Cooling management effects on dry matter intake, metabolic hormones levels and welfare parameters in dairy cows during heat stress.

This research paper addresses the hypothesis that intensive cooling management during the summer improves the secretion of metabolic hormones in dairy cows. To test this hypothesis, we characterized the effect of different cooling managements on the different ghrelin isoforms and leptin secretion of 20 Israeli-Holstein dairy cows during 5 weeks during heat stress. The cows were divided into two groups: one was exposed to 5 cooling sessions per day (5 CS) and the other to 8 cooling sessions per day (8 CS). Blood was collected and leptin and ghrelin isoforms level were radioimmunoassayed. Analysis of the interaction between coolings and the week of the experiment showed that the 8 CS group consumed more food and produced more milk, although neither difference was statistically significant. In addition, the 8 CS group exhibited higher blood levels of acyl-ghrelin and leptin as compared to the 5 CS group. Conversely, the blood levels of total ghrelin were lower in the cows exposed to 8 CS as compared to cows from the 5 CS treatment. Furthermore, a significant correlation was found only between total ghrelin levels and the weeks, but not with other parameters examined. We further compared digestibility as well as stress parameters between the groups. We found that the 8 CS group cows ruminated and lay down more hours during a day and simultaneously had better activity time. No significant difference was detected between groups in milk yield and digestibility parameters. Our results suggest that intensive cooling management during the hot season influences the levels of metabolic hormones in the circulation and helps to mitigate the detrimental effect of heat stress on dairy cow welfare and production.

Comparison of the immune responses associated with experimental bovine mastitis caused by different strains of Escherichia coli.

We studied the mammary immune response to different mammary pathogenic Escherichia coli (MPEC) strains in cows, hypothesising that the dynamics of response would differ. E. coli is a major aetiologic agent of acute clinical bovine mastitis of various degrees of severity with specific strains being associated with persistent infections. We compared challenge with three distinct pathogenic MPEC strains (VL2874, VL2732 and P4), isolated from different forms of mastitis (per-acute, persistent and acute, respectively). A secondary objective was to verify the lack of mammary pathogenicity of an environmental isolate (K71) that is used for comparison against MPEC in genomic and phenotypic studies. Twelve cows were challenged by intra-mammary infusion with one of the strains. Cellular and chemokine responses and bacterial culture follow-up were performed for 35 d. All cows challenged by any of the MPEC strains developed clinical mastitis. Differences were found in the intensity and duration of response, in somatic cell count, secreted cytokines (TNF-α, IL-6 and IL-17) and levels of milk leucocyte membrane Toll-like receptor 4 (TLR4). A sharp decrease of TLR4 on leucocytes was observed concomitantly to peak bacterial counts in milk. Intra-mammary infusion of strain K71 did not elicit inflammation and bacteria were not recovered from milk. Results suggest some differences in the mammary immune response to distinct MPEC strains that could be correlated to their previously observed pathogenic traits. This is also the first report of an E. coli strain that is non-pathogenic to the bovine mammary gland.

Alternative Traits for Genetic Evaluation of Mastitis Based on Lifetime Merit.

Genetic selection has achieved little progress in reducing mastitis incidence. Mastitis traits are problematic due to the lack of sensitivity of the data and reliance on clinical diagnosis, often missing subclinical cases, and/or on monthly somatic cell count (SCC) measurements. The current measure for mastitis is the lactation average of the somatic cells score (LSCS). We studied two datasets: (1) 148 heifers divided into non-intramammary infected, sub-clinically infected and clinical mastitis groups; (2) data from 89,601 heifers from Israeli Holsteins through the same period divided into "udder healthy" (UH) and "non-healthy" (UNH) by a threshold of SCC 120,000 cells/mL in all nine monthly milk recordings. In study 1, non-infected heifers had significantly (p < 0.05) more partum, production days and overall lifetime milk production compared to clinical and sub-clinically infected. In study 2, UH heifers (20.3%) had significantly higher (p < 0.01) lifetime milk, production days, and lactations. Subdividing datasets by sires, the same analyses detected differences in percentages of UH daughters between the sire groups. Lifetime milk production correlated (r = +0.83, p < 0.001) with udder health status. SCC threshold of less than 120,000 cells/mL during all first lactation measurements indicated healthy udder, providing a valuable insight that this dichotomous trait is advantageous for calculating lifetime net-merit index (NM$) over LSCS.

