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We evaluated the between-cow (b-cow) variation and repeatability in omasal and milk fatty acids (FA) related to methane (CH4) emission. The dataset was originated from 9 studies with rumen-cannulated dairy cows conducted using either a switch-back or a Latin square design. Production of CH4 per mole of VFA (Y_CH4VFA) was calculated based on VFA stoichiometry. Experiment, diet within experiment, period within experiment, and cow within experiment were considered as random factors. Empirical models were developed between the variables of interest by univariate and bivariate mixed model regression analysis. The variation associated with diet was higher than the b-cow variation with low repeatability (< 0.25) for milk odd- and branch-chain FA (OBCFA). Similarly, for de novo synthesized milk FA, diet variation was ~ 3-fold greater than the b-cow variation; repeatability for these FA was moderate to high (0.34-0.58). Also, for both cis-9 C18:1 and cis-9 cis-12 cis-15 C18:3 diet variation was more than double the b-cow variation, but repeatability was moderate. Among the de novo milk FA, C4:0 was positively related with stoichiometric Y_CH4VFA, while for OBCFA, anteiso C15:0 and C15:0 were negatively related with it. Notably, when analyzing the relationship between omasal FA and milk FA we observed positive intercept estimates for all the OBCFA, which may indicate endogenous post-ruminal synthesis of these FA, most likely in the mammary gland. For milk iso C13:0, iso C15:0, anteiso C15:0, and C15:0 were positively influenced by omasal proportion of their respective FA and by energy balance. In contrast, the concentration of milk C17:0, iso C18:0, C18:0, cis-11 C18:1, and cis-9 cis-12 cis-15 C18:3 were positively influenced by omasal proportion of their respective FA but negatively related to calculated energy balance. Our findings demonstrate that for most milk FA examined, a larger variation is attributed to diet than b-cow differences with low to moderate repeatability. While some milk FA were positively or negatively related with Y_CH4VFA, there was a pronounced effect of calculated energy balance on these estimates. Additionally, even though OBCFA have been indicated as markers of rumen function, our results suggest that endogenous synthesis of these FA may occur, which therefore, may limit the utilization of milk FA as a proxy for CH4 predictions for cows fed the same diet.

Lupus is a debilitating multi-organ autoimmune disease clinically typified by periods of flare and remission. Exposing lupus-prone female NZBWF1 mice to crystalline silica (cSiO2), a known human autoimmune trigger, mimics flaring by inducing interferon-related gene (IRG) expression, inflammation, ectopic lymphoid structure (ELS) development, and autoantibody production in the lung that collectively accelerate glomerulonephritis. cSiO2-triggered flaring in this model can be prevented by supplementing mouse diet with the ω-3 polyunsaturated fatty acid (PUFA) docosahexaenoic acid (DHA). A limitation of previous studies was the use of purified diet that, although optimized for rodent health, does not reflect the high American intake of saturated fatty acid (SFA), ω-6 PUFAs, and total fat. To address this, we employed here a modified Total Western Diet (mTWD) emulating the 50th percentile U.S. macronutrient distribution to discern how DHA supplementation and/or SFA and ω-6 reduction influences cSiO2-triggered lupus flaring in female NZBWF1 mice. Six-week-old mice were fed isocaloric experimental diets for 2 wks, intranasally instilled with cSiO2 or saline vehicle weekly for 4 wks, and tissues assessed for lupus endpoints 11 wks following cSiO2 instillation. In mice fed basal mTWD, cSiO2 induced robust IRG expression, proinflammatory cytokine and chemokine elevation, leukocyte infiltration, ELS neogenesis, and autoantibody production in the lung, as well as early kidney nephritis onset compared to vehicle-treated mice fed mTWD. Consumption of mTWD containing DHA at the caloric equivalent to a human dose of 5 g/day dramatically suppressed induction of all lupus-associated endpoints. While decreasing SFA and ω-6 in mTWD modestly inhibited some disease markers, DHA addition to this diet was required for maximal protection against lupus development. Taken together, DHA supplementation at a translationally relevant dose was highly effective in preventing cSiO2-triggered lupus flaring in NZBWF1 mice, even against the background of a typical Western diet.

