Activation of the endocannabinoid system (ECS) elicits negative effects on the reproductive system in mammals. Omega-3 (n-3) fatty acid (FA) supplementation lowers ECS activation and has anti-inflammatory effects. Thus, we hypothesized that supplementing cows with n-3 FA will downregulate components of the ECS and immune system in preovulatory follicles and in the endometrium. Twenty-four multiparous Holstein dairy cows were supplemented from d 256 of pregnancy to d 70 postpartum as follows: (i) control (CTL; n = 12) prepartum with 250 g/d/cow calcium salts of FA and postpartum at 1.6% of the diet (DM basis); (ii) FLX (n = 12) prepartum with 700 g/d/cow of extruded flaxseed supplement rich in α-linolenic acid (ALA, C18:3n-3), and postpartum at 6.4% of diet (DM basis) of the same supplement. Ovaries were monitored at 30 DIM, and following estrous cycle synchronization we aspirated the follicular fluids (FF) of follicles ≥7 mm, separated the granulosa cells (GC), and performed endometrium biopsies at 58 ± 5 DIM. The FF were analyzed for concentrations of estradiol (E2) and progesterone (P4), and E2-active follicles were declared when E2/P4 was >1. The FA and endocannabinoid (eCB) profiles were determined in plasma and in the reproductive tissues. Proteomic analyses and mRNA abundances were determined in GC and endometrium. Supplementation of n-3 FA increased the proportion of total n-3 FA and decreased the omega-6 (n-6) to n-3 FA ratio in plasma, FF and GC compared with CTL. In plasma and FF, n-3 FA supplementation decreased the proportion of the n-6 FA eCB precursor arachidonic acid (AA; C20:4n-6), and increased the abundance of the n-3 FA-derived eCB eicosapentaenoyl ethanolamide compared with CTL. In the endometrium, n-3 FA supplementation reduced the abundance of the n-6 FA-derived eCB 2-arachidonoylglycerol (2-AG) compared with CTL. Proteomic analysis of GC showed that n-3 FA supplementation increased the abundance of FA-binding-protein-5, which is involved in intracellular transport of eCB, as well as the abundances of the cytokine-receptor-like-factor-2 and glutathione-S-transferase-LANCL1, whereas it reduced the abundances of several complement proteins: complement factors I, D, H, complement components C7 chain and C8 β chain, and complement component 1 Q subcomponent-binding protein, mitochondrial (C1QBP). In addition, the abundance of superoxide-dismutase (SOD3) was lower in FLX GC than in CTL. In the endometrium, n-3 FA supplementation decreased the abundance of a few immune-related proteins. In the GC, n-3 FA supplementation reduced the relative mRNA abundances of type 1 and type 2 cannabinoids receptors compared with CTL. Across treatments, a positive correlation was found between the relative abundance in FF of the eCB anandamide with C7, C1QBP and SOD3 in GC; while FF 2-AG had a negative correlation with them. Overall, in line with our premise, dietary n-3 FA supplementation attenuated the levels of some eCB and reduced the expression of several proteins and genes related to the ECS and immune system in the preovulatory follicle and in the endometrium, which may be part of the etiology of the positive effects of n-3 FA on the reproductive system in dairy cows.

We examined the effects of a supplement of plant polyphenols extracts of green tea, capsicum, and fenugreek, and electrolytes ([Na+, K+]; AXT, Axion ThermoPlus, CCPA, France] during summer heat load on production, welfare, and oxidative stress proteins in adipose tissue (AT) of dairy cows. A total of 42 multiparous mid-lactation cows were divided into 3 groups during summer, and were fed for 2 wk either a standard milking cow diet (CTL, n = 14) or diets supplemented with 100 g/d of AXT (100AXT, n = 14), or 150 g/d of AXT (150AXT, n = 14), while being cooled 5 times a day. Then, half of the cows from each dietary treatment were cooled (CL) or not cooled (NCL) for 2 wk, after which the cooled and uncooled groups were switched for additional 2 wk. Cows were milked 3 times a day, and milk composition was analyzed at the end of each 2-wk period. Vaginal temperature (VT) was measured for 3 consecutive days in each period. Biopsies of subcutaneous AT were taken from 10 NCL cows (5 each of CTL and 150AXT) at the end of the period and examined by liquid chromatography-tandem mass spectrometry proteomics analysis. Data were analyzed with PROC MIXED of SAS (version 9.2, SAS Institute Inc.). The model included the effects of dietary treatment, cooling regimen, period, and their interactions. Protein and mRNA abundances and proteomic data (P ≤ 0.05 and fold change [FC] ± 1.5) were analyzed by t-test. Milk yields and 4% fat-corrected milk (FCM) were higher in 100AXT than in CTL; milk components were not different. Dry matter intake (DMI) was higher in 100AXT than in CTL. The effect of cooling and the interactions of period × cooling were significant for DMI, 4% FCM, energy-corrected milk, and milk/DMI. The proportion of time that VT was >39°C was lower in 100AXT and in 150AXT than in CTL. Daily rumination time was greater in 150AXT than in CTL, and lying time was greater in 100AXT and 150AXT than in CTL. Proteomics of AT demonstrated that 150AXT had increased abundances of peroxidasin (FC = 1.6), microsomal glutathione S-transferase 2 (FC = 2.5), and heme oxygenase 1 (FC = 3.6) compared with CTL. Top enriched canonical pathways included acute phase response signaling, Nrf2-mediated oxidative stress response, and lipopolysaccharide (LPS)/IL-1-mediated inhibition of RXR function. Immunoblots of AT showed a higher abundance of the transient receptor potential vanilloid 1 and of LPS binding protein in AT of 150AXT compared with CTL. Supplementation of AXT increased DMI, milk, and 4% FCM, lowered VT, improved welfare indices, and enriched the AT with Nrf2-oxidative stress response and acute phase response proteins in heat-stressed dairy cows.

