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Birth of viable puppies derived from breeding cloned female dogs with a cloned male
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Theriogenology 72 (2009) 721–730 www.theriojournal.com
Birth of viable puppies derived from breeding cloned female dogs with a cloned male J.E. Park a, S.G. Hong a, J.T. Kang a, H.J. Oh a, M.K. Kim b, M.J. Kim b, H.J. Kim a, D.Y. Kim c,*, G. Jang a, B.C. Lee a,* a
Department of Theriogenology & Biotechnology, College of Veterinary Medicine, Seoul National University, Seoul 151-742, Republic of Korea b Department of Animal Science and Biotechnology, College of Agriculture and Life Sciences, Chungngam National University, Daejeon 305-764, Republic of Korea c Department of Veterinary Pathology, College of Veterinary Medicine, Seoul National University, Seoul 151-742, Republic of Korea Received 19 December 2008; received in revised form 30 April 2009; accepted 5 May 2009
Abstract Since the establishment of production of viable cloned dogs by somatic cell nucleus transfer, great concern has been given to the reproductive abilities of these animals (Canis familiaris). Therefore, we investigated reproductive activity of cloned dogs by (1) performing sperm analysis using computer-assisted sperm analysis and early embryonic development, (2) assessing reproductive cycling by measuring serum progesterone (P4) levels and performing vaginal cytology, and (3) breeding cloned dogs using artificial insemination. Results showed that most parameters of sperm motility in a cloned male dog were within the reference range, and in vivo–matured oocytes from a noncloned female were successfully fertilized by spermatozoa from a cloned male dog and develop normally to the 8-cell stage. Three cloned female dogs displayed normal patterns of P4 levels and morphologic changes of the vaginal epithelium. Two cloned female dogs became pregnant using semen from a cloned male dog and successfully delivered 10 puppies by natural labor. In conclusion, these data demonstrated that both cloned male and female dogs are fertile, and their puppies are currently alive and healthy with normal growth patterns. # 2009 Published by Elsevier Inc. Keywords: Artificial insemination; Cloned dogs; Pregnancy; Reproductive ability; Sperm motility
1. Introduction Somatic cell nucleus transfer (SCNT) has the potential to enhance the expansion of elite genetics, and, to date, a number of cloned livestock (sheep, goat, and cows) as well as laboratory animals (mice and rabbit) have been generated by SCNT [1]. Whereas * Corresponding authors. Tel.: +82 2 880 1269; fax: +82 2 873 1269. E-mail addresses: [email protected] (D.Y. Kim), [email protected] (B.C. Lee). 0093-691X/$ – see front matter # 2009 Published by Elsevier Inc. doi:10.1016/j.theriogenology.2009.05.007
cloned animals have been shown to display a normal range of fertility [2–5], the productive capacity of cloned dogs is yet to be determined. The first cloned male dog, ‘‘Snuppy’’ (an Afghan hound), generated at our laboratory [6] is currently 3 yr 7 mo old and is healthy. Three cloned Afghan hound females that were generated later [7] have displayed signs of estrus initiation lately, indicating that they may have reached reproductive maturity. In this study, the reproductive competences of these cloned dogs (Canis familiaris) were investigated.
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2. Materials and methods
2.4. Artificial insemination
2.1. Reagents
AI was performed at the first observed estrus either by intrauterine insemination at laparotomy [12] or by transcervical insemination during endoscopic visualization of the cervix [13,14]. Due to limitations of cage facilities, only two of the three female cloned dogs (Females A, B, and C) were used. For intrauterine insemination, Female A was placed under general anesthesia and semen was introduced into both uterine horns. The total insemination volume was adjusted to 1 mL by dilution with a buffered extender [12]. Transcervical intrauterine insemination was accomplished on the standing bitch (Female C) with the aid of a rigid fiberoptic endoscope and a urinary catheter without sedation. The endoscope was introduced into the vagina and advanced until the external os of the cervix could be visualized. Subsequently, a 5-Fr canine urinary catheter was manipulated into the cervical opening and inserted into the uterus. After insemination, the endoscope was removed, and the bitch’s hindquarters were elevated for 5 to 10 min to minimize backflow of semen.
Unless otherwise indicated, reagents were purchased from Sigma-Aldrich Corp. (St. Louis, MO, USA). 2.2. Semen analysis of a cloned male dog Semen was collected from a cloned male dog [6] by an experienced operator using digital manipulation [8,9]. The procedure was performed in the presence of a cloned female dog in estrus to arouse the cloned male dog into attaining a complete erection. Semen quality was evaluated using a computer-assisted sperm analyzer (CASA; Medical Supply, Seoul, Republic of Korea), a microscope (JP/BX50; Olympus, Tokyo, Japan), and a CCD Camera (HVR-2000C; Hyvision System Inc., Kyunggi, Republic of Korea). The sperm motility and other sperm motion variables including curvilinear velocity (VCL), straight line velocity (VSL), average path velocity (VAP), beat cross-frequency (BCF), mean angular displacement (MAD), wobble (WOB; VAP VCL), dance (DNC; VCL ALH), and amplitude of lateral head displacement (ALH) were measured. Ejaculates with sperm motility of 80% and a concentration of 200 106 spermatozoa/mL were used for artificial insemination (AI). To evaluate the fertilizing capability of spermatozoa from the cloned male, insemination into the uterine horn close to the oviduct of a noncloned female dog with progesterone (P4) reaching 4.0 to about 7.5 ng/mL was performed via a surgical approach [6,9]. On the 4th day after insemination, fertilized embryos were collected by surgically flushing the oviduct.
2.5. Pregnancy diagnosis and monitoring Pregnancy in the two cloned females was detected on the 23rd to 25th days after AI using a SONOACE 9900 (Medison Co., LTD, Seoul, Republic of Korea) ultrasound scanner with an attached 7.0-MHz lineararray transducer. Subsequently, maintenance of pregnancy was monitored weekly. To identify the exact number of fetuses and for assessment of the size relationships between the fetuses and the maternal pelvic canal, survey radiographs were taken at the nearterm stage of pregnancy.
2.3. Hormonal assay and vaginal cytology in female cloned dogs
2.6. Microsatellite marker analyses for genotyping
For measuring serum P4, 5 mL blood was collected daily from the cephalic vein, and serum was separated by centrifugation at 6000 rpm for 10 min. Sera were analyzed using a DSL-3900 ACTIVE Progesterone Coated-Tube Radioimmunoassay Kit (Diagnostic Systems Laboratories Inc., Webster, USA) with the assistance of the Neodin Veterinary Laboratory (Seoul, Republic of Korea; http://www.vetlab.co.kr). The day on which the serum P4 level reached 4.0 ng/mL was regarded as the day of ovulation [6,7,10,11]. Specimens for vaginal cytology were obtained on sterile cotton swabs, which were rolled on glass slides and stained with Diff-Quik (Sysmex Co., Kobe, Japan).
