RESUMEN
Canine oocytes ovulated at prophase complete meiosis and continue to develop in presence of a high progesterone concentration in the oviduct. Considering that meiotic competence of canine oocyte is accomplished in the oviductal environment, we postulate that hormonal milieu resulting from the circulating progesterone concentration may affect oocyte maturation and early development of embryos. From 237 oocyte donors, 2620 oocytes were collected and their meiotic status and morphology were determined. To determine optimal characteristics of the mature oocytes subjected to nuclear transfer, a proportion of the meiotic status of the oocytes were classified in reference to time post-ovulation as well as progesterone (P4) level. A high proportion of matured oocytes were collected from >126h (55.5%) post-ovulation or 40-50ngmL-1 (46.4%) group compared to the other groups. Of the oocyte donors that provided mature oocytes in vivo, there was no correlation between serum progesterone of donors and time post ovulation, however, time post-ovulation were significantly shorter for <30ng/mL group (P<0.05). Using mature oocytes, 1161 cloned embryos were reconstructed and transferred into 77 surrogates. In order to determine the relationship between pregnancy performance and serum progesterone level, embryos were transferred into surrogates showing various P4 serum levels. The highest pregnancy (31.8%) and live birth cloning efficacy (2.2%) rates were observed when the embryos were transferred into surrogates with circulating P4 levels were from 40 to 50ngmL-1. In conclusion, measurement of circulating progesterone of female dog could be a suitable an indicator of the optimal time to collect quality oocyte and to select surrogates for cloning.
Asunto(s)
Clonación de Organismos/veterinaria , Perros/fisiología , Técnicas de Maduración In Vitro de los Oocitos/veterinaria , Técnicas de Transferencia Nuclear , Oocitos/fisiología , Animales , Transferencia de Embrión/veterinaria , Femenino , Ovulación , Embarazo , Progesterona/sangreRESUMEN
Dog cloning offers a substantial potential because of the advancements in assisted reproductive technology and development of the human disease model in line with the transgenic technique. However, little is known about the development of the canine cloned embryo during the preimplantation period. The aim of this study was to investigate the most efficient method and time for collecting cloned canine preimplantation embryos and to ascertain the developmental timeline of cloned canine embryos. Two hundred cloned embryos were created and transferred into 11 surrogates. The preimplantation stage cloned embryos were then collected on Days 7, 8, and 9 using an ovariohysterectomy or the Foley balloon catheter method. The recovery rate of reconstructed embryos was 63.6% and 60.6% for the ovariohysterectomy and Foley balloon catheter methods, respectively. Although significant differences were observed in the early developmental stages (one-cell and 16-cell stages), no significant difference was observed in the blastocyst stage. Significantly higher blastocyst rate was observed when the embryos were collected on Day 8 (11.4%) than on Day 7 (0.0%; P < 0.05). At the proximal uterine horn on Day 7, no embryos at any stage were found, whereas on Days 8 and 9, blastocysts were found. We have observed a 63% initial pregnancy rate at 25 to 30 days after embryo transfer and a 50% full-term pregnancy rate, whereas 6.3% of the puppies were born, and 5.5% were born live among the total transferred embryos. Our results suggest that cloned embryos can develop to blastocysts by Day 8, and full-term pregnancy can be achieved after embryo transfer in canine.
