Inbreeding and effective population size in French dairy sheep: Comparison between genomic and pedigree estimates.

Before availability of dense SNP data, genetic diversity was characterized and managed with pedigree-based information. Besides this classical approach, 2 methodologies have been proposed in recent years to characterize and manage diversity from dense SNP data: the SNP-by-SNP approach and the alternative based on runs of homozygosity (ROH). The establishment of criteria to identify ROH is a current constraint in the literature dealing with ROH. The objective of this study was, using a medium-density SNP chip, to quantify by 3 methods (pedigree, SNP-by-SNP, and ROH) the genetic diversity on 5 selected French dairy sheep subpopulations and breeds and to assess the effect of the definition of ROH on these estimates. The data set available included individuals from the breeds Basco-Béarnaise, Manech Tête Noire, Manech Tête Rousse, and 2 subpopulations of Lacaune: Lacaune Confederation and Lacaune Ovitest. Animals were genotyped with the Illumina OvineSNP50 BeadChip (Illumina Inc., San Diego, CA). After filtering, the genomic data included 38,287 autosomal SNP and 8,700 individuals, which comprised 72,803 animals in the pedigree. The results indicated that no significant differences were observed in effective population size estimates obtained from pedigree or genomic (SNP-by-SNP or ROH) information. In general, estimates of effective population size were above 200 in Lacaune Confederation and Lacaune Ovitest subpopulations and below 200 in Basco-Béarnaise, Manech Tête Noire, and Manech Tête Rousse breeds. The minimum length that constituted a ROH, the minimum number of SNP that constituted a ROH, as well as the minimum density and the maximum distance allowed between 2 homozygous SNP are ROH-defining factors with important implications in the estimation of the rate of inbreeding. The ROH-based rates of inbreeding in concordance with those obtained from pedigree information require a specific set of values. This particular set of values is different from that identified to obtain ROH-based rates of inbreeding similar to those obtained on a SNP-by-SNP basis. Factors to define ROH do not change the results much unless extreme values are considered, although further research on ROH-based inbreeding is still required.

Prediction of genetic gain in finite populations with heterogeneous predicted breeding values accuracies.

The algebraic expression of the genetic selection differential (expected genetic superiority of breeders after a selection on their Predicted Breeding Values) was derived when a limited number of individuals were selected from a limited sample of candidates on the basis of their predicted genetic value, with heterogeneous reliabilities. A formula is proposed for situations in which these reliabilities can be clustered in a few classes. We show that the expected genetic selection differential increases with the number of classes, the mean reliability being constant. In the panel of cases simulated, this increase reached up to 18% of the values obtained in the homogeneous situation. We used the proposed formulae to estimate selection differentials and compared it numerically with performing simulations. In terms of speed of computation, our algebraic formulae performed better than simulations in populations of limited size.

CLIP Test: a new fast, simple and powerful method to distinguish between linked or pleiotropic quantitative trait loci in linkage disequilibria analysis.

An important question arises when mapping quantitative trait loci (QTLs) for genetically correlated traits: is the correlation due to pleiotropy (a single QTL affecting more than one trait) and/or close linkage (different QTLs that are physically close to each other and influence the traits)? In this article, we propose the Close Linkage versus Pleiotropism (CLIP) test, a fast, simple and powerful method to distinguish between these two situations. The CLIP test is based on the comparison of the square of the observed correlation between a combination of apparent effects at the marker level to the minimal value it can take under the pleiotropic assumption. A simulation study was performed to estimate the power and alpha risk of the CLIP test and compare it to a test that evaluated whether the confidence intervals of the two QTLs overlapped or not (CI test). On average, the CLIP test showed a higher power (68%) to detect close-linked QTLs than the CI test (43%) and a same alpha risk (4%).

Quantitative trait loci for resistance to infection in sheep using a live Salmonella Abortusovis vaccine.

Quantitative trait loci (QTL) mapping for susceptibility to a Salmonella Abortusovis vaccinal strain was performed using an experimental design involving 30 Romane sheep sire families (1216 progenies). Nine QTL corresponding to bacterial load, weight variations and antibody response criteria were mapped on eight chromosomes, including the major histocompatibility complex area on chromosome 20. Surprisingly, none was found to be significant in the SLC11A1 region (formerly NRAMP1) that has been shown to influence Salmonella susceptibility in other species.