Retrospective evaluation of udder recovery of cows with subclinical mastitis following treatment with acoustic pulse technology (APT) on commercial dairy farms and its economic impact.

Retrospective evaluation of udder recovery following treatment of the inflamed quarter with acoustic pulse technology (APT) of cows with subclinical mastitis was done on 4 Israeli commercial dairy farms. Here, we evaluated the APT treatment as a tool to manage subclinical mastitis and its economic consequences in commercial farms. Recovery of the infected glands following APT treatment was compared to the customary no-treatment (NT) for cows with subclinical mastitis. Over 2 years, 467 cows with subclinical mastitis were identified. Subclinical mastitis was defined by elevated somatic cell count (SCC; >1 × 106 cells/mL) in the monthly test-day milk sample; 222 cows were treated with APT and 245 cows were not treated and served as control. Differences between treatment groups in culling, milk quality, milk yield and bacterial elimination were analyzed. After treatment, cure from bacteria was calculated only for cows with pre-isolated bacteria. The percentage of sampled cows determined as cured (no bacterial finding) in the NT group was 32.7% (35/107) (30.9% Gram negative; 32.4% Gram positive) and in the APT-treated group, 83.9% (42/55) (89.4% Gram negative; 80.6% Gram positive). Culling rate due to mastitis was significantly lower (>90%) in the APT-treated vs. NT group. Recovery was 66.0% in the APT group compared to 11.5% in the NT group at 90 d post-treatment. Average milk volume per cow in the APT-treated group was 16.1% higher compared to NT cows. Based on the study, savings incurred by using APT to treat only subclinical cows per 100-cow herd can total $15,106/y, or $309 per treated subclinically infected cow.

Intramammary rapamycin administration to calves induces epithelial stem cell self-renewal and latent cell proliferation and milk protein expression.

Mammary epithelial stem cells differentiate to create the basal and luminal layers of the gland. Inducing the number of differentiating bovine mammary stem cells may provide compensating populations for the milk-producing cells that die during lactation. Inhibition of mTOR activity by rapamycin signals self-renewal of intestinal stem cells, with similar consequences in the mouse mammary gland and in bovine mammary implants maintained in mice. The implementation of these results in farm animals for better mammary development and production was studied in 3-month-old calves. mTOR activity decreased by ~50% in mammary epithelial cells subjected to 3-week rapamycin administration, with no negative consequences on mammary morphology or ß-casein expression. Subsequently, stem cell self-renewal was induced, reflected by a higher propagation rate of cultures from rapamycin-treated glands compared to respective controls and higher expression of selected markers. Followed by 4-day estrogen and progesterone administration, rapamycin significantly induced proliferation rate. Higher numbers of basal and luminal PCNA+ cells were detected in small ducts near the elongating sites as compared to large ducts, in which only luminal cells were affected. Rapamycin administration resulted in induction of individual milk protein genes' expression, which was negatively correlated to their endogenous levels. The inductive effect of rapamycin on luminal cell number was confirmed in organoid cultures, but milk protein expression decreased, probably due to lack of oscillation in rapamycin levels. In conclusion, intramammary rapamycin administration is an effective methodology to reduce mTOR activity in bovine mammary epithelial cells and consequently, induce stem cell self-renewal. The latent positive effect of rapamycin on epithelial cell proliferation and its potential to improve milk protein expression in calves may have beneficial implications for mature cows.

Proteome dataset of peripheral blood mononuclear cells in postpartum dairy cows supplemented with different sources of omega-3 fatty acids.

