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Cloning dogs and cats: Where do we stand? (Proceedings)

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Aug 01, 2009

Assisted reproductive technologies in small animals are not a 21st century invention; the first artificial insemination in dogs was performed in 1780 and the first description of an oocyte at the microscopic level was that of a canine oocyte, in 1827. However, economic realities have prevented rapid development of reproductive technologies in small animals compared to domestic large animal species. What are the advanced reproductive technologies currently used or proposed in small animals and how likely are they to be commercially available in the near future?

Reproductive physiology and early embryogenesis

The ovaries contain thousands of follicles, each of which contains an egg or ovum. As each estrous cycle begins, a cohort of follicles is selected to begin development. Development is promoted by release of hormones from the hypothalamus (gonadotropin releasing hormone [GnRH]) and pituitary (follicle stimulating hormone [FSH] and luteinizing hormone [LH]). As the follicle develops, it secretes estrogen, which causes the physical and behavioral signs of early heat, or proestrus. In the bitch, estrogen concentrations fall about 9 days after the onset of proestrus; at this time, the bitch will stand to be bred (standing heat or estrus) and a surge of LH is released, causing ovulation. Immature eggs are released from the follicles into the uterine tube, where they undergo two more cell divisions before fertilization can occur. In the queen, estrous behavior occurs when circulating estrogen concentrations are high and copulation stimulates release of GnRH and subsequent ovulation of mature oocytes into the uterine tube.

The egg released into the oviduct is surrounded by the zona pellucida and by a layer of granulosa cells from the follicle, the cumulus oophorus. Spermatozoa introduced into the reproductive tract of the bitch undergo capacitation, a calcium-dependent process involving the acrosome reaction on the head of the spermatozoon and achievement of hypermotility. Capacitated spermatozoa digest the layer of cells surrounding the egg and invade the zona pellucida. As soon as one spermatozoon binds to the inner layer of the zona pellucida, entry of other spermatozoa is blocked by an electrochemical reaction so only one spermatozoon fertilizes each egg. Cell division begins immediately.

Repeated doubling of cells occurs (2 cells – 4 cells – 8 cells – 16 cells) with concomitant changes in cell size and placement. The 16-cell stage is called a morula. The 16 to 64 cell stage is called a blastocyst. The blastocyst is a hollow sphere lined with blastomeres (embryonic cells) and filled with fluid. The blastocyst is divided into the inner cell mass, a group of blastomeres at one pole of the blastocyst will go on to form the embryo itself and two of the fetal membranes (yolk sac and allantois), and the trophoblasts, cells lining the outer surface of the blastocyst that go on to form the other two fetal membranes (chorion and amnion).

Points of technological intervention

No one has demonstrated ability to complete development in an artificial environment – at some point, the embryo must be placed in the uterus of a surrogate dam. The transfer of the embryo usually is accomplished with surgical placement of multiple morulas or blastocysts into the uterus of a recipient dam synchronized to be at the same point in the estrous cycle as the donor dam. It has been demonstrated that the recipient need not be the same species as the embryo but to date, closely related species have been used. This is of value in maintaining threatened or endangered canid and felid species, using domestic bitches and queens as surrogate dams.

Where does the embryo come from? Traditionally, donor dams were allowed to cycle and were bred, and embryos were retrieved from their uterine tubes or uterus surgically. These embryos were then transferred to a recipient dam. This simple form of embryo transfer has been reported successful in dogs and cats since the last 1970s. However, this simple form of embryo transfer does not gain us much in small animals. In large animal species, the donor dam can be superovulated, in which hormone therapy is used to cause release of a larger number of oocytes than normal during one estrous cycle and those many fertilized embryos are transferred into multiple recipient dams. Superovulation has not been demonstrated to work well in bitches and queens, who already release a relatively large number of oocytes during each estrus.

Instead, what people would like to be able to do is to retrieve multiple oocytes from a donor female, fertilize (in vitro fertilization = IVF) and mature them (in vitro maturation = IVM) outside of the donor dam and then implant the embryos produced into multiple recipients. This can be done by surgically retrieving oocytes from ovaries of donor animals and, in cats, by harvesting oocytes from ovaries excised from animals in the event of an untimely death. The oocyte is exposed to capacitated spermatozoa for fertilization. An even more advanced technique, intracytoplasmic sperm injection (ICSI) involves placement of a single capacitated spermatozoon into the oocyte. Maturation through early cleavage is easy in cats but very difficult in dogs. Unique features of the reproductive physiology of the bitch that impair IVM include ovulation of an immature oocyte, ovulation into a low-estrogen / high-progesterone environment, and necessity of continued presence of cumulus cells for ongoing maturation to occur.

Embryos also can be produced by nuclear transfer, commonly termed cloning. In this technique, the nucleus of a single cell from the donor is transferred into an oocyte that has had its DNA removed, that new oocyte fertilized and matured in vitro, and the subsequent embryo transferred into a recipient dam at the morula or blastocyst stage. The beauty of this technique is that any cell of the donor can be used; all reports of cloning in dogs and cats to date used fibroblasts retrieved from skin biopsies from the ear.

The final point to consider is the speed at which this must occur. In human and large animal medicine, embryos can be frozen at various points in development and later thawed and successfully implanted into a recipient dam. This permits creation of many embryos from a given donor and birth of her offspring over an extended period of time.