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

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), KH(2)PO(4) (0.25 vs. 1.0 mM), and the ratio of CaCl(2) to MgSO(4)-7H(2)O (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 KH(2)PO(4), 2.0 mM CaCl(2), 1.0 mM MgSO(4), 1.5 mM glucose, 6.0 mM L-lactate, 0.1 mM pyruvate, and 0x EAAs with 25.0 mM NaHCO(3), 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.

Reversible meiotic arrest in feline oocytes

“…Cumulus-oocyte complexes containing multiple, compact layers of cumulus cells and an oocyte with a uniformly dark cytoplasm were washed twice in FOCMH before culturing in maturation medium. The medium used for maturation was FOCM (Herrick et al 2007; Table 1) containing 6.0 mM glucose and supplemented with 0.5Â MEM essential amino acids, 1.0Â MEM vitamins, 0.6 mM cysteine, 0.1 mM cysteamine, 10 mg mL À1 insulin, 5.5 mg mL À1 transferrin and 5.0 ng mL À1 selenium (Herrick et al 2010). Cumulus-oocyte complexes were cultured in 6% CO 2 in air for 6 to 36 h depending on the experiment.…”

“…Denuded zygotes were washed and placed into FOCM for culture (6% CO 2 , 5% O 2 and 89% N 2 , 48 h). On Day 3 of culture the proportion of embryos that had cleaved was evaluated and all cleaved embryos were moved to fresh FOCM with 5% (v/v) fetal calf serum instead of BSA and cultured (6% CO 2 , 5% O2 and 89% N2) for 96 h. On Day 7 of culture (,168 h after IVF), the proportion of embryos that had developed to the blastocyst stage was determined and all blastocysts were fixed for determination of total cell number (Pursel et al 1985;Herrick et al 2007). Only embryos that were classified as a blastocyst based on morphology and contained at least 50 cells were considered to be blastocysts for calculation of developmental parameters.…”

Sex determining of cat embryo and some feline species

“…(Spindler et al, 2006;Herrick, 2007). Each ovary was sliced repeatedly with a scalpel blade to release cumulus-oocyte complexes (COCs) into a 90-mm culture dish containing tissue culture medium (TCM-199) with 25 mM HEPES supplemented with 4 mg/ml bovine serum albumin (BSA).…”

“…• C. Next, the uterus was flushed with SOF and the recovered embryos were evaluated and frozen (Kajta, 1998;Herrick et al, 2007).…”

Foreword: A perspective on the role of emerging technologies for the propagation of companion animals, non-domestic and endangered species

“…A better understanding of and insight into regulatory processes is essential when using ART to create optimal conditions during several critical time points in development. In this respect, the careful stepwise development of a feline embryo culture medium in domestic cats, resulting in improved embryo development and viability, was successful when applied to in vitro embryo culture of non-domestic small cat species (Herrick et al 2006a(Herrick et al , 2006b(Herrick et al , 2007.…”