Docosahexaenoic acid elevates trans-18:1 isomers but is not directly converted into trans-18:1 isomers in ruminal batch cultures

Pathways of docosahexaenoic (DHA) biohydrogenation are not known; however, DHA is metabolized by ruminal microorganisms. The addition of DHA to the rumen alters the fatty acid profile of the rumen and milk and leads to increased trans-18:1 isomers, particularly trans-11 18:1. This study included 2 in vitro experiments to identify if the increase in trans-11 C18:1 was due to DHA being converted into trans-11 18:1 or if DHA stimulated trans-11 products from biohydrogenation of other fatty acids. In each experiment, ruminal microorganisms collected from a lactating Holstein cow were incubated in 10-mL batch cultures for 0, 6, 24, and 48 h and a uniformly ¹³C-labeled DHA was added to the cultures at 0 h as a metabolic tracer. Experiment 1 tested 0.5% DHA supplementation and experiment 2 examined 1, 2, and 3% DHA supplementation to determine if the level of DHA effected its conversion into trans-11 18:1. In both experiments, any fatty acid that was enriched with the ¹³C label was determined to arise from DHA. Palmitic (C16:0), stearic (C18:0), all trans-18:1, eicosanoic (C20:0), and docosanoic (C22:0) acids were examined for enrichment. In experiment 1, the amount of trans-18:1 isomers increased 0.415 mg from 0 to 48 h; however, no label was found in trans-18:1 at any time. Docosanoic acid was highly enriched at 24 h and 48 h to 20.2 and 16.3%. Low levels of enrichment were found in palmitic and stearic acids. In experiment 2, trans-18:1 isomers increased 185, 256, and 272% from 0 to 48 h when DHA was supplemented at 1, 2, and 3%, respectively; however, as in experiment 1, no enrichment occurred of any trans-18:1 isomer. In experiment 2, low levels of label were found in palmitic and stearic acids. Enrichment of docosanoic acid decreased linearly with increased DHA supplementation. These studies showed that trans-18:1 fatty acids are not produced from DHA, supporting that DHA elevates trans-18:1 by modifying biohydrogenation pathways of other polyunsaturated fatty acids.     

Conjugated alpha-linolenic acid isomers in bovine milk and muscle

Conjugated linolenic acids (CLnA) are octadecatrienoic fatty acid isomers with at least 2 conjugated double bonds. Various CLnA isomers occur naturally, and some isomers could be formed by ruminants from dietary α-linolenic acid. Ruminant biohydrogenation of polyunsaturated fatty acids gives rise to the formation of numerous metabolites having conjugated or nonconjugated structures. The objectives of this study were to identify and characterize CLnA isomers in milk fat and muscle lipid extracts from cattle fed a high-forage diet. The analysis of total fatty acid methyl esters revealed levels of total CLnA of 0.39% in a single milk lipid extract and 0.34% in a single muscle lipid extract. Fatty acid methyl esters were fractionated by argentation thin-layer chromatography. A fraction containing dienoic fatty acids as well as CLnA isomers was isolated and analyzed. The double bond positions of CLnA isomers (cis-9, trans-11, cis-15 and cis-9, trans-13, cis-15 18:3) were confirmed by mass spectrometry of their 4,4-dimethyloxazoline derivatives. Mass spectra of the cis-9, trans-13, cis-15 18:3 isomer was characterized by an intense ion at m/z 236 attributable to the formation of 2 stabilized allylic radical fragments, whereas this intense ion corresponding to the stabilized radical fragments was located at m/z 262 for the cis-9, trans-11, cis-15 18:3 isomer. The gap of 12 amu between m/z 250 and 262 confirmed the occurrence of a double bond in position Δ¹³. Configuration of the double bonds of standards having similar mass spectra and gas-liquid chromatographic retention times was confirmed by ¹H nuclear magnetic resonance. We also showed that both CLnA isomers were found in the muscle lipid extract, whereas only the cis-9, trans-11, cis-15 18:3 isomer was identified in the milk lipid extract. This study appears to be the first to identify 2 CLnA isomers in bovine muscle lipid extract.     

