Estimating direct genetic and maternal effects affecting rabbit growth and feed efficiency with a factorial design.

The aim of this experiment was to evaluate the significance of neonatal environment on feed efficiency. For that purpose, rabbits from a line selected for residual feed intake (RFI) during 10 generations (G10 kits) were cross-fostered with non-selected control does (i.e., G0 line), and reciprocally. In parallel, sibs were fostered by mothers from their original line. Nine hundred animals were raised in individual (N = 456) or collective (N = 320) cages. Traits analysed in this study were body weight at 32 days and at 63 days, average daily gain (ADG), feed intake between weaning and 63 days (FI), feed conversion ratio (FCR) and RFI. The maternal environment offered by does from the line selected for RFI deteriorated the FCR of the kits, independently of their line of origin, during fattening (+0.08 ± 0.02) compared to FCR of kits nursed by G0 does. The line, the type of housing and the batch were significant effects for all the measured traits: G10 kits were lighter than their G0 counterparts at 32 days (-82.9 ± 9 g, p < 0.0001) and at 63 days (-161 ± 16 g, p < 0.0001). They also had a lower ADG (-2.36 ± 0.36 g/day, p < 0.0001), RFI (-521 ± 24 g/day, p < 0.0001) and a lower FI (-855 ± 31 g, p < 0.0001), resulting in a more desirable feed efficiency (FCR: -0.35 ± 0.02). There was no significant difference in the contrast of G10 and G0 performances between collective and individual/digestive cages (p > 0.22): -2.35 g/day versus 2.94 g/day for ADG, -0.39 versus -0.40 for FCR, -577 g versus -565 g for RFI and -879 g versus -859 g for FI, respectively). Thus, no genotype-by-environment (housing) interaction is expected at the commercial level, that is, no re-ranking of the animals due to collective housing.

Genome-Wide Identification of the Mutation Underlying Fleece Variation and Discriminating Ancestral Hairy Species from Modern Woolly Sheep.

The composition and structure of fleece variation observed in mammals is a consequence of a strong selective pressure for fiber production after domestication. In sheep, fleece variation discriminates ancestral species carrying a long and hairy fleece from modern domestic sheep (Ovis aries) owning a short and woolly fleece. Here, we report that the "woolly" allele results from the insertion of an antisense EIF2S2 retrogene (called asEIF2S2) into the 3' UTR of the IRF2BP2 gene leading to an abnormal IRF2BP2 transcript. We provide evidence that this chimeric IRF2BP2/asEIF2S2 messenger 1) targets the genuine sense EIF2S2 RNA and 2) creates a long endogenous double-stranded RNA which alters the expression of both EIF2S2 and IRF2BP2 mRNA. This represents a unique example of a phenotype arising via a RNA-RNA hybrid, itself generated through a retroposition mechanism. Our results bring new insights on the sheep population history thanks to the identification of the molecular origin of an evolutionary phenotypic variation.

The highly prolific phenotype of Lacaune sheep is associated with an ectopic expression of the B4GALNT2 gene within the ovary.

Prolific sheep have proven to be a valuable model to identify genes and mutations implicated in female fertility. In the Lacaune sheep breed, large variation in litter size is genetically determined by the segregation of a fecundity major gene influencing ovulation rate, named FecL and its prolific allele FecL(L) . Our previous work localized FecL on sheep chromosome 11 within a locus of 1.1 Mb encompassing 20 genes. With the aim to identify the FecL gene, we developed a high throughput sequencing strategy of long-range PCR fragments spanning the locus of FecL(L) carrier and non-carrier ewes. Resulting informative markers defined a new 194.6 kb minimal interval. The reduced FecL locus contained only two genes, insulin-like growth factor 2 mRNA binding protein 1 (IGF2BP1) and beta-1,4-N-acetyl-galactosaminyl transferase 2 (B4GALNT2), and we identified two SNP in complete linkage disequilibrium with FecL(L) . B4GALNT2 appeared as the best positional and expressional candidate for FecL, since it showed an ectopic expression in the ovarian follicles of FecL(L) /FecL(L) ewes at mRNA and protein levels. In FecL(L) carrier ewes only, B4GALNT2 transferase activity was localized in granulosa cells and specifically glycosylated proteins were detected in granulosa cell extracts and follicular fluids. The identification of these glycoproteins by mass spectrometry revealed at least 10 proteins, including inhibin alpha and betaA subunits, as potential targets of B4GALNT2 activity. Specific ovarian protein glycosylation by B4GALNT2 is proposed as a new mechanism of ovulation rate regulation in sheep, and could contribute to open new fields of investigation to understand female infertility pathogenesis.