Asunto(s)
Clonación de Organismos , Perros/embriología , Histerectomía/veterinaria , Resultado del Embarazo , Preñez , Recolección de Tejidos y Órganos , Animales , Transferencia de Embrión/métodos , Transferencia de Embrión/veterinaria , Femenino , EmbarazoRESUMEN
Somatic cell nuclear transfer (SCNT) provides an excellent model for studying epigenomic reprogramming during mammalian development. We mapped the whole genome and whole methylome for potential anomalies of mutations or epimutations in SCNT-generated dogs with XY chromosomal sex but complete gonadal dysgenesis, which is classified as 78, XY disorder of sex development (DSD). Whole genome sequencing revealed no potential genomic variations that could explain the pathogenesis of DSD. However, extensive but stochastic anomalies of genome-wide DNA methylation were discovered in these SCNT DSD dogs. Persistent abnormal hypermethylation of the SRY gene was observed together with its down-regulated mRNA and protein expression. Failure of SRY expression due to hypermethylation was further correlated with silencing of a serial of testis determining genes, including SOX9, SF1, SOX8, AMH and DMRT1 in an early embryonic development stage at E34 in the XY(DSD) gonad, and high activation of the female specific genes, including FOXL2, RSPO1, CYP19A1, WNT4, ERα and ERß, after one postnatal year in the ovotestis. Our results demonstrate that incomplete demethylation on the SRY gene is the driving cause of XY(DSD) in these XY DSD dogs, indicating a central role of epigenetic regulation in sex determination.
Asunto(s)
Metilación de ADN , Trastornos del Desarrollo Sexual/genética , Técnicas de Transferencia Nuclear/efectos adversos , Proteína de la Región Y Determinante del Sexo/genética , Animales , Clonación de Organismos , Modelos Animales de Enfermedad , Trastornos del Desarrollo Sexual/etiología , Trastornos del Desarrollo Sexual/metabolismo , Perros , Epigénesis Genética , Femenino , Disgenesia Gonadal/etiología , Disgenesia Gonadal/genética , Disgenesia Gonadal/metabolismo , Masculino , Embarazo , Procesos de Determinación del Sexo , Procesos Estocásticos , Testículo/embriología , Testículo/metabolismo , Cromosoma Y/genéticaRESUMEN
Using in vivo-flushed oocytes from a homogenous dog population and subsequent embryo transfer after nuclear transfer, we studied the effects of donor cells collected from 10 different breeds on cloning efficiency and perinatal development of resulted cloned puppies. The breeds were categorized into four groups according to their body weight: small (≤9 kg), medium (>9-20 kg), large (>20-40 kg), and ultra large (>40 kg). A total of 1611 cloned embryos were transferred into 454 surrogate bitches for production of cloned puppies. No statistically significant differences were observed for initial pregnancy rates at Day 30 of embryo transfer for the donor cells originated from different breeds. However, full-term pregnancy rates were 16.5%, 11.0%, 10.0%, and 7.1% for the donor cells originated from ultra-large breed, large, medium, and small breeds, respectively, where pregnancy rate in the ultra-large group was significantly higher compared with the small breeds (P < 0.01). Perinatal mortality until weaning was significantly higher in small breeds (33.3%) compared with medium, large, or ultra-large breeds where no mortality was observed. The mean birth weight of cloned pups significantly increased proportional to breed size. The highest litter size was examined in ultra-large breeds. There was no correlation between the number of embryo transferred and litter size. Taken together, the efficiency of somatic cell cloning and fetal survival after embryo transfer may be affected significantly by selecting the appropriate genotype.
Asunto(s)
Clonación de Organismos/veterinaria , Perros/genética , Perros/fisiología , Fibroblastos , Animales , Animales Recién Nacidos , Peso Corporal , Transferencia de Embrión/veterinaria , Embrión de Mamíferos/citología , Embrión de Mamíferos/embriología , Femenino , Genotipo , Masculino , EmbarazoRESUMEN
Canines are considered the most authentic model for studying multifactorial human diseases, as these animals typically share a common environment with man. Somatic cell nuclear transfer (SCNT) technology along with genetic engineering of nuclear donor cells provides a unique opportunity for examining human diseases using transgenic canines. In the present study, we generated transgenic canines that overexpressed the human amyloid precursor protein (APP) gene containing well-characterized familial Alzheimer's disease (AD) mutations. We successfully obtained five out of six live puppies by SCNT. This was confirmed by observing the expression of green fluorescence protein in the body as a visual transgenic marker and the overexpression of the mutated APP gene in the brain. The transgenic canines developed AD-like symptoms, such as enlarged ventricles, an atrophied hippocampus, and ß-amyloid plaques in the brain. Thus, the transgenic canines we created can serve as a novel animal model for studying human AD.