QTL detection from regression analysis of 'generalized de-regressed proof' information.

QTL detection using the regression of phenotypes on transmission probability is largely used when large families are available. In three generations designs, the use of a 'de-regressed proof' as a phenotype to be analysed was proposed by Weller et al. (1990) and Tribout et al. (2008). Our work generalizes this approach. A score (that we define as a 'generalized de-regressed proof') is described, which combines performance phenotypes recorded in multigenerational offspring of genotyped individuals. Estimation of the QTL effect on this score with a simple regression is unbiased. The link between this score and the BLUP animal model of the polygenic effect is demonstrated. The theory is developed and two simple examples illustrate how this technique can be implemented.

The levels of artificial insemination and missing sire information make genomic selection not always beneficial in meat sheep.

Numerous meat sheep breeding programs in developed and developing countries are characterized by incomplete sire information and a predominant use of natural matings. These two parameters potentially affect the benefit of genomic selection (GS), especially for the selection of a late-in-life trait. Using stochastic simulations, the genetic gains obtained using genomic and conventional strategies for a maternal trait were evaluated in meat sheep population. Natural mating and artificial insemination (AI)-based designs, inspired by the current diversity of designs used for French meat sheep breeds, were modeled and three genomic strategies were tested and compared with a conventional selection strategy: parentage assignment, GS based on a male or a male and female reference population. Genomic selection based on a male reference population did not always outperform conventional selection. Its benefit depended on the design, the level of missing information on dam sires, and the level of AI. Genomic selection based on a male and female reference population always outperformed the conventional selection strategy, even if only 25 % of the females in the nucleus were genotyped.

Ensilicated tetanus antigen retains immunogenicity: in vivo study and time-resolved SAXS characterization.

Our recently developed ensilication approach can physically stabilize proteins in silica without use of a pre-formed particle matrix. Stabilisation is done by tailor fitting individual proteins with a silica coat using a modified sol-gel process. Biopharmaceuticals, e.g. liquid-formulated vaccines with adjuvants, frequently have poor thermal stability; heating and/or freezing impairs their potency. As a result, there is an increase in the prevalence of vaccine-preventable diseases in low-income countries even when there are means to combat them. One of the root causes lies in the problematic vaccine 'cold chain' distribution. We believe that ensilication can improve vaccine availability by enabling transportation without refrigeration. Here, we show that ensilication stabilizes tetanus toxin C fragment (TTCF), a component of the tetanus toxoid present in the diphtheria, tetanus and pertussis (DTP) vaccine. Experimental in vivo immunization data show that the ensilicated material can be stored, transported at ambient temperatures, and even heat-treated without compromising the immunogenic properties of TTCF. To further our understanding of the ensilication process and its protective effect on proteins, we have also studied the formation of TTCF-silica nanoparticles via time-resolved Small Angle X-ray Scattering (SAXS). Our results reveal ensilication to be a staged diffusion-limited cluster aggregation (DLCA) type reaction. An early stage (tens of seconds) in which individual proteins are coated with silica is followed by a subsequent stage (several minutes) in which the protein-containing silica nanoparticles aggregate into larger clusters. Our results suggest that we could utilize this technology for vaccines, therapeutics or other biopharmaceuticals that are not compatible with lyophilization.

Thermal resilience of ensilicated lysozyme via calorimetric and in vivo analysis.

Ensilication is a novel method of protein thermal stabilisation using silica. It uses a modified sol-gel process which tailor fits a protective silica shell around the solvent accessible protein surface. This, electrostatically attached, shell has been found to protect the protein against thermal influences and retains its native structure and function after release. Here, we report the calorimetric analysis of an ensilicated model protein, hen egg-white lysozyme (HEWL) under several ensilication conditions. DSC, TGA-DTA-MS, CD, were used to determine unfolding temperatures of native, released and ensilicated lysozyme to verify the thermal resilience of the ensilicated material. Our findings indicate that ensilication protects against thermal fluctuations even at low concentrations of silica used for ensilication. Secondly, the thermal stabilisation is comparable to lyophilisation, and in some cases is even greater than lyophilisation. Additionally, we performed a mouse in vivo study using lysozyme to demonstrate the antigenic retention over long-term storage. The results suggest that protein is confined within the ensilicated material, and thus is unable to unfold and denature but is still functional after long-term storage.

