Female dogs only ovulate once or twice a year, and after the ovum are released, require 48 to 72 hours in the oviduct to mature. At this point, they can be harvested for use in producing embryos outside of the donor dog’s reproductive system.
Ovulation is determined by collecting blood or observing signs of heat such as discharge from the female dog’s vulva. Day five to six from release, the egg, or oocyte, is mature and ready for fertilization. Ovum may be harvested by performing a spay on the female donor dog at this point and dissecting the oviduct to obtain mature oocytes. Oocytes are stored in a medium with mineral oil in preparation for fertilization with donor sperm.
Sperm collected from donor male dogs is kept in a medium where magnesium levels are appropriately maintained at different stages so as to promote development of sperm, making them viable for fertilization of eggs. Sperm is tested to ensure viability before using for IVF.
Sperm is introduced to the oocytes and storage media with a pipette, and left for approximately 14 hours to allow for fertilization of the oocytes. At this point the zygotes are washed with fresh medium to remove excess sperm and cells and the zygotes returned to the culture. Embryos are then observed 48 hours post-fertilization and may then be frozen for future use or continued in fresh medium and observed for development before use.
Cryopreserved, frozen, embryos are then thawed and placed in an appropriate medium for preservation prior to use. Host females are observed for estrus and when naturally occurring estrus is present, embryos are transferred on the appropriate synchronized day. For example, an 8 or 10 day old fertilized egg, embryo, would be implanted 8 to 10 days after estrus in the host female dog’s uterine horn. To affect transfer, laparotomy is performed to expose the uterine horn or oviducts and an incision is made into the cranial area of the uterine horn or oviduct where embryos can be transferred with a pipette.