Parentage analysis was performed on blood samples taken from the puppies that were produced by AI, from the cloned male dog, and from the cloned female dogs as well. To extract genomic DNA, blood samples were incubated overnight with a lysis buffer (0.05 M Tris, 0.05 M ethylenediamine tetraacetic acid [EDTA], 0.5% sodium dodecyl sulfate [SDS], pH 8.0) supplemented with 400 mg proteinase K, followed by phenol extraction and ethanol precipitation. The following nine markers were selected for analysis: PEZ02, PEZ06, PEZ08, PEZ15, PEZ20, CPH04, CPH07, CPH09, and CPH12. Isolated genomic DNA samples were dissolved in 50 mL TE (Tris-EDTA) and used for
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microsatellite assay with nine specific markers originally derived from dogs [15–17]. Length variations were assayed by polymerase chain reaction (PCR) amplification with fluorescently labeled locus-specific primers and PAGE (polyacrylamide gel electrophoresis) on an automated DNA sequencer (ABI 3130xl; Applied Biosystems, Foster City, CA, USA). Proprietary software (GeneScan and Genotyper; Applied Biosystems) was used to estimate the PCR product sizes in nucleotides. 2.7. Growth and hematologic profiles of the offspring (F1 puppies) To compare growth rates of puppies, their weights were monitored daily for the first month of life, then weekly until the dogs were 3 mo of age. Blood samples were collected from the puppies via jugular vein cannulation at 1 mo of age for a complete blood count (CBC) and serum biochemistry. Blood samples (3 mL) were collected in anticoagulant tubes, and serum samples were obtained by centrifugation at 6000 rpm for 10 min. For serum biochemistry, alanine aminotransferase (ALT), aspartate aminotransferase (AST), alkaline
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phosphatase (ALP), blood urea nitrogen (BUN), creatine, glucose, total bilirubin, albumin, total protein, phosphorus and calcium were examined. Another 1 mL blood was collected into an evacuated tube containing EDTA to measure red blood cell (RBC) count, white blood cell (WBC) count, hemoglobin concentration (Hb), packed cell volume (PCV), mean corpuscular volume (MCV), mean corpuscular hemoglobin (MCH), and mean corpuscular hemoglobin concentration (MCHC). For determination of CBC, a Celltac-a hematology analyzer (Nihon Kohden, Tokyo, Japan) was used. Serum biochemistry profile was determined using a Selectra 2 chemistry analyzer (Merck, Amsterdam, The Netherlands) according to the manufacturer’s protocols. The mean weight of puppies was compared with average growth data of an Afghan litter from birth to 12 wk [18,19], and the hematologic and serum chemistry values of puppies were compared with reference data [20]. 2.8. Experimental design The purpose of this study was to investigate reproductive competence of cloned dogs, and experiments were performed as shown in Figure 1. In a cloned
Fig. 1. Schematic illustration of the experimental design.
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Fig. 2. (A) Spermatozoa collected from a cloned male dog and (B) fertilized embryos.
male dog, semen was collected and analyzed by CASA, and in vivo fertilization was performed using a noncloned female dog to evaluate the fertilization ability of sperm. In three female dogs, after detecting onset of estrus, ovulation timing was determined by P4 concentration and vaginal cytology, and AI was carried out by two technical methods (intrauterine/transcervical). Because this was an observational study with no control group, a successful outcome was defined as a successful pregnancy and delivery of puppies without complications. The parameters measured in spermatozoa of the cloned male dog, changes in P4 concentration and the day of first observed estrus in cloned female dogs were compared with reference data for normal dogs [13,21–23]. The health status of offspring sired by cloned dogs was evaluated by body weight gain and blood analysis. 3. Results 3.1. Fertility of cloned male dog
Table 1 Analysis of ejaculated fresh semen from a cloned male dog by computer-assisted motility analyzer. Parameters
Reference range*
Cloned male dog
Motility (%) VCL (mm/sec) VSL (mm/sec) VAP (mm/sec) BCF (Hz) MAD (degree) WOB (%) DNC (mm2/sec) ALH (mm)
92–98 139–207 61–71 73–98 9–14 17–22 47–59 860–1765 6–9
97 233 123 128 8 19 555 2096 9
VCL, curvilinear velocity; VSL, straight line velocity; VAP, average path velocity; BCF, beat cross-frequency; MAD, mean angular displacement; WOB, wobble (VAP VCL); DNC, dance (VCL ALH); ALH, lateral head displacement. * Reference data from Kim et al. [12].
ficial in parallel with the changes in P4 level (Fig. 4). The P4 concentrations of clones A, B and C on the estimated day of ovulation were 5.81, 5.45, and 5.43 ng/ mL, respectively.
Semen analysis and AI were used to determine the fertility of the cloned male dog. Semen analysis was performed using fresh semen (Fig. 2) by CASA. Results showed that motility, BCF, MAD, and ALH were all within the reference ranges (Table 1). Spermatozoa inseminated into the oviducts of noncloned female dogs successfully fertilized ova as was evidence by formation of the 8-cell stage embryos (Fig. 2). 3.2. Fertility of cloned female dogs The first estrus was exhibited by cloned Females A, B and C at the ages of 627, 604, and 716 d, respectively, which were within the reference range [21]. The changes in serum P4 level during the estrus cycle in these dogs were also within the reference range (Fig. 3). Vaginal epithelial cells changed from basal to super-
Fig. 3. Progesterone changes in three cloned female dogs. The hormone was measured every day after finding vaginal discharge as a sign of estrus.
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After having evidence of the fertility of the cloned male dog and the exhibition of estrus cycling in the cloned female dogs, we undertook AI using fresh semen. To exclude individual sample differences, an aliquot of semen (10 mL) was assessed for motility
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and number of sperm using a phase-contrast microscope before using collected semen for AI. The two inseminated cloned dogs, Females A and C, were both diagnosed as pregnant and successfully gave birth to litters (four and six puppies, respectively) by
Fig. 4. Representative pictures of vaginal cytology in one of three cloned female dogs. Vaginal smear samples were collected every day. Corresponding P4 concentrations (ng/mL) on the day of smear were as follows: (A) 0.61 ng/mL, (B) 0.69 ng/mL, (C) 0.89 ng/mL, (D) 2.75 ng/mL, (E) 5.81 ng/mL, (F) 7.80 ng/mL, (G) 13.39 ng/mL, (H) 20.94 ng/mL. Artificial insemination was performed on Day H.
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Fig. 5. Representative pictures of pregnant female dogs and their puppies. (A) Ultrasonogram of fetus (arrow) in the fetal vesicle on Day 38 after AI. Fetal head and ribs are identifiable. Fetal heartbeat was recognized. (B) Lateral radiograph of pregnant cloned dog at near term. The alignments of the six fetal skull bones (arrowheads) as well as the arrangement of the cervical through lumbar vertebrae and the pelvis with the appendages are identified. (C) Four puppies born from Female A and all the F1 puppies were raised under the care of their mothers. (D) F1 puppies from a cloned female dog at 23 d old.
natural delivery (Fig. 5). The puppies were all born alive, exhibiting no congenital abnormalities. Microsatellite analysis of genomic DNA extracted from blood cells confirmed that the puppies originated
from the cloned male and female dogs (Table 2). The F1 puppies were cared for by their biological dam until they were weaned at approximately 30 d of age.
