Yves Farizon

Starch and oil in the donor cow diet and starch in substrate differently affect the in vitro ruminal biohydrogenation of linoleic and linolenic acids

Trans isomers of fatty acids exhibit different health properties. Among them, trans-10,cis-12 conjugated linoleic acid has negative effects on milk fat production and can affect human health. A shift from the trans-11 to the trans-10 pathway of biohydrogenation (BH) can occur in the rumen of dairy cows receiving high-concentrate diets, especially when the diet is supplemented with highly unsaturated fat sources. The differences of BH patterns between linoleic acid (LeA) and linolenic acid (LnA) in such ruminal conditions remain unknown; thus, the aim of this work was to investigate in vitro the effects of starch and sunflower oil in the diet of the donor cows and starch level in the incubates on the BH patterns and efficiencies of LeA and LnA. The design was a 4 × 4 Latin square design with 4 cows, 4 periods, and 4 diets with combinations of 21 or 34% starch and 0 or 5% sunflower oil. The rumen content of each cow during each period was incubated with 4 substrates, combining 2 starch levels and either LeA or LnA addition. Capillary electrophoresis single-strand conformation polymorphism of incubates showed that dietary starch decreased the diversity of the bacterial community and the high-starch plus oil diet modified its structure. High-starch diets poorly affected isomerization and first reduction of LeA and LnA, but decreased the efficiencies of trans-11,cis-15-C18:2 and trans C18:1 reduction. Dietary sunflower oil increased the efficiency of LeA isomerization but decreased the efficiency of trans C18:1 reduction. An interaction between dietary starch and dietary oil resulted in the highest trans-10 isomers production in incubates when the donor cow received the high-starch plus oil diet. The partition between trans-10 and trans-11 isomers was also affected by an interaction between starch level and the fatty acid added to the incubates, showing that the trans-10 shift only occurred with LeA, whereas LnA was mainly hydrogenated via the more usual trans-11 pathway, whatever the starch level in the substrate, although the bacterial communities were not different between LeA and LnA incubates. In LeA incubates, trans-10 isomer production was significantly related to the structure of the bacterial community.

Temperature and duration of heating of sunflower oil affect ruminal biohydrogenation of linoleic acid in vitro

Sunflower oil heated at 110 or 150 degrees C for 1, 3, or 6h was incubated with ruminal content in order to investigate the effects of temperature and duration of heating of oil on the ruminal biohydrogenation of linoleic acid in vitro. When increased, these 2 parameters acted together to decrease the disappearance of linoleic acid in the media by inhibiting the isomerization of linoleic acid, which led to a decrease in conjugated linoleic acids and trans-C18:1 production. Nevertheless, trans-10 isomer production increased with heating temperature, suggesting an activation of Delta(9)-isomerization, whereas trans-11 isomer production decreased, traducing an inhibition of Delta(12)-isomerization. The amount of peroxides generated during heating was correlated with the proportions of biohydrogenation intermediates so that they might explain, at least in part, the observed effects. The effects of heating temperature and duration on ruminal bacteria community was assessed using capillary electrophoresis single-strand conformation polymorphism. Ruminal bacterial population significantly differed according to heating temperature, but was not affected by heating duration. Heating of fat affected ruminal biohydrogenation, at least in part because of oxidative products generated during heating, by altering enzymatic reactions and bacterial population.

Effect of chemical form, heating, and oxidation products of linoleic acid on rumen bacterial population and activities of biohydrogenating enzymes.

Heating polyunsaturated fatty acids (PUFA) produces oxidation products, such as hydroperoxides, aldehydes, and oxypolymers, which could be responsible at least in part for modification of PUFA rumen biohydrogenation (BH). Three in vitro experiments were conducted to investigate the effects of linoleic acid (cis-9,cis-12-C18:2) oxidation products on BH. In the first experiment, we studied the effects of free linoleic acid (FLA), heated FLA (HFLA, at 150 °C for 6h), triacylglycerols of linoleic acid (TGLA), heated TGLA (HTGLA, at 150 °C for 6h), 13-hydroperoxide (13HPOD), trans-2-decenal (T2D), and hexanal (HEX) on BH in vitro after 6 and 24h of incubation. In the second experiment, aldehydes differing in chain length and degree of unsaturation [pentanal, HEX, heptanal, nonanal, T2D, trans-2,trans-4-decadienal (T2T4D)] were incubated in vitro for 5h in rumen fluid. In the third experiment, 9-hydroperoxide (9HPOD), 13HPOD, HEX, or T2T4D were incubated for 1h in rumen fluid inactivated with chloramphenicol to investigate their effects on enzyme activity. In experiment 1, heat treatment of TGLA generated TGLA oxypolymers, did not affect cis-9,cis-12-C18:2 disappearance, but did decrease BH intermediates, especially trans-11 isomers. Heating FLA decreased cis-9,cis-12-C18:2 disappearance and cis-9,trans-11-CLA and trans-11-C18:1 production. Treatment with HEX and T2D did not affect cis-9,cis-12-C18:2 disappearance and barely affected production of BH intermediates. The bacterial community was affected by 13HPOD compared with FLA and HFLA, in parallel with an increase in trans-10 isomer production after a 6-h incubation. After 24h of incubation, 13HPOD decreased trans-11 isomer production, but to a lesser extent than HFLA. In experiment 2, some weak but significant effects were observed on BH, unrelated to chain length or degree of unsaturation of aldehydes; the bacterial community was not affected. In experiment 3, 9HPOD inhibited Δ(9)-isomerization, and both 9HPOD and 13HPOD inhibited Δ(12)-isomerization. We concluded that oxypolymers did not affect cis-9,cis-12-C18:2 disappearance. Heating both esterified and free cis-9,cis-12-C18:2 greatly altered Δ(12)-isomerization. Aldehydes had few effects. Hydroperoxides are responsible, at least in part, for the effects of fat heating: 13HPOD increased trans-10 isomer production (probably by affecting the bacterial community) and decreased trans-11 isomer production by inhibiting Δ(12)-isomerase activity, whereas 9HPOD inhibited both isomerases.

