Mammary Fatty Acid Research Articles

Fatty Acid Desaturation Is Suppressed in Mir-26a/b Knockout Goat Mammary Epithelial Cells by Upregulating INSIG1.

MicroRNA-26 (miR-26a and miR-26b) plays a critical role in lipid metabolism, but its endogenous regulatory mechanism in fatty acid metabolism is not clear in goat mammary epithelial cells (GMECs). GMECs with the simultaneous knockout of miR-26a and miR-26b were obtained using the CRISPR/Cas9 system with four sgRNAs. In knockout GMECs, the contents of triglyceride, cholesterol, lipid droplets, and unsaturated fatty acid (UFA) were significantly reduced, and the expression of genes related to fatty acid metabolism was decreased, but the expression level of miR-26 target insulin-induced gene 1 (INSIG1) was significantly increased. Interestingly, the content of UFA in miR-26a and miR-26b simultaneous knockout GMECs was significantly lower than that in wild-type GMECs and miR-26a- and miR-26b-alone knockout cells. After decreasing INSIG1 expression in knockout cells, the contents of triglycerides, cholesterol, lipid droplets, and UFAs were restored, respectively. Our studies demonstrate that the knockout of miR-26a/b suppressed fatty acid desaturation by upregulating the target INSIG1. This provides reference methods and data for studying the functions of miRNA families and using miRNAs to regulate mammary fatty acid synthesis.

Fatty Acid Transfer from Blood to Milk Is Disrupted in Mothers with Low Milk Production, Obesity, and Inflammation

Obesity is associated with chronic inflammation and is a risk factor for insufficient milk production. Inflammation-mediated suppression of LPL could inhibit mammary uptake of long-chain fatty acids (LCFAs; >16 carbons). In an ancillary case-control analysis, we investigated whether women with low milk production despite regular breast emptying have elevated inflammation and disrupted transfer of LCFAs from plasma into milk. Data and specimens from a low milk supply study and an exclusively breastfeeding control group were analyzed, with milk production measured by 24-h test-weighing at 2-10 wk postpartum. Low milk supply groups were defined as very low (VL; <300mL/d; n=23) or moderate (MOD; ≥300mL/d; n=20) milk production, and compared with controls (≥699mL/d; n=18). Serum and milk fatty acids (weight% of total) were measured by GC, serum and milk TNF-α by ELISA, and serum high-sensitivity C-reactive protein (hsCRP) by clinical analyzer. Group differences were assessed by linear regression models, chi-square exact tests, and Kruskal-Wallis nonparametric tests. VL cases, as compared with MOD cases and controls, had higher prevalence of elevated serum hsCRP (>5mg/L; 57%, 15%, and 22%, respectively; P=0.004), detectable milk TNF-α (67%, 32%, and 33%, respectively; P=0.04), and obesity (78%, 40%, and 22%, respectively; P=0.003). VL cases had lower mean±SD LCFAs in milk (60%±3%) than MOD cases (65%±4%) and controls (66%±5%) (P<0.001). Milk and serum LCFAs were strongly correlated in controls (r=0.82, P<0.001), but not in the MOD (r=0.25, P=0.30) or VL (r=0.20, P=0.41) groups (Pint<0.001). Mothers with very low milk production have significantly higher obesity and inflammatory biomarkers, lower LCFAs in milk, and disrupted association between plasma and milk LCFAs. These data support the hypothesis that inflammation disrupts normal mammary gland fatty acid uptake. Further research should address impacts of inflammation and obesity on mammary fatty acid uptake for milk production.

Effects of dietary branched-chain amino acid supplementation on serum and milk metabolome profiles in dairy cows during early lactation.

The 3 branched-chain AA (BCAA), Val, Leu, and Ile, are essential AA used by tissues as substrates for protein synthesis and energy generation. In addition, BCAA are also involved in modulating cell signaling pathways, such as nutrient sensing and insulin signaling. In our previous study, dietary BCAA supplementation was shown to improve protein synthesis and glucose homeostasis in transition cows. However, a more detailed understanding of the changes in metabolic pathways associated with an increased BCAA availability is desired to fine-tune nutritional supplementation strategies. Multiparous Holstein cows (n = 20) were enrolled 28 d before expected calving and assigned to either the BCAA treatment (n = 10) or the control group (n = 10). Cows assigned to BCAA were fed 550 g/d of rumen-protected BCAA mixed with 200 g/d of dry molasses from calving until 35 DIM, whereas the cows assigned to the control were fed only 200 g/d of dry molasses. Serum samples were collected on d 10 before expected calving, as well as on d 4 and d 21 postpartum. Milk samples were collected on d 14 postpartum. From a larger cohort, we selected 20 BCAA-supplemented cows with the greatest plasma urea nitrogen concentration, as an indicator for greater BCAA availability, for the metabolomics analysis herein. Serum and milk samples were subjected to a liquid chromatography-mass spectrometry-based assay, detecting and measuring the abundance of 241 serum and 211 milk metabolic features, respectively. Multivariable statistical analyses revealed that BCAA supplementation altered the metabolome profiles of both serum and milk samples. Increased abundance of serum phosphocholine and glutathione and of milk Val, Ile, and Leu, and decreased abundance of milk acyl-carnitines were associated with BCAA supplementation. Altered phosphocholine and glutathione abundances point to altered hepatic choline metabolism and antioxidant balance, respectively. Altered milk acyl-carnitine abundances suggest changes in mammary fatty acid metabolism. Dietary BCAA supplementation was associated with a range of alterations in serum and milk metabolome profiles, adding to our understanding of the role of BCAA availability in modulating dairy cow protein, lipid, and energy metabolism on a whole-body level and how it affects milk composition.

