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.

[1]  G. Seidel,et al.  Cryopreservation procedures for Day 7-8 equine embryos. , 2010, Equine veterinary journal. Supplement.

[2]  W. Allen,et al.  Influence of co-culture during maturation on the developmental potential of equine oocytes fertilized by intracytoplasmic sperm injection (ICSI). , 2001, Reproduction.

[3]  W. Allen,et al.  Effects of different activation treatments on fertilization of horse oocytes by intracytoplasmic sperm injection. , 2000, Journal of reproduction and fertility.

[4]  K. Hamano,et al.  Gender preselection in cattle with intracytoplasmically injected, flow cytometrically sorted sperm heads. , 1999, Biology of reproduction.

[5]  J. Mizuno,et al.  Intracellular calcium responses in bovine oocytes induced by spermatozoa and by reagents. , 1998, Theriogenology.

[6]  G. Evans,et al.  Cleavage, development and competence of sheep embryos fertilized by intracytoplasmic sperm injection and in vitro fertilization. , 1998, Theriogenology.

[7]  A. Hossaini,et al.  Intracytoplasmic sperm injection of in vitro-matured equine oocytes. , 1997, Biology of reproduction.

[8]  G. Lacalandra,et al.  Intracytoplasmic sperm injection (ICSI) versus conventional IVF on abattoir-derived and in vitro-matured equine oocytes. , 1997, Theriogenology.

[9]  G. Lacalandra,et al.  In vitro maturation and fertilization of equine oocytes recovered during the breeding season. , 1996, Theriogenology.

[10]  K. Betteridge.  Phylogeny, ontogeny and embryo transfer , 1995 .

[11]  M. Grøndahl,et al.  Structural and endocrine aspects of equine oocyte maturation in vivo , 1995, Molecular reproduction and development.

[12]  Y. Okada,et al.  In vitro fertilization rate of horse oocytes with partially removed zonae. , 1994, Theriogenology.

[13]  G. Seidel,et al.  Methods for collecting and maturing equine oocytes in vitro , 1993 .

[14]  J. Robl,et al.  Sperm, inositol trisphosphate, and thimerosal-induced intracellular Ca2+ elevations in rabbit eggs. , 1993, Developmental biology.

[15]  Zsolt Peter Nagy,et al.  High fertilization and implantation rates after intracytoplasmic sperm injection , 1993 .

[16]  B. Ball,et al.  Development to blastocysts of one- to two-cell equine embryos after coculture with uterine tubal epithelial cells. , 1993, American journal of veterinary research.

[17]  K. Hinrichs,et al.  In vitro maturation of horse oocytes: characterization of chromatin configuration using fluorescence microscopy. , 1993, Biology of reproduction.

[18]  P. Devroey,et al.  Pregnancies after intracytoplasmic injection of single spermatozoon into an oocyte , 1992, The Lancet.

[19]  B. Ball,et al.  Survival of equine embryos co-cultured with equine oviductal epithelium from the four- to eight-cell to the blastocyst stage after transfer to synchronous recipient mares. , 1992, Theriogenology.

[20]  J. Zhang,et al.  In vitro fertilization of horse follicular oocytes matured in vitro , 1990, Molecular reproduction and development.

[21]  C. Brown,et al.  Characterization of proteins secreted by sheep oviduct epithelial cells and their function in embryonic development. , 1989, Development.

[22]  A. Enders,et al.  The ovulated ovum of the horse: cytology of nonfertilized ova to pronuclear stage ova. , 1987, Biology of reproduction.

[23]  O. Knudsen.  Percutaneous tendon splitting--method and results. , 1976, Equine veterinary journal.

[24]  T. A. Venkitasubramanian,et al.  Ovarian lipids in guinea-pigs. , 1973, Journal of reproduction and fertility.

[25]  H. R. Tervit,et al.  Successful culture in vitro of sheep and cattle ova. , 1972, Journal of reproduction and fertility.

[26]  R. Godke,et al.  Production of live foals from sperm-injected oocytes harvested from pregnant mares. , 2000, Journal of reproduction and fertility. Supplement.

[27]  A. Trounson,et al.  Pregnancies produced from fertile and infertile stallions by intracytoplasmic sperm injection (ICSI) of single frozen-thawed spermatozoa into in vivo matured mare oocytes. , 2000, Journal of reproduction and fertility. Supplement.

[28]  K. Hinrichs.  Production of embryos by assisted reproduction in the horse. , 1998, Theriogenology.

[29]  E. Squires,et al.  A pregnancy after intracytoplasmic sperm injection into equine oocytes matured in vitro , 1996 .

[30]  S. Hochi,et al.  Effect of glucose in the culture medium on development of horse oocytes matured and microfertilized in vitro. , 1995, Reproduction, fertility, and development.

[31]  S. Iwasaki,et al.  Investigation of Various Conditions in Microfertilization of Bovine Oocytes and Subsequent Changes in Nuclei and Development to Embryos , 1993 .

[32]  J. Parrish,et al.  In vitro maturation of equine oocytes , 1992 .

[33]  E. Palmer,et al.  Establishment of equine oviduct cell monolayers for co-culture with early equine embryos. , 1991, Journal of reproduction and fertility. Supplement.

[34]  F. Longo.  Morphogenesis of the Mammalian Egg Cortex , 1991 .

[35]  B. Ball,et al.  In vitro development of 4- to 8-cell equine embryos cocultured with trophoblastic vesicles or oviductal explants , 1990 .

[36]  J. Parrish,et al.  In vitro fertilization of in vitro-matured equine oocytes. , 1990 .

[37]  D. A. Freeman,et al.  Coculture of day-2 equine embryos with oviductal or uterine tissue , 1990 .

[38]  B. Bavister.  Role of oviductal secretions in embryonic growth in vivo and in vitro , 1988 .

[39]  M. Tischner,et al.  Evaluation of mare oocyte collection methods and stallion sperm penetration of zona-free hamster ova. , 1987, Journal of reproduction and fertility. Supplement.

[40]  J. Fulka,et al.  Culture of horse oocytes in vitro. , 1981, Journal of reproduction and fertility.