Integrating data from spontaneous and induced trans-10 shift of ruminal biohydrogenation reveals discriminant bacterial community changes at the OTU level.

Introduction: Microbial digestion is of key importance for ruminants, and disturbances can affect efficiency and quality of products for human consumers. Ruminal biohydrogenation of dietary unsaturated fatty acids leads to a wide variety of specific fatty acids. Some dietary conditions can affect the pathways of this transformation, leading to trans-10 fatty acids rather than the more usual trans-11 fatty acids, this change resulting in milk fat depression in dairy cows. Materials and methods: We combined data from an induced and spontaneous trans-10 shift of ruminal biohydrogenation, providing new insight on bacterial changes at different taxonomic levels. A trans-10 shift was induced using dietary addition of concentrate and/or unsaturated fat, and the spontaneous milk fat depression was observed in a commercial dairy herd. Results and discussion: Most changes of microbial community related to bacteria that are not known to be involved in the biohydrogenation process, suggesting that the trans-10 shift may represent the biochemical marker of a wide change of bacterial community. At OTU level, sparse discriminant analysis revealed strong associations between this change of biohydrogenation pathway and some taxa, especially three taxa belonging to [Eubacterium] coprostanoligenes group, Muribaculaceae and Lachnospiraceae NK3A20 group, that could both be microbial markers of this disturbance and candidates for studies relative to their ability to produce trans-10 fatty acids.

Enzymatic Study of Linoleic and Alpha-Linolenic Acids Biohydrogenation by Chloramphenicol-Treated Mixed Rumen Bacterial Species.

In the rumen, dietary polyunsaturated fatty acids (PUFA) are reduced by a multistage reaction called biohydrogenation (BH). BH leads to a high proportion of saturated fat in ruminant products, but also products some potential bioactive intermediates like conjugated linoleic and linolenic acids. BH is composed of two kinds of reactions: first an isomerization of PUFA followed by reductions (two for linoleic acid, C18:2n-6; three for α-linolenic acid, C18:3n-3). There is little knowledge about BH enzymes as BH bacterial species are the subject of a lot of studies. Nevertheless, both aspects must be explored to control BH and enhance the fatty acids profile of ruminant products. In the present study, an alternative approach was developed to study the enzymes produced in vivo by mixed ruminal bacteria, using inactivation of bacteria by chloramphenicol, an inhibitor of protein synthesis in prokaryotes, before in vitro incubation. To study C18:2n-6 and C18:3n-3 BH several experiments were used: (1) with different incubation durations (0 to 3) to estimate average rates and efficiencies of all BH reactions, and intermediates production; and (2) with different initial quantities of PUFA (0.25 to 2 mg) to estimate Michaelis-Menten enzymatic parameters, Km and Vmax. A last experiment explored the effect of pH buffer and donor cow diet on C18:2n-6 isomerization pathways. Concerning C18:2n-6 BH, this study confirmed the high saturability of its isomerization, the inhibition of both trans11 and trans10 pathways by a low pH, and the last reduction to stearic acid as the limiting-step. Concerning C18:3n-3, its BH was faster than C18:2n-6, in particular its isomerization (Vmax = 3.4 vs. 0.6 mM/h, respectively), and the limiting-step was the second reduction to t11-C18:1. Besides, our mixed isomerases had a higher affinity for C18:2n-6 than for C18:3n-3 (Km = 2.0 × 10-3 vs. 4.3 × 10-3 M, respectively), but due to their high saturability by C18:2n-6, they had a lower efficiency to isomerize C18:2n-6 than C18:3n-3. Chloramphenicol-treated ruminal fluid would be a meaningful method to study the BH enzymes activities.

Whole blood transcriptome analysis reveals potential competition in metabolic pathways between negative energy balance and response to inflammatory challenge.