Effect of lipid supplementation on the endogenous synthesis of milk cis-9,trans-11 conjugated linoleic acid in dairy sheep and goats: A tracer assay with 13C-vaccenic acid

A major proportion of milk rumenic acid (RA; cis-9,trans-11 CLA) is synthesized through mammary Δ9-desaturation of vaccenic acid (VA; trans-11 18:1). Diet composition may determine the relative contribution of this endogenous synthesis to milk RA content, with effects that might differ between ruminant species. However, this hypothesis is mostly based on estimated values, proxies of stearoyl-CoA desaturase (SCD) activity, and indirect comparisons between publications in the literature. With the aim of providing new insights into this issue, in vivo Δ9-desaturation of 13C-labeled VA (measured via milk 13C-VA and -RA secretion) was directly compared in sheep and goats fed a diet without lipid supplementation or including 2% of linseed oil. Four Assaf sheep and 4 Murciano-Granadina goats were used in a replicated 2 × 2 crossover design to test the effects of the 2 dietary treatments during 2 consecutive 25-d periods. On d 22 of each period, 500 mg of 13C-VA were i.v. injected to each animal. Dairy performance, milk fatty acid profile, including isotope analysis, and mammary mRNA abundance of genes coding for SCD were examined on d 21 to 25 of each period. Supplementation with linseed oil improved milk fat concentration and increased the content of milk VA and RA. However, the isotopic tracer assay suggested no variation in the relative proportion of VA desaturated to milk RA, and the percentage of this CLA isomer deriving from SCD activity would remain constant regardless of dietary treatment. These results put into question a major effect of lipid supplementation on the endogenous synthesis of milk RA and support that mammary Δ9-desaturation capacity would not represent a limiting factor when designing feeding strategies to increase milk RA content. The lack of diet-induced effects was common to caprines and ovines, but inherent interspecies differences in mammary lipogenesis were found. Thus, the higher proportions of VA desaturation and endogenous synthesis of milk RA in sheep supported a greater SCD activity compared with goats, a finding that was not associated with the similar mRNA abundance of SCD1 in the 2 species. On the other hand, transfer efficiency of the isotopic tracer to milk was 37% higher in caprine than in ovine, suggesting a greater efficiency in mammary fatty acid uptake from plasma in caprine.

Comprehensive Transcriptome Profiling of Dairy Goat Mammary Gland Identifies Genes and Networks Crucial for Lactation and Fatty Acid Metabolism.

Milk fatty acids secreted by the mammary gland are one of the most important determinants of the nutritional value of goat milk. Unlike cow milk, limited data are available on the transcriptome-wide changes across stages of lactation in dairy goats. In this study, goat mammary gland tissue collected at peak lactation, cessation of milking, and involution were analyzed with digital gene expression (DGE) sequencing to generate longitudinal transcript profiles. A total of 51,299 unigenes were identified and further annotated to 12,763 genes, of which 9,131 were differentially expressed across various stages of lactation. Most abundant genes and differentially expressed genes (DEGs) were functionally classified through clusters of euKaryotic Orthologous Groups (KOG), Gene Ontology (GO), and Kyoto Encyclopedia of Genes and Genomes (KEGG) databases. A total of 16 possible expression patterns were uncovered, and 13 genes were deemed novel candidates for regulation of lactation in the goat: POLG, SPTA1, KLC, GIT2, COPS3, PDP, CD31, USP16/29/37, TLL1, NCAPH, ABI2, DNAJC4, and MAPK8IP3. In addition, PLA2, CPT1, PLD, GGA, SRPRB, and AP4S1 are proposed as novel and promising candidates regulating mammary fatty acid metabolism. “Butirosin and neomycin biosynthesis” and “Glyoxylate and dicarboxylate metabolism” were the most impacted pathways, and revealed novel metabolic alterations in lipid metabolism as lactation progressed. Overall, the present study provides new insights into the synthesis and metabolism of fatty acids and lipid species in the mammary gland along with more detailed information on molecular regulation of lactogenesis. The major findings will benefit efforts to further improve milk quality in dairy goats.

