Current state in biotechnology in canine and feline reproduction

Animal Reproduction Science 60–61 Ž2000. 375–387 www.elsevier.comrlocateranireprosci

Current state in biotechnology in canine and feline reproduction W. Farstad ) Department of Reproduction and Forensic Medicine, Norwegian School of Veterinary Science, P.O. Box 8146 Dep., N-0033 Oslo, Norway

Abstract Biotechnology has proceeded much further in cats than in canines, although the pregnancy rate after in vitro maturation ŽIVM., IVC and embryo transfer ŽET. is still relatively low. The use of AI with frozen–thawed semen as a breeding tool to overcome breeding incompatibility or to preserve male genetic material has been limited in felines in contrast to the situation in domestic dogs and foxes. In many research scenarios and endangered felid species programs, the in vitro production of feline embryos with subsequent transfer has complemented the use of AI. Improvement of IVM, in vitro fertilization ŽIVF. and embryo culture coupled with ovarian tissue grafting, cryobanking of follicles, oocytes, semen, or embryos, with subsequent ET into surrogate females, may render this technology feasible for use in endangered wild felids. In canines, reliable systems for in vitro production of embryos, embryo cryopreservation and transfer are yet to be developed. The refinement of invasive fertilization techniques, such as intracytoplasmic sperm injection ŽICSI., may eventually provide a tool for removal of recipient oocyte nuclei and transfer of selected embryonic or somatic cell donor nuclei into domestic cat ooplasm, thereby providing a tool for genetic modification, or for preservation of valuable genetic material. q 2000 Elsevier Science B.V. All rights reserved. Keywords: Canine; Feline; Artificial insemination; In vitro fertilization; Embryo transfer

1. Introduction Domestic cats Ž Felis catus . and dogs Ž Canis familiaris. primarily serve as companion animals, and breeding has not been subject to planning on a large-scale basis as in )

Tel.: q47-22964855; fax: q47-22597081. E-mail address: [email protected] ŽW. Farstad..

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other domestic animals. Most breeding is within small kennels with a small number of breeding animals, with the exception of large colonies of cats and dogs bred exclusively for biomedical research. In dogs, the export and import of semen and live breeding animals have shown a steady increase, and semen banks have been established both in research institutions and by private companies ŽFarstad, 1996.. In the cat, however, exchange of breeding animals occurs within laboratory animal research institutions, but commercial initiatives relating to trade with frozen semen are scarce. The semi-domestic fox breeds, the red fox Ž Vulpes Õulpes . and the blue fox Ž Alopex lagopus . have been farmed for pelts mainly in Northern America, the Nordic countries, Russia, the Baltic states and Poland since the early 1900s ŽNes et al., 1987., and in this context planned breeding has been carried out to a larger extent than in dogs and cats. Also, international trade with live breeding animals was extensive during the 1960s and 1970s, and during the last 5 years, frozen silver fox semen has been exported from Norway to Canada with the birth of live pups ŽFougner, 1999, personal communication.. There has been relatively limited interest in conserving the wild members of the canid family by means of assisted reproduction — in many cases because most of them reproduce well both in the wild and in captivity, but also because progress in reproductive biotechnology has encountered major problems particularly concerning in vitro models for female gametes and embryos. At present, both the gray wolf, red wolf Ž C. rufus ., Mexican wolf Ž C. lupus baileyi ., Egyptian wolf Ž Lycaon pictus ., Ethiopian wolf Ž C. simensis ., South American Savannah dog Ž Speothos Õenaticus ., maned wolf Ž Chrysaocon brachyarus. and two fox species: the San Joaquin kit Ž V. macrotis . and the Northern swift fox Ž V. Õelox hebes ., are considered to be threatened by extinction ŽGottelli et al., 1994; Goodrowe et al., 1998; IUCN 1996; CITES, 1997, 1998.. In Scandinavia, the wild polar fox Ž A. lagopus . is considered vulnerable. Thus, with limited space in the wild and in zoological institutions, the need for strategies involving multidisciplinary action for enhancing conservation of these species is increasing rapidly. Most of the 36 wild species of felids are classified as threatened, vulnerable or endangered ŽNowell and Jackson, 1996., maybe with the exception of the Northern European lynx Ž Lynx lynx ., which is again hunted, although by restricted licences, in Norway and Sweden. In wild cats, research for biotechnology development is well underway with the establishment of conservation programs in which assisted reproduction plays an important part ŽWildt et al., 1992.. Research on maturation, fertilization and embryo development in vitro, as well as embryo transfer ŽET. and cryopreservation has increased rapidly during the last decade in domestic cats ŽGoodrowe et al., 1988, 1989, 1991; Johnston et al., 1989; Donoghue et al., 1992; Luvoni and Oliva, 1993; Pope et al., 1993, 1994, 1997, 1998; Schramm and Bavister, 1995; Wood and Wildt, 1997; Wood et al., 1995; Wolfe and Wildt, 1996; Luvoni et al., 1997; Howard, 1999. as well as in wild felids ŽDonoghue et al., 1993, 1996; Howard et al., 1992, 1997a; Swanson et al., 1996a; Pope et al., 1993; Jewgenow et al., 1997.. In most of these studies, the domestic cat has served as a convenient research model species for endangered felids. Further, the possibilities for using domestic cats as models for human disease and for studies of inherited genetic disorders stimulate the use of biotechniques in felines. Thus, the current state of reproductive biotechnology is somewhat different for canines and

