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CLONING AND STEM CELLS Volume 6, Number 3, 2004 © Mary Ann Liebert, Inc.

Birth of African Cloned Kittens Born from Domestic

MARTHA C. GÓMEZ,1,2 C. EARLE POPE,1 ANGELICA GIRALDO,1,2 LESLIE A. LYONS,3 REBECCA F. HARRIS,1 AMY L. KING,1 ALEX COLE,1 ROBERT A. GODKE,2 and BETSY L. DRESSER1,4

ABSTRACT

In the present study, we used the African Wildcat ( silvestris lybica) as a somatic cell donor to evaluate the in vivo developmental competence, after transfer into domestic re- cipients, of cloned embryos produced by the fusion of African Wildcat (AWC) fibroblast cell nuclei with domestic cat cytoplasts. Cloned embryos were produced by fusion of a single AWC somatic cell to in vivo or in vitro enucleated domestic cat cytoplasts. When the two sources of oocytes were compared, fusion rate was higher using in vivo–matured oocytes as recipient cytoplasts, but cleavage rate was higher after reconstruction of in vitro–matured oocytes. To determine the number of reconstructed embryos required per domestic cat recip- ient to consistently establish pregnancies, AWC cloned embryos were transferred within two

groups: recipients (n 24) receiving 25 embryos and recipients (n 26) receiving 30 em- bryos. Twelve recipients (46.2%) receiving 30 embryos were diagnosed to be pregnant, while no pregnancies were established in recipients receiving 25 NT embryos. Also, to determine the influence of length of in vitro culture on pregnancy rate, we compared oviductal transfer on day 1 and uterine transfer on day 5, 6, or 7. Pregnancy rates were similar after transfer of embryos on day 1 (6/12; 50.0%), day 5 (4/9; 44.4%), or day 6 (2/5; 40.0%) to synchronous recip- ients, but the number of fetuses developing after transfer of embryos on day 1 (n 17), ver- sus day 5 (n 4) or day 6 (n 3) was significantly different. Of the 12 pregnant recipients, nine (75%) developed to term and fetal resorption or abortion occurred in the other three (25%) from day 30 to 48 of gestation. Of a total of 17 cloned kittens born, seven were stillborn, eight died within hours of delivery or up to 6 weeks of age, and two are alive and healthy. Peri- natal mortality was due to lung immaturity at premature delivery, placental separation and bacterial septicemia. Subsequent DNA analysis of 12 cat-specific microsatellite loci confirmed that all 17 kittens were clones of the AWC donor male. These AWC kittens represent the first wild carnivores to be produced by nuclear transfer.

INTRODUCTION gered. In fact, 36 of 37 of this family are at risk of extinction. The population decline can HE SURVIVAL OF MOST SPECIES in the be attributed to poaching and human destruction Tfamily is considered as threatened or endan- of natural ecosystems. As populations decrease,

1Audubon Center for Research of Endangered Species, New Orleans, Louisiana 70131 USA. 2Department of Sciences, LSU Agricultural Center, Baton Rouge, Louisiana 70803 USA. 3School of Veterinary Medicine, University of California Davis, Davis, California 95616 USA. 4Department of Biological Sciences, University of New Orleans, New Orleans, Louisiana 70148 USA.