Short communication: Effect of supplemental zinc source with and without evaporative cooling on systemic and mammary metabolism of lactating dairy cows during summer

The negative effects of heat stress partly result from disturbed systemic metabolic responses and possibly altered mammary gland metabolism of lactating dairy cows. Our previous research reported that supplemental dietary Zn sources may affect milk fat synthesis of lactating cows during summer. Thus, our objective was to evaluate the systemic and mammary metabolism of cows fed 2 supplemental Zn sources under 2 environmental conditions. Multiparous lactating Holstein cows (n = 72; days in milk: 99.7 ± 13.4 d; parity: 2.9 ± 0.3) were randomly assigned to 4 treatments in a 2 × 2 factorial arrangement. Treatments included 2 different environments: cooled (CL) using fans and misters or noncooled (NC), and 2 supplemental Zn sources: 75 mg of Zn hydroxychloride/kg of DM (IOZ) or 35 mg of Zn hydroxychloride/kg of DM + 40 mg of Zn-Met complex/kg of DM (ZMC). The 168-d experiment was divided into baseline and environmental challenge phases, 84 d each. During the baseline phase, all cows were cooled and fed respective dietary treatments, and during the environmental challenge phase cows continued receiving the same diets but NC cows were deprived of cooling. Temperature-humidity index averaged 77.6 ± 3.8 and 77.8 ± 3.8 for CL and NC pens, respectively, during the environmental challenge phase. Plasma was collected before the baseline phase and at 1, 3, 5, 12, 22, 26, 41, 54, 61, 68, 75, and 81 d of the environmental challenge phase for metabolites and insulin analyses. Mammary biopsies were collected before the baseline phase and at 7 and 56 d of the environmental challenge phase to measure mRNA abundance of proteins related to mammary metabolism. Compared with CL, NC reduced plasma glucose, nonesterified fatty acids, β-hydroxybutyrate, and triglyceride concentrations, but increased insulin concentration. Cows fed ZMC had greater plasma triglyceride concentration than IOZ. Treatments had no effect on mRNA abundance of protein related to mammary fatty acid and glucose metabolism except that NC cows had greater mammary mRNA abundance of 6-phosphogluconate dehydrogenase and ATP-dependent 6-phosphofructokinase than CL cows. In conclusion, deprivation of evaporative cooling influenced the metabolism of lactating dairy cows but dietary Zn source had no apparent effect.

Branched chain amino acids alter fatty acid profile in colostrum of sows fed a high fat diet

BackgroundBranched chain amino acids (BCAAs) are important substrates for milk protein synthesis in the mammary gland, and are tightly related to lipid metabolism. No study has been performed examining the role of BCAAs with high fat diets on milk fat synthesis. This study was designed to investigate the effect of dietary BCAAs on growth performance of piglets, progeny body weight, and milk fat composition in sows fed a high fat diet. Four diets (CON = control diet; HF = high fat diet with 8% soybean oil; HF-MB=HF plus 0.39% BCAAs; HF-HB=HF plus 0.78% BCAAs) were fed to sows from late gestation to weaning.ResultsCompared to HF, BCAAs (HF-MB and HF-HB) increased the litter weight (P < 0.05) and overall litter weight gain (P < 0.05) at weaning and increased colostrum fat content by 27.3–35.8% (P < 0.01). Fatty acid profiles between the two doses of BCAAs were similar. Compared with HF, HF-MB tended to decrease the percentage of C18:3n3 (P = 0.063) and increased the percentage of C18:1n9c (P = 0.03). In addition, BCAAs in HF-MB increased the concentration of total fatty acid by 22.1% in colostrum (P = 0.03) but decreased that in serum at parturition by 53.2% (P = 0.027). The fatty acids in colostrum that increased with BCAAs were C15:0, C17:0, C20:3n6, C20:4n6, C20:5n3 and C22:6n3 (P = 0.00~0.04). Colostrum fatty acids of C20:0, C21:0, C22:0, C16:1, C20:1, C18:1n9c also tended to be increased (0.05 < P < 0.1) with BCAAs. The change in sow serum fatty acid profile due to BCAAs was different from that in colostrum.ConclusionsBCAAs in high fat diet of sows altered the fatty acid composition in colostrum and enhanced litter growth. Our study indicated that BCAAs supplementation can enhance mammary fatty acid uptake and mammary fat synthesis and that supplemental BCAAs and fat in late gestation and lactation diets for sows can improve reproductive performance.

MiR-497 regulates fatty acid synthesis via LATS2 in bovine mammary epithelial cells.