Identification of new quantitative trait Loci (other than the PRNP gene) modulating the scrapie incubation period in sheep.

Although susceptibility to scrapie is largely controlled by the PRNP gene, we have searched for additional genomic regions that affect scrapie incubation time in sheep, using two half-sib families with a susceptible PRNP genotype and naturally infected by scrapie. Quantitative trait loci were detected on OAR6 and OAR18.

Ensilication Improves the Thermal Stability of the Tuberculosis Antigen Ag85b and an Sbi-Ag85b Vaccine Conjugate.

There is an urgent need for the development of vaccine thermostabilisation methodologies as the maintenance of a continuous and reliable cold chain remains a major hurdle to the global distribution of safe and effective vaccines. Ensilication, a method that encases proteins in a resistant silica cage has been shown to physically prevent the thermal denaturation of a number of model proteins. In this study we investigate the utility of this promising approach in improving the thermal stability of antigens and vaccine conjugates highly relevant to the development of candidate tuberculosis vaccines, including antigen 85b conjugated with the Staphylococcus aureus-protein based adjuvant Sbi. Here we analyse the sensitivity of these constructs to thermal denaturation and demonstrate for the first time the benefits of ensilication in conferring these vaccine-relevant proteins with protection against temperature-induced loss of structure and function without the need for refrigeration. Our results reveal the potential of ensilication in facilitating the storage and transport of vaccines at ambient temperatures in the future and therefore in delivering life-saving vaccines globally, and in particular to remote areas of developing countries where disease rates are often highest.

Optimal mating strategies to manage a heterozygous advantage major gene in sheep.

Some mutations (or 'major genes') have a desirable effect in heterozygous carriers but an undesirable effect in homozygous carriers. When these mutations affect a trait of significant economic importance, their eradication, depending on their effect and frequency, may be counterproductive. This is especially the case of major genes affecting the ovulation rate and thus the prolificacy in meat sheep populations. To manage such situations, a mating design based on the major genotypes of reproducers has to be optimized. Both the effect of the major gene and the cost of genotyping candidates at this locus influence the expected genetic progress and profitability of the breeding plan. The aim of this study was to determine the optimal combination of matings that maximizes profitability at the level of the whole population (nucleus + commercial flocks). A deterministic model was developed and, using sequential quadratic programming methodology, the optimal strategy (optimal combination of matings) that maximized the economic gain achieved by the population across a range of genotype effects and genotyping costs was determined. The optimal strategy was compared with simpler and more practical strategies based on a limited number of parental genotype mating types. Depending on the genotype effect and genotyping costs, the optimal strategy varied, such that either the heterozygous frequency and/or polygenic gain was maximized with a large number of animals genotyped, or when genotyping costs were higher, the optimization led to lower heterozygous frequency and/or polygenic gain with fewer animals genotyped. Comparisons showed that some simpler strategies were close to the optimal strategy. An overlapping model was then derived as an application of the real case of the French Lacaune meat sheep OVI-TEST breeding program. Results showed that a practical strategy based on mating non-carriers to heterozygous carriers was only slightly less effective than the optimal strategy, with a reduction in efficiency from 3% to 8%, depending on the genotyping costs. Based on only two different parental genotype mating types, this strategy would be easy to implement.

Genetic and economic effects of the increase in female paternal filiations by parentage assignment in sheep and goat breeding programs.