The mammary gland has an incredible level of organization and a remarkable ability to convert circulating nutrients into milk components. This review highlights four areas of high interest in the biology of milk synthesis where advances over the last quarter-century have resulted in new understanding or revealed new opportunities. First, advances in our understanding of the mechanisms of milk secretion has led to a substantial increase in our knowledge of the intracellular origin of lipid droplets and the identity and potential function of milk fat globule membrane proteins in milk-lipid secretion. Second, recent breakthroughs have advanced our understanding of the nutritional regulation of milk fat and highlighted the interrelations between dietary components, digestive processes in the rumen, and the regulation of mammary synthesis of milk fat. Third, nutritional quality is becoming increasingly important in food choices because of consumer awareness of the links between diet and health. The traditional nutritional value of milk and dairy products is well established, but recent discoveries have identified a number of “bioactive” components in milk with potential to improve human health. Finally, the concept of genetic engineering and the use of animals as “bioreactors” and the “pharming” of proteins not normally found in milk have gained recognition, with the dairy industry ideally suited to take advantage of advances in these areas.

Citrate is a normal constituent of milk that affects milk-processing characteristics. It is an intermediate in the tricarboxylic acid cycle and plays an indirect role in fat synthesis by providing reducing equivalents in the form of NADPH. The objective of this study was to investigate variation in citrate with stage of lactation and de novo fatty acid synthesis, without confounding dietary effects. Twenty-four cows were fed the same diet, and milk citrate and fatty acids were determined over a 10-d period. Eight cows were in early lactation [13±1.8 d in milk (DIM; mean ±standard error], 8 in midlactation (130 ±4.6 DIM), and 8 in late lactation (283 ±3.4 DIM). For cows in early, mid, and late lactation, milk yield was 34.4, 34.4, and 21.4 L/d [standard error of difference (SED) 1.78]; milk fat was 50.4, 40.3, and 41.4g/L (3.68); milk citrate was 11.3, 9.7, and 10.1 mmol/L (0.64); the ratio of 4–14C:18–20C fatty acids was 0.9, 1.3, and 1.2 (0.07). Activity of the fatty acid synthase enzyme system (EC 2.3.1.85) was calculated as acetate used for chain elongation (ACE); ACE (mol/d) for cows in early, mid, and late lactation, was 7.3, 11.1, and 8.1 (SED 1.05). For individual cows, citrate (mmol/L) = 14.3 − 0.44 ×ACE (r2 = 0.58). We propose that ACE provides a more accurate indication of synthase activity than do fatty acid ratios or yields. This study confirms the hypothesis that variation in milk citrate with stage of lactation is related to de novo synthesis of fatty acids and that the relationship is independent of diet and milk yield.

A rapid method for milk lipid separation followed by transmethylation to produce fatty acid methyl esters from bovine milk samples is presented. Fat is separated by a nonsolvent method using centrifugation. The method was compared with the popular hexane:isopropanol solvent extraction method, and fatty acid proportions were statistically identical for both methods. In 108 replicates, variance accounted for by using the 2 methods was of a similar magnitude to variance due to repeat separations or repeat injections onto the gas chromatography column. It is concluded that the proposed method is accurate, simple, rapid, safe, economical, and especially suitable for large numbers of samples.

Under certain dietary situations, rumen biohydrogenation results in the production of unique fatty acids that inhibit milk fat synthesis. The first of these to be identified was trans-10, cis-12 conjugated linoleic acid (CLA), but others are postulated to contribute to diet-induced milk fat depression (MFD). Our objective was to examine the potential role of trans-9, cis-11 CLA in the regulation of milk fat. In a preliminary study, we used gas-liquid and high-performance liquid chromatography techniques to examine milk fat samples from a diet-induced MFD study and found that an increase in trans-9, cis-11 CLA corresponded to the decrease in milk fat yield. We investigated this further using a CLA enrichment of 9, 11 isomers to examine the biological effect of trans-9, cis-11 CLA on milk fat synthesis. Four rumen-fistulated Holstein cows were randomly assigned in a 4 x 4 Latin square experiment involving 5-d treatment periods and abomasal infusion of 1) ethanol (control), 2) a 9, 11 CLA mix (containing 32% trans-9, cis-11, 29% cis-9, trans-11, and 17% trans-9, trans-11), 3) a trans-9, trans-11 CLA supplement, and 4) a trans-10, cis-12 CLA supplement (positive control). The trans-9, trans-11 CLA and trans-10, cis-12 CLA supplements were of high purity (>90%), and all supplements were infused at a rate to provide 5 g/d of the CLA isomer of interest. Milk yield and dry matter intake did not differ among treatments. Compared with the control treatment, milk fat yield was reduced by 15% for the 9, 11 CLA mixture and by 27% for the trans-10, cis-12 CLA treatment. We also found that trans-9, trans-11 CLA had no effect on milk fat yield, and previous research has shown that milk fat yield is unaltered when cows are infused with cis-9, trans-11 CLA. When all treatments were considered, results suggested that trans-9, cis-11 was the CLA isomer in the 9, 11 CLA mix responsible for the reduction in milk fat synthesis, although the magnitude was less than that observed for trans-10, cis-12 CLA. Interestingly, trans-9, trans-11 CLA altered the milk fat desaturase index, further demonstrating that alterations in desaturase can occur independently of effects on milk fat synthesis. Overall, our investigations identified that an increase in milk fat content of trans-9, cis-11 CLA was associated with diet-induced MFD and provided evidence of a role for this isomer in MFD based on the 15% reduction in milk fat yield with abomasal infusion of a CLA enrichment that supplied 5 g/d of trans-9, cis-11 CLA.