Asunto(s)
Enfermedad de Alzheimer/patología , Precursor de Proteína beta-Amiloide/genética , Proteínas Mutantes/metabolismo , Mutación/genética , Animales , Animales Modificados Genéticamente , Encéfalo/metabolismo , Encéfalo/patología , Línea Celular , Línea Celular Tumoral , Núcleo Celular/metabolismo , Modelos Animales de Enfermedad , Perros , Fibroblastos/metabolismo , Vectores Genéticos/metabolismo , Proteínas Fluorescentes Verdes/metabolismo , Humanos , Immunoblotting , Reacción en Cadena de la Polimerasa , Regiones Promotoras Genéticas/genética , Reproducibilidad de los Resultados , Antígenos Thy-1/genética , Transgenes/genéticaRESUMEN
Interspecies somatic cell nuclear transfer (iSCNT) is an emerging assisted reproductive technology (ART) for preserving Nature's diversity. The scarcity of oocytes from some species makes utilisation of readily available oocytes inevitable. In the present study, we describe the successful cloning of coyotes (Canis latrans) through iSCNT using oocytes from domestic dogs (Canis lupus familiaris or dingo). Transfer of 320 interspecies-reconstructed embryos into 22 domestic dog recipients resulted in six pregnancies, from which eight viable offspring were delivered. Fusion rate and cloning efficiency during iSCNT cloning of coyotes were not significantly different from those observed during intraspecies cloning of domestic dogs. Using neonatal fibroblasts as donor cells significantly improved the cloning efficiency compared with cloning using adult fibroblast donor cells (P<0.05). The use of domestic dog oocytes in the cloning of coyotes in the present study holds promise for cloning other endangered species in the Canidae family using similar techniques. However, there are still limitations of the iSCNT technology, as demonstrated by births of morphologically abnormal coyotes and the clones' inheritance of maternal domestic dog mitochondrial DNA.
Asunto(s)
Clonación de Organismos/veterinaria , Coyotes/genética , Especies en Peligro de Extinción , Fibroblastos/fisiología , Técnicas de Transferencia Nuclear/veterinaria , Oocitos/fisiología , Animales , Animales Endogámicos , Células Cultivadas , Clonación de Organismos/efectos adversos , Anomalías Congénitas/etiología , Anomalías Congénitas/veterinaria , Coyotes/fisiología , Cruzamientos Genéticos , ADN Mitocondrial/metabolismo , Perros , Transferencia de Embrión/veterinaria , Femenino , Nacimiento Vivo/veterinaria , Masculino , Repeticiones de Microsatélite , Técnicas de Transferencia Nuclear/efectos adversos , Recuperación del Oocito/veterinaria , Embarazo , República de Corea , Mortinato/veterinariaRESUMEN
Dogs are useful models for studying human metabolic diseases such as type 2 diabetes mellitus due to similarities in physiology, anatomy and life styles with humans. Somatic cell nuclear transfer (SCNT) facilitates the production of transgenic dogs. In this study, we generated transgenic dogs expressing the phosphoenolpyruvate carboxykinase (PEPCK) gene, which is closely involved in the pathogenesis of type 2 diabetes mellitus. In addition, we assessed the cloning efficiency associated with adult or fetal (cloned or natural mating) fibroblasts as a nuclear source. Cloning efficiency was determined by the fusion, pregnancy and cloning rates. The fusion rates were significantly high for fibroblasts from cloned fetuses, but the pregnancy and cloning rates were relatively high for cells from normal fetuses. Based on these data, fetal fibroblasts were selected as the nuclear donor for SCNT and genetically engineered to overexpress the PEPCK gene and dual selection marker genes controlled by the PEPCK promoter. The transgenic cells were introduced into oocytes and transferred into five recipient dogs, resulting in two pregnancies. Finally, three puppies were born and confirmed by microsatellite analysis to be genetically identical to the donor. One puppy successfully overexpressed PEPCK mRNA and protein in the liver. This canine disease model may be useful for studying the pathogenesis and/or therapeutic targets of type 2 diabetes mellitus.
