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Abstract Essential fatty acids (EFA) and conjugated linoleic acids (CLA) are unsaturated fatty acids with immune-modulatory effects, yet their synergistic effect is poorly understood in dairy cows. This study aimed at identifying differentially abundant proteins (DAP) and their associated pathways in dairy cows supplied with a combination of EFA and CLA during the transition from antepartum (AP) to early postpartum (PP). Sixteen Holstein cows were abomasally infused with coconut oil as a control (CTRL) or a mixture of EFA (linseed + safflower oil) and CLA (Lutalin, BASF) (EFA + CLA) from − 63 to + 63 days relative to parturition. Label-free quantitative proteomics was performed on plasma samples collected at days − 21, + 1, + 28, and + 63. During the transition time, DAP, consisting of a cluster of apolipoproteins (APO), including APOE, APOH, and APOB, along with a cluster of immune-related proteins, were related to complement and coagulation cascades, inflammatory response, and cholesterol metabolism. In response to EFA + CLA, specific APO comprising APOC3, APOA1, APOA4, and APOC4 were increased in a time-dependent manner; they were linked to triglyceride-enriched lipoprotein metabolisms and immune function. Altogether, these results provide new insights into metabolic and immune adaptation and crosstalk between them in transition dairy cows divergent in EFA + CLA status.

Abstract The impact of nutritional modification to increase functional polyunsaturated fatty acids (PUFA), such as n-3 and n-6 fatty acids (FA) or conjugated linoleic acid (CLA), on milk proteome profile during early lactation remains largely unknown. We used an untargeted proteomics approach to investigate the impact of lactation day and PUFA supplementation on the proteome signature in skimmed milk over the course of early lactation. Sixteen Holstein dairy cows received abomasal infusion of saturated FA (CTRL) or a mixture of essential FA and CLA (EFA + CLA group) from − 63 to + 63 days relative to parturition. Using quantitative proteomics, 479 unique proteins were identified in skimmed milk at days 1, 28, and 63 postpartum. The top discriminating proteins between transition milk (day 1) and mature milk (days 28 and 63), including members of complements (i.e. C2 and C5), growth factor (TGFB2), lipoproteins (i.e. APOE and APOD), and chaperones (i.e. ST13 and CLU), are associated with calves’ immune system and gut development. The EFA + CLA supplementation moderately affected a few proteins associated with regulating mammary glands’ lipogenesis through the (re)assembly of lipoprotein particles, possibly under the PPAR signaling pathway. Collectively, skimmed milk proteome is dynamically regulated initially by cow’s metabolic and physiological changes and to a lesser extent by nutritional PUFA modifications.

Fatty acids are known for their regulatory role in inflammation and oxidative stress. The present study investigated 38 calves born from dams, abomasally supplemented with coconut oil, essential fatty acids (EFA), conjugated linoleic acid (CLA) or EFA + CLA, according to immunological traits and the oxidative and anti-oxidative status for the first 5 days of life. On day 2 of life, plasma total bilirubin, cholesterol, interleukin 1-β and ferric ion reducing anti-oxygen power (FRAP) were lower in calves with than without maternal EFA supplementation, and FRAP additionally on day 4. On day 3, the concentrations of reactive oxygen metabolites were higher in calves with than without maternal EFA supplementation and additionally on day 5 together of retinol. Total leucocyte counts were decreased in the EFA group compared to the CLA group on day 5. Lymphocyte proportions decreased from day 1 to 5 only in the EFA + CLA group. On day 2, plasma total protein was higher in CLA and EFA + CLA than in EFA calves. Similarly, CLA calves had higher interleukin 1-β concentrations compared to EFA + CLA calves. FRAP was decreased by CLA on day 4. Overall, the maternal fatty acid supply affected the inflammatory response and the oxidative and anti-oxidative status of the neonatal offspring.

The overall goal of this study was to evaluate if intermediary energy metabolism of cows fed with trans-10, cis-12 conjugated linoleic acid (CLA) was modified such that milk-energy compounds were produced with less intermediary energy expenditure as compared to control cows. Published data on supplemented CLA were assembled. The extent was calculated to which the trans-10, cis-12 CLA isomer has an impact on glucose and energy conversion in the mammary gland by modifying glucose equivalent supply and energy required for fatty acid (FA) and fat synthesis, and if this will eventually lead to an improved glucose and energy status of CLA-supplemented high-yielding dairy cows. A possible relationship between CLA supplementation level and milk energy yield response was also studied. Calculations were conducted separately for orally and abomasally administered CLA and based on energy required for supply of glucose equivalents, i.e. lactose, glycerol and NADPH₂. Further, modifications of milk FA profile due to CLA supplementation were considered when energy expenditures for FA and fat synthesis were quantified. Differences in yields between control and CLA groups were transformed into glucose energy equivalents. Only abomasal infusion (r² = 0·31) but not oral CLA administration (r² = 0·11) supplementation to dairy cow diets resulted in less glucose equivalent energy. Modifications of milk FA profiles also saved energy but the relationship with CLA supplementation was weaker for abomasal infusion (r² = 0·06) than oral administration (r² = 0·38). On average, 10 g/d of abomasally infused trans-10, cis-12 CLA saved 1·1 to 2·3 MJ net energy expressed as glucose equivalents, whereas both positive and negative values were observed when the trans-10, cis-12 CLA was fed to the cows.This study revealed a weak to moderate dose-dependent relationship between the amount of trans-10, cis-12 CLA administered and the amount of energy in glucose equivalents and energy for the synthesis of milk fat conserved from milk ingredient synthesis. Because abomasal infusion of the trans-10, cis-12 CLA more consistently conserved energy in glucose equivalents compared with oral CLA intake, rumen protection of the fed CLA products appears incomplete. Milk fat synthesis showed an energy saving with a weak dose-dependent relationship when CLA was supplemented orally or by abomasal infusion.