In sheep and goat breeding programs, the proportion of females for which the sire is known (known paternity rate [KPR]) can be very low. In this context, paternity assignment using SNP is an attractive tool. The annual genetic gain (AGG) is impacted by the accuracy of the EBV. In populations with a low KPR, the number of known relatives for a given individual is low, and the EBV that are based on this information are imprecise. However, the impact of partially known paternal filiations, in terms of potential genetic and economic losses, has never been quantitatively evaluated in situations where natural mating is the main reproductive mode. A deterministic model was developed to assess, for a panel of real breeding programs, the influence of the female KPR on the AGG and economic benefit. First, males were divided into categories according to their status (natural mating or AI sire) and breeding cycle and females according to parity, sire status (including unknown sire), and breeding cycle of the sire. Second, a demographic model described, for each category, the accumulation of known records for individuals and their close relatives. The output from this model was used to compute the average accuracy of the EBV per category. Then, a genetic model based on the gene flow between categories over time was described. Using the average accuracy of EBV per category, it provided the asymptotic AGG of the nucleus given its KPR. In the economic studies, changes to the mean genetic values in the nucleus and the commercial population after an increase in KPR and various gain:cost ratios (monetary gain due to an extra genetic SD of the selected trait divided by the cost of 1 assignment) were considered. Relative profit and payback periods were computed. We showed that SNP-based parentage assignment aimed at increasing the female KPR was not always profitable and that the type of breeding program and the size of the commercial population should be taken into consideration. Notably, achieving a profit was largely dependent on obtaining a favorable gain:cost ratio. The maximum supplementary AGG (16.9%) was obtained for breeding programs using only natural mating. In such programs without AI, a gain:cost ratio of 5 was needed to make assignment profitable at the nucleus level whereas a gain:cost ratio of 2 was sufficient if the nucleus represented a third of the total population.

Economic evaluation of genomic selection in small ruminants: a sheep meat breeding program.

Recent genomic evaluation studies using real data and predicting genetic gain by modeling breeding programs have reported moderate expected benefits from the replacement of classic selection schemes by genomic selection (GS) in small ruminants. The objectives of this study were to compare the cost, monetary genetic gain and economic efficiency of classic selection and GS schemes in the meat sheep industry. Deterministic methods were used to model selection based on multi-trait indices from a sheep meat breeding program. Decisional variables related to male selection candidates and progeny testing were optimized to maximize the annual monetary genetic gain (AMGG), that is, a weighted sum of meat and maternal traits annual genetic gains. For GS, a reference population of 2000 individuals was assumed and genomic information was available for evaluation of male candidates only. In the classic selection scheme, males breeding values were estimated from own and offspring phenotypes. In GS, different scenarios were considered, differing by the information used to select males (genomic only, genomic+own performance, genomic+offspring phenotypes). The results showed that all GS scenarios were associated with higher total variable costs than classic selection (if the cost of genotyping was 123 euros/animal). In terms of AMGG and economic returns, GS scenarios were found to be superior to classic selection only if genomic information was combined with their own meat phenotypes (GS-Pheno) or with their progeny test information. The predicted economic efficiency, defined as returns (proportional to number of expressions of AMGG in the nucleus and commercial flocks) minus total variable costs, showed that the best GS scenario (GS-Pheno) was up to 15% more efficient than classic selection. For all selection scenarios, optimization increased the overall AMGG, returns and economic efficiency. As a conclusion, our study shows that some forms of GS strategies are more advantageous than classic selection, provided that GS is already initiated (i.e. the initial reference population is available). Optimizing decisional variables of the classic selection scheme could be of greater benefit than including genomic information in optimized designs.

A centuries-long epidemic of scrapie in British sheep?

The apparent persistence of scrapie in British sheep for more than 250 years is difficult to explain. Susceptibility to scrapie is associated with particular alleles at a single locus, the PrP gene. As the only known effect of these alleles is to confer susceptibility to a fatal disease, natural selection is expected to reduce their frequency, as has been observed in practice during scrapie outbreaks in single sheep flocks. Susceptibility alleles, and hence scrapie itself, are therefore expected to become rare, yet the disease remains widespread. We suggest that the paradox of scrapie's persistence can be explained by the exceptionally long time-scales inherent in the epidemiology of the disease. It is proposed that scrapie should be regarded as epidemic in British sheep but, unlike more familiar epidemics, which have time-scales of months or years, the scrapie epidemic has a time-scale of centuries. This interpretation implies that scrapie should eventually disappear from the sheep population.

Mapping quantitative trait loci in crosses between outbred lines using least squares.