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felines, and in the following, a review on recent progress will be given for each of these carnivore families.

2. Canines Canines are monoestrous. Seasonality is not obvious in most breeds of dogs, except for the Basenji, in which females experience estrus during the autumn in the northern hemisphere. The blue Ž A. lagopus . and red foxes Ž V. Õulpes . are seasonal with their breeding season during January–March. Ovulation occurs 1–2 days after the preovulatory LH peak at the beginning of estrus in both dogs and foxes. In bitches and vixens, preovulatory luteinization of follicles occurs, exposing oocytes to increasing concentrations of progesterone, as opposed to the situation in many other domestic mammals, where estrogen dominates the preovulatory follicular environment. In most mammals, ovulation of the oocyte occurs when the oocyte has reached the metaphase of the second meiotic division. In canines, the oocyte is ovulated at the beginning of the first meiotic division, and the germinal vesicle is broken down shortly after ovulation. Subsequent stages of oocyte maturation occur in the oviduct ŽHolst and Phemister, 1971; Farstad et al., 1989.. 2.1. Oocyte maturation, fertilization and embryonic deÕelopment in Õitro Canine oocytes may resume meiosis spontaneously in vitro using adaptations of bovine and porcine in vitro maturation ŽIVM. techniques, although at a much lower rate and efficiency than most other domestic animal oocytes. In canids, IVM has shown limited success with maturation rates varying from 0% to 58% for oocytes matured to Metaphase I, Anaphase I and Metaphase II ŽMIrAIrMII.; maturation is usually around 20% ŽMII., in a variety of different culture systems and media ŽMahi and Yanagimaci, 1976; Robertson et al., 1992; Nickson et al., 1993; Yamada et al., 1992, 1993; Bolamba et al., 1997; Hewitt and England 1997, 1998a, 1999a; Hewitt et al., 1998; Theiss, 1997; Metcalfe, 1999.. Most oocytes used for IVM experiments in the dog have been collected from random sources, usually from animals undergoing ovariohysterectomy. Hence, oocytes from prepubertal, anestrous, luteal phases of pregnant and non-pregnant animals, as well as proestrous and estrous females have been used with no apparent effect of the stage of estrous cycle ŽNickson et al., 1993; Hewitt and England, 1997; Theiss, 1997; Metcalfe, 1999., whereas the age of the donor animal ŽNickson et al., 1993; Hewitt and England, 1998a., oocyte size ŽTheiss, 1997; Hewitt and England, 1998a; Srsen et al., 1998. and nuclear and cumulus morphology ŽNickson et al., 1993. all influence IVM rates. In foxes, the IVM of ovarian oocytes, collected either from preovulatory or anestrus follicles, have resulted in maturation rates similar to those in the bitch Ži.e., 80% — 100 GVBD, 25% MII. ŽKrogenæs et al., 1993; Wen et al., 1994.. Recently, maturation rates to MII have been improved for blue fox oocytes collected from anestrous animals Ž40% MII. ŽSrsen et al., 1998.. Canine oocytes may undergo IVM in intact follicles dissected from the ovary ŽBolamba et al., 1998.. The production of antral follicles from small preantral follicles has been attempted using bitch ovarian tissue