Genetic control of multiple births in low ovulating mammalian species.

In mammals, litter size is a highly variable trait. Some species such as humans or cattle are monotocous, with one or sometimes two newborns per birth, whereas others, the polytocous species such as mice or pigs, are highly prolific and often produce a dozen newborns at each farrowing. In monotocous species, however, two or three newborns per birth may sometime be unwanted. In more polytocous species such as sheep or pigs, litter size is studied in order to increase livestock prolificacy. By contrast, twinning rates in humans or cattle may increase birth difficulties and health problems in the newborns. In this context, the aim of our review was to provide a clearer understanding of the genetic and physiological factors that control multiple births in low-ovulating mammalian species, with particular focus on three species: sheep, cattle, and humans, where knowledge of the ovulation rate in one may enlighten findings in the others. This article therefore reviews the phenotypic and genetic variability observed with respect to ovulation and twinning rates. It then presents the QTL and major genes that have been identified in each species. Finally, we draw a picture of the diversity of the physiological mechanisms underlying multiple ovulation. Although several major genes have been discovered in sheep, QTL detection methods in humans or cattle have suggested that the determinism of litter size is complex and probably involves several genes in order to explain variations in the number of ovulations.

Determination of anti-Müllerian hormone concentrations in blood as a tool to select Holstein donor cows for embryo production: from the laboratory to the farm.

High between-animal variability in the number of embryos produced by multiple ovulation and embryo transfer (MOET) and ovum pick-up and in vitro production (OPU-IVP) methods remains a major limit to the development of embryo biotechnologies in cattle. The measurement of anti-Müllerian hormone (AMH) endocrine concentrations in cows can help to predict their follicular and ovulatory responses to gonadotrophin treatment. The present study aimed to provide practical information for a simple prognostic method based on AMH measurement in Holstein cows. Accurate AMH concentrations could be measured with ELISA in blood or plasma. In cows undergoing repeated OPU protocols over 1 year, the AMH concentrations measured in plasma samples collected before each gonadotrophin treatment were found to be highly repeatable and were tightly correlated with follicular responses. From data obtained at both an experimental station and farm settings, it was possible to propose AMH cut-off values to identify low-responding cows. Gonadotrophin-stimulated cows producing fewer than 15 large follicles at oestrus and fewer than 10 embryos in MOET protocols could be discarded efficiently with plasma AMH concentrations below 87 and 74 pg mL(-1), respectively. In conclusion, we propose a prognostic method based on a single AMH measurement to improve the results of embryo biotechnologies.

Regulation of anti-Müllerian hormone production in domestic animals.

In mammals, anti-Müllerian hormone (AMH) expression is detected in the granulosa cells of all growing follicles and is highest in healthy small antral follicles, which contribute most significantly to AMH endocrine levels. AMH is a reliable endocrine marker of this population of gonadotrophin-responsive follicles in ruminants and, over the longer term, plasma AMH concentrations are characteristic of individual animals. In the cow, plasma AMH concentrations follow specific dynamic profiles throughout the prepubertal period, the oestrous cycle and the change from gestation to the post partum period, with the alterations most likely reflecting numerical changes in the population of high AMH-producing follicles. In granulosa cells, bone morphogenetic proteins (BMP) enhance AMH gene expression and AMH synthesis, with these effects antagonised by FSH. BMP could both support follicular growth and contribute significantly to the induction and/or maintenance of AMH expression in small growing follicles. AMH expression decreases sharply in large follicles when they become oestrogenic, suggesting a role for FSH and/or oestradiol in these changes, but the underlying mechanisms remain hypothetical. A better understanding of the factors and mechanisms regulating AMH production is needed to propose new strategies for managing the reserve of primordial and small growing follicles, as well as for improving embryo production.

Variability in Global DNA Methylation Rate Across Tissues and Over Time in Sheep.

