Morphokinetic Profiling Suggests That Rapid First Cleavage Division Accurately Predicts the Chances of Blastulation in Pig In Vitro Produced Embryos.

The study of pig preimplantation embryo development has several potential uses: from agriculture to the production of medically relevant genetically modified organisms and from rare breed conservation to acting as a physiologically relevant model for progressing human and other (e.g., endangered) species' in vitro fertilisation technology. Despite this, barriers to the widespread adoption of pig embryo in vitro production include lipid-laden cells that are hard to visualise, slow adoption of contemporary technologies such as the use of time-lapse incubators or artificial intelligence, poor blastulation and high polyspermy rates. Here, we employ a commercially available time-lapse incubator to provide a comprehensive overview of the morphokinetics of pig preimplantation development for the first time. We tested the hypotheses that (a) there are differences in developmental timings between blastulating and non-blastulating embryos and (b) embryo developmental morphokinetic features can be used to predict the likelihood of blastulation. The abattoir-derived oocytes fertilised by commercial extended semen produced presumptive zygotes were split into two groups: cavitating/blastulating 144 h post gamete co-incubation and those that were not. The blastulating group reached the 2-cell and morula stages significantly earlier, and the time taken to reach the 2-cell stage was identified to be a predictive marker for blastocyst formation. Reverse cleavage was also associated with poor blastulation. These data demonstrate the potential of morphokinetic analysis in automating and upscaling pig in vitro production through effective embryo selection.

Successful recovery of motile and viable boar sperm after vitrification with different methods (pearls and mini straws) using sucrose as a cryoprotectant.

Vitrification of sperm by direct contact with liquid nitrogen is increasing in popularity as an alternative to conventional (slow) freezing. Although slow freezing is very challenging in boar sperm cryopreservation, this is currently the standard method used. We compared vitrification in "pearls" and in "mini straws" using the in vitro fertilization media Porcine Gamete Media with 0.3 M sucrose with the standard (slow) method used to preserve boar sperm. Both vitrification methods reduced the viability of the sperm sample more than slow freezing (42.2 ± 4.3% total motility and 71.4 ± 2.3% alive), however, both protocols allowed for the successful recovery of the sperm samples. By comparing two different methods of vitrification and two different methods of post-thaw preparation we were able to determine the optimal vitrification-thaw protocol for boar sperm. When comparing pearls and mini-straws, the smaller liquid volume associated with pearls had a positive effect on the survivability of the samples, reducing sperm DNA damage (1.2 ± 0.2% vs. 5.1 ± 0.1.7%) and preserving motility (26.15 ± 2.8% vs 9.39 ± 0.9%) after thawing. In conclusion, the pearl method was the most suitable of the vitrification techniques for use with boar sperm.

Preimplantation Genetic Testing for Aneuploidy (PGT-A) Reveals High Levels of Chromosomal Errors in In Vivo-Derived Pig Embryos, with an Increased Incidence When Produced In Vitro.

Preimplantation genetic testing for aneuploidy (PGT-A) is widespread, but controversial, in humans and improves pregnancy and live birth rates in cattle. In pigs, it presents a possible solution to improve in vitro embryo production (IVP), however, the incidence and origin of chromosomal errors remains under-explored. To address this, we used single nucleotide polymorphism (SNP)-based PGT-A algorithms in 101 in vivo-derived (IVD) and 64 IVP porcine embryos. More errors were observed in IVP vs. IVD blastocysts (79.7% vs. 13.6% p < 0.001). In IVD embryos, fewer errors were found at blastocyst stage compared to cleavage (4-cell) stage (13.6% vs. 40%, p = 0.056). One androgenetic and two parthenogenetic embryos were also identified. Triploidy was the most common error in IVD embryos (15.8%), but only observed at cleavage, not blastocyst stage, followed by whole chromosome aneuploidy (9.9%). In IVP blastocysts, 32.8% were parthenogenetic, 25.0% (hypo-)triploid, 12.5% aneuploid, and 9.4% haploid. Parthenogenetic blastocysts arose from just three out of ten sows, suggesting a possible donor effect. The high incidence of chromosomal abnormalities in general, but in IVP embryos in particular, suggests an explanation for the low success of porcine IVP. The approaches described provide a means of monitoring technical improvements and suggest future application of PGT-A might improve embryo transfer success.