The use of genetic maps based upon molecular markers has allowed the dissection of some of the factors underlying quantitative variation in crosses between inbred lines. For many species crossing inbred lines is not a practical proposition, although crosses between genetically very different outbred lines are possible. Here we develop a least squares method for the analysis of crosses between outbred lines which simultaneously uses information from multiple linked markers. The method is suitable for crosses where the lines may be segregating at marker loci but can be assumed to be fixed for alternative alleles at the major quantitative trait loci (QTLs) affecting the traits under analysis (e.g., crosses between divergent selection lines or breeds with different selection histories). The simultaneous use of multiple markers from a linkage group increases the sensitivity of the test statistic, and thus the power for the detection of QTLs, compared to the use of single markers or markers flanking an interval. The gain is greater for more closely spaced markers and for markers of lower information content. Use of multiple markers can also remove the bias in the estimated position and effect of a QTL which may result when different markers in a linkage group vary in their heterozygosity in the F1 (and thus in their information content) and are considered only singly or a pair at a time. The method is relatively simple to apply so that more complex models can be fitted than is currently possible by maximum likelihood. Thus fixed effects of background genotype can be fitted simultaneously with the exploration of a single linkage group which will increase the power to detect QTLs by reducing the residual variance. More complex models with several QTLs in the same linkage group and two-locus interactions between QTLs can similarly be examined. Thus least squares provides a powerful tool to extend the range of crosses from which QTLs can be dissected whilst at the same time allowing flexible and realistic models to be explored.

Large-scale investigation of the parameters in response to Eimeria maxima challenge in broilers.

Coccidiosis, a parasitic disease of the intestinal tract caused by members of the genera Eimeria and Isospora, is one of the most common and costly diseases in chicken. The aims of this study were to assess the effect of the challenge and level of variability of measured parameters in chickens during the challenge with Eimeria maxima. Furthermore, this study aimed to investigate which parameters are the most relevant indicators of the health status. Finally, the study also aimed to estimate accuracy of prediction for traits that cannot be measured on large scale (such as intestinal lesion score and fecal oocyst count) using parameters that can easily be measured on all animals. The study was performed in 2 parts: a pilot challenge on 240 animals followed by a large-scale challenge on 2,024 animals. In both experiments, animals were challenged with 50,000 Eimeria maxima oocysts at 16 d of age. In the pilot challenge, all animals were measured for BW gain, plasma coloration, hematocrit, and rectal temperature and, in addition, a subset of 48 animals was measured for oocyst count and the intestinal lesion score. All animals from the second challenge were measured for BW gain, plasma coloration, and hematocrit whereas a subset of 184 animals was measured for intestinal lesion score, fecal oocyst count, blood parameters, and plasma protein content and composition. Most of the parameters measured were significantly affected by the challenge. Lesion scores for duodenum and jejunum (P < 0.001), oocyst count (P < 0.05), plasma coloration for the optical density values between 450 and 490 nm (P < 0.001), albumin (P < 0.001), α1-globulin (P < 0.01), α2-globulin (P < 0.001), α3-globulin (P < 0.01), and ß2-globulin (P < 0.001) were the most strongly affected parameters and expressed the greatest levels of variation. Plasma protein profiles proved to be a new, reliable parameter for measuring response to Eimeria maxima. Prediction of intestinal lesion score and fecal oocyst count using the other parameters measured was not very precise (R2 < 0.7). The study was successfully performed in real raising conditions on a large scale. Finally, we observed a high variability in response to the challenge, suggesting that broilers' response to Eimeria maxima has a strong genetic determinism, which may be improved by genetic selection.

Number of mature follicles ovulating after a challenge of human chorionic gonadotropin in different breeds of sheep at different physiological stages.

