Potential for low-forage diets to maintain milk production in forage-limited situations.

We determined the effect of feeding diets similar in neutral detergent fiber (NDF), starch, and crude protein (CP) with different amounts of forage on the yields of milk and milk components of mid-lactation dairy cows. Thirty-two Holstein cows (132 ± 68 d in milk) were used in a crossover design with 2 consecutive 28-d periods, with sample and data collection during the final 5 d of each period. Treatment diets were (1) control diet (CON) containing high forage (55.5% diet dry matter [DM]; forage NDF 19.2% diet DM) and no supplemental fatty acids or AA; and (2) low-forage (LF) diet containing low forage (36.6% diet DM; forage NDF 12.7% diet DM), including supplemental fat (1.43% diet DM; 82% C16:0-enriched supplement) and rumen-protected methionine and lysine. Diets were balanced for similar NDF (∼30.2% diet DM), starch (∼26.7% diet DM), and CP (∼16.2% diet DM). There was no effect of treatment on milk yield, milk fat content, or body weight. Compared with CON, LF increased DM intake (30.8 vs. 31.8 kg/d), milk fat yield (1.78 vs. 1.84 kg/d), milk protein yield (1.47 vs. 1.56 kg/d), milk protein content (3.24% vs. 3.41%), energy-corrected milk (48.3 vs. 50.2 kg/d), and body condition score (3.2 vs. 3.3). Our results demonstrate that feeding a low-forage diet supplemented with a C16:0-enriched fatty acid supplement and AA increased DM intake and the yields of milk fat and protein, without changes in body weight. The effect of a low-forage diet without supplemental fatty acids and AA was not tested.

Oleic acid abomasal infusion limits lipolysis and improves insulin sensitivity in adipose tissue from periparturient dairy cows.

Excessive adipose tissue (AT) lipolysis around parturition in dairy cows is associated with impaired AT insulin sensitivity and increased incidence of metabolic diseases. Supplementing cows with oleic acid (OA) reduces circulating biomarkers of lipolysis and improves energy balance. Nevertheless, it is unclear if OA alters lipid trafficking in AT. In the liver and skeletal muscle, OA improves mitochondrial function and promotes lipid droplet formation by activating perilipin 5 (PLIN5) and peroxisome proliferator-activated receptor α (PPARα). However, it is unknown if this mechanism occurs in AT. The objective of this study was to determine the effect of OA on AT lipolysis, systemic and AT insulin sensitivity, and AT mitochondrial function in periparturient dairy cows. Twelve rumen-cannulated Holstein cows were infused abomasally following parturition with ethanol (CON) or OA (60 g/d) for 14 d. Subcutaneous AT samples were collected at 11 ± 3.6 d before calving (-12 d), and 6 ± 1.0 d (7 d) and 13 ± 1.4 d (14 d) after parturition. An intravenous glucose tolerance test was performed on d 14. Adipocyte morphometry was performed on hematoxylin and eosin-stained AT sections. The antilipolytic effect of insulin (1 µg/L) was evaluated using an ex vivo explant culture following lipolysis stimulation. PLIN5 and PPARα transcription and translation were determined by real-time quantitative PCR and capillary electrophoresis, respectively. RNA sequencing was used to evaluate the transcriptomic profile of mitochondrial gene networks. In CON cows, postpartum lipolysis increased the percentage of smaller (<3,000 µm2) adipocytes at 14 d compared with -12 d. However, OA limited adipocyte size reduction at 14 d. Likewise, OA decreased lipolysis plasma markers nonesterified free fatty acids and ß-hydroxybutyrate at 5 and 7 d. Over the 14-d period, compared with CON, OA increased the concentration of plasma insulin and decreased plasma glucose. During the glucose tolerance test, OA decreased circulating glucose concentration (at 10, 20, 30, 40 min) and the glucose clearance rate. Moreover, OA increased insulin at 10 and 20 min and tended to increase it at 30 min. Following lipolysis stimulation, OA improved the antilipolytic effect of insulin in the AT at 14 d. PLIN5 and PPARA gene expression decreased postpartum regardless of treatment. However, OA increased PLIN5 protein expression at 14 d and increased PPARA at 7 and 14 d. Immunohistochemical analysis of AT and RNA sequencing data showed that OA increased the number of mitochondria and improved mitochondrial function. However, OA had no effect on production and digestibility. Our results demonstrate that OA limits AT lipolysis, improves systemic and AT insulin sensitivity, and is associated with markers of mitochondrial function supporting a shift to lipogenesis in AT of periparturient dairy cows.

Cannabinoid-1 receptor activation modulates lipid mobilization and adipogenesis in the adipose tissue of dairy cows.