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insularization eventually results in subpopula- nancy rates in species such as the pig, up to 62 tion extinctions caused by detrimental genetic reconstructed embryos are transferred into each and demographic effects (Nowell and Jackson, recipient (Polejaeva et al., 2000). In the domestic 1996a). cat, we reported that more pregnancies occurred Domestic cats are a useful research model to after uterine transfer of 12 IVF/IVC-derived develop assisted reproductive technologies for embryos (Pope et al., 1993). In vivo developmen- the conservation of endangered felids. During the tal potential of cloned embryos may be even more last two decades, the feasibility of oocyte recov- susceptible to the stressful environment of in vitro ery (in vivo maturation), in vitro maturation culture. Although in vitro development of cat em- (IVM), in vitro fertilization (IVF), in vitro embryo bryos, as measured by blastocyst development culture (IVC) and embryo transfer (ET) to recip- and number of cells per embryo, has improved ients has been demonstrated (Pope, 2000; Luvoni, (Pope et al., 1999; Gómez et al., 2001, 2003), the 2000). The domestic cat can also serve as a suc- presence of perturbations affecting embryo via- cessful recipient of embryos from closely related bility such as nutritional imbalances, oxidative small non-domestics cats as shown by the birth stress, and defective genomic imprinting must be of Indian cat (Pope et al., 1993) and African acknowledged (Leese, 2002). Wildcat kittens (Pope et al., 2000) after transfer of In the present study, we used the African Wild- IVF-derived embryos. cat (Felis silvestris lybica) as a somatic cell donor Recently, the feasibility of producing viable do- to evaluate the in vivo developmental compe- mestic cat offspring by nuclear transfer was dem- tence, after transfer into domestic cat recipients, onstrated (Shin et al., 2002). Regarding endan- of cloned embryos produced by the fusion of gered felids, nuclear transfer is a potentially AWC fibroblast cell nuclei with domestic cat cy- valuable technique for assuring the continuation toplasts. Specifically, our primary goals were to of species with few remaining numbers of ani- (1) determine the minimal number of recon- mals. In fact, could maintain genetic vari- structed embryos required per recipient to estab- ability in endangered felids when there are few lish pregnancy; (2) evaluate the influence of in in a founder population by conserving vitro culture interval on establishment and main- the maximum number of alleles for future breed- tenance of fetal development in recipients after ing projects (Seidel, 2001). transfer of reconstructed embryos; and (3) evalu- Inter-species nuclear transfer involves the ate factors affecting pre- and post-natal fetal sur- transfer of a donor cell nucleus of one species into vival and describe characteristics of the kittens. an enucleated oocyte of another species. Since a limited number of endangered felid oocytes are available, the ooplasm of the domestic cat oocyte MATERIALS AND METHODS can be used as the recipient cytoplast of a somatic cell nucleus of an endangered cat. Recently, we Animals have shown that domestic cat ooplasm is a com- patible host for somatic cell nuclei from an AWC Domestic cats used as oocyte donors and em- and the reconstructed embryos do undergo de- bryo recipients were group-housed in environ- velopment in vitro (Gómez et al., 2003). Although mentally controlled rooms with a 14-h/10-h the feasibility of inter-species nuclear transfer has light/dark cycle at 20–26°C. The antibody de- been demonstrated only three live endangered fined cats were purchased from Liberty Research, have been produced: gaur (Lanza et al., Inc. (Waverly, NY). The AWC male was housed 2000), mouflon (Loi et al., 2001), and banteng in a separate room under the same conditions. (Jansen et al., 2004) after transfer of NT embryos The rooms were cleaned, and cats were fed once into domestic ruminant recipients. daily (Science Diet, Hill Pet Nutrition). Fresh wa- Incomplete nuclear reprogramming is a major ter was available at all times. All animal proce- constraint to the in vivo developmental potential dures were approved by the Institutional Animal of cloned embryos (Kikyo and Wolffe, 2000). Care and Use Committee of the Audubon Center Most cloned blastocysts, regardless of species, are for Research of Endangered Species as required not able to maintain fetal development to term by the Health Research Extension Act of 1985 (Colman, 2000). Therefore, to maximize preg- (Public Law 99-158). 5241_e03_p247-258 9/30/04 9:34 AM Page 249

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Chemicals Cumulus cells of in vivo– and in vitro–matured oocytes were removed by vortexing in 1 mg/mL Chemicals were purchased from Sigma Chem- of hyaluronidase (type IV) for 5 min, followed by ical Co. (St. Louis, MO), unless otherwise stated. gentle mechanical pipetting. Denuded oocytes were placed in Tyrode’s solution (Irvine Scien- Establishment of adult AWC cell line tific, Santa Ana, CA) with 1% MEM nonessential amino acids, 3 mg/mL BSA, 15 mM NaHCO3, The cell line was generated from skin tissue col- 0.36 mM pyruvate, 2.2 mM calcium lactate, 1 mM lected by biopsy from a 3-year-old male AWC. glutamine, and 50 g/mL gentamicin (IVC-1 2 Collected tissue was finely cut into 1-mm pieces medium) at 38°C in 5% CO2 until further use. and plated in a 75-cm2 tissue-culture flask (Nunc, Denmark) containing 7 mL of Dulbecco Modified Eagles Medium (DMEM) supplemented with 50 g/mL of gentamicin and 10% (v/v) fetal bovine Nuclear transfer serum (FBS; Hyclone, Logan, UT) and cultured The protocol for nuclear transfer has been de- at 38°C in 5% CO2/air. After 7–10 days of incu- scribed previously (Gómez et al., 2003). Briefly, bation, monolayer outgrowths with fibroblastic- denuded metaphase II oocytes were incubated like morphology were disaggregated with 2.5 for 15 min at 38°C in Ca2-free and Mg2-free mg/mL of pronase, re-suspended in DMEM with modified Tyrode’s salt solution supplemented 10% FBS and 10% (v/v) dimethyl sulfoxide, and with 1% MEM nonessential amino acids, 3 cooled at 1.0°C/min to 80°C (Mr. Frosty; Nal- mg/mL BSA, 30 mM NaHCO3, 0.36 mM pyru- gene, Rochester, NY) before storage in liquid ni- vate, 1 mM glutamine, 50 g/mL gentamicin trogen (LN2). To prepare fibroblasts as donor cells (ECM medium), 20 g/mL Hoechst 33342 and 20 for NT, cells were thawed and cultured in DMEM g/mL cytochalasin B (CCB). After incubation, containing 10% FBS for 1–3 passages. Then, when oocytes were enucleated in ECM medium in cells reached 80% confluency, FBS was reduced which supplemental NaHCO3 was reduced to 15 to 0.5% and cells were cultured for an additional mM and to which 15 mM Hepes, 20 g/mL CCB 5 days (serum starvation) before cryopreserva- and 2 mg/mL sucrose were added. The first po- tion and storage in LN2. lar body and 10% of the underlying cytoplasm were drawn into an enucleation pipette (20 m, OD). Removal of the metaphase II plate was con- Oocyte maturation firmed by brief exposure to epifluorescence mi- For in vitro maturation, cumulus–oocyte com- croscopy. plexes (COC) were recovered from ovaries of do- A vial containing AWC fibroblast cells syn- mestic cats within 2–5 h after ovariohysterec- chronized in G0/G1 phase by serum starvation tomy. Selected COCs were cultured in modified (Gómez et al., 2003) was thawed prior to being TCM-199 containing 1 IU/mL hCG, 0.5 IU/mL used for NT, and a single AWC fibroblast cell was eCG, 10 g/mL epidermal growth factor, and 3 introduced into the perivitelline space of each mg/mL BSA (Fraction V, fatty acid free; Sero- enucleated oocyte. For fusion, each NT couplet, 2 logical Proteins, Kankakee, IL) for 24 h in 5% CO2, in a solution of 0.3 M mannitol and 0.1 mM Mg , 5% O2 and 90% N2 at 38°C (Gómez et al., 2003). was placed between two stainless-steel electrodes To obtain in vivo–matured oocytes, domestic spaced 120 m apart (LF-101; Nepa Gene, Tokyo, cats were treated with a total of 3–4 IU of porcine- Japan) that were controlled by micromanipula- follicle stimulating hormone (FSH, Sioux Bio- tors. Membrane fusion was induced by applying chemical, Sioux Center, IA) given in decreasing a 3-sec AC pre-pulse of 20V, 1 MHz followed by daily doses (s.c.) for 4 days, followed by 3 IU of two 30-sec DC pulses of 240V/mm at intervals porcine-luteinizing hormone (im; LH, Sioux Bio- of 0.5 sec. Following the fusion pulses, couplets chemical) on day 5. At 24–26 h after LH injection, were washed and cultured in IVC-1 medium sup- oocytes were collected by laparoscopic aspiration plemented with 7.8 mM calcium lactate (IVC-1 of mature ovarian follicles (Gómez et al., 2000). Ca2) for 30 min., at which time fusion was eval- In vivo–matured oocytes were used for NT im- uated visually by confirming the presence or ab- mediately after aspiration. sence of the donor cell in the perivitelline space. 5241_e03_p247-258 9/30/04 9:34 AM Page 250

