The extent of nitrogen isotopic fractionation in rumen bacteria is associated with changes in rumen nitrogen metabolism.

Nitrogen use efficiency is an important index in ruminants and can be indirectly evaluated through the N isotopic discrimination between the animal and its diet (Δ15Nanimal-diet). The concentration and source of N may determine both the extent of the N isotopic discrimination in bacteria and N use efficiency. We hypothesised that the uptake and release of ammonia by rumen bacteria will affect the natural 15N enrichment of the bacterial biomass over their substrates (Δ15Nbacteria-substrate) and thereby further impacting Δ15Nanimal-diet. To test this hypothesis, two independent in vitro experiments were conducted using two contrasting N sources (organic vs inorganic) at different levels either in pure rumen bacteria culture incubations (Experiment #1) or in mixed rumen cultures (Experiment #2). In Experiment #1, tryptone casein or ammonium chloride were tested at low (1 mM N) and high (11.5 mM N) concentrations on three rumen bacterial strains (Fibrobacter succinogenes, Eubacterium limosum and Xylanibacter ruminicola) incubated in triplicate in anaerobic batch monocultures during 48h. In Experiment #2 mixed rumen cultures were incubated during 120 h with peptone or ammonium chloride at five different levels of N (1.5, 3, 4.5, 6 and 12-mM). In experiment #1, Δ15Nbacteria-substrate was lowest when the ammonia-consumer bacterium Fibrobacter succinogenes was grown on ammonium chloride, and highest when the proteolytic bacterial strain Xylanibacter ruminicola was grown on tryptone. In experiment #2, Δ15Nbacteria-substrate was lower with inorganic (ammonium chloride) vs organic (peptone) N source. A strong negative correlation between Δ15Nbacteria-substrate and Rikenellaceae_RC9_gut_group, a potential fibrolytic rumen bacterium, was detected. Together, our results showed that Δ15Nbacteria-substrate may change according to the balance between synthesis of microbial protein from ammonia versus non-ammonia N sources and confirm the key role of rumen bacteria as modulators of Δ15Nanimal-diet.

Untargeted metabolomics confirms the association between plasma branched chain amino acids and residual feed intake in beef heifers.

This study explored plasma biomarkers and metabolic pathways underlying feed efficiency measured as residual feed intake (RFI) in Charolais heifers. A total of 48 RFI extreme individuals (High-RFI, n = 24; Low-RFI, n = 24) were selected from a population of 142 heifers for classical plasma metabolite and hormone quantification and plasma metabolomic profiling through untargeted LC-MS. Most efficient heifers (Low-RFI) had greater (P = 0.03) plasma concentrations of IGF-1 and tended to have (P = 0.06) a lower back fat depth compared to least efficient heifers. However, no changes were noted (P ≥ 0.10) for plasma concentrations of glucose, insulin, non-esterified fatty acids, ß-hydroxybutyrate and urea. The plasma metabolomic dataset comprised 3,457 ions with none significantly differing between RFI classes after false discovery rate correction (FDR > 0.10). Among the 101 ions having a raw P < 0.05 for the RFI effect, 13 were putatively annotated by using internal databases and 6 compounds were further confirmed with standards. Metabolic pathway analysis from these 6 confirmed compounds revealed that the branched chain amino acid metabolism was significantly (FDR < 0.05) impacted by the RFI classes. Our results confirmed for the first time in beef heifers previous findings obtained in male beef cattle and pointing to changes in branched-chain amino acids metabolism along with that of body composition as biological mechanisms related to RFI. Further studies are warranted to ascertain whether there is a cause-and-effect relationship between these mechanisms and RFI.

Protein metabolism, body composition and oxygen consumption in young bulls divergent in residual feed intake offered two contrasting forage-based diets.