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.

Follicular fluid steroid hormones and in vitro embryo development in Duroc and Landrace pigs.

The Duroc sire line has a smaller litter size compared to the Landrace dam line and we have previously observed fewer surface follicles on Duroc ovaries one day after weaning. In that same study, a broader cumulus expansion and faster nuclear maturation were observed for Duroc oocytes at 20 h of in vitro maturation (IVM), while Landrace oocytes showed more advanced stages of cortical granule distributions. However, no differences between breeds were observed after the final IVM period. The aim of this study was to assess subsequent in vitro embryo production (IVP) in Duroc and Landrace. Furthermore, follicle diameter and steroid hormone levels in follicular fluid (FF) were measured to study possible relation to oocyte developmental competence. Follicular phase sow ovaries were collected one day after weaning and follicle size of the 10 largest follicles were measured per ovary before aspiration. Cumulus-oocyte complexes (COCs) were matured in vitro, and cumulus expansion was analysed by assessing individual COC areas at 0 and 20 h. Fertilization of Duroc and Landrace oocytes was performed with sperm from both a Duroc and a Landrace boar. A larger follicle diameter was observed for Landrace animals (5.7 vs. 4.8 mm, P < 0.0001) and individual COC area was additionally larger at 0 h after aspiration (P < 0.0001) compared to Duroc. Contrary, cumulus expansion from 0 to 20 h of maturation was broader for Duroc oocytes than for Landrace (407 ± 67% vs. 319 ± 31%, P < 0.0001). After fertilization, cleavage rate was higher for Duroc oocytes, and the highest blastocyst yield was obtained for Duroc oocytes fertilized with the Landrace sperm. Steroid hormone analysis of the follicular fluid showed differences in the pathways between breeds with a higher total level of estrogens (P = 0.01) and aromatase products/substrates ratio (P < 0.01) in Landrace compared to Duroc. In conclusion, results suggest that Duroc oocytes have a better in vitro oocyte developmental competence when cultured under the same in vitro conditions and breed differences in steroidogenesis were found in the early follicular phase.

Genetic aspects of piglet survival and related traits: a review.

In livestock, mortality in general, and mortality of the young, is societal worries and is economically relevant for farm efficiency. Genetic change is cumulative; if it exists for survival of the young and genetic merit can be estimated with sufficient accuracy, it can help alleviate the pressure of mortality. Lack of survival is a moving target; livestock production is in continuous change and labor shortage is a given. There is now ample evidence of clear genetic variance and of models able to provide genomic predictions with enough accuracy for selection response. Underlying traits such as birth weight, uniformity in birth weight, gestation length, number of teats, and farrowing duration all show genetic variation and support selection for survival or, alternatively, be selected for on their own merit.

Piglet survival is under genetic control and there are clear differences between individuals in their ability to live. Animals that do not survive their first weeks will obviously not reproduce as this is natural selection. Animals that survive still harbor relevant genetic differences. The genomic toolset, the use of genetic markers, makes it possible to link each animal to all others in the population, alive or dead, creating good opportunities for selection. Piglet survival depends on the genetic make-up of 1) the piglet itself, is it vital and heavy enough, 2) of the mother, are the piglets born at term, with low variation in birth weight, and 3) of the sow nursing the piglets, often the mother, does she allow the piglets to drink enough colostrum and milk of enough quality? This review explores the black box approach, complex statistical analysis of very large scale genomic recording of survival data, and it explores the biological approach, the influences of gestation length, birth weight, uniformity, number of teats, colostrum, etc., on birth weight. There is little doubt that genetic selection can increase survival of piglets. The challenge is to do this selection in balance with other production traits, such as litter size and body composition.

Effect of two 'progressively motile sperm-oocyte' ratios on porcine in vitro fertilization and embryo development.

