Divergent selection on milk somatic cell count in goats improves udder health and milk quality with no effect on nematode resistance.

Milk somatic cell count (SCC) is commonly higher in goats than in cattle and sheep. Furthermore, the ability of milk SCC to predict mastitis is considered lower in goats than in cattle and sheep, and the relevance of somatic cell score (SCS)-based selection in this species has been questioned. To address this issue, we created 2 divergent lines of Alpine goats using artificially inseminated bucks with extreme estimated breeding values for SCS. A total of 287 goats, 158 in high- and 129 in low-SCS lines, were scrutinized for mastitis infections. We subjected 2,688 milk samples to conventional bacteriological analyses on agarose and bacterial counts were estimated for positive samples. The SCS, milk yield, fat content, and protein content were recorded every 3 wk. Clinical mastitis was systematically noted. A subset of 40 goats (20 from each line) was subsequently challenged with Haemonchus contortus and monitored for anemia (blood packed cell volume) and fecal egg counts to see if SCS-based selection had an indirect effect on resistance to gastrointestinal nematodes. Milk production traits, including milk quantity, fat content, and protein content, were similar in both goat lines. In contrast, the raw milk SCC almost doubled between the lines, with 1,542,000 versus 855,000 cells/mL in the high- and low-SCS lines, respectively. The difference in breeding value for SCS between lines was 1.65 genetic standard deviation equivalents. The Staphylococcus spp. most frequently isolated from milk were S. xylosus, S. caprae, S. epidermidis, and S. aureus. The frequency of positive bacteriology samples was significantly higher in the high-SCS line (49%) than in the low-SCS line (33%). The highest odds ratio was 3.49 (95% confidence interval: 11.95-6.25) for S. aureus. The distribution of bacterial species in positive samples between lines was comparable. The average quantity of bacteria in positive samples was also significantly higher in high-SCS goats (69 ± 80 growing colonies) than in low-SCS goats (38 ± 62 growing colonies). Clinical cases were rare and equally distributed between high- (n = 4; 2.5%) and low-SCS (n = 3; 2.3%) lines. Furthermore, the larger the amounts of bacteria in milk the higher the SCS level. Conversely, goats with repeatedly culture-negative udders exhibited the lowest SCC levels, with an average of below 300,000 cells/mL. We therefore confirmed that SCS is a relevant predictor of intramammary infection and hygienic quality of milk in goats and can be used for prophylactic purposes. After challenge with H. contortus, goats were anemic with high fecal egg counts but we found no difference between the genetic lines. This result provides initial evidence that resistance to mastitis or to gastrointestinal nematodes infections is under independent genetic regulation. Altogether, this monitoring of the goat lines indicated that SCS-based selection helps to improve udder health by decreasing milk cell counts and reducing the incidence of infection and related bacterial shedding in milk. Selection for low SCC should not affect a goat's ability to cope with gastrointestinal nematodes.

Goat domestication and breeding: a jigsaw of historical, biological and molecular data with missing pieces.

Domestic goats (Capra hircus) are spread across the five continents with a census of 1 billion individuals. The worldwide population of goats descends from a limited number of bezoars (Capra aegagrus) domesticated 10 000 YBP (years before the present) in the Fertile Crescent. The extraordinary adaptability and hardiness of goats favoured their rapid spread over the Old World, reaching the Iberian Peninsula and Southern Africa 7000 YBP and 2000 YBP respectively. Molecular studies have revealed one major mitochondrial haplogroup A and five less frequent haplogroups B, C, D, F and G. Moreover, the analysis of autosomal and Y-chromosome markers has evidenced an appreciable geographic differentiation. The implementation of new molecular technologies, such as whole-genome sequencing and genome-wide genotyping, allows for the exploration of caprine diversity at an unprecedented scale, thus providing new insights into the evolutionary history of goats. In spite of a number of pitfalls, the characterization of the functional elements of the goat genome is expected to play a key role in understanding the genetic determination of economically relevant traits. Genomic selection and genome editing also hold great potential, particularly for improving traits that cannot be modified easily by traditional selection.

Development of a SNP panel dedicated to parentage assignment in French sheep populations.