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grafting to host ovaries of SCID mice. Graft establishment and some follicular recruitment occurred, even though the production of antral follicles was not obtained ŽMetcalfe, 1999.. A refinement of this technique may enable the use of ovaries from valuable animals for further production of oocytes posthumously. Canine embryos have been produced after fertilization in vitro of in vivo matured ŽRenton et al., 1991; Farstad et al., 1993a., and from in vitro matured oocytes, but in the latter development beyond the eight-cell stage has not been reported ŽYamada et al., 1993; Metcalfe, 1999.. An in vitro ‘‘block’’ to development has been described for many species in which in vitro fertilization ŽIVF. has been attempted Žfor a review on developmental ‘‘block’’ in vitro of mouse, hamster, sheep and cow embryos, see McGinnis and Youngs, 1992; Sparks et al., 1992; for cat: Swanson et al., 1996a.. The maternal embryonic transition constitutes a critical phase of embryo development. In foxes and dogs, structural studies and cultivation with 3 H-uridine, indicate that activation of the embryonic genome occurs at the six- to eight-cell stage in foxes and the eight-cell stage in dog embryos ŽFarstad et al., 1993b; Bysted and Greve, 2000.. The scarcity of reports in the literature of attempts to modify the culture conditions in vitro for IVM-derived embryos after the eight-cell stage may indicate that some difficulties have been encountered in propagating development past this stage, but too little information is available to conclude that such an in vitro block exists in dog oocytes. To date, no reports of production of live young after IVF from either in vivo- or in vitro matured dog or fox oocytes exist in the literature. 2.2. Sperm treatment, cryopreserÕation and assisted fertilization The in vitro capacitation of canine sperm may be achieved in canine capacitation medium ŽMahi and Yanangimaci, 1976. or in modified Tyrode’s solution ŽFarstad et al., 1993a,b; Hewitt and England, 1999b.. Calcium ionophore A23187 can promote capacitation and the acrosome reaction in a similar manner as Ca2q in vitro ŽSzasz et al., 1997; Hewitt and England, 1998c.. The reports on IVF rates in dogs or foxes are few, but cleavage rates of 5–20% and pronuclear formation in 20–37% of oocytes have been reported ŽFarstad et al., 1993a,b; Nickson et al., 1993; Yamada et al., 1993; Metcalfe, 1999.. Intracytoplasmic sperm injection ŽICSI. has been attempted with chilled dog sperm, with the formation of male pronuclei in 8% of oocytes, but no cleavage occurred ŽFulton et al., 1998.. The cryopreservation of dog and fox semen has been frequently reviewed, and a variety of freezing regimens, extenders and thawing protocols for dog and fox semen have been published ŽEngland, 1993; Farstad, 1996; Rota, 1998.. Recently, modifications of the commonly used TRIS egg yolk extender by the addition of Equex STM paste improved post-thaw survival of dog sperm during incubation at 388C and produced an overall pregnancy rate after vaginal or intrauterine ŽIU. AI of 84% ŽRota et al., 1997, 1999.. Differences between canine species with respect to cooling tolerance may exist, since freezing trials with blue foxes Ž A. lagopus . and red wolves Ž C. rufus . have resulted in a significant reduction in the percentage of intact acrosomes after freezing and thawing ŽFarstad et al., 1992; Goodrowe et al., 1998, 2000.. Dual fluorescent staining techniques, binding tests and oocyte penetration assays have been developed for

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dog sperm, which may also be usable for the assessment of sperm function in vitro for other canines ŽHay et al., 1997; Hewitt and England, 1998b,c; Mayenco-Aguirre and Peres-Cortez, 1998, Strom ¨ Holst, 1999.. Red wolf sperm bind to domestic dog oocytes ŽGoodrowe, 1999, personal communication.. 2.3. Artificial insemination and ET IU artificial insemination may be carried out non-surgically using either endoscopic visualization or transcervical catheterization, or surgically by laparoscopy Žfor review, see Farstad, 2000.. Results in foxes and dogs using IU non-surgical AI are good, and both birth rates and litter sizes approach those of natural mating Žsee reviews Farstad, 1996, 1998.. The ET of in vivo-derived embryos has been carried out surgically both in the silver fox and the dog resulting in live young, but with low success rates ŽKinney et al., 1979; Tsutsui et al., 1989; Jalkanen and Lindeberg, 1998.. Recently, ET has been carried out in the blue fox, using the IU catheter developed for artificial insemination in foxes ŽLindeberg, 1999, personal communication.. To date, the birth of live young from cryopreserved canine embryos has not been reported. However, in the blue fox, freezing of embryos recently resulted in the observation of implantation sites in naturally synchronized recipient females after ET ŽLindeberg, 1999, personal communication.. The refinement of freezing regimens, improvement of donor-recipient synchronization, in vitro handling of embryos and transfer techniques may soon render both cryobanking of embryos and ET feasible in foxes.

