Laurence Bernard

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.

Expression and Nutritional Regulation of Lipogenic Genes in the Ruminant Lactating Mammary Gland

The effect of nutrition on milk fat yield and composition has largely been investigated in cows and goats, with some differences for fatty acid (FA) composition responses and marked species differences in milk fat yield response. Recently, the characterization of lipogenic genes in ruminant species allowed in vivo studies focused on the effect of nutrition on mammary expression of these genes, in cows (mainly fed milk fat-depressing diets) and goats (fed lipid-supplemented diets). These few studies demonstrated some similarities in the regulation of gene expression between the two species, although the responses were not always in agreement with milk FA secretion responses. A central role for trans-10 C18:1 and trans-10, cis-12 CLA as regulators of milk fat synthesis has been proposed. However, trans-10 C18:1 does not directly control milk fat synthesis in cows, despite the fact that it largely responds to dietary factors, with its concentration being negatively correlated with milk fat yield response in cows and, to a lesser extent, in goats. Milk trans-10, cis-12 CLA is often correlated with milk fat depression in cows but not in goats and, when postruminally infused, acts as an inhibitor of the expression of key lipogenic genes in cows. Recent evidence has also proven the inhibitory effect of the trans-9, cis-11 CLA isomer. The molecular mechanisms by which nutrients regulate lipogenic gene expression have yet to be well identified, but a central role for SREBP-1 has been outlined as mediator of FA effects, whereas the roles of PPARs and STAT5 need to be determined. It is expected that the development of in vitro functional systems for lipid synthesis and secretion will allow future progress toward (1) the identification of the inhibitors and activators of fat synthesis, (2) the knowledge of cellular mechanisms, and (3) the understanding of differences between ruminant species.

Effect of sunflower-seed oil and linseed oil on tissue lipid metabolism, gene expression, and milk fatty acid secretion in Alpine goats fed maize silage–based diets

Lipid in the diet is known to enhance milk fat secretion and alter milk fatty acid composition in lactating goats. In the current experiment, the contribution of peripheral tissue and mammary gland lipid metabolism to changes in milk fat composition from plant oils was examined. Fourteen Alpine goats in midlactation were used in a 3 x 3 Latin square design with 28-d experimental periods. Treatments comprised maize silage-based diets containing no additional oil (M), sunflower-seed oil (MSO; 6.1% of diet DM), or linseed oil (MLO; 6.2% of diet DM). Compared with the control, milk yield was greater in goats fed MSO (3.37 and 3.62 kg/d, respectively), whereas MLO enhanced milk fat content (+3.9 g/kg), resulting in a 14% increase in milk fat secretion. Both MSO and MLO increased milk lactose secretion by 12 and 8%, respectively, compared with M. Relative to the control, plant oils decreased C10 to C16 secretion (32 and 24%, respectively, for MSO and MLO) and enhanced C18 output in milk (ca. 110%). Diets MSO and MLO increased cis-9 18:1 secretion in milk by 25 and 31%, respectively, compared with M. The outputs of trans-11 18:1 and cis-9, trans-11 18:2 in milk were increased 8.34- and 6.02-fold for MSO and 5.58- and 3.71-fold for MLO compared with M, and MSO increased trans-10 18:1 and trans-10, cis-12 18:2 secretion. Plant oils decreased milk fat cis-9 14:1/14:0; cis-9 16:1/16:0; cis-9 18:1/18:0; and cis-9, trans-11 18:2/trans-11 18:1 concentration ratios but had no effect on mammary stearoyl-CoA desaturase mRNA or activity. Furthermore, changes in milk fatty acid secretion were not associated with alterations in mammary acetyl-CoA carboxylase mRNA and activity, abundance of mRNA encoding for lipoprotein lipase and fatty acid synthase, or malic enzyme and glycerol-3-phosphate dehydrogenase activity in mammary tissue. Mammary lipoprotein lipase activity was increased with MSO relative to MLO. Treatments had no effect on glucose-6-phosphate dehydrogenase, malic enzyme, glycerol-3-phosphate dehydrogenase activity, or mRNA abundance and/or activity of lipoprotein lipase, acetyl-CoA carboxylase, fatty acid synthase, and stearoyl-CoA desaturase in liver or adipose tissue. In conclusion, inclusion of sunflower-seed oil and linseed oil in maize silage-based diets alters milk fatty acid secretion in goats via mechanisms independent of changes in mammary, hepatic, or adipose tissue lipogenic gene expression. Furthermore, data provided indications that the regulation of mammary lipogenic responses to plant oils on starch-rich diets differs between the caprine and bovine.

