Male fetuses negatively affect the vitality of the litter and the dam's metabolic and physiological state in multifetal pregnant ewe.

In sheep, ~30% of fetuses do not survive till parturition, and 17.7% of the multifetal pregnancies experience partial litter loss (PLL). In humans, multifetal pregnancies are associated with a higher risk of perinatal mortality. Therefore, the objectives were to examine the association between partial litter loss, fetal sex, dam's metabolic and physiological state, and pregnancy outcome in multifetal pregnant ewes. The study includes two parts. The first was a retrospective study, in which we analyzed data of 675 lambings and examined the PLL incidence according to male ratio (MR) for all litter sizes (range 2-6). Lambings were categorized as having a low male ratio (LMR; <50% males) or a high male ratio (LMR; >50% males). In the second part, we monitored 24 ewes from 80 to 138 days in pregnancy every 10 days, and then daily until lambing, by ultrasound scanning for maternal heart rate (HR), and Doppler ultrasound for litter vitality. Blood samples were taken from dams on the days of scanning. Male ratio strongly affected PLL, where the general survival rate (for all lambings) was reduced from 90% in LMR lambings to 85% in HMR lambings. The odds ratio for PLL in HMR vs. LMR litters was 1.82. Birth body weight and the survival rate of female was higher in LMR than HMR lambings, with no differences for male lambs in both parameters. In the second part, dams' HR during the last trimester was 9.4% higher in LMR than in HMR pregnancies, with no differences in fetuses' HR. The plasma glucose and insulin concentrations were not significantly different between groups, but plasma ß-hydroxybutyrate and nonesterified fatty acid concentrations were, respectively, 31% and 20% lower in HMR vs. LMR ewes. In conclusion, male fetuses negatively affect pregnancy outcomes and influence dams' metabolic and physiological state in sheep.

Alpha-linolenic acid modulates systemic and adipose tissue-specific insulin sensitivity, inflammation, and the endocannabinoid system in dairy cows.

Metabolic disorders are often linked to alterations in insulin signaling. Omega-3 (n-3) fatty acids modulate immunometabolic responses; thus, we examined the effects of peripartum n-3 on systemic and adipose tissue (AT)-specific insulin sensitivity, immune function, and the endocannabinoid system (ECS) in dairy cows. Cows were supplemented peripartum with saturated fat (CTL) or flaxseed supplement rich in alpha-linolenic acid (ALA). Blood immunometabolic biomarkers were examined, and at 5-8 d postpartum (PP), an intravenous glucose-tolerance-test (GTT) and AT biopsies were performed. Insulin sensitivity in AT was assessed by phosphoproteomics and proteomics. Peripartum n-3 reduced the plasma concentrations of Interleukin-6 (IL-6) and IL-17α, lowered the percentage of white blood cells PP, and reduced inflammatory proteins in AT. Systemic insulin sensitivity was higher in ALA than in CTL. In AT, the top canonical pathways, according to the differential phosphoproteome in ALA, were protein-kinase-A signaling and insulin-receptor signaling; network analysis and immunoblots validated the lower phosphorylation of protein kinase B (Akt), and lower abundance of insulin receptor, together suggesting reduced insulin sensitivity in ALA AT. The n-3 reduced the plasma concentrations of ECS-associated ligands, and lowered the abundances of cannabinoid-1-receptor and monoglycerol-lipase in peripheral blood mononuclear cells PP. Peripartum ALA supplementation in dairy cows improved systemic insulin sensitivity and immune function, reduced ECS components, and had tissue-specific effects on insulin-sensitivity in AT, possibly counter-balancing the systemic responses.

Is pregnancy loss initiated by embryonic death or luteal regression? Profiles of pregnancy-associated glycoproteins during elevated progesterone and pregnancy loss.