Asunto(s)
Diabetes Mellitus Tipo 2/genética , Modelos Animales de Enfermedad , Perros , Proteínas Serina-Treonina Quinasas/genética , Animales , Animales Modificados Genéticamente , Línea Celular , Diabetes Mellitus Tipo 2/enzimología , Perros/genética , Transferencia de Embrión/métodos , Femenino , Fibroblastos/metabolismo , Expresión Génica , Orden Génico , Marcación de Gen , Vectores Genéticos , Humanos , Técnicas de Transferencia Nuclear , Regiones Promotoras Genéticas , Proteínas Serina-Treonina Quinasas/metabolismoRESUMEN
The present study was undertaken to evaluate two activation methods for somatic cell nuclear transfer (SCNT), namely, fusion and simultaneous activation (FSA, fusion medium contains calcium), versus fusion followed by chemical activation (F+CA, fusion medium does not contain calcium), and to evaluate the effects of parity of recipient dogs on the success of SCNT. Oocytes retrieved from outbred dogs were reconstructed with adult somatic cells collected from an 11-year-old female dog named Missy. In the FSA method, oocytes were fused and activated at the same time using two DC pulses of 1.75 kV/cm for 15 microsec. In the F+CA method, oocytes were fused with two DC pulses of 1.75 kV/cm for 15 microsec, and then activated 1 h after fusion by 10 microM calcium ionophore for 4 m and cultured for 4 h in 1.9 mM 6-dimethylaminopurine for postactivation. Activation method had a significant impact on the production efficiency of cloned dogs. There was a significant difference in full-term pregnancy rate and percentage of live puppies between the two methods (6.3% and 38.5% for FSA and F+CA, respectively). In our study, four out of five live offspring produced by F+CA survived versus FSA, which did not result in any surviving puppies. Overall, as few as 14 dogs and 54 reconstructed embryos were needed to produce a cloned puppy. In addition, the parity of recipient bitches had no effect on the success of SCNT in canine species. Both the nullipara and multipara bitches produced live puppies following SCNT-ET.
Asunto(s)
Clonación de Organismos/veterinaria , Perros/genética , Técnicas de Transferencia Nuclear/veterinaria , Animales , Calcio/química , Clonación de Organismos/métodos , Genotipo , Repeticiones de Microsatélite/genética , Oocitos/fisiologíaRESUMEN
The present study was undertaken to evaluate two enucleation methods for somatic cell nuclear transfer (SCNT), and to standardize the optimum number of embryos for transfer to each recipient for canines. Oocytes retrieved from outbreed dogs were reconstructed with adult somatic cells from a male Beagle dog. A total of 134 or 267 oocytes were enucleated either by aspiration or squeezing method, fused with two DC pulses of 1.75 kV/cm for 15 micros electrical stimulation, chemically activated after 1h of fusion using 10 microM calcium ionophore for 4 min and cultured 4h in 1.9 mM 6-dimethylaminopurine. Finally, 103 or 214 embryos for aspiration or squeezing method were transferred to 6 or 11 naturally synchronized recipients, respectively. A total of 53, 317 and 342 embryos were transferred to 7, 17 and 12 recipients for the group of 4-10, 11-25 and 26-40 embryos, respectively. There was no difference between fusion rate (76.87% vs. 80.15%), full term pregnancy rate (16.66% vs. 27.27%) and percent of live puppies born (0.97% vs. 1.87%) for aspiration and squeezing method (P>0.05). Production efficiency of cloned dogs was significantly affected by the number of embryos transferred to each recipient. No pregnancy was established for the group of 4-10 embryos (n=7) and 26-40 embryos (n=12) while pregnancy was detected in 23.53% recipients received a group of 11-25 embryos (n=17). Among them, five (1.76%) live puppies were born (P<0.05). These data show an increase in the overall efficiency of SCNT in canine species.