Table 2 Microsatellite genotypes of 13 dogs, including the cloned male dog (Father), two cloned female dogs (Females A and C), and live F1 offspring (A1 to A3, C1 to C6).
Father Female A Puppy A1 Puppy A2 Puppy A3 Female C Puppy C1 Puppy C2 Puppy C3 Puppy C4 Puppy C5 Puppy C6
PEZ02
PEZ06
PEZ08
PEZ15
PEZ20
CPH04
CPH07
CPH09
CPH12
121/121 121/121 121/121 121/121 121/121 121/121 121/121 121/121 121/121 121/121 121/121 121/121
180/180 180/184 180/180 180/180 180/180 180/184 180/180 180/184 180/180 180/180 180/184 180/180
230/230 230/246 230/230 230/230 230/230 230/246 230/230 230/230 230/230 230/230 230/230 230/230
204/204 204/208 204/208 204/204 204/204 204/208 204/208 204/208 204/204 204/204 204/204 204/208
174/174 178/178 174/178 174/178 174/178 178/178 174/178 174/178 174/178 174/178 174/178 174/178
139/139 139/141 139/141 139/139 139/141 139/141 139/139 139/141 139/141 139/139 139/139 139/139
162/168 162/162 162/162 162/162 162/168 162/162 162/168 162/162 162/168 162/168 162/162 162/162
147/147 139/147 139/147 147/147 139/147 139/147 147/147 139/147 139/147 147/147 139/147 139/147
202/202 191/202 191/202 202/202 202/202 191/202 191/202 202/202 202/202 191/202 202/202 191/202
Nine canine-specific markers were used for microsatellite assay.
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Table 3 F1 puppies sired by a cloned male dog by AI. Cloned female dog
Method of insemination
Pregnancy length (d)
Name of puppy
Sex
Birth weight (g)
Phenotype; coat color
Female A
Intrauterine insemination (surgical)
60
A1
Female
410
Cream white
60
A2 A3 A4* C1
Female Male Female Male
460 510 360 460
Gray with shaded mask Gold with shaded mask Black Brown brindle
C2 C3 C4 C5 C6
Female Male Female Female Female
430 480 310 380 420
Black and tans Domino Domino Gold with shaded mask Black and tans
Female C
*
Transcervical insemination
This puppy died within 2 wk after birth due to small body size and anorexia.
The mean birth weights of F1 puppies from the two cloned females were 435 32.27 g and 413.33 24.99 g (Table 3). The sex ratio and coat color of the 10 puppies is shown in Table 3. The surviving (9 of 10) F1 puppies showed consistent weight gain with no significant difference among them and was within the reference ranges (Fig. 6). Individual hematologic and serum biochemical data at 1 mo of age are listed in Tables 4 and 5. All values for the F1 puppies were within laboratory reference ranges for healthy Afghan dogs. 4. Discussion Since the first cloned mammal ‘‘Dolly’’ was born in 1997 [22], there have been concerns regarding the reproductive competence of cloned animals. Several groups have examined the perinatal and postnatal development and reproductive characteristics of cloned animals and demonstrated their fertility [2,23–27]. In the current study, we demonstrated that cloned dogs are fertile and reproductively healthy.
To determine the fertility of a male dog, semen samples were analyzed by CASA and a noncloned female dog inseminated. Several parameters of sperm motility, including BCF, MAD, and ALH, were within the reference ranges. Interestingly, however, VCL, VSL, VAP, WOB, and DNC were higher than reference ranges. These CASA parameters are related to velocity of spermatozoa, as they are one of the parameters affecting the outcome of in vitro fertilization and the ability of spermatozoa to penetrate cervical mucus and fuse with an oocyte [28,29]. It is unlikely that this is a unique feature of a cloned dog, but further characterization is needed using an increased number of cloned dogs or comparing the sperm quality of the cloned dogs with that of the donor. The donor dog has been neutered, and, therefore, the experiment could not be performed at the moment. Nonetheless, the fact that all the embryos collected were at the 8-cell stage 4 d after insemination was in agreement with a previous report that spermatozoa produced by a cloned male dog can generate the same
Fig. 6. The body weight change patterns of F1 puppies from birth to 3 mo of age. A1 to A4: puppies born between cloned male dog and Female A. C1 to C6: puppies born between cloned male dog and Female C. Puppy A4 died within 2 wk after birth due to small body size and anorexia. Data from Niblock [19].
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Table 4 Age-specific reference ranges and results of hematologic tests in F1 offspring at 1 mo of age. Parameter (units) 3
WBC (10 /mL) RBC (106/mL) Hb (g/dL) PCV (%) MCV (fL) MCH (pg) MCHC (g/dL)
Reference range*
Female A puppies
Female C puppies
A1
A2
A3
Mean SE
C1
C2
C3
C4
C5
C6
Mean SE
15.7 4.4 4.7 0.4 10.3 0.9 31.4 2.4 65.8 2.3 21.8 1.2 32.6 1.8
15.8 5.43 11.3 33.9 62.4 20.8 33.3
13.3 4.93 10.1 31.7 64.3 20.5 31.9
19.8 4.79 10.4 30.8 64.3 21.7 33.8
16.3 1.9 5.1 0.2 10.6 0.4 32.1 0.9 63.7 0.6 21.0 0.4 33.0 0.6
16.4 4.74 10.1 30.6 64.6 21.3 33
17.1 4.5 9.7 29 64.4 21.6 33.4
17.3 5.15 10.7 32.5 63.1 20.8 32.9
14.8 4.14 8.6 26.9 65 20.8 32
13.6 4.74 10.2 30.3 63.9 21.5 33.7
16.8 4.68 9.8 30.5 65.2 20.9 32.1
16.0 0.6 4.7 0.1 9.9 0.3 30.0 0.8 64.4 0.3 21.2 0.1 32.9 0.3
WBC, white blood cell count; RBC, red blood cell count; Hb, hemoglobin concentration; PCV, packed cell volume; MCV, mean corpuscular volume; MCH, mean corpuscular hemoglobin; MCHC, mean corpuscular hemoglobin concentration. * Data from Shifrine et al. [20].