Because progesterone (P4) is essential for pregnancy establishment and maintenance, we investigated the effect of increased concentrations of P4 on embryonic attachment and concentrations of pregnancy-associated glycoproteins (PAG). Additionally, we investigated the relationships among luteal regression, pregnancy loss, and PAG concentrations in cows undergoing pregnancy loss by d 33 of pregnancy. Lactating dairy cows were allocated into control (n = 40) and human chorionic gonadotropin (hCG; 3,300 IU on d 7 and 13 to promote greater circulating P4; GnRH = d 0; n = 46) groups. Progesterone was measured daily from d 7 to 33, and PAG was measured daily from d 17 to 33; both hormones were also measured on d 47 and 61. An increase in PAG >10% compared with d 17 was considered a marker for pregnancy. The gold standard for pregnancy diagnosis was ultrasound evaluation of embryonic heartbeat on d 33. Statistical analyses were done with PROC MIXED from SAS Institute Inc. Concentrations of P4 were greater from d 8 onward in the hCG group. Concentrations of PAG did not differ between groups from d 17 to 33, suggesting no effect of increased P4 on hastening embryonic attachment and placental development. Nevertheless, PAG was greater in the hCG group on d 47 and 61, suggesting greater placental area or PAG secretory capacity. Pregnancy loss between d 20 and 33 occurred in 24.6% of cows. About 50% of pregnancy loss was due to luteal regression and about 50% was due to conceptus failure; that is, a decrease in PAG in the absence of luteal regression. In conclusion, increased P4 does not hasten embryonic attachment or early placental development but it leads to increased PAG in the second half of the second month of gestation. Additionally, pregnancy loss seems to be initiated by either corpus luteum regression or conceptus failure.

The form more than the fatty acid profile of fat supplements influences digestibility but not necessarily the production performance of dairy cows.

The form of a lipid supplement, its degree of saturation, and its fatty acid (FA) profile greatly influence digestibility and cow productive response. The objective in this study was to examine the effect of fat supplements that differ in their form or FA profile on nutrient digestibility and cow performance. Forty-two mid-lactation cows (128 ± 53 d) were assigned to 3 treatment groups according to milk yield, days in milk, and body weight. For 13 wk, the cows were fed rations that contained (on a dry matter basis) (1) 2.4% of calcium salts of fatty acids (CSFA) consisting of 45% palmitic acid (PA) and 35% oleic acid (OA; CS45:35); (2) 2.4% of CSFA consisting of 80% PA and 10% OA (CS80:10); or (3) 2.0% of free FA consisting of 80% PA and 10% OA (FF80:10). Rumen samples were taken to measure the ammonia and volatile FA concentrations, and fecal samples were taken to measure the digestibility. Preplanned comparisons were CS45:35 versus CS80:10 to assess 2 CSFA supplements with different FA profiles, and CS80:10 versus FF80:10 to assess similar FA profiles in different forms. Compared with CS45:35, CS80:10 decreased the milk yields, increased the fat percentage, and tended to increase the energy-corrected milk (ECM) yields. The fat percentage of milk was highest in the FF80:10 cows (4.02%), intermediate in the CS80:10 cows (3.89%), and lowest in the CS45:35 cows (3.75%). Compared with CS80:10, FF80:10 increased milk yields (50.1 vs. 49.4 kg/d, respectively), tended to increase fat percentage, and increased 4% fat-corrected milk (4% FCM; 49.1 vs. 47.7 kg/d, respectively) and ECM yields (49.5 vs. 48.2 kg/d, respectively). Treatment had no effect on dry matter intake (DMI), and compared with CS80:10 cows, the calculated energy balance was lower in the FF80:10 cows. The 4% FCM/DMI and ECM/DMI ratios were higher in the FF80:10 group compared with the CS80:10 group. Compared with the CS80:10 cows, the FF80:10 cows had a lower rumen pH, higher propionate, lower acetate/propionate ratio, and higher total VFA. Compared with CS45:35 cows, the apparent total-tract digestibilities of neutral detergent fiber and acid detergent fiber were higher in CS80:10 cows; whereas, the apparent total-tract digestibilities of dry matter, organic matter, protein, neutral detergent fiber, and acid detergent fiber were higher in the CS80:10 cows compared with the FF80:10 cows. Compared with the CS80:10 group, the apparent digestibility of total FA was 13.0 percentage points lower in the FF80:10 cows (79.1 vs. 66.1%, respectively), and similarly, the digestibilities of 16-carbon and 18-carbon FA were lower in the FF80:10 cows than in the CS80:10 cows. In conclusion, the form, more than the FA profile of fat supplements, influenced digestibility. Further, the CSFA supplements were more digestible than the free fatty acids, regardless of the FA profile. However, energy partitioning toward production appeared to be higher in the FF80:10 cows, although the digestibility of nutrients was lower than in the CSFA product with a similar FA profile.