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After 2–3 h of culture in IVC-1 Ca2, activa- embryos near the lower tip was threaded through tion was induced by placing fused couplets be- the tom cat catheter, and the embryos were ex- tween two electrodes in a fusion chamber con- pelled into the oviduct using positive pressure taining 3 mL of a solution of 0.3 M mannitol, 0.1 from a 1-mL syringe. mM Mg2, and 0.05 mM Ca2 and exposing them The recipients of day 5, 6, and 7 embryos were to two 60-sec DC pulses of 120 V/mm. Then, gonadotropin-treated females that had under- couplets were incubated in 30-L droplets of gone laparoscopic oocyte aspiration 5–7 days pre- IVC-1 medium supplemented with 10 g/mL cy- viously. Briefly, one uterine horn was exterior- cloheximide and 5 g/mL CCB at 38°C in 5% CO2 ized through a 1.5-cm mid-ventral incision and in air for 4 h. punctured with a sterile 16-gauge round-tipped, short bevel trocar 1 cm from the anterior tip. Embryos were aspirated in 50 L of IVC-2 Embryo culture medium into a 14.5-cm tom cat catheter using a After activation, reconstructed embryos were 1-mL plastic syringe. The catheter was then cultured in 500 L of IVC-1 medium in 5% CO2, threaded 4–5 cm into the uterine lumen before de- 5% O2, and 90% N2 at 38°C. Some couplets were positing the embryos (Pope et al., 1994). transferred to the oviducts of recipient females on day 1. The remaining couplets were cultured un- Pregnancy detection and parturition til day 2 or 3, at which time, after uncleaved cou- plets were recorded and removed, embryos were Each recipient was checked for pregnancy sta- placed into 500 L of fresh IVC-1 medium con- tus using abdominal ultrasonography on day taining 1% MEM essential amino acids (IVC-1 21–23 after ovulation or oocyte aspiration. Preg- EAA). Then, on day 5, the number of morulae nant recipients were monitored by ultrasono- was recorded and all embryos were moved into graphy on a weekly basis until day 60, at which Tyrode’s solution containing 1% NEAA, 2% EAA, time daily ultrasonographic examinations were 10% FBS, and the same supplements as in IVC-1 made to check for fetal movement and heart beat. medium (IVC-2 medium) and cultured until Pregnant recipients were allowed to complete

transfer to a recipient. gestation, at which time kittens were delivered by Caesarean section, either (1) as an intervention after onset of vaginal bleeding and/or occurrence Embryo transfer of labor symptoms or (2) on a pre-scheduled day Cloned embryos were transferred into the before symptoms of parturition were seen. In an oviducts or uteri of 50 gonadotropin-treated do- effort to reduce the onset of premature parturi- mestic short hair (DSH) recipients. Day 1 em- tion, some pregnant cats (n 3) were orally ad- bryos were transferred to the oviducts of recipi- ministered altrenogest (0.08 mg/kg, Regu-Mate, ents, some of which had served as oocyte donors Hoechst; Kustritz, 2001) daily from days 55 to on day 0 (n 2) and others of which served as 65–67 of gestation. Also, in an attempt to reduce recipients only after induction of ovulation (n perinatal kitten mortality (n 7) cats were 14) using a lower dose of FSH (1–3 IU) and a treated with four injections (im), 12 h apart, of higher dose of LH (5 IU) than the oocyte donors betamethasone (0.1 mg/kg, CelestoneSoluspan, received. For oviductal embryo transfer, the ab- Schering) starting at varying times after day 60 of dominal cavity was visualized via a 5-mm endo- gestation. scope inserted just above the umbilicus, and a 16 gauge 6.35 cm stainless steel trocar/cannula Microsatellite analysis was inserted transabdominally near the midline 5 cm below the umbilicus. Then, a 14.5-cm tom To determine the clonal status of all kittens, cat catheter (3.5 French, Sherwood Medical, St. DNA was extracted using standard phenol/chlo- Louis, MO) was passed through the 16-gauge roform techniques from the fibroblast cells and cannula and directed through the infundibulum blood of the AWC donor, blood of the recipient overlaying the ovary and into the upper am- dam, umbilical cord blood of live kittens, or pullary portion of the oviduct. A 50-cm length of spleen of dead kittens. Standard sodium hydrox- sterile polyethylene tubing (PE 10, no. 427400, ide isolation was used to isolate DNA from cheek Becton Dickinson, Sparks, MD) containing the swabs of live kittens. Feline derived microsatel- 5241_e03_p247-258 9/30/04 9:34 AM Page 251

