treatment group or pooled standard deviation). The SMD can be categorized as having a small effect (0.20), a moderate effect (0.50), and a large effect (0.80). The meta-regression identified the potential sources of heterogeneity, including the body weight of cows, experimental duration/duration of Cr supplementation, blood sampling time (3 wk before parturition until 4 wk after parturition categorized as the transition period, else as the nontransition period), and form of Cr complexes. Blood glucose did not differ significantly between control and Cr-supplemented cows with an estimated SMD of µ = 0.0071 (95% confidence interval [CI]: -0.212 to 0.226). The effect of Cr supplementation on blood insulin was also nonsignificant with an SMD of µ = 0.0007 (95% CI: -0.191 to 0.193). Cows receiving Cr supplements had significantly higher levels of glucagon than controls (95% CI: 0.116 to 0.489), with an estimated SMD = 0.303. Combined transition and nontransition data suggest Cr supplementation did not affect the concentration of NEFA. However, in transition cows, Cr supplementation significantly decreased blood NEFA levels as compared with controls (95% CI: -0.522 to -0.0039), with estimated SMD = -0.263. The estimated SMD was µ = -0.1983 (95% CI: -0.734 to 0.337) for cortisol and -0.0923 (95% CI: -0.316 to 0.131) for total protein. In summary, Cr supplementation in the transition cows decreased NEFA concentration. Blood glucose, insulin, cortisol, and STP concentrations were unaffected. However, Cr supplementation increased glucagon concentration.

Evaluating enteric methane emissions within a herd of genetically divergent grazing dairy cows.

Enteric methane (CH4) emissions of 3 genetic groups (GG) of dairy cows were recorded across the grazing season (early March to late October). The 3 GG were (1) high economic breeding index (EBI) Holstein-Friesian (HF) representative of the top 1% of dairy cows in Ireland at the time of the study (elite), (2) national average (NA) EBI, which were representative of the average HF dairy cow in Ireland, and (3) purebred Jersey (JE) cows. Enteric CH4 was recorded using GreenFeed technology. Seasonal variation in CH4 was observed, with the lowest daily CH4 emissions and CH4 expressed per unit of dry matter intake occurring in spring (253 g/d and 15.56 g/kg, respectively), intermediate in summer (303 g/d and 18.26 g/kg, respectively), and greatest in autumn (324 g/d and 19.80 g/kg, respectively). Seasonal variation was also observed in the proportion of gross energy intake converted to CH4 (Ym); in the spring the Ym was lowest at 0.046, increasing to 0.053 and 0.058 in the summer and autumn, respectively. There was no difference in daily CH4 between the elite and NA, whereas JE had lower CH4 emissions compared with the elite. When expressed per unit of milk solids (fat + protein yield; MS), the elite and JE produced 6.8% and 9.7% less CH4 per kilogram of MS, respectively, compared with NA. There was no difference between the GG for CH4 per unit of DMI or the Ym. This research emphasizes the variation in CH4 emissions across the grazing season and among cows of differing genetic merit for CH4 emission intensities but not for CH4 per unit of DMI or the Ym.

Effect of intramammary infection and inflammation on milk protein profile assessed at the quarter level in Holstein cows.

In this study we wanted to investigate the associations between naturally occurring subclinical intramammary infection (IMI) caused by different etiological agents (i.e., Staphylococcus aureus, Streptococcus agalactiae, Streptococcus uberis, and Prototheca spp.), in combination with somatic cell count (SCC), on the detailed milk protein profile measured at the individual mammary gland quarter. An initial bacteriological screening (time 0; T0) conducted on individual composite milk from 450 Holstein cows reared in 3 herds, was performed to identify cows with subclinical IMI. We identified 78 infected animals which were followed up at the quarter level at 2 different sampling times: T1 and T2, 2 and 6 wk after T0, respectively. A total of 529 quarter samples belonging to the previously selected animals were collected at the 2 sampling points and analyzed with a reversed phase HPLC (RP-HPLC) validated method. Specifically, we identified and quantified 4 caseins (CN), namely αS1-CN, αS2-CN, κ-CN, and ß-CN, and 3 whey protein fractions, namely ß-lactoglobulin, α-lactalbumin, and lactoferrin (LF), which were later expressed both quantitatively (g/L) and qualitatively (as a percentage of the total milk nitrogen content, % N). Data were analyzed with a hierarchical linear mixed model with the following fixed effects: days in milk (DIM), parity, herd, SCC, bacteriological status (BACT), and the SCC × BACT interaction. The random effect of individual cow, nested within herd, DIM and parity was used as the error term for the latter effects. Both IMI (i.e., BACT) and SCC significantly reduced the proportion of ß-CN and αS1-CN, ascribed to the increased activity of both milk endogenous and microbial proteases. Less evident alterations were found for whey proteins, except for LF, which being a glycoprotein with direct and undirect antimicrobial activity, increased both with IMI and SCC, suggesting its involvement in the modulation of both the innate and adaptive immune response. Finally, increasing SCC in the positive samples was associated with a more marked reduction of total caseins at T1, and αS1-CN at T2, suggesting a synergic effect of infection and inflammation, more evident at high SCC. In conclusion, our work helps clarify the behavior of protein fractions at quarter level in animals having subclinical IMI. The inflammation status driven by the increase in SCC, rather the infection, was associated with the most significant changes, suggesting that the activity of endogenous proteolytic enzymes related to the onset of inflammation might have a pivotal role in directing the alteration of the milk protein profile.

Serum metabolome differences associated with subclinical intramammary infection caused by Streptococcus agalactiae and Prototheca spp. in multiparous dairy cows.

Mastitis is one of the most significant diseases in dairy cows and causes several economic losses. Somatic cell count (SCC) is often used as an indirect diagnostic tool for mastitis, especially for subclinical mastitis (SCM) where no symptoms or signs can be detected. Streptococcus agalactiae is one of the main causes of contagious mastitis, and Prototheca spp. is an alga-inducing environmental mastitis that is not always correlated with increased milk SCC. The aim of this study was to evaluate the changes in the metabolomic profile of blood in relation to subclinical intramammary infection (IMI) in dairy cows. In addition, differences resulting from the etiologic agent causing mastitis were also considered. Forty Holstein-Friesian dairy cows in mid and late lactation were enrolled in this cross-sectional design study. Based on the bacteriological examination of milk, the animals were divided into 3 groups: group CTR (control group; n = 16), group A (affected by SCM with IMI caused by Strep. agalactiae; n = 17), and group P (affected by SCM with IMI caused by Prototheca spp.; n = 7). Blood samples from the jugular vein were collected in tubes containing clot activator; the serum aliquot was stored until metabolomic analysis by 1H-nuclear magnetic resonance spectroscopy. Statistical analysis was conducted by fitting a linear model with the group as the fixed effect and SCC as the covariate. Forty-two metabolites were identified, and among them 10 were significantly different among groups. Groups A and P showed greater levels of His and lactose and lower levels of acetate, Asn, and dimethylamine compared with group CTR. Group A showed high levels of Val, and group P showed high levels of Cit and methylguanidine, as well as lower levels of 3-hydroxybutyrate, acetone, allantoin, carnitine, citrate, and ethanol. These metabolites were related to ruminal fermentations, energy metabolism, urea synthesis and metabolism, immune and inflammatory response, and mammary gland permeability. These results suggest systemic involvement with subclinical IMI and that the metabolic profile of animals with SCM undergoes changes related to the etiological agent of mastitis.