Hepes buffered tcm 199 medium

Sheep ovaries were obtained from a local slaughterhouse and transferred at 37 °C to the laboratory within 1–2 h from slaughter. Cumulus-Oocyte Complexes (COCs) were aspirated using a 21 G needles in the presence of 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) buffered TCM-199 medium (Gibco, Life Technologies, Milan, Italy) and 0.005% (w:v) heparin. Only COCs having at least 2 or 3 layers of compact cumulus cells were selected for IVM, which was performed in 4-well dishes containing 500 μl of IVM medium per well. The IVM medium was composed of bicarbonate-buffered TCM-199 (Gibco) containing 2 mM glutamine, 0.3 mM sodium pyruvate, 100 μM cysteamine, 10% fetal bovine serum (FBS) (Gibco), 5 μg/ml follicle stimulating hormone (FSH; Ovagen, ICP, Auckland, New Zealand), 5 μg/ml luteinizing hormone (LH) and 1 μg/ml 17 β-estradiol. Maturation was completed in a humidified atmosphere at 38.5 °C and 5% CO2 in air for 24 h, as previously described^{34 (link)}. After IVM, only MII oocytes with an expanded cumulus and normal morphology were selected for Intracytoplasmic Sperm Injection (ICSI). Cumulus was removed by fast pipetting of COCs into a 500 μl of 300 U/ml of hyaluronidase solution in Hepes-buffered TCM-199 + 0.4% BSA (w:v) (H199). Then, oocytes were 3 times washed in H199 and incubated into a Petri dish, until injection.

In vitro maturation was performed as previously described^{25 (link)}. Briefly, sheep ovaries were collected from local slaughterhouses and transferred to our laboratory at 37 °C within 1 h. Oocytes were aspirated with 21 G needles in HEPES-buffered TCM-199 medium (Gibco, Life Technologies, Milan, Italy) supplemented with 0.005% (w:v) heparin. Cumulus-Oocyte Complexes (COCs) were cultures in 4 wells-dishes containing 0.5 ml IVM medium, composed by bicarbonate-buffered TCM-199 (Gibco) containing 2 mM glutamine, 0.3 mM sodium pyruvate, 100 μM cysteamine, 10% (v:v) fetal bovine serum (FBS) (Gibco), 5 μg/ml follicle stimulating hormone (FSH) (Ovagen, ICP, Auckland, New Zealand), 5 μg/ml luteinizing hormone (LH) and 1 μg/ml β-estradiol, in a humidified atmosphere at 38.5 °C and 5% CO₂ in air for 24 h. All the process (from oocyte pick-up to the start of IVM) it ends within 1.5 h.