Cumulus Cell and Oocyte Gene Expression in Prepubertal Gilts and Sows Identifies Cumulus Cells as a Prime Informative Parameter of Oocyte Quality.

Cumulus cells (CCs) are pivotal during oocyte development. This study aimed to identify novel marker genes for porcine oocyte quality by examining the expression of selected genes in CCs and oocytes, employing the model of oocytes from prepubertal animals being of reduced quality compared to those from adult animals. Total RNA was extracted either directly after follicle aspiration or after in vitro maturation, followed by RT-qPCR. Immature gilt CCs accumulated BBOX1 transcripts, involved in L-carnitine biosynthesis, to a 14.8-fold higher level (p < 0.05) relative to sows, while for CPT2, participating in fatty acid oxidation, the level was 0.48 (p < 0.05). While showing no differences between gilt and sow CCs after maturation, CPT2 and BBOX1 levels in oocytes were higher in gilts at both time points. The apparent delayed lipid metabolism and reduced accumulation of ALDOA and G6PD transcripts in gilt CCs after maturation, implying downregulation of glycolysis and the pentose phosphate pathway, suggest gilt cumulus-oocyte complexes have inadequate ATP stores and oxidative stress balance compared to sows at the end of maturation. Reduced expression of BBOX1 and higher expression of CPT2 in CCs before maturation and higher expression of G6PD and ALDOA after maturation are new potential markers of oocyte quality.

Liquid storage of porcine in vitro-produced blastocysts; a practical approach for short storage.

Commercial application of embryo transfer in pig breeding is dependent on the storage of embryos. The aim of this study was to assess the embryo quality of in vitro-produced blastocysts after 3 h liquid storage at 37°C in CO2-free medium by evaluating morphology, in vitro developmental capacity and apoptosis. Blastocysts at days 5 and 6 post-fertilization were randomly allocated to the storage group (HEPES-buffered NCSU-23 medium including bovine serum albumin in a portable embryo transport incubator at 37°C) or a control group (porcine blastocyst medium in a conventional culture incubator). Thereafter, blastocysts were evaluated for morphology and stained to assess apoptosis straight after the 3 h storage period or after a further 24 h conventional incubation. There was no significant difference between the storage and control group after 3 h storage and the further 24 h conventional incubation for any of the parameters, nor for apoptosis straight after the 3 h storage. Embryos that reached the blastocyst stage at day 5 showed less apoptosis (6.6% vs 10.9%, P = 0.01) and a trend for a higher rate of developmental capacity (70.6% vs 51.5%, P = 0.089) than embryos reaching the blastocyst stage on day 6. In conclusion, in vitro-produced porcine blastocysts can be stored for 3 h at physiological temperature in transportable incubators using a CO2-independent medium without compromising quality.

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.

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.

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.

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.