Oxylipids are derived from polyunsaturated fatty acids (PUFA) in cellular membranes and the relative abundance or balance may contribute to disease pathogenesis. Previous studies documented unique oxylipid profiles from cows with either coliform or Streptococcus uberis mastitis, suggesting that lipid mediator biosynthesis may be dependent on the type of microbial-derived agonist. Changing the fatty acid content of peripheral blood leukocytes also may be critical to the relative expression of oxylipid profiles and the outcome of bacterial infection. No information is available in dairy cows describing how changing cellular PUFA content will modify oxylipids in the context of a microbial agonist challenge. Therefore, the hypothesis for the current study was that PUFA supplementation would change bovine leukocyte fatty acid content and respective oxylipid profiles from ex vivo microbial agonist-challenged leukocytes. Fatty acid content of leukocytes and plasma was quantified in (1) samples from cows not supplemented with PUFA, (2) cows supplemented with linoleic acid (LnA), and (3) cows supplemented with α-linolenic acid (ALA). Plasma oxylipids were assessed after S. uberis or lipopolysaccharide exposure and was compared with unstimulated oxylipid profiles. Fatty acid supplementation with ALA significantly increased ALA content of blood leukocytes and plasma relative to LnA. Fatty acid supplementation affected several S. uberis-induced oxylipids, but only S. uberis-induced 15-oxoETE was greater with ALA supplementation compared with LnA. Notably, only LPS-induced 5,6 LXA4 was altered with fatty acid supplementation, but no significant effect of LnA vs. ALA treatment was identified. Future studies are needed to understand how leukocyte activation and membrane PUFA availability collectively contribute to differential oxylipid profiles.

The antilipogenic effects of trans-10, cis-12 CLA are widely reported across monogastric species. However, abomasal infusions of this CLA isomer have been shown to increase expression of lipogenic genes in the adipose of lactating dairy cows. It is not clear if this is a result of energy repartitioning due to a decrease in milk fat synthesis or a direct effect of trans-10, cis-12 CLA on adipocytes. Our objective was to examine the effects of trans-10, cis-12 CLA on cultured adipocytes derived from subcutaneous adipose of lactating dairy cows (n = 4). Adipose samples were digested with collagenase type II and cells from the stromal vascular fraction were cultured in DMEM/F12 supplemented with 10% fetal bovine serum. Preadipocytes were obtained from outgrowth of plastic adherent cells and seeded in assay plates. After reaching confluence, cells were induced to differentiate and maintained in the plates for 10 d. From d 2 to 10, the medium was supplemented with one of two treatments: 50 μM trans-10, cis-12 CLA (T10C12) or 50 μM cis-9, trans-11 CLA (C9T11). On d 10, intracellular triglyceride was quantified using an AdipoRed assay and RNA was extracted to analyze gene expression using RT-qPCR. Statistical analysis was performed using linear mixed models. Fold changes (FC) in gene expression are presented relative to the C9T11 treatment. Conjugated linoleic acid supplementation did not affect triglyceride content of the adipocytes (P = 0.47), but the ratio of triglyceride content of adipocytes to preadipocytes indicated differentiation occurred in both treatment groups (T10C12 = 60.0 and C9T11 = 55.0). T10C12 decreased expression of the lipogenic genes ACACA (FC = 0.75; P = 0.01), ELOVL6 (FC = 0.73; P = 0.03), and SCD1 (FC = 0.52; P = 0.03). Expression of C/EBPβ, FAS, GAPDH, LPL, and PPARγ were not affected by treatment (P ≥ 0.43). In contrast to what has been observed in murine-derived 3T3-L1 cells, T10C12 increased the expression of the fatty acid transport gene FABP4 (FC = 2.06; P = 0.01) and tended to increase the expression of DGAT1 (FC = 1.07; P = 0.06), which is associated with triglyceride synthesis. Although trans-10, cis-12 CLA decreased the expression of several lipogenic genes, it did not inhibit lipid accumulation. This suggests that cultured adipocytes derived from dairy cows respond differently to trans-10, cis-12 CLA than those derived from monogastric species.