Whole intact rapeseeds or sunflower oil in high-forage or high-concentrate diets affects milk yield, milk composition, and mammary gene expression profile in goats

This study aimed to ascertain the response of goat mammary metabolic pathways to concentrate and lipid feeding in relation to milk fatty acid (FA) composition and secretion. Sixteen midlactation multiparous goats received diets differing in forage-to-concentrate ratio [high forage (HF) 64:36, and low forage (LF) 43:57] supplemented or not with lipids [HF with 130 g/d of oil from whole intact rapeseeds (RS) and LF with 130 g/d of sunflower oil (SO)] in a 4 x 4 Latin square design. Milk yield, milk composition, FA profile, and FA secretion were measured, as well as the expression profiles of key genes in mammary metabolism and of 8,382 genes, using a bovine oligonucleotide microarray. After 3 wk of treatment, milk, lactose, and protein yields were lower with HF-RS than with the other diets, whereas treatment had no effect on milk protein content. Milk fat content was higher with the HF-RS and LF-SO diets than with the HF and LF diets, and SO supplementation increased milk fat yield compared with the LF diet. Decreasing the forage-to-concentrate ratio from 64:36 to 43:57 had a limited effect on goat milk FA concentrations and secretions. Supplementing the LF diet with SO changed almost all the FA concentrations, including decreases in medium-chain saturated FA and large increases in trans C18:1 and C18:2 isomers (particularly trans-11 C18:1 and cis-9, trans-11 conjugated linoleic acid), without significant changes in C18:0 and cis-9 C18:1, whereas supplementing the HF diet with RS led to a strong decrease in short- and medium-chain saturated FA and a very strong increase in C18:0 and cis-9 C18:1, without significant changes in trans C18:1 and conjugated linoleic acid. Despite the decreases in milk lactose and protein yields observed with HF-RS, and despite the decrease in milk medium-chain FA and the increase in C18 FA secretion with RS or SO supplementation, none of the dietary treatments had any effect on mammary mRNA expression of the key genes involved in lactose (e.g., alpha-lactalbumin), protein (e.g., beta-casein), and lipid metabolism (e.g., lipoprotein lipase) after 3 wk of treatment. In addition, transcriptome analysis did not provide evidence of treatments inducing significant changes in the expression of specific genes in the mammary gland. However, 2-way hierarchical clustering analysis highlighted different global mammary expression profiles between diets, showing that the gene expression profiles corresponding to the same diet were gathered by common groups of genes. This experiment suggests that after 3 wk of dietary treatment, other factors, such as substrate availability for mammary metabolism, could play an important role in contributing to milk FA responses to changes in diet composition in the goat.

Selection of reference genes for quantitative real-time PCR normalisation in adipose tissue, muscle, liver and mammary gland from ruminants.