This article contains raw and processed data related to research published by Kra et al. [1]. There is a scarce knowledge on the proteome of peripheral blood mononuclear cells (PBMC) during the transition period in dairy cows. In human research, proteomics PBMC is used in order to gain insight into inflammatory diseases and syndromes. Dietary fats, and specifically omega-3 (n-3) FA, can moderate the immune fluctuation caused by parturition through improvements of the immune function [2]. Therefore, this study aim was to characterize the changes that may occur in proteome of PBMC during transition, as influenced by different n-3 FA supplementation. Proteomics data of PBMC was obtained from postpartum dairy cows supplemented peripartum with either encapsulated saturated fat (CTL), encapsulated flaxseed oil that is enriched with ALA (α-linolenic acid; FLX) or encapsulated fish oil that is enriched with EPA (eicosapentaenoic acid) and DHA (docosahexaenoic acid; FO).The analysis was done by liquid chromatography-mass spectrometry from PBMCs protein extraction. The cells were collected from six cows per treatment during the 1st week postpartum. Quantification of differential abundance between groups was done using MS1 intensity based label-free. Label-free quantitative shotgun proteomics was used for characterization. This novel dataset of proteomics data from PBMC contains 3807 proteins; 44, 42 and 65 were differently abundant (P ≤ 0.05 and FC ± 1.5), in FLX vs. CTL, FO vs. CTL and FLX vs. FO, respectively; these findings are discussed in our recent research article (Kra et al., 2021). The present dataset of PBMC proteome adds new information regarding the effects of n-3 FA on the immune system, while providing reference for PBMC proteome in postpartum dairy cows.

An evaluation of casein hydrolyzate in combination with antibiotic for bacterial cure and subsequent increase in milk yield in dairy cows.

BACKGROUND: A 3-yr study examined whether prepartum treatment with casein hydrolyzate in combination with antibiotic, as routinely used in Israel for dry cow therapy, improved bacterial cure and increased milk yield in subsequent lactations in comparison with treatment with antibiotic alone. The vast majority of bacterial isolates in samples collected prior to drying-off comprised coagulase-negative staphylococci, mostly as Staph. chromogenes. RESULTS: Bacterial cure associated with the combined treatment was 73.8% in cows, significantly higher than the 51.7% cure recorded when cows were treated only with antibiotic. During the study, the annual milk yield of non-casein hydrolyzate treated and treated control cows increased at ~2% per year, which is consistent with the national annual increase attributed to genetic selection. In cows treated with casein hydrolyzate the increase was 9% (above the 2% expected) in the first lactation after the treatment, and 6.3% (above the 4% expected for 2 years) in the second lactation after treatment. These increases were significantly higher than those in the controls and those expected through genetic improvement. CONCLUSIONS: Treatment with casein hydrolyzate at dry-off was shown to be a viable mean to eliminate existing environmental bacterial infection, and to improve milk yield in the next lactation.

Management of high cows-share-contribution of SCC to the bulk milk tank by acoustic pulse technology (APT).

A cow with mastitis has a high somatic cell count (SCC) in its milk. Cow-share-contribution of somatic cells to the bulk milk tank (BMTSCC) refers to the relative addition made by each cow's milk to the bulk tank's SCC. Since bulk milk is graded and priced according to the BMTSCC, high-yielding cows with mastitis are the main contributors to penalizations in milk price. The benefits of acoustic pulse technology (APT) application to tissues are well documented, including its anti-inflammatory effect and restoration of tissue function by triggering natural healing processes. An APT-based device was developed specifically for treating mastitis in dairy cows. It enables rapid and deep penetration of the acoustic pulses over a large area of the udder in a single session. A study was performed on six farms with a total of 3,900 cows. One unit of cow-share-contribution equaled the addition of 1,000 cells to each mL of the bulk milk volume above the mean BMTSCC. A total of 206 cows were selected: 103 were treated with APT and 103 served as controls. All of the cows contributed over 1.5 units to the BMTSCC at the time of treatment. Seventy-five days after APT treatment, 2 of the 103 treated cows (1.9%) were culled, compared to 19 (18.5%) of the 103 control cows, as well as infected quarter dry-off in 5 others (4.85%). Overall success was defined as a decrease of >75% in cow-share-contribution from treatment time in two of the three monthly milk recordings following treatment. Results indicated 57.3% success for the APT-treated cows vs. 14.6% for the untreated control groups. Highest share-contribution provide an additional tool for the farmer's decision of how to control BMTSCC. Because the cow-share-contribution value is relative to herd size and BMTSCC, this study included a similar number of cows, with similar SCC and milk yield from each of the six herds.