Both mRNA and miRNA play an important role in the regulation of mammary fatty acid metabolism and milk fat synthesis. Although studies have shown a strong transcriptional control of fatty acid metabolism, less is known about the regulatory mechanisms of milk fat synthesis as a function of miRNA-mRNA interactions. In this study, we carried out transcriptome sequencing using mammary tissues from the early lactation period, peak lactation, mid-lactation and late lactation in dairy cows and identified key genes regulating milk fatty acid metabolism. A total of 32 differentially co-expressed gene were screened out. Large tumor suppressor kinase 2 (LATS2) was chosen for further study using luciferase reporter assays, qRT-PCR and western blotting. The aim was to demonstrate that miR-497 is an upstream regulator of LATS2, i.e. miR-497 and LATS2 are a potential miRNA/mRNA regulatory pair. The results indicated that miR-497 could inhibit the production of triglycerides (TAG) and unsaturated fatty acids in bovine mammary epithelial cells (BMECs). In contrast, LATS2 can promote the production of TAG and unsaturated fatty acids. "Rescue" experiments further verified the miR-497/LATS2 regulatory network. Overall, data underscored that the miR-497/LATS2 pathway exerts control on milk fat metabolism and provides a theoretical approach for improving milk quality via genetic means.

Regulation of Stearoyl-Coenzyme A Desaturase 1 by trans-10,cis-12 Conjugated Linoleic Acid via SREBP1 in Primary Goat Mammary Epithelial Cells

trans-10, cis-12 Conjugated linoleic acid (t10c12-CLA) is a biohydrogenation intermediate in the rumen that inhibits mammary fatty acid de novo synthesis in lactating dairy goats. However, the underlying molecular pathways in milk-lipid metabolism affected by t10c12-CLA are not completely understood. The present study investigated the lipid-regulation mechanisms in goat mammary epithelial cells (GMECs) in response to t10c12-CLA. Gene-expression analysis indicated sterol-regulatory-element-binding transcription factor1 ( SREBF1) and its putative target gene stearoyl-CoA desaturase ( SCD1) were down-regulated (fold changes of 0.33 ± 0.04, P < 0.05, and 0.19 ± 0.01, P < 0.01, respectively). Concentrations of cellular palmitoleic acid (C16:1) and oleic acid (C18:1) were decreased (1.12 ± 0.05 vs 1.69 ± 0.11% and 15.70 ± 0.44 vs 24.97 ± 0.82%, respectively, P < 0.01), whereas those of linoleic acid (C18:2) were increased (5.00 ± 0.14 vs 3.81 ± 0.25%, P < 0.05); the desaturation indices of C16 and C18 were decreased in response to t10c12-CLA treatment (6.90 ± 0.05 vs 8.00 ± 0.30% and 61.41 ± 0.65 vs 67.73 ± 1.33%, respectively, P < 0.05). A luciferase-activity assay indicated that deletion of the sterol-response-element (SRE) site and the nuclear-factor (NF-Y) site in the SCD1-promoter region (-511/+65 bp) suppressed the regulatory effect of t10c12-CLA. Overexpression of SREBF1 partly counteracted the inhibitory effect of t10c12-CLA on de novo fatty acid synthesis. Overall, t10c12-CLA causes an inhibition of fatty acid synthesis and desaturation and regulates SCD1 expression by affecting the binding of SREBP1 protein to the SRE and NF-Y sites.

Dog mammary tumour phospholipid fatty acid profile: Possible implications for tumour grading and prognosis

Dog mammary tumour phospholipid fatty acid profile: Possible implications for tumour grading and prognosis

Investigating mutual relationship among milk fatty acids by multivariate factor analysis in dairy cows

The interpretation of mutual relationship among milk fatty acids (FA) is not easy due to the high number of FA contained in milk fat and to the high degree of correlation among them. Multivariate analysis includes different statistical approaches that could help explaining complex pattern of variables. In this study, Multivariate Factor Analysis (MFA) was used to decompose the correlation matrix of 47 FA and milk production traits (milk yield and protein and fat content) measured in 300 Italian Holstein Friesian cows reared in the North of Italy in 23 commercial dairy farms, representative of the intensive dairy system. MFA was able to extract seven latent factors with specific biologic meaning: secretion of Long Chain FA (KLCFA), mammary FA de novo synthesis (Km), rumen biohydrogenation (Kbh), synthesis of odd chain FA (Ko), synthesis of branched chain FA (Kb), mammary desaturation activity (Kd), milk yield (Kmy). According to the pattern of communalities of the factor analysis, C18:3c9c12c15 was the only FA, along with C18:2t11c15, to be uncorrelated with the other variables and it seemed to be excluded by the metabolic pattern described by the seven factors. The desaturation products of the SCD enzyme were independently associated to three latent factors, suggesting new insights in the regulation of SCD activity. Factors were considered as new quantitative phenotypes related to prominent features of milk FA profile. With the aim of evaluating the feeding regimen and animal effects, latent factors were analysed with a mixed model, which considered the fixed effect of lactation stage, parity, some feeding regimen characteristics and the random effect of bull. Lactation stage significantly affected Km and Kmy factors. In perspective, the seven factors extracted by applying MFA analysis to milk FA composition could be considered as new and more informative traits to test the effect of endogenous and exogenous variation factors.