Glucose-6-Phosphate Dehydrogenase Activity in Milk May Serve as a Non-Invasive Metabolic Biomarker of Energy Balance in Postpartum Dairy Cows.

Negative energy balance (EB) postpartum is associated with adverse outcomes in dairy cows; therefore, non-invasive biomarkers to measure EB are of particular interest. We determined whether specific metabolites, oxidative stress indicators, enzyme activity, and fatty acid (FA) profiles in milk can serve as indicators of negative EB. Forty-two multiparous Holstein dairy cows were divided at calving into 2 groups: one was milked 3 times daily and the other, twice a day for the first 30 d in milk (DIM). Cows were classified retrospectively as being in either negative EB (NEB, n = 19; the mean EB during the first 21 DIM were less than the overall median of -2.8 Mcal/d), or in positive EB (PEB, n = 21; the mean EB was ≥-2.8 Mcal/d). The daily milk yield, feed intake, and body weight were recorded individually. Blood samples were analyzed for metabolites and stress biomarkers. Milk samples were taken twice weekly from 5 to 45 DIM to analyze the milk solids, the FA profile, glucose, glucose-6-P (G6P), G6P-dehydrogenase (G6PDH) activity, malic and lactic acids, malondialdehyde (MDA), and oxygen radical antioxidant capacity (ORAC). The NEB cows produced 10.5% more milk, and consumed 7.6% less dry matter than the PEB cows. The plasma glucose concentration was greater and ß-hydroxybutyrate was lower in the PEB vs. the NEB cows. The average concentrations of milk glucose, G6P, malic and lactic acids, and MDA did not differ between groups; however, the G6PDH activity was higher and ORAC tended to be higher in the milk of NEB vs. the PEB cows. The correlation between milk G6PDH activity and EB was significant (r = -0.39). The percentages of oleic acid and total unsaturated FA in milk were higher for the NEB vs. the PEB cows. These findings indicate that G6PDH activity in milk is associated with NEB and that it can serve as a non-invasive candidate biomarker of NEB in postpartum cows, that should be validated in future studies.

Reducing dietary sodium of dairy cows fed a low-roughages diet affect intake and feed efficiency, but not yield.

Wastewater from dairy farms has become a major environmental and economical concern. Sodium residue in treated and untreated wastewater from dairy farms used for irrigation can lead to soil and groundwater salinization, with the risk of soil degradation. We examined the effect of reducing sodium fed to mid to late lactating cows from 0.61% (high sodium [HS]) to 0.45% (low sodium [LS]) of dry matter on dry matter intake (DMI), milk and milk-component yields, eating behavior, apparent total track digestibility, feed efficiency, and sodium excretion into the environment. We randomly assigned 28 multiparous high-yielding ( > 35 kg milk/d) cows to 1 of 2 treatment groups (LS or HS) in a crossover design, with 7 d of adaptation and 28 d of data collection. Reducing sodium in the diet reduced sodium intake from 171 to 123 g/d while lowering sodium excreted in the manure by 22%. Energy corrected milk (ECM) yield (37.4 kg/d) and sodium excretion in the milk (33.7 g/d) were similar for both groups. The DMI of LS cows was lower than that of HS cows (27.3 vs. 28 kg/d) and consequently, feed efficiency of the LS cows was higher (1.40 vs. 1.35 ECM/DMI). Eating rate, meal and visit frequency, and eating time were similar for both treatments; meal and visit duration were longer for the HS cows, and meal and visit sizes tended to be larger. Digestibility of DM and amylase-treated neutral detergent fiber remained similar. Based on the results of this study, and discussed considerations, we recommend lowering the dietary sodium content for mid to late lactating cows in commercial herds to 0.52% of DM, in order to reduce sodium excretion to the environment via urine.