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lite loci were amplified as previously reported by consistently establish pregnancies, AWC cloned Raymond et al. (1999) using standard ABI fluo- embryos were transferred into domestic cat re- rescent chemistries on an ABI 377. The results cipients within two groups: (1) recipients (n 24) were analyzed using the software STRand that received fewer than 25 embryos (mean (Hughes). 19.4; range 8–25), and (2) recipients (n 26) that received more than 30 embryos (mean Statistical analyses 41.7; range 31–59). Also, to determine the in- fluence of length of in vitro culture on pregnancy The Chi square test was used to test the dif- rate we compared the transfer of day 1 embryos ferences between treatments. Statistical differ- into the oviduct (n 588) with transfer of day 5 ence was set at the p 0.05 level. (n 467), day 6 (n 355), and day 7 (n 142) embryos into the uterus. Embryos were at the 1–2-cell stage on day 1, morulae on day 5 and day RESULTS 6, and morulae and blastocysts on day 7 of cul- ture. Embryo production after NT Twelve of 26 (46.2%) recipient cats receiving A total of 2,565 in vivo (n 1751) and in vitro more than 30 NT embryos were diagnosed to be (n 814) matured domestic cat oocytes were me- pregnant when examined by ultrasonography at chanically enucleated and electrically fused to 21–23 days after induction of ovulation or aspi- frozen-thawed AWC fibroblasts. Fusion effi- ration of follicular oocytes. In contrast, none of ciency was higher when in vivo–matured oocytes the recipients receiving less than 25 embryos (n (1696/1751; 96.8%) were used as recipient cyto- 24) were diagnosed pregnant (Table 1). Although, plasts as compared with in vitro–matured oocytes no differences were observed in pregnancy rates (736/814, 90.4%, p 0.05). In contrast, cleavage after transfer of 30 embryos on day 1 (6/12, rate of embryos reconstructed with in vitro–ma- 50.0%), day 5 (4/9, 44.4%), or day 6 (2/5, 40.0%) tured oocytes (435/511, 85.1%) was higher than to synchronous recipients, a significant difference that of embryos reconstructed with in vivo–ma- was found in the number of fetuses implanted af-

tured oocytes (944/1194, 79.1%, p 0.05). Some ter transfer embryos on day 1 (n 17) versus day reconstructed couplets (n 727) were not in- 5 (n 4) or day 6 (n 3; p 0.05; Table 1). Six of cluded in the cleavage rate evaluation because 7 (85.7%) pregnant cats with multiple fetuses they were transferred to domestic cat recipients were recipients of day 1 embryos. on day 1 before cleavage occurred. Birth of AWC cloned kittens and Embryo transfer and pregnancies histological analysis To determine the number of reconstructed em- Of the 12 pregnant recipients, nine (75%) preg- bryos that should be transferred per recipient to nancies developed to term (60 days). In the

TABLE 1. EFFECTS OF TRANSFER DAY AND NUMBER OF EMBRYOS ON OCCURRENCE OF PREGNANCY IN DOMESTIC CAT RECIPIENTS RECEIVING AFRICAN WILDCAT (AWC) CLONED EMBRYOS

AWC NT embryos Average per recipient Recipients

Embryos per recipient ET, day Total, nn SD Total, n Pregnant, n (%) Fetuses, n

25 7 142 15.7 5.5 9 0 (0) 6 172 19.1 4.4 9 0 (0) 5 50 25.0 0 2 0 (0) 1 102 25.5 0.5 4 0 (0) 30 6 183 36.6 4.1 5 2 (40.0) 3 5 417 46.3 12.1 9 4 (44.4) 4 1 486 40.5 10.5 12 6 (50.0) 17 Total 1552 50 12 (24.0) 24 5241_e03_p247-258 9/30/04 9:34 AM Page 252

