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Intracytoplasmic sperm injection (ICSI) for the treatment of canine infertility
366
INTRACYTOPLASMIC
SPERM INJECTION (RX) FOR THE TREATMENT OF CANINEINFERTILITY
R. M. Fultont, L. Keskintepe2, B. S. Durrants, and R. A. Fayrer-Hoskenl iDepartment of Large Animal Medicine and Department of Physiology and Phattnacology, College of Veterinary Medicine, University of Georgia, Athens, Georgia 30662 USA *Augusta Reproductive Biology Associates, 812 Chafee Avenue, Augusta, Georgia 36964 USA WXES, Zoological Society of San Diego, P.O. Box 551, San Diego, California 92112-0551 USA Increasing numbers of valuable purebred dogs are being presented with reproductive problems that may only be resolved by assisted reproductive technology. Veterinarians and dog breeders are seeking services similar to those offered at human infertility laboratories, such as intracytoplasmic sperm injection (ICSI). The aim of this research was to develop ICSI for the dog. The ultimate goal is to combine ICSI with in vitro oocyte maturation, and embryo transfer for the treatment of infertility in companion animals. Furthermore, there will be applications for conservation of endangered carnivores. Oocytes were harvested by collagenase/DNase enzymatic digestion of fresh ovaries collected from spayed bitches. The oocytes were cultured in TCM-199 with 100 U/ml penicillin, 0.1 mg/ml streptomycin, 15 @ml FSH, 5 ng/ml LH and 10% fetal calf serum under oil for 48 hours at 38’C and 5% CO2 in air. Maturation was determined by polar body formation. Oocytes were denuded of cumulus cells by repeated passage through a small bore glass pipet before transport in a 38’C water bath to the injection facility. Canine semen was collected by digital manipulation, washed in TCM- 199 by centrifugation, resuspended in an egg yolk extender, cooled in an Equitainer to mimic clinical semen cooling, and held at 5’C, overnight, prior to injection. On the day of injection, the semen was prepared by centrifugation for 20 minutes through a 4590% Percoll gradient. The sperm cells were harvested from the layer between the two concentrations of Percoll. Selected spermatozoa were placed in a 10 pl droplet of 10% polyvinyl pyrrolidone (PVP). Individual sperm cells were immobilized by breaking the tail with a strike of the tip of the injection pipet. A single spermatozoon was aspirated tail first into the pipet which was then transferred to a second droplet of medium containing one or two matured ova. Each ovum was secured with the polar body at the 12 o’clock position by gentle suction of a holding pipet. Excess fluid was removed from the injection pipet immediately before penetration of the zona at the 3 o’clock position. A small amount of ooplasm was aspirated before injection of the spermatozoon and removal of the pipet. For control oocytes, the injection pipet contained media but no spermatozoon. Oocytes were placed back into TCM-199 and incubated at 38’C and 5% CO2 in air for 12 hours. Oocytes were fxed in acetic acid:ethanol, stained with 2% acetic acid:orcein and examined by light microscopy for evidence of male and female pronuclei. Decondensed sperm chromatin with the female pronucleus was observed in 42.1% (16/38) of the injected oocytes, and 7.8% (3/38) of ova produced both male and female pronuclei. Sperm nuclear material was not decondensed in the remainder of the spermatozoon injected oocytes. No change was evident in the control oocytes. Based on the success of the procedure in other species (human, mouse, goat, cow, sheep, and pig), higher fertilization rates in the dog may be expected in future experiments. The technique will provide zygotes for cleavage experiments and embryos for culture to the blastocyst stage. These resultant embryos will be transferred to synchronized recipients as a treatment for canine infertility.
INTRACYTOPLASMIC
SPERM INJECTION (RX) FOR THE TREATMENT OF CANINEINFERTILITY
R. M. Fultont, L. Keskintepe2, B. S. Durrants, and R. A. Fayrer-Hoskenl iDepartment of Large Animal Medicine and Department of Physiology and Phattnacology, College of Veterinary Medicine, University of Georgia, Athens, Georgia 30662 USA *Augusta Reproductive Biology Associates, 812 Chafee Avenue, Augusta, Georgia 36964 USA WXES, Zoological Society of San Diego, P.O. Box 551, San Diego, California 92112-0551 USA Increasing numbers of valuable purebred dogs are being presented with reproductive problems that may only be resolved by assisted reproductive technology. Veterinarians and dog breeders are seeking services similar to those offered at human infertility laboratories, such as intracytoplasmic sperm injection (ICSI). The aim of this research was to develop ICSI for the dog. The ultimate goal is to combine ICSI with in vitro oocyte maturation, and embryo transfer for the treatment of infertility in companion animals. Furthermore, there will be applications for conservation of endangered carnivores. Oocytes were harvested by collagenase/DNase enzymatic digestion of fresh ovaries collected from spayed bitches. The oocytes were cultured in TCM-199 with 100 U/ml penicillin, 0.1 mg/ml streptomycin, 15 @ml FSH, 5 ng/ml LH and 10% fetal calf serum under oil for 48 hours at 38’C and 5% CO2 in air. Maturation was determined by polar body formation. Oocytes were denuded of cumulus cells by repeated passage through a small bore glass pipet before transport in a 38’C water bath to the injection facility. Canine semen was collected by digital manipulation, washed in TCM- 199 by centrifugation, resuspended in an egg yolk extender, cooled in an Equitainer to mimic clinical semen cooling, and held at 5’C, overnight, prior to injection. On the day of injection, the semen was prepared by centrifugation for 20 minutes through a 4590% Percoll gradient. The sperm cells were harvested from the layer between the two concentrations of Percoll. Selected spermatozoa were placed in a 10 pl droplet of 10% polyvinyl pyrrolidone (PVP). Individual sperm cells were immobilized by breaking the tail with a strike of the tip of the injection pipet. A single spermatozoon was aspirated tail first into the pipet which was then transferred to a second droplet of medium containing one or two matured ova. Each ovum was secured with the polar body at the 12 o’clock position by gentle suction of a holding pipet. Excess fluid was removed from the injection pipet immediately before penetration of the zona at the 3 o’clock position. A small amount of ooplasm was aspirated before injection of the spermatozoon and removal of the pipet. For control oocytes, the injection pipet contained media but no spermatozoon. Oocytes were placed back into TCM-199 and incubated at 38’C and 5% CO2 in air for 12 hours. Oocytes were fxed in acetic acid:ethanol, stained with 2% acetic acid:orcein and examined by light microscopy for evidence of male and female pronuclei. Decondensed sperm chromatin with the female pronucleus was observed in 42.1% (16/38) of the injected oocytes, and 7.8% (3/38) of ova produced both male and female pronuclei. Sperm nuclear material was not decondensed in the remainder of the spermatozoon injected oocytes. No change was evident in the control oocytes. Based on the success of the procedure in other species (human, mouse, goat, cow, sheep, and pig), higher fertilization rates in the dog may be expected in future experiments. The technique will provide zygotes for cleavage experiments and embryos for culture to the blastocyst stage. These resultant embryos will be transferred to synchronized recipients as a treatment for canine infertility.