124 Seroprevalence characterization of porcine follicular fluid from adult sows and prepubertal gilts

The inclusion of reproductive fluids (RFs) from specific phases of the estrous cycle in the culture medium has demonstrated improved developmental kinetics in porcine embryos, higher numbers of blastomeres in pig blastocysts, and fewer irregularly expressed genes compared with embryos produced with standard in vitro media (Cánovas et al. 2017 eLife 6, e23670). However, because RFs are not immune to sanitary risks, it is essential to detect the most important viruses in each batch of fluid before use. As it is not feasible to determine the seroprevalence status of the animals from which different reproductive fluids are collected, we proposed to characterize the seroprevalence of reproductive fluids in this study. Specifically, samples of porcine follicular fluid (PFF) from adult females from farms vaccinated against the main diseases affecting the porcine species were compared with samples collected from unvaccinated fattening animals. Both vaccinated and unvaccinated animals were slaughtered in abattoirs. Additionally, the effect of PFF from both sources on IVP was tested. Ovaries were collected from adult sows (S) and prepubertal gilts (G), the latter selected based on ovarian morphology (absence of corpora lutea or albicantia), at a local abattoir. PFF was aspirated from follicles of 2–8 mm, centrifuged to remove cellular debris, frozen at −80°C, and lyophilized. PFF samples were tested for the presence of Circovirus type 2, PRRS type 1, PRRS type 2, and Parvovirus. Samples were considered positive with Cq < 37; doubtful/non-conclusive with Cq > 37.0 (presence of DNA traits or nonspecific reaction); or negative with Cq = 0. This experiment was repeated seven times. Subsequently, we pooled the negative PFFs and performed pig IVP comparing the addition of Sʹ or Gʹ PFF to the IVM medium. For this, cumulus-oocyte complexes were aspirated from 2- to 8-mm follicles, selected based on morphology, and cultured for 22 h in maturation medium supplemented with 0.57 mM cysteine, 1 mM dibutyryl cAMP, 5 mg mL⁻¹ insulin, 50 mM β-mercaptoethanol, 10 IU mL⁻¹ eCG, 10 IU mL⁻¹ hCG, and 10% PFF (vol/vol), followed by 22 h in fresh maturation medium without dibutyryl cAMP, eCG, and hCG. Following IVM, frozen-thawed boar sperm, from the same boar and ejaculate, were selected by swim-up in PIG-SUM-LYO medium (EmbryoCloud), and oocytes and spermatozoa were co-incubated for 22 h in PIG-IVF-LYO medium, after which embryos were transferred to PIG-IVC1-LYO medium for 24 h, when cleavage was evaluated. After this, 2-cell embryos were transferred to PIG-IVC2-LYO medium until Day 7 of culture. Embryo stage was observed on Day 6 post-insemination. A total of three replicates were conducted, with 50 oocytes per group per replicate. Statistical analysis was performed using Student’s t-test (P < 0.05). In GPFF, the prevalence of Parvovirus was 85.7%, Circovirus type 2 was 57.1%, PRRS 1 was 14.3%, and PRRS 2 was not detected. Only 14.3% of the samples were negative for the tested panel. Conversely, SPFF was found to be 100% negative for all the viruses screened. Regarding IVP, cleavage rate (%) on Day 2 was significantly higher with SPFF (79.2 ± 3.6) compared with GPFF (59.2 ± 4.), while the blastocyst formation rate (%) on Day 6 was similar in SPFF (25.3 ± 4.4) and GPFF (23.9 ± 4.4). There were no significant differences in blastocyst cell numbers between groups (48.8 ± 3.7 and 49.7 ± 5.2 for GPFF and SPFF, respectively). The results indicate that collecting follicular fluid samples from adult sows is an effective way to prevent the presence of undesirable viruses in culture media supplemented with reproductive fluids. Furthermore, the supplementation of porcine maturation media with porcine follicular fluid from adult females is validated.

Funded by Fundación Séneca (22247/PDC/23), PLEC2022-009246/AEI/10.13039/501100011033, Investigo Program and NextGenerationEU/PRTR.