3. Felines 3.1. Oocyte maturation Generally, cats are seasonally polyestrous carnivores with sexual activity during the months of increasing day length, and sexual inactivity during the months of declining day lengths. Contrary to canines and most other domestic animals, cats are Žusually. reflex ovulators, i.e. oocytes are ovulated 24–48 h after the post-coital LH release. The oocytes are ovulated as secondary oocytes in metaphase II. Goodrowe et al. Ž1988. first demonstrated that unovulated follicular oocytes after IVF were able to sustain development to term with the birth of live kittens. IVM rates are relatively high in cat oocytes Ž40–60%. depending on the quality of the oocyte and the type of hormonal supplementation. The stage of the estrous cycle and supplementation of maturation media with gonadotrophins ŽGoodrowe et al., 1991; Schramm and Bavister, 1995; Wood et al., 1995; Pope et al., 1997. and quality of the cumulus oocyte complex ŽWood and Wildt, 1997. influence in vitro development. The highest incidence of MII can be expected after 40–48 h of IVM, similar to the time period from mating to ovulation in the queen ŽGoodrowe et al., 1989.. However, other studies have found that most oocytes reach MII within the first 24 h of IVM, and insemination at 40 h or later does not result in development to blastocysts ŽLuvoni and Oliva, 1993; Wolfe and Wildt, 1996.. The average rate of oocytes that complete maturation to MII in vitro is consistently higher in

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felines than in canines, and maturation occurs more rapidly Ž40–60% MII, 24 h IVM vs. 0–58% AIrMIrMII, 48–72 h IVM, respectively., but IVM rates are still lower than in most farm animal IVM systems Ž) 80%, 24 h IVM.. IVM has also been successful in some non-domestic felids ŽJohnston et al., 1991.. 3.2. Sperm treatment and assisted fertilization The quality of ejaculated sperm differs within felid species. Some breeds with low genetic variability have a high incidence of teratospermia and high number of sperm with acrosomal defects, such as the cheetah Ž Acinonyx jubatus . ŽWildt et al., 1992.. The structural mechanisms relating to the acrosome and functional defects in protein phosphorylation of some wild feline sperm may be the cause of decreased sperm function ŽGoodrowe, 1999, personal communication.. Homologous or heterologous zona binding systems and oocyte penetration assays have been developed for feline sperm ŽGoodrowe and Hay, 1993; Swanson et al., 1998; Nelson et al., 1999.. Fertilization rates after in vitro fertilization of domestic cat oocytes varies between 40% and 50% of in vitro matured and 60–80% of in vivo matured oocytes ŽPope, 1999, personal communication.. In vitro fertilization has been successful in the domestic cat in terms of production of both embryos and live offspring ŽGoodrowe et al., 1989; Hoffert et al., 1997.. In a few non-domestic felids, such as the tiger Ž Panthera tigris) and Indian Desert cat Ž F. silÕestris., offspring have been obtained ŽPope et al., 1989; Donoghue et al., 1990; for review, see Howard, 1999.. Lately, blastocysts have been obtained from in vivo matured oocytes collected from gonadotrophin stimulated domestic queens, fertilized in vitro by any of the following: Ž1. co-incubation with sperm, Ž2. subzonal insemination ŽSUZI. or Ž3. ICSI ŽPope et al., 1998.. Earlier attempts at comparing SUZI and ICSI were in favor of SUZI ŽPope et al., 1995., but improvements in sperm injection technique and visualization of ooplasm by centrifugation of oocytes improved results considerably in favor of ICSI. ICSI is the most invasive of the assisted fertilization techniques: it allows fertilization with immobile Žteratogeneic. sperm, and also enables fusion between nuclei at different stages of development. Thus, refining methods for injection of sperm or other DNA containing material into the oocyte may spur the development of nuclear transfer techniques in felids, and thereby provide the possibility for genetic modification, as well as for conservation of nuclear material. Cat ooplasm could be a potential host for somatic cell nuclei from endangered species of felids, as suggested by the bovine model, which demonstrated that bovine oocyte cytoplasm supports embryo development of nuclear transfer produced embryos from many species ŽDominko et al., 1999.. 3.3. Embryo culture in Õitro In vivo matured oocytes readily develop to the blastocyst stage after in vitro fertilization and culture. The developmental rate of in vivo matured in vivo fertilized embryos to the morula stage in TCM Ž50–90%., and the rate of blastocyst formation was 50–66% Žmean rate 64.7%. depending on the developmental stage of the zygoterearly cleavage stage embryo at flushing of the gonadotrophin stimulated donors

