J. van Baal

Effects of feeding rapeseed oil, soybean oil, or linseed oil on stearoyl-CoA desaturase expression in the mammary gland of dairy cows

Stearoyl-CoA desaturase (SCD) is an important enzyme in the bovine mammary gland, and it introduces a double bond at the Δ(9) location of primarily myristoyl-, palmitoyl-, and stearoyl-CoA. The main objective of this study was to compare the effects of various fatty acids (FA) typically present in dairy cow rations on the expression of SCD1 and SCD5 in the mammary gland of dairy cows. Twenty-eight Holstein-Friesian cows were randomly assigned to 1 of 4 dietary treatments. The dietary treatments were a basal diet supplemented (dry matter basis) with 2.7% rapeseed oil as a source of C18:1 cis-9; 2.7% soybean oil as a source of C18:2 cis-9,12; 2.7% linseed oil as a source of C18:3 cis-9,12,15; or 2.7% of a 1:1:1 mixture of the 3 oils. The oil supplements were included in the concentrate, which was fed together with corn silage and grass silage. In addition, cows were grazing on pasture, consisting mainly of perennial ryegrass, during the day. Biopsies from the mammary gland were taken and analyzed for mRNA expression of SCD1 and SCD5 by using quantitative real-time PCR. Milk yield as well as milk protein and fat contents did not differ among the 4 dietary treatments. Dietary supplementation with rapeseed oil and linseed oil increased proportions of C18:1 cis-9 and C18:3 cis-9,12,15 in blood plasma, respectively, compared with the other treatments. Supplementation with soybean oil and linseed oil increased milk FA proportions of C18:2 cis-9,12 and C18:3 cis-9,12,15, respectively, but supplementation with rapeseed oil did not increase C18:1 cis-9 in milk. Mammary SCD1 expression was reduced by supplementation of soybean oil compared with rapeseed oil and linseed oil. In contrast, SCD5 expression did not differ among the 4 treatments. The C16 and C18 desaturation indices, representing proxies for SCD activity, were lower for the soybean oil diet compared with the diet supplemented with a mixture of the 3 oils. In conclusion, our study shows that mammary SCD1 expression is significantly downregulated in dairy cows by feeding unprotected soybean oil compared with rapeseed oil or linseed oil, and this is partially reflected by the lower desaturase indices in the milk. Furthermore, mammary SCD5 expression appears to be differently regulated than expression of SCD1.

Effect of rumen-protected choline on performance, blood metabolites, and hepatic triacylglycerols of periparturient dairy cattle

The effects of a dietary supplement of rumen-protected choline on feed intake, milk yield, milk composition, blood metabolites, and hepatic triacylglycerol were evaluated in periparturient dairy cows. Thirty-eight multiparous cows were blocked into 19 pairs and then randomly allocated to either one of 2 treatments. The treatments were supplementation either with or without (control) rumen-protected choline. Treatments were applied from 3 wk before until 6 wk after calving. Both groups received the same basal diet, being a mixed feed of grass silage, corn silage, straw, and soybean meal, and a concentrate mixture delivered through transponder-controlled feed dispensers. For all cows, the concentrate mixture was gradually increased from 0 kg/day (wk -3) to 0.9 kg of dry matter (DM)/d (day of calving) and up to 8.1 kg of DM/d on d 17 postcalving until the end of the experiment. Additionally, a mixture of 60 g of a rumen-protected choline supplement (providing 14.4 g of choline) and of 540 g of soybean meal or a (isoenergetic) mixture of 18 g of palm oil and 582 g of soybean meal (control) was offered individually in feed dispensers. Individual feed intake, milk yield, and body weight were recorded daily. Milk samples were analyzed weekly for fat, protein, and lactose content. Blood was sampled at wk -3, d 1, d 4, d 7, d 10, wk 2, wk 3, and wk 6 and analyzed for glucose, nonesterified fatty acids, and β-hydroxybutyric acid. Liver biopsies were taken from 8 randomly selected pairs of cows at wk -3, wk 1, wk 4, and wk 6 and analyzed for triacylglycerol concentration. We found that choline supplementation increased DM intake from 14.4 to 16.0 kg/d and, hence, net energy intake from 98.2 to 109.1 MJ/d at the intercept of the lactation curve at 1 day in milk (DIM), but the effect of choline on milk protein yield gradually decreased during the course of the study. Choline supplementation had no effect on milk yield, milk fat yield, or lactose yield. Milk protein yield was increased from 1.13 to 1.26 kg/d at the intercept of the lactation curve at 1 DIM, but the effect of choline on milk protein yield gradually decreased during the course of the study. Choline supplementation was associated with decreased milk fat concentration at the intercept of the lactation curve at 1 DIM, but the effect of choline on milk fat concentration gradually decreased as lactation progressed. Choline supplementation had no effect on energy-corrected milk yield, energy balance, body weight, body condition score, and measured blood parameters. Choline supplementation decreased the concentration of liver triacylglycerol during the first 4 wk after parturition. Results from this study suggest that hepatic fat export in periparturient dairy cows is improved by choline supplementation during the transition period and this may potentially decrease the risk for metabolic disorders in the periparturient dairy cow.