In vitro biohydrogenation of 13C-labeled α-linolenic acid in response to ruminal alterations associated with diet-induced milk fat depression in ewes

The basis for marine lipid-induced milk fat depression (MFD) has not been established yet, but recent reports suggest the putative contribution of shifts in the ruminal metabolism of α-linolenic acid (ALA). To test this hypothesis, an isotopic tracer approach was used in batch cultures of rumen microorganisms with inoculum collected from cannulated ewes fed either a total mixed ration without lipid supplementation (control inoculum) or the same diet supplemented with 2% of fish oil, which is known to cause MFD in lactating sheep (FO-MFD inoculum). The [1-¹³C]ALA was added at a dose of 1% of incubated dry matter and the proportions of ¹³C-labeled fatty acids (FA) were examined after 24 h of incubation, using complementary gas chromatography and gas chromatography-combustion isotope ratio mass spectrometry (GC-C-IRMS) analyses. Expected differences in FA profiles were confirmed between control and FO-MFD inocula (e.g., large decreases in 18:0 and increases in most 18:1 and 18:2 intermediates, particularly trans isomers, to fish oil supply). The biohydrogenation of ¹³ALA was extensive and yielded multiple metabolites, with a total of 48 chromatographic peaks showing ¹³C enrichment, regardless of the inoculum type. However, although ALA was biohydrogenated through common pathways under standard or MFD conditions, large changes in the accumulation of ¹³C-labeled FA suggest important differences in the relative contribution of each specific route. First, increased accumulation of trans-11-containing FA in FO-MFD incubations was accompanied by a general repression of the trans-13/14 pathway (supported by lower trans-13+14 18:1 or trans-11,trans-13 18:2 proportions), together with a lower production of cis FA (e.g., cis-9, -12, and -15 18:1 and some cis,cis 18:2). Results also downplayed the relevance of the trans-11 to trans-10 shift as an effective marker of diet-induced MFD in sheep, and challenged the involvement of some trans-10 intermediates (e.g., trans-10 18:1 and trans-10,cis-15 18:2) in the low-fat milk syndrome in this species. Conversely, increased abundance of most 18:3 intermediates (including some unidentified isomers) might be representative of ruminal alterations related to fish oil supplementation in ewes. Further research is necessary to examine the potential association between these findings and MFD in lactating animals.     

The non-glyceride saponifiables of shea butter

Triterpene alcohols and sterols were found to be present in a commercial sample of shea butter (the seed fat of Butyrospermum parkii, Sapotaceae) as esters of both cinnamic and fatty acids. These non-glyceride saponifiables represented 6% by weight of the shea butter under study, whereas free triterpene alcohols and sterols accounted for only 1 % by weight. Cinnamic acid was found neither in the free form, nor esterified to glycerol. Saponification of the non-glyceride saponifiables yielded the triterpene alcohols α-amyrin, butyrospermol, lupeol, β-amyrin and germanicol as major components. Other alcohols identified were parked, 24-methylenelanost-9(11)-en-3-ol, dammaradienol, 24-methylenedammarenol and the two sterols α-spinasterol and Δ⁷-stigmastenol. Esterified to these alcohols were cinnamic (trans and cis), palmitic, stearic, oleic, linoleic and arachidic acids. The fatty acid composition of the non-glyceride saponifiables is different from that of the glycerides. No selectivity was observed in the esterification of these fatty acids by triterpene alcohols and sterols. Investigation of the products from acid catalysed transmethylation has led to two new experimental findings: (i) the difference in reaction rate of fatty acid and cinnamic acid esters and (ii) the formation of methoxylated artifacts from alcohols having unsaturation, in the C¹⁷ side chain, between C²⁴ and C²⁵ by the addition of methanol across the double bond. Analysis of the lipids extracted from shea kernels confirmed that the cinnamic acid esters were only present as non-glyceride saponifiables.     

Composition of adipose tissue triglycerides of neonatal and year-old lambs

Triglyccrides were isolated from perinephric (internal) and subcutaneous (external) adipose tissue obtained from neonatal lambs and from lambs which had been fed, until they were a year old, on a semi-synthetic ration or on a diet of grass cubes. the triglycerides went analysed for their fatty acid composition (including trans unsaturated acids) and for the intramolecular distribution of these acids between the primary and secondary alcoholic groups of the glycerol moiety. Whereas in the year-old lambs (and in adult sheep previously examined), the triglycerides of internal adipose tissue had a higher content of stearic acid and trans unsaturated acid than those of external tissues, the triglycerides from the perinephric and subcutaneous tissues of the neonatal lambs were very similar in fatty acid composition. Palmitic acid and C₁₈ mono-unsaturated acid together constituted more than 80% of the total acids. This composition resembles that of the subcutaneous triglycerides of the grown animal and suggests that, at all stages of growth, the triglycerides of external tissues are largely the result of endogenous synthesis. The fatty acids of the adipose tissues of the neonatal animals did not contain any of the acids of exogenous origin, such as those with trans double bonds, which characterise the triglycerides of the growing and mature animals, particularly those of the internal depots. Nevertheless, the intramolecular disposition of the fatty acids in the triglycerides formed in utero was similar to that previously observed in triglycerides from both the internal and external depots of the adult sheep. Saturated acids (palmitic and stearic acids) predominated amongst those esterified in the 1-and 3-positions of the glycerol moiety and unsaturated acids (almost entirely oleic acid) were the major components esterified in the 2-position. While the triglycerides from corresponding body sites in the two groups of year-old lambs were generally quite similar with respect to their content of palmitic acid, stearic acid and C₁₈ mono-unsaturated acid, the contribution of trans isomer to the total C₁₈ mono-unsaturated acid was considerably greater in the tissues (particularly perinephric tissue) of the animals fed on grass cube than in the tissues of those given the semi-synthetic ration. This difference between the two groups of lambs was associated with a corresponding difference in the proportions of C₁₈ trans unsaturated acid in the lipids of the rumen contents of the animals.     