Amplified adipose tissue (AT) lipolysis and suppressed lipogenesis characterize the periparturient period of dairy cows. The intensity of lipolysis recedes with the progression of lactation; however, when lipolysis is excessive and prolonged, disease risk is exacerbated and productivity compromised. Interventions that minimize lipolysis while maintaining adequate supply of energy and enhancing lipogenesis may improve periparturient cows' health and lactation performance. Cannabinoid-1 receptor (CB1R) activation in rodent AT enhances the lipogenic and adipogenic capacity of adipocytes, yet the effects in dairy cow AT remain unknown. Using a synthetic CB1R agonist and an antagonist, we determined the effects of CB1R stimulation on lipolysis, lipogenesis, and adipogenesis in the AT of dairy cows. Adipose tissue explants were collected from healthy, nonlactating and nongestating (NLNG; n = 6) or periparturient (n = 12) cows at 1 wk before parturition and at 2 and 3 wk postpartum (PP1 and PP2, respectively). Explants were treated with the ß-adrenergic agonist isoproterenol (1 µM) in the presence of the CB1R agonist arachidonyl-2'-chloroethylamide (ACEA) ± the CB1R antagonist rimonabant (RIM). Lipolysis was quantified based on glycerol release. We found that ACEA reduced lipolysis in NLNG cows; however, it did not exhibit a direct effect on AT lipolysis in periparturient cows. Inhibition of CB1R with RIM in postpartum cow AT did not alter lipolysis. To evaluate adipogenesis and lipogenesis, preadipocytes isolated from NLNG cows' AT were induced to differentiate in the presence or absence of ACEA ± RIM for 4 and 12 d. Live cell imaging, lipid accumulation, and expressions of key adipogenic and lipogenic markers were assessed. Preadipocytes treated with ACEA had higher adipogenesis, whereas ACEA+RIM reduced it. Adipocytes treated with ACEA and RIM for 12 d exhibited enhanced lipogenesis compared with untreated cells (control). Lipid content was reduced in ACEA+RIM but not with RIM alone. Collectively, our results support that lipolysis may be reduced by CB1R stimulation in NLNG cows but not in periparturient cows. In addition, our findings demonstrate that adipogenesis and lipogenesis are enhanced by activation of CB1R in the AT of NLNG dairy cows. In summary, we provide initial evidence which supports that the sensitivity of the AT endocannabinoid system to endocannabinoids, and its ability to modulate AT lipolysis, adipogenesis, and lipogenesis, vary based on dairy cows' lactation stage.

Abomasal infusion of different exogenous emulsifiers alters fatty acid digestibility and milk fat yield of lactating dairy cows.

We evaluated the effects of abomasal infusion of emulsifiers on fatty acid (FA) digestibility and milk production of lactating dairy cows. All emulsifiers examined were polysorbates, nonionic surfactants, consisting of a polyethoxylated sorbitan esterified with FA. The polysorbates tested in this study consisted of the same polyethoxylated sorbitan base but differed by the FA esterified to it. Eight rumen-cannulated multiparous cows (89 ± 13 d in milk) were assigned to a treatment sequence in 4 × 4 Latin squares with 18-d periods consisting of 7 d of washout and 11 d of infusion. Treatments were abomasal infusions of water only (CON) or 30 g/d of different emulsifiers as follows: polysorbate-C16:0 (T40), polysorbate-C18:0+C16:0 (T60), and polysorbate-C18:1 (T80). Emulsifiers were dissolved in water and delivered at 6-h intervals (total daily infusion was divided into 4 equal infusions per day). Cows were fed the same diet that contained (% diet dry matter) 32.1% neutral detergent fiber, 15.7% crude protein, 25.8% starch, and 3.32% FA (including 1.92% FA from a saturated FA supplement containing 34.2% C16:0 and 47.7% C18:0). The T80 treatment increased total FA digestibility compared with CON (5.40 percentage units) and T60 (3.90 percentage units) and tended to increase it compared with T40. Also, T40 tended to increase and T80 increased (4.80 percentage units) 16-carbon FA digestibility compared with CON. The T80 treatment increased 18-carbon FA digestibility compared with the other treatments. The T40 treatment tended to increase and T80 increased total FA absorption compared with CON (53 g/d) and T60 (52 g/d). Both T40 and T80 increased the absorption of 16-carbon FA compared with CON and T60. The T60 treatment did not differ from CON for any digestibility variable. Both T40 and T80 increased the yields of milk fat, 3.5% fat-corrected milk, and de novo, mixed, and preformed milk FA compared with CON. In conclusion, not all emulsifiers increased FA digestibility. Compared with CON, T80 increased the digestibility and absorption of total, 16-, and 18-carbon FA. The T40 treatment tended to increase and T80 increased total FA absorption and the yields of milk fat and 3.5% FCM compared with CON. Milk fat yield was increased by increases in de novo, mixed, and preformed milk FA. In our short-term infusion study, results suggest that the predominant FA present in the polysorbate affects its ability to improve FA digestibility. Overall, FA digestibility and absorption were improved the most when cows received the T80 treatment.

Lipopolysaccharide induces lipolysis and insulin resistance in adipose tissue from dairy cows.

