Driving gut microbiota enterotypes through host genetics.

BACKGROUND: Population stratification based on interindividual variability in gut microbiota composition has revealed the existence of several ecotypes named enterotypes in humans and various animal species. Enterotypes are often associated with environmental factors including diet, but knowledge of the role of host genetics remains scarce. Moreover, enterotypes harbor functionalities likely associated with varying abilities and susceptibilities of their host. Previously, we showed that under controlled conditions, 60-day-old pig populations consistently split into two enterotypes with either Prevotella and Mitsuokella (PM enterotype) or Ruminococcus and Treponema (RT enterotype) as keystone taxa. Here, our aim was to rely on pig as a model to study the influence of host genetics to assemble enterotypes, and to provide clues on enterotype functional differences and their links with growth traits. RESULTS: We established two pig lines contrasted for abundances of the genera pairs specifying each enterotype at 60 days of age and assessed them for fecal microbiota composition and growth throughout three consecutive generations. Response to selection across three generations revealed, per line, an increase in the prevalence of the selected enterotype and in the average relative abundances of directly and indirectly selected bacterial genera. The PM enterotype was found less diverse than the RT enterotype but more efficient for piglet growth during the post-weaning period. Shotgun metagenomics revealed differentially abundant bacterial species between the two enterotypes. By using the KEGG Orthology database, we show that functions related to starch degradation and polysaccharide metabolism are enriched in the PM enterotype, whereas functions related to general nucleoside transport and peptide/nickel transport are enriched in the RT enterotype. Our results also suggest that the PM and RT enterotypes might differ in the metabolism of valine, leucin, and isoleucine, favoring their biosynthesis and degradation, respectively. CONCLUSION: We experimentally demonstrated that enterotypes are functional ecosystems that can be selected as a whole by exerting pressure on the host genetics. We also highlight that holobionts should be considered as units of selection in breeding programs. These results pave the way for a holistic use of host genetics, microbiota diversity, and enterotype functionalities to understand holobiont shaping and adaptation. Video Abstract.

Genic and non-genic SNP contributions to additive and dominance genetic effects in purebred and crossbred pig traits.

The present research has estimated the additive and dominance genetic variances of genic and intergenic segments for average daily gain (ADG), backfat thickness (BFT) and pH of the semimembranosus dorsi muscle (PHS). Further, the predictive performance using additive and additive dominance models in a purebred Piétrain (PB) and a crossbred (Piétrain × Large White, CB) pig population was assessed. All genomic regions contributed equally to the additive and dominance genetic variations and lead to the same predictive ability that did not improve with the inclusion of dominance genetic effect and inbreeding in the models. Using all SNPs available, additive genotypic correlations between PB and CB performances for the three traits were high and positive (> 0.83) and dominance genotypic correlation was very inaccurate. Estimates of dominance genotypic correlations between all pairs of traits in both populations were imprecise but positive for ADG-BFT in CB and BFT-PHS in PB and CB with a high probability (> 0.98). Additive and dominance genotypic correlations between BFT and PHS were of different sign in both populations, which could indicate that genes contributing to the additive genetic progress in both traits would have an antagonistic effect when used for exploiting dominance effects in planned matings.

Correlation Networks Provide New Insights into the Architecture of Testicular Steroid Pathways in Pigs.

Steroid metabolism is a fundamental process in the porcine testis to provide testosterone but also estrogens and androstenone, which are essential for the physiology of the boar. This study concerns boars at an early stage of puberty. Using a RT-qPCR approach, we showed that the transcriptional activities of several genes providing key enzymes involved in this metabolism (such as CYP11A1) are correlated. Surprisingly, HSD17B3, a key gene for testosterone production, was absent from this group. An additional weighted gene co-expression network analysis was performed on two large sets of mRNA-seq to identify co-expression modules. Of these modules, two containing either CYP11A1 or HSD17B3 were further analyzed. This comprehensive correlation meta-analysis identified a group of 85 genes with CYP11A1 as hub gene, but did not allow the characterization of a robust correlation network around HSD17B3. As the CYP11A1-group includes most of the genes involved in steroid synthesis pathways (including LHCGR encoding for the LH receptor), it may control the synthesis of most of the testicular steroids. The independent expression of HSD17B3 probably allows part of the production of testosterone to escape this control. This CYP11A1-group contained also INSL3 and AGT genes encoding a peptide hormone and an angiotensin peptide precursor, respectively.