Periparturient cows that experience severe adipose tissue lipolysis are at a higher risk for inflammatory and metabolic diseases. Fetuin-A (FetA) is a glycoprotein that inhibits insulin signaling and enhances inflammatory responses in adipose tissues, which are known to exacerbate lipolytic responses in humans and rodents. However, little is known about its role during lipolysis and its use as a biomarker for metabolic stress and lactation performance in dairy cows. Our objective was to determine the dynamics of serum and adipose FetA concentrations and its association with metabolic markers during negative energy balance (NEB)–induced lipolysis at different stages of lactation. In Experiment 1, 26 multiparous cows were followed through the transition period. Blood samples and subcutaneous adipose tissue were collected at dry-off (DO; −51 ± 3 d), close-up (CU; −14 ± 2 d), and early lactation (EL; 7 ± 0.5 d). In Experiment 2, FetA response to lipolysis was evaluated independently of parturition-associated metabolic challenges using midlactation cows (119–210 DIM) assigned to one of two feeding protocols: ad libitum (AL; n = 3; +EB = 3.2 ± 0.66 Mcal/d) or feed restricted (FR; n = 3; EB = −13.3 ± 0.5 Mcal/d). Blood and subcutaneous adipose tissue were collected after a 4-d period of feed restriction. FetA was determined by ELISA and western blot. Data were analyzed using a repeated measures mixed model. Serum and adipose FetA concentrations were affected by lactation stage. In Experiment 1, serum FetA concentrations were lower at EL (DO: 1.31 ± 0.06 mg/mL; CU: 1.27 ± 0.09 mg/mL; and EL: 1.14 ± 0.06 mg/mL; P < 0.05) when NEFA concentration was greatest (DO: 0.34 ± 0.02 mEq/L; CU: 0.63 ± 0.2 mEq/L; and EL: 1.19 ± 0.14 mEq/L; P < 0.05). Unlike in serum, adipose FetA expression decreased at CU (relative band density; DO: 1.5 ± 0.4; CU: 0.2 ± 0.02; EL: and 1.6 ± 0.6; P < 0.05). Circulating FetA concentration was higher in overconditioned dry cows (BCS ≥ 3.75; P < 0.05) and was positively associated with BCS (R2 = 0.24, P < 0.0001) and BCS loss (R2 = 0.43, P = 0.0005) during the transition period. Cows with high BCS and increased serum FetA concentrations at DO had lower serum glucose concentrations at EL (P < 0.05). In Experiment 2, despite the feed restriction-induced lipolysis (NEFA; FR = 0.47 ± 0.05 mEq/L and AL = 0.09 ± 0.08 mEq/L), neither serum nor adipose FetA concentrations were affected in midlactation cows (P > 0.05). These results demonstrate that serum and adipose FetA concentrations during lipolytic states are determined by lactation stage and BCS around parturition. Fetuin-A is a potential novel biomarker for metabolic stress induced by lipolysis during the transition period. Future work will determine the mechanisms by which FetA affects lipolytic and inflammatory responses in adipose tissues of transition dairy cows.

We hypothesized that stressing cows during gestation (prenatal stress, PNS) would increase the insulin sensitivity of their progeny. Specifically, 12 prenatally stressed (dams transported for 2h on d 60, 80, 100, 120, and 140 of gestation) and 12 Control (dams not transported) yearling bulls of similar BW (298.50 ± 5.95 kg) and balanced for temperament (4 temperamental and 8 calm per group) were subjected to an iv glucose tolerance test to compare insulin responsiveness and glucose clearance. Following 12 h off feed the bulls were fitted with jugular vein catheters and placed in individual stanchions. After a 2h acclimation period, bulls were administered a glucose tolerance test (0.5 mL/kg BW of 50% dextrose solution). Blood samples were collected at 10min intervals for 40 min and then at 20min intervals until 180 min postchallenge. Serum concentrations of glucose and insulin were determined by enzymatic assay and ELISA, respectively. Data were analyzed using mixed models procedures of SAS with repeated measures. Basal concentrations of insulin (P< 0.53) and glucose (P< 0.35) were not affected by PNS. Serum insulin increased in both CON and PNS bulls within 10 min of glucose administration with a tendency for PNS bulls to reach the insulin peak earlier (P= 0.09) and return to basal insulin earlier (P< 0.01) than CON bulls (Table 066). Glucose concentration wasn't affected by treatment (P= 0.14) or by the interaction of treatment by time (P= 0.53). Time to reach basal glucose concentration was unaffected by treatment (P= 0.73). The area under the response curve was greater for glucose (P< 0.01) and lower for insulin (P< 0.01) in the PNS bulls compared to CON bulls. The insulin to glucose ratio was lower (P<0.06) in PNS bulls.These results support our hypothesis that PNS increased sensitivity to insulin. Glucose and insulin response variables to a glucose challenge in PNS and Control bulls. Glucose and insulin response variables to a glucose challenge in PNS and Control bulls.