Recent studies highlighted the influence of epigenetic marks in the variability of many complex traits, both in plants and animals. These studied focused only on specific sites of the genome having differentially methylated profiles among individuals and/or tissues. In contrast, we recently used the methylation rate of the entire genome as a unique measure considered as a novel quantitative phenotype in sheep. This phenotype named global DNA methylation rate (GDMR), measured by luminometric assay, integrates the methylation level of each CpG dinucleotide within the 6 million of CCGG sites along the ovine genome. GDMR measured in blood previously showed moderate heritability of 0.20 and provided evidence for a genetic determinism. The main objective of the present study was to better characterize the GDMR phenotype in various tissues and investigate its variability in several breeds of sheep reared in the same environment. GDMR was measured on blood samples collected monthly from 59 growing male and female lambs (24 Romane, 23 Blackbelly and 12 Charollais), between birth and 4 months of age. Blood GDMR was on average around 80% and was influenced by the sampling date (p < 0.001), the breed (p = 0.002) and the sex (p = 0.002). In addition, GDMR was determined in 12 somatic (frontal lobe, pituitary gland, heart, lung, sub cutaneous and perirenal adipose tissue, skeletal muscle, liver, spleen, adrenal gland, medulla and cortical kidney) and 6 reproductive tissues (ovary, oviduct, uterus, testis, epididymis and seminal vesicle). GDMR was on average 70% in somatic tissues but marked variation was observed depending on the tissue. The GDMR measured in blood was higher than that measured in other somatic tissues, and is not a good proxy of less accessible tissues. Female reproductive tissues had a 10% higher GDMR than male reproductive tissues. We demonstrated a significant influence of the breed on blood GDMR, certainly reflecting the influence of different genetic backgrounds. The effect of the breed on GDMR may be related to their specific abilities to adapt to and live in different conditions.

Endocrine characterization of the reproductive axis in highly prolific lacaune sheep homozygous for the FecLL mutation.

A prolific allele named FecL(L) is known to segregate in the meat breed of the French Lacaune sheep on the basis of ovulation rate record. To gain more knowledge about the physiological effects of FecL(L), ewes homozygous for FecL(L) (L/L) were compared to wild-type ewes (+/+) for ovarian phenotype and reproductive endocrine profiles. At the ovarian level, the increased ovulation rate in L/L ewes was associated with an increased number of antral follicles of greater than 3 mm and with preovulatory follicles being, on average, 1 mm smaller. Intrafollicular estradiol and testosterone concentrations were not significantly different between the two genotypes. In contrast, L/L large follicles (>or=6 mm) had lower intrafollicular progesterone concentration. At the molecular level, expressions of ovarian markers, such as CYP19A1, CYP11A1, CYP17A1, LHR, and INHA, were not significantly different between the two genotypes. In contrast, FSHR and STAR mRNA levels increased in granulosa cells from L/L ewes. Plasma concentrations of estradiol, luteinizing hormone (LH), follicle-stimulating hormone (FSH), and progesterone measured across a synchronized estrous cycle revealed a significant increase in estradiol levels during the follicular phase, a precocious LH surge, and an increase in progesterone level during the luteal phase of L/L ewes compared to +/+ ewes. Circulating concentrations of FSH were not different between the two genotypes. The precocious LH surge was associated with an increase in frequency of LH pulsatility during the follicular phase. At the pituitary level, mRNA levels for LHB, FSHB, GNRHR, and ESR1 were not significantly different between the two genotypes. In contrast, ESR2 mRNA expression was decreased in L/L ewes compared to +/+ ewes. Based on ovarian phenotype and endocrine profiles, these findings suggest that the mutation in the FecL gene affects ovarian function in a different way compared to other known prolific mutations affecting the bone morphogenetic protein signaling system in the ovine species.

Genetic Determinism Exists for the Global DNA Methylation Rate in Sheep.

Recent studies showed that epigenetic marks, including DNA methylation, influence production and adaptive traits in plants and animals. So far, most studies dealing with genetics and epigenetics considered DNA methylation sites independently. However, the genetic basis of the global DNA methylation rate (GDMR) remains unknown. The main objective of the present study was to investigate genetic determinism of GDMR in sheep. The experiment was conducted on 1,047 Romane sheep allocated into 10 half-sib families. After weaning, all the lambs were phenotyped for global GDMR in blood as well as for production and adaptive traits. GDMR was measured by LUminometric Methylation Analysis (LUMA) using a pyrosequencing approach. Association analyses were conducted on some of the lambs (n = 775) genotyped by using the Illumina OvineSNP50 BeadChip. Blood GDMR varied among the animals (average 70.7 ± 6.0%). Female lambs had significantly higher GDMR than male lambs. Inter-individual variability of blood GDMR had an additive genetic component and heritability was moderate (h 2 = 0.20 ± 0.05). No significant genetic correlation was found between GDMR and growth or carcass traits, birthcoat, or social behaviors. Association analyses revealed 28 QTLs associated with blood GDMR. Seven genomic regions on chromosomes 1, 5, 11, 17, 24, and 26 were of most interest due to either high significant associations with GDMR or to the relevance of genes located close to the QTLs. QTL effects were moderate. Genomic regions associated with GDMR harbored several genes not yet described as being involved in DNA methylation, but some are already known to play an active role in gene expression. In addition, some candidate genes, CHD1, NCO3A, KDM8, KAT7, and KAT6A have previously been described to be involved in epigenetic modifications. In conclusion, the results of the present study indicate that blood GDMR in domestic sheep is under polygenic influence and provide new insights into DNA methylation genetic determinism.