Intense and protracted adipose tissue (AT) fat mobilization increases the risk of metabolic and inflammatory periparturient diseases in dairy cows. This vulnerability increases when cows have endotoxemia-common during periparturient diseases such as mastitis, metritis, and pneumonia-but the mechanisms are unknown. Fat mobilization intensity is determined by the balance between lipolysis and lipogenesis. Around parturition, the rate of lipolysis surpasses that of lipogenesis, leading to enhanced free fatty acid release into the circulation. We hypothesized that exposure to endotoxin (ET) increases AT lipolysis by activation of classic and inflammatory lipolytic pathways and reduction of insulin sensitivity. In experiment 1, subcutaneous AT (SCAT) explants were collected from periparturient (n = 12) Holstein cows at 11 ± 3.6 d (mean ± SE) before calving, and 6 ± 1 d and 13 ± 1.4 d after parturition. Explants were treated with the endotoxin lipopolysaccharide (LPS; 20 µg/mL; basal = 0 µg/mL) for 3 h. The effect of LPS on lipolysis was assessed in the presence of the ß-adrenergic agonist and promoter of lipolysis isoproterenol (ISO; 1 µM; LPS+ISO). In experiment 2, SCAT explants were harvested from 24 nonlactating, nongestating multiparous Holstein dairy cows and exposed to the same treatments as in experiment 1 for 3 and 7 h. The effect of LPS on the antilipolytic responses induced by insulin (INS = 1 µL/L, LPS+INS) was established during ISO stimulation [ISO+INS, LPS+ISO+INS]. The characterization of lipolysis included the quantification of glycerol release and the assessment of markers of lipase activity [adipose triglyceride lipase (ATGL), hormone-sensitive lipase (HSL), and phosphorylated HSL Ser563 (pHSL)], and insulin pathway activation (AKT, pAKT) using capillary electrophoresis. Inflammatory gene networks were evaluated by real-time quantitative PCR. In periparturient cows, LPS increased AT lipolysis by 67 ± 12% at 3 h across all time points compared with basal. In nonlactating cows, LPS was an effective lipolytic agent at 3 h and 7 h, increasing glycerol release by 115 ± 18% and 68.7 ± 16%, respectively, relative to basal. In experiment 2, LPS enhanced ATGL activity with minimal HSL activation at 3 h. In contrast, at 7 h, LPS increased HSL phosphorylation (i.e., HSL activity) by 123 ± 11%. The LPS-induced HSL lipolytic activity at 7 h coincided with the activation of the MEK/ERK inflammatory pathway. In experiment 2, INS reduced the lipolytic effect of ISO (ISO+INS: -63 ± 18%) and LPS (LPS+INS: -45.2 ± 18%) at 3 h. However, the antilipolytic effect of INS was lost in the presence of LPS at 7 h (LPS+INS: -16.3 ± 16%) and LPS+ISO+INS at 3 and 7 h (-3.84 ± 23.6% and -21.2 ± 14.6%). Accordingly, LPS reduced pAKT:AKT (0.11 ± 0.07) compared with basal (0.18 ± 0.05) at 7 h. Our results indicated that exposure to LPS activated the classic and inflammatory lipolytic pathways and reduced insulin sensitivity in SCAT. These data provide evidence that during endotoxemia, dairy cows may be more susceptible to lipolysis dysregulation and loss of adipocyte sensitivity to the antilipolytic action of insulin.

In Utero Exposure to trans-10, cis-12 Conjugated Linoleic Acid Modifies Postnatal Development of the Mammary Gland and its Hormone Responsiveness.

We previously showed that dietary trans-10, cis-12 conjugated linoleic acid (10,12 CLA) stimulates estrogen-independent mammary growth in young ovariectomized mice. Here we investigated the effects of in utero or postnatal exposure to cis-9, trans-11 (9,11 CLA) and 10,12 CLA on postnatal development of the mammary gland and its responsiveness to ovarian steroids. In the first experiment we fed dams different CLA prior to and during gestation, then cross fostered female pups onto control fed dams prior to assessing the histomorphology of their mammary glands. Pregnant dams in the second experiment were similarly exposed to CLA, after which their female pups were ovariectomized then treated with 17ß-estradiol (E), progesterone (P) or E + P for 5 days. In a third experiment, mature female mice were fed different CLA for 28 days prior to ovariectomy, then treated with E, P or E + P. Our data indicate that 10,12 CLA modifies the responsiveness of the mammary glands to E or E + P when exposure occurs either in utero, or postnatally. These findings underline the sensitivity of the mammary glands to dietary fatty acids and reinforce the potential for maternal nutrition to impact postnatal development of the mammary glands and their risk for developing cancer.

Replacing stearic acid with oleic acid in supplemental fat blends improves fatty acid digestibility of lactating dairy cows.