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three (25%) other pregnancies, a total of five fe- four experienced respiratory failure shortly after tuses were present at day 21–23, four of which delivery and resuscitation efforts were not suc- were reabsorbed between days 30 and 40, and one cessful, one kitten lived for 36 h and one kitten which was spontaneously aborted on day 48 of had died in utero at 45 days of gestation. gestation (Table 2). Analysis of the kitten that died 36 h after de- Six of the nine term pregnancies, consisting of livery showed multifocal moderate to severe a total of eight fetuses, were not hormonally acute ulcerative enterocolitis associated with rod- treated during the last 7–10 days of pregnancy shaped bacteria, and squames were present (Table 2). Two of these recipients each had a sin- within the alveolar spaces of the lungs. These re- gle kitten that died in uteri within 24 h of de- sults suggest that the kitten had complications livery by Caesarean section on days 64 and 67 of caused by bacterial septicemia of clostridial ori- gestation. Histological analysis of the two kittens gin. showed no indications of infectious disease. No The kitten that died in utero on 45 days of inflammatory or other degenerative lesions were gestation showed gross physical abnormalities, recognizable in the tissue sections and all organs including incomplete closure of the ventral body appeared to be developing normally. One of the wall musculature with abdominal organ exteri- two kittens had moderate haemorrhage within orization, severe cerebella and cerebellum apla- the choroids plexus of the brain, possibly due to sia and no recognizable central nervous system. hypoxia that occurred after placental separation. All the other kittens that died shortly after de- The other four recipients delivered six kittens livery had a normal physical appearance except by Caesarean section on days 62–67 of pregnancy for one that showed incomplete closure of the after vaginal bleeding occurred. Of the six kittens, ventral body wall musculature with abdominal

TABLE 2. INVIVODEVELOPMENT OF AFRICAN WILDCAT (AWC) CLONED EMBRYOS AFTER OVIDUCTAL OR UTERINE TRANSFER

Pregnant

recipients Total Fetuses AWC kittens (days 48–70) Embryos fetuses reabsorbed ET transferred days days Delivery n day n 21–23 30–40 day Stillborn Live 36 h Healthy Weight, g

15 411 1 21 422 2 3 1 56 2 1 48 1 — 4 6 39 1 — 64 1c 90.4 5 5 35 1 — 67 1c 108.7 6a 1594 3 62 1e 60.5 7a 6 42 2 — 62 1 95.4 1f 102.2 8a 5 43 1 — 62 1 77.0 9a 1 34 2 — 67 1 107.0 1d,e 50.0 10b 5 39 1 — 69 1 98.3 11b 1 40 5 — 65 1 92.5 1.g 73.0 1c 86.9 1d — 1e 71.9 12b 1352 — 671c 111.0 1f 105.2 Total 24 7 7 7 3 88.7

aPregnant cats showed vaginal bleeding. bPregnant cats treated with progestagen and betamethasone. c Died in uteri just before delivery. dDied in utero on day 45–50 of gestation. eKittens with body wall defect. fKittens died within 36–72 h after birth. gDied at 6 weeks of age. 5241_e03_p247-258 9/30/04 9:34 AM Page 253

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organ exteriorization. Histological evaluation of ents that were not hormonally treated during the the lungs of the kittens revealed variable collapse final stage of gestation. of alveolar spaces and presence of epithelial Two kittens, currently 7 and 3 months of age squames within the lumen of alveolar spaces, in- (Fig. 1) are healthy and developing normally. Of dicating that alveoli were not inflated and the the other two kittens, one died at 56 h after birth lungs were immature. Thus, prenatal and early and one died at 6 weeks of age. Histological anal- neonatal mortality appears to have been primar- ysis of the kitten that died at 56 h after birth in- ily due to lung immaturity resulting from the dicated a diffuse moderate septic and supurative spontaneous initiation of premature parturition. pneumonia with abnormally high bacterial Three pregnant cats that were hormonally counts in several organs. The bacteria may have treated during the last 10–12 days of gestation de- gained entry through the urachus and extended livered eight kittens by Caesarean section. The into the urinary bladder. four kittens that survived were born on 6 August The kitten that died at 6 weeks of age had a 2003, 15 November 2003, and 12 January 2004 at congenital megaesophagus and acute supurative 69, 65, and 67 days of gestation, respectively pneumonia. The primary cause of death was (Table 2). pneumonia, likely a result of aspiration of for- Of the four kittens that did not survive, two mula during bottle-feeding. died in utero within hours of delivery on days 64 As shown in Table 2, the birth average weight and 66. One kitten that died within a few min- of live kittens (92.3 g; range 71.9–105.0 g) was utes after delivery had incomplete closure of the similar to the average weight of kittens that died ventral body wall musculature with abdominal during the peri- and post-parturient period (87.4 organ exteriorization. Another kitten died in utero g; range 50.0–111.0 g). Phenotypically, all on 50 days of gestation and was being resorbed cloned kittens (n 17) were African . at delivery. Subsequent DNA analysis of 12 cat-specific mi- Pathology analysis of one kitten that died in crosatellite loci performed with “blind” testing at utero on day 64 revealed diffuse adrenal conges- the School of Veterinary Medicine, University of tion and mild nephrosis. Necrosis of the renal California, Davis, confirmed that they were iden- tubular epithelium could be induced by endo- tical to DNA of the AWC donor male (“Jazz”; toxic shock. The hypoxia may have been caused Table 3). by cardiovascular collapse and release of nephro- toxins before death. Therefore, prenatal death Placental analysis was possibly due to hypoxia that occurred after placenta separation and is probably analogous to A total of 11 placentae were analyzed. Macro- the prenatal death occurring in pregnant recipi- scopic analyses showed that five placentae had

