1 ml syringe

The right ovary was immediately frozen in liquid nitrogen and stored at −80 °C until further analysis. Left ovaries were placed in plastic bags in a water bath at 37 °C. The 15 largest follicles of the left ovary were aspirated within 5 h after collection, using a 21G × 5/8 needle and 1 mL syringe (VWR, Amsterdam, The Netherlands). These are assumed to represent approximately half of the ovulatory follicle pool, as ovulation rates in modern sows are around 25 [14] (link). After recovery of the cumulus–oocyte complexes (COCs), the follicular fluid content of the 15 largest follicles was pooled, collected in a tube and allowed to settle for 5 min. The supernatant was removed and centrifuged at 1900×g at 4 °C for 30 min to separate cells from the follicular fluid. The total volume of follicular fluid was assessed by reverse pipetting and was subsequently stored at −80 °C until further analysis.
The recovered COCs were used for in vitro maturation (IVM) and in vitro fertilization (IVF). The COC recovery rate was 64.7 ± 5.6 vs. 72.2 ± 3.9 for RES and FF, respectively.

S. pyogenes strains in late-log phase (OD_{600 nm} = 0.4) were spun down (6000× g, 3 min, RT) and washed once with 1 mL of phosphate-buffered saline (PBS). Bacterial suspensions were passed 10 times through a 27 Gauge syringe insert (NeoLab, Heidelberg, Germany) in a 1 mL syringe (VWR) to separate the cocci chains, and 10⁶ BLaER1 cells were infected at MOI 1. Plates were centrifuged (300× g, 5 min, RT) to synchronize the infection. P/S was added after 1 h to a final concentration of 1% to inhibit bacterial replication and excessive cell death. After overnight incubation, plates were centrifuged (500× g, 5 min, RT), after which supernatants were collected and stored at −20 °C until use. Supernatants were thawed on ice and IL-6 and IL-1β concentrations were measured using Enzyme-Linked ImmunoSorbent Assay (ELISA) kits (Thermofisher Scientific, Darmstadt, Germany) according to the manufacturer’s protocol. ELISA data were analyzed by extrapolating values of each sample from the standard curve using a log-log fit. The standard curve was generated for each plate by plotting the logarithm of the absorbance of the standards against the logarithm of the concentrations of each standard. In contrast, fresh supernatants were used to measure cell death using the Lactate Dehydrogenase (LDH)-Cytotoxicity Assay Kit II (Abcam, Cambridge, UK) according to the manufacturer’s instructions.