160 EFFECTS OF EXTRACELLULAR ION CONCENTRATIONS ON IN VITRO DEVELOPMENT OF DOMESTIC CAT IVF EMBRYOS

Abstract

Domestic cat embryos typically are cultured in media formulated for somatic cells or embryos from rodents or livestock species. Under these conditions, blastocyst development has been inconsistent and delayed relative to embryos grown in vivo, and embryo viability following transfer has been low. Our goal is to systematically define the culture requirements of the feline embryo to improve embryo development and viability. The objective of this study was to determine the ionic (NaCl, KCl, KH₂PO₄, and CaCl₂ : MgSO₄) preferences of domestic cat IVF embryos. Anestral female cats were injected (i.m.) with 150 IU eCG followed 84 h later by 100 IU hCG. Oocytes were recovered via laparoscopic follicular aspiration approximately 24 h post-hCG injection (Day 0). Semen was collected from one of two males by means of an artificial vagina and washed once in HEPES-buffered IVF medium. Mature cumulus-oocyte complexes were co-incubated with 2.5–5 × 10⁵ motile sperm mL⁻¹ in IVF medium (100 mM NaCl, 4.0 mM KCl, 1.0 mM KH₂ PO₄, 2.0 mM CaCl₂, 1.0 mM MgSO₄-7H₂O, 25.0 mM NaHCO₃, 3.0 mM glucose, 0.1 mM pyruvate, 6.0 mM L-lactate, 1.0 mM glutamine, 0.1 mM taurine, 1 × MEM nonessential amino acids, 50 μg mL⁻¹ gentamicin, and 4.0 mg mL⁻¹ BSA) for 19 to 22 h in 6% CO₂ in air (38.7°C). Cumulus cells were removed and embryos cultured (8–11 embryos/50 μL drop; 6% CO₂, 5% O₂, 89% N₂, 38.7°C) in media containing 100.0 or 120.0 mM NaCl, 4.0 or 8.0 mM KCl, 0.25 or 1.0 mM KH₂PO₄, and 1.0 mM : 2.0 mM or 2.0 mM : 1.0 mM CaCl₂ : MgSO₄ (2 × 2 × 2 × 2 factorial design). The remaining components of the culture medium were identical to the IVF medium (but w/o gentamicin). Development to the blastocyst stage by Day 6, metabolism (glycolysis and pyruvate) of each blastocyst, and final cell number (Hoechst 33342 staining) of all embryos were evaluated. Final cell number of cleaved embryos and development to the blastocyst stage were analyzed using analysis of variance in the GLIMMIX macro of SAS. A total of 236 oocytes were inseminated, yielding 128 cleaved embryos (54%), including 6 blastocysts (4.7% of cleaved embryos). Cell number was not (P > 0.05) affected by NaCl, KCl, or KH₂PO₄ concentrations, but tended (P = 0.057) to be higher after culture in 2.0 mM : 1.0 mM CaCl₂ : MgSO₄. Treatments did not significantly affect (P > 0.05) development to the blastocyst stage, but numerically more blastocysts were produced in 100.0 mM NaCl (4/6), 8.0 mM KCl (5/6), or 1.0 mM KH₂PO₄ (5/6). Both CaCl₂ : MgSO₄ ratios resulted in 3 blastocysts. Blastocysts contained 61.08 ± 5.1 (mean ± SEM, n = 6) cells and actively metabolized glucose (glycolysis, 3.7 ± 0.8 pmol/embryo/3 h or 0.06 ± 0.01 pmol/cell/3 h) and pyruvate (0.75 ± 0.27 pmol/embryo/3 h or 0.013 ± 0.005 pmol/cell/3 h). These results suggest that the ionic composition of culture media influences the in vitro development of cat IVF embryos. (Supported by NIH grant RR15388.)