Protein metabolism and body composition have been identified as major determinants of residual feed intake (RFI) in beef cattle fed high-starch fattening diets. This study aimed to evaluate if these two identified RFI determinants in beef cattle are the same across two contrasting silage-based diets. During two consecutive years, an 84-day feed efficiency test (Test A) immediately followed by a second 112-day feed efficiency test (Test B) was carried out using a total of 100 animals offered either one of two diets (either corn silage- or grass silage-based) over 196 days. At the end of Test A, the 32 animals most divergent for RFI (16 extreme RFI animals per diet, eight low RFI and eight high RFI) were identified and evaluated during Test B for their i) N use efficiency (NUE; N retention/N intake) calculated either from a 10-d nitrogen balance trial or from estimations based on body composition changes occurring during the whole experiment (Test A and Test B; 196 days), ii) carcass and whole-body protein turnover rates analysed through the 3-methyl-histidine urinary excretion and the N isotopic turnover rates of urine, respectively, and iii) body composition measured at the slaughterhouse at the end of Test B. Oxygen consumption was measured during Test B for the 100 animals by two GreenFeed systems. Irrespective of the diet, efficient RFI animals tended (P = 0.08) to improve their NUE when N retention was estimated for 196 days or when considering their lower urinary urea-N to total N ratio (P = 0.03). In contrast, NUE calculated during the 10-d N balance showed no differences (P = 0.65) across RFI groups suggesting that this method may not be suitable to capture small NUE differences. Efficient RFI individuals presented higher dressing percentage and muscle deposition in the carcass (P = 0.003) but lighter rumen (P = 0.001), and a trend for lower oxygen consumption (P = 0.08) than inefficient RFI animals irrespective of the diet. Lower protein degradation rates of skeletal muscle and lower protein synthesis rates of plasma proteins were found in efficient RFI cattle but only with the corn silage-based diet (RFI × Diet; P = 0.02). The higher insulinaemia associated with the corn silage-based diet (P = 0.001) seemed to be a key metabolic feature explaining the positive association between protein turnover and RFI only in this diet. Feed N was more efficiently used for growth by efficient RFI animals regardless of the diet but lower protein turnover rates in efficient RFI animals were only observed with corn silage-based diets.

Common and diet-specific metabolic pathways underlying residual feed intake in fattening Charolais yearling bulls.

Residual feed intake (RFI) is one of the preferred traits for feed efficiency animal breeding. However, RFI measurement is expensive and time-consuming and animal ranking may depend on the nature of the diets. We aimed to explore RFI plasma biomarkers and to unravel the underlying metabolic pathways in yearling bulls fed either a corn-silage diet rich in starch (corn diet) or a grass-silage diet rich in fiber (grass diet). Forty-eight extreme RFI animals (Low-RFI, n = 24, versus High-RFI, n = 24, balanced per diet) were selected from a population of 364 Charolais bulls and their plasma was subjected to a targeted LC-MS metabolomic approach together with classical metabolite and hormonal plasma analyses. Greater lean body mass and nitrogen use efficiency, and lower protein turnover were identified as common mechanisms underlying RFI irrespective of the diet. On the other hand, greater adiposity and plasma concentrations of branched-chain amino acids (BCAA) together with lower insulin sensitivity in High-RFI animals were only observed with corn diet. Conversely, greater plasma concentrations of BCAA and total triglycerides, but similar insulin concentrations were noted in efficient RFI cattle with grass diet. Our data suggest that there are diet-specific mechanisms explaining RFI differences in fattening Charolais yearling bulls.

Plasma concentrations of branched-chain amino acids differ with Holstein genetic strain in pasture-based dairy systems.

In pasture-based systems, there are nutritional and climatic challenges exacerbated across lactation; thus, dairy cows require an enhanced adaptive capacity compared with cows in confined systems. We aimed to evaluate the effect of lactation stage (21 vs. 180 days in milk, DIM) and Holstein genetic strain (North American Holstein, NAH, n = 8; New Zealand Holstein, NZH, n = 8) on metabolic adaptations of grazing dairy cows through plasma metabolomic profiling and its association with classical metabolites. Although 67 metabolites were affected (FDR < 0.05) by DIM, no metabolite was observed to differ between genetic strains while only alanine was affected (FDR = 0.02) by the interaction between genetic strain and DIM. However, complementary tools for time-series analysis (ASCA analysis, MEBA ranking) indicated that alanine and the branched-chain amino acids (BCAA) differed between genetic strains in a lactation-stage dependent manner. Indeed, NZH cows had lower (P-Tukey < 0.05) plasma concentrations of leucine, isoleucine and valine than NAH cows at 21 DIM, probably signaling for greater insulin sensitivity. Metabolic pathway analysis also revealed that, independently of genetic strains, AA metabolism might be structurally involved in homeorhetic changes as 40% (19/46) of metabolic pathways differentially expressed (FDR < 0.05) between 21 and 180 DIM belonged to AA metabolism.