Rumen biohydrogenation-derived fatty acids in milk fat from grazing dairy cows supplemented with rapeseed, sunflower, or linseed oils

The effects of supplementation with rapeseed, sunflower, and linseed oils (0.5 kg/d; good sources of oleic, linoleic, and linolenic acids, respectively) on milk responses and milk fat fatty acid (FA) profile, with special emphasis on rumen-derived biohydrogenation intermediates (BI), were evaluated in a replicated 4 × 4 Latin square study using 16 grazing dairy cows. The dietary treatments were 1) control diet: 20-h access to grazing pasture supplemented with 5 kg/d of corn-based concentrate mixture (96% corn; CC); 2) RO diet: 20-h access to grazing supplemented with 4.5 kg/d of CC and 0.5 kg of rapeseed oil; 3) SO diet: 20-h access to grazing supplemented with 4.5 kg/d of CC and 0.5 kg of sunflower oil; and 4) LO diet: 20-h access to grazing supplemented with 4.5 kg/d of CC and 0.5 kg of linseed oil. Milk fatty acids were converted to methyl esters and analyzed by gas-liquid chromatography and silver-ion HPLC. Dietary treatments had no effect on milk production or on milk protein content and milk protein production. Supplementation with rapeseed and sunflower oils lowered milk fat content and milk fat production, but linseed oil had no effect. Inclusion of dietary vegetable oils promoted lower concentrations of short-chain (including 4:0) and medium-chain FA (including odd- and branched-chain FA) and 18:3n-3, and higher concentrations of C₁₈ FA (including stearic and oleic acids). The BI concentration was higher with the dietary inclusion of vegetable oils, although the magnitude of the concentration and its pattern differed between oils. The RO treatment resulted in moderate increases in BI, including trans 18:1 isomers and 18:2 trans-7,cis-9, but failed to increase 18:1 trans-11 and 18:2 cis-9,trans-11. Sunflower oil supplementation resulted in the highest concentrations of the 18:1 trans-10, 18:1 cis-12, and 18:2 trans-10,trans-12 isomers. Concentrations of 18:1 trans-11 and 18:2 cis-9,trans-11 were higher than with the control and RO treatments but were similar to the LO treatment. Concentration of BI in milk fat was maximal with LO, having the highest concentrations of some 18:1 isomers (i.e., trans-13/14, trans-15, cis-15, cis-16), most of the nonconjugated 18:2 isomers (i.e., trans-11,trans-15, trans-11,cis-15, cis-9,cis-15, and cis-12,cis-15), and conjugated 18:2 isomers (i.e., trans-11,cis-13, cis-12,trans-14, trans-11,trans-13, trans-12,trans-14, and trans-9,trans-11), and all conjugated 18:3 isomers. The LO treatment induced the highest amount and diversity of BI without decreasing milk fat concentration, as the RO and SO treatments had, suggesting that the BI associated with 18:3n-3 intake may not be the major contributors to inhibition of mammary milk fat synthesis.     

Synthesis of the suspected trans-11,cis-13 conjugated linoleic acid isomer in ruminant mammary tissue by FADS3-catalyzed Δ13-desaturation of vaccenic acid

The octadecadienoic conjugated linoleic acid (CLA) isomer with trans-11 and cis-13 double bonds (trans-11,cis-13 CLA) has been described in ruminant milk. For now, this specific CLA is suspected to derive exclusively from ruminal biohydrogenation of dietary α-linolenic acid. However, in rodents, the fatty acid desaturase 3 (FADS3) gene was recently shown to code for an enzyme able to catalyze the unexpected Δ13-desaturation of vaccenic acid, producing a Δ11,13-CLA with all the structural characteristics of the trans-11,cis-13 isomer, although no commercial standard exists for complete conclusive identification. Because the FADS3 gene has already been reported in bovine animals, we hypothesized in the present study that an alternative direct FADS3-catalyzed Δ13-desaturation of vaccenic acid in mammary tissue may therefore co-exist with α-linolenic acid biohydrogenation to explain the final ruminant milk trans-11,cis-13 CLA presence. Here, we first confirm that the FADS3 gene is present in ruminant mammal genomic sequence databases. Second, we demonstrate that the Δ11,13-CLA found in milk fat and the highly probable trans-11,cis-13 CLA isomer produced by rodent FADS3 possess exactly the same structural characteristics. Then, we show that bovine mammary MAC-T and BME-UV epithelial cells express both FADS3 and stearoyl-CoA desaturase 1 (SCD1) mRNA and are able to synthesize both the suspected trans-11,cis-13 CLA and cis-9,trans-11CLA (rumenic acid) isomers when incubated with vaccenic acid. Finally, the concomitant presence of the suspected trans-11,cis-13 CLA isomer with FADS3 mRNA was shown in goat mammary tissue, whereas both were conversely very low or even absent in goat liver. Therefore, this study provides several lines of evidence that, by analogy with rumenic acid, trans-11,cis-13 CLA may originate both from ruminal biohydrogenation and from direct FADS3-catalyzed Δ13-desaturation of vaccenic acid in mammary tissue.     