Proteomic analysis of peripheral blood mononuclear cells and inflammatory status in postpartum dairy cows supplemented with different sources of omega-3 fatty acids.

We examined the effects of dietary n-3 fatty acids on the proteome of peripheral blood mononuclear cells (PBMC) in transition dairy cows. Forty-two dry cows were divided into three groups supplemented with: saturated fat (CTL); flaxseed oil (FLX); or fish oil (FO). PBMC were collected from five cows per group at week 1 postpartum for proteomic analysis. The n-3 fatty acid content in plasma and PBMC was higher in FLX and FO than in CTL cows. In PBMC, 3807 proteins were quantified and 44, 42 and 65 were differently abundant in FLX vs. CTL, FO vs. CTL and FLX vs. FO, respectively. In FLX vs. CTL, the abundance of the p65-subunit-of-transcription-factor NF-κB was higher, whereas albumin, C4b-binding protein and complement factor H levels were lower. In FLX vs. FO, complement factors B and H and hemopexin were higher. The top canonical pathway enriched in FLX compared to other groups was acute-phase-response signaling. The percentage of CD25+ blood cells was lower in FLX and FO at 1 week postpartum, and gene expression of NF-κB in white blood cells was lower in FLX than in CTL. Dietary sources of n-3 fatty acids differentially affected the proteome of PBMC, possibly altering the inflammatory status. SIGNIFICANCE: The transition dairy cow experiences a variable degree of systemic subacute inflammation, and proteomics of peripheral blood mononuclear cells (PBMC) may contribute to obtain insight into this process. Omega-3 fatty acids can moderate the immunological effect, and therefore we examined the effects of these fatty acids from flaxseed (FLX) or fish oils (FO) on the proteome of PBMC at week 1 postpartum. More than 3800 proteins were quantified, and in cows supplemented with FLX, enrichment of the acute-phase-signaling and complement systems were apparent in the PBMC compared to CTL and FO PBMC. This information may be useful to further explore the mechanism by which dietary omega-3 fatty acids affect the immune system in postpartum dairy cows.

Basal Levels of CD18 Antigen Presenting Cells in Cow Milk Associate with Copy Number Variation of Fc Gamma Receptors.

Differentiation of cells by flow cytometry provides informative somatic cell counts (SCCs) that allow analyzing leukocyte population patterns in udder infections of different etiologies. Postulating that this approach also enhances the statistical power to detect genetic variants linked to cell levels in milk of healthy mammary glands, we used monoclonal antibodies anti-CD18, anti-CD4, anti--CD14, and anti-PMN to count cells presenting these surface antigens, and performed a genome-wide association study of these counts in 125 Israeli Holsteins genotyped using SNP BeadChips. We identified an informative haplotype of 15 SNPs in the centromeric end of BTA3 that was strongly associated with CD18 cells (p < 2.3 × 10-9). Within this region, examination of the network of genes interacting with ITGB2 (CD18) indicated an Fc-γ-receptor gene cluster, including FCGR2A (CD32). Sanger-sequence analysis of FCGR2s-linked exon 3 variation to CD18 counts. Meta-analysis of RNA-Seq data revealed a significant negative correlation (R = -0.51) between expression of CD32 and CD18 in milk. Assembly of DNA-Seq reads uncovered FCGR copy-number variation and a variant, designated V7, was abundant in dairy cattle, probably reflecting adaptation to selection pressure for low SCC in Holstein milk.

Stochasticity constrained by deterministic effects of diet and age drive rumen microbiome assembly dynamics.