Gene networks for three feed efficiency criteria reveal shared and specific biological processes.

BACKGROUND: French beef producers suffer from the decrease in profitability of their farms mainly because of the continuous increase in feed costs. Selection for feed efficiency in beef cattle represents a relevant solution to face this problem. However, feed efficiency is a complex trait that can be assessed by three major criteria: residual feed intake (RFI), residual gain (RG) and feed efficiency ratio (FE), which involve different genetic determinisms. An analysis that combines phenotype and whole-genome sequence data provides a unique framework for genomic studies. The aim of our study was to identify the gene networks and the biological processes that are responsible for the genetic determinism that is shared between these three feed efficiency criteria. RESULTS: A population of 1477 French Charolais young bulls was phenotyped for feed intake (FI), average daily gain (ADG) and final weight (FW) to estimate RFI, RG and FE. A subset of 789 young bulls was genotyped on the BovineSNP50 single nucleotide polymorphism (SNP) array and imputed at the sequence level using RUN6 of the 1000 Bull Genomes Project. We conducted a genome-wide association study (GWAS) to estimate the individual effect of 8.5 million SNPs and applied an association weight matrix (AWM) approach to analyse the results, one for each feed efficiency criterion. The results highlighted co-association networks including 626 genes for RFI, 426 for RG and 564 for FE. Enrichment assessment revealed the biological processes that show the strongest association with RFI, RG and FE, i.e. digestive tract (salivary, gastric and mucin secretion) and metabolic processes (cellular and cardiovascular). Energetic functions were more associated with RFI and FE and cardio-vascular and cellular processes with RG. Several hormones such as apelin, glucagon, insulin, aldosterone, the gonadotrophin releasing hormone and the thyroid hormone were also identified, and these should be tested in future studies as candidate biomarkers for feed efficiency. CONCLUSIONS: The combination of network and pathway analyses at the sequence level led to the identification of both common and specific mechanisms that are involved in RFI, RG and FE, and to a better understanding of the genetic determinism underlying these three criteria. The effects of the genes involved in each of the identified processes need to be tested in genomic evaluations to confirm the potential gain in reliability of using functional variants to select animals for feed efficiency.

The origin of N isotopic discrimination and its relationship with feed efficiency in fattening yearling bulls is diet-dependent.

Nitrogen (N) isotopic discrimination (i.e. the difference in natural 15N abundance between the animal proteins and the diet; Δ15N) is known to correlate with N use efficiency (NUE) and feed conversion efficiency (FCE) in ruminants. However, results from the literature are not always consistent across studies, likely due to isotopic discrimination pathways that may differ with the nature of diets. The objective of the present study was to assess at which level, from rumen to tissues, Δ15N originates and becomes related to NUE and FCE in fattening yearling bulls when they are fed two contrasted diets. Twenty-four Charolais yearling bulls were randomly divided into two groups and fed during 8 months, from weaning to slaughter, either 1) a high starch diet based on corn silage supplying a balanced N to energy ratio at the rumen level (starch) or 2) a high fiber diet based on grass silage supplying an excess of rumen degradable N (fiber). All animals were slaughtered and samples of different digestive pools (ruminal, duodenal, ileal and fecal contents), animal tissues (duodenum, liver and muscle), blood and urine were collected for each animal. Ruminal content was further used to isolate liquid-associated bacteria (LAB), protozoa and free ammonia, while plasma proteins were obtained from blood. All samples along with feed were analyzed for their N isotopic composition. For both diets, the digestive contribution (i.e. the N isotopic discrimination occurring before absorption) to the Δ15N observed in animal tissues accounted for 65 ± 11%, leaving only one third to the contribution of post-absorptive metabolism. Concerning the Δ15N in digestive pools, the majority of these changes occurred in the rumen (av. Δ15N = 2.12 ± 0.66‰), with only minor 15N enrichments thereafter (av. Δ15N = 2.24 ± 0.41‰), highlighting the key role of the rumen on N isotopic discrimination. A strong, significant overall relationship (n = 24) between Δ15N and FCE or NUE was found when using any post-absorptive metabolic pool (duodenum, liver, or muscle tissues, or plasma proteins; 0.52 < r < 0.73; P ≤ 0.01), probably as these pools reflect both digestive and post-absorptive metabolic phenomena. Fiber diet compared to starch diet had a lower feed efficiency and promoted higher (P ≤ 0.05) Δ15N values across all post-absorptive metabolic pools and some digestive pools (ruminal, duodenal, and ileal contents). The within-diet relationship (n = 12) between Δ15N and feed efficiency was not as strong and consistent as the overall relationship, with contrasted responses between the two diets for specific pools (diet x pool interaction; P ≤ 0.01). Our results highlight the contrasted use of N at the rumen level between the two experimental diets and suggests the need for different equations to predict FCE or NUE from Δ15N according to the type of diet. In conclusion, rumen digestion and associated microbial activity can play an important role on N isotopic discrimination so rumen effect related to diet may interfere with the relationship between Δ15N and feed efficiency in fattening yearling bulls.