The objective of our study was to determine the effects of altering the ratio of stearic (C18:0; SA) and oleic (cis-9 C18:1; OA) acids in supplemental fatty acid (FA) blends on FA digestibility and milk yield of dairy cows. Eight multiparous Holstein cows (mean ± SD; 157 ± 11.8 d in milk) were randomly assigned to treatment sequence in a replicated 4 × 4 Latin square design with 14-d periods. Digestibility and production data were collected during the last 4 d of each period. The treatments were an unsupplemented control diet (CON), and 3 diets incorporating FA supplement blends at 1.4% of diet dry matter (DM) containing (as a % of total FA) 50% SA and 10% OA, 40% SA and 20% OA, or 30% SA and 30% OA. The FA blends were balanced to contain 33% palmitic, 5% linoleic, and <0.5% linolenic acids. The FA supplements replaced soyhulls in the CON diet. Preplanned contrasts were as follows: (1) overall effect of FA treatments [CON vs. the average of the FA-supplemented diets; (50:10 + 40:20 + 30:30)/3], (2) the linear effect of OA inclusion in the supplemental FA blend, and (3) the quadratic effect of OA inclusion in the supplemental FA blend. There was no effect of treatment on DM intake, but the replacement of soyhulls in the FA treatments decreased neutral detergent fiber intake. Overall, compared with CON, FA treatments increased DM and neutral detergent fiber digestibility, and increasing OA within FA treatments quadratically increased digestibility of DM and neutral detergent fiber. Overall, FA treatments increased the intake of total, 16-carbon, and 18-carbon FA, decreased the digestibility of total and 18-carbon FA, but increased absorption of total, 16-carbon, and 18-carbon FA. Within FA treatments, increasing OA linearly increased the digestibility of total, 16-carbon, and 18-carbon FA, as well as the absorption of total, 16-carbon, and 18-carbon FA. Overall, FA treatments increased the yields of milk, energy-corrected milk, and milk fat, and tended to increase milk protein yield. Compared with CON, FA treatments had no effect on the yield of de novo milk FA and increased the yields of mixed and preformed milk FA. Within FA treatments, increasing OA did not affect the yields of milk or milk components, linearly decreased the yield of de novo FA, and quadratically affected the yield of mixed and preformed milk FA. Overall, FA treatments increased plasma nonesterified fatty acids but did not affect ß-hydroxybutyrate or insulin. Within FA treatments, increasing OA quadratically affected plasma nonesterified fatty acids, and tended to linearly increase ß-hydroxybutyrate and quadratically affect insulin. In conclusion, supplemental FA blends containing different ratios of SA and OA did not affect DM intake but increased the yields of milk and milk components. Supplemental FA blends also increased digestibility of DM and neutral detergent fiber and decreased digestibility of total and 18-carbon FA compared with CON. Although increasing OA within FA supplements did not alter milk production, increasing OA within FA supplements increased total, 16-carbon, and 18-carbon FA digestibility and FA absorption. Further research is required to determine longer term effects of SA and OA on nutrient digestion and partitioning and opportunities for maintaining or improving FA digestibility with increasing SA intake and availability in the small intestine.

Omega-3 Polyunsaturated Fatty Acid Intervention Against Established Autoimmunity in a Murine Model of Toxicant-Triggered Lupus.

Workplace exposure to respirable crystalline silica dust (cSiO2) has been etiologically linked to the development of lupus and other human autoimmune diseases. Lupus triggering can be recapitulated in female NZBWF1 mice by four weekly intranasal instillations with 1 mg cSiO2. This elicits inflammatory/autoimmune gene expression and ectopic lymphoid structure (ELS) development in the lung within 1 week, ultimately driving early onset of systemic autoimmunity and glomerulonephritis. Intriguingly, dietary supplementation with docosahexaenoic acid (DHA), an ω-3 polyunsaturated fatty acid (PUFA) found in fish oil, beginning 2 week prior to cSiO2 challenge, prevented inflammation and autoimmune flaring in this novel model. However, it is not yet known how ω-3 PUFA intervention influences established autoimmunity in this murine model of toxicant-triggered lupus. Here we tested the hypothesis that DHA intervention after cSiO2-initiated intrapulmonary autoimmunity will suppress lupus progression in the NZBWF1 mouse. Six-week old NZWBF1 female mice were fed purified isocaloric diet for 2 weeks and then intranasally instilled with 1 mg cSiO2 or saline vehicle weekly for 4 consecutive weeks. One week after the final instillation, which marks onset of ELS formation, mice were fed diets supplemented with 0, 4, or 10 g/kg DHA. One cohort of mice (n = 8/group) was terminated 13 weeks after the last cSiO2 instillation and assessed for autoimmune hallmarks. A second cohort of mice (n = 8/group) remained on experimental diets and was monitored for proteinuria and moribund criteria to ascertain progression of glomerulonephritis and survival, respectively. DHA consumption dose-dependently increased ω-3 PUFA content in the plasma, lung, and kidney at the expense of the ω-6 PUFA arachidonic acid. Dietary intervention with high but not low DHA after cSiO2 treatment suppressed or delayed: (i) recruitment of T cells and B cells to the lung, (ii) development of pulmonary ELS, (iii) elevation of a wide spectrum of plasma autoantibodies associated with lupus and other autoimmune diseases, (iv) initiation and progression of glomerulonephritis, and (v) onset of the moribund state. Taken together, these preclinical findings suggest that DHA supplementation at a human caloric equivalent of 5 g/d was an effective therapeutic regimen for slowing progression of established autoimmunity triggered by the environmental toxicant cSiO2.

Transcriptomic profiling of adipose tissue inflammation, remodeling, and lipid metabolism in periparturient dairy cows (Bos taurus).

BACKGROUND: Periparturient cows release fatty acid reserves from adipose tissue (AT) through lipolysis in response to the negative energy balance induced by physiological changes related to parturition and the onset of lactation. However, lipolysis causes inflammation and structural remodeling in AT that in excess predisposes cows to disease. The objective of this study was to determine the effects of the periparturient period on the transcriptomic profile of AT using NGS RNAseq. RESULTS: Subcutaneous AT samples were collected from Holstein cows (n = 12) at 11 ± 3.6 d before calving date (PreP) and at 6 ± 1d (PP1) and 13 ± 1.4d (PP2) after parturition. Differential expression analyses showed 1946 and 1524 DEG at PP1 and PP2, respectively, compared to PreP. Functional Enrichment Analysis revealed functions grouped in categories such as lipid metabolism, molecular transport, energy production, inflammation, and free radical scavenging to be affected by parturition and the onset of lactation (FDR < 0.05). Inflammation related genes such as TLR4 and IL6 were categorized as upstream lipolysis triggers. In contrast, FASN, ELOVL6, ACLS1, and THRSP were identified as upstream inhibitors of lipid synthesis. Complement (C3), CXCL2, and HMOX1 were defined as links between inflammatory pathways and those involved in the generation of reactive oxygen species. CONCLUSIONS: Results offer a comprehensive characterization of gene expression dynamics in periparturient AT, identify upstream regulators of AT function, and demonstrate complex interactions between lipid mobilization, inflammation, extracellular matrix remodeling, and redox signaling in the adipose organ.