The Effect of Short-Wavelength White LED Illumination throughout the Night on the Milk Fatty Acid Profile of High-Yielding Dairy Cows.

Fatty acid levels in milk vary between day and night milking. Many dairy cows are still kept under white light-emitting diode (W-LED) illumination throughout the night, although it is known to disrupt endogenous circadian rhythms. We investigated the effects of whole-night W-LED illumination (125 lux) on milk yield and circadian composition, compared to a natural light−dark (LD) cycle of 10 h light. Mid−late lactation cows (n = 34) that were exposed to natural LD cycle showed circadian variation in milk fat composition, characterized by higher health-promoting monounsaturated fatty acid (MUFA; 24.2 ± 0.4 vs. 23.2 ± 0.4 g/100 g fat, p < 0.001) and lower saturated fatty acid levels (71.2 ± 0.4 vs. 72.5 ± 0.4, p < 0.001) at 13:30 h (day milk) than at 03:30 h (night milk). Compared to natural LD (n = 16), W-LED (n = 18) did not affect milk production or milk fat yields, yet abolished the milking time variation in milk fat composition towards a less healthy fatty acid profile. This lowered MUFA levels of day milk (23.8 ± 0.4 vs. 26.7 ± 0.4, p < 0.01). Therefore, W-LED has no commercial advantage over the tested natural LD cycle, and conversely, even shows circadian disruption. Accordingly, a natural LD cycle of 10 h light is preferable over W-LED from the perspective of cost savings, the cows' well-being, and preserving the natural milk fat profile, as the nutritional value of the day milk is slightly higher.

Effects of omega-3 supplementation on components of the endocannabinoid system and metabolic and inflammatory responses in adipose and liver of peripartum dairy cows.

BACKGROUND: Dietary supplementation of omega-3 fatty acids can reduce the activation of the endocannabinoid system (ECS) by decreasing the availability of arachidonic acid, thus lowering endocannabinoids (eCBs) levels. The ECS is a modulator of energy metabolism, stress response and inflammation in mammals, yet there is little information on the roles of the ECS in transition dairy cows. During the periparturient period, the adipose tissue and liver are the main metabolic organs that participate in the adaptations of dairy cows to onset of lactation; however, exceeded adipose tissue lipolysis and accumulation of lipids in the liver have adverse effects on cows' physiology. Here we aimed to examine whether omega-3 supplementation during the transition period will modulate ECS activation and affect metabolic and inflammatory indices in postpartum dairy cows, by supplementing twenty-eight transition Holstein dairy cows with either saturated fat (CTL) or encapsulated flaxseed oil (FLX). Components of the ECS, metabolic and inflammatory markers were measured in blood, liver, and subcutaneous adipose tissue. RESULTS: FLX supplementation reduced feed intake by 8.1% (P < 0.01) and reduced plasma levels of arachidonic acid (by 44.2%; P = 0.02) and anandamide (by 49.7%; P = 0.03) postpartum compared to CTL. The mRNA transcription levels of the cannabinoid receptor 1 (CNR1/CB1) tended to be lower (2.5 folds) in white blood cells of FLX than in CTL (P = 0.10), and protein abundance of ECS enzyme monoacylglycerol lipase was higher in peripheral blood mononuclear cells of FLX than in CTL (P = 0.04). In adipose tissue, palmitoylethanolamide levels were lower in FLX than in CTL (by 61.5%; P = 0.02), relative mRNA transcription of lipogenic genes were higher, and the protein abundance of cannabinoid receptor 2 (P = 0.08) and monoacylglycerol lipase (P = 0.10) tended to be higher in FLX compared to CTL. Hepatic 2-arachidonoylglycerol tended to be higher (by 73.1%; P = 0.07), and interlukin-6 mRNA transcription level was 1.5 folds lower in liver of FLX than in CTL (P = 0.03). CONCLUSIONS: Nutritional supplementation of omega-3 fatty acids seems to partly modulate ECS activation, which could be related to lower feed intake. The altered ECS components in blood, adipose tissue and liver are associated with moderate modulations in lipid metabolism in the adipose and inflammation in liver of peripartum dairy cows.