How to Improve Meat Quality and Welfare in Entire Male Pigs by Genetics.

Giving up surgical castration is desirable to avoid pain during surgery but breeding entire males raises issues on meat quality, particularly on boar taint, and aggression. It has been known for decades that boar taint is directly related to sexual development in uncastrated male pigs. The proportion of tainted carcasses depends on many factors, including genetics. The selection of lines with a low risk of developing boar taint should be considered as the most desirable solution in the medium to long term. It has been evidenced that selection against boar taint is feasible, and has been set up in a balanced way in some pig populations to counterbalance potential unfavorable effects on reproductive performances. Selection against aggressive behaviors, though theoretically feasible, faces phenotyping challenges that compromise selection in practice. In the near future, new developments in modelization, automatic recording, and genomic data will help define breeding objectives to solve entire male meat quality and welfare issues.

Omics Application in Animal Science-A Special Emphasis on Stress Response and Damaging Behaviour in Pigs.

Increasing stress resilience of livestock is important for ethical and profitable meat and dairy production. Susceptibility to stress can entail damaging behaviours, a common problem in pig production. Breeding animals with increased stress resilience is difficult for various reasons. First, studies on neuroendocrine and behavioural stress responses in farm animals are scarce, as it is difficult to record adequate phenotypes under field conditions. Second, damaging behaviours and stress susceptibility are complex traits, and their biology is not yet well understood. Dissecting complex traits into biologically better defined, heritable and easily measurable proxy traits and developing biomarkers will facilitate recording these traits in large numbers. High-throughput molecular technologies ("omics") study the entirety of molecules and their interactions in a single analysis step. They can help to decipher the contributions of different physiological systems and identify candidate molecules that are representative of different physiological pathways. Here, we provide a general overview of different omics approaches and we give examples of how these techniques could be applied to discover biomarkers. We discuss the genetic dissection of the stress response by different omics techniques and we provide examples and outline potential applications of omics tools to understand and prevent outbreaks of damaging behaviours.

Characterization of Autozygosity in Pigs in Three-Way Crossbreeding.

Crossbreeding in livestock can be used to increase genetic diversity. The resulting increase in variability is related to the heterozygosity of the crossbred animal. The evolution of diversity during crossbreeding can be assessed using genomic data. The objective of this study was to describe patterns of runs of homozygosity (ROH) in animals resulting from three-way crossbreeding, from parental pure lines, and in their crossbred offspring. The crossbreeding scheme consisted of a first crossbreeding Pietrain boars and Large White sows, after which the offspring of the Pietrain × Large White were crossed with Duroc boars. The offspring of the second crossbreeding are called G0, the offspring of G0 boars and G0 sows are called G1. All the animals were genotyped using the Illumina SNP60 porcine chip. After filtering, analyses were performed with 2,336 animals and 48,579 autosomal single nucleotide polymorphism (SNP). The mean ROH-based inbreeding coefficients were shown to be 0.27 ± 0.05, 0.23 ± 0.04, and 0.26 ± 0.04 for Duroc, Large White, and Pietrain, respectively. ROH were detected in the Pietrain × Large White crossbred but the homozygous segments were fewer and smaller than in their parents. Similar results were obtained in the G0 crossbred. However, in the G1 crossbreds the number and the size of ROH were higher than in G0 parents. Similar ROH hotspots were detected on SSC1, SSC4, SSC7, SSC9, SSC13, SSC14, and SSC15 in both G0 and G1 animals. Long ROH (>16 Mb) were observed in G1 animals, suggesting regions with low recombination rates. The conservation of these homozygous segments in the three crossbred populations means that some haplotypes were shared between parental breeds. Gene annotation in ROH hotspots in G0 animals identified genes related to production traits including carcass composition and reproduction. These findings advance our understanding of how to manage genetic diversity in crossbred populations.

Prospects for the Analysis and Reduction of Damaging Behaviour in Group-Housed Livestock, With Application to Pig Breeding.