How complex communities assemble through the animal's life, and how predictable the process is remains unexplored. Here, we investigate the forces that drive the assembly of rumen microbiomes throughout a cow's life, with emphasis on the balance between stochastic and deterministic processes. We analyse the development of the rumen microbiome from birth to adulthood using 16S-rRNA amplicon sequencing data and find that the animals shared a group of core successional species that invaded early on and persisted until adulthood. Along with deterministic factors, such as age and diet, early arriving species exerted strong priority effects, whereby dynamics of late successional taxa were strongly dependent on microbiome composition at early life stages. Priority effects also manifest as dramatic changes in microbiome development dynamics between animals delivered by C-section vs. natural birth, with the former undergoing much more rapid species invasion and accelerated microbiome development. Overall, our findings show that together with strong deterministic constrains imposed by diet and age, stochastic colonization in early life has long-lasting impacts on the development of animal microbiomes.

Physiological response of mammary glands to Escherichia coli infection:A conflict between glucose need for milk production and immune response.

The mammary immune and physiological responses to distinct mammary-pathogenic E. coli (MPEC) strains were studied. One gland in each of ten cows were challenged intra-mammary and milk composition (lactose, fat, total protein, casein), biochemical (glucose, glucose-6-phosphate (Glu6P), oxalate, malate, lactate, pyruvate and citrate, malate and lactate dehydrogenases, lactate dehydrogenase (LDH), nitrite, lactic peroxidase, catalase, albumin, lactoferrin, immunoglobulin) and clotting parameters were followed for 35 days post-challenge. Challenge lead to clinical acute mastitis, with peak bacterial counts in milk at 16-24 h post-challenge. Biochemical and clotting parameters in milk reported were partially in accord with lipopolysaccharide-induced mastitis, but increased Glu6P and LDH activity and prolonged lactate dehydrogenase and Glu6P/Glu alterations were found. Some alterations measured in milk resolved within days after challenge, while others endured for above one month, regardless of bacterial clearance, and some reflected physiological responses to mastitis such as the balance between aerobic and anaerobic metabolism (citrate to lactate ratios). The results suggest that E. coli mastitis can be divided into two stages: an acute, clinical phase, as an immediate response to bacterial infection in the mammary gland, and a chronic phase, independent of bacteria clearance, in response to tissue damage caused during the acute phase.

Milk quality and milk transformation parameters from infected mammary glands depends on the infecting bacteria species.

The current study measured the influence of milk of subclinically infected glands by different bacteria species on the cow's milk. The effects of bacterial infection or inflammation on gland milk yield were related to the bacteria species that caused the infection. The volume of milk of the inflamed gland from the cow's milk yield was significantly lower (P<0.001) for the glands previously infected by Escherichia coli (PIEc) and those infected with Streptococcus dysgalactiae. Coagulation properties, rennet clotting time (RCT) and curd firmness (CF) also depended on the bacteria causing the infection. RCT values of all the inflamed glands were significantly longer (P<0.001) and CF values were significantly lower than that of the healthy ones. Moreover, in the whole milk, CF was also significantly lower and not proportional to the volume of the milk from the inflamed gland of the cow's milk. Calculation of the predicted 40% dry matter curd weight (PCW) on the cow level, including the healthy and inflamed glands or the healthy glands alone, showed that for 10 of 13 PIEc cows, the presence of the affected gland's milk in the whole cow milk resulted in a lower PCW value. Likewise, 7 of 20 cows infected by S. dysgalactiae had negative delta values. Unlike the latter bacteria, PCW from milk of glands infected with CNS increased, although in a lower magnitude than in the healthy glands. No correlation was found between logSCC in the whole cow milk (healthy and inflamed glands) and PCW.

Experimental model of toxin-induced subclinical mastitis and its effect on disruption of follicular function in cows.