Negative energy balance (NEB) during early lactation results in extensive adipose tissue lipolysis in dairy cows. However, it is not clear if specific adipose depots or fatty acids (FA) are preferentially mobilized. Our objective was to characterize the FA profile of adipose depots and milk fat following feed restriction-induced NEB. Twelve multiparous late lactation ( > 200 DIM) Holstein cows, in two experimental blocks, were subjected to treatments consisting of ad libitum feed intake (ADLIB; n = 6) or feed restriction (RESTR; n = 6) resulting in an energy balance of −13.3 ± 0.5 Mcal/d over 4 d. Milk samples were analyzed for FA composition and collected on d 4. Following the treatment period, all cows were slaughtered and tissue samples were collected from 6 adipose depots: omental, subcutaneous flank, tailhead, perirenal, inguinal, and sternal. Statistical analysis of adipose and milk FA composition was performed using linear mixed models. RESTR increased the C14 desaturase index (cis-9 C14:1/(cis-9 C14:1 + C14:0)) of the sternal and tailhead depots (P < 0.05) and the C16 desaturase index (cis-9 C16:1/(cis-9 C16:1 + C16:0)) of the tailhead depot (P < 0.01). RESTR decreased C18:0 content of the tailhead depot (8.7 vs. 12.8 g/100 g FA; P = 0.01). Across all depots, RESTR increased the cis-9 C14:1 content of adipose tissue (P = 0.04). RESTR decreased daily yield of de novo-synthesized FA in milk (P = 0.02) but the yields of 16-carbon and preformed FA were not affected by treatment (P ≥ 0.20). Compared to ADLIB, RESTR increased the C18 desaturase index (cis-9 C18:1/(cis-9 C18:1 + C18:0)) (0.73 vs. 0.66) and the C16 desaturase index (0.08 vs. 0.05) of milk fat (P < 0.01). RESTR increased the daily yields of cis-9 C16:1 and cis-9 C18:1 in milk fat compared to ADLIB (both P < 0.05), while the yields of C16:0 and C18:0 were not affected by treatment (P ≥ 0.24). RESTR increased total monounsaturated FA yield in milk (P = 0.03), but treatment did not alter total saturated FA or polyunsaturated FA yields (P ≥ 0.11). Alterations in adipose FA composition, suggesting mobilization of saturated FA, occurred only in the subcutaneous adipose depots, and most dramatically in the tailhead. However, increased desaturase activity in the mammary gland most likely prevented a subsequent increase in saturated FA yield of milk.