The reliability of reverse transcription quantitative real-time PCR (RT-qPCR) depends on normalising the mRNA abundance using carefully selected, stable reference genes. Our aim was to propose sets of reference genes for normalisation in bovine or caprine adipose tissue (AT), mammary gland, liver and muscle. All of these tissues contribute to nutrient partitioning and metabolism and, thus, to the profitability of ruminant productions (i.e. carcasses, meat and milk). In this study, eight commonly used reference genes that belong to different functional classes (CLN3, EIF3K, MRPL39, PPIA, RPLP0, TBP, TOP2B and UXT) were analysed using the geNorm procedure to determine the most stable reference genes in bovine and/or caprine tissues. Abundances and rankings of reference genes varied between tissues, species and the combination of tissues and/or species. Therefore, we proposed 29 sets of reference genes that differed depending on the tissue and/or species. As examples of the 29 sets, EIF3K, TOP2B and UXT were proposed as the most stable reference genes in bovine AT; UXT, EIF3K and RPLP0 were the most stable reference genes in bovine and caprine AT. The optimal number of reference genes for data normalisation was 3 for 27 of the proposed 29 sets. In two of the 29 sets, four to five reference genes were necessary for data normalisation when the number of studied tissues was increased. For example, UXT, EIF3K, TBP, TOP2B and CLN3 were required for data normalisation in bovine mammary gland, AT, muscle and liver. We have evaluated some of our proposed sets of reference genes for the normalisation of CD36 gene expression. Normalisation using the three most stable reference genes has revealed downregulation of CD36 gene expression in bovine mammary gland by a concentrate-based diet that is supplemented with sunflower oil and upregulation of CD36 gene expression in caprine liver by including a rapidly degradable starch in the diet. The dietary regulation of the gene expression of CD36 has been erased by normalisation with the least stable reference genes, which may result in misinterpretation of CD36 gene regulation. To conclude, our results provide valuable reference gene sets for other studies that aim to measure tissue and/or species-specific mRNA abundance in ruminants.

Effect of sunflower-seed oil or linseed oil on milk fatty acid secretion and lipogenic gene expression in goats fed hay-based diets.

Plant oils in the diet are known to alter milk fat composition owing to changes in the supply of fatty acid precursors and/or activity of lipogenic enzymes in the mammary gland. Thirteen mid-lactating Alpine goats were used in a 3 x 3 Latin square design with 28-d periods to evaluate possible mechanisms regulating milk fat synthesis and fatty acid composition on grass hay-based diets containing none (H) or 55 g/kg diet dry matter of sunflower-seed oil (HSO) or linseed oil (HLO). Inclusion of oils in the diet had no effect on milk yield but enhanced (P<0.05) milk fat secretion. Compared with the control, HLO and HSO decreased (P<0.05) C10-C16 secretion and increased (P<0.05) C18 output in milk, responses that were accompanied by reductions in milk fat cis-9 14:1/14:0, cis-9 18:1/18:0 and cis-9, trans-11 18:2/cis-9 18:1 concentration ratios. Plant oil supplements decreased (P<0.05) mammary stearoyl-CoA desaturase (SCD) activity but had no effect on SCD mRNA. Treatments had no effect on glucose-6-phosphate dehydrogenase, malic enzyme and glycerol-3-phosphate dehydrogenase activity, or mRNA abundance and/or activity of lipoprotein lipase, acetyl-CoA carboxylase and fatty acid synthase in mammary, hepatic or adipose tissue. The results provided little support for milk fatty acid secretion responses to HLO and HSO being mediated via changes in mammary, hepatic or adipose mRNA abundance or in the activity of key lipogenic enzymes. In conclusion, plant oils in the diet enhance milk fat synthesis, alter milk fatty acid composition and specifically inhibit mammary SCD activity in the goat. Furthermore, the results suggest that the regulation of mammary lipogenesis in response to plant oils appears related to factors other than altered mammary gene expression or potential lipogenic enzyme activity.

Expression and nutritional regulation of lipogenic genes in mammary gland and adipose tissues of lactating goats.