Conjugated linoleic acids (CLAs) modulate the fatty acid composition in dairy cow milk, which represents the most important nutrient source of neonatal calves. In turn, dietary fatty acids are known to influence the gut microbiota. The current preliminary study investigated effects of a maternal fatty acid supplementation (MFAS) during transition period with coconut oil (CON, control), CLA (Lutalin®), or CLA + EFA (Lutalin® + essential fatty acids–linseed oil; safflower oil) on physico-chemical characteristics of jejunal content and microbiota of 5-day-old calves. MFAS of CLA + EFA increased α-linolenic, eicosapentaenoic, docosapentaenoic, and n-3 fatty acid proportions in jejunum compared to the other groups ( P < 0.05). Proportions of n-6 and polyunsaturated fatty acids increased by MFAS of CLA + EFA compared to CON ( P < 0.05). Most abundant phyla in the jejunum were Proteobacteria, Firmicutes , and Bacteroidota . CLA + EFA decreased the relative abundance of Diplorickettsiales ( Proteobacteria ) compared to CON and CLA ( P < 0.05). CLA calves showed a lower abundance of Enterobacterales ( Proteobacteria ) compared to CON calves ( P = 0.001). The abundance of Veillonellales-Selenomonadales and RF39 ( Firmicutes ) decreased in CLA + EFA calves compared to CON calves ( P < 0.05). Bacteroidales ( Bacteroidota ) decreased in CLA + EFA calves compared to CLA calves ( P < 0.05). The relative abundance of Cyanobacteria and Euryarchaeota decreased and the abundance of Chloroflexi increased in CLA + EFA calves compared to CON and CLA calves ( P < 0.05). MFAS alters the fatty acid composition and microbial milieu in the intestinal content of neonatal calves due to their ability to modulate colostral fatty acid composition of dams.

Conjugated linoleic acid (CLA) isomers are known for their health-promoting effects in mammals and metabolic functions in dairy cows and are synthesized in the forestomach depending on essential fatty acid (EFA) intake. The current preliminary study investigated effects of a maternal fatty acid supplementation (MFAS) during late pregnancy and early lactation with coconut oil (CON, control), CLA (Lutalin®), or CLA + EFA (Lutalin® linseed oil; safflower oil) on plasma fatty acid composition and T and B cell subsets in mesenteric lymph nodes (MLN) and the small intestine of 5-day-old calves. MFAS of CLA + EFA increased α-linolenic, eicosapentaenoic, docosapentaenoic, and n -3 fatty acid proportions in calf plasma fat on days 1 and 5 after birth ( P < 0.05). On day 5, CLA and CLA + EFA calves showed higher plasma fat trans -10, cis -12 CLA proportions, and CLA calves had higher plasma cis -9, trans -11 CLA proportions compared with CON calves ( P < 0.1). MFAS of CLA tended to increase CD4 + T cell subsets in MLN and increased CD21 + B cell subsets in ileal lamina propria compared with CON but decreased CD2 + T cell subsets in jejunal lamina propria ( P < 0.05). CLA + EFA decreased CD4 + T cell subsets in MLN compared with CLA ( P < 0.05). MFAS of CLA seemed to affect the intestinal adaptive immune system of calves, but additional EFA supplementations reversed CLA effects. Possible direct CLA and EFA effects or whether changes in milk composition affected this immune modulation must be clarified in further studies.