Genome-Wide Identification of a Regulatory Mutation in BMP15 Controlling Prolificacy in Sheep.

The search for the genetic determinism of prolificacy variability in sheep has evidenced several major mutations in genes playing a crucial role in the control of ovulation rate. In the Noire du Velay (NV) sheep population, a recent genetic study has evidenced the segregation of such a mutation named FecL L . However, based on litter size (LS) records of FecL L non-carrier ewes, the segregation of a second prolificacy major mutation was suspected in this population. In order to identify this mutation, we have combined a case/control genome-wide association study with ovine 50k SNP chip genotyping, whole genome sequencing, and functional analyses. A new single nucleotide polymorphism (OARX:50977717T > A, NC_019484) located on the X chromosome upstream of the BMP15 gene was evidenced to be highly associated with the prolificacy variability (P = 1.93E-11). The variant allele was called FecX N and shown to segregate also in the Blanche du Massif Central (BMC) sheep population. In both NV and BMC, the FecX N allele frequency was estimated close to 0.10, and its effect on LS was estimated at +0.20 lamb per lambing at the heterozygous state. Homozygous FecX N carrier ewes were fertile with increased prolificacy in contrast to numerous mutations affecting BMP15. At the molecular level, FecX N was shown to decrease BMP15 promoter activity and supposed to impact BMP15 expression in the oocyte. This regulatory action was proposed as the causal mechanism for the FecX N mutation to control ovulation rate and prolificacy in sheep.

Use of combined in silico expression data and phylogenetic analysis to identify new oocyte genes encoding RNA binding proteins in the mouse.

During folliculogenesis, oocytes accumulate maternal mRNAs in preparation for the first steps of early embryogenesis. The processing of oocyte mRNAs is ensured by heterogeneous nuclear ribonucleoproteins (hnRNPs) genes that encode RNA binding proteins implied in mRNA biogenesis, translation, alternative splicing, nuclear exportation, and degradation. In the present work, by combining phylogenetic analyses and, when available, in silico expression data, we have identified three new oocyte-expressed genes encoding RNA binding proteins by using two strategies. Firstly, we have identified mouse orthologs of the Car1 gene, known to be involved in regulation of germ cell apoptosis in C. elegans, and of the Squid gene, required for the establishment of anteroposterior polarity in the Drosophila oocyte. Secondly, we have identified, among genes whose ESTs are highly represented in oocyte libraries, a paralog of Poly(A) binding protein--Interacting Protein 2 (Paip2) gene, known to inhibit the interaction of the Poly(A)-Binding Protein with Poly(A) tails of mRNAs. For all of these genes, the expression in oocyte was verified by in situ hybridization. Overall, this work underlines the efficiency of in silico methodologies to identify new genes involved in biological processes such as oogenesis.

Variation in Recombination Rate and Its Genetic Determinism in Sheep Populations.

Recombination is a complex biological process that results from a cascade of multiple events during meiosis. Understanding the genetic determinism of recombination can help to understand if and how these events are interacting. To tackle this question, we studied the patterns of recombination in sheep, using multiple approaches and data sets. We constructed male recombination maps in a dairy breed from the south of France (the Lacaune breed) at a fine scale by combining meiotic recombination rates from a large pedigree genotyped with a 50K SNP array and historical recombination rates from a sample of unrelated individuals genotyped with a 600K SNP array. This analysis revealed recombination patterns in sheep similar to other mammals but also genome regions that have likely been affected by directional and diversifying selection. We estimated the average recombination rate of Lacaune sheep at 1.5 cM/Mb, identified ∼50,000 crossover hotspots on the genome, and found a high correlation between historical and meiotic recombination rate estimates. A genome-wide association study revealed two major loci affecting interindividual variation in recombination rate in Lacaune, including the RNF212 and HEI10 genes and possibly two other loci of smaller effects including the KCNJ15 and FSHR genes. The comparison of these new results to those obtained previously in a distantly related population of domestic sheep (the Soay) revealed that Soay and Lacaune males have a very similar distribution of recombination along the genome. The two data sets were thus combined to create more precise male meiotic recombination maps in Sheep. However, despite their similar recombination maps, Soay and Lacaune males were found to exhibit different heritabilities and QTL effects for interindividual variation in genome-wide recombination rates. This highlights the robustness of recombination patterns to underlying variation in their genetic determinism.