BACKGROUND: The efficiency of breeding programs partly relies on the accuracy of the estimated breeding values which decreases when pedigrees are incomplete. Two reproduction techniques are mainly used by sheep breeders to identify the sires of lambs: animal insemination and natural matings with a single ram per group of ewes. Both methods have major drawbacks, notably time-consuming tasks for breeders, and are thus used at varying levels in breeding programs. As a consequence, the percentage of known sires can be very low in some breeds and results in less accurate estimated breeding values. RESULTS: In order to address this issue and offer an alternative strategy for obtaining parentage information, we designed a set of 249 SNPs for parentage assignment in French sheep breeds and tested its efficiency in one breed. The set was derived from the 54 K SNP chip that was used to genotype the thirty main French sheep populations. Only SNPs in Hardy-Weinberg equilibrium, displaying the highest Minor Allele Frequency across all the thirty populations and not associated with Mendelian errors in verified family trios were selected. The panel of 249 SNPs was successfully used in an on-farm test in the BMC breed and resulted in more than 95% of lambs being assigned to a unique sire. CONCLUSION: In this study we developed a SNP panel for assignment that achieved good results in the on-farm testing. We also raised some conditions for optimal use of this panel: at least 180 SNPs should be used and a minute preparation of the list of candidate sires. Our panel also displays high levels of MAF in the SheepHapMap breeds, particularly in the South West European breeds.

Genetic parameter estimation for major milk fatty acids in Alpine and Saanen primiparous goats.

Genetic parameters for 18 fatty acids or groups of fatty acids (FA), milk production traits, and somatic cell score (SCS) were estimated by restricted maximum likelihood with a repeatability animal model, using 45,259 test-day records from the first lactations of 13,677 Alpine and Saanen goats. Fatty acid data were collected as part of an extensive recording scheme (PhénoFinLait), and sample testing was based on mid-infrared spectra estimates. The total predicted FA content in milk was approximately 3.5% in Alpine and Saanen goats. Goat milk fat showed similar saturated FA to cattle and sheep, but higher contents of capric (C10:0) FA (~ 9.7 g/100g of milk fat). Heritability estimates ranged from 0.18 to 0.49 for FA and estimates were generally higher when FA were expressed in g/100g of milk fat compared with g/100g of milk. In general, the 3 specific short- and medium-chain goat FA, caproic acid (C6:0), caprylic acid (C8:0), and especially capric (C10:0) acid, had among the highest heritability estimates (from 0.21 to 0.37; average of 0.30). Heritability estimates for milk yield, fat and protein contents, and SCS were 0.22, 0.23, 0.39, 0.09, and 0.24, 0.20, 0.40, and 0.15, in Alpine and Saanen goats, respectively. When FA were expressed in g/100g of milk, genetic correlations between fat content and all FA were high and positive. Genetic correlations between the fat content and FA groups expressed in g/100g of fat led to further investigation of the association between fat content and FA profile within milk fat. Accordingly, in both Saanen and Alpine breeds, no significant genetic correlations were found between fat content and C16:0, whereas the correlations between fat content and specific goat FA (C6:0 to C10:0) were positive (0.17 to 0.59). In addition, the genetic correlation between fat content and C14:0 was negative (-0.17 to -0.35). The values of the genetic correlations between protein content and individual FA were similar, although genetic correlations between protein content and FA groups were close to zero. Genetic correlations of milk yield or SCS with the FA profile were weak. Results for genetic parameters for FA, however, should be further validated, because the low predicting ability of certain FA using mid-infrared spectra and the limited calibration data set might have resulted in low accuracy. In conclusion, our results indicated substantial genetic variation in goat milk FA that supported their amenability for genetic selection. In addition, selection on protein and fat contents is not expected to have an undesirable effect on the FA profile in regard to specificity of goat products and human health.

A pilot study on transcriptome data analysis of folliculogenesis in pigs.

Three different stages of pig antral follicles have been studied in a granulosa-cell transcriptome analysis on nylon microarrays (1152 clones). The data have been generated from seven RNA follicle pools and several technical replicates were made. The objective of this paper was to state the feasibility of a transcriptomic protocol for the study of folliculogenesis in the pig. A statistical analysis was chosen, relying on the linear mixed model (LMM) paradigm. Low variability within technical replicates was hence checked with a LMM. Relevant genes that might be involved in the studied process were then selected. For the most significant genes, statistical methods such as principal component analysis and unsupervised hierarchical clustering were applied to assess their relevance, and a random forest analysis proved their predictive value. The selection of genes was consistent with previous studies and also allowed the identification of new genes whose role in pig folliculogenesis will be further investigated.