We investigated the effects of seasonal heat stress and the degree of body weight (BW) loss postpartum (PP) on immune and inflammatory markers in transition dairy cows. Blood sampled twice weekly during the transition period (3 wk prepartum to 3 wk PP) from 12 peripartum Holstein cows in summer and 12 in winter was analyzed for plasma nonesterified fatty acids, the oxidative stress marker malondialdehyde (MDA), haptoglobin, and tumor necrosis factor-α (TNF-α). Complete blood counts and white blood cell (WBC) subpopulations (CD4, CD8, CD25, WC1, and CD335) were examined weekly during the transition period. Adipose tissue biopsies were taken at 7 d PP from summer-calving cows. In cows calving during seasonal heat stress, plasma TNF-α concentrations were 4.9-fold higher prepartum and 5.1-fold higher PP, and MDA concentrations were higher prepartum and PP than in winter. Hematocrit was lower prepartum and tended to be lower PP in summer compared with winter, and after adjusting to hematocrit values fewer WBC prepartum (-17%) and PP (-22%), lower monocytes prepartum (-37%) and PP (-49%), and fewer neutrophils (-25%) PP were found in summer compared with winter-calving cows. The percentages of cytotoxic T cells (CD8) and natural killer cells (CD335) were lower, as well as prepartum gamma delta-T cells (WC1) in summer compared with winter-calving cows. Moreover, lower red blood cell counts prepartum and mean platelet volume and red blood cell distribution width prepartum and PP, as well as higher hemoglobin prepartum and PP, were found in summer than winter-calving cows. In summer, cows that lost more BW PP had higher plasma MDA and TNF-α concentrations, fewer basophil numbers prepartum, a decrease in the percentage of cells expressing the IL-2 receptor (CD25), increased ratio of T helper cells (CD4):CD8, and a tendency for increased WC1 percentages in blood prepartum, as well as a higher percentage of WC1 PP and higher abundance of TNF-α and lower lipopolysaccharide-binding protein in adipose tissue compared with cows that lost less BW PP. In conclusion, seasonal heat stress during the peripartum period was associated with lower WBC counts coupled with increased levels of subacute inflammation, indicating dysregulation of immune function, whereas the degree of BW loss had minor effects. This suggests that heat load is a more potent stressor associated with immune function than the variation in the degree of BW loss PP in high-yielding peripartum dairy cows.