The balance of n-3 and n-6 fatty acids (FA) in immune system tissues can influence the degree of inflammatory responses in dairy cattle. Linoleic acid (C18:2 n-6) and linolenic acid (C18:3 n-3) are the most abundant n-6 and n-3 FA in lactating dairy cow rations, and are associated with pro-inflammatory and anti-inflammatory responses, respectively. Our objective was to evaluate the incorporation of these FA, and their downstream oxidized FA (oxylipids), into plasma and white blood cells (WBC) following supplementation. Six mid-lactation dairy cows were abomasally infused 4x/d for 7-d treatment periods with 7-d washout intervals in a replicated balanced Latin square design with 3 treatments: 1) CON = ethanol carrier, 2) LA = 45 g/d C18:2 n-6, and 3) LNA = 45 g/d C18:3 n-3. Blood was collected on d 7 of the treatment periods and analyzed for WBC and plasma lipid fraction FA and plasma oxylipid composition. Yields of milk and milk components were calculated for d 6 and d 7 of the treatment periods. Statistical analysis was performed using linear mixed models. Dry matter intake was not affected by treatment (P = 0.68). LA treatment increased the yield of milk and milk protein compared to CON and LNA (P ≤ 0.05). LNA treatment increased milk fat concentration compared to CON and LA (P ≤ 0.05). The concentration of C18:3 n-3 in WBC was increased by LNA (0.86 g/100 g FA; P ≤ 0.05), compared to LA (0.39 g/100 g FA) and CON (0.34 g/100 g FA), but C18:2 n-6 was unaffected by treatment (P = 0.15). LNA increased C18:3 n-3 (3.17 g/100 g FA) and C20:5 n-3 (0.43 g/100 g FA) in the phospholipid fraction of plasma, compared to CON and LA (P ≤ 0.01), while LA increased C18:2 n-6 (38.7 g/100 g FA), compared to the other treatments (P < 0.01). Plasma phospholipid C20:4 n-6 concentration was not altered by treatment (P = 0.65). LNA decreased C20:4 n-6-derived 8,9-DiHETrE (P < 0.01) and tended to decrease C18:2 n-6-derived 12,13 EpOME in plasma (P = 0.09). When C18:3 n-3 and C18:2 n-6 were abomasally infused at the same dose, C18:3 n-3 had a greater influence on the profile of plasma FA and oxylipids and the FA composition of WBC. These changes have the potential to mediate inflammatory responses in cattle at risk of infection.

The objective of this study was to estimate the heritability of 3 measures of temperament in Brahman and Brahman-influenced calves (n = 1,209). Individual animal pen scores (PS) were determined by a trained observer who evaluated groups of 5 or 4 calves at a time for willingness to be approached by a human. Exit velocity (EV) was the rate (m/s) at which each calf exited a squeeze chute. Temperament score (TS) was calculated individually as (PS + EV)/2. Temperament was evaluated at 5 different times of record (28 d preweaning, weaning, 28 d postweaning, 56 d postweaning, and yearling). Contemporary groups (n = 34) comprised calves of the same sex born in the same season of the same year. There were an average of 36 calves per contemporary group and group size ranged from 3 to 78 calves. Average weaning age (186 d) ranged from 105 to 304 d. Calves were born from 2002 through 2012. Random effects included additive genetic and the permanent environmental variance. The fixed effects analyzed were age of dam, sex of calf, contemporary group, fraction of Brahman (2 levels: 1 and 0.5), age of calf at record, and weaning age. At weaning, the mean PS was 2.68 ± 0.1, the mean EV was 2.41 ± 0.1, and the mean TS was 2.48 ± 0.1. The PS was affected by fraction of Brahman (P = 0.034) and tended to be affected by age of dam (P = 0.06). The EV was affected by contemporary group (P < 0.001) and tended to be affected by weaning age (P = 0.074). Contemporary group affected TS (P < 0.001). All 3 methods of temperament evaluation were affected by time of record (P < 0.001). The regression coefficients for PS, EV, and TS were 0.0023 ± 0.0014, 0.0022 ± 0.0012, and 0.0015 ± 0.0012 m·s(-1)·d(-1) of age, respectively. Estimates of maternal genetic effects were always 0 and omitted from final models. Estimates of heritability were 0.27 ± 0.1, 0.49 ± 0.1, and 0.43 ± 0.1 for EV, PS, and TS, respectively. Estimates of permanent environmental variances as proportions of phenotypic variance were 0.33 ± 0.1, 0.23 ± 0.1, and 0.33 ± 0.1 for EV, PS, and TS, respectively. There appears to be sufficient additive genetic variance for selective improvement of temperament characteristics in Brahman cattle.