The Effects of High-Fat Diets from Calcium Salts of Palm Oil on Milk Yields, Rumen Environment, and Digestibility of High-Yielding Dairy Cows Fed Low-Forage Diet.

Instability in grain prices led to continuing worldwide growth in the proportion of fat supplements in lactating cows' rations. However, fat supplementation was associated with decreases in feed intake, rumen fermentation, and feed digestibility. The present objectives were to test the effects of high-fat diets from calcium salts of palm oil fatty acids (CS-PFA) in lactating cow rations containing high proportions of concentrate, on feed intake, milk yields, rumen environment, and digestibility. Forty-two multiparous mid-lactation dairy cows were assigned to three treatments, designated as low fat (LF), moderate fat (MF), and high fat (HF) that contained (on DM basis), respectively, (i) 4.7% total fat with 1.7% CS-PFA, (ii) 5.8% total fat with 2.8% CS-PFA, and (iii) 6.8% total fat with 3.9% CS-PFA. Rumen samples were collected for pH, ammonia, and volatile fatty acid (VFA) measurements, and fecal grab samples were collected for digestibility measurements. A numerical trend of decreasing dry matter intake with increasing CS-PFA in diet was observed: 28.7, 28.5, and 28.1 kg/day in LF, MF, and HF, respectively (p < 0.20). No differences between treatments were observed in milk yields and milk-fat percentages, but protein percentage in milk tended to fall with increasing dietary CS-PFA content (p < 0.08), which resulted in 6.4% smaller protein yields in the HF than in the LF group (p < 0.01). Milk urea nitrogen was 15.3% higher in HF than in LF cows (p < 0.05). Rumen pH was higher at all sampling times in the MF and HF than in the LF cows. Concentrations of propionic acid and total VFA were higher in LF than in MF and HF cows. The apparent total-tract digestibility of dry matter was higher with LF than with HF (p < 0.002), and that of organic matter was lowest with the HF diet (p < 0.005). The apparent NDF digestibility declined with increasing dietary fat content, and it was 8.5 percentage points lower in HF than in LF cows (p < 0.009). Apparent fat digestibility increased with increasing dietary fat content, and it was higher by 10.4 percentage points in the HF than in the LF group (p < 0.004). In conclusion, diets with high concentrate-to-forage ratios, containing up to 6.8% total fat and 3.9% CS-PFA, negatively affected rumen fermentation and NDF digestibility in high-yielding dairy cows; however, the effects on yields were minor, indicating that, under specific circumstances, the inclusion of large amounts of CS-PFA in dairy cows' rations with low fiber content is feasible.

Carryover effects of feeding bulls with an omega-3-enriched-diet-From spermatozoa to developed embryos.

