Submission Number: MON-4-18-23

Abstract Number: 532

COMPARISON OF GARDNER'S G1/G2 SEQUENTIAL MEDIA AND BUFFALO RAT LIVER (BRL) CELL CO-CULTURE FOR BOVINE IN-VITRO EMBRYO PRODUCTION.

Monica L Hall-Woods ¹, Rebecca L Krisher ³, Michelle Lane ⁴, David K Gardner ⁴ and Cheryl S Asa ²

Saint Louis University, St. Louis, MO, USA ¹
Saint Louis Zoo, St. Louis, MO, USA ²
Purdue University, West Lafayette, IN, USA ³
Colorado Center for Reproductive Medicine, Englewood, CO, USA ⁴

Abstract:
Much debate has occurred over the use of somatic cell co-culture versus defined media for domestic cattle in-vitro embryo culture. Detailed studies on the metabolic and physiological requirements of in-vitro derived embryos have allowed the development of superior cell-free media that reflect the nutrient and environmental changes necessary for embryonic development. This study was designed to compare in-vitro produced domestic cattle embryos using Gardner's G1/G2 sequential semi-defined media and buffalo rat liver (BRL) cell co-culture. Oocytes (n = 278) collected from abattoir ovaries were cultured at 38.5C in 5% CO₂ in air for 24 hours in 250 l of maturation medium (TCM 199 supplemented with 10% FCS, 0.01 U/ml each bFSH and bLH, 27.5 g/ml pyruvate and 50 ng/ml epidermal growth factor). Frozen-thawed semen was separated using a swim-up procedure. Oocytes were inseminated in 425 l of fertilization medium (Tyrode's albumin, lactate and pyruvate (TALP) medium supplemented with 2 g/ml heparin, 20 M penicillamine, 10 M hypotaurine, 1 M epinephrine and 1 10⁶ sperm/ml) and cultured at 38.5C in 5% CO₂ in air for 18 hours. Presumptive zygotes were cultured either for 168 hrs on monolayers of BRL cells at 38.5C in 5% CO₂ in air or for 72 hours in G1 medium followed by 96 hours in G2 medium at 38.5C in 5% CO₂, 10% O₂, 85% N₂. Both culture treatments supported similar cleavage (75.2%, G1/G2; 75.2%, BRL) and percent blastocyst development of cleaved embryos (46.1%, G1/G2; 36.9%, BRL) on day 8 post-insemination. However, G1/G2 produced significantly more hatched blastocysts (15.7%, G1/G2; 1.3%, BRL; P = 0.01). Also, G1/G2 supported morulae and blastocysts with significantly higher (P < 0.05) mean cell numbers than BRL cells, with mean blastocyst cell numbers of 137.7 8.6 and 108.8 8.7 for G1/G2 and BRL cells, respectively. These results indicate that more higher quality domestic cattle embryos can be produced using sequential semi-defined media rather than BRL cell co-culture. .

Keywords: embryo culture

This abstract is being presented at: 8:00 AM in session:
IVF /ART II