Milk production responses to altering the dietary ratio of palmitic and oleic acids varies with production level in dairy cows.

We evaluated the effects of altering the dietary ratio of palmitic (C16:0; PA) and oleic (cis-9 C18:1; OA) acids on production responses of cows with a wide range of milk production (32 to 65 kg/d) in a crossover design experiment with a preliminary period. Thirty-two multiparous Holstein cows (144 ± 54 d in milk) were assigned randomly to a treatment sequence. Treatments were diets supplemented with fatty acid (FA) blends (1.5% of diet dry matter) that provided 80% C16:0 + 10% cis-9 C18:1 (PA) and 60% C16:0 + 30% cis-9 C18:1 (PA+OA). The corn silage and alfalfa-based diets contained 20.0% forage neutral detergent fiber (NDF), 28.5% starch, and 17.1% crude protein. Treatment periods were 21 d with the final 5 d used for data and sample collection. Treatment did not affect dry matter intake (DMI), milk yield, energy-corrected milk (ECM), body weight, or body weight change. The PA+OA diet increased total, 16-carbon, and 18-carbon FA digestibility compared with the PA diet. Compared with PA+OA, PA increased fat yield (1.97 vs. 1.91 kg/d) and protein yield (1.61 vs. 1.55 kg/d). The PA diet also increased the yield of de novo (448 vs. 428 g/d) and mixed (749 vs. 669 g/d) milk FA and decreased the yield of preformed FA (605 vs. 627 g/d) compared with PA+OA. Interactions were detected between treatment and preliminary milk yield for DMI, total FA intake, 16-carbon FA intake, ECM, 3.5% fat-corrected milk (linear interaction), and a tendency for milk yield (linear interaction); lower-producing cows (<45 kg/d) had increased DMI and ECM on the PA diet, whereas higher-producing cows (>55 kg/d) had increased DMI and ECM on the PA+OA diet. A linear interaction was detected between treatment and preliminary milk yield for mixed milk FA yield (linear interaction) and a tendency for de novo milk FA yield (linear interaction). Our results demonstrate that feeding a fat supplement containing more cis-9 C18:1 replacing C16:0 increased production responses (DMI, milk yield, and ECM) in higher-producing cows, but decreased production responses in lower-producing cows.

Colostrum supplementation with n-3 fatty acids alters plasma polyunsaturated fatty acids and inflammatory mediators in newborn calves.

Calves may experience increased oxidative stress at birth through activation of metabolic and respiratory processes. Reducing oxidative stress may enhance calf viability in early life. Our objective was to determine the dose response to fish and flaxseed oil when supplemented in colostrum on concentrations of plasma fatty acid (FA), FA metabolites, and index of oxidative stress during the critical first week of life in calves to understand how supplementing n-3 FA may decrease oxidative stress. We hypothesized that n-3 FA supplemented in colostrum in a linear dose-dependent fashion would associate with increased plasma n-3 FA concentrations and decreased oxidative stress. Twenty-four male and female Holstein calves were randomly assigned to receive 0, 30, 60, or 120 mL of a 1:1 fish to flaxseed oil supplement in colostrum. All calves received 2.8 L of previously frozen colostrum (≥22% Brix) with their respective treatment within 6 h after birth. Blood was sampled before first feeding after birth and on d 1, 2, 4, 7, and 14 d of age to assess oxidant status and plasma free PUFA, phospholipid FA, and oxylipid concentrations. Health indicators were observed daily. Indicators of general health and growth were unaffected by treatment. Supplemented calves exhibited greater concentrations of n-3 FA in plasma as free and phospholipid FA and some n-3 and n-6 FA-derived oxylipids in the first week of life in a linear fashion with increasing supplemental dose. Fish and flaxseed oil treatments did not alter oxidant status but overall decreased isoprostane concentrations in plasma, indicating oxidative stress was decreased. Together, these responses indicate that the fish and flaxseed oil supplement was antiinflammatory. In conclusion, supplementing colostrum with 30, 60, and 120 mL of a 1:1 mixture of fish and flaxseed oil linearly increased plasma concentrations of n-3 FA and metabolites and decreased biomarkers of oxidative stress, but did not alter oxidant status or affect health or growth. Our findings suggest neonatal calves may benefit from n-3 FA supplementation in colostrum to encourage a greater antiinflammatory state.

Requisite Omega-3 HUFA Biomarker Thresholds for Preventing Murine Lupus Flaring.