Table 5 Age-specific reference ranges and results of serum biochemical tests in F1 offspring at 1 mo of age. Parameter (units)
ALT (U/L) AST (U/L) ALP (U/L) BUN (mg/dL) Creatinine (mg/dL) Glucose (mg/dL) Bilirubin (mg/dL) Albumin (g/dL) Total protein (g/dL) Calcium (mg/dL) Phosphate (mg/dL)
Reference rangey
37 12.2 44 10.2 162 39.9 12 2.3 0.6 0.04 83 12.8 0.2 0.2 3.0 0.14 5.0 0.23 10.6 0.34 8.8 0.47
Female A puppies
Female C puppies
A1
A2
A3
Mean SE
C1
C2
C3
C4
C5
C6
Mean SE
42 35 208 9 0.6 117 0.4 2.4 3.7 10.7 9.1
36 32 197 5 0.6 105 0.2 2.3 3.6 10.6 8.7
33 29 222 7 0.6 113 0.2 2.3 3.6 10.8 9.4
37.0 2.2 32.0 1.4 209.0 5.9 7.0 0.9 0.6 0.0 111.7 2.9 0.3 0.1 2.3 0.05 3.6 0.03 10.7 0.05 9.1 0.2
25 24 201 8 0.6 128 0.3 2.2 3.7 10.5 10.1
34 38 270 8 0.6 134 0.7 2.4 4 10.8 10.2
32 24 200 8 0.6 120 0.3 2.2 3.2 9.8 8.8
33 30 159 7 0.6 123 0.3 2.5 3.9 11.2 10.3
33 43 306 7 0.6 112 0.5 2.6 4.2 10.6 10.8
31 29 170 8 0.6 114 0.3 2.3 3.6 10.4 9.6
31.3 1.3 31.3 3.1 217.7 23.7 7.7 0.2 0.6 0.0 121.8 3.4 0.4 0.1 2.4 0.1 3.8 0.1 10.6 0.2 10.0 0.3
ALT, alanine aminotransferase; AST, aspartate aminotransferase; ALP, alkaline phosphatase; BUN, blood urea nitrogen. y Four-month-old beagle blood chemistry values (Covance Research Products Inc., Cumberland, VA, USA).
developmental stage of embryos with noncloned dogs [30]. Even though the cloned male dog used in this study exhibited libido along with other mating behavior, his inexperience in the clasping technique made it difficult to use natural mating for testing fertility. It is well documented that one of the most common mating problems of the dog is caused by inexperience, and AI is the best alternative [21,31]. For such reason, the AI method was used in this study utilizing fresh semen from the cloned male dog, which resulted in pregnancy and successful births in both female clones. The body weights of the puppies were monitored to evaluate growth because it is widely assumed that birth weight and body weight gains are important survival determinants in most mammalian species. The birth weights of puppies derived from cloned dogs were within reference ranges and showed consistent weight gains without any disorders. Additionally, as further evidence of the
puppies’ health, hematologic and serum biochemical measurements were performed to provide fundamental biological data on physiologic status, and the values obtained were mostly within the reference ranges, except for some hematology and serum parameters. It is widely assumed that age appeared to influence several serum biochemical parameters in young dogs [20,32,33], thus we think the F1 puppies did not reveal any abnormal values in blood analysis results, similar to the results of evaluation in litters produced by mating pairs of other cloned dams and sires [2,24,34,35]. These results appear to support the hypothesis that the offspring from cloned dogs have similar growth performance and health to those of naturally bred puppies. In conclusion, the current study demonstrates that both cloned male and female dogs are fertile, allowing the application of cloning technology for the purpose of expanding an elite canine gene pool in the dog.
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Acknowledgments We appreciate the assistance of Drs. JunYoung Kim and MinCheol Choi of the Veterinary Teaching Hospital at Seoul National University with ultrasonography and x-ray filming. We thank Dr. Barry D. Bavister (Wayne State University) for his valuable editing of the manuscript and Dr. CheMyong Jay Ko (University of Kentucky) for critical suggestions regarding this project. This study was financially supported by KOSEF (grant no. M10625030005-09N250300510, approval number by ILAR (institute of laboratory animal resources) in SNU; SNU-090508-5), the SNU foundation (Benefactor; RNL BIO), TS Corporation, and the Korean MEST through the BK21 program for Veterinary Science. References [1] Campbell KH, Fisher P, Chen WC, Choi I, Kelly RD, Lee JH, Xhu J. Somatic cell nuclear transfer: Past, present and future perspectives. Theriogenology 2007;68(Suppl 1):S214–31. [2] Martin M, Adams C, Wiseman B. Pre-weaning performance and health of pigs born to cloned (fetal cell derived) swine versus non-cloned swine. Theriogenology 2004;62:113–22. [3] Batchelder CA, Bertolini M, Mason JB, Moyer AL, Hoffert KA, Petkov SG, et al. Perinatal physiology in cloned and normal calves: hematologic and biochemical profiles. Cloning Stem Cells 2007;9:83–96. [4] Batchelder CA, Bertolini M, Mason JB, Moyer AL, Hoffert KA, Petkov SG, et al. Perinatal physiology in cloned and normal calves: physical and clinical characteristics. Cloning Stem Cells 2007;9:63–82. [5] Panarace M, Aguero JI, Garrote M, Jauregui G, Segovia A, Cane L, et al. How healthy are clones and their progeny: 5 years of field experience. Theriogenology 2007;67:142–51. [6] Lee BC, Kim MK, Jang G, Oh HJ, Yuda F, Kim HJ, et al. Dogs cloned from adult somatic cells. Nature 2005;436:641. [7] Jang G, Kim MK, Oh HJ, Hossein MS, Fibrianto YH, Hong SG, et al. Birth of viable female dogs produced by somatic cell nuclear transfer. Theriogenology 2007;67:941–7. [8] Kustritz MV, Hess M. Effect of administration of prostaglandin F2alpha or presence of an estrous teaser bitch on characteristics of the canine ejaculate. Theriogenology 2007;67:255–8. [9] Traas AM, Kustritz MV. Effect of administrating oxytocin or prostaglandin F2alpha on characteristics of the canine ejaculate. Can Vet J 2004;45:999–1002. [10] Hase M, Hori T, Kawakami E, Tsutsui T. Plasma LH and progesterone levels before and after ovulation and observation of ovarian follicles by ultrasonographic diagnosis system in dogs. J Vet Med Sci 2000;62:243–8. [11] Bouchard GF, Solorzano N, Concannon PW, Youngquist RS, Bierschwal CJ. Determination of ovulation time in bitches based on teasing, vaginal cytology, and elisa for progesterone. Theriogenology 1991;35:603–11. [12] Kim HJ, Oh HJ, Jang G, Kim MK. Birth of puppies after intrauterine and intratubal insemination with frozen-thawed canine semen. J Vet Sci 2007;8:75–80.