The impact of omega-3 nutritional manipulation on semen cryosurvival and quality post thawing is controversial. Our aim was to examine how feeding bulls with omega-3 supplementation from different sources affects the spermatozoa quality parameters. Fifteen Israeli Holstein bulls were fed for 13 weeks with a standard ration top-dressed with encapsulated-fat supplementation: fish or flaxseed oil or saturated fatty acids (control). Ejaculates were collected before, during, and after the feeding trial. Frozen-thawed samples were evaluated by a flow cytometer for spermatozoa viability, mitochondrial membrane potential, the level of reactive oxygen species (ROS), acrosome membrane integrity, DNA fragmentation, phosphatidylserine translocation, and membrane fluidity. Both fish and flaxseed oil treatment resulted in lower ROS levels vs. control groups, during and after the feeding trial. Fewer spermatozoa with damaged acrosomes were observed in the fish oil group after the feeding trial. The spermatozoa membrane fluidity was altered in both the fish and flaxseed oil groups throughout the feeding trial, but only in the flaxseed oil group after the feeding trial. The proportion of spermatozoa with fragmented DNA was lower in the flaxseed oil group after the feeding trial. The spermatozoa fertilization competence did not differ between groups however, blastocyst formation rate was higher in the fish and flaxseed oil groups relative to the control. This was associated with differential gene expression in the blastocysts. Overall, the omega-3-enriched food improved the spermatozoa characteristics; this was further expressed in the developing blastocysts, suggesting a carryover effect from the spermatozoa to the embryos.

Effects of Environmental Heat Load on Endocannabinoid System Components in Adipose Tissue of High Yielding Dairy Cows.

Environmental heat load (HL) adversely affects the performance of dairy cows. The endocannabinoid system (ECS) regulates metabolism and the stress response, thus we hypothesized that HL may affect the ECS of dairy cows. Our objective was to determine the levels of endocannabinoids (eCBs) and gene and protein expressions of the ECS components in adipose tissue (AT) and plasma of early postpartum (PP) and late-lactation cows. In addition, we examined eCBs in milk, and studied the interaction of eCBs with bovine cannabinoids receptors CB1 and CB2. In the first experiment, plasma and AT were sampled from cows calving during summer (S, n = 9) or winter (W, n = 9). Dry matter intake (DMI) and energy balance (EB) were lower in S vs. W, and relative gene expressions of transient-receptor-potential-cation-channel-subfamily-V-member-1 (TRPV1), the cannabinoid receptors CNR1 (CB1) and CNR2 (CB2), and monoglyceride lipase (MGLL) were decreased in AT of S compared to W. Protein abundance of peroxisome proliferator-activated-receptor-alpha (PPAR-α) was decreased, while tumor-necrosis factor-α (TNF-α) was increased in AT of S vs. W. Other components of the ECS were not different between S and W calving cows. To study whether the degree of HL may affect the ECS, we performed a second experiment with 24 late-lactation cows that were either cooled (CL) or not cooled (heat-stressed; HS) during summer. DMI was lower in HS vs. CL, AT protein abundance of PPAR-α was lower, and TRPV1 tended to be lower in HS vs. CL, but other components of the ECS were not different between groups. Milk levels of 2-arachidonoylglycerol (2-AG) tended to increase in HS vs. CL. Additionally, modeling of the bovine cannabinoid receptors demonstrated their binding to anandamide and 2-AG. Environmental HL, possibly via lower intake, is associated with limited alterations in ECS components in AT of dairy 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.

Proteome dataset of liver from dairy cows experiencing negative or positive energy balance at early lactation.

This article contains raw and processed data related to research published by Swartz et al. [1]. We present proteomics data from liver of postpartum dairy cows that were obtained by liquid chromatography-mass spectrometry following protein extraction. Differential abundance between liver of cows experiencing either negative energy balance (NEB, n = 6) or positive energy balance (PEB, n = 4) at 17 ± 3 days in lactation was quantified using MS1 intensity based label-free. There is a paucity of studies examining the associations of NEB with the liver proteome in early lactation dairy cows. Therefore, our objective was to examine the differences in the liver proteome in periparturient dairy cows experiencing naturally occurring NEB compared to cows in PEB. In this study, multiparous Holstein dairy cows were milked either 2 or 3 times daily for the first 30 days in milk (DIM) to alter energy balance, and were classified retrospectively as NEB (n = 18) or PEB (n = 22). We collected liver biopsies from 10 cows (n = 5 from each milking frequency), that were retrospectively classified according to their energy balance (NEB, n = 6; PEB, n = 4). The liver proteome was characterized using label-free quantitative shotgun proteomics. This novel dataset contains 2,741 proteins were identified, and 68 of those were differentially abundant between NEB and PEB (P ≤ 0.05 and FC± 1.5); these findings are discussed in our recent research article [1]. The present dataset of liver proteome can be used as either biological markers for disease or therapeutic targets to improve metabolic adaptations to lactation in postpartum dairy cattle. Data are available via ProteomeXchange with identifier PXD028124.