Natural 15N abundance in specific amino acids indicates associations between transamination rates and residual feed intake in beef cattle.

Improving the ability of animals to convert feed resources into food for humans is needed for more sustainable livestock systems. Genetic selection for animals eating less while maintaining their performance (i.e., low residual feed intake [RFI]) appears a smart strategy but its effectiveness relies on high-throughput animal phenotyping. Here, we explored plasma nitrogen (N) isotope ratios in an attempt to identify easily superior young bulls in terms of RFI. For this, 48 Charolais young bulls fed two contrasting diets (corn vs. grass silage diets) were selected from a larger population as extreme RFI animals (24 low-RFI vs. 24 high-RFI) and their plasma analyzed for natural 15N abundance (δ15N) in the whole protein (bulk protein) and in the individual protein-bound amino acids (PbAA). For the first time, we showed that the δ 15N in plasma bulk protein differed (P = 0.007) between efficient (low-RFI) and inefficient (high-RFI) cattle regardless of diet. Furthermore, most analyzed PbAA followed the same trend as the bulk protein, with lower (P < 0.05) δ 15N values in more efficient (low-RFI) compared with less efficient (high-RFI) cattle, again regardless of diet. The only three exceptions were Phe, Met, and Lys (P > 0.05) for which the first metabolic reaction before being catabolized does not involve transamination, a pathway known naturally to enrich AAs in 15N. The contrasted isotopic signatures across RFI groups only in those PbAA undergoing transamination are interpreted as differences in transamination rates and N-use efficiency between low- and high-RFI phenotypes. Natural isotopic N signatures in bulk proteins and specific PbAA can be proposed as biomarkers of RFI in growing beef cattle fed different diets. However, the current study cannot delineate whether this effect only occurs post-absorption or to some extent also in the rumen. Our data support the conclusion that most efficient cattle in terms of RFI upregulate N conservation mechanisms compared with less efficient cattle and justify future research on this topic.

New Approach Studying Interactions Regarding Trade-Off between Beef Performances and Meat Qualities.

The beef cattle industry is facing multiple problems, from the unequal distribution of added value to the poor matching of its product with fast-changing demand. Therefore, the aim of this study was to examine the interactions between the main variables, evaluating the nutritional and organoleptic properties of meat and cattle performances, including carcass properties, to assess a new method of managing the trade-off between these four performance goals. For this purpose, each variable evaluating the parameters of interest has been statistically modeled and based on data collected on 30 Blonde d'Aquitaine heifers. The variables were obtained after a statistical pre-treatment (clustering of variables) to reduce the redundancy of the 62 initial variables. The sensitivity analysis evaluated the importance of each independent variable in the models, and a graphical approach completed the analysis of the relationships between the variables. Then, the models were used to generate virtual animals and study the relationships between the nutritional and organoleptic quality. No apparent link between the nutritional and organoleptic properties of meat (r = -0.17) was established, indicating that no important trade-off between these two qualities was needed. The 30 best and worst profiles were selected based on nutritional and organoleptic expectations set by a group of experts from the INRA (French National Institute for Agricultural Research) and Institut de l'Elevage (French Livestock Institute). The comparison between the two extreme profiles showed that heavier and fatter carcasses led to low nutritional and organoleptic quality.

Fat accretion measurements strengthen the relationship between feed conversion efficiency and Nitrogen isotopic discrimination while rumen microbial genes contribute little.