Lupus is a systemic autoimmune disease typified by uncontrolled inflammation, disruption of immune tolerance, and intermittent flaring - events triggerable by environmental factors. Preclinical and clinical studies reveal that consumption of the marine ω-3 highly unsaturated fatty acids (HUFAs) eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) might be used as a precision nutrition intervention to lessen lupus symptoms. The anti-inflammatory and pro-resolving effects of ω-3 HUFAs are inextricably linked to their presence in membrane phospholipids. The ω-3 HUFA score, calculated as [100 × (ω-3 HUFAs/(ω-3 HUFAs + ω-6 HUFAs))] in red blood cells (RBCs), and the Omega-3 Index (O3I), calculated as [100 × ((DHA+EPA)/total fatty acids)] in RBCs, are two biomarkers potentially amenable to relating tissue HUFA balance to clinical outcomes in individuals with lupus. Using data from three prior preclinical DHA supplementation studies, we tested the hypothesis that the ω-3 HUFA score and the O3I inversely correlate with indicators of autoimmune pathogenesis in the cSiO2-triggered lupus flaring model. The three studies employed both low and high fat rodent diets, as well as more complex diets emulating the U.S. dietary pattern. The ω-3 HUFA scores in RBCs were comparatively more robust than the O3I at predicting HUFA balances in the kidney, liver, spleen, and lung. Importantly, increases in both the ω-3 HUFA score (>40%) and the O3I (>10%) were strongly associated with suppression of cSiO2-triggered (1) expression of interferon-regulated genes, proinflammatory cytokine production, leukocyte infiltration, and ectopic lymphoid structure development in the lung, (2) pulmonary and systemic autoantibody production, and (3) glomerulonephritis. Collectively, these findings identify achievable ω-3 HUFA scores and O3I thresholds that could be targeted in future human intervention studies querying how ω-3 HUFA consumption influences lupus and other autoimmune diseases.

Effects of commercially available palmitic and stearic acid supplements on nutrient digestibility and production responses of lactating dairy cows.

We evaluated the effects of commercially available fatty acid (FA) supplements containing palmitic (C16:0) and stearic acid (C18:0) on nutrient digestibility and production responses of dairy cows. Thirty-six mid-lactation (146 ± 55 d in milk) multiparous Holstein cows were randomly assigned to twelve 3 × 3 balanced truncated Latin squares, with 3 treatments and 2 consecutive 35-d periods, with the final 5 d used for sample and data collection. Treatments were (1) a control diet containing no supplemental FA (CON), (2) a control diet supplemented with a commercially available C16:0 supplement (PA), and (3) a control diet supplemented with a commercially available C16:0 and C18:0 supplement (MIX). Supplements were fed at 1.5% dry matter and replaced soyhulls in CON. The statistical model included the random effect of cow nested within square and the fixed effects of treatment, period, square, and their interactions. Preplanned contrasts were (1) overall effect of FA treatments [CON vs. the average of the FA treatments (FAT); 1/2 (PA + MIX)], and (2) effect of FA supplement (PA vs. MIX). Treatment had no effects on dry matter intake, body weight, or body weight change. Compared with CON, FAT decreased digestibilities of total FA and 18-carbon FA but did not affect dry matter and neutral detergent fiber digestibility. Compared with MIX, PA increased dry matter and neutral detergent fiber digestibilities by 3.6 and 4.8 percentage units, respectively. The PA also increased total FA and 18-carbon FA digestibilities but did not alter 16-carbon FA digestibility compared with MIX. Using a Lucas test, we estimated apparent digestibility coefficients of 0.768 and 0.553 for the PA and MIX supplements, respectively. Compared with CON, FAT increased milk yield and tended to increase energy-corrected milk, but did not affect yield of milk fat or milk protein. The PA increased energy-corrected milk and milk fat yield but had no effect on milk protein yield compared with MIX. Our results indicate that dairy cows producing around 45 kg of milk respond better to a FA supplement enriched in C16:0 compared with a supplement containing both C16:0 and C18:0, which is likely due in part to PA increasing FA and neutral detergent fiber digestibility compared with MIX.

Lipolysis modulates the biosynthesis of inflammatory lipid mediators derived from linoleic acid in adipose tissue of periparturient dairy cows.

Oxidized linoleic acid metabolites (OXLAM) are products of adipocyte lipolysis with the potential to modulate adipose tissue (AT) lipid metabolism and inflammation. In periparturient cows, linoleic acid is preferentially mobilized from AT during lipolysis by hormone-sensitive lipase (HSL) compared with other polyunsaturated fatty acids. Enzymatic and nonenzymatic reactions generate OXLAM from linoleic acid. Among OXLAM, 9-, 10-, and 12-hydroxy-octadecadienoic acids (HODE) are associated with pro-inflammatory responses, whereas 9- and 13-oxo-octadecadienoic acids (oxoODE) and 13-HODE can facilitate inflammation resolution and promote lipogenesis. This study evaluated the effect of HSL activity on OXLAM biosynthesis using subcutaneous AT explants collected from multiparous dairy cows at 10 d before and again at 10 and 24 d after calving. Explants were treated for 3 h without or with the ß-adrenergic agonist isoproterenol (ISO; 1 µM; MilliporeSigma, Burlington, MA) to induce HSL activity. The contribution of HSL to OXLAM biosynthesis was determined by inhibiting its activity with CAY10499 (2 µM; Cayman Chemical, Ann Arbor, MI). After treatments, media and explants were collected for lipidomic analysis using HPLC-tandem mass spectroscopy. Results indicated that ISO increased the biosynthesis of 9-, 12-, and 13-HODE and 9-oxoODE, and this effect was reduced at 24 d after calving. Inhibiting HSL activity partially reversed ISO effects on HODE and 9-oxoODE. Our ex vivo model demonstrated for the first time a direct effect of HSL activity on the biosynthesis of OXLAM in AT, especially at 10 d before and 10 d after calving. The biosynthesis of anti-inflammatory OXLAM is limited during the first weeks after parturition and may promote AT inflammation and lipolytic responses to negative energy balance. These results indicate that HSL activity releases linoleic acid for OXLAM biosynthesis in concentrations of a magnitude that may bypass the need for the activation of phospholipases linked with the inflammatory cascade and thus supports, in part, lipolysis-driven inflammation within AT of periparturient cows.