Innovations in the breeding and management of pigs are needed to improve the performance and welfare of animals raised in social groups, and in particular to minimise biting and damage to group mates. Depending on the context, social interactions between pigs can be frequent or infrequent, aggressive, or non-aggressive. Injuries or emotional distress may follow. The behaviours leading to damage to conspecifics include progeny savaging, tail, ear or vulva biting, and excessive aggression. In combination with changes in husbandry practices designed to improve living conditions, refined methods of genetic selection may be a solution reducing these behaviours. Knowledge gaps relating to lack of data and limits in statistical analyses have been identified. The originality of this paper lies in its proposal of several statistical methods for common use in analysing and predicting unwanted behaviours, and for genetic use in the breeding context. We focus on models of interaction reflecting the identity and behaviour of group mates which can be applied directly to damaging traits, social network analysis to define new and more integrative traits, and capture-recapture analysis to replace missing data by estimating the probability of behaviours. We provide the rationale for each method and suggest they should be combined for a more accurate estimation of the variation underlying damaging behaviours.

Pedigree and genomic evaluation of pigs using a terminal-cross model.

BACKGROUND: In crossbreeding schemes, within-line selection of purebreds is performed mainly to improve the performance of crossbred descendants under field conditions. The genetic correlation between purebred and crossbred performance is an important parameter to be assessed because purebred performance can be a poor predictor of the performance of crossbred offspring. With the availability of high-density markers, the feasibility of using crossbred information to evaluate purebred candidates can be reassessed. This study implements and applies a single-step terminal-cross model (GEN) to real data to estimate the genetic parameters of several production and quality traits in pigs. METHODS: Piétrain sires were mated with Piétrain and Large White dams to produce purebred and crossbred male half-sib piglets; growth rate, feed conversion ratio, lean meat, pH of longissimus dorsi muscle, drip loss and intramuscular fat content were recorded on all half-sibs. Animals were genotyped using the Illumina Porcine SNP60 BeadChip. The genetic correlation between purebred and crossbred performance was estimated separately for each trait. Purebred animals were evaluated using an animal model, whereas the additive genetic effect of a crossbred individual was decomposed into the additive effects of the sire and dam and a Mendelian sampling effect that was confounded with the residual effect. Genotypes of the Piétrain animals were integrated in the genetic evaluation by using a single-step procedure. As benchmarks, we used a model that was identical to GEN but only accounted for pedigree information (PED) and also two univariate single-step models (GEN_UNI) that took either purebred or crossbred performance into account. RESULTS: Genetic correlations between purebred and crossbred performance were high and positive for all traits (>0.69). Accuracies of estimated breeding values of genotyped sires and purebred offspring that were obtained with the GEN model outperformed both those obtained with the PED and the GEN_UNI models. The use of genomic information increased the predictive ability of the GEN model, but it did not substantially outperform the GEN_UNI models. CONCLUSIONS: We present a single-step terminal-cross model that integrates genomic information of purebred and crossbred performance by using available software. It improves the theoretical accuracy of genetic evaluations in breeding programs that are based on crossbreeding.

Time course of the response to ACTH in pig: biological and transcriptomic study.

BACKGROUND: HPA axis plays a major role in physiological homeostasis. It is also involved in stress and adaptive response to the environment. In farm animals in general and specifically in pigs, breeding strategies have highly favored production traits such as lean growth rate, feed efficiency and prolificacy at the cost of robustness. On the hypothesis that the HPA axis could contribute to the trade-off between robustness and production traits, we have designed this experiment to explore individual variation in the biological response to the main stress hormone, cortisol, in pigs. We used ACTH injections to trigger production of cortisol in 120 juvenile Large White (LW) pigs from 28 litters and the kinetics of the response was measured with biological variables and whole blood gene expression at 4 time points. A multilevel statistical analysis was used to take into account the longitudinal aspect of the data. RESULTS: Cortisol level reached its peak 1 h after ACTH injection. White blood cell composition was modified with a decrease of lymphocytes and monocytes and an increase of granulocytes (F D R<0.05). Basal level of cortisol was correlated with birth and weaning weights. Microarray analysis identified 65 unique genes of which expression responded to the injection of ACTH (adjusted P<0.05). These genes were classified into 4 clusters with distinctive kinetics in response to ACTH injection. The first cluster identified genes strongly correlated to cortisol and previously reported as being regulated by glucocorticoids. In particular, DDIT4, DUSP1, FKBP5, IL7R, NFKBIA, PER1, RGS2 and RHOB were shown to be connected to each other by the glucocorticoid receptor NR3C1. Most of the differentially expressed genes that encode transcription factors have not been described yet as being important in transcription networks involved in stress response. Their co-expression may mean co-regulation and they could thus provide new patterns of biomarkers of the individual sensitivity to cortisol. CONCLUSIONS: We identified 65 genes as biological markers of HPA axis activation at the gene expression level. These genes might be candidates for a better understanding of the molecular mechanisms of the stress response.