A B

FIG. 1. African wildcat cloned kittens (A) “Ditteaux” born on 6th August 2003, at two months after birth. (B) “Miles” and Otis” born on 15th November 2003, at seven days after birth. 5241_e03_p247-258 9/30/04 9:34 AM Page 254

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TABLE 3. ANALYSIS OF FELINE GENETIC MARKERS lung immaturity at premature delivery and bac- terial septicemia. AWC cloned kittens AWC donor (n 17) We obtained pregnancies only in recipients “Jazz” that received 30 NT embryos. A possible ex- Feline Cheek Umbilical planation for the high number of embryos re- markers Fibroblasts Blood cells cord quired for producing pregnancies is that most of FCA075 132/134 132/134 132/134 132/134 the reconstructed embryos had inadequate nu- FCA097 140/146 140/146 140/146 140/146 clear remodeling or incomplete reprogramming FCA201 153/155 153/155 153/155 153/155 (Gómez et al., 2003). We previously demonstrated FCA224 160/178 160/178 160/178 160/178 in AWC reconstructed embryos that 18% were FCA229 158/162 158/162 158/162 158/162 FCA293 179/187 179/187 179/187 179/187 fragmented and unable to undergo chromatin re- FCA305 191/201 191/201 191/201 191/201 modeling, 38% arrested at the 8–10-cell stages and FCA441 158/166 158/166 158/166 158/166 44% and 24% reached the morula and blastocysts FCA018 233/233 233/233 233/233 233/233 stages, respectively (Gómez et al., 2003). In this FCA145 174/174 174/174 174/174 174/174 FCA651 135/135 135/135 135/135 135/135 study, NT embryos were reconstructed from the FCA674 148/148 148/148 148/148 148/148 same AWC somatic cell line and oocytes were similar in type and source to those used in pre- Values represent the sizes of the two alleles of the amplified microsatellites markers. vious NT experiments. Then, from the mean number of embryos transferred on day 1 (40.5), day 5 (46.3), and day 6 (36.6), we can estimate that patches of serous-lacy tissue replacing normal at least 10 embryos should develop to the blasto- placental tissue. Microscopic analysis of these five cyst stage. Based on these values, we suggest that, placentae showed that areas of the placental ep- using our current methods, to consistently estab- ithelium were reduced in thickness and had lish pregnancies in domestic cats receiving AWC shorter and thicker papillary projections or NT-derived embryos, at least 30 embryos should merely a thin band of edematous fibrous con- be transferred per recipient. If it is assumed that nective tissue. These observations indicate the NT-derived cat blastocysts may be less develop- ocurrence of multifocal placental atrophy with fi- mentally competent than IVF-derived cat blasto- brous tissue replacement. Curiously, these ab- cysts, these numbers directly correlate with the normal placentae allowed development of kittens minimal number of IVF derived embryos re- to term, including two kittens that survived. The quired to reliably establish pregnancies in do- placenta of a kitten that died in uteri before de- mestic cat recipients. livery by Caesarean section showed excessive ab- Domestic cat recipients receiving day-7 blasto- normal mineralization and intermittent serosan- cysts (mean 7.4) and morulae (mean 8.3) on guineous exudates. These abnormalities were not day 7 post-oocyte retrieval did not establish preg- present in all the placental sections analyzed; nancies. During this portion of the study we were some sections were histologically normal. transferring an average of 10 blastocysts per cat. We do not think that the longer in vitro culture interval was the primary reason for the develop- DISCUSSION mental failure because we have produced do- mestic cat kittens after transfer of either 10 or 11 In the present study, we demonstrated that IVF-derived day-7 blastocysts per recipient (Pope AWC kittens can be produced after transfer of et al., 2003). Further experiments should deter- embryos derived by fusion of adult somatic cells mine if pregnancies can be established after trans- from one species with enucleated oocytes of a ferring AWC cloned blastocysts. closely related species (domestic cat). We have In , more lambs were produced after shown that, under our current laboratory condi- early transfer (day 2) of IVF embryos as compared tions, transfer of at least 30 NT embryos per re- to later transfer at day 6 (Walmsley et al., 2004). cipient was required for establishing a preg- Similarly, in the present study, a higher percent- nancy. Also, the length of in vitro culture affected age of NT embryos developed into fetuses when the number of embryos that successfully under- the transfer was done after a shorter period of cul- went fetal development. The primary causes of ture (day 1, n 17/486, 3.5%) as compared with peri-natal mortality were placental separation, embryos that were cultured for longer periods 5241_e03_p247-258 9/30/04 9:34 AM Page 255