Linoleic and α-linolenic acid as precursor and inhibitor for the synthesis of long-chain polyunsaturated fatty acids in liver and brain of growing pigs.

Studies suggested that in human adults, linoleic acid (LA) inhibits the biosynthesis of n-3 long-chain polyunsaturated fatty acids (LC-PUFA), but their effects in growing subjects are largely unknown. We used growing pigs as a model to investigate whether high LA intake affects the conversion of n-3 LC-PUFA by determining fatty acid composition and mRNA levels of Δ5- and Δ6 desaturase and elongase 2 and -5 in liver and brain. In a 2 × 2 factorial arrangement, 32 gilts from eight litters were assigned to one of the four dietary treatments, varying in LA and α-linolenic acid (ALA) intakes. Low ALA and LA intakes were 0.15 and 1.31, and high ALA and LA intakes were 1.48 and 2.65 g/kg BW0.75 per day, respectively. LA intake increased arachidonic acid (ARA) in liver. ALA intake increased eicosapentaenoic acid (EPA) concentrations, but decreased docosahexaenoic acid (DHA) (all P < 0.01) in liver. Competition between the n-3 and n-6 LC-PUFA biosynthetic pathways was evidenced by reductions of ARA (>40%) at high ALA intakes. Concentration of EPA (>35%) and DHA (>20%) was decreased by high LA intake (all P < 0.001). Liver mRNA levels of Δ5- and Δ6 desaturase were increased by LA, and that of elongase 2 by both ALA and LA intakes. In contrast, brain DHA was virtually unaffected by dietary LA and ALA. Generally, dietary LA inhibited the biosynthesis of n-3 LC-PUFA in liver. ALA strongly affects the conversion of both hepatic n-3 and n-6 LC-PUFA. DHA levels in brain were irresponsive to these diets. Apart from Δ6 desaturase, elongase 2 may be a rate-limiting enzyme in the formation of DHA.

Ileal and faecal protein digestibility measurement in humans and other non-ruminants – a comparative species view

A comparative non-ruminant species view of the contribution of the large intestinal metabolism to inaccuracies in nitrogen and amino acid absorption measurements is provided to assess potential implications for the determination of crude protein/amino acid digestibility in adult humans consuming lower digestible protein sources. Most of the amino acids in the hindgut are constituents of the microorganisms and significant microbial metabolism of dietary and endogenous amino acids occurs. Bacterial metabolism of nitrogen-containing compounds leads to a significant disappearance of nitrogen in the large intestine. Literature data show that some 79 % of the nitrogen entering the large intestine of the horse is absorbed. For dogs, sows, and growing pigs these estimates are 49, 34 and 16 %, respectively. The coefficient of gut differentiation of humans compares closely to that of dogs while the coefficient of fermentation in humans is the lowest of all non-ruminant species and closest to that of cats and dogs. Large intestinal digesta transit times of humans compare closest to adult dogs. Significant amino acid metabolism has been shown to occur in the large intestine of the adult dog. Use of the growing pig as an animal model is likely to underestimate the fermentation of amino acids in the human large intestine. Based on the significant degree of fermentation of nitrogen-containing components in the large intestine of several non-ruminant species, it can be expected that determination of amino acid digestibility at a faecal level in humans consuming low quality proteins would not provide accurate estimates of the amino acids absorbed by the intestine.

