Microbiability of milk composition and genetic control of microbiota effects in sheep.

Recently, high-dimensional omics data are becoming available in larger quantities, and models have been developed that integrate them with genomics to understand in finer detail the relationship between genotype and phenotype, and thus improve the performance of genetic evaluations. Our objectives are to quantify the effect of the inclusion of microbiome data in the genetic evaluation for dairy traits in sheep, through the estimation of the heritability, microbiability, and how the microbiome effect on dairy traits decomposes into genetic and nongenetic parts. In this study we analyzed milk and rumen samples of 795 Lacaune dairy ewes. We included, as phenotype, dairy traits and milk fatty acids and proteins composition; as omics measurements, 16S rRNA rumen bacterial abundances; and as genotyping, 54K SNP chip for all ewes. Two nested genomic models were used: a first model to predict the individual contributions of the genetic and microbial abundances to phenotypes, and a second model to predict the additive genetic effect of the microbial community. In addition, microbiome-wide association studies for all dairy traits were applied using the 2,059 rumen bacterial abundances, and the genetic correlations between microbiome principal components and dairy traits were estimated. Results showed that in general the inclusion of both genetic and microbiome effect did not improve the fit of the model compared with the model with the genetic effect only. In addition, for all dairy traits the total heritability was equal to the direct heritability after fitting microbiota effects, due to a microbiability being almost zero for most dairy traits and heritability of the microbial community was very close to zero. Microbiome-wide association studies did not show operational taxonomic units with major effect for any of the dairy traits evaluated, and the genetic correlations between the first 5 principal components and dairy traits were low to moderate. So far, we can conclude that, using a substantial data set of 795 Lacaune dairy ewes, rumen bacterial abundances do not provide improved genetic evaluation for dairy traits in sheep.

Rumen microbiota and dietary fat: a mutual shaping.

Although fat content in usual ruminant diets is very low, fat supplements can be given to farm ruminants to modulate rumen activity or the fatty acid (FA) profile of meat and milk. Unsaturated FAs, which are dominant in common fat sources for ruminants, have negative effects on microbial growth, especially protozoa and fibrolytic bacteria. In turn, the rumen microbiota detoxifies unsaturated FAs (UFAs) through a biohydrogenation (BH) process, transforming dietary UFAs with cis geometrical double-bonds into mainly trans UFAs and, finally, into saturated FAs. Culture studies have provided a large amount of data regarding bacterial species and strains that are affected by UFAs or involved in lipolysis or BH, with a major focus on the Butyrivibrio genus. More recent data using molecular approaches to rumen microbiota extend and challenge these data, but further research will be necessary to improve our understanding of fat and rumen microbiota interactions.

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.

Effects of pH and fermentative substrate on ruminal metabolism of fatty acids during short-term in vitro incubation.

The ruminal biohydrogenation of c9,c12-18:2 can be affected by the fibre/starch ratio of the diet and the ruminal pH. The objectives of this study were to examine independently in vitro the effects of fermentation substrate (hay vs. corn starch) and buffer pH (6 vs. 7) on the biohydrogenation of c9,c12-18:2 carried out by grape seed oil, focusing on its t11 and t10 pathways, using 6-h ruminal incubations. The experimental design was a 2 × 2 factorial arrangement. Fermentation substrate and pH affected the C18 fatty acid balance in incubated media, but few interactions were observed. Compared with starch, hay as the fermentation substrate favoured the production of 18:0 (×2.3), all trans-18:1 isomers (×12.6) and CLA (×6.1), except c9,t11-CLA, and the disappearance of unsaturated C18 fatty acids, but decreased the production of odd and branched chain fatty acids. Compared with pH 6 buffer, pH 7 buffer resulted in higher c9,c12-18:2 disappearance and CLA production. For c9,t11-CLA, an interaction was noticed between the two factors, leading to the highest production in cultures incubated on hay with the 7 pH buffer. Compared with starch, hay as fermentation substrate favoured the activity of t11 producers, which are fibrolytic bacteria, and the production of t10 isomers, possibly due to the presence of potential t10 producers in hay. Low pH resulted in a decreased t11 isomers production and in a slightly increased t10 isomers production, probably due to a modulation of enzymatic or bacterial activity.

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.

Starch plus sunflower oil addition to the diet of dry dairy cows results in a trans-11 to trans-10 shift of biohydrogenation.

