Supplementation of porcine in vitro maturation medium with FGF2, LIF, and IGF1 enhances cytoplasmic maturation in prepubertal gilts oocytes and improves embryo quality.

In porcine in vitro production (IVP) systems, the use of oocytes derived from prepubertal gilts, whilst being commercially attractive, remains challenging due to their poor developmental competence following in vitro maturation (IVM). Follicular fluid contains important growth factors and plays a key role during oocyte maturation; therefore, it is a common supplementation for porcine IVM medium. However, follicular fluid contains many poorly characterized components, is batch variable, and its use raises biosecurity concerns. In an effort to design a defined IVM system, growth factors such as cytokines have been previously tested. These include leukaemia inhibitory factor (LIF), fibroblast growth factor 2 (FGF2), and insulin-like growth factor 1 (IGF1), the combination of which is termed 'FLI'. Here, using abattoir-derived oocytes in a well established porcine IVP system, we compared follicular fluid and FLI supplementation during both IVM and embryo culture to test the hypothesis that FLI can substitute for follicular fluid without compromising oocyte nuclear and cytoplasmic maturation. We demonstrate that in oocytes derived from prepubertal gilts, FLI supplementation enhances oocyte meiotic maturation and has a positive effect on the quality and developmental competence of embryos. Moreover, for the first time, we studied the effects of follicular fluid and FLI combined showing no synergistic effects.

Balancing selection at a premature stop mutation in the myostatin gene underlies a recessive leg weakness syndrome in pigs.

Balancing selection provides a plausible explanation for the maintenance of deleterious alleles at moderate frequency in livestock, including lethal recessives exhibiting heterozygous advantage in carriers. In the current study, a leg weakness syndrome causing mortality of piglets in a commercial line showed monogenic recessive inheritance, and a region on chromosome 15 associated with the syndrome was identified by homozygosity mapping. Whole genome resequencing of cases and controls identified a mutation causing a premature stop codon within exon 3 of the porcine Myostatin (MSTN) gene, similar to those causing a double-muscling phenotype observed in several mammalian species. The MSTN mutation was in Hardy-Weinberg equilibrium in the population at birth, but significantly distorted amongst animals still in the herd at 110 kg, due to an absence of homozygous mutant genotypes. In heterozygous form, the MSTN mutation was associated with a major increase in muscle depth and decrease in fat depth, suggesting that the deleterious allele was maintained at moderate frequency due to heterozygous advantage (allele frequency, q = 0.22). Knockout of the porcine MSTN by gene editing has previously been linked to problems of low piglet survival and lameness. This MSTN mutation is an example of putative balancing selection in livestock, providing a plausible explanation for the lack of disrupting MSTN mutations in pigs despite many generations of selection for lean growth.

Genetic selection against intrauterine growth retardation in piglets: a problem at the piglet level with a solution at the sow level.

BACKGROUND: In polytocous livestock species, litter size and offspring weight act antagonistically; in modern pig breeds, selection for increased litter size has resulted in lower mean birth weights, an increased number of small piglets and an increased number of those affected by varying degrees of intrauterine growth retardation (IUGR). IUGR poses life-long challenges, both mental, with morphological brain changes and altered cognition, and physical, such as immaturity of organs, reduced colostrum intake and weight gain. In pigs, head morphology of newborn piglets is a good phenotypic marker for identifying such compromised piglets. Growth retardation could be considered as a property of the dam, in part due to either uterine capacity or insufficiency. A novel approach to this issue is to consider the proportion of IUGR-affected piglets in a litter as an indirect measure of uterine capacity. However, uterine capacity or sufficiency cannot be equated solely to litter size and thus is a trait difficult to measure on farm. RESULTS: A total of 21,159 Landrace × Large White or Landrace × White Duroc piglets (born over 52 weeks) with recorded head morphology and birth weights were followed from birth until death or weaning. At the piglet level, the estimated heritability for IUGR (as defined by head morphology) was low at 0.01 ± 0.01. Piglet direct genetic effects of birth weight (h2 = 0.07 ± 0.02) were strongly negatively correlated with head morphology (- 0.93), in that IUGR-affected piglets tended to have lower birth weights. At the sow level, analysis of the proportion of IUGR-affected piglets in a litter gave a heritability of 0.20 ± 0.06, with high and negative genetic correlations of the proportion of IUGR-affected piglets with average offspring birth weight (- 0.90) and with the proportion of piglets surviving until 24 h (- 0.80). CONCLUSIONS: This suggests that the proportion of IUGR-affected piglets in a litter is a suitable indirect measure of uterine capacity for inclusion in breeding programmes that aim at reducing IUGR in piglets and improving piglet survival.

The production of pig preimplantation embryos in vitro: Current progress and future prospects.

Human assisted reproductive technology procedures are routinely performed in clinics globally, and some of these approaches are now common in other mammals such as cattle. This is currently not the case in pigs. Given that the global population is expected to increase by over two billion people between now and 2050, the demand for meat will also undoubtedly increase. With this in mind, a more sustainable way to produce livestock; increasing productivity and implementing methods that will lead to faster genetic selection, is imperative. The establishment of routine and production scale pig embryo in vitro production could be a solution to this problem. Producers would be able to increase the overall number of offspring born, animal transportation would be more straightforward and in vitro produced embryos could be produced from the gametes of selected elite. Here we review the most recent developments in pig embryology, outline the current barriers and key challenges that exist, and outline research priorities to surmount these difficulties.