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.

Characterization of the liver proteome in dairy cows experiencing negative energy balance at early lactation.

Negative energy balance (NEB) is associated with metabolic disorders in early lactation dairy cows. Therefore, our objective was to characterize the liver proteome in cows experiencing either NEB or positive energy balance (PEB). Forty-two multiparous Holstein dairy cows were milked either 2 or 3 times daily for the first 30 days in milk (DIM) to alter EB, and were classified retrospectively as NEB (n = 18) or PEB (n = 22). Liver biopsies were collected from 10 cows (n = 5 from each milking frequency) at 17 ± 3 DIM (NEB, n = 6; PEB, n = 4). The liver proteome was characterized using label-free quantitative shotgun proteomics and Ingenuity Pathway Analysis used to identify key affected canonical pathways. Overall, 2741 proteins were identified, and 68 of those were differentially abundant (P ≤ 0.05 and FC ± 1.5). ENO3 (FC = 10.3, P < 0.01) and FABP5 (FC = -12.5, P = 0.045) were the most dramatically upregulated and downregulated proteins, respectively, in NEB cows. Numerous mitochondrial proteins (NDUFA5, NDUFS3, NDUFA6, COX7A2L, COX6C, and COA5) were differentially abundant. Canonical pathways associated with NEB were LPS/IL-1 mediated inhibition of RXR function, oxidative phosphorylation, and mitochondrial dysfunction. Additionally, cows experiencing NEB had less hepatic IL10 transcript abundance than PEB. Together, NEB was associated with altered hepatic inflammatory status, likely due to oxidative stress from mitochondrial dysfunction. SIGNIFICANCE: Our manuscript describes the associations of negative energy balance with the liver proteome in early lactation dairy cows, when metabolic stress and the incidence of diseases is increased. Specifically, we found associations of negative energy balance with shifts in hepatic protein abundance involved in fatty acid uptake, impaired anti-inflammatory responses, and mitochondrial dysfunction. Moving forward, differentially abundant proteins found in this study may be useful as either biological markers for disease or therapeutic targets to improve metabolic adaptations to lactation in postpartum dairy cattle.

High-dose vitamin B1 therapy prevents the development of experimental fatty liver driven by overnutrition.

Fatty liver is an abnormal metabolic condition of excess intrahepatic fat. This condition, referred to as hepatic steatosis, is tightly associated with chronic liver disease and systemic metabolic morbidity. The most prevalent form in humans, i.e. non-alcoholic fatty liver, generally develops due to overnutrition and sedentary lifestyle, and has as yet no approved drug therapy. Previously, we have developed a relevant large-animal model in which overnourished sheep raised on a high-calorie carbohydrate-rich diet develop hyperglycemia, hyperinsulinemia, insulin resistance, and hepatic steatosis. Here, we tested the hypothesis that treatment with thiamine (vitamin B1) can counter the development of hepatic steatosis driven by overnutrition. Remarkably, the thiamine-treated animals presented with completely normal levels of intrahepatic fat, despite consuming the same amount of liver-fattening diet. Thiamine treatment also decreased hyperglycemia and increased the glycogen content of the liver, but it did not improve insulin sensitivity, suggesting that steatosis can be addressed independently of targeting insulin resistance. Thiamine increased the catalytic capacity for hepatic oxidation of carbohydrates and fatty acids. However, at gene-expression levels, more-pronounced effects were observed on lipid-droplet formation and lipidation of very-low-density lipoprotein, suggesting that thiamine affects lipid metabolism not only through its known classic coenzyme roles. This discovery of the potent anti-steatotic effect of thiamine may prove clinically useful in managing fatty liver-related disorders.This article has an associated First Person interview with the joint first authors of the paper.