Negative Energy Balance (NEB) is considered to increase susceptibility to mastitis. The objective of this study was to improve our understanding of the underlying mechanisms by comparing transcriptomic profiles following NEB and a concomitant mammary inflammation. Accordingly, we performed RNA-seq analysis of blood cells in energy-restricted ewes and control-diet ewes at four different time points before and after intra mammary challenge with phlogogenic ligands. Blood leucocytes responded to NEB by shutting down lipid-generating processes, including cholesterol and fatty acid synthesis, probably under transcriptional control of SREBF 1. Furthermore, fatty acid oxidation was activated and glucose oxidation and transport inhibited in response to energy restriction. Among the differentially expressed genes (DEGs) in response to energy restriction, 64 genes were also differential in response to the inflammatory challenge. Opposite response included the activation of cholesterol and fatty acid synthesis during the inflammatory challenge. Moreover, activation of glucose oxidation and transport coupled with the increase of plasma glucose concentration in response to the inflammatory stimuli suggested a preferential utilization of glucose as the energy source during this stress. Leucocyte metabolism therefore undergoes strong metabolic changes during an inflammatory challenge, which could be in competition with those induced by energy restriction.

Microbial ecology of the rumen evaluated by 454 GS FLX pyrosequencing is affected by starch and oil supplementation of diets.

To provide a comprehensive examination of the bacterial diversity in the rumen content of cows fed different diets, high-throughput 16S rRNA gene-based pyrosequencing was used. Four rumen fistulated nonlactating Holstein cows received 12 kg of dry matter per day of four diets based on maize silage during four periods: the low-starch diet (22% starch, 3% fat); the high-starch diet, supplemented with wheat plus barley (35% starch, 3% fat); the low-starch plus oil diet, supplemented with 5% of sunflower oil (20% starch, 7.6% fat) and the high-starch plus oil diet (33% starch, 7.3% fat). Samples were taken after 12 days of adaptation, 5 h postfeeding. Whatever the diet, bacterial community of sieved rumen contents was dominated by Firmicutes and Bacteroidetes. Lachnospiraceae, Ruminococcaceae, Prevotellaceae, and Rikenellaceae families were highly present and were clearly affected by cow diet. The highest abundance of Prevotellaceae and the lowest abundance of Ruminococcaceae and Rikenellaceae were found with the high-starch plus oil diet. Dietary starch increased the relative abundance of only three genera: Barnesiella, Oribacterium and Olsenella, but decreased the relative abundances of several genera, with very significant effects for Rikenellaceae_RC9 and Butyrivibrio-Pseudobutyrivibrio. Oil alone had a limited effect, but interestingly, starch plus oil addition differently affected the bacterial populations compared to starch addition without oil.

Validation of a new point-of-care assay for determination of ß-carotene concentration in bovine whole blood and plasma.

BACKGROUND: ß-Carotene is an important precursor of vitamin A, and is associated with bovine fertility. ß-Carotene concentrations in plasma are used to optimize ß-carotene supplementation in cattle, but measurement requires specialized equipment to separate plasma and extract and measure ß-carotene, either using spectrophotometry or high performance liquid chromatography (HPLC). OBJECTIVE: The objective of this study was to validate a new 2-step point-of-care (POC) assay for measuring ß-carotene in whole blood and plasma. METHODS: ß-carotene concentrations in plasma from 166 cows were measured using HPLC and compared with results obtained using a POC assay, the iCheck-iEx-Carotene test kit. Whole blood samples from 23 of these cattle were also evaluated using the POC assay and compared with HPLC-plasma results from the same 23 animals. The POC assay includes an extraction vial (iEx Carotene) and hand-held photometer (iCheck Carotene). RESULTS: Concentrations of ß-carotene in plasma measured using the POC assay ranged from 0.40 to 15.84 mg/L (n = 166). No differences were observed between methods for assay of plasma (mean ± SD; n = 166): HPLC-plasma 4.23 ± 2.35 mg/L; POC-plasma 4.49 ± 2.36 mg/L. Similar good agreement was found when plasma analyzed using HPLC was compared with whole blood analyzed using the POC system (n = 23): HPLC-plasma 3.46 ± 2.12 mg/L; POC-whole blood 3.67 ± 2.29 mg/L. CONCLUSIONS: Concentrations of ß-carotene can be measured in blood and plasma from cattle easily and rapidly using a POC assay, and results are comparable to those obtained by the highly sophisticated HPLC method. Immediate feedback regarding ß-carotene deficiency facilitates rapid and appropriate optimization of ß-carotene supplementation in feed.