Effects of the heating process of soybean oil and seeds on fatty acid biohydrogenation in vitro

Heating fat is an efficient way to alter ruminal biohydrogenation (BH) and milk fat quality. Nevertheless, results are variable among studies and this could be due to various heating conditions differently affecting BH. The objectives of this study were to determine the effect of type and duration of heating of soybean oil or seeds on BH in vitro. Ruminal content cultures were incubated to first investigate the effects of roasting duration (no heating, and 0.5- and 6-h roasting) at 125°C and its interaction with fat source (soybean seeds vs. soybean oil), focusing on linoleic acid BH and its intermediates: conjugated linoleic acid (CLA) and trans-C18:1. Additionally, we compared the effects of seed extrusion with the 6 combinations of unheated and roasted oils and seeds. None of the treatments was efficient to protect linoleic acid from BH. Soybean oil resulted in higher trans-11 isomer production than seeds: 5.7 and 1.2 times higher for cis-9,trans-11 CLA and trans-11 C18:1, respectively. A 125°C, 0.5-h roasting increased trans-11 isomer production by 11% compared with no heating and 6-h roasted fat. Extrusion of seeds was more efficient to increase trans-11 C18:1 production than seed roasting, leading to values similar to oils. For other fatty acids, including cis-9,trans-11 CLA, extrusion resulted in similar balances to seeds (mainly 0.5-h-roasted seeds). Extruded oilseeds would be more efficient than roasted seeds to produce trans-11 C18:1; nevertheless, effects of conditions of extrusion need to be explored.

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.

Somatic cell count–based selection reduces susceptibility to energy shortage during early lactation in a sheep model

During the transition from late gestation to early lactation ruminants experience a negative energy balance (NEB), which is considered to increase susceptibility to mammary infections. Our previous study in 2 divergent lines of sheep selected for high and low somatic cell score (SCS) suggested an association between the response to NEB and genetic susceptibility to mastitis. Forty-eight early-lactation primiparous dairy ewes from the 2 SCS genetic lines were allocated to 2 homogeneous subgroups-an NEB group, which was energy restricted and received 60% of the energy requirements for 15 d, and a control-fed group-to obtain 4 balanced groups of 12 ewes: high-SCS positive energy balance, low-SCS positive energy balance, high-SCS NEB, and low-SCS NEB. High-SCS ewes showed greater weight loss and increased plasmatic concentrations of β-hydroxybutyrate and nonesterified fatty acids than low-SCS ewes when confronted with an induced NEB. The aim of this study was to further characterize this interaction by combining transcriptomic and phenotypic data with a generalized partial least squares discriminant analysis using mixOmics package framework. A preliminary analysis using 3 blocks of phenotypes (fatty acids, weight and production, blood metabolites) revealed a high correlation between fat-to-protein ratio, β-hydroxybutyrate, and nonesterified fatty acids concentrations with milk long-chain fatty acid yields. These phenotypes allowed good discrimination of the energy-restricted high-SCS ewes and confirmed a high level of adipose tissue mobilization in this group. A second analysis, which included RNA-seq data, revealed high correlations between the long-chain fatty acid yields in milk and PDK4, CPT1A, SLC25A20, KLF10, and KLF11 expression, highlighting the relationship between mobilization of body reserves and enhanced fatty acids utilization for energy production in blood cells. Finally, analysis of milk composition measured in 1,025 ewes from the 2 genetic lines over 10 yr confirmed significant higher fat-to-protein ratio in high-SCS ewes in early lactation. Altogether, our results strongly confirmed a genetic link between susceptibility to mastitis and metabolic adaptation to energy shortage. Improving genetic resistance to mastitis using SCS should be accompanied by a favorable effect on the response to metabolic stress, especially in highly stressful early lactation. Moreover, this study suggests that the fat-to-protein ratio could be used as a low-cost tool for monitoring energy balance and ketosis during this critical phase of lactation.