In a previous study, we showed that access to willow fodder decreased somatic cell counts (SCC) in the milk of local Mamber goats grazing in brushland at the end of lactation. To test whether the consumption of willow affects the cells of the immune system, Alpine crossbred dairy goats grazing in the same environment were either offered free access to freshly cut willow fodder (W, n = 24) or not (C, n = 24) for 2 weeks. The willow fodder contained 7.5 g/kg DM of salicin. The other major secondary compounds were catechin, myricitrin, hyperin and chlorogenic acid (2.2, 2.6, 1.0 and 0.75 g/kg DM, respectively). Udder health status was determined before the experiment, and each of the two groups included five (W) or six (C) goats defined as infected, as established by microbial cfu in milk, and 19 (W) or 18 (C) non-infected goats. Goats ingested, on average, 600 g of DM from willow (25% of food intake), resulting in minor changes in dietary quality compared to the controls, as established by faecal near-IR spectrometry. Throughout the 2 weeks of experiment, differences between groups in dietary CP contents were minor and affected neither by infection nor by access to willow; the dietary percentage of neutral detergent fibre (NDF) decreased in C and increased in W; dietary acid detergent fibre (ADF) increased; and the dietary tannin contents decreased for both treatments. However, milking performance and milk quality attributes in both W and C goats were similar. Initial SCC and milk neutrophil (cluster of differentiation (CD)18+ and porcine granulocyte (PG)68) cell counts were higher in infected than in non-infected goats; counts decreased significantly in W but not in C uninfected goats. The percentage of CD8+ T-cells increased in all C goats, while in the W group, a significant increase was found only for infected goats. The consumption of willow mitigated an increase in CD8+ in blood and triggered an increase in CD8+ in milk, suggesting an immune-regulatory effect independent of udder status. To our knowledge, this is the first report of a direct nutraceutical effect of fodder ingestion on the immune status of goats.

Reducing milk production during early lactation might be of interest to improve the energy balance (EB) of high-yielding dairy cows. Therefore, the objective of this study was to determine how reducing the milking frequency (MF) of high-yielding dairy cows from thrice to twice a day during the first 30 d in milk (DIM) affects yields, intake, efficiency, metabolic status, and carryover effects. To this end, 42 multiparous cows were divided into 2 groups according to their previous lactation performance, parity, and body weight. The control cows were milked 3 times a day (3ML) and the treated cows were milked twice a day (2ML) until 30 DIM and then both groups were milked 3 times a day. Milk samples were taken twice a week from 2 or 3 consecutive milkings until 45 DIM for analysis of milk solids, and both groups were followed until 100 DIM to determine the carryover effects of MF until 30 DIM. Individual dry matter intake (DMI), milk yield, and body weight were recorded daily. Blood samples were taken 3 times weekly from 14 d prepartum until 45 DIM. Milk yield during the first 30 DIM was 8.6% higher (49.3 and 45.4 kg/d, respectively), milk fat percentage was lower (3.96 and 4.27%, respectively), and the yields of all milk solids were higher in the 3ML cows than in the 2ML cows. Dry matter intake and 4% fat-corrected milk were similar between groups. The EB during the first 30 DIM was lower in the 3ML cows than in the 2ML cows, and milk yield, but not 4% fat-corrected milk yield, per unit of DMI was higher in the 3ML cows. No differences were observed between groups from 31 to 100 DIM in milk yield (∼56.3 kg/d for both groups), milk solids yield, DMI, or milk/DMI; however, fat percentage was lower and EB was higher in the 3ML cows. Blood glucose concentrations between 0 and 30 DIM were lower and β-hydroxybutyrate concentrations were higher in the 3ML cows than in the 2ML cows, but nonesterified fatty acids concentrations were lower, which may be attributed to the lower clearance frequency of nonesterified fatty acids from the blood stream in the 2ML cows. A lower proportion of the 3ML cows (10%) ovulated ≤15 DIM compared with the 2ML cows (40%), with no beneficial effects on preovulatory follicle characteristics. Reducing the MF from thrice to twice a day during the first 30 DIM improved EB and metabolic status, with only minor effects on production.

This study aimed to investigate sodium salicylate (SS) treatment effects on the proteome of adipose tissue (AT) in postpartum cows. Twenty Holstein cows were assigned to control (CON, n = 10) or SS (n = 10) provided via drinking water (2.3 g/L) during the first 7 d of lactation. Subcutaneous AT was collected on d 7 of treatment and label-free quantitative shotgun proteomics and immunoblotting were analyzed in a subset of 5 AT per group. Eighty out of 1422 proteins (5.6%) were differentially abundant between CON and SS [fold change +/- 1.5, P < 0.05]. Top canonical pathways differing between CON and SS (Ingenuity) were complement system, interleukin-10 signaling, and acute phase response signaling. The abundances of complement Clr, ClqC, ClqB and C6 were greater in SS than CON. Regarding IL-10 signaling, the abundances of BLVRB, STAT3, and lipopolysaccharide binding protein (LBP) were greater in SS AT compared to CON. Immunoblots revealed increased abundance of paraoxanase-1 and tumor necrosis factor-alpha, as well as a tendency for greater abundance of cluster differentiation 172a in SS AT, which may indicate of increased macrophage infiltration. SS treatment postpartum likely promotes inflammatory signaling in AT of dairy cows, perhaps due to immune cell recruitment. Significance: This work demonstrates that treating early lactating cows with sodium salicylate, an anti-inflammatory agent that has been shown to have metabolic effects and increase milk production in dairy cows, affects the proteome of subcutaneous adipose tissue in early lactating dairy cows. Unexpectedly, sodium salicylate treatment enriched inflammatory pathways of the complement system, cytokine signaling, and acute phase response, as revealed by proteomic analysis of subcutaneous adipose tissues from cows at 7 d postpartum. These findings imply that SS treatment during the first 7 d of lactation likely promotes inflammatory signaling in AT of the dairy cow, perhaps due to immune cell recruitment. Tissue-specific impacts of systemic sodium salicylate requires further scrutiny.