This study establishes an experimental model for subclinical mastitis induced by Gram-positive (G+) exosecretions of Staphylococcus aureus origin or Gram-negative (G-) endotoxin of Escherichia coli origin to examine its effects on follicular growth and steroid concentrations in Holstein dairy cows. Cows were synchronized with the Ovsynch protocol followed by a series of follicular cycles that included GnRH and PGF2α doses administered every 8 days. Cows received small intramammary doses of either G+ (10 µg, n = 10) or G- (0.5 µg, n = 6) toxin, or saline (n = 6; uninfected control) every 48 hours for 20 days. Follicular fluids were aspirated from preovulatory follicles before (aspiration one: control), at the end of (aspiration two: immediate effect), and 16 days after the end of (aspiration three: carryover effect) toxin exposure. During the 3 weeks of subclinical mastitis induced by G+ or G-, no local inflammatory signs were detected in the mammary gland and no systemic symptoms were noted: body temperatures of the treated cows did not differ from controls; plasma cortisol and haptoglobin concentrations were not elevated and did not differ among groups. Somatic cell count was higher in the treated groups than in controls, and higher in the G- versus G+ group. For analysis of reproductive responses, cows were further classified as nonaffected or affected based on an more than 20% decline in follicular androstenedione concentration in aspiration two or three relative to the first, control aspiration. Most G- (5/6) and 40% of G+ (4/10) cows were defined as affected by induced mastitis. An immediate decrease in the number of medium-size follicles was recorded on Day 4 of the induced cycle, toward the end of the 20-day mastitis induction, in the affected G+ compared with uninfected control group (1.0 ± 0.5 vs. 3.0 ± 0.4 follicles; P < 0.05); the affected G- and nonaffected G+ subgroups exhibited a similar numerical decline in the number of follicles. A carryover (but not immediate) decrease to 51% and 62% in follicular estradiol concentrations in G- affected group and G+ affected group was detected relative to controls (P < 0.05). The nonaffected G+ subgroup did not differ from its control counterparts. Based on the current experimental model, subclinical IMI induced by G+ or G- toxin disrupts follicular functions, and it seems that the ovarian pool of early antral follicles is susceptible to subclinical mastitis.

Real-time evaluation of milk quality as reflected by clotting parameters of individual cow's milk during the milking session, between day-to-day and during lactation.

Real-time analysis of milk coagulation properties as performed by the AfiLab™ milk spectrometer introduces new opportunities for the dairy industry. The study evaluated the performance of the AfiLab™ in a milking parlor of a commercial farm to provide real-time analysis of milk-clotting parameters -Afi-CF for cheese manufacture and determine its repeatability in time for individual cows. The AfiLab™ in a parlor, equipped with two parallel milk lines, enables to divert the milk on-line into two bulk milk tanks (A and B). Three commercial dairy herds of 220 to 320 Israeli Holstein cows producing ∼11 500 l during 305 days were selected for the study. The Afi-CF repeatability during time was found significant (P < 0.001) for cows. The statistic model succeeded in explaining 83.5% of the variance between Afi-CF and cows, and no significant variance was found between the mean weekly repeated recordings. Days in milk and log somatic cell count (SCC) had no significant effect. Fat, protein and lactose significantly affected Afi-CF and the empirical van Slyke equation. Real-time simulations were performed for different cutoff levels of coagulation properties where the milk of high Afi-CF cutoff value was channeled to tank A and the lower into tank B. The simulations showed that milk coagulation properties of an individual cow are not uniform, as most cows contributed milk to both tanks. Proportions of the individual cow's milk in each tank depended on the selected Afi-CF cutoff. The assessment of the major causative factors of a cow producing low-quality milk for cheese production was evaluated for the group that produced the low 10% quality milk. The largest number of cows in those groups at the three farms was found to be cows with post-intramammary infection with Escherichia coli and subclinical infections with streptococci or coagulase-negative staphylococci (∼30%), although the SCC of these cows was not significantly different. Early time in lactation together with high milk yield >50 l/day, and late in lactation together with low milk yield<15 l/day and estrous (0 to 5 days) were also important influencing factors for low-quality milk. However, ∼50% of the tested variables did not explain any of the factors responsible for the cow producing milk in the low - 10% Afi-CF.