Hyperglycemia-stimulating diet induces liver steatosis in sheep.

Hepatic steatosis is strongly associated with chronic liver disease and systemic metabolic disorder. Adipose lipolysis is a recognized principal source of intrahepatic fat in various metabolic disorders, including non-alcoholic fatty liver disease. We hypothesized that, in the premorbid state, hepatic de novo lipogenesis (DNL) driven by excess carbohydrates abundance might play a more significant role. We employed a novel nutritional model in sheep of two distinct carbohydrates abundances. During 4 months of the dietary treatment, lambs were monitored for metabolic and terminal liver parameters. Lambs grown on the high-calorie (HC) diet were consistently more hyperglycemic and hyperinsulinemic than lambs grown on the lower-calorie (LC) diet (P < 0.0001). As a result, the HC lambs developed systemic- (HOMA-IR of 7.3 vs. 3.1; P < 0.0001), and adipose- (ADIPO-IR of 342.7 vs. 74.4; P < 0.0001) insulin resistance, significant adiposity (P < 0.0001), and higher plasma triglycerides (P < 0.05). Circulating leukocytes in the HC lambs had higher mRNA expression levels of the proinflammatory markers CCL2 (P < 0.01) and TNF-alpha (P < 0.04), and IL1B trended higher (P < 0.1). Remarkably, lambs on the HC diet developed substantial liver steatosis (mean fat content of 8.1 vs. 5.3% in the LC group; P < 0.0001) with a higher histological steatosis score (2.1 vs. 0.4; P < 0.0002). Hepatic steatosis was most-strongly associated with blood glucose and insulin levels but negatively correlated with circulating fatty acids-indicating a more significant contribution from hepatic DNL than from adipose lipolysis. Sheep may prove an attractive large-animal model of fatty liver and metabolic comorbidities resulting from excess carbohydrate-based energy early in life.

Improved Folch Method for Liver-Fat Quantification.

Fatty liver represents a significant metabolic pathology of excess intrahepatic fat in domestic animals and humans. Quantification of hepatic-fat content is therefore essential for diagnosis and investigation of liver and metabolic disease. However, the reproducibility of hepatic steatosis analysis is often low due to subjective and technical factors. We hypothesized that improvement in tissue-lipids extraction efficiency would contribute to the accuracy and precision of liver-fat determination. To test it, we investigated the effect of standardized tissue sonication on liver-fat quantification by the Folch method in sheep. Liver samples from grownup lambs of lean (n = 16) and fatty (n = 15) livers, and from pregnant ewes (n = 6) who died from pregnancy toxemia (PT), were used for hepatic-fat content determination with or without tissue sonication. In the grown lambs, an average hepatic-fat content of 6.6% was determined in sonicated compared to 5.1% in non-sonicated specimens (P = 0.0002). Similarly, in ewes with PT, an average of 12.5% was determined with sonication compared to 10.8% without it (P = 0.0006), and the reproducibility was higher with sonication (CV of 3.1 vs. 6.1%, respectively). Thus, tissue sonication improved the efficiency of liver-lipids extraction and was significant to the accuracy and precision of hepatic-fat determination. Enzymatic quantification of triglycerides was moderately correlated with the results obtained gravimetrically (r = 0.632, P < 0.005). The reported data provide reliable reference values for pregnancy toxemic sheep. The significant improvement in liver-fat quantification observed with the reported revised protocol is likely applicable to most mammals and humans.