While the effect of long-chain fatty acids on adipose tissue (AT) lipogenic activities has been described in non-lactating ruminants (Vernon, 1977), little is known about their effects on the mammary gland and the AT in lactating animals. However, in cows in mid lactation, duodenal rapeseed oil infusion decreased the rate of fatty acid (FA) synthesis in AT and increased milk yield of long-chain FA (18[ratio ]1, 18[ratio ]2 and 18[ratio ]3) and decreased medium-chain FA (14[ratio ]0 and 16[ratio ]0), suggesting a depressive effect of fat feeding on mammary lipid synthesis de novo (Chilliard et al. 1991). On the other hand, in goat species, the addition of vegetable lipids to the diet led to an increase in the milk fat content and yield (Chilliard et al. 2003) suggesting that the possible negative effect of long-chain FA on FA synthesis in the lactating mammary gland could be more than compensated by increasing the supply of FA brought to the mammary gland for milk synthesis. Elsewhere, AT from various anatomical sites are characterized by different FA composition in goat (Bas et al. 1987) together with different patterns of lipogenic gene expression in sheep (Barber et al. 2000). These results suggest that each AT site is characterized by a specific metabolism. However, in lactating ruminants, few data are available on the extent of expression and regulation of genes coding for lipogenic enzymes in AT. Therefore, the current study was performed in three lipogenic tissues of lactating goats, namely the mammary gland, an internal AT site (perirenal AT) and an external AT site (subcutaneous AT).

Effects of polyunsaturated fatty acids from plant oils and algae on milk fat yield and composition are associated with mammary lipogenic and SREBF1 gene expression

The main aim of the present study was to examine the effects of long-term supplementing diets with saturated or unprotected polyunsaturated fatty acids from two different plant oils rich in either n-3 or n-6 fatty acids (FAs) plus docosahexaenoic acid (DHA)-rich algae on mammary gene expression and milk fat composition in lactating dairy cows. Gene expression was determined from mammary tissue and milk epithelial cells. Eighteen primiparous German Holstein dairy cows in mid-lactation were randomly assigned into three dietary treatments that consist of silage-based diets supplemented with rumen-stable fractionated palm fat (SAT; 3.1% of the basal diet dry matter, DM), or a mixture of linseed oil (2.7% of the basal diet DM) plus DHA-rich algae (LINA; 0.4% of the basal diet DM) or a mixture of sunflower oil (2.7% of the basal diet DM) plus DHA-rich algae (SUNA; 0.4% of the basal diet DM), for a period of 10 weeks. At the end of the experimental period, the cows were slaughtered and mammary tissues were collected to study the gene expression of lipogenic enzymes. During the last week, the milk yield and composition were determined, and milk was collected for FA measurements and the isolation of milk purified mammary epithelial cells (MECs). Supplementation with plant oils and DHA-rich algae resulted in milk fat depression (MFD; yield and percentage). The secretion of de novo FAs in the milk was reduced, whereas the secretion of trans-10,cis-12-CLA and DHA were increased. These changes in FA secretions were associated in mammary tissue with a joint down-regulation of mammary lipogenic enzyme gene expression (stearoyl-CoA desaturase, SCD1; FA synthase, FASN) and expression of the regulatory element binding transcription factor (SREBF1), whereas no effect was observed on lipoprotein lipase (LPL) and glycerol-3-phosphate acyltransferase 1, mitochondrial (GPAM). A positive relationship between mammary SCD1 and SREBF1 mRNA abundances was observed, suggesting a similar regulation for these genes. Such data on mammary gene expression in lactating cows presenting MFD contribute to strengthen the molecular mechanisms that govern milk fat synthesis in the mammary glands. In purified MEC, the dietary treatments had no effect on gene expressions. Differences between mammary tissue and milk purified MEC gene expression were attributed to the effect of lipid supplements on the number of milk purified MEC and its RNA quality, which are determinant factors for the analysis of gene expression using milk cells.

Effects of fish oil and starch added to a diet containing sunflower-seed oil on dairy goat performance, milk fatty acid composition and in vivo Δ9-desaturation of [13C]vaccenic acid.

