Four well plate

IVM media were supplemented with 10% v/v of the FF obtained either from cyclic or prepubertal pigs and of known fatty acids concentration. FF was aspirated from 3–5 mm ovarian follicles and collected to 1,5 ml tubes (Eppendorf, Germany) and centrifuged at 12000 rpm for 1 min. Supernatant was transferred to a new 1.5 ml tubes, frozen in liquid nitrogen and stored at −80 °C.
In vitro maturation of COC was performed in NCSU-23 (North Carolina State University Medium-23). Every COC group was incubated in 500 µl of IVM medium in four-well plates (Nunc, New York, USA) in HeraCell 150 incubator (Thermo Scientific) under conditions: 5% CO₂ in atmosphere, 39 °C and maximum humidity. The first 20 h of IVM included IVM medium supplemented with hormones: 10U PMSG (pregnant mare serum gonadothropin, Chorulon, MSD Animal Health, Netherlands) and 10U hCG (Folligon, MSD Animal Health, Netherlands). Next step included transfer of COCs to fresh, equilibrated medium without hormones and incubation for 24 hours.
After IVM all oocytes with first polar body and no degenerative changes were denuded and transferred to PBS with 0.2% PVP (polyvinylpyrrolidone), frozen or fixed for further analyses. Oocytes subjected for embryo production were denuded in fresh equilibrated NCSU23 medium.

Tissue culture plates (four-well-plates; Nunc) were coated with poly-D-lysine (0.1 mg/ml, molecular weight 300 000 Da; Sigma), rinsed with distilled water and air-dried. To prepare AC cultures, postnatal rats were killed by decapitation, retinae were rapidly dissected from the eyecups and incubated at 37°C for 30 min in a digestion solution containing papain (10 U/ml; Worthington) and L-cysteine (0.3 mg/ml; Sigma) in Neurobasal (NB) medium (Gibco). They were then rinsed with NB medium and triturated in 1 ml NB medium. Dissociated cells were passed through a cell strainer (40 mm) and 300 µl cell suspension in NB medium containing B27 supplement (1:50; Gibco), penicillin/streptomycin (0.2 mg/ml; Biochrom) and L-Glutamine (0.5 mM; Sigma) were added to each well. Cells remained in culture for 2 hours or 2 days and were fixed in 4% paraformaldehyde (PFA) solution in PBS for 25 min and then in 100% methanol (Sigma) for 10 min. AC were stained with antibodies against ChAT (1∶100) and MLP (1∶400).

The ovaries were collected from the slaughterhouse, kept in a physiological saline solution containing penicillin and streptomycin at 20–25 °C, and transported to the laboratory within 2 h. The cumulus-oocyte complexes (COCs) were collected, followed by in vitro maturation according to our previous study [50 (link)]. Briefly, COCs were collected from 3- to 8 mm diameter antral follicles using a syringe needle, picked up and cleaned in washing buffer (M199 + 1% FBS) three times under a stereoscopic microscope. The COCs were then cultured in four-well plates (Nunc) in 500 µL of oocyte maturation media at 38.5 °C under 5% CO₂. After 24 h of IVM, the bovine oocytes were transferred by pipette into 1-mg/mL hyaluronidase to remove cumulus cells. The first polar body extrusions were calculated as the maturation rate of the oocytes.

Embryos were warmed using Embryo Thaw Media Kit following the manufacturer’s instructions (Fujifilm Irvine Scientific, Cat. No. 90124). The day before warming, Continuous Single Culture-NX Complete medium (90168, Fujifilm Irvine Scientific) was equilibrated overnight at 37°C, 5% CO₂. On the day of warming (day 1), the straw that contains the embryo was defrosted at room temperature for 30 s and immersed in prewarmed (37 °C) water for 1 min until the ice melted. The embryo was then transferred into T-1 (5 min), T-2 (5 min), T-3 (10 min) solutions for slow warming, and finally into Multipurpose Handling Medium (90163, Fujifilm Irvine Scientific) for recovery. All these incubation steps were done using four well plates (Nunc) and 1 ml per solution. Warmed embryos were finally incubated in drops of pre-equilibrated Continuous Single Culture-NX Complete medium under mineral oil (9305, Irvine Scientific). Culture conditions are the following: 37°C, 21% O_2, and 5% CO_2. Embryos were incubated for a total of 48 hr until reaching the morula stage (day 4). Embryos were fixed with 4% PFA at day 4 for immunofluorescence analysis.

Porcine and bovine blastocysts were fixed in 4% PFA for 30 min at 37 °C in four-well plates (Nunc). PFA was removed by washing the embryos twice in PBS with 0.2% PVP and stored at 4 °C for no longer than two weeks. Embryo permeabilization was performed with 0.2% Triton X-100 solution for 30 min at room temp. (RT) and washed 2× afterwards in 0.2% PVP/PBS. Lipid droplets were stained with fluorescent dye—20 μg/mL BODIPY 493/503 (Thermo Scientific, Carlsbad, CA, USA) at room temperature for one hour. The chromatin of blastomeres were visualized by staining with 0.5 μg/mL DAPI (4′,6-diamidino-2-phenylindole; Vector Laboratories, Burlingame, CA, USA). Embryos were mounted on glass slide with single concave (Comex, PL), coverslipped and analyzed using confocal microscope Zeiss LSM 880 using 488 nm filter with band pass 500–550 nm for BODIPY 493/503 (Laser Argon2) and 420–480 nm for DAPI (Laser Diode 405). LD were assessed through several optical sections (Z-stack) captured every 5 μm to exclude double positioning of the same structures on two stacks. Objective (LD LCl Plan Apochromat 40×/1.2 Imm Korr DIC 27; Zeiss, Germany), pinhole, filters, offset settings were kept constant throughout the experiments. LD counting using ImageJ software (NIH, Bethesda, MD, USA) was previously described.

Porcine ovaries were collected from a local slaughter house and kept in saline at 32–37 °C. Antral follicles (3–5 mm indiameter) were aspirated with an 18-gauge needle. Aspirated oocytes with an evenly granulated cytoplasm and at least three uniform layers of compact cumulus cells were selected and cultured in four-well plates (Nunc, Naperville, IL, USA) containing 500 μL of maturation medium that was TCM199 (Gibco) based medium plus 0.05 μg/ml EGF, 0.5 μg/ml LH and FSH, at 39 °C in 5 % CO₂ in air [17 (link)]. The rates of polarbody extrusion were calculated from 16 h–42 h of IVM. Matured porcine oocyte was obtained at 33 h and 42 h for further experiments.