Low-grade inflammation has been implicated as a contributor to metabolic disease during the transition to lactation. In previous work, administration of sodium salicylate (SS) for 7 d led to hypoglycemia in mature dairy cows in early lactation. The purpose of this study was to identify the mode of action underlying this response to SS. Twenty mature (parity 3) cows were assigned alternately at time of calving to either control or SS treatments; the control received a molasses placebo in drinking water, whereas SS received 2.3 g/L of SS with the molasses carrier in drinking water for 7 d after parturition. Blood samples were collected daily. A glucose turnover assay was performed on d 7, followed by liver, muscle, and adipose tissue biopsies. There were no treatment effects on intake of dry matter or water. Tumor necrosis factor α mRNA abundance tended to be decreased by SS in adipose tissue but not in muscle or liver, and plasma haptoglobin and adiponectin concentrations were not altered by treatment. Treatment did not significantly alter plasma glucose or insulin concentrations, but plasma glucagon concentration tended to be increased by SS and the insulin:glucagon molar ratio was significantly decreased. Cows on SS had a tendency for a 25% decrease in glucose turnover rate compared with control cows. However, there were no differences in transcript abundance of pyruvate carboxylase (PC) or glucose-6-phosphatase (G6PC) in liver or of glucose transporter 4 (GLUT4) in any of the tissues. Finally, SS did not alter insulin receptor substrate-1 phosphorylation in muscle or adipose, but tended to increase phosphorylation of AMP-activated protein kinase and decrease protein kinase B phosphorylation in adipose tissue. These findings may be explained by enhanced hepatic insulin sensitivity leading to posttranscriptional suppression of gluconeogenesis and adaptive responses to decreased glucose supply in the pancreas and adipose tissue.

We hypothesized that the biochemical properties of milk, specifically the concentrations of lactose, glucose, glucose 6-phosphate, citrate and malate reflect evolutional adaptations of distinct reproductive strategies in women, cows and mice. The concentration of lactose was highest in women's milk, lowest in mouse milk and intermediate in cow milk. Glucose concentration paralleled lactose concentrations. The ratio of glucose 6-phosphate to glucose in mouse milk was twofold greater and significantly higher than that in women's milk. Although the concentration of citrate in mouse milk was lower than in women's and cows' milk, it defied the long-held notion that mouse milk does not contain citrate. Malate concentration was highest in mouse milk. The gross composition of milk and lactation length in the studied species indicated that they represent three reproduction-strategy archetypes.

Environmental heat stress and metabolic stress during transition from late gestation to lactation are main factors limiting production in dairy cattle, and there is a complex interaction between them. Many proteins expressed in adipose tissue are involved in metabolic responses to stress. We aimed to investigate the effects of seasonal heat stress on adipose proteome in late-pregnant cows, and to identify biomarkers of heat stress. Late pregnant cows during summer heat stress (S, n = 18), or during the winter season (W, n = 12) were used. Subcutaneous adipose tissue biopsies sampled 14 days prepartum from S (n = 10) and W (n = 8) were analyzed by intensity based, label-free, quantitative shotgun proteomics (nano-LC-MS/MS). Plasma concentrations of malondialdehyde and cortisol were higher in S than in W cows. Proteomic analysis revealed that 107/1495 proteins were differentially abundant in S compared to W (P < 0.05 and fold change of at least +/- 1.5). Top canonical pathways in S vs. W adipose were Nrf2-mediated oxidative stress response, acute-phase response, and FXR/RXR and LXR/RXR activation. Novel biomarkers of heat stress in adipose tissue were found. These findings indicate that seasonal heat stress has a unique effect on adipose tissue in late-pregnant cows.Significance: This work shows that seasonal heat stress increases plasma concentrations of the oxidative stress marker malondialdehyde and cortisol in transition dairy cows. As many proteins expressed in the adipose tissue are involved in metabolic responses to stress, we investigated the effects of heat stress on the proteome of adipose tissue from late-pregnant cows during summer or winter seasons. We demonstrated that heat stress enriches several stress-related pathways, such as the Nrf2-mediated oxidative stress response and the acute phase response in adipose tissues. Thus, environmental heat stress has a unique effect on adipose tissue in late-pregnant cows, as part of the regulatory adaptations to chronic heat load during the summer season. In addition, this study presents the widest available dataset of adipose tissue proteome in dairy cows, and revealed several novel biomarkers of heat stress in adipose tissue of dairy cows, the use of which awaits further validation. (C) 2017 Elsevier B.V. All rights reserved.