Effects of fat source and dietary sodium bicarbonate plus straw on the conjugated linoleic acid content of milk of dairy cows.

The effects of fat source (0.7 kg of fatty acids from extruded soybeans or palmitic acid), of sodium bicarbonate (0.3 kg) plus straw (1 kg) and the interaction of these treatments on the content of conjugated linoleic acid (CLA) in the milk of dairy cows were examined. During nine weeks a group of 10 cows received a ration with palmitic acid and bicarbonate plus straw (ration PAB). During three periods of three weeks a second group of 10 cows received successively a ration with extruded soybeans and bicarbonate plus straw (ration ESB), a ration with palmitic acid without bicarbonate or straw (ration PA), and a ration with extruded soybeans without bicarbonate or straw (ration ES). Rations ES and ESB increased the content of polyunsaturated fatty acids in milk, but decreased milk fat content, compared to rations PAB and PA. Ration ESB led to the greatest milk CLA content, by a synergy between the high amount of dietary fat, and the action of bicarbonate plus straw, favouring trans11 isomers of CLA and C18:1, presumably via a ruminal pH near neutrality. Ration ES favoured trans10 isomers, not desaturated in the mammary gland, so that the milk CLA content was lower than with ration ESB, and resulted in the lowest milk fat content. In conclusion, a ration supplemented with both extruded soybeans and bicarbonate plus straw, was an efficient way to increase the CLA content in the milk of dairy cows.

Rationale and design of the TRANSFACT project phase I: a study to assess the effect of the two different dietary sources of trans fatty acids on cardiovascular risk factors in humans.

BACKGROUND: Detrimental effects of consumption of industrial trans fatty acids (TFA) from partially hydrogenated vegetable oils (PHVO) on cardiovascular disease (CVD) risk factors are well documented. However, very little information is available on the effect of natural sources of TFA coming from milk fat, dairy products and ruminant meat. In fact, due to the naturally low level of TFA in milk fat, it is almost impossible to conduct a clinical trial with a limited number of subjects (<200). METHODOLOGY: To compare the effects of industrial and natural dietary sources of TFA, two specific test fats have been designed and produced. A substantial amount of milk fat (130 kg) enriched in TFA has been produced by modification of the cow's diet and selection of cows with the highest TFA content. The level obtained was approximately 4- to 7-fold higher than typically present in milk fat (approximately 20 instead of 3-6 g/100 g of total fatty acids). The control fat is composed of PHVO balanced in saturated fatty acids (lauric, myristic and palmitic). Both experimental fats contain about 20-22% of monounsaturated TFA and the volunteers' daily experimental fat intake (54 g), will represent about 12.0 g/day of TFA or 5.4% of the daily energy (based on 2000 kcal/day). These two test fats have been incorporated into food items and will be provided to 46 healthy subjects under a randomised, double blind, controlled, cross-over design. The primary outcome is high-density lipoprotein cholesterol (HDL-C), which is an independent risk factor for CVD. Other parameters such as low-density lipoprotein cholesterol (LDL-C), very low-density lipoprotein cholesterol (VLDL-C), and HDL-C level and subclasses will be also to be evaluated. CONCLUSION: We have shown that it is technically feasible to perform a clinical trial on the comparative effects of natural and industrial sources of TFA isomers on CVD risk factors. Results are expected by mid-2006.