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Toward a Feline-Optimized Culture Medium: Impact of Ions, Carbohydrates, Essential Amino Acids, Vitamins, and Serum on Development and Metabolism of In Vitro Fertilization-Derived Feline Embryos Relative to Embryos Grown In Vivo¹

Center for Conservation and Research of Endangered Wildlife,³ Cincinnati Zoo and Botanical Garden, Cincinnati, Ohio 45220 Department of Animal Sciences,⁴ Purdue University, West Lafayette, Indiana 47907 Department of Biological Sciences,⁵ University of Cincinnati, Cincinnati, Ohio 45221

ABSTRACT

The objective of this study was to define the physiologic needs of domestic cat embryos to facilitate development of a feline-specific culture medium. In a series of factorial experiments, in vivo-matured oocytes (n = 2040) from gonadotropin-treated domestic cats were inseminated in vitro to generate embryos (n = 1464) for culture. In the initial study, concentrations of NaCl (100.0 vs. 120.0 mM), KCl (4.0 vs. 8.0 mM), KH₂PO₄ (0.25 vs. 1.0 mM), and the ratio of CaCl₂ to MgSO₄-7H₂O (1.0:2.0 mM vs. 2.0:1.0 mM) in the medium were evaluated during Days 1–6 (Day 0: oocyte recovery and in vitro fertilization [IVF]) of culture. Subsequent experiments assessed the effects of varying concentrations of carbohydrate (glucose, 1.5, 3.0, or 6.0 mM; L-lactate, 3.0, 6.0, or 12.0 mM; and pyruvate, 0.1 or 1.0 mM) and essential amino acids (EAAs; 0, 0.5, or 1.0x) in the medium during Days 1–3 and Days 3–6 of culture. Inclusion of vitamins (0 vs. 1.0x) and fetal calf serum (FCS; 0 vs. 5% [v/v]) in the medium also was evaluated during Days 3–6. Development and metabolism of IVF embryos on Day 3 or Day 6 were compared to age-matched in vivo embryos recovered from naturally mated queens. A feline-optimized culture medium (FOCM) was formulated based on these results (100.0 mM NaCl, 8.0 mM KCl, 1.0 mM KH₂PO₄, 2.0 mM CaCl₂, 1.0 mM MgSO₄, 1.5 mM glucose, 6.0 mM L-lactate, 0.1 mM pyruvate, and 0x EAAs with 25.0 mM NaHCO₃, 1.0 mM alanyl-glutamine, 0.1 mM taurine, and 1.0x nonessential amino acids) with 0.4% (w/v) BSA from Days 0–3 and 5% FCS from Days 3–6. Using this medium, ~70% of cleaved embryos developed into blastocysts with profiles of carbohydrate metabolism similar to in vivo embryos. Our results suggest that feline embryos have stage-specific responses to carbohydrates and are sensitive to EAAs but are still reliant on one or more unidentified components of FCS for optimal blastocyst development.

assisted reproductive technology, early development, embryo, in vitro fertilization

¹Supported by 5R24RR15388-5 from the National Institute of Health to W.F.S.

Correspondence: ²Jason R. Herrick, C.R.E.W./Cincinnati Zoo and Botanical Garden, 3400 Vine St., Cincinnati, OH 45220. FAX: 513 569 8213; e-mail: jason.herrick{at}cincinnatizoo.org