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ŽKanda et al., 1995.. Blastocysts have been produced by IVC of in vitro matured and IVF cat oocytes, and the rate of blastocyst formation varied between 10 and 50% ŽWolfe and Wildt, 1996; Wood and Wildt, 1997; Pope et al., 1997; Freistedt et al., 1999; Swanson et al., 1999.. Live kittens have been born after transfer of embryos produced by complete in vitro generation, i.e. IVM, IVF and IVC ŽPope et al., 1997.. Until recently, a high percentage of felid embryos produced in vitro experienced a developmental block at the morula to blastocyst stage, i.e. somewhat later than the in vitro block observed in other mammals ŽDonoghue et al., 1990; Johnston et al., 1991; Roth et al., 1994; Swanson et al., 1996b; Hoffert et al., 1997.. A difference in metabolism Žglycolysis. was demonstrated between in vivo and IVM cat oocytes that may partially explain why IVM oocytes may have developmental difficulties after IVF was compared with in vivo matured oocytes ŽSpindler and Wildt, 1999.. A recent study showed that both supplementation of cysteine to the maturation medium and reduction of the O 2 atmosphere significantly improved in vitro development to the blastocyst stage ŽPope et al., 1999.. Thus, as in bovine IVF, in vitro matured cat oocytes do not develop to blastocysts as readily as their in vivo matured counterparts, but the difference is no longer strikingly large due to improvements in culture conditions ŽSwanson et al., 1998.. Results in the vicinity of 30–40% blastocysts from IVM oocytes vs. 40–50% from in vivo matured oocytes can be expected on day 7 ŽPope, 1999, personal communication.. 3.4. Short- and long-term preserÕation of gametes, follicles and embryos Reports on the of use of frozen semen for the exchange of feline genetic material has been limited ŽHoward et al., 1997b.. Cat semen may be chilled to 48C and stored for 24–48 h in a TesT buffer Žbased on a trishydroxymethyl amino methane sulphonic acid buffer, Tes. at pH 7.4, and subsequently used for AI or in vitro insemination ŽAxner, 1998.. Buffers, such as TesT and Tris Žtrishydroxy methylamino methane., have been used with 4% glycerol or dimethylsulphoxide ŽDMSO. and 20% egg yolk, yielding no differences between the tested extenders. High cryoprotectant concentrations Ži.e., 8%. compromised cat sperm ŽNelson et al., 1999.. Pelleted freezing has often been the standard method ŽHoward, 1986.. Freezing in straws has been found to be equal to freezing in pellets ŽWood et al., 1993.. A pregnancy rate of only 10% was obtained in cats after the use of frozen–thawed semen with vaginal deposition of semen ŽPlatz et al., 1978., and vaginal inseminations in wild felids have been unsuccessful ŽHoward, 1999.. Offspring from IU laparoscopic AI with frozen–thawed semen have been obtained in ocelot Ž F. pardalis ., leopard cat Ž F. bengalensis., cheetah Ž Aci. jubatus ., snow leopard Ž P. uncia., clouded leopard Ž Neofelis nebulosa. and tiger Žfor review see, Howard, 1999.. Cat oocytes collected from ovaries and exposed to up to 72 h in refrigerated storage matured to MII at normal rates Ž50–60%.. Oocytes collected from ovaries that had been stored for 24 h developed to blastocysts, showing that cold storage of feline oocytes does not compromise their ability to sustain development in vitro ŽWolfe and Wildt, 1996., and cold storage up to 48 h results in moderate gross degeneration of oocytes ŽWood et al., 1997.. Immature oocytes Žgerminal vesicle stage. have been successfully cryopreserved and have resumed and completed meiosis after slow freezing with