Genetic and environmental relationships of detailed milk fatty acids profile determined by gas chromatography in Brown Swiss cows

The aim of this study was to characterize the profile of 47 fatty acids, including conjugated linoleic acid (CLA), 13 fatty acid groups, and 5 Δ9-desaturation indices in milk samples from Brown Swiss cows. The genetic variation was assessed and the statistical relevance of the genetic background for each trait was evaluated using the Bayes factor test. The additive genetic, herd-date, and residual relationships were also estimated among all single fatty acids and groups of fatty acids. Individual milk samples were collected from 1,158 Italian Brown Swiss cows and a detailed analysis of fat percentages and milk fatty acid compositions was performed by gas chromatography. Bayesian animal models were used for (co)variance components estimation. Exploitable genetic variation was observed for most of the de novo synthesized fatty acids and saturated fatty acids, except for C4:0 and C6:0, whereas long-chain fatty acids and unsaturated fatty acids (including CLA) were mainly influenced by herd-date effects. Herd-date effect explained large portions of the total phenotypic variance for C18:2 cis-9,cis-12 (0.668), C18:3 cis-9,cis-12,cis-15 (0.631), and the biohydrogenation and elongation products of these fatty acids. The desaturation ratios showed higher heritability estimates than the individual fatty acids, except for CLA desaturation index (0.098). Among the medium-chain fatty acids, C12:0 had greater heritability than C14:0 (0.243 vs. 0.097, respectively). Both C14:0 and C16:0 showed negative additive genetic correlations with the main monounsaturated and polyunsaturated fatty acids of milk fat, suggesting that their synthesis in the mammary gland may be influenced by the presence of unsaturated fatty acids. No correlation was observed between C4:0 and the other short-chain fatty acids (except for C6:0), confirming the independence of C4:0 from de novo mammary fatty acid synthesis. Among the genetic correlations dealing with potentially beneficial fatty acids, C18:0 was positively correlated with vaccenic and rumenic acids and negatively with linoleic acid. Finally, fatty acids C6:0 through C14:0 showed relevant correlations due to unknown environmental effects, suggesting the potential existence of genetic variances in micro-environmental sensitivity. This study allowed us to acquire new knowledge about the genetic and the environmental relationships among fatty acids. Likewise, the existence of genetic variation for most of de novo synthetized fatty acids and saturated fatty acids was also observed. Overall, these results provide useful information to combine feeding with genetic selection strategies for obtaining a desirable milk fatty acids profile, depending on the origin of fatty acids in milk.

Perspective on calf and mammary gland development through changes in the bovine milk proteome over a complete lactation

Milk contains all the nutrients for the growth and development of the neonate. However, milk composition is not constant during lactation. To study the changes of the milk proteome over lactation, filter-aided sample preparation combined with dimethyl labeling followed by liquid chromatography tandem mass spectrometry was used to identify and quantify milk proteins from 4 cows. A total of 229 proteins were identified, of which 219 were quantified. An 80% overlap was found in identified and quantified proteins between the 4 individual cows during lactation. Over lactation, the number of quantified proteins changed slightly (less than 10%), whereas the concentration of proteins changed considerably. Transport proteins involved in lipid synthesis (fatty acid–binding protein, perilipin-2, butyrophilin) increased, whereas proteins related to cholesterol transport (apolipoprotein E) decreased. The changes of lipid synthesis proteins are in accordance with the increased milk fat yield over lactation, indicating the increase of de novo mammary fatty acid synthesis as lactation advances. The high abundance of immune-related proteins in early lactation indicates the important role of these proteins for immune system development of calves. The increase in immune-related proteins (immunoglobulins, osteopontin, lactoferrin) and the decrease of proteins related to milk component synthesis (α-lactalbumin, β-lactoglobulin, fatty acid–binding protein, perilipin-2, butyrophilin) in late lactation can be associated with the protection of the mammary gland. In conclusion, the changes of proteins with different biological functions reflect not only the changing needs of calves but also the development and protection of the mammary gland over lactation.