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[13] Pretzer SD, Lillich RK, Althouse GC. Single, transcervical insemination using frozen-thawed semen in the Greyhound: a case series study. Theriogenology 2006;65:1029–36. [14] Cremonesi F, Salamon L, Groppetti D, Pecile A. Results of a single transcervical endoscopic insemination using frozen semen in the bitch. Vet Res Commun 2005;29(Suppl 2):187–9. [15] Parker HG, Kruglyak L, Ostrander EA. Molecular genetics: DNA analysis of a putative dog clone. Nature 2006;440: E1–2. [16] Cargill EJ, Clark LA, Steiner JM, Murphy KE. Multiplexing of canine microsatellite markers for whole-genome screens. Genomics 2002;80:250–3. [17] Richmana M, Mellersha CS, Andre´b C, Galibertb F, Ostrander EA. Characterization of a minimal screening set of 172 microsatellite markers for genomewide screens of the canine genome. J Biochem Biophys Methods 2001;30:137–49. [18] Race L. Afghan Hounds, The World of Dogs. Kingdom Books; 1999. pp. 182-183. [19] Niblock M. The Afghan Hound; A Definitive Study. Arco Publishing; 1980. pp. 335-355. [20] Shifrine M, Munn SL, Rosenblatt LS, Bulgin MS, Wilson FD. Hematologic changes to 60 days of age in clinically normal beagles. Lab Anim Sci 1973;23:894–8. [21] Johnston SD, Kustritz MVR, Olson PNS. Canine and Feline Theriogenology. WB Saunders; 2001. pp. 18-19. [22] Wilmut I, Schnieke AE, McWhir J, Kind AJ, Campbell KH. Viable offspring derived from fetal and adult mammalian cells. Nature 1997;385:810–3. [23] Kasai K, Sano F, Miyashita N, Watanabe S, Nagai T. Comparison of the growth performances of offspring produced by a pair of cloned cattle and their nuclear donor animals. J Reprod Dev 2007;53:135–42. [24] Watanabe S, Nagai T. Health status and productive performance of somatic cell cloned cattle and their offspring produced in Japan. J Reprod Dev 2008;54:6–17. [25] Chavatte-Palmer P, Remy D, Cordonnier N, Richard C, Issenman H, Laigre P, et al. Health status of cloned cattle at different ages. Cloning Stem Cells 2004;6:94–100. [26] Choi EG, Yin XJ, Lee HS, Kim LH, Shin HD, Kim NH, Kong IK. Reproductive fertility of cloned male cats derived from adult somatic cell nuclear transfer. Cloning Stem Cells 2007;9:281– 90. [27] Tamashiro KL, Wakayama T, Blanchard RJ, Blanchard DC, Yanagimachi R. Postnatal growth and behavioral development of mice cloned from adult cumulus cells. Biol Reprod 2000;63:328–34. [28] Verstegen J, Iguer-Ouada M, Onclin K. Computer assisted semen analyzers in andrology research and veterinary practice. Theriogenology 2002;57:149–79. [29] Hirano Y, Shibahara H, Obara H, Suzuki T, Takamizawa S, Yamaguchi C, et al. Relationships between sperm motility characteristics assessed by the computer-aided sperm analysis (CASA) and fertilization rates in vitro. J Assist Reprod Genet 2001;18:213–8. [30] Abe Y, Suwa Y, Yanagimoto-Ueta Y, Suzuki H. Preimplantation development of embryos in labrador retrievers. J Reprod Dev 2008;54:135–7. [31] Simpson GM, Egland GCW. BSAVA Manual of Small Animal Reproduction and Neonatology. British Small Animal Veterinary Association; 1998. pp. 97-100. [32] Harper EJ, Hackett RM, Wilkinson J, Heaton PR. Age-related variations in hematologic and plasma biochemical test results in
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Beagles and Labrador Retrievers. J Am Vet Med Assoc 2003;223:1436–42. [33] Mundim AV, Coelho AO, Horteˆncio SM, Guimara˜es EC, Espindola FS. Influence of age and sex on the serum biochemical profile of Doberman dogs in the growth phase. Comp Clin Pathol 2007;16:41–6.
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Theriogenology 72 (2009) 721–730 www.theriojournal.com
Birth of viable puppies derived from breeding cloned female dogs with a cloned male J.E. Park a, S.G. Hong a, J.T. Kang a, H.J. Oh a, M.K. Kim b, M.J. Kim b, H.J. Kim a, D.Y. Kim c,*, G. Jang a, B.C. Lee a,* a
Department of Theriogenology & Biotechnology, College of Veterinary Medicine, Seoul National University, Seoul 151-742, Republic of Korea b Department of Animal Science and Biotechnology, College of Agriculture and Life Sciences, Chungngam National University, Daejeon 305-764, Republic of Korea c Department of Veterinary Pathology, College of Veterinary Medicine, Seoul National University, Seoul 151-742, Republic of Korea Received 19 December 2008; received in revised form 30 April 2009; accepted 5 May 2009
Abstract Since the establishment of production of viable cloned dogs by somatic cell nucleus transfer, great concern has been given to the reproductive abilities of these animals (Canis familiaris). Therefore, we investigated reproductive activity of cloned dogs by (1) performing sperm analysis using computer-assisted sperm analysis and early embryonic development, (2) assessing reproductive cycling by measuring serum progesterone (P4) levels and performing vaginal cytology, and (3) breeding cloned dogs using artificial insemination. Results showed that most parameters of sperm motility in a cloned male dog were within the reference range, and in vivo–matured oocytes from a noncloned female were successfully fertilized by spermatozoa from a cloned male dog and develop normally to the 8-cell stage. Three cloned female dogs displayed normal patterns of P4 levels and morphologic changes of the vaginal epithelium. Two cloned female dogs became pregnant using semen from a cloned male dog and successfully delivered 10 puppies by natural labor. In conclusion, these data demonstrated that both cloned male and female dogs are fertile, and their puppies are currently alive and healthy with normal growth patterns. # 2009 Published by Elsevier Inc. Keywords: Artificial insemination; Cloned dogs; Pregnancy; Reproductive ability; Sperm motility
1. Introduction Somatic cell nucleus transfer (SCNT) has the potential to enhance the expansion of elite genetics, and, to date, a number of cloned livestock (sheep, goat, and cows) as well as laboratory animals (mice and rabbit) have been generated by SCNT [1]. Whereas * Corresponding authors. Tel.: +82 2 880 1269; fax: +82 2 873 1269. E-mail addresses: [email protected] (D.Y. Kim), [email protected] (B.C. Lee). 0093-691X/$ – see front matter # 2009 Published by Elsevier Inc. doi:10.1016/j.theriogenology.2009.05.007
cloned animals have been shown to display a normal range of fertility [2–5], the productive capacity of cloned dogs is yet to be determined. The first cloned male dog, ‘‘Snuppy’’ (an Afghan hound), generated at our laboratory [6] is currently 3 yr 7 mo old and is healthy. Three cloned Afghan hound females that were generated later [7] have displayed signs of estrus initiation lately, indicating that they may have reached reproductive maturity. In this study, the reproductive competences of these cloned dogs (Canis familiaris) were investigated.