Effect of rumen-protected choline supplementation on liver and adipose gene expression during the transition period in dairy cattle

We previously reported that supplementation of rumen-protected choline (RPC) reduces the hepatic triacylglycerol concentration in periparturient dairy cows during early lactation. Here, we investigated the effect of RPC on the transcript levels of lipid metabolism-related genes in liver and adipose tissue biopsies, taken at wk -3, 1, 3, and 6 after calving, to elucidate the mechanisms underlying this RPC-induced reduction of hepatic lipidosis. Sixteen multiparous cows were blocked into 8 pairs and randomly allocated to either 1 of 2 treatments, with or without RPC. Treatments were applied from 3 wk before to 6 wk after calving. Both groups received a basal diet and concentrate mixture. One group received RPC supplementation, resulting in an intake of 14.4 g of choline per day, whereas controls received an isoenergetic mixture of palm oil and additional soybean meal. Liver and adipose tissue biopsies were taken at wk -3, 1, 3, and 6 to determine the mRNA abundance of 18 key genes involved in liver and adipose tissue lipid and energy metabolism. Milk samples were collected in wk 1, 2, 3, and 6 postpartum for analysis of milk fatty acid (FA) composition. The RPC-induced reduction in hepatic lipidosis could not be attributed to altered lipolysis in adipose tissue, as no treatment effect was observed on the expression of peroxisome proliferator-activated receptor γ, lipoprotein lipase, or FA synthase in adipose tissue, or on the milk FA composition. Rumen-protected choline supplementation increased the expression of FA transport protein 5 and carnitine transporter SLC22A5 in the liver, suggesting an increase in the capacity of FA uptake and intracellular transport, but no treatment effect was observed on carnitine palmitoyl transferase 1A, transporting long-chain FA into mitochondria. In the same organ, RPC appeared to promote apolipoprotein B-containing lipoprotein assembly, as shown by elevated microsomal triglyceride transfer protein expression and apolipoprotein B100 expression. Cows supplemented with RPC displayed elevated levels of glucose transporter 2 mRNA and a reduced peak in pyruvate carboxylase mRNA immediately after calving, showing that supplementation also resulted in improved carbohydrate metabolism. The results from this study suggest that RPC supplementation reduces liver triacylglycerol by improved FA processing and very-low-density lipoprotein synthesis, and RPC also benefits hepatic carbohydrate metabolism.

Effects of short- and long-chain fatty acids on the expression of stearoyl-CoA desaturase and other lipogenic genes in bovine mammary epithelial cells

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.

Influence of nutrition on feline calcium oxalate urolithiasis with emphasis on endogenous oxalate synthesis

The prevalence of calcium oxalate (CaOx) uroliths detected in cats with lower urinary tract disease has shown a sharp increase over the last decades with a concomitant reciprocal decrease in the occurrence of struvite (magnesium ammonium phosphate) uroliths. CaOx stone-preventative diets are available nowadays, but seem to be marginally effective, as CaOx urolith recurrence occurs in patients fed these diets. In order to improve the preventative measures against CaOx urolithiasis, it is important to understand its aetiopathogenesis. The main research focus in CaOx formation in cats has been on the role of Ca, whereas little research effort has been directed towards the role and origin of urinary oxalates. As in man, the exogenous origin of urinary oxalates in cats is thought to be of minor importance, although the precise contribution of dietary oxalates remains unclear. The generally accepted dietary risk factors for CaOx urolithiasis in cats are discussed and a model for the biosynthetic pathways of oxalate in feline liver is provided. Alanine:glyoxylate aminotransferase 1 (AGT1) in endogenous oxalate metabolism is a liver-specific enzyme targeted in the mitochondria in cats, and allows for efficient conversion of glyoxylate to glycine when fed a carnivorous diet. The low peroxisomal activity of AGT1 in cat liver is compatible with the view that felids utilised a low-carbohydrate diet throughout evolution. Future research should focus on understanding de novo biosynthesis of oxalate in cats and their adaptation(s) in oxalate metabolism, and on dietary oxalate intake and absorption by cats.