Effect of forage conservation method on plasma lipids, mammary lipogenesis, and milk fatty acid composition in lactating cows fed diets containing a 60:40 forage-to-concentrate ratio

The effects of forage conservation method on plasma lipids, mammary lipogenesis, and milk fat were examined in 2 complementary experiments. Treatments comprised fresh grass, hay, or untreated (UTS) or formic acid treated silage (FAS) prepared from the same grass sward. Preparation of conserved forages coincided with the collection of samples from cows fed fresh grass. In the first experiment, 5 multiparous Finnish Ayrshire cows (229 d in milk) were used to compare a diet based on fresh grass followed by hay during 2 consecutive 14-d periods, separated by a 5-d transition during which extensively wilted grass was fed. In the second experiment, 5 multiparous Finnish Ayrshire cows (53 d in milk) were assigned to 1 of 2 blocks and allocated treatments according to a replicated 3×3 Latin square design, with 14-d periods to compare hay, UTS, and FAS. Cows received 7 or 9kg/d of the same concentrate in experiments 1 and 2, respectively. Arterial concentrations of triacylglycerol (TAG) and phospholipid were higher in cows fed fresh grass, UTS, and FAS compared with hay. Nonesterified fatty acid (NEFA) concentrations and the relative abundance of 18:2n-6 and 18:3n-3 in TAG of arterial blood were also higher in cows fed fresh grass than conserved forages. On all diets, TAG was the principle source of fatty acids (FA) for milk fat synthesis, whereas mammary extraction of NEFA was negligible, except during zero-grazing, which was associated with a lower, albeit positive calculated energy balance. Mammary FA uptake was higher and the synthesis of 16:0 lower in cows fed fresh grass than hay. Conservation of grass by drying or ensiling had no influence on mammary extraction of TAG and NEFA, despite an increase in milk fat secretion for silages compared with hay and for FAS than UTS. Relative to hay, milk fat from fresh grass contained lower 12:0, 14:0, and 16:0 and higher S3,R7,R11,15-tetramethyl-16:0, cis-9 18:1, trans-11 18:1, cis-9,trans-11 18:2, 18:2n-6, and 18:3n-3 concentrations. Even though conserved forages altered mammary lipogenesis, differences in milk FA composition were relatively minor, other than a higher enrichment of S3,R7,R11,15-tetramethyl-16:0 in milk from silages compared with hay. In conclusion, differences in milk fat composition on fresh grass relative to conserved forages were associated with a lower energy balance, increased uptake of preformed FA, and decreased synthesis of 16:0 de novo in the mammary glands, in the absence of alterations in stearoyl-coenzyme A desaturase activity.

Effect of dietary fat blend enriched in oleic or linoleic acid and monensin supplementation on dairy cattle performance, milk fatty acid profiles, and milk fat depression

The effect of feeding increasing levels of oleic and linoleic acid both independently and together, with or without monensin, on milk fat depression was evaluated. Fifty-six Holstein cows were blocked by parity and then were divided by milk production into 2 groups (high or low) of 14 cows each within each parity block. A cow pair of 1 high and 1 low production cow within each parity block was fed in a single electronic feeding gate. Gates (n=28) were considered the experimental unit and were assigned to monensin (17.5g/t of dry matter) or control as the main plot (n=14 each). The 7 cow pairs in each of the fixed effect groups were further assigned to a sequence of fat blend diets as split plot. Seven fat blend treatments in the split plot 7×7 Latin square were no added fat (no fat) and diets with increasing levels of oleic or linoleic acid: low C18:1+low C18:2 (LOLL); low C18:1+medium C18:2 (LOML); low C18:1+high C18:2 (LOHL); medium C18:1+low C18:2 (MOLL); medium C18:1+medium C18:2 (MOML); and high C18:1+low C18:2 (HOLL). Monensin feeding did not affect milk yield or concentration and yield of milk fat. Feeding monensin decreased the proportion of C <16, increased the proportion of total C18, increased the proportion and yield of trans-10 C18:1, and increased the proportion of trans-10,cis-12 conjugated linoleic acid in milk fatty acids (FA). As dietary C18:1 or C18:2 increased beyond the concentration present in LOLL, milk fat concentration, milk fat yield, and proportion and yield of milk C <16 all decreased, and the proportion and yield of milk trans-10 C18:1 increased. A quadratic effect on milk fat concentration and yield was noticed for C18:2 feeding, but not for C18:1 feeding. When dietary contents of total FA and FA other than C18:1 and C18:2 were similar, C18:2-rich diets decreased milk fat concentration and yield compared with C18:1-rich diets (LOML vs. MOLL, and LOHL vs. HOLL), indicating that C18:2 is more potent than C18:1 for depressing milk fat. Increasing dietary FA content from no fat to LOLL, which increased primarily C18:1 and C18:2 with small increases in C18:0 and C16:0, decreased the secretion of C <16 but increased total C18 secretion in milk. This suggests that biohydrogenation intermediates act to decrease mammary FA synthesis at low levels of added C18:1 and C18:2. No significant monensin × fat interactions were detected for the milk composition parameters analyzed; however, a monensin × fat interaction was found for milk fat trans-10 C18:1 proportion.

