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.

Abstract The quality of porcine oocytes for use in IVF is commonly graded according to the number of layers of cumulus cells (CCs) surrounding the oocyte; together these form the cumulus oocyte complex (COC). At least three compact layers of CCs is regarded as important for efficient IVP. To test this, oocytes were scored according to cumulus investment, with grade A representing COCs with three or more cumulus layers including granulosa cell‐cumulus oocyte complexes, grade B those with an intact corona radiata surrounded by another layer of cumulus cells and grades C and D representing COCs with lower CC investment. These oocytes were then monitored for in vitro maturation (IVM), as assessed by tubulin immunostaining for meiotic progression, the development of a cortical granule ring, and by glutathione levels. Results indicate that grading correlates closely with nuclear maturation and cytoplasmic maturation, suggesting that grading oocytes by cumulus investment is a reliable method to predict IVM success. Importantly, Grade A and B oocytes showed no significant differences in any measure and hence using a cut‐off of two or more CC layers may be optimal. We also determined the effect of assisted aspiration for oocyte retrieval, comparing the effect of needle size and applied pressure on the retrieval rate. These data indicated that both variables affected oocyte recovery rates and the quality of recovered oocytes. In combination, these experiments indicate that grade A and B oocytes have a similar developmental potential and that the recovery of oocytes of these grades is maximised by use of an 18‐gauge needle and 50 mmHg aspiration pressure.

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.

Citation for published version: Matika, O, Robledo Sanchez, D, Pong-Wong, R, Woolliams, J, Bishop, S, Riggio, V, Hoste, A, Walling, GA, Archibald, A & Houston, R 2018, 'A premature stop mutation in the porcine myostatin gene is a candidate causative variant for a recessive leg weakness syndrome and affects muscle depth', Paper presented at 11th World Congress on Genetics Applied to Livestock Production, Auckland, New Zealand, 11/02/18 16/02/18 pp. 297.

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.

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.

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.

Artificial insemination is routinely used in commercial pig breeding, for which the use of high quality semen samples is imperative. Currently, semen quality is determined manually by morphological assessment. This method leads to high inter-operator variability due to its subjective nature. The development of a semi-automated software-based approach to assess sperm morphology would enable faster identification of morphological defects and permit identification of subtle differences that may affect fertilisation success. Here we have used a novel method to comprehensively analyse pig sperm nuclear morphology in greater detail than was previously possible. Semen samples from 50 fertile and 50 sub-fertile samples that had been previously manually categorised as fertile or sub-fertile were analysed using this new method, with at least 200 fixed and DAPI (4',6-diamidino-2-phenylindole) stained sperm heads imaged per sample. Differences in sperm nuclear morphology were observed between fertile and sub-fertile samples; specifically, fertile samples were associated with higher mean nuclear area, a consequence of a greater head width and a lower variability between sperm heads. This novel, unbiased and fast analysis method demonstrates a significant difference in sperm head morphology between fertile and sub-fertile animals, and has the potential to be further developed and used as a tool for sperm morphology assessment in the pig breeding industry.

SummaryBalanced chromosomal aberrations have been shown to affect fertility in most species studied, often leading to hypoprolificacy (reduced litter size) in domestic animals such as pigs. With an increasing emphasis in modern food production on the use of a small population of high quality males for artificial insemination, the potential economic and environmental costs of hypoprolific boars, bulls, rams etc. are considerable. There is therefore a need for novel tools to facilitate rapid, cost‐effective chromosome translocation screening. This has previously been achieved by standard karyotype analysis; however, this approach relies on a significant level of expertise and is limited in its ability to identify subtle, cryptic translocations. To address this problem, we developed a novel device and protocol for translocation screening using subtelomeric probes and fluorescence in situ hybridisation. Probes were designed using BACs (bacterial artificial chromosomes) from the subtelomeric region of the short (p‐arm) and long (q‐arm) of each porcine chromosome. They were directly labelled with FITC or Texas Red (p‐arm and q‐arm respectively) prior to application of a ‘Multiprobe’ device, thereby enabling simultaneous detection of each individual porcine chromosome on a single slide. Initial experiments designed to isolate BACs in subtelomeric regions led to the discovery of a series of incorrectly mapped regions in the porcine genome assembly (from a total of 82 BACs, only 45 BACs mapped correctly). Our work therefore highlights the importance of accurate physical mapping of newly sequenced genomes. The system herein described allows for robust and comprehensive analysis of the porcine karyotype, an adjunct to classical cytogenetics that provides a valuable tool to expedite efficient, cost effective food production.

The past 10–15 years have seen a revolution in the field of genomics, first with the human genome project, followed by those of key model and agricultural species (chicken, pig, cattle, sheep) and, most recently, ? 60 de novo avian genome assemblies. The ultimate aim of a genome assembly is to create a contiguous unbroken length of sequence from p- to q-terminus to facilitate studies of gene mapping, trait linkage, phylogenomics, and gross genomic organization/change. Chromosome rearrangements are biologically relevant both in the context of reduction in reproductive capability of individual animals and in the establishment in reproductive isolation as species evolve and diverge. Moreover, a karyotype effectively represents a low-resolution map of the genome of any species. In investigating all these aspects, FISH remains the tool of choice, and this study describes a step change in its use.

The aim of the work was to assess the effects of three dietary protein regimes on pig performance and nitrogen (N) excretion, in particular, whether performance can be maintained in lean, fast growing pigs when protein levels are reduced to limit N excretion. Entire male pigs of a lean genotype (Pietrain × Large White × Landrace), 192 in total in four batches, were grown from 40 to 115 kg in pens with four pigs per pen. The diets were: (i) a high-protein control regime; (ii) a low-protein regime in which protein was reduced by ~2 percentage units in each growth stage, but with levels of five essential amino acids the same as in the control (LP1); (iii) an even lower protein regime in which levels of essential amino acids were not maintained beyond 60 kg (LP2). The LP2 regime was designed to promote intramuscular fat deposition rather than efficient growth. Excretion of N was reduced by 17% and 19% in LP1 and LP2, respectively, compared with the control. Average daily gain was lower and feed conversion ratio higher in LP2 than the other regimes, as expected. The control and LP1, which differed in protein but not essential amino acid levels, produced broadly similar results for performance, but pigs in LP1 had poorer feed conversion than control pigs, which could be due to slightly greater fat deposition. The results show the difficulty in maintaining consistently high levels of performance in fast-growing, lean pigs when dietary protein levels are reduced.

The effects on compositional changes across the growing-finishing period (40-115 kg) of feeding pig diets with different protein and amino acid levels were investigated using CT scanning (at 60, 85 and 115 kg live weight). Pigs of a lean commercial genotype were fed a commercial control regime (C), or a low protein regime with either high (LP1) or low (LP2) essential amino acid levels, all balanced for net energy. In vivo CT measurements agreed well with post-slaughter sample joint dissection results for carcass tissue weights/proportions, and CT-measured muscle density predicted intramuscular fat accurately. Pigs on C and LP1 regimes did not differ significantly in composition during growth. However, pigs on the LP2 regime had significantly more fat (in carcass, internal and intra-muscular depots) and less muscle, from 85 kg onwards. Although fat levels differed depending on diet regime (LP2>others), proportions of fat in different body depots were unaffected.