Correction: Silica-Triggered Autoimmunity in Lupus-Prone Mice Blocked by Docosahexaenoic Acid Consumption.

[This corrects the article DOI: 10.1371/journal.pone.0160622.].

Impact of uterine macrophage phenotype on placental retention in dairy cows.

Reproductive diseases affect 25% of dairy cows in the US and often develop from retention of the placenta. It is well established that expulsion of the placenta is a highly regulated inflammatory process, but the mechanisms by which dysregulation of uterine immune responses impair this process are poorly understood. In healthy non-ruminants, pro-inflammatory M1 macrophages are predominant in uterine tissue after parturition. However, macrophage phenotype in the postpartum bovine uterus is unknown. Our study compared macrophage phenotypes in the uterine caruncles of multiparous dairy cows that during the first day postpartum either retained (RET, n = 5) or had normal expulsion (NOR, n = 5) of placenta. Immune cells were sorted magnetically from the caruncular endometrial cell fraction using the CD172a marker and monocyte/macrophage population was characterized using flow cytometry. Transcriptional and protein expression studies were performed on uterine caruncles. Compared to NOR, RET samples showed a lower CD14+/CD16+ expression (P < 0.05) in caruncle monocyte/macrophage population. As opposed to NOR, RET further demonstrated greater expression of anti-inflammatory M2 macrophage associated genes CD206, C-type lectin domain family 7 member A (CLEC7A), and RNASE6. In addition, caruncles from RET showed decreased signal transducer and activator of transcription 3 (STAT3) activation, an important promoter of proteolytic activity, compared to NOR. Our studies demonstrate that there is an overall lower number of macrophage populations in the caruncle of cows with RET placenta and these are polarized towards M2 phenotype. Excessive accumulation of M2 macrophages may lead to reduced trafficking of immune cells into the caruncle thus impairing the inflammatory, phagocytic and proteolytic processes that lead to placental expulsion.

The contribution of hormone sensitive lipase to adipose tissue lipolysis and its regulation by insulin in periparturient dairy cows.

Hormone sensitive lipase (HSL) activation is part of the metabolic adaptations to the negative energy balance common to the mammalian periparturient period. This study determined HSL contribution to adipose tissue (AT) lipolysis and how insulin regulates its activity in periparturient dairy cows. Subcutaneous AT (SCAT) samples were collected at 11 d prepartum (dry) and 11 (fresh) and 24 d (lactation) postpartum. Basal and stimulated lipolysis (ISO) responses were determined using explant cultures. HSL contribution to lipolysis was assessed using an HSL inhibitor (CAY). Basal lipolysis was higher in SCAT at dry compared with fresh. CAY inhibited basal lipolysis negligibly at dry, but at fresh and lactation it reduced basal lipolysis by 36.1 ± 4.51% and 43.1 ± 4.83%, respectively. Insulin inhibited lipolysis more pronouncedly in dry compared to fresh. Results demonstrate that HSL contribution to basal lipolysis is negligible prepartum. However, HSL is a major driver of SCAT lipolytic responses postpartum. Lower basal lipolysis postpartum suggests that reduced lipogenesis is an important contributor to fatty acid release from SCAT. Loss of adipocyte sensitivity to the antilipolytic action of insulin develops in the early lactation period and supports a state of insulin resistance in AT of cows during the first month postpartum.

Short communication: Effects of body fat mobilization on macrophage infiltration in adipose tissue of early lactation dairy cows.

Intense lipolysis triggers an inflammatory response within adipose tissue characterized by adipose tissue macrophage (ATM) infiltration; however, the mechanisms triggering this process are poorly characterized in transition dairy cows. The aim of this study was to determine the association between ATM infiltration and body fat mobilization in the transition period, markers of excessive lipolysis, and adipose tissue expression of genes related to chemotactic and inflammatory responses. Subcutaneous adipose tissue samples were taken from the tailhead of 9 multiparous Holstein cows, 27 ± 2.2 d (far-off) and 10 ± 1.5 d (close-up) before and 9 ± 0.3 d after calving (fresh). Blood samples were collected by coccygeal venipuncture 2 h before adipose sample collections. Body condition score (BCS) was assessed independently by 3 experienced technicians at every time point. Based on BCS loss intensity between the close-up and fresh period, cows were divided into 2 groups: low BCS loss (LBCSL, change in BCS <0.25 units, n = 5) and high BCS loss (HBCSL, change in BCS >0.25 units, n = 4). Although none of the LBCSL cows had a health event, all cows in the HBCSL group suffered from one or more clinical disorder (retained placenta, milk fever, or ketosis) in the transition period. The number of ATM was determined by immunohistochemistry, and expression of selected chemotactic and inflammatory genes was determined by reverse-transcription quantitative real-time PCR in subcutaneous adipose tissue samples. The proportion of ATM in subcutaneous adipose tissue increased in HBCSL during the postpartum period. The proportion of ATM was not associated with serum ß-hydroxybutyrate or free fatty acid concentrations on the day of adipose tissue collection. The ATM infiltration in the fresh period was associated with local expression of the chemotactic genes, C-C motif chemokine ligand 22 (CCL22), osteopontin (SPP1), and the receptor for SPP1, cluster of differentiation 44 (CD44). This supports a potential chemotactic role of CCL22 and SPP1 for ATM in bovine adipose tissue. None of the genes encoding pro- or anti-inflammatory mediators, tumor necrosis factor (TNF), IL6, and IL10 were associated with the proportion of ATM. Our results indicate that ATM infiltration of subcutaneous adipose tissue is associated with body fat mobilization in early-lactation dairy cows and supports a role for ATM in the adaptation of adipose tissues to the metabolic challenges of the transition period.