The ovulatory response of ewes from breeds that differ widely in prolificacy (Ile-de-France, ++ Booroola Merino, Romanov, F+ Booroola Merino and F+ Booroola Romanov with adult ovulation rates of about 1.5, 1.2, 3, 3 and 3.5 respectively) to 750 IU of hCG given at different physiological stages (before puberty, during anestrus or during the luteal phase) was compared. In all except one experiment, hCG induced ovulation in 73 to 98% of the lambs, indicating that follicles sensitive to LH were present at all stages studied. Ranking of the breeds according to hCG-induced ovulation rate in prepuberal lambs was similar to that based on adult ovulation rate. Furthermore, hCG induced more ovulations in prepuberal F+ than in ++ lambs (3.7 +/- 1.4 vs 1.7 +/- .8 at 4.5 mo of age) as well as in anestrous ewes (F + at 3.1 +/- 1.8 vs ++ at 1.6 +/- .7). Within ewes, the correlation between hCG-induced ovulation rate and mature ovulation rate was positive in nonprolific breeds but not significant in prolific breeds. We conclude that 1) the number of hCG-induced ovulations can be used to identify sheep that are carriers of the Booroola gene and 2) the mechanisms responsible for a number of large ovulatory follicles typical of a breed are present at stages (prepuberal, anestrus, luteal phase) other than the follicular phase.

Influence of the three RN genotypes on chemical composition, enzyme activities, and myofiber characteristics of porcine skeletal muscle.

An experiment was conducted to evaluate the effects of the RN genotype on skeletal muscle characteristics in pigs sharing otherwise the same polygenic background. Animals were genotyped for RN on the basis of RTN (Rendement Technologique Napole) records using segregation analysis methods. Samples of longissimus (L) and semispinalis capitis (S) muscles were taken from 39 rn+/rn+, 38 RN-/rn+ and 37 RN-/RN- pigs slaughtered at 108 +/- 8.6 kg live weight. Activities of lactate dehydrogenase (LDH), citrate synthase (CS), and beta-hydroxy-acyl-coenzyme A dehydrogenase (HAD) were measured on both muscles to assess glycolytic, oxidative, and lipid beta-oxidation capacities, respectively. Histological examinations and chemical analyses were performed on L muscle. The energetic metabolism of the white L muscle was more oxidative in RN-/RN- than in rn+/rn+ pigs, as shown by increased CS and HAD activities (P < .001), decreased LDH activity (P < .001), larger cross-sectional area of IIA (P < .05) and IIB-red (P < .05) fibers, higher relative area of IIA fibers ( P < .05), and lower relative area of IIB-white fibers (P < .001). No significant difference was found between heterozygous and homozygous carriers of the RN- allele, except for CS activity, which was lower in RN-/rn+ than in RN-/RN- pigs. In L muscle, the RN- allele led to a large increase in glycolytic potential (+3.5 phenotypic SD between homozygotes) and lightness (+.7 SD), and a decrease in ultimate pH, dry matter, and protein contents (-1.7 to -2 phenotypic SD for these three traits), with an almost completely dominant effect. No differences were found between genotypes for intramuscular fat and hydroxyproline contents. In the red S muscle, the presence of RN- had no influence on enzyme activities. These results indicate that the RN genotype greatly influences compositional and histochemical traits and metabolic enzyme activities in a muscle type-dependent manner, with a completely or incompletely dominant effect of the RN- allele.

Effect of two candidate genes on the Salmonella carrier state in fowl.

Selection for increased resistance to Salmonella carrier-state (defined as the persistency of the bacteria 4 wk after inoculation) could reduce the risk for the consumer of food toxi-infections. The effects of two genomic regions on chromosomes 7 and 17 harboring two genes, NRAMP1 (SLC11A1) and TLR4, known to be involved in the level of chicken infection 3 d after inoculation by Salmonella were thus tested on a total of 331 hens orally inoculated at the peak of lay with 10(9) bacteria. The animals and their parents were genotyped for a total of 10 microsatellite markers mapped on chromosomes 7 and 17. Using maximum likelihood analysis and interval mapping, it was found that the SLC11A1 region was significantly involved in the control of the probability of spleen contamination 4 wk after inoculation. Single nucleotide polymorphisms (SNP) within the SLC11A1 and TLR4 gene were tested on those animals as well as on a second batch of 279 hens whose resistance was assessed in the same conditions. As the former was significantly associated with the risk of spleen contamination and the number of contaminated organs, SLC11A1 appears to be involved in the control of resistance to Salmonella carrier state. The involvement of the TLR4 gene was also highly suspected as a significant association between SNP within the gene, and the number of contaminated organs was detected.