The effect of co-culture on the development of in vitro matured equine oocytes after intracytoplastic sperm injection.
It is clear that, in the horse, there are many weak links in the process of in vitro embryo production; an optimal culture system for equine oocytes does not exist, and related data are conflicting. Therefore, the ability of 3 different culture systems to support embryonic development of ICSI horse oocytes was examined. Oocytes (n = 261) suitable for culture were collected from 55 ovaries and divided, according to cumulus morphology, into 2 categories: expanded cumulus and compacted cumulus. Oocytes with expanded and compacted cumulus were cultured for in vitro maturation in TCM 199 + 10% FCS + 0.1 iu/ml FSH/LH at 38.5 degrees C under 5% CO2 in air for 24 and 40 h, respectively. Oocytes (n = 149) reached metaphase II and were subjected to ICSI with frozen semen and then incubated in 3 different culture systems: A) TCM 199 + 10% FCS alone or B) on granulosa cell monolayer, C) SOF + MEM amino acids + 0.8% BSA. Cultural conditions were 39 degrees C and 5% CO2 in air for A and B, while a gas mixture (5% CO2, 5% O2, 90% N2) was used for C. The fertilisation rate was 32%. The cleavage rate in Group A was 74.4% (32/43); 18 embryos reached 2-6 cell stage, eight 8-16 cell, four 16-32 cell and two >32 cell. In Group B, the cleavage rate was 73.5% (36/49) with better results in embryonic development; 14 reached 2-6 cell stage, eighteen 8-16 cell, twelve 16-32 cell and five >32 cell. In Group C, the cleavage rate was significantly lower then in A and B; only 15 of 47 ICSI oocytes (39.1%) cleaved with maximum development to 2-6 cell stage. The remaining oocytes (68.1%) degenerated during culture. In conclusion, IVM horse oocytes can be fertilised in vitro with high efficiency with ICSI and co-culture systems showed to be superior in supporting in vitro embryo culture compared to simple ones. The identification of the factors beneficial to in vitro embryo development provided by the somatic cells could be important to optimise the embryo culture systems for equine embryos.
Rosati
I
I
Department of Animal Biology, University of Sassari, Italy.
Berlinguer
F
F
Bogliolo
L
L
Leoni
G
G
Ledda
S
S
Naitana
S
S
D000818:Animals; D002970:Cleavage Stage, Ovum; D018920:Coculture Techniques; D003470:Culture Media; D004622:Embryo, Mammalian / Q000502:physiology; D005314:Embryonic and Fetal Development; D005187:Fallopian Tubes / Q000166:cytology; D005260:Female; D005307:Fertilization in Vitro / Q000662:veterinary; D006107:Granulosa Cells / Q000502:physiology*; D006736:Horses / Q000196:embryology / Q000502:physiology*; D008297:Male; D008677:Metaphase; D009865:Oocytes / Q000502:physiology*; D011247:Pregnancy; D020554:Sperm Injections, Intracytoplasmic / Q000379:methods / Q000662:veterinary*
D003470:Culture Media