Fetuin-A: A negative acute-phase protein linked to adipose tissue function in periparturient dairy cows.

Fetuin-A (FetA) is a free fatty acid transporter and an acute-phase protein that enhances cellular lipid uptake and lipogenesis. In nonruminants, FetA is involved in lipid-induced inflammation. Despite FetA importance in lipid metabolism and inflammation, its expression and dynamics in adipose tissue (AT) of dairy cows are unknown. The objectives of this study were to (1) determine serum and AT FetA dynamics over the periparturient period and in mid-lactation cows in negative energy balance (NEB) after a feed restriction protocol and (2) characterize how an inflammatory challenge affects adipocyte FetA expression. Blood and subcutaneous AT were collected from 16 cows with high (≥3.75, n = 8) or moderate (≤3.5, n = 8) body condition score (BCS) at -26 ± 7 d (far off) and -8 ± 5 d (close up) before calving and at 10 ± 2 d after parturition (early lactation) and from 14 nonpregnant mid-lactation cows (>220 d in milk) after a feed restriction protocol. Serum FetA concentrations were 0.89 ± 0.13 mg/mL at far off, 0.96 ± 0.13 mg/mL at close up, and 0.77 ± 0.13 mg/mL at early lactation and were 1.09 ± 0.09 and 1.17 ± 0.09 mg/mL in feed-restricted and control cows, respectively. Serum and AT FetA contents decreased at the onset of lactation when lipolysis was higher. No changes in AT and serum FetA were observed after feed restriction induced NEB in mid-lactation cows. Prepartum BCS had no effect on serum FetA, but AT expression of AHSG, the gene encoding FetA, was reduced in periparturient cows with high BCS at dry-off throughout all time points. Circulating FetA was positively associated with serum albumin and calcium and with BCS variation over the periparturient period. The dynamics of AHSG expression were analogous to the patterns of lipogenic markers ABDH5, ELOVL6, FABP4, FASN, PPARγ, and SCD1. Expression of AHSG and FetA protein in AT was inversely correlated with AT proinflammatory markers CD68, CD44, SPP1, and CCL2. In vitro, bovine adipocytes challenged with lipopolysaccharide downregulated FetA protein expression. Adipocytes treated with FetA had lower CCL2 expression compared with those exposed to lipopolysaccharide. Overall, FetA is a systemic and local AT negative acute-phase protein linked to AT function in periparturient cows. Furthermore, FetA may support physiological adaptations to NEB in periparturient cows.

Periparturient lipolysis and oxylipid biosynthesis in bovine adipose tissues.

The periparturient period of dairy cows is characterized by intense lipolysis in adipose tissues (AT), which induces the release of free fatty acids (FFA) into circulation. Among FFA, polyunsaturated fatty acids are susceptible to oxidation and can modulate inflammatory responses during lipolysis within AT. Linoleic and arachidonic acid oxidized products (oxylipids) such as hydroxy-octadecadienoic acids (HODE) and hydroxy-eicosatetraenoic acids (HETE), were recently identified as products of lipolysis that could modulate AT inflammation during lipolysis. However, the effect of lipolysis intensity during the transition from gestation to lactation on fatty acid substrate availability and subsequent AT oxylipid biosynthesis is currently unknown. We hypothesized that in periparturient dairy cows, alterations in AT and plasma fatty acids and oxylipid profiles coincide with changes in lipolysis intensity and stage of lactation. Blood and subcutaneous AT samples were collected from periparturient cows at -27±7 (G1) and -10±5 (G2) d prepartum and at 8±3 d postpartum (PP). Targeted lipidomic analysis was performed on plasma and AT using HPLC-MS/MS. We report that FFA concentrations increased as parturition approached and were highest at PP. Cows exhibiting high lipolysis rate at PP (FFA>1.0 mEq/L) had higher body condition scores at G1 compared to cows with low lipolysis rate (FFA<1.0 mEq/L). Concentrations of plasma linoleic and arachidonic acids were increased at PP. In AT, 13-HODE, and 5-, 11- and 15-HETE were increased at PP compared to G1 and G2. Concentrations of beta hydroxybutyrate were positively correlated with those of 13-HODE and 15-HETE in AT. Plasma concentrations of 5- and 20-HETE were increased at PP. These data demonstrate that prepartum adiposity predisposes cows to intense lipolysis post-partum and may exacerbate AT inflammation because of increased production of pro-inflammatory oxylipids including 5- and 15-HETE and 13-HODE. These results support a role for certain linoleic and arachidonic acid-derived oxylipids as positive and negative modulators of AT inflammation during periparturient lipolysis.