Economic aspects of implementing genomic evaluations in a pig sire line breeding scheme.

BACKGROUND: Replacing pedigree-based BLUP evaluations by genomic evaluations in pig breeding schemes can result in greater selection accuracy and genetic gains, especially for traits with limited phenotypes. However, this methodological change would generate additional costs. The objective of this study was to determine whether additional expenditures would be more profitably devoted to implementing genomic evaluations or to increasing phenotyping capacity while retaining traditional evaluations. METHODS: Stochastic simulation was used to simulate a population with 1050 breeding females and 50 boars that was selected for 10 years for a breeding goal with two uncorrelated traits with heritabilities of 0.4. The reference breeding scheme was based on phenotyping 13 770 candidates per year for trait 1 and 270 sibs of candidates per year for trait 2, with selection based on pedigree-based BLUP estimated breeding values. Increased expenditures were allocated to either increasing the phenotyping capacity for trait 2 while maintaining traditional evaluations, or to implementing genomic selection. The genomic scheme was based on two training populations: one for trait 2, consisting of phenotyped sibs of the candidates whose number increased from 1000 to 3430 over time, and one for trait 1, consisting of the selection candidates. Several genomic scenarios were tested, where the size of the training population for trait 1, and the number of genotyped candidates pre-selected based on their parental estimated breeding value, varied. RESULTS: Both approaches resulted in higher genetic trends for the population breeding goal and lower rates of inbreeding compared to the reference scheme. However, even a very marked increase in phenotyping capacity for trait 2 could not match improvements achieved with genomic selection when the number of genotyped candidates was large. Genotyping just a limited number of pre-selected candidates significantly reduced the extra costs, while preserving most of the benefits in terms of genetic trends and inbreeding. Implementing genomic evaluations was the most efficient approach when major expenditure was possible, whereas increasing phenotypes was preferable when limited resources were available. CONCLUSIONS: Economic decisions on implementing genomic evaluations in a pig nucleus population must take account of population characteristics, phenotyping and genotyping costs, and available funds.

New investigations around CYP11A1 and its possible involvement in an androstenone QTL characterised in Large White pigs.

BACKGROUND: Previously, in boars with extreme androstenone levels, differential expression of the CYP11A1 gene in the testes has been characterised. CYP11A1 is located in a region where a QTL influencing boar fat androstenone levels has been detected in a Large White pig population. Clarifying the role of CYP11A1 in boar taint is important because it catalyses the initial step of androstenone synthesis and also of steroid synthesis. RESULTS: A genome-wide association study located CYP11A1 at approximately 1300 kb upstream from SNP H3GA0021967, defining the centre of the region containing the QTL for androstenone variation. In this study, we partially sequenced the CYP11A1 gene and identified several new single nucleotide polymorphisms (SNP) within it. Characterisation of one animal, heterozygous for CYP11A1 testicular expression but homozygous for a haplotype of a large region containing CYP11A1, revealed that variation of CYP11A1 expression is probably regulated by a mutation located downstream from the SNP H3GA0021967. We analysed CYP11A1 expression in LW families according to haplotypes of the QTL region's centre. Effects of haplotypes on CYP11A1 expression and on androstenone accumulation were not concordant. CONCLUSION: This study shows that testicular expression of CYP11A1 is not solely responsible for the QTL influencing boar fat androstenone levels. As a conclusion, we propose to refute the hypothesis that a single mutation located near the centre of the QTL region could control androstenone accumulation in fat by regulating the CYP11A1 expression.

Genetic and metabolic aspects of androstenone and skatole deposition in pig adipose tissue: a review.