Changes in fermentation and animal performance during recovery from classical diet-induced milk fat depression using corn with differing rates of starch degradability

Diet-induced milk fat depression (MFD) is a multifactorial disorder that can be triggered by a variety of conditions. Feeding high amounts of starch and unsaturated fatty acids has been shown to reduce milk fat yield and composition, as well as alter ruminal biohydrogenation patterns. However, little is known about how starch degradability in the rumen influences recovery from diet-induced MFD and if production of milk fat–inhibiting isomers will persist following an episode of MFD. The objective of this study was to evaluate production performance and ruminal fermentation in cows recovering from MFD when corn with a low or high starch degradability is fed. Six ruminally fistulated Holstein cows were used in a crossover design with 2 periods. During each period, MFD was induced for 10 d by feeding a diet with low fiber, high starch, and high unsaturated fatty acid. The polyunsaturated fatty acid concentration of the diet during the induction phase was modified primarily through inclusion of soybean oil. Following induction, cows were switched to either a high degradable starch recovery diet (HDS) or a low degradable starch recovery diet (LDS) for 18 d. The 7-h starch degradability was 66.5% for LDS and 87.8% for HDS. Milk was collected every 3 d for component and fatty acid analysis. On d 0, 4, 7, 10, 16, 22, and 28 of each period, ruminal pH and rumen fluid were collected every 2 h. Milk fat yield and composition was reduced during MFD induction and progressively increased by day in both HDS and LDS during recovery. Dry matter intake was similar among treatments and increased steadily over time during recovery. Preformed fatty acids were greater for HDS-fed animals, and de novo fatty acid in milk fat was greater for LDS-fed animals. Milk trans-10 C18:1 tended to be greater for HDS, and trans-10,cis-12 conjugated linoleic acid was significantly greater for HDS. cis-9,trans-11 conjugated linoleic acid was not affected by starch degradability during recovery. Total volatile fatty acids, butyrate, and valerate tended to differ or differed with recovery treatment, but ruminal pH and ammonia concentration were unaffected. The HDS diet responded similarly to the LDS diet during recovery with regard to milk fat percentage, but milk and fat yield tended to consistently be lower in HDS. When considering approaches to ameliorate diet-induced MFD, the degradability of the starch within rations should be evaluated. Although animal performance was similar, some trans fatty acid isomers were persistent in the milk through the recovery phase with HDS-fed animals, suggesting that milk fat synthesis might be potentially inhibited and biohydrogenation pathways modified in the rumen following an episode of MFD.     

Effect of pH and level of concentrate in the diet on the production of biohydrogenation intermediates in a dual-flow continuous culture