Genetic control of complex traits, with a focus on reproduction in pigs.

Reproductive traits are complex, and desirable reproductive phenotypes, such as litter size or semen quality, are true polygenetic traits determined by multiple gene regulatory pathways. Each individual gene contributes to the overall variation in these traits, so genetic improvements can be achieved using conventional selection methodology. In the past, a pedigree-based-relationship matrix was used; this is now replaced by a combination of pedigree-based- and genomic-relationship matrices. The heritability of reproductive traits is low to moderate, so large-scale data recording is required to identify specific, selectable attributes. Male reproductive traits-including ejaculate volume and sperm progressive motility-are moderately heritable, and could be used in selection programs. A few high-merit artificial-insemination boars can impact many sow populations, so additional knowledge about male reproduction-specifically pre-pubertal detection of infertility and the technologies of semen cryopreservation and sex sorting-should further improve global breeding efforts. Conversely, female pig reproduction is currently a limiting factor of genetic improvement. Litter size and farrowing interval are the main obstacles to increasing selection intensity and to reducing generation interval in a breeding program. Age at puberty and weaning-to-estrus interval can be selected for, thereby reducing the number of non-productive days. The number of piglets born alive and litter weights are also reliably influenced by genetic selection. Characterization of genotype-environment interactions will provide opportunities to match genetics to specific farm systems. Continued investment to understand physiological models for improved phenotyping and the development of technologies to facilitate pig embryo production for genetic selection are warranted to ensure optimal breeding in future generations.

Genome wide analysis reveals single nucleotide polymorphisms associated with fatness and putative novel copy number variants in three pig breeds.

BACKGROUND: Obesity, excess fat tissue in the body, can underlie a variety of medical complaints including heart disease, stroke and cancer. The pig is an excellent model organism for the study of various human disorders, including obesity, as well as being the foremost agricultural species. In order to identify genetic variants associated with fatness, we used a selective genomic approach sampling DNA from animals at the extreme ends of the fat and lean spectrum using estimated breeding values derived from a total population size of over 70,000 animals. DNA from 3 breeds (Sire Line Large White, Duroc and a white Pietrain composite line (Titan)) was used to interrogate the Illumina Porcine SNP60 Genotyping Beadchip in order to identify significant associations in terms of single nucleotide polymorphisms (SNPs) and copy number variants (CNVs). RESULTS: By sampling animals at each end of the fat/lean EBV (estimate breeding value) spectrum the whole population could be assessed using less than 300 animals, without losing statistical power. Indeed, several significant SNPs (at the 5% genome wide significance level) were discovered, 4 of these linked to genes with ontologies that had previously been correlated with fatness (NTS, FABP6, SST and NR3C2). Quantitative analysis of the data identified putative CNV regions containing genes whose ontology suggested fatness related functions (MCHR1, PPARα, SLC5A1 and SLC5A4). CONCLUSIONS: Selective genotyping of EBVs at either end of the phenotypic spectrum proved to be a cost effective means of identifying SNPs and CNVs associated with fatness and with estimated major effects in a large population of animals.

Novel approach for deriving genome wide SNP analysis data from archived blood spots.

BACKGROUND: The ability to transport and store DNA at room temperature in low volumes has the advantage of optimising cost, time and storage space. Blood spots on adapted filter papers are popular for this, with FTA (Flinders Technology Associates) Whatman™TM technology being one of the most recent. Plant material, plasmids, viral particles, bacteria and animal blood have been stored and transported successfully using this technology, however the method of porcine DNA extraction from FTA Whatman™TM cards is a relatively new approach, allowing nucleic acids to be ready for downstream applications such as PCR, whole genome amplification, sequencing and subsequent application to single nucleotide polymorphism microarrays has hitherto been under-explored. FINDINGS: DNA was extracted from FTA Whatman™TM cards (following adaptations of the manufacturer's instructions), whole genome amplified and subsequently analysed to validate the integrity of the DNA for downstream SNP analysis. DNA was successfully extracted from 288/288 samples and amplified by WGA. Allele dropout post WGA, was observed in less than 2% of samples and there was no clear evidence of amplification bias nor contamination. Acceptable call rates on porcine SNP chips were also achieved using DNA extracted and amplified in this way. CONCLUSIONS: DNA extracted from FTA Whatman cards is of a high enough quality and quantity following whole genomic amplification to perform meaningful SNP chip studies.

On the mapping of quantitative trait loci at marker and non-marker locations.

Previous studies have noted that the estimated positions of a large proportion of mapped quantitative trait loci (QTLs) coincide with marker locations and have suggested that this indicates a bias in the mapping methodology. In this study we predict the expected proportion of QTLs with positions estimated to be at the location of a marker and further examine the problem using simulated data. The results show that the higher proportion of putative QTLs estimated to be at marker positions compared with non-marker positions is an expected consequence of the estimation methods. The study initially focused on a single interval with no QTLs and was extended to include multiple intervals and QTLs of large effect. Further, the study demonstrated that the larger proportion of estimated QTL positions at the location of markers was not unique to linear regression mapping. Maximum likelihood produced similar results, although the accumulation of positional estimates at outermost markers was reduced when regions outside the linkage group were also considered. The bias towards marker positions is greatest under the null hypothesis of no QTLs or when QTL effects are small. This study discusses the impact the findings could have on the calculation of thresholds and confidence intervals produced by bootstrap methods.