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(day 5, n 4/417, 1.0%; day 6, n 3/183, 1.6%). higher fusion rates with in vivo–matured cyto- From these results, we suggest that the develop- plasts and higher cleavage rates with in vitro–ma- mental potential of some NT embryos is “res- tured cytoplasts. In a previous study, we found cued” by transferring them to recipient cats as un- no differences in blastocyst development in vitro cleaved couplets and two-cell embryos; thereby when in vivo–matured and in vitro–matured cat minimizing the detrimental effects of in vitro cul- oocytes were used as recipient cytoplasts of AWC ture (Leese, 2002). Although more embryos im- cells (Gómez et al., 2003). A primary purpose of planted after transfer on day 1, fetal mortality by the present study was to find out if AWC kittens day 48 of gestation also increased (6/17; 35.3%) could be produced by transfer of NT embryos to as compared with embryos transferred on day 5 domestic cat recipients. Because we were unable (1/4; 25.0%) or day 6 (0/3; 0%). Also, three kit- to produce NT pregnancies after transferring tens that were born after embryo transfer on day 17 embryos per recipient in a previous study 1 had incomplete closure of the ventral body wall (Gómez et al., 2003), in the present study in or- musculature with abdominal organ exterioriza- der to transfer 25 embryos per recipient, for tion. Because few fetuses were evaluated, we can- most transfers it was necessary to combine re- not conclude that the increase in fetal mortality constructed embryos derived from both in or the physical abnormalities were directly re- vitro–matured and in vivo–matured domestic cat lated to the shorter period of culture. However, cytoplasts. The one domestic cat recipient receiv- we can speculate that losses occurring after trans- ing NT embryos derived from in vivo–matured fer on day 1 are not found after the transfer of cytoplasts did establish pregnancy and produce embryos on day 5 or 6 because the developmen- a live kitten. We do not know if the other NT kit- tal potential of such embryos is sufficiently re- tens originated from embryos derived from in duced by the extended in vitro culture interval vivo–matured or in vitro–matured cytoplasts. Cer- that they are incapable of further development in tainly, the comparative developmental compe- utero. tence of in vitro–matured versus in vivo–matured Although embryo viability was demonstrated, cat cytoplasts requires investigation. embryo survival after transfer was quite modest Several developmental abnormalities occur in (1.0–3.5%) and was similar to the overall effi- pregnancies from somatic cell nuclear trans- ciency of cloning reported in other species fer–derived embryos, including a high rate of (1.0–4.0%; Wilmut and Paterson, 2003). In addi- abortion during early gestation and a number of tion to incomplete reprogramming of the differ- perinatal complications. One complication we en- entiated nucleus, another factor that may con- countered in some cats was the occurrence of tribute to low cloning efficiency is variation in vaginal bleeding by day 62–67 of pregnancy with- oocyte quality. Both in vivo–matured and in out overt signs of labor. Although the kittens vitro–matured oocytes have been used as recipi- were delivered within the normal gestation pe- ent cytoplasts for production of cloned animals. riod of domestic cats (62–71 days; Herron, 1977) For example, in sheep, higher blastocyst de- and African Wildcats (56–63 days; Nowell and velopment rates, pregnancy rates and embryo Jackson, 1996b), the kittens died shortly after de- survival were reported when in vivo–matured livery from respiratory failure, a condition usu- oocytes were used as donor cytoplasts (Wells et ally associated with premature delivery. al., 1997). In contrast, in goats no differences were In domestic cats, serum progesterone levels be- found in fusion rates, embryo development and gin a slow decline by day 50 of pregnancy, pro- in the number of cloned kids produced from gressing to a precipitous decrease during the last oocytes harvested from FSH-stimulated donor week prior to parturition (Verhage et al., 1976). animals or from in vitro–matured oocytes recov- Although we did not measure serum progester- ered from abbatoir ovaries (Reggio et al., 2001). one levels in pregnant recipients, it seems possi- In pigs, in vivo–matured oocytes have been used ble that, since the recipient domestic cats were extensively as cytoplast recipients (Miyoshi et al., gestating AWC cloned fetuses, feto-placental en- 2003). Walker et al. (2002) reported that in docrine signaling may have been inadequate. As vitro–matured pig oocytes could also be used to a result, progesterone levels may have shown an produce cloned piglets; however, a direct com- abnormally early decrease, starting a cascade of parison between the two sources of cytoplasts has pre-parturient events that resulted in vaginal not been done. In the present study, we found bleeding. Consequently, with the last three preg- 5241_e03_p247-258 9/30/04 9:34 AM Page 256