Milk fat depression in cows fed high-grain diets has been related to an increase in the concentration of trans-10 C_18:1 and trans-10,cis-12 conjugated linoleic acid (CLA) in milk. These fatty acids (FA) are produced as a result of the alteration in rumen biohydrogenation of dietary unsaturated FA. Because a reduction in ruminal pH is usually observed when high-concentrate diets are fed, the main cause that determines the alteration in the biohydrogenation pathways is not clear. The effect of pH (6.4 vs. 5.6) and dietary forage to concentrate ratios (F:C; 70:30 F:C vs. 30:70 F:C) on rumen microbial fermentation, effluent FA profile, and DNA concentration of bacteria involved in lipolysis and biohydrogenation processes were investigated in a continuous culture trial. The dual-flow continuous culture consisted of 2 periods of 8 d (5 d for adaptation and 3 d for sampling), with a 2 × 2 factorial arrangement of treatments. Samples from solid and liquid mixed effluents were taken for determination of total N, ammonia-N, and volatile fatty acid concentrations, and the remainder of the sample was lyophilized. Dry samples were analyzed for dry matter, ash, neutral and acid detergent fiber, FA, and purine contents. The pH 5.6 reduced organic matter and fiber digestibility, ammonia-N concentration and flow, and crude protein degradation, and increased nonammonia and dietary N flows. The pH 5.6 decreased the flow of C_18:0, trans-11 C_18:1 and cis-9, trans-11 CLA, and increased the flow of trans-10 C_18:1, C_18:2n-6, C_18:3n-3, trans-11,cis-15 C_18:2 and trans-10,cis-12 CLA in the 1 h after feeding effluent. The pH 5.6 reduced Anaerovibrio lipolytica (32.7 vs. 72.1 pg/10 ng of total DNA) and Butyrivibrio fibrisolvens vaccenic acid subgroup (588 vs. 1,394 pg/10 ng of total DNA) DNA concentrations. The high-concentrate diet increased organic matter and fiber digestibility, nonammonia and bacterial N flows, and reduced ammonia-N concentration and flow. The high-concentrate diet reduced trans-11 C_18:1 and trans-10 C_18:1, and increased C_18:2n-6, C_18:3n-3 and trans-10,cis-12 CLA proportions in the 1 h after feeding effluent. The increase observed in trans-10,cis-12 CLA proportion in the 1 h after feeding effluent due to the high-concentrate diet was smaller that that observed at pH 5.6. Results indicate that the pH is the main cause of the accumulation of trans-10 C_18:1 and trans-10, cis-12 CLA in the effluent, but the trans-10,cis-12 CLA proportion can be also affected by high levels of concentrate in the diet.     

Interaction of molasses and monensin in alfalfa hay- or corn silage-based diets on rumen fermentation, total tract digestibility, and milk production by Holstein cows

Sugar supplementation can stimulate rumen microbial growth and possibly fiber digestibility; however, excess ruminal carbohydrate availability relative to rumen-degradable protein (RDP) can promote energy spilling by microbes, decrease rumen pH, or depress fiber digestibility. Both RDP supply and rumen pH might be altered by forage source and monensin. Therefore, the objective of this study was to evaluate interactions of a sugar source (molasses) with monensin and 2 forage sources on rumen fermentation, total tract digestibility, and production and fatty acid composition of milk. Seven ruminally cannulated lactating Holstein cows were used in a 5 × 7 incomplete Latin square design with five 28-d periods. Four corn silage diets consisted of 1) control (C), 2) 2.6% molasses (M), 3) 2.6% molasses plus 0.45% urea (MU), or 4) 2.6% molasses plus 0.45% urea plus monensin sodium (Rumensin, at the intermediate dosage from the label, 16 g/909 kg of dry matter; MUR). Three chopped alfalfa hay diets consisted of 1) control (C), 2) 2.6% molasses (M), or 3) 2.6% molasses plus Rumensin (MR). Urea was added to corn silage diets to provide RDP comparable to alfalfa hay diets with no urea. Corn silage C and M diets were balanced to have 16.2% crude protein; and the remaining diets, 17.2% crude protein. Dry matter intake was not affected by treatment, but there was a trend for lower milk production in alfalfa hay diets compared with corn silage diets. Despite increased total volatile fatty acid and acetate concentrations in the rumen, total tract organic matter digestibility was lower for alfalfa hay-fed cows. Rumensin did not affect volatile fatty acid concentrations but decreased milk fat from 3.22 to 2.72% in corn silage diets but less in alfalfa hay diets. Medium-chain milk fatty acids (% of total fat) were lower for alfalfa hay compared with corn silage diets, and short-chain milk fatty acids tended to decrease when Rumensin was added. In whole rumen contents, concentrations of trans-10, cis-12 C_18:2 were increased when cows were fed corn silage diets. Rumensin had no effect on conjugated linoleic acid isomers in either milk or rumen contents but tended to increase the concentration of trans-10 C_18:1 in rumen samples. Molasses with urea increased ruminal NH₃-N and milk urea N when cows were fed corn silage diets (6.8 vs. 11.3 and 7.6 vs. 12.0 mg/dL for M vs. MU, respectively). Based on ruminal fermentation characteristics and fatty acid isomers in milk, molasses did not appear to promote ruminal acidosis or milk fat depression. However, combinations of Rumensin with corn silage-based diets already containing molasses and with a relatively high nonfiber carbohydrate:forage neutral detergent fiber ratio influenced biohydrogenation characteristics that are indicators of increased risk for milk fat depression.     