Abstract The effect of an exogenous amylase on postruminal digestion of starch infused into the abomasum of cattle was studied. Four rumen-cannulated heifers were fed 5.5 kg DM/d of a diet without starch, and assigned randomly to a crossover design. The experiment consisted of 2 periods lasting 23 d each with 10 d for adaptation to the diet followed by 13 d of abomasal infusion and sample collection. During the first 3 d of each infusion phase, isotonic saline solution was infused (1 liter/h) for measurement of baseline values in feces, followed by daily infusions of 880 g DM corn starch (1 kg/10 liters of water) without or with the addition of 2% of amylase. Titanium dioxide (10 g/d) was ruminally administered for estimation of fecal excretion. Digestion of starch in small intestine was calculated as the difference between the amounts of infused starch, disappeared from hindgut and fecal excretion. The apparent disappearance of starch from the hindgut was estimated based on the increment of microbial nitrogen (N) excretion due to starch infusion (1 g microbial N/100 g fermented starch) compared to baseline values. The concentration of purine bases in feces was used to estimate excretion of microbial N. Microbial N excretion increased with starch infusion (P < 0.05) but was not influenced by amylase (P = 0.81). Starch disappearance from the small intestine was not improved by amylase (P = 0.78) and averaged 85%. Amylase affected neither blood concentration of glucose (P = 0.80) nor of insulin (P = 0.26), but glucagon was lower without (P < 0.0001) than with amylase. The infusion of starch increased fecal excretion of total VFA (acetate, propionate, and butyrate) by 53% (P < 0.05), which indicates increased carbohydrate fermentation in the hindgut and incomplete digestion of starch in the small intestine. However, the excretion of total VFA was not affected by amylase (P = 0.66). Lactate excretion was higher at the second day of starch infusion (P < 0.05) without than with amylase, which suggests lower flow of starch from the small intestine to the hindgut due to a possible effect of amylase addition in animals not adapted to starch digestion. However, lactate excretion returned near to baseline values within 2 d, which was probably due to increase of lactate-utilizing bacteria and the adaptation of the microbial population in the hindgut. Further studies with higher starch levels and addition of amylase are recommended.

Phosphoproteomics is a cutting-edge technique that can be utilized to explore adipose tissue (AT) metabolism by quantifying the repertoire of phospho-peptides (PP) in AT. Dairy cows were supplemented with conjugated linoleic acid (CLA, n = 5) or a control diet (CON, n = 5) from 63 d prepartum to 63 d postpartum; cows were slaughtered at 63 d postpartum and AT was collected. We performed a quantitative phosphoproteomics analysis of subcutaneous (SC) and omental (OM) AT using nanoUPLC-MS/MS and examined the effects of CLA supplementation on the change in the phosphoproteome. A total of 5919 PP were detected in AT, and the abundance of 854 (14.4%) were differential between CON and CLA AT (p ≤ 0.05 and fold change ± 1.5). The abundance of 470 PP (7.9%) differed between OM and SC AT, and the interaction treatment vs. AT depot was significant for 205 PP (3.5% of total PP). The integrated phosphoproteome demonstrated the up- and downregulation of PP from proteins related to lipolysis and lipogenesis, and phosphorylation events in multiple pathways, including the regulation of lipolysis in adipocytes, mTOR signaling, insulin signaling, AMPK signaling, and glycolysis. The differential regulation of phosphosite on a serine residue (S777) of fatty acid synthase (FASN) in AT of CLA-supplemented cows was related to lipogenesis and with more phosphorylation sites compared to acetyl-coenzyme A synthetase (ACSS2). Increased protein phosphorylation was seen in acetyl-CoA carboxylase 1 (ACACA;8 PP), FASN (9 PP), hormone sensitive lipase (LIPE;6 PP), perilipin (PLIN;3 PP), and diacylglycerol lipase alpha (DAGLA;1 PP) in CLA vs. CON AT. The relative gene expression in the SC and OM AT revealed an increase in LIPE and FASN in CLA compared to CON AT. In addition, the expression of DAGLA, which is a lipid metabolism enzyme related to the endocannabinoid system, was 1.6-fold higher in CLA vs. CON AT, and the expression of the cannabinoid receptor CNR1 was reduced in CLA vs. CON AT. Immunoblots of SC and OM AT showed an increased abundance of FASN and a lower abundance of CB1 in CLA vs. CON. This study presents a complete map of the SC and the OM AT phosphoproteome in dairy cows following CLA supplementation and discloses many unknown phosphorylation sites, suggestive of increased lipid turnover in AT, for further functional investigation.

Diets of dairy cows are often based on maize silage (MS), delivering lower amounts of n-3 fatty acids (FA) compared to grass silage-based diets. The fatty acid composition of the cell membrane can affect the cell function. We evaluated the effects of an MS-based diet on bovine red blood cell (RBC) membrane FA composition and dietary effects on controlled ATP release of RBC. In trial 1, German Holstein cows were fed an MS-based total mixed ration for 24 weeks. The FA composition of RBC membranes from repeatedly taken blood samples was analysed in addition to the abundance of the RBC membrane protein flotillin-1, which is involved in, for example, cell signalling. In trial 2, four rumen fistulated MS-fed cows were abomasally infused in a 4 × 4 Latin square model with three successively increasing lipid dosages (coconut oil, linseed–safflower oil mix (EFA; rich in n-3 FA), Lutalin®, providing conjugated linoleic acids (CLA) or the combination of the supplements, EFA + CLA) for six weeks, followed by a three-week washout period. In trial 2, we analysed RBC ATP release, flotillin-1, and the membrane protein abundance of pannexin-1, which is involved in ATP release as the last part of a signalling cascade. In trial 1, the total amount of n-3 FA in RBC membranes decreased and the flotillin-1 abundance increased over time. In trial 2, the RBC n-3 FA amount was higher after the six-week infusion period of EFA or EFA + CLA. Furthermore, depending on the dosage of FA, the ATP release from RBC increased. The abundance of flotillin-1 and pannexin-1 was not affected in trial 2. It is concluded that changes of the membrane FA composition influence the RBC function, leading to altered ATP release from intact bovine RBC.