Adipose tissue has a central role in the regulation of metabolism in dairy cows, and many proteins expressed in this tissue are involved in metabolic responses to stress (Peinado et al., 2012) [1]. Environmental heat stress is one of the main stressors limiting production in dairy cattle (Fuquay, 1981; West, 2003) [2], [3], and there is a complex interaction between heat stress and the transition period from late pregnancy to onset of lactation, which is manifested in heat-stressed late-gestation cows (Tao and Dahl, 2013) [4]. We recently defined the proteome of adipose tissue in peripartum dairy cows, identifying 586 proteins of which 18.9% were differentially abundant in insulin-resistant compared to insulin-sensitive adipose tissue (Zachut, 2015) [5]. That study showed that proteomic techniques constitute a valuable tool for identifying novel biomarkers in adipose tissue that are related to metabolic adaptation to stress in dairy cows. The objective of the present work was to examine the adipose tissue proteome under thermo-neutral or seasonal heat stress conditions in late pregnant dairy cows. We have collected subcutaneous adipose tissue biopsies from 10 late pregnant dairy cows during summer heat stress and from 8 late pregnant dairy cows during winter season, and identified and quantified 1495 proteins in the adipose tissues. This dataset of adipose tissue proteome from dairy cows adds novel information on the variety of proteins that are abundant in this tissue during late pregnancy under thermo-neutral as well as heat stress conditions. Differential abundance of 107 (7.1%) proteins was found between summer and winter adipose. These results are discussed in our recent research article (Zachut et al., 2017) [6].

Nuclear factor erythroid 2-related factor 2 (Nrf2) is a critical regulator of key aspects of the antioxidant defense pathway under chronic stress. Oxidative stress and Nrf2 affect adipose tissue (AT) function. Heat stress at late pregnancy affects the physiology and performance in subsequent lactation. We investigated the effects of seasonal heat load on the proteome of adipose tissue in late pregnant dairy cows. Adipose tissue biopsies were obtained from 18 multiparous late pregnant dry cows at 14 d before expected calving during summer (S, n = 10) or winter (W, n = 8). Cows were also divided retrospectively according to BW loss during the first month postpartum to HWL–high weight loss (n = 9), and LWL–low weight loss (n = 9). Blood samples were collected twice a week for oxidative stress marker malondialdehyde (MDA) and cortisol concentrations. Proteins were analyzed by intensity based, label-free quantitative shotgun proteomics at Weizmann Institute of Science (Rehovot, Israel). Proteins were extracted and subjected to in-solution tryptic digestion, followed by nanoflow liquid chromatography coupled to high-resolution tandem mass spectrometry (nanoLC-MS/MS). Quantitative data were extracted using Genedata Expressionist data analysis package and proteins identified using Mascot search engine. Proteomics data, after logarithmic transformation, were analyzed by two-way ANOVA for effects of season (S vs. W), subgroup (HWL vs. LWL), and their interaction. Both pre- and postpartum, S cows had higher plasma MDA and cortisol concentrations compared with W (P < 0.005). Proteomic analysis quantified 1496 proteins in AT, from which the abundance of 132 (8.8%) proteins was differential in S vs. W [Fold change (FC) ± 1.5 and P < 0.05]. One of the top canonical pathways affected by season was Nrf2-mediated oxidative stress response (Ingenuity Pathway Analysis, Qiagen); the abundance of ubiquitin-conjugating enzyme E2 K (P < 0.006) and stress-induced-phosphoprotein 1 (P < 0.001) was elevated, while mitogen-activated protein kinase kinase 1 (P < 0.02), ferritin (P < 0.02), glutathione S-transferase Mu 1 (P < 0.04) and microsomal glutathione S-transferase 1 (P < 0.02) decreased in S vs. W adipose. These findings imply that Nrf2-mediated oxidative stress response plays a main role in the reaction of AT to counterbalance the increased oxidative stress under heat stress conditions in late pregnant dairy cows.

Schematic model to explain the changes in milk G6P/glucose ratio in early lactation.