Combined effects of trans-10,cis-12 conjugated linoleic acid, propionate, and acetate on milk fat yield and composition in dairy cows

Diets inducing milk fat depression (MFD) are known to alter ruminal lipid metabolism, leading to the formation of specific isomers [such as trans-10,cis-12 conjugated linoleic acid (CLA)] that inhibit milk fat synthesis in lactating dairy cows. However, ruminal outflow of these isomers does not fully account for the decreases in milk fat synthesis observed during diet-induced MFD. The high-concentrate diets inducing MFD also induce a greater production of propionate, suggesting a possible inhibition of milk fat by propionate associated with trans-10,cis-12-CLA during MFD. The present experiment aimed to study the combined effects of propionate and trans-10,cis-12-CLA (both inhibitors of milk fat synthesis) on milk fat secretion and the effects of the combination of 2 nutrients with opposite effects (acetate and propionate). Six Holstein cows were used in a 6 × 6 Latin square design with 21-d periods (14 d of nutrient infusion). The treatments were control; ruminal infusion of 1,500 g/d of acetate (A); ruminal infusion of 800 g/d of propionate (P); duodenal infusion of 1.60 g/d of trans-10,cis-12-CLA (CLA); ruminal infusion of 750 g/d of acetate + 400 g/d of propionate (A+P); and duodenal infusion of 1.60 g/d of trans-10,cis-12-CLA + ruminal infusion of 800 g/d of propionate (CLA+P). The amounts of nutrients infused were chosen to induce a similar variation in milk fat content. Treatments A and P decreased dry matter intake. Compared with the control, P and CLA treatments decreased milk fat content and yield by 9% and 15% on average. Treatment A increased milk fat content by 6.5% but did not modify milk fat yield (because of a decrease in milk yield). The effects of A and P, and CLA and P on milk fat and fatty acid percentages and yield were additive (A+P and CLA+P treatments). With a same dose of trans-10,cis-12-CLA, the additional supply of propionate induced a decrease in milk fat 40% higher than that induced by trans-10,cis-12-CLA alone. The milk fatty acid profile obtained with CLA+P was similar to those observed with high-concentrate diets inducing MFD. In conclusion, under our experimental conditions, the effects of the 3 nutrients were additive on mammary lipogenesis, regardless of their separate effects. We also show that propionate could contribute to the milk fat reductions unaccounted for by trans-10,cis-12-CLA during MFD induced by high-concentrate diets.     

Effects of direct-fed Bacillus subtilis and Bacillus licheniformis on production performance and milk fatty acid profile in dairy cows

The aim of the study was to determine the effect of a Bacillus-based direct-fed microbial on performance of mid-lactating Holstein dairy cows and on their milk fatty acid composition. Six multiparous cows fitted with a rumen cannula were used in a randomized replicated crossover design. Cows received 200 g/d of either whey powder as a control or BioPlus 2B (Chr. Hansen), a commercial direct-fed microbial providing Bacillus subtilis and Bacillus licheniformis, representing a daily dose of 6.4 × 10¹¹ cfu, and using whey powder as a carrier. The 2 experimental periods lasted 14 d and were separated by a 7-d washout interval. Samples were collected on d 0, 13, and 14 of each period. Data from d 0 were used as covariate. Significance was declared at P ≤ 0.05 and tendency at 0.05 < P ≤ 0.10. There was a 10-fold increase in the relative concentration of bacteria from the Bacillus subtilis group in the rumen when feeding direct-fed Bacillus compared with control. Treatment did not affect ruminal pH, NH₃-N, or concentrations of acetate, propionate, and butyrate. However, direct-fed Bacillus increased ruminal concentrations of isovalerate and isobutyrate (tendency). Treatments did not affect lactation performance. Supplying direct-fed Bacillus enhanced milk relative concentration of anteiso 13:0 by 27.3% and of anteiso 15:0 by 6.5% and tended to increase concentrations of iso 14:0 (+41.8%) relative to control. When expressed on a yield basis, direct-fed Bacillus increased the secretion of anteiso 13:0 and decreased that of 11:0, 15:0, 17:0 (tendency), and cis-9 17:1. These variations, although limited in magnitude, indicate that milk branched-chain fatty acid composition is sensitive to ruminal microbiota modifications without changes in chemical composition of the diet.     

Seed fats of some New Zealand Juncaceae

The seed fats of nine species of Juncus L. and five varieties of Luzula DC, both of the family Juncaceae, have been shown to be somewhat similar to one another in fatty acid composition but are nevertheless distinguishable. They differ from those of the family Agavaceae¹ in containing the following range of fatty acids: linoleic acid 28-58%, oleic acid 22-55%, palmitic acid 6-19%, stearic acid 1-4%, linolenic acid 0-7%, and small amounts of C₁₂, C₁₄, C₁₅, C₁₇,C₁₉, C₂₀, and C₂₄ acids.     