Trans fatty acids (FA), exhibit different biological properties. Among them, cis-9,trans-11 conjugated linoleic acid has some interesting putative health properties, whereas trans-10,cis-12 conjugated linoleic acid has negative effects on cow milk fat production and would negatively affect human health. In high-yielding dairy cows, a shift from trans-11 to trans-10 pathway of biohydrogenation (BH) can occur in the rumen of cows receiving high-concentrate diets, especially when the diet is supplemented with unsaturated fat sources. To study this shift, 4 rumen-fistulated nonlactating Holstein cows were assigned to a 4×4 Latin square design with 4 different diets during 4 periods. Cows received 12 kg of dry matter per day of 4 diets based on corn silage during 4 successive periods: a control diet (22% starch, <3% crude fat on DM basis), a high-starch diet supplemented with wheat plus barley (35% starch, <3% crude fat), a sunflower oil diet supplemented with 5% of sunflower oil (20% starch, 7.6% crude fat), and a high-starch plus sunflower oil diet (33% starch, 7.3% crude fat). Five hours after feeding, proportions of trans-11 BH isomers greatly increased in the rumen content with the addition of sunflower oil, without change in ruminal pH compared with the control diet. Addition of starch to the control diet had no effect on BH pathways but decreased ruminal pH. The addition of a large amount of starch in association with sunflower oil increased trans-10 FA at the expense of trans-11 FA in the rumen content, revealing a trans-11 to trans-10 shift. Interestingly, with this latter diet, ruminal pH did not change compared with a single addition of starch. This trans-11 to trans-10 shift occurred progressively, after a decrease in the proportion of trans-11 FA in the rumen, suggesting that this shift could result from a dysbiosis in the rumen in favor of trans-10-producing bacteria at the expense of those producing trans-11 or a modification of bacterial activities.

Predicting feed efficiency traits in growing lambs from their ruminal microbiota.

Selecting feed-efficient sheep could improve the sustainability of this livestock production. However, most sheep breeding companies cannot afford to record feed intake to select feed-efficient animals. Past studies underlined the potential of omics data, including microbiota metabarcoding data, as proxies for feed efficiency. The study involved 277 Romane lambs from two lines divergently selected for residual feed intake (RFI). There were two objectives: check the consequences of selecting for feed efficiency over the rumen microbiota, and assess the predictive ability of the rumen microbiota for host traits. The study assessed two contrasting diets (concentrate diet and mixed diet) and two microbial groups (prokaryotes and eukaryotes). Discriminant analyses did not highlight any significant effect of sheep selection for residual feed intake on the rumen microbiota composition. Indeed, prokaryotic and eukaryotic microbiota compositions poorly discriminated the RFI lines, with averaged balanced error rates ranging from 45% to 55%. Correlations between host traits (feed efficiency and production traits) and their predictions from microbiota data varied between -0.07 and 0.56, depending on the trait, diet and sequencing. Feed intake was the most accurately predicted trait. However, predictions from fixed effects and BW were more accurate than or as accurate as predictions from the microbiota. Environmental effects can greatly affect the variability of microbiota compositions. Considering batch and environmental effects should be paramount when the predictive ability of the microbiota is assessed. This study argues why metabarcoding the rumen microbiota is not the best way to predict meat sheep production traits: fixed effects and BW were more cost-effective proxies and they led to similar or better predictive accuracies than microbiota metabarcoding (16S and 18S sequencing).

Effects of oil and natural or synthetic vitamin E on ruminal and milk fatty acid profiles in cows receiving a high-starch diet.

Among trans fatty acids, trans-10,cis-12 CLA has negative effects on cow milk fat production and can affect human health. In high-yielding dairy cows, a shift from the trans-11 to the trans-10 pathway of biohydrogenation (BH) can occur in the rumen of cows receiving high-concentrate diets, especially when the diet is supplemented with unsaturated fat sources. In some but not all experiments, vitamin E has been shown to control this shift. To ascertain the effects of vitamin E on this shift of BH pathway, 2 studies were conducted. The first study explored in vitro the effects of addition of natural (RRR-α-tocopherol acetate) and synthetic (dl-α-tocopherol acetate) vitamin E. Compared with control and synthetic vitamin E, the natural form resulted in a greater trans-10/trans-11 ratio; however, the effect was very low, suggesting that vitamin E was neither a limiting factor for rumen BH nor a modulator of the BH pathway. An in vivo study investigated the effect of natural vitamin E (RRR-α-tocopherol) on this shift and subsequent milk fat depression. Six rumen-fistulated lactating Holstein cows were assigned to a 2×2 crossover design. Cows received 20-kg DM of a control diet based on corn silage with 22% of wheat, and after 2 wk of adaptation, the diet was supplemented with 600 g of sunflower oil for 2 more weeks. During the last week of this 4-wk experimental period, cows were divided into 2 groups: an unsupplemented control group and a group receiving 11 g of RRR-α-tocopherol acetate per day. A trans-10 shift of ruminal BH associated with milk fat depression due to oil supplementation of a high-wheat diet was observed, but vitamin E supplementation of dairy cows did not result in a reversal toward a trans-11 BH pathway, and did not restore milk fat content.

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.