Role of trans fatty acids in the nutritional regulation of mammary lipogenesis in ruminants

Fat is an important constituent contributing to the organoleptic, processing and physical properties of ruminant milk. Understanding the regulation of milk fat synthesis is central to the development of nutritional strategies to enhance the nutritional value of milk, decrease milk energy secretion and improve the energy balance of lactating ruminants. Nutrition is the major environmental factor regulating the concentration and composition of fat in ruminant milk. Feeding low-fibre/high-starch diets and/or lipid supplements rich in polyunsaturated fatty acids induce milk fat depression (MFD) in the bovine, typically increase milk fat secretion in the caprine, whereas limited data in sheep suggest that the responses are more similar to the goat than the cow. Following the observation that reductions in milk fat synthesis during diet-induced MFD are associated with increases in the concentration of specific trans fatty acids in milk, the biohydrogenation theory of MFD was proposed, which attributes the causal mechanism to altered ruminal lipid metabolism leading to increased formation of specific biohydrogenation intermediates that exert anti-lipogenic effects. Trans-10, cis-12 conjugated linoleic acid (CLA) is the only biohydrogenation intermediate to have been infused at the abomasum over a range of experimental doses (1.25 to 14.0 g/day) and shown unequivocally to inhibit milk fat synthesis in ruminants. However, increases in ruminal trans-10, cis-12 CLA formation do not explain entirely diet-induced MFD, suggesting that other biohydrogenation intermediates and/or other mechanisms may also be involved. Experiments involving abomasal infusions (g/day) in lactating cows have provided evidence that cis-10, trans-12 CLA (1.2), trans-9, cis-11 CLA (5.0) and trans-10 18:1 (92.1) may also exert anti-lipogenic effects. Use of molecular-based approaches have demonstrated that mammary abundance of transcripts encoding for key lipogenic genes are reduced during MFD in the bovine, changes that are accompanied by decrease in sterol response element binding protein 1 (SREBP1) and alterations in the expression of genes related to the SREBP1 pathway. Recent studies indicate that transcription of one or more adipogenic genes is increased in subcutaneous adipose tissue in cows during acute or chronic MFD. Feeding diets of similar composition do not induce MFD or substantially alter mammary lipogenic gene expression in the goat. The available data suggests that variation in mammary fatty acid secretion and lipogenic responses to changes in diet composition between ruminants reflect inherent interspecies differences in ruminal lipid metabolism and mammary specific regulation of cellular processes and key lipogenic enzymes involved in the synthesis of milk fat triacylglycerides.

Recent advances in the regulation of milk fat synthesis

In addition to its economic value, milk fat is responsible for many of milk’s characteristics and can be markedly affected by diet. Diet-induced milk fat depression (MFD) was first described over a century ago and remains a common problem observed under both intensive and extensive management. The biohydrogenation theory established that MFD is caused by an inhibition of mammary synthesis of milk fat by specific fatty acids (FA) produced as intermediates in ruminal biohydrogenation. During MFD, lipogenic capacity and transcription of key lipid synthesis genes in the mammary gland are down-regulated in a coordinated manner. Our investigations have established that expressions of sterol response element-binding protein 1 (SREBP1) and SREBP-activation proteins are down-regulated during MFD. Importantly, key lipogenic enzymes are transcriptionally regulated via SREBP1. Collectively, these results provide strong evidence for SREBP1 as a central signaling pathway in the regulation of mammary FA synthesis. Spot 14 is also down-regulated during MFD, consistent with a lipogenic role for this novel nuclear protein. In addition, SREBP1c and Spot 14 knock-out mice exhibit reduced milk fat similar to the magnitude and pattern of MFD in the cow. Application of molecular biology approaches has provided the latest chapter in the regulation of milk fat synthesis and is reviewed along with a brief background in nutritional regulation of milk fat synthesis in ruminants.

Long-Chain Fatty Acid Metabolism in Dairy Cows: A Meta-Analysis of Milk Fatty Acid Yield in Relation to Duodenal Flows and De Novo Synthesis