Pigeon peas as a supplement for lactating dairy cows fed corn silage-based diets

Holstein rumen-cannulated cows [n = 7; initial body weight (BW) 640.56 ± 71.43 kg] were fed a corn silage basal diet with 1 of 3 concentrates (C = control; P10 = 10% pigeon peas; P20 = 20% pigeon peas). Cows were randomly assigned to treatments in a replicated 3 × 3 Latin square and individually fed using Calan gates. Each experimental period was 21 d with 7 d for adaption and 14 d for sample collection. Ruminal fluid samples were taken the last day of each experimental period and analyzed for pH, ammonia, long-chain fatty acids, and volatile fatty acids (VFA). Consecutive a.m. and p.m. milk samples were taken during the last 2 wk of the 21-d period and analyzed for fat, protein, long-chain fatty acids, and somatic cell count. Dry matter intake (kg/d) was reduced during the second period and was greater for P10 diets. Milk protein was greater for cows fed P20 compared with P10. Energy-corrected milk was greater for cows fed the control diet compared with P10. Treatment had no effect on milk yield. Ruminal fluid pH decreased over sampling times; however, pH remained at or above 5.5. Diets did not affect ruminal fluid pH; however, pH was different for sampling periods. Ruminal ammonia decreased until 8 h postfeeding at which time it peaked consistent with changes in ammonia concentrations that usually peak 3 to 5 h postfeeding on diets high in plant proteins. Dietary treatments altered ruminal fluid VFA with reduced concentrations of acetate and greater concentrations of propionate for control diet, resulting in reduced acetate:propionate ratio. Isobutyrate exhibited an hour by treatment interaction, in which isobutyrate decreased until 8 h postfeeding and then tended to be greater for P10 than for other treatments. Animals fed the P10 diet had greater concentrations of ruminal isovalerate. Ruminal cis-9,trans-11 and trans-10,cis-12 conjugated linoleic acid (CLA) isomers were not affected by dietary treatments. The P10 diet had greatest ruminal synthesis of cis-9,trans-11, but control cows had greatest ruminal synthesis of trans-10,cis-12. Milk CLA isomers were similar among treatments. Trends were observed for greater cis-9,trans-11 and trans-10,cis-12 for the P10 diet. Pigeon peas may be used as a protein supplement in dairy diets without affecting milk production, dry matter intake, or ruminal environment when they replace corn and soybean meal.     

Efficient generation of 2E-hexenal by a hydroperoxide lyase from mung bean seedlings

2E-hexenal was generated with a high molar conversion rate by the incubation of a hydroperoxide lyase containing extract from mung bean seedlings and its substrate, 13-hydroperoxy-9Z, 11E, 15Z-octadecatrienoic acid (13-HPOT). Various parameters affected the yield of 2E-hexenal and the conversion rate: hydroperoxide lyase activity was especially, pronounced in seedlings older than 10 days. The cleavage of 13-HPOT by a solubilized enzyme extract proceeded best at pH 6.5. Time-course studies showed that the majority of flavour compounds was already generated during the first 10 min of the incubation period. Using optimized reaction conditions, a maximum yield of 1062 mg kg⁻¹ 2E-hexenal was obtained with 32.6 mmol kg⁻¹ (10.1 g kg⁻¹) 13-HPOT. However, the conversion rate was highest (maximum 86.3%) at a low substrate concentration, indicating a suicidal behaviour of the hydroperoxide lyase. Enzymatically hydrolyzed and dioxygenated linseed oil was a suitable alternative to pure linolenic acid.     

Occurrence of C16:1 isomers in milk fats from ewes fed with different dietary lipid supplements

The trans as well as the cis C16:1 isomer profiles were established in 43 ewe milk fats supplemented with different dietary lipid sources representative of the variety of unsaturated fatty acids found in nature such as olive, sunflower, linseed and fish oils. Fractionation by silver-ion solid phase extraction facilitated a rapid separation of the trans, cis and saturated FA before gas chromatography analysis took place. C16:1 isomers with a double bond in positions 7, 9 and 13 in the cis group and 8 and 9 in the trans fraction were the most abundant. Dietary lipid supplementation produced a noticeable increase in the total trans C16:1 content and elevated correlations were observed between trans-8 C16:1 and trans-10 C18:1 as well as trans-9 C16:1 and trans-11 C18:1. These results support the idea that altering the trans C18:1 profile affects trans C16:1 isomer composition consistent with the β-oxidation products from the trans C18:1 isomers.     