In vivo gene expression in granulosa cells during pig terminal follicular development.

Ovarian antral follicular development is clearly dependent on pituitary gonadotrophins FSH and LH. Although the endocrine mechanism that controls ovarian folliculogenesis leading to ovulation is quite well understood, the detailed mechanisms and molecular determinants in the different follicular compartments remain to be clarified. The aim of this study was to identify the genes differentially expressed in pig granulosa cells along the terminal ovarian follicle growth, to gain a comprehensive view of these molecular mechanisms. First, we developed a specific micro-array using cDNAs from suppression subtractive hybridization libraries (345 contigs) obtained by comparison of three follicle size classes: small, medium and large antral healthy follicles. In a second step, a transcriptomic analysis using cDNA probes from these three follicle classes identified 79 differentially expressed transcripts along the terminal follicular growth and 26 predictive genes of size classes. The differential expression of 18 genes has been controlled using real-time PCR experiments validating the micro-array analysis. Finally, the integration of the data using Ingenuity Pathways Analysis identified five gene networks providing descriptive elements of the terminal follicular development. Specifically, we observed: (1) the down-expression of ribosomal protein genes, (2) the genes involved in lipid metabolism and (3) the down-expression of cell morphology and ion-binding genes. In conclusion, this study gives new insight into the gene expression during pig terminal follicular growth in vivo and suggested, in particular, a morphological change in pig granulosa cells accompanying terminal follicular growth.

Identification of differential gene expression in in vitro FSH treated pig granulosa cells using suppression subtractive hybridization.

FSH, which binds to specific receptors on granulosa cells in mammals, plays a key role in folliculogenesis. Its biological activity involves stimulation of intercellular communication and upregulation of steroidogenesis, but the entire spectrum of the genes regulated by FSH has yet to be fully characterized. In order to find new regulated transcripts, however rare, we have used a Suppression Subtractive Hybridization approach (SSH) on pig granulosa cells in primary culture treated or not with FSH. Two SSH libraries were generated and 76 clones were sequenced after selection by differential screening. Sixty four different sequences were identified, including 3 novel sequences. Experiments demonstrated the presence of 25 regulated transcripts.A gene ontology analysis of these 25 genes revealed (1) catalytic; (2) transport; (3) signal transducer; (4) binding; (5) anti-oxidant and (6) structural activities. These findings may deepen our understanding of FSH's effects. Particularly, they suggest that FSH is involved in the modulation of peroxidase activity and remodelling of chromatin.

Conserved synteny and gene order difference between human chromosome 12 and pig chromosome 5.

A comparative map of human chromosome 12 (HSA 12) and pig chromosome 5 (SSC 5) was constructed using ten pig expressed sequence tags (ESTs). These ESTs were isolated from primary granulosa cell cultures by differential display (EST b10b), or from a granulosa cDNA library (VIIIE1, DRIM, N*9, RIIID2 and RVIC1) or from a small intestine cDNA library (ATPSB, ITGB7, MYH9, and STAT2). Also used were two Traced Orthologous Amplified Sequence Tags (TOASTs) (LALBA, TRA1), one microsatellite-associated gene (IGF1) and finally five human YACs selected for their cytogenetic position, with a view to increasing the number of informative markers for the comparison. Large-insert clones were obtained by screening a pig bacterial artificial chromosome (BAC) library with specific primers for each EST and TOAST and for IGF1. These BACs were used as probes for fluorescent in situ hybridisation (FISH) both on porcine and human metaphases. In addition, the human YACs were FISH mapped on pig chromosomes. This allowed us to refine and, in some cases, to correct the previous mapping obtained with a somatic cell hybrid panel. While these data confirm chromosome painting results showing that the distal part of SSC 5p arm is conserved on HSA 22, while the rest of the chromosome corresponds to HSA 12, they also demonstrate gene-order differences between human and pig. In addition, it was also possible to determine the position of the synteny breakpoint.

Pregnancy-associated plasma protein-A (PAPP-A) in ovine, bovine, porcine, and equine ovarian follicles: involvement in IGF binding protein-4 proteolytic degradation and mRNA expression during follicular development.