Alteration of gene expression in mammary gland tissue of dairy cows in response to dietary unsaturated fatty acids

The aim of this study was to determine the effects of supplementing unprotected dietary unsaturated fatty acids (UFAs) from different plant oils on gene expression in the mammary gland of grazing dairy cows. A total of 28 Holstein-Friesian dairy cows in mid-lactation were blocked according to parity, days in milk, milk yield and fat percentage. The cows were then randomly assigned to four UFA sources based on rapeseed, soybean, linseed or a mixture of the three oils for 23 days, after which, all 28 cows were switched to a control diet for an additional 28 days. On the last day of both periods, mammary gland biopsies were taken to study genome-wide differences in gene expression on Affymetrix GeneChip® Bovine Genome Arrays (no. 900493) by ServiceXS (Leiden, The Netherlands). Supplementation with UFAs resulted in increased milk yield but decreased milk fat and protein percentages. Furthermore, the proportion of de novo fatty acids (FAs) in the milk was reduced, whereas that of long-chain FAs increased. Applying a statistical cut-off of false discovery rate of q-values <0.05 together with an absolute fold change of 1.3, a total of 972 genes were found to be significantly affected through UFA supplementation, indicating that large transcriptional adaptations occurred in the mammary gland when grazing dairy cows were supplemented with unprotected dietary UFA. Gene sets related to cell development and remodeling, apoptosis, nutrient metabolic process, as well as immune system response were predominantly downregulated during UFA supplementation. Such molecular knowledge on the physiology of the mammary gland might provide the basis for further functional research on dairy cows.

Dietary effects of linseed on fatty acid composition of milk and on liver, adipose and mammary gland metabolism of periparturient dairy cows

During the transition period in dairy cows, drastic adaptations within and between key tissues and cell types occur in a coordinated manner to support late gestation, the synthesis of large quantities of milk and metabolic homoeostasis. The start of lactation coincides with an increase of triacylglycerols in the liver, which has been associated with several economically important diseases in dairy cows (i.e. hepatic lipidiosis, mastitis). The polyunsaturated fatty acids have been used to improve liver metabolism and immune function in the mammary gland. Therefore, the effects of dietary linseed supplementation on milk quality and liver, adipose and mammary gland metabolism of periparturient dairy cows were studied in 14 cows that were randomly assigned to control or linseed supplementation. Animals were treated from 3 weeks antepartum until 6 weeks post-partum. Linseed did not modify dry matter intake, but increased milk yield and lactose yield, and decreased milk fat concentration, which coincided with lower proportion of C16 and higher proportions of stearic acid, conjugated linoleic acid and α-linolenic acid in milk fat. Linseed supplementation did not significantly change the expression of key lipid metabolism genes in liver and adipose tissues, except of glucose transporter 2 (GLUT2) in liver, which was increased in cows supplemented with linseed, suggesting that more glucose was secreted and probably available for lactose synthesis compared with cows fed control diet. Large adaptations of transcription occurred in the mammary gland when dairy cows were supplemented with linseed. The main affected functional modules were related to energy metabolism, cell proliferation and remodelling, as well as the immune system response.

Comparison between stearoyl-CoA desaturase expression in milk somatic cells and in mammary tissue of lactating dairy cows

Stearoyl-CoA desaturase (SCD) is an important enzyme in the bovine mammary gland, where it inserts a cis-double bond at the Δ9 position in a wide range of fatty acids. Investigating SCD expression in the bovine mammary gland generally requires invasive biopsy to obtain mammary tissue. The aim of this study was to evaluate the use of milk somatic cells as a non-invasive alternative to biopsy for measuring mammary SCD expression in dairy cows. Both milk somatic cells and mammary tissue were collected from 14 Holstein-Friesian cows and used for analysis of SCD expression by real-time PCR. The SCD5 mRNA levels in mammary tissue compared with SCD1 were low, and for several milk somatic cell samples, SCD5 expression was even below the limit of detection. A significant relationship was found between SCD1 expression in milk somatic cells and in mammary tissue. In addition, SCD1 expression in milk somatic cells was significantly related to Δ9-desaturase indices in milk, which are commonly used as an indicator of SCD1 activity within the mammary gland. Our study showed that milk somatic cells can be used as a source of mRNA to study SCD1 expression in dairy cows, offering a non-invasive alternative to mammary tissue samples obtained by biopsy.