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DMSO, although the meiotic resumption rate was half the rate of unfrozen controls ŽLuvoni et al., 1997.. Isolated preantral ovarian cat follicles also have been cryopreserved, showing that a small subpopulation of these follicles survive and are functionally intact after freezing with conventional cryoprotectants ŽJewgenow et al., 1998.. The first report on the birth of live kittens after ET of cryopreserved, in vivo-derived embryos was reported in 1988 ŽDresser et al., 1988. and later, from cryopreserved embryos produced in vitro from in vivo matured oocytes ŽPope et al., 1994. and after in IVM, IVF and IVC-derived cryopreserved cat embryos ŽPope et al., 1997.. The cryopreservation of domestic cat embryos can be carried out in a variety of cryoprotectants. Modifications of freezing regimens for bovine and mouse embryos have provided promising results ŽPope et al., 1994, 1997; Swanson et al., 1999.. 3.5. Artificial breeding techniques Artificial insemination in domestic cats has mostly been used in research, in which cats have served as model species. Laparoscopic insemination is often used ŽHoward et al., 1992, 1997; Donoghue et al., 1993, 1996; Barone et al., 1994; Swanson et al., 1996a.. Deep vaginal insemination may be done in domestic cats by inserting a French Tom cat catheter as far as possible into the vagina Žsee Axner, ´ 1998.. A method for non-surgical IU ET has been described, which may equally well be used for intrauterine AI ŽSwanson and Godke, 1994.. Pregnancy rates are higher after IU Žlaparoscopic. AI both in domestic and captive wild cats Žsee Axner, ´ 1998; Howard, 1999.. The time of insemination in relation to gonadotrophin stimulation and anaesthesia influences pregnancy rates. The best time for AI when anaesthesia is used, is after ovulation has occurred ŽHoward, 1999.. ET has been carried out in both domestic and wild cats with both fresh and cryopreserved in vivo and in vitro-derived embryos ŽDresser et al., 1988; Pope et al., 1989, 1994, 1997; Donoghue et al., 1990; Kanda et al., 1995; Swanson et al., 1998.. Surgical, laparoscopic and transcervical ETs have resulted in live offspring, albeit at a relatively low rate in terms of survival of transferred embryos ŽSwanson et al., 1998.. Transfer to either the oviduct or the uterus has resulted in pregnancies. Lately, transferring 5–8 morulae or blastocysts per recipient improved overall pregnancy rate as well as pregnancy rate per recipient ŽSwanson et al., 1999..

4. Conclusion The understatement that ‘‘cats are not small dogs’’ seems appropriate with respect to biotechnology. Although both canines and felines are carnivores, basic differences exist in their reproductive and gamete physiology, which should caution direct comparisons between the two families. Reproductive biotechnology has proceeded much further in cats than in canines. Improvement of IVM, IVF, ICSI, and embryo culture, using ovarian tissue grafting, cryobanking of follicles, oocytes, semen or embryos, with subsequent ET into surrogate females, may render this technology feasible for use in valuable pet animals and endangered wild relatives. In canines, reliable systems for the

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in vitro production of embryos, embryo cryopresevation and transfer are yet to be developed, but progress has been significant during the last 2–3 years.

Acknowledgements The author would like to express her sincere gratitude to Liisa Jalkanen and Heli Lindeberg at the University of Kuopio, Juankoski Fur Animal Research Station, Kuopio, Finland, for sharing yet unpublished data on non-surgical embryo transfer in blue foxes and trials with frozen fox embryos. The same goes also to the Danish group: Britta V. Bysted, Torben Greve and Poul Hyttel at the Royal Agricultural University, Copenhagen, for providing submitted, yet unpublished material on genome activation in dog embryos. The author is also grateful to Karen L. Goodrowe, Toronto Zoo, Ontario, Canada, and Earle Pope, Cincinnati Zoo, OH, USA, who are both active in canine and feline reproductive biotechnology research, for valuable discussions.

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