IGF binding protein-4 (IGFBP-4) proteolytic degradation is a common feature of preovulatory follicles from human, ovine, bovine, porcine, and equine ovary. In all these species, the protease is a zinc-dependent metalloprotease and its ability to degrade IGFBP-4 is IGF dependent. The human intrafollicular IGFBP-4-degrading protease has recently been identified as pregnancy-associated plasma protein-A (PAPP-A). The aim of this study was to investigate whether PAPP-A is also involved in IGFBP-4 degradation in ovine, bovine, porcine, and equine preovulatory follicles and to study the expression of PAPP-A mRNA in bovine and porcine granulosa cells from different classes of follicles. Immunoneutralization and immunoprecipitation with polyclonal antibodies raised against human PAPP-A inhibited IGFBP-4 proteolytic degradation in preovulatory follicular fluid from the four species studied. As previously reported for the intrafollicular proteolytic activity degrading IGFBP-4, recombinant human PAPP-A generated in vitro 17- and 10-kDa IGFBP-4-proteolytic fragments. Recombinant PAPP-A activity was also shown to be IGF dependent and was inhibited by heparin-binding domain-containing peptides. In all mammalian species studied, the PAPP-A sequences showed high degree of identity. Moreover, the PAPP-A gene was localized on porcine chromosome 1 (1q29-1q213), in agreement with the localization of human PAPP-A gene on human chromosome 9q33.1. In bovine and porcine ovaries, real-time quantitative RT-PCR showed that PAPP-A mRNA expression in granulosa cells was maximal in fully differentiated follicles and was positively correlated with expression of P450 aromatase and LH receptor mRNAs. Overall, these data show that PAPP-A is responsible for IGFBP-4 degradation in ovine, bovine, porcine, and equine preovulatory follicles. The high expression of PAPP-A mRNA in granulosa cells from large, differentiated follicles suggest that it is a new functional marker of follicular development.

Functional study and regional mapping of 44 hormono-regulated genes isolated from a porcine granulosa cell library.

cDNA clones from a pig granulosa cell cDNA library were isolated by (differential hybridisation for follicle stimulating hormone (FSH) regulation in granulosa cells in a previous study. The clones that did not match any known sequence were studied for their expression in granulosa cells (treated or not by FSH) and in fresh isolated ovarian follicles mainly by comparative RT-PCR analysis. These results give functional data on genes that may be implicated in follicular growing. These ESTs have been localised on the porcine genome, using a somatic cell hybrid panel, providing new type I markers on the porcine map and information on the comparative map between humans and pigs.

Regional localisations of VIM, HSD3b, ACTA1 and PGM1 in pigs.

The comparative map between human and pig has progressed rapidly over the past 2 years. Nevertheless, some points still need to be clarified, particularly the correspondences between human chromosome 10 (HSA10) and porcine chromosome 10 (SSC10) and between human chromosome 1 (HSA1) and porcine chromosomes. The gene codings for vimentin (VIM) carried by HSA10 and three genes carried by HSA1 (hydroxy delta 5 steroid dehydrogenase 3 beta: HSD3B: alpha actin 1: ACTA1: and phosphoglucomutase 1: PGM1) were selected and the regional localisations on pig chromosomes were determined using a well-characterised somatic cell hybrid panel.

A first catalog of genes involved in pig ovarian follicular differentiation.

As a first step toward the characterization of genetic expression in pig ovaries, we have selected 238 clones by differential hybridization from a pig granulosa cell cDNA library, using probes prepared from RNA extracted from either untreated or FSH-treated cells and, in order to generate expressed sequence tags (ESTs), we have performed 3' and 5' single-pass sequencing of these clones. Sequences of the 3' end of the 167 clones that produced informative sequence data were first compared with each other, revealing a redundancy level of 21%. Sequences from the 136 unique clones were analyzed for similarities with sequence data included in Genbank and EMBL databases. Among these unique clones, 54 (40%) matched significantly with sequences from either Genbank of EMBL: 4 with known genes in pig, 35 matched with previously reported human genes, and 15 with other mammalian genes. Eighty-two clones (60%) showed no significant match with any gene or DNA sequence in the Genbank and EMBL databases and thus may represent new pig transcripts.