Sperm motility and viability of cryopreserved semen vary between boars and straws, which influences the outcomes of in vitro embryo production (IVEP). However, progressive motility is usually not considered during IVEP. The aim of this study was to assess fertilization with a 500:1 and 250:1 'progressively motile sperm to oocyte' ratio on IVEP outcomes using semen from three Duroc and three Landrace boars. Frozen-thawed sperm was centrifuged through a 45/90% Percoll® density gradient and sperm quality parameters were assessed. In vitro matured oocytes were fertilized at the two ratios, a portion was stained 10-12 h after start of fertilization to analyze fertilization and polyspermy, while the remaining zygotes were cultured up to day 7. The 500:1 ratio resulted in a higher fertilization and blastocyst yield on day 6 compared with the 250:1 ratio, but no effect of ratio was observed for polyspermy, cleavage rate or blastocyst cell number. Individual differences between boars were observed for fertilization, cleavage and blastocyst rates, but not for the other IVEP outcomes. In conclusion, a higher fertilization and blastocyst yield was obtained with the 500:1 ratio compared with the 250:1 ratio, while polyspermy level was consistent across ratios. Differences in IVEP outcomes were still observed between the individual boars although adjusted for progressive motility. Promising blastocyst yields and high total blastocyst cell counts were obtained with sperm from both breeds.

The role of apoptosis in cryopreserved animal oocytes and embryos.

Cryopreservation of both gametes and embryos, both for storage and for the preservation of their developmental capacity is a critical aspect of assisted reproductive technology. The survival of reproductive material following cryopreservation protocols is not only vital to clinical applications in the human in vitro fertilisation clinic, but is also important in the in vitro production of livestock embryos. The ability to routinely cryopreserve oocytes and embryos of livestock species has the potential to improve animal welfare, reduce environmental impact, and reduce the associated costs for breeding companies through the reduction of live animal transportation. Unfortunately, frozen oocytes and embryos are regularly documented to contain a higher proportion of apoptotic cells compared to their non-frozen counterparts, with freezing procedures thought to trigger apoptotic pathways of cell death. Comparisons between frozen and non-frozen samples also show changes in the gene expression of apoptotic factors such as Bcl-2 and Bax in response to cryopreservation. Apoptotic inhibition has the potential to improve cryosurvival, and how to achieve this is subject to debate. Here, we review how exposure to low temperatures during cryopreservation may be responsible for the abnormal activation of apoptotic pathways in mammalian oocytes and embryos, and discuss the ways in which they can be influenced to improve cryopreservation protocols, particularly in agriculturally important species.

Ovarian characteristics and in vitro nuclear and cytoplasmic oocyte maturation in Duroc and Landrace pigs.

Differences in total number of piglets born per litter are observed between the Norwegian Duroc (ND) sire and Norwegian Landrace (NL) dam line. The aim of this study was to evaluate ovarian characteristics, and in vitro nuclear and cytoplasmic oocyte maturation in both breeds. One day after weaning, follicular phase ovaries were collected. Ovary length and weight were measured and the number of follicles (< 3 mm and 3-8 mm) was counted. Cumulus-oocyte complexes (COCs) were collected and matured for 48 hr. To assess cumulus expansion, COC area was analysed at 0 and 20 hr. Nuclear maturation and cortical granule (CG) distribution were analysed at 20 and 48 hr, and total glutathione (GSH) was measured at 48 hr to further elucidate cytoplasmic maturation. In first parity sows, a smaller ovary length and fewer 3 to 8 mm follicles were observed in ND compared to NL. For all sows, ND COCs covered a significantly smaller area at 0 hr, but a higher cumulus expansion ratio was observed at 20 hr compared to NL (364 ± 46% versus. 278 ± 27%, p < 0.001). At 20 hr, more ND oocytes exhibited advanced stages of nuclear maturation, while more NL oocytes showed advanced stages of CG distribution. Nuclear maturation to MII stage at 48 hr did not differ between ND and NL oocytes (90.1% and 87.7%, respectively). Moreover, no significant differences were observed for GSH content or CG distribution after maturation. In conclusion, differences with regard to ovarian characteristics as well as to cumulus expansion, and nuclear and cytoplasmic oocyte maturation at 20 hr were observed between the breeds. Further studies are required to determine if this subsequently affects in vitro fertilization and embryo development.

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.