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2. Materials and methods
2.4. Artificial insemination
2.1. Reagents
AI was performed at the first observed estrus either by intrauterine insemination at laparotomy [12] or by transcervical insemination during endoscopic visualization of the cervix [13,14]. Due to limitations of cage facilities, only two of the three female cloned dogs (Females A, B, and C) were used. For intrauterine insemination, Female A was placed under general anesthesia and semen was introduced into both uterine horns. The total insemination volume was adjusted to 1 mL by dilution with a buffered extender [12]. Transcervical intrauterine insemination was accomplished on the standing bitch (Female C) with the aid of a rigid fiberoptic endoscope and a urinary catheter without sedation. The endoscope was introduced into the vagina and advanced until the external os of the cervix could be visualized. Subsequently, a 5-Fr canine urinary catheter was manipulated into the cervical opening and inserted into the uterus. After insemination, the endoscope was removed, and the bitch’s hindquarters were elevated for 5 to 10 min to minimize backflow of semen.
Unless otherwise indicated, reagents were purchased from Sigma-Aldrich Corp. (St. Louis, MO, USA). 2.2. Semen analysis of a cloned male dog Semen was collected from a cloned male dog [6] by an experienced operator using digital manipulation [8,9]. The procedure was performed in the presence of a cloned female dog in estrus to arouse the cloned male dog into attaining a complete erection. Semen quality was evaluated using a computer-assisted sperm analyzer (CASA; Medical Supply, Seoul, Republic of Korea), a microscope (JP/BX50; Olympus, Tokyo, Japan), and a CCD Camera (HVR-2000C; Hyvision System Inc., Kyunggi, Republic of Korea). The sperm motility and other sperm motion variables including curvilinear velocity (VCL), straight line velocity (VSL), average path velocity (VAP), beat cross-frequency (BCF), mean angular displacement (MAD), wobble (WOB; VAP VCL), dance (DNC; VCL ALH), and amplitude of lateral head displacement (ALH) were measured. Ejaculates with sperm motility of 80% and a concentration of 200 106 spermatozoa/mL were used for artificial insemination (AI). To evaluate the fertilizing capability of spermatozoa from the cloned male, insemination into the uterine horn close to the oviduct of a noncloned female dog with progesterone (P4) reaching 4.0 to about 7.5 ng/mL was performed via a surgical approach [6,9]. On the 4th day after insemination, fertilized embryos were collected by surgically flushing the oviduct.
2.5. Pregnancy diagnosis and monitoring Pregnancy in the two cloned females was detected on the 23rd to 25th days after AI using a SONOACE 9900 (Medison Co., LTD, Seoul, Republic of Korea) ultrasound scanner with an attached 7.0-MHz lineararray transducer. Subsequently, maintenance of pregnancy was monitored weekly. To identify the exact number of fetuses and for assessment of the size relationships between the fetuses and the maternal pelvic canal, survey radiographs were taken at the nearterm stage of pregnancy.
2.3. Hormonal assay and vaginal cytology in female cloned dogs
2.6. Microsatellite marker analyses for genotyping
For measuring serum P4, 5 mL blood was collected daily from the cephalic vein, and serum was separated by centrifugation at 6000 rpm for 10 min. Sera were analyzed using a DSL-3900 ACTIVE Progesterone Coated-Tube Radioimmunoassay Kit (Diagnostic Systems Laboratories Inc., Webster, USA) with the assistance of the Neodin Veterinary Laboratory (Seoul, Republic of Korea; http://www.vetlab.co.kr). The day on which the serum P4 level reached 4.0 ng/mL was regarded as the day of ovulation [6,7,10,11]. Specimens for vaginal cytology were obtained on sterile cotton swabs, which were rolled on glass slides and stained with Diff-Quik (Sysmex Co., Kobe, Japan).
Parentage analysis was performed on blood samples taken from the puppies that were produced by AI, from the cloned male dog, and from the cloned female dogs as well. To extract genomic DNA, blood samples were incubated overnight with a lysis buffer (0.05 M Tris, 0.05 M ethylenediamine tetraacetic acid [EDTA], 0.5% sodium dodecyl sulfate [SDS], pH 8.0) supplemented with 400 mg proteinase K, followed by phenol extraction and ethanol precipitation. The following nine markers were selected for analysis: PEZ02, PEZ06, PEZ08, PEZ15, PEZ20, CPH04, CPH07, CPH09, and CPH12. Isolated genomic DNA samples were dissolved in 50 mL TE (Tris-EDTA) and used for
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microsatellite assay with nine specific markers originally derived from dogs [15–17]. Length variations were assayed by polymerase chain reaction (PCR) amplification with fluorescently labeled locus-specific primers and PAGE (polyacrylamide gel electrophoresis) on an automated DNA sequencer (ABI 3130xl; Applied Biosystems, Foster City, CA, USA). Proprietary software (GeneScan and Genotyper; Applied Biosystems) was used to estimate the PCR product sizes in nucleotides. 2.7. Growth and hematologic profiles of the offspring (F1 puppies) To compare growth rates of puppies, their weights were monitored daily for the first month of life, then weekly until the dogs were 3 mo of age. Blood samples were collected from the puppies via jugular vein cannulation at 1 mo of age for a complete blood count (CBC) and serum biochemistry. Blood samples (3 mL) were collected in anticoagulant tubes, and serum samples were obtained by centrifugation at 6000 rpm for 10 min. For serum biochemistry, alanine aminotransferase (ALT), aspartate aminotransferase (AST), alkaline
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phosphatase (ALP), blood urea nitrogen (BUN), creatine, glucose, total bilirubin, albumin, total protein, phosphorus and calcium were examined. Another 1 mL blood was collected into an evacuated tube containing EDTA to measure red blood cell (RBC) count, white blood cell (WBC) count, hemoglobin concentration (Hb), packed cell volume (PCV), mean corpuscular volume (MCV), mean corpuscular hemoglobin (MCH), and mean corpuscular hemoglobin concentration (MCHC). For determination of CBC, a Celltac-a hematology analyzer (Nihon Kohden, Tokyo, Japan) was used. Serum biochemistry profile was determined using a Selectra 2 chemistry analyzer (Merck, Amsterdam, The Netherlands) according to the manufacturer’s protocols. The mean weight of puppies was compared with average growth data of an Afghan litter from birth to 12 wk [18,19], and the hematologic and serum chemistry values of puppies were compared with reference data [20]. 2.8. Experimental design The purpose of this study was to investigate reproductive competence of cloned dogs, and experiments were performed as shown in Figure 1. In a cloned
Fig. 1. Schematic illustration of the experimental design.