This study is a meta-analysis of the response of milk long-chain fatty acid (FA) yield and composition to lipid supply, based on published experiments reporting duodenal FA flows or duodenal lipid infusions and milk FA composition (i.e., 39 experiments reporting 139 experimental treatments). Analysis of these data underlined the interdependence between milk yields of C18 and short- and medium-chain (C4 to C16) FA. Lipid supplementation (producing an increase in duodenal C18 flow) decreased linearly milk C4 to C16 yield (−0.26g of C4 to C16 produced per gram of duodenal C18 flow increase) and increased quadratically milk C18 yield. When these 2 effects increased the percentage of C18 in milk FA up to a threshold value (around 52% of total FA), then milk C18 yield was limited by C4 to C16 yield, decreasing the C18 transfer efficiency from duodenum to milk with high-lipid diets. Moreover, for a given duodenal C18 flow, a decrease in milk C4 to C16 yield induced a decrease in milk C18 yield. Despite high variations in C18 transfer efficiency between duodenum and milk, for a given experimental condition, the percentages of C18 FA in milk total C18 could be predicted from their percentages in duodenal C18, and the percentages at the duodenum and in milk were very similar when mammary desaturation was taken into account (i.e., considering the sums of substrates and products of mammary desaturase). The estimated amounts of 18:0, trans-11–, and trans-13–18:1 desaturated by the mammary gland were a linear function of their mammary uptake, and mammary desaturation was responsible for 80, 95, and 81%, respectively, of the yield of their products (i.e., cis-9–18:1; cis-9, trans-11–, and cis-9, trans-13–18:2). Thus, mammary FA desaturation capacity did not seem to be a limiting factor in the experimental conditions published so far.

Regulation of Fat Synthesis by Conjugated Linoleic Acid: Lactation and the Ruminant Model , ,

Conjugated linoleic acid (CLA) isomers effect an impressive range of biological processes including the ability to inhibit milk fatty acid synthesis. Although this has been demonstrated in several mammals, research has been most extensive with dairy cows. The first isomer shown to affect milk fat synthesis during lactation was trans-10, cis-12 CLA, and its effects have been well characterized including dose-response relationships. Recent studies have tentatively identified 2 additional CLA isomers that regulate milk fat synthesis. Regulation by CLA occurs naturally in dairy cows when specific CLA isomers produced as intermediates in rumen biohydrogenation act to inhibit milk fat synthesis; this physiological example of nutritional genomics is referred to as diet-induced milk fat depression. Molecular mechanisms for the reduction in mammary lipid synthesis involve a coordinated down-regulation of mRNA expression for key lipogenic enzymes associated with the complementary pathways of milk fat synthesis. Results provide strong evidence of a role for sterol response element-binding protein 1 and Spot 14 in this translational regulation. Effects of CLA on body fat accretion have also been investigated in nonlactating animals, but CLA effects on mammary fatty acid synthesis occur at an order-of-magnitude lower dose and appear to involve very different mechanisms than those proposed for the antiobesity effects of CLA. Overall, results demonstrate the unique value of cows as a model to investigate the role of CLA in the regulation of milk fat synthesis during lactation.

Gene networks driving bovine milk fat synthesis during the lactation cycle

BackgroundThe molecular events associated with regulation of milk fat synthesis in the bovine mammary gland remain largely unknown. Our objective was to study mammary tissue mRNA expression via quantitative PCR of 45 genes associated with lipid synthesis (triacylglycerol and phospholipids) and secretion from the late pre-partum/non-lactating period through the end of subsequent lactation. mRNA expression was coupled with milk fatty acid (FA) composition and calculated indexes of FA desaturation and de novo synthesis by the mammary gland.ResultsMarked up-regulation and/or % relative mRNA abundance during lactation were observed for genes associated with mammary FA uptake from blood (LPL, CD36), intracellular FA trafficking (FABP3), long-chain (ACSL1) and short-chain (ACSS2) intracellular FA activation, de novo FA synthesis (ACACA, FASN), desaturation (SCD, FADS1), triacylglycerol synthesis (AGPAT6, GPAM, LPIN1), lipid droplet formation (BTN1A1, XDH), ketone body utilization (BDH1), and transcription regulation (INSIG1, PPARG, PPARGC1A). Change in SREBF1 mRNA expression during lactation, thought to be central for milk fat synthesis regulation, was ≤2-fold in magnitude, while expression of INSIG1, which negatively regulates SREBP activation, was >12-fold and had a parallel pattern of expression to PPARGC1A. Genes involved in phospholipid synthesis had moderate up-regulation in expression and % relative mRNA abundance. The mRNA abundance and up-regulation in expression of ABCG2 during lactation was markedly high, suggesting a biological role of this gene in milk synthesis/secretion. Weak correlations were observed between both milk FA composition and desaturase indexes (i.e., apparent SCD activity) with mRNA expression pattern of genes measured.ConclusionA network of genes participates in coordinating milk fat synthesis and secretion. Results challenge the proposal that SREBF1 is central for milk fat synthesis regulation and highlight a pivotal role for a concerted action among PPARG, PPARGC1A, and INSIG1. Expression of SCD, the most abundant gene measured, appears to be key during milk fat synthesis. The lack of correlation between gene expression and calculated desaturase indexes does not support their use to infer mRNA expression or enzyme activity (e.g., SCD). Longitudinal mRNA expression allowed development of transcriptional regulation networks and an updated model of milk fat synthesis regulation.