Stearoyl-CoA desaturase (SCD) in the bovine mammary gland introduces a cis-double bond at the Δ9 position in a wide range of fatty acids (FA). Several long-chain polyunsaturated fatty acids (PUFA) inhibit expression of SCD, but information on the effect of short-chain fatty acids on mammary SCD expression is scarce. We used a bovine mammary cell line (MAC-T) to assess the effect of acetic acid (Ac) and β-hydroxybutyric acid (BHBA) in comparison with the effect of various long-chain fatty acids on the mRNA expression of the lipogenic enzymes SCD, acetyl-CoA carboxylase (ACACA), fatty acid synthase (FASN) and their associated gene regulatory proteins sterol regulatory element binding transcription factor 1 (SREBF1), insulin-induced gene 1 protein (INSIG1) and peroxisome proliferator-activated receptor alpha (PPARA)and peroxisome proliferator-activated receptor delta (PPARD) by quantitative real-time PCR. MAC-T cells were treated for 12 h without FA additions (CON) or with either 5 mM Ac, 5 mM BHBA, a combination of 5 mM Ac + 5 mM BHBA, 100 μM C16:0, 100 μM C18:0, 100 μM C18:1 cis-9, 100 μM C18:1 trans-11, 100 μM C18:2 cis-9,12 or 100 μM C18:3 cis-9,12,15. Compared with control, mRNA expression of SCD1 was increased by Ac (+61%) and reduced by C18:1 cis-9 (−61%), C18:2 cis-9,12 (−84%) and C18:3 cis-9,12,15 (−88%). In contrast to native bovine mammary gland tissue, MAC-T cells did not express SCD5. Expression of ACACA was increased by Ac (+44%) and reduced by C18:2 cis-9,12 (−48%) and C18:3 cis-9,12,15 (−49%). Compared with control, FASN expression was not significantly affected by the treatments. The mRNA level of SREBF1 was not affected by Ac or BHBA, but was reduced by C18:1 cis-9 (−44%), C18:1 trans-11 (−42%), C18:2 cis-9,12 (−62%) and C18:3 cis-9,12,15 (−68%) compared with control. Expression of INSIG1 was downregulated by C18:0 (−37%), C18:1 cis-9 (−63%), C18:1 trans-11 (−53%), C18:2 cis-9,12 (−81%) and C18:3 cis-9,12,15 (−91%). Both PPARA and PPARD expression were not significantly affected by the treatments. Our results show that Ac upregulated mRNA expression of SCD1 and ACACA in MAC-T cells. The opposite effect of the PUFA C18:2 cis-9,12 and C18:3 cis-9,12,15 on the these genes and the failure of Ac to mimic the PUFA-inhibited SREBF1 and INSIG1 mRNA expression, suggest that Ac can stimulate mammary lipogenesis via a transcriptional regulatory mechanism different from PUFA.

The efficacy of conjugated linoleic acid (CLA) supplements containing trans-10, cis-12 for reducing milk fat synthesis has been well documented in dairy cows, but studies with other ruminant species are less convincing, and there have been no investigations of this in sheep. Therefore, the current study was designed to determine whether trans-10, cis-12 CLA would inhibit milk fat synthesis in sheep. Twenty multiparous ewes in early lactation were paired and randomly allocated to 2 treatments: grass hay plus concentrate either unsupplemented (control) or supplemented with lipid-encapsulated CLA to provide 2.4 g/d of trans-10, cis-12 CLA. The CLA dose was based on published responses of dairy cows extrapolated to ewes on a metabolic body weight basis. The experimental design was a 2-period crossover with 10-d treatment periods separated by a 10-d interval. Compared with the control, CLA supplementation reduced milk fat content from 6.4 to 4.9% and reduced fat yield from 95 to 80 g/d. The CLA treatment also increased milk yield from 1,471 to 1,611 g/d and increased protein yield from 68 to 73 g/d. Milk protein content and DMI were unaffected by treatment. The reduction in milk fat yield was due to decreases in both de novo fatty acid synthesis and uptake of preformed fatty acids. Milk fat content of trans-10, cis-12 CLA was < 0.01 and 0.12 g/100 g of fatty acids for the control and CLA treatments, respectively. The transfer efficiency of trans-10, cis-12 CLA from the dietary supplement into milk fat was 3.8%. Results of the present study demonstrate that a CLA supplement containing trans-10, cis-12 CLA reduces milk fat synthesis in lactating sheep in a manner similar to dairy cows when fed at an equivalent dose (metabolic body weight basis). Furthermore, the nutrients spared by the reduction in milk fat coincided with an increase in milk and milk protein yield.