High levels of androstenone and skatole in fat tissues are considered the primary causes of boar taint, an unpleasant odour and flavour of the meat from non-castrated male pigs. The aim of this article is to review our current knowledge of the biology and genetic control of the accumulation of androstenone and skatole in fat tissue. Two QTL mapping studies have shown the complexity of the genetic control of these traits. During the last ten years, several authors have taken a more physiological approach to investigate the involvement of genes controlling the metabolism of androstenone and skatole. Although some authors have claimed the identification of candidate genes, it is more appropriate to talk about target genes. This suggests that genes affecting androstenone and skatole levels will have to be sought for among specific or non-specific transcription factors interacting with these target genes.

Relationships between sensory and physicochemical measurements in meat of rabbit from three different breeding systems using canonical correlation analysis.

Meat from rabbits reared either according to a standard (STAND) or a high quality norm (LABEL) or a low growth breeding (RUSSE) system were submitted to a sensory evaluation and to a large set of physicochemical measurements (weight of retail cuts, colour parameters, ultimate pH, femur flexure test, Warner-Bratzler shear test, water holding capacities and cooking losses). STAND rabbit meat exhibited the most juicy meat in back and in leg (p<0.01). Leg tenderness significantly decreased (p<0.001) in the rank order STAND>LABEL>RUSSE. Canonical correlation analysis showed strong correlations between physicochemical and sensory variables (R(2)=0.73 and 0.68 between the two first pairs of canonical variates). Especially, sensory tenderness and WB shear test variables assessed on raw longissimus muscle (LL) were correlated. Fibrous attribute in back was correlated with cooking loss in LL. When analysed separately only RUSSE rabbits exhibited the same relations between variables as those calculated in whole dataset.

Identification of QTL with effects on intramuscular fat content and fatty acid composition in a Duroc x Large White cross.

BACKGROUND: Improving pork quality can be done by increasing intramuscular fat (IMF) content. This trait is influenced by quantitative trait loci (QTL) sought out in different pig populations. Considering the high IMF content observed in the Duroc pig, it was appealing to determine whether favourable alleles at a major gene or QTL could be found. The detection was performed in an experimental F2 Duroc x Large White population first by segregation analysis, then by QTL mapping using additional molecular information. RESULTS: Segregation analysis provided evidence for a major gene, with a recessive Duroc allele increasing IMF by 1.8% in Duroc homozygous pigs. However, results depended on whether data were normalised or not. After Box-Cox transformation, likelihood ratio was indeed 12 times lower and no longer significant. The QTL detection results were partly consistent with the segregation analysis. Three QTL significant at the chromosome wide level were evidenced. Two QTL, located on chromosomes 13 and 15, showed a high IMF Duroc recessive allele with an overall effect slightly lower than that expected from segregation analysis (+0.4 g/100 g muscle). The third QTL was located on chromosome 1, with a dominant Large White allele inducing high IMF content (+0.5 g/100 g muscle). Additional QTL were detected for muscular fatty acid composition. CONCLUSION: The study presented results from two complementary approaches, a segregation analysis and a QTL detection, to seek out genes involved in the higher IMF content observed in the Duroc population. Discrepancies between both methods might be partially explained by the existence of at least two QTL with similar characteristics located on two different chromosomes for which different boars were heterozygous. The favourable and dominant allele detected in the Large White population was unexpected. Obviously, in both populations, the favourable alleles inducing high IMF content were not fixed and improving IMF by fixing favourable alleles using markers can then be applied both in Duroc and LW populations. With QTL affecting fatty acid composition, combining an increase of IMF content enhancing monounsaturated fatty acid percentage would be of great interest.

Comparison of sarcoplasmic proteomes between two groups of pig muscles selected for shear force of cooked meat.

Two-dimensional electrophoresis was used to compare Longissimus sarcoplasmic protein abundance between two groups (tough meat and tender meat), defined on the basis of extreme Warner-Bratzler shear force values measured on cooked pork. Fourteen protein spots differed in quantity (P<0.05) between the two groups and were identified. Adypocyte fatty acid binding protein and acyl-CoA binding protein involved in lipid traffic and in the control of gene expression regulating cell proliferation and differentiation, and Enoyl-CoA hydratase, aldose reductase and triosephosphate isomerase indirectly related to lipid metabolism were overrepresented in the tender group. The tender group was further characterized by increased levels of proteins involved in protein folding and polymerization (initiation factor elf-3beta, chaperonin subunit 2, profilin II). The results suggest that the lower post-cooking shear force could at least in part be related to muscle adipogenetic and/or myogenetic status of which the possible underlying mechanisms are discussed.