Enrichment of the conjugated linoleic acid content of bovine milk fat by dry fractionation

This study investigated the effect of fat fractionation on the conjugated linoleic acid (cis-9, trans-11-C_18 : 2) content of bovine milk fat. Anhydrous milk fat was fractionated into hard and soft fractions using controlled cooling and agitation. Fractionation of milk fat pre-melted at 60°C using a temperature programme of 33–10°C and a cooling rate of 0.58°C h⁻¹ yielded a soft fraction containing 63.2% more conjugated linoleic acid (2.22 g 100 g⁻¹ FAME), which was also enriched in polyunsaturated fatty acids and vaccenic acid (trans-11-C_18 : 1) compared with the parent fat. Agitation following fractionation was found to have a negative effect on the conjugated linoleic acid content of the soft fraction. Refractionation of the soft fraction did not increase the yield of conjugated linoleic acid. The conjugated linoleic acid and trans fatty acid content of 26 selected food products ranging in milk fat content from 0 to 100% is reported. Conjugated linoleic acid concentrations ranged from 0 to 16.2 mg g⁻¹ fat and were generally lower than the trans fatty acid content which ranged from 0 to 155.7 mg g⁻¹ fat. Spreads containing vegetable oils contained higher trans fatty acid and lower conjugated linoleic acid contents than milk fat-containing products. This study highlights that a milk fat fraction enriched in conjugated linoleic acid may be achieved by dry fractionation.     

A canonical discriminant analysis to study the association between milk fatty acids of ruminal origin and milk fat depression in dairy cows

Although milk fat depression (MFD) has been observed and described since the beginning of the last century, all the molecular and biochemical mechanisms involved are still not completely understood. Some fatty acids (FA) originating during rumen biohydrogenation have been proposed as causative elements of MFD. However, contradictory results were obtained when studying the effect of single FA on MFD. An alternative could be the simultaneous evaluation of the effect of many FA using a multivariate approach. The aim of this study was to evaluate the relationship between individual milk FA of ruminal origin and MFD using canonical discriminant analysis, a multivariate technique able to distinguish 2 or more groups on the basis of a pool of variables. In a commercial dairy herd, a diet containing 26% starch on a DM basis induced an unintentional MFD syndrome in 14 cows out of 40. Milk yielded by these 14 animals showed a fat content lower than 50% of the ordinary value, whereas milk production and protein content were normal. The remaining 26 cows secreted typical milk fat content and therefore were considered the control group, even though they ate the same diet. The stepwise discriminant analysis selected 14 milk FA of ruminal origin most able to distinguish the 2 groups. This restricted pool of FA was used, as variables, in a run of the canonical discriminant analysis that was able to significantly discriminate between the 2 groups. Out of the 14 FA, 5 conjugated linoleic acid isomers (C18:2 trans-10,trans-12, C18:2 trans-8,trans-10, C18:2 trans-11,cis-13, C18:2 cis-9,cis-11, C18:2 cis-10,cis-12) and C15:0 iso were more related to the control group, whereas C18:2 trans-10,cis-12, C16:1 trans-6–7, C16:1 trans-9, C18:1 trans-6–8, C18:1 trans-9, C18:1 trans-10, C18:1 cis-11, and C18:3n-3 were positively associated with the MFD group, allowing a complete discrimination. On the basis of these results, we can conclude that (1) the shift of ruminal biohydrogenation from C18:1 trans-11 to C18:1 trans-10 seemed to be strongly associated with MFD; (2) at the same time, other C18:1 trans isomers showed a similar association; (3) on the contrary, conjugated linoleic acid isomers other than C18:2 trans-10,cis-12 seemed to be associated with a normal fat secretion. Results confirmed that MFD is the consequence of a combined effect of the outflow of many ruminal FA, which collectively affect mammary fat synthesis. Because the animals of the 2 groups were fed the same diet, these results suggested that factors other than diet are involved in the MFD syndrome. Feeding behavior (i.e., ability to select dietary ingredients in a total mixed ration), rumen environment and the composition of ruminal bacteria are additional factors able to modify the products of rumen biohydrogenation. Results of the present work confirmed that the multivariate approach can be a useful tool to evaluate a metabolic pathway that involves several parameters, providing interesting suggestions about the role of some FA involved in MFD. However, results about the MFD syndrome obtained in the present research require a deep molecular investigation to be confirmed.     

Nickel–boron alloy catalysts reduce the formation of Trans fatty acids in hydrogenated soybean oil

Soybean oil was hydrogenated by a group of amorphous alloy nanocatalysts of nickel and boron (Ni–B). The Ni–B alloy catalysts showed activities dependent on synthesis conditions. The trans-fat contents in products hydrogenated by one Ni–B catalyst were approximately half of those when a commercial nickel catalyst was used. The Ni–B catalyst had a lower selectivity towards formation of trans-fatty acids but a higher selectivity towards formation of stearic acid than the commercial nickel catalyst. The increases in trans-fatty acids correlated linearly with the increases in C18:1 (oleic and elaidic acids) for nickel-containing catalysts. The incorporation of boron in nickel-containing catalysts increased the selectivity ratio (for forming stearic acid) but did not change the linolenic selectivity. Because of the simplicity to synthesize amorphous catalysts, this work may lead to future development of novel alloy nanocatalysts to synergistically reduce the formation of both trans- and saturated fatty acids in hydrogenated products.