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Fig. 2. (A) Spermatozoa collected from a cloned male dog and (B) fertilized embryos.
male dog, semen was collected and analyzed by CASA, and in vivo fertilization was performed using a noncloned female dog to evaluate the fertilization ability of sperm. In three female dogs, after detecting onset of estrus, ovulation timing was determined by P4 concentration and vaginal cytology, and AI was carried out by two technical methods (intrauterine/transcervical). Because this was an observational study with no control group, a successful outcome was defined as a successful pregnancy and delivery of puppies without complications. The parameters measured in spermatozoa of the cloned male dog, changes in P4 concentration and the day of first observed estrus in cloned female dogs were compared with reference data for normal dogs [13,21–23]. The health status of offspring sired by cloned dogs was evaluated by body weight gain and blood analysis. 3. Results 3.1. Fertility of cloned male dog
Table 1 Analysis of ejaculated fresh semen from a cloned male dog by computer-assisted motility analyzer. Parameters
Reference range*
Cloned male dog
Motility (%) VCL (mm/sec) VSL (mm/sec) VAP (mm/sec) BCF (Hz) MAD (degree) WOB (%) DNC (mm2/sec) ALH (mm)
92–98 139–207 61–71 73–98 9–14 17–22 47–59 860–1765 6–9
97 233 123 128 8 19 555 2096 9
VCL, curvilinear velocity; VSL, straight line velocity; VAP, average path velocity; BCF, beat cross-frequency; MAD, mean angular displacement; WOB, wobble (VAP VCL); DNC, dance (VCL ALH); ALH, lateral head displacement. * Reference data from Kim et al. [12].
ficial in parallel with the changes in P4 level (Fig. 4). The P4 concentrations of clones A, B and C on the estimated day of ovulation were 5.81, 5.45, and 5.43 ng/ mL, respectively.
Semen analysis and AI were used to determine the fertility of the cloned male dog. Semen analysis was performed using fresh semen (Fig. 2) by CASA. Results showed that motility, BCF, MAD, and ALH were all within the reference ranges (Table 1). Spermatozoa inseminated into the oviducts of noncloned female dogs successfully fertilized ova as was evidence by formation of the 8-cell stage embryos (Fig. 2). 3.2. Fertility of cloned female dogs The first estrus was exhibited by cloned Females A, B and C at the ages of 627, 604, and 716 d, respectively, which were within the reference range [21]. The changes in serum P4 level during the estrus cycle in these dogs were also within the reference range (Fig. 3). Vaginal epithelial cells changed from basal to super-
Fig. 3. Progesterone changes in three cloned female dogs. The hormone was measured every day after finding vaginal discharge as a sign of estrus.
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After having evidence of the fertility of the cloned male dog and the exhibition of estrus cycling in the cloned female dogs, we undertook AI using fresh semen. To exclude individual sample differences, an aliquot of semen (10 mL) was assessed for motility
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and number of sperm using a phase-contrast microscope before using collected semen for AI. The two inseminated cloned dogs, Females A and C, were both diagnosed as pregnant and successfully gave birth to litters (four and six puppies, respectively) by
Fig. 4. Representative pictures of vaginal cytology in one of three cloned female dogs. Vaginal smear samples were collected every day. Corresponding P4 concentrations (ng/mL) on the day of smear were as follows: (A) 0.61 ng/mL, (B) 0.69 ng/mL, (C) 0.89 ng/mL, (D) 2.75 ng/mL, (E) 5.81 ng/mL, (F) 7.80 ng/mL, (G) 13.39 ng/mL, (H) 20.94 ng/mL. Artificial insemination was performed on Day H.
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Fig. 5. Representative pictures of pregnant female dogs and their puppies. (A) Ultrasonogram of fetus (arrow) in the fetal vesicle on Day 38 after AI. Fetal head and ribs are identifiable. Fetal heartbeat was recognized. (B) Lateral radiograph of pregnant cloned dog at near term. The alignments of the six fetal skull bones (arrowheads) as well as the arrangement of the cervical through lumbar vertebrae and the pelvis with the appendages are identified. (C) Four puppies born from Female A and all the F1 puppies were raised under the care of their mothers. (D) F1 puppies from a cloned female dog at 23 d old.
natural delivery (Fig. 5). The puppies were all born alive, exhibiting no congenital abnormalities. Microsatellite analysis of genomic DNA extracted from blood cells confirmed that the puppies originated
from the cloned male and female dogs (Table 2). The F1 puppies were cared for by their biological dam until they were weaned at approximately 30 d of age.
Table 2 Microsatellite genotypes of 13 dogs, including the cloned male dog (Father), two cloned female dogs (Females A and C), and live F1 offspring (A1 to A3, C1 to C6).
Father Female A Puppy A1 Puppy A2 Puppy A3 Female C Puppy C1 Puppy C2 Puppy C3 Puppy C4 Puppy C5 Puppy C6
PEZ02
PEZ06
PEZ08
PEZ15
PEZ20
CPH04
CPH07
CPH09
CPH12
121/121 121/121 121/121 121/121 121/121 121/121 121/121 121/121 121/121 121/121 121/121 121/121
180/180 180/184 180/180 180/180 180/180 180/184 180/180 180/184 180/180 180/180 180/184 180/180
230/230 230/246 230/230 230/230 230/230 230/246 230/230 230/230 230/230 230/230 230/230 230/230
204/204 204/208 204/208 204/204 204/204 204/208 204/208 204/208 204/204 204/204 204/204 204/208
174/174 178/178 174/178 174/178 174/178 178/178 174/178 174/178 174/178 174/178 174/178 174/178
139/139 139/141 139/141 139/139 139/141 139/141 139/139 139/141 139/141 139/139 139/139 139/139
162/168 162/162 162/162 162/162 162/168 162/162 162/168 162/162 162/168 162/168 162/162 162/162
147/147 139/147 139/147 147/147 139/147 139/147 147/147 139/147 139/147 147/147 139/147 139/147
202/202 191/202 191/202 202/202 202/202 191/202 191/202 202/202 202/202 191/202 202/202 191/202
Nine canine-specific markers were used for microsatellite assay.
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Table 3 F1 puppies sired by a cloned male dog by AI. Cloned female dog
Method of insemination
Pregnancy length (d)
Name of puppy
Sex
Birth weight (g)
Phenotype; coat color
Female A
Intrauterine insemination (surgical)
60
A1
Female
410
Cream white
60
A2 A3 A4* C1
Female Male Female Male
460 510 360 460
Gray with shaded mask Gold with shaded mask Black Brown brindle
C2 C3 C4 C5 C6
Female Male Female Female Female
430 480 310 380 420
Black and tans Domino Domino Gold with shaded mask Black and tans
Female C
*
Transcervical insemination
This puppy died within 2 wk after birth due to small body size and anorexia.
The mean birth weights of F1 puppies from the two cloned females were 435 32.27 g and 413.33 24.99 g (Table 3). The sex ratio and coat color of the 10 puppies is shown in Table 3. The surviving (9 of 10) F1 puppies showed consistent weight gain with no significant difference among them and was within the reference ranges (Fig. 6). Individual hematologic and serum biochemical data at 1 mo of age are listed in Tables 4 and 5. All values for the F1 puppies were within laboratory reference ranges for healthy Afghan dogs. 4. Discussion Since the first cloned mammal ‘‘Dolly’’ was born in 1997 [22], there have been concerns regarding the reproductive competence of cloned animals. Several groups have examined the perinatal and postnatal development and reproductive characteristics of cloned animals and demonstrated their fertility [2,23–27]. In the current study, we demonstrated that cloned dogs are fertile and reproductively healthy.