The effect of conjugated linoleic acid (CLA) supplements containing trans-10, cis-12 for reducing milk fat synthesis has been well described in dairy cows and sheep. Studies on lactating goats, however, remain inconclusive. Therefore, the current study investigated the efficacy of a lipid-encapsulated trans-10, cis-12 CLA supplement (LE-CLA) on milk production and milk fatty acid profile in dairy goats. Thirty multiparous Alpine lactating goats in late lactation were used in a 3 x 3 Latin square design (14-d treatment periods separated by 14-d intervals). Does were fed a total mixed ration of Bermuda grass hay, dehydrated alfalfa pellets, and concentrate. Does were randomly allocated to 3 treatments: A) unsupplemented (control), B) supplemented with 30 g/d of LE-CLA (low dose; CLA-1), and C) supplemented with 60 g/d of LE-CLA (high dose; CLA-2). Milk yield, dry matter intake, and milk protein content and yield were unaffected by treatment. Compared with the control, milk fat yield was reduced 8% by the CLA-1 treatment and 21% by the CLA-2 treatment, with milk fat content reduced 5 and 18% by the CLA-1 and CLA-2 treatments, respectively. The reduction in milk fat yield was due to decreases in both de novo fatty acid synthesis and uptake of preformed fatty acids. Milk fat content of trans-10, cis-12 CLA was 0.03, 0.09, and 0.19 g/100 g of fatty acids for the control, CLA-1, and CLA-2 treatments, respectively. The transfer efficiency of trans-10, cis-12 CLA from the 2 levels of CLA supplement into milk fat was not different between treatments and averaged 1.85%. In conclusion, trans-10, cis-12 CLA reduced milk fat synthesis in lactating dairy goats in a manner similar to that observed for lactating dairy cows and dairy sheep. Dose-response comparisons, however, suggest that the degree of reduction in milk fat synthesis is less in dairy goats compared with dairy cows and dairy sheep.

Trans fatty acids (FA) arise in ruminant-derived foods as a consequence of rumen biohydrogenation and are of interest because of their biological effects and potential role in chronic human diseases. Our objective was to compare 2 trans FA, elaidic acid (EA; trans-9 18:1) and vaccenic acid (VA; trans-11 18:1), with oleic acid (OA; cis-9 18:1) relative to plasma lipid transport and mammary utilization for milk fat synthesis. Three ruminally cannulated, Holstein dairy cows, 259 ± 6 DIM (mean ± SEM), were randomly assigned in a 3 x 3 Latin square design. Treatments were a 4-d abomasal infusion of 1) OA (45.5 g/d), 2) EA (41.7 g/d), and 3) VA (41.4 g/d). Milk samples were collected at each milking and blood samples were collected at the start and end of each treatment period. The proportions of total plasma FA associated with each plasma lipid fraction at baseline (pretreatment) were 62.6 ± 0.6% phospholipids, 26.1 ± 0.6% cholesterol esters, 9.8 ± 0.4% triglycerides, and 1.5 ± 0.1% nonesterified fatty acids; these values were unaffected by treatment. There were striking differences in the FA composition of the individual plasma lipid fractions and in the distribution of specific 18-carbon FA among the lipid fractions. Infusion of treatment isomers caused their specific increase in the various plasma lipid fractions but had no effect on milk production variables, including milk fat yield and content. Transfer efficiency of infused OA, EA, and VA to milk fat averaged 65.5 ± 3.0%, 59.7 ± 1.5%, and 54.3 ± 0.6%, respectively. For the VA infusion, 24.6 ± 1.1% of the transfer was accounted for by the increased yield of cis-9, trans-11 conjugated linoleic acid in milk fat, consistent with its endogenous synthesis from VA via the mammary enzyme Δ⁹-desaturase. Notably, linoleic acid (18:2n-6) and linolenic acid (18:3n-3) accounted for 47.7% of total plasma FA, but only 2.6% of FA in milk. Overall, results demonstrate clear differences in plasma transport and mammary uptake and utilization of 18-carbon FA, and these relate to the location, orientation, and number of double bonds.