The potential benefits on human health have prompted an interest in developing nutritional strategies for specifically increasing rumenic acid (RA) in ruminant milk. The aims of the present study were to (i) compare two dietary treatments with lipid supplements on milk yield and composition, (ii) measure the in vivo delta9-desaturation of vaccenic acid (VA) to RA using 13C-labelled VA and (iii) determine the effect of the dietary treatments on this variable. Treatments were 90 g sunflower-seed oil (SO) per d or 60 g sunflower-seed oil and 30 g fish oil per d plus additional starch (SFO), in a grassland hay-based diet given to eight Alpine goats in a 2 x 2 cross-over design with 21 d experimental periods. Milk yield and composition were similar between treatments. Goats fed SFO had higher milk 6 : 0-16 : 0 concentration, lower milk sigmaC18 concentrations and showed no effect on milk VA and RA, compared with SO. At the end of the experiment, intravenous injection of 1.5 g [13C]VA followed by measurements of milk lipid 13C enrichment showed that in vivo 31.7 and 31.6 % of VA was delta9-desaturated into milk RA in the caprine with the SO and SFO treatments, respectively. The expression of genes encoding for delta9-desaturase (or stearoyl-CoA desaturase; SCD1, SCD5) in mammary tissues and four milk delta9-desaturation ratios were similar between treatments. In conclusion, the present study provides the first estimates of in vivo endogenous synthesis of RA (63-73 % of milk RA) from VA in goats, and shows no difference between the two lipid supplements compared.

Comparison of the nutritional regulation of milk fat secretion and composition in cows and goats

A study with 2 ruminant species (goats and cows) with inherent differences in lipid metabolism was performed to test the hypothesis that milk fat depression (MFD) due to marine lipid supplements or diets containing high amounts of starch and plant oil is caused by different mechanisms and that each ruminant species responds differently. Cows and goats were allocated to 1 of 3 groups (4 cows and 5 goats per group) and fed diets containing no additional oil (control) or supplemented with fish oil (FO) or sunflower oil and wheat starch (SOS) according to a 3 × 3 Latin square design with 26-d experimental periods. In cows, milk fat content was lowered by FO and SOS (-31%), whereas only FO decreased milk fat content in goats (-21%) compared with the control. Furthermore, FO and SOS decreased milk fat yield in cows, but not in goats. In both species, FO and SOS decreased the secretion of C16 FA output. However, SOS increased milk secretion of >C16 FA in goats. Compared with the control, SOS resulted in similar increases in milk trans-10,cis-12 conjugated linoleic acid (CLA) in both species, but caused a 2-fold larger increase in trans-10 18:1 concentration in cows than for goats. Relative to the control, responses to FO in both species were characterized by a marked decrease in milk concentration of 18:0 (-74%) and cis-9 18:1 (-62%), together with a ~5-fold increase in total trans 18:1, but the proportionate changes in trans-10 18:1 were lower for goats. Direct comparison of animal performance and milk FA responses to FO and SOS treatments demonstrated interspecies differences in mammary lipogenesis, suggesting a lower sensitivity to the inhibitory effects of trans-10,cis-12 CLA in goats and that ruminal biohydrogenation pathways are more stable and less prone to diet-induced shifts toward the formation of trans-10-containing intermediates in goats compared with cows. Even though a direct cause and effect could not be established, results suggest that regulation of milk fat synthesis during FO-induced MFD may be related to a shortage of 18:0 for endogenous mammary cis-9 18:1 synthesis, increase in the incorporation of trans FA in milk triacylglycerols, and limitations in the synthesis of FA de novo to maintain milk fat melting point. However, the possible contribution of biohydrogenation intermediates with putative antilipogenic effects in the mammary gland, including trans-9,cis-11 CLA, trans-10 18:1, or cis-11 18:1 to FO-induced MFD cannot be excluded.