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nant recipients, in an effort to reduce perinatal hypoglycemia. This disorder involves the dereg- kitten mortality, a synthetic progestagen (al- ulation of several genes, including insulin-like trenogest) was orally administered. Altrenogest growth factor, type 2 (IGF2) and cyclin-depen- has been shown to maintain gestation in ovariec- dent kinase inhibitor 1C (CDKN1C; Murrel et al., tomized mares and (Hinrichs et al., 1986; 2004). The product of IGF2 is a major fetal growth Eilts et al., 1994). Since pregnancy was success- factor, and is associated with overgrowth in fe- fully maintained (until elective Caesarean sec- tuses derived from sheep embryos cultured in tion) in cats treated with the synthetic progesta- vitro before transfer to recipients (Young et al., gen, luteal insufficiency may be implicated in the 2001). Recently, in humans, an association was occurrence of premature vaginal bleeding. Ma- found between babies produced by in vitro tech- ternal administration of betamethasone is widely nology and the incidence of B-WS (DeBaun et al., used to improve fetal lung maturation (Senat, 2003). In our study, a major abnormality found in 2002; Celik et al., 2002). Recipients administered three cloned kittens was incomplete closure of both synthetic progestagen and betamethasone ventral body wall musculature with exterioriza- during the latter part of gestation produced a to- tion of abdominal organs; however, pathology as- tal of five live kittens. Thus, while the improve- sociated with overgrowth was not observed. ment in fetal survival rate cannot be conclusively Likewise, knockout mice for CDKN1C have been attributed to the treatment combination of pro- found to manifest an anterior wall defect but do gestagen and betamethasone, the present results not exhibit fetal overgrowth or other features of do provide at least circumstantial evidence of B-WS (Fitzpatrick et al., 2002). Whether body wall their effectiveness. pathology is caused by gene deregulation or is Placental abnormalities are a major cause of merely a consequence of another wall anomaly is mortality in cloned animals and likely contribute not known. to a number of perinatal complications in farm The influence of possible mitochondrial het- animals (Cibelli et al., 2002). Abnormal placentae eroplasmy and/or a deleterious mtDNA effect on have been reported in cloned mice, cattle and neonatal death of the cloned AWC kittens cannot sheep (Shimozawa et al., 2003; Hill et al., 2000; be discounted. Indeed, in future studies the pre- Hartwich et al., 2003) and may be a result of aber- cise mitochondrial inheritance pattern in AWC rant gene expression (Suemizu et al., 2003). In our cloned kittens will be evaluated to determine its study, histological analysis of placental tissue re- influence on survival rate. vealed a reduction in thickness and multifocal at- The effectiveness of our cloning procedure was rophy. In fact, some kittens died from hypoxia af- shown by the birth of healthy AWC kittens. These ter placental separation, with respiratory failure births represent the first wild carnivores to be as a secondary consequence. Accordingly, it is produced by NT and further expand the grow- plausible that placental anomalies/dysfunction ing list of species in which cloned offspring have were either directly or indirectly responsible for been produced. Our success may be attributable the perinatal deaths of the cloned kittens. To im- to improvements in the embryo culture system prove success rates and reduce neonatal deaths, (Gómez et al., 2003), refinements made to the nu- basic studies are needed to identify factors caus- clear transfer procedure (Gómez et al., 2003), in- ing abnormal placentation. creasing the number of embryos transferred, re- Developmental anomalies occur with a greater ducing the culture interval before transfer and frequency in cloned animals than in the average modification of current protocols successfully population. Diagnostic pathology of cloned used in other species to manage pregnancy and lambs that died after birth revealed several improve viability of offspring (Ptak et al., 2002). cloning-related abnormalities, some of which While there are major limitations to current were analogous to congenital human diseases cloning technologies, knowledge about nuclear (Rhind et al., 2003). For example, four of eight transfer procedures is advancing rapidly and cloned lambs exhibited fetal overgrowth and a technology is improving. Researchers should be body wall defect. These abnormalities are linked accessible to the possibilities offered by nuclear to Beckwith-Wiedemann syndrome (B-WS), a hu- transfer as one of the assisted reproduction tech- man genetic syndrome characterized by abdom- niques potentially useful for the conservation of inal wall defects, visceromegaly, macroglossia, endangered species, which in some cases, may of- pre- and postnatal overgrowth and neonatal fer the prospect of species continuation rather 5241_e03_p247-258 9/30/04 9:34 AM Page 257

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than extinction. Future research should focus on velopment of in vitro matured, in vitro fertilized do- defining and understanding the mechanisms re- mestic cat embryos following cryopreservation, culture sponsible for the abnormalities, so that efficiency and transfer. Theriogenology 60, 239–251. is improved and neonatal death rate reduced. We Hartwich, K.M., Walker, S.K., Jurisevic, A.M., et al. (2003). Evidence of impaired placental function in cloned fetal expect that advances in understanding the mo- sheep during late gestation. Theriogenology 59, 256 [ab- lecular events for reprogramming of the donor str]. genome will contribute to clarifying the deriva- Herron, M.A. (1977). Feline reproduction. Vet. Clin. North tion of developmental defects in cloned embryos. Am. 7, 715–722. Hill, J.R., Burghardt, R.C., Jones, K., et al. (2000). Evidence for placental abnormality as the major cause of mor- tality in first-trimester somatic cell cloned bovine fe- ACKNOWLEDGMENTS tuses. Biol. Reprod. 63, 1787–1794. Hinrichs, K., Sertich, P.L., and Kenny R.M. (1986). Use of We are grateful to Stella Sullivan for her ex- altrenogest to prepare ovariectomized mares as embryo cellent cat care and hand rearing some of the transfer recipients. Theriogenology 26, 455–460. cloned kittens. In addition, we thank Dr. Karen Janssen, D.L., Edwards, M.L., Koster, J.A., et al. (2004). Miller, Dr. Steve Miller, Barbara Vincent and the Postnatal management of chryptordich banteng calves Audubon Nature Institute veterinary staff for cloned by nuclear transfer utilizing frozen fibroblast cultures and enucleated cow ova. Reprod. Fertil. Dev. their help with the surgery procedures; Jackie 16, 224(abst). Coulon for coordinating and collecting ovaries Kikyo, N., and Wolffe, A.P. (2000). Reprogramming nu- from the veterinary clinics; and the staff and in- clei: insights from cloning, nuclear transfer and het- terns from the Species Survival Center for the 24 erokaryons. J. Cell Sci. 113, 11–20. h per day cat observations towards the end of Kustritz, R. (2001). Use of supplemental progesterone in each pregnancy. management of canine pregnancy. In: Recent Advances in Small Animal Reproduction. P.W. Concannon, G. En- gland, and J. Verstegen, eds. (Ithaca: International Vet- erinary Information Service), p. A1220.0401. REFERENCES Lanza, R.P., Cibelli, J.B., Diaz, F., et al. (2000). Cloning of

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