Semen production in two rabbit lines divergently selected for 63-d body weight.

Thirty-one bucks from two lines divergently selected for 63-d body weight (low, L and high, H) were solicited every week (twice at a 15min interval) during 18 weeks resulting in 482 ejaculates. While differing markedly on adult body weight (L: 4650g versus H: 5925g), both lines had the same testis weight. Libido did not differ between the lines. The proportion of ejaculates suitable for insemination was markedly higher in the L line (66.5% versus 44.2%). Mass motility and the volume of the ejaculates were higher in the L line while the sperm concentration was higher in the H line. Overall, the total number of spermatozoa per ejaculate was similar in both lines but the efficient number of spermatozoa per ejaculate, a synthetic criterion taking into account the ability of the ejaculate for insemination was higher in the L line (229 versus 170x10(6)). The L line had higher values of average path velocity, linearity and curvilinear velocity but a lower value of beat cross frequency. In the L line, both ejaculates had the same concentration, while in the H line, the first ejaculate was more concentrated than the second one. Some male reproductive traits are therefore genetically related to body weight.

A mutation creating a potential illegitimate microRNA target site in the myostatin gene affects muscularity in sheep.

Texel sheep are renowned for their exceptional meatiness. To identify the genes underlying this economically important feature, we performed a whole-genome scan in a Romanov x Texel F2 population. We mapped a quantitative trait locus with a major effect on muscle mass to chromosome 2 and subsequently fine-mapped it to a chromosome interval encompassing the myostatin (GDF8) gene. We herein demonstrate that the GDF8 allele of Texel sheep is characterized by a G to A transition in the 3' UTR that creates a target site for mir1 and mir206, microRNAs (miRNAs) that are highly expressed in skeletal muscle. This causes translational inhibition of the myostatin gene and hence contributes to the muscular hypertrophy of Texel sheep. Analysis of SNP databases for humans and mice demonstrates that mutations creating or destroying putative miRNA target sites are abundant and might be important effectors of phenotypic variation.

Proteome analysis of the sarcoplasmic fraction of pig semimembranosus muscle: implications on meat color development.

Two-dimensional electrophoresis was used to investigate sarcoplasmic protein expression in pig Semimembranosus muscles sampled 20 min after slaughter. Two groups (light and dark) of 12 animals were selected from 1000 pigs, based on meat L values measured 36 h postmortem. Twenty-two proteins or fragments (p < 0.05) were differentially expressed. Muscles leading to darker meat had a more oxidative metabolism, indicated by more abundant mitochondrial enzymes of the respiratory chain, hemoglobin, and chaperone or regulator proteins (HSP27, alphaB-crystallin, and glucose-regulated protein 58 kDa). Conversely, enzymes of glycolysis were overexpressed in the lighter group. Such samples were also characterized by higher levels of glutathione S-transferase omega, which can activate the RyR calcium channels, and higher levels of cyclophilin D. This protein pattern is likely to have severe implications on postmortem metabolism, namely, acceleration of ATP depletion and pH fall and subsequent enhanced protein denaturation, well-known to induce discoloration.

Divergent selection on 63-day body weight in the rabbit: response on growth, carcass and muscle traits.

The effects of selection for growth rate on weights and qualitative carcass and muscle traits were assessed by comparing two lines selected for live body weight at 63 days of age and a cryopreserved control population raised contemporaneously with generation 5 selected rabbits. The animals were divergently selected for five generations for either a high (H line) or a low (L line) body weight, based on their BLUP breeding value. Heritability (h2) was 0.22 for 63-d body weight (N = 4754). Growth performance and quantitative carcass traits in the C group were intermediate between the H and L lines (N = 390). Perirenal fat proportion (h2 = 0.64) and dressing out percentage (h2 = 0.55) ranked in the order L < H = C (from high to low). The weight and cross-sectional area of the Semitendinosus muscle, and the mean diameter of the constitutive myofibres were reduced in the L line only (N = 140). In the Longissimus muscle (N = 180), the ultimate pH (h2 = 0.16) and the maximum shear force reached in the Warner-Braztler test (h2 = 0.57) were slightly modified by selection.