To determine the fertility of a male dog, semen samples were analyzed by CASA and a noncloned female dog inseminated. Several parameters of sperm motility, including BCF, MAD, and ALH, were within the reference ranges. Interestingly, however, VCL, VSL, VAP, WOB, and DNC were higher than reference ranges. These CASA parameters are related to velocity of spermatozoa, as they are one of the parameters affecting the outcome of in vitro fertilization and the ability of spermatozoa to penetrate cervical mucus and fuse with an oocyte [28,29]. It is unlikely that this is a unique feature of a cloned dog, but further characterization is needed using an increased number of cloned dogs or comparing the sperm quality of the cloned dogs with that of the donor. The donor dog has been neutered, and, therefore, the experiment could not be performed at the moment. Nonetheless, the fact that all the embryos collected were at the 8-cell stage 4 d after insemination was in agreement with a previous report that spermatozoa produced by a cloned male dog can generate the same
Fig. 6. The body weight change patterns of F1 puppies from birth to 3 mo of age. A1 to A4: puppies born between cloned male dog and Female A. C1 to C6: puppies born between cloned male dog and Female C. Puppy A4 died within 2 wk after birth due to small body size and anorexia. Data from Niblock [19].
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Table 4 Age-specific reference ranges and results of hematologic tests in F1 offspring at 1 mo of age. Parameter (units) 3
WBC (10 /mL) RBC (106/mL) Hb (g/dL) PCV (%) MCV (fL) MCH (pg) MCHC (g/dL)
Reference range*
Female A puppies
Female C puppies
A1
A2
A3
Mean SE
C1
C2
C3
C4
C5
C6
Mean SE
15.7 4.4 4.7 0.4 10.3 0.9 31.4 2.4 65.8 2.3 21.8 1.2 32.6 1.8
15.8 5.43 11.3 33.9 62.4 20.8 33.3
13.3 4.93 10.1 31.7 64.3 20.5 31.9
19.8 4.79 10.4 30.8 64.3 21.7 33.8
16.3 1.9 5.1 0.2 10.6 0.4 32.1 0.9 63.7 0.6 21.0 0.4 33.0 0.6
16.4 4.74 10.1 30.6 64.6 21.3 33
17.1 4.5 9.7 29 64.4 21.6 33.4
17.3 5.15 10.7 32.5 63.1 20.8 32.9
14.8 4.14 8.6 26.9 65 20.8 32
13.6 4.74 10.2 30.3 63.9 21.5 33.7
16.8 4.68 9.8 30.5 65.2 20.9 32.1
16.0 0.6 4.7 0.1 9.9 0.3 30.0 0.8 64.4 0.3 21.2 0.1 32.9 0.3
WBC, white blood cell count; RBC, red blood cell count; Hb, hemoglobin concentration; PCV, packed cell volume; MCV, mean corpuscular volume; MCH, mean corpuscular hemoglobin; MCHC, mean corpuscular hemoglobin concentration. * Data from Shifrine et al. [20].
Table 5 Age-specific reference ranges and results of serum biochemical tests in F1 offspring at 1 mo of age. Parameter (units)
ALT (U/L) AST (U/L) ALP (U/L) BUN (mg/dL) Creatinine (mg/dL) Glucose (mg/dL) Bilirubin (mg/dL) Albumin (g/dL) Total protein (g/dL) Calcium (mg/dL) Phosphate (mg/dL)
Reference rangey
37 12.2 44 10.2 162 39.9 12 2.3 0.6 0.04 83 12.8 0.2 0.2 3.0 0.14 5.0 0.23 10.6 0.34 8.8 0.47
Female A puppies
Female C puppies
A1
A2
A3
Mean SE
C1
C2
C3
C4
C5
C6
Mean SE
42 35 208 9 0.6 117 0.4 2.4 3.7 10.7 9.1
36 32 197 5 0.6 105 0.2 2.3 3.6 10.6 8.7
33 29 222 7 0.6 113 0.2 2.3 3.6 10.8 9.4
37.0 2.2 32.0 1.4 209.0 5.9 7.0 0.9 0.6 0.0 111.7 2.9 0.3 0.1 2.3 0.05 3.6 0.03 10.7 0.05 9.1 0.2
25 24 201 8 0.6 128 0.3 2.2 3.7 10.5 10.1
34 38 270 8 0.6 134 0.7 2.4 4 10.8 10.2
32 24 200 8 0.6 120 0.3 2.2 3.2 9.8 8.8
33 30 159 7 0.6 123 0.3 2.5 3.9 11.2 10.3
33 43 306 7 0.6 112 0.5 2.6 4.2 10.6 10.8
31 29 170 8 0.6 114 0.3 2.3 3.6 10.4 9.6
31.3 1.3 31.3 3.1 217.7 23.7 7.7 0.2 0.6 0.0 121.8 3.4 0.4 0.1 2.4 0.1 3.8 0.1 10.6 0.2 10.0 0.3
ALT, alanine aminotransferase; AST, aspartate aminotransferase; ALP, alkaline phosphatase; BUN, blood urea nitrogen. y Four-month-old beagle blood chemistry values (Covance Research Products Inc., Cumberland, VA, USA).
developmental stage of embryos with noncloned dogs [30]. Even though the cloned male dog used in this study exhibited libido along with other mating behavior, his inexperience in the clasping technique made it difficult to use natural mating for testing fertility. It is well documented that one of the most common mating problems of the dog is caused by inexperience, and AI is the best alternative [21,31]. For such reason, the AI method was used in this study utilizing fresh semen from the cloned male dog, which resulted in pregnancy and successful births in both female clones. The body weights of the puppies were monitored to evaluate growth because it is widely assumed that birth weight and body weight gains are important survival determinants in most mammalian species. The birth weights of puppies derived from cloned dogs were within reference ranges and showed consistent weight gains without any disorders. Additionally, as further evidence of the
puppies’ health, hematologic and serum biochemical measurements were performed to provide fundamental biological data on physiologic status, and the values obtained were mostly within the reference ranges, except for some hematology and serum parameters. It is widely assumed that age appeared to influence several serum biochemical parameters in young dogs [20,32,33], thus we think the F1 puppies did not reveal any abnormal values in blood analysis results, similar to the results of evaluation in litters produced by mating pairs of other cloned dams and sires [2,24,34,35]. These results appear to support the hypothesis that the offspring from cloned dogs have similar growth performance and health to those of naturally bred puppies. In conclusion, the current study demonstrates that both cloned male and female dogs are fertile, allowing the application of cloning technology for the purpose of expanding an elite canine gene pool in the dog.
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