The use of biomarkers for feed conversion efficiency (FCE), such as Nitrogen isotopic discrimination (Δ15N), facilitates easier measurement and may be useful in breeding strategies. However, we need to better understand the relationship between FCE and Δ15N, particularly the effects of differences in the composition of liveweight gain and rumen N metabolism. Alongside measurements of FCE and Δ15N, we estimated changes in body composition and used dietary treatments with and without nitrates, and rumen metagenomics to explore these effects. Nitrate fed steers had reduced FCE and higher Δ15N in plasma compared to steers offered non-nitrate containing diets. The negative relationship between FCE and Δ15N was strengthened with the inclusion of fat depth change at the 3rd lumbar vertebrae, but not with average daily gain. We identified 1,700 microbial genes with a relative abundance >0.01% of which, 26 were associated with Δ15N. These genes explained 69% of variation in Δ15N and showed clustering in two distinct functional networks. However, there was no clear relationship between their relative abundances and Δ15N, suggesting that rumen microbial genes contribute little to Δ15N. Conversely, we show that changes in the composition of gain (fat accretion) provide additional strength to the relationship between FCE and Δ15N.

Exploration of Biological Markers of Feed Efficiency in Young Bulls.

The efficiency with which ruminants convert feed to desirable products is difficult to measure under normal commercial settings. We explored the use of potential biological markers from easily obtainable samples, that is, blood, hair, and feces, to characterize potential causes of divergent efficiency when considered as residual feed intake (RFI) or feed conversion efficiency (FCE). A total of 54 Charolais bulls, 20 in period 1 and 34 in period 2, were examined for individual dry matter intake (DMI) and growth. Bulls were offered a diet of 70:30 wrapped grass silage to concentrate for 99 d. At the conclusion of the test period, blood samples were collected for the determination of vitamins B2 and B6, and plasma used for the determination of metabolites, natural isotopic 15N abundance (15N NIA, expressed as δ15N ‰) and fractionation (Δ15Nplasma proteins-diet and Δ13Cplasma proteins-diet) and near-infrared spectroscopy (NIRS). Feces were analyzed by NIRS. Bulls were slaughtered at 15-17 months of age and carcass characteristics determined. Bulls were ranked according to RFI with extremes (SD ± 0.5; n = 31) classified as either efficient (Neg-RFI) or inefficient (Pos-RFI). Extreme bulls were then classified for FCE (high vs low FCE), changing the groups. Pos-RFI bulls consumed 14% more feed than Neg-RFI bulls for the same level of weight gain. Low FCE bulls tended to eat more, but had lower weight gains than high FCE bulls. No differences were detected in carcass conformation, fat scores, hot carcass weight, or dressing percentage. Yet, heart and bladder weights were heavier in Pos-RFI, and rumen weight tended to be heavier in Pos-RFI bulls. RFI did not affect bulk 15N or 13C fractionation. A negative correlation was observed between FCE and Δ15Nplasma proteins-diet. Inefficient bulls (Pos-RFI) had higher δ15N in glycine compared to Neg-RFI bulls. Similarly, metabolomic analysis showed a tendency for concentrations of glycine and sarcosine to be elevated in Pos-RFI bulls, whereas aspartic acid and carnosine tended to be elevated, and serine tended to be lower in High FCE. Among vitamins, only flavin adenine dinucleotide concentration was higher in the blood of bulls with High FCE. These results suggest that the two feed efficiency metrics differ in the underlying mechanisms of metabolism, where RFI is driven by differences in the energetic requirements of visceral organs and the extent of AA catabolism.

Natural (15)N Abundance in Key Amino Acids from Lamb Muscle: Exploring a New Horizon in Diet Authentication and Assessment of Feed Efficiency in Ruminants.

Natural (15)N abundance (δ(15)N) varies between individual amino acids (AAs). We hypothesized that δ(15)N of nontransaminating and essential AAs ("source" AAs, such as phenylalanine) present in animal tissues could be used as a marker of dietary origin, whereas δ(15)N of transaminating AAs ("trophic" AAs, such as glutamic acid) could give more detailed insights into animal feed efficiency. Two diets based on dehydrated Lucerne pellets were tested in growing lambs, which promoted different feed efficiencies. No dietary effects were noted on δ(15)N of any AAs analyzed in lamb muscle. In addition, δ(15)N of phenylalanine was unexpectedly similar to that of glutamic acid, suggesting that δ(15)N of AAs is significantly derived from the metabolism of the rumen microbiota and, thus, are not suited for diet authentication in ruminants. In contrast, the δ(15)N of transaminating AAs facilitates an improved prediction of animal feed efficiency compared to the classical isotopic bulk N analysis.