Asunto(s)
Clonación de Organismos/veterinaria , Perros/embriología , Transferencia de Embrión/veterinaria , Técnicas de Transferencia Nuclear/veterinaria , Animales , Clonación de Organismos/métodos , Perros/genética , Estimulación Eléctrica , Transferencia de Embrión/métodos , Femenino , Fibroblastos/ultraestructura , Genotipo , Masculino , Repeticiones de Microsatélite/genética , Oocitos/ultraestructura , Embarazo , Resultado del EmbarazoRESUMEN
To improve the efficiency of somatic cell nuclear transfer (SCNT) in dogs, we evaluated whether or not the interval between fusion and activation affects the success rate of SCNT. Oocytes retrieved from outbred dogs were reconstructed with adult somatic cells from a male or female Golden Retriever. In total, 151 and 225 reconstructed oocytes were transferred to 9 and 14 naturally synchronized surrogates for male and female donor cells, respectively. Chromosomal morphology was evaluated in 12 oocytes held for an interval of 2 hr between fusion and activation and 14 oocytes held for an interval of 4 hr. Three hundred seventy-six and 288 embryos were transferred to 23 and 16 surrogates for the 2 and 4 hr interval groups, respectively. Both the male (two pregnant surrogates gave birth to three puppies) and female (one pregnant surrogate gave birth to one puppy) donor cells gave birth to live puppies (P > 0.05). In the 2 hr group, significantly more reconstructed oocytes showed condensed, metaphase-like chromosomes compared to the 4 hr group (P < 0.05). A significantly higher pregnancy rate and a greater number of live born puppies were observed in the 2 hr group (13.0% and 1.1%, respectively) compared to the 4 hr group (0%) (P < 0.05). In total, three surrogate dogs carried pregnancies to term and four puppies were born. These results demonstrate that decreasing the interval between fusion and activation increases the success rate of clone production and pregnancy. These results may increase the overall efficiency of SCNT in the canine family.
Asunto(s)
Clonación de Organismos , Perros/genética , Técnicas de Cultivo de Embriones/veterinaria , Embrión de Mamíferos/fisiología , Técnicas de Transferencia Nuclear/veterinaria , Animales , Interpretación Estadística de Datos , Perros/fisiología , Técnicas de Cultivo de Embriones/métodos , Embrión de Mamíferos/ultraestructura , Femenino , Masculino , Repeticiones de Microsatélite/genética , Oocitos/fisiología , Embarazo , Índice de Embarazo , Factores de TiempoRESUMEN
The aim of this study was to examine the effects of modifications to a standard slow freezing protocol on the viability of in vitro produced bovine embryos. Bovine oocytes were matured, fertilized with frozen-thawed semen, and presumptive zygotes cultured in defined two-step culture media. The standard freezing medium was 1.5M ethylene glycol (EG), 0.1M sucrose, 10% fetal bovine serum (FBS) in Dulbecco's phosphate buffered saline (D-PBS). A preliminary trial showed that in vitro produced embryos cryopreserved in this medium had a survival rate of 74.6% at 24h and 53.5% at 48 h post-thaw. Experiment 1 studied the effects of omitting the sucrose supplement or replacing it with 0.1M xylose. In Experiment 2, the effects of partial (0%, 25% or 50%) or total (100%) replacement of sodium chloride with choline chloride in the cryopreservation medium were examined (the medium with 100% replacement was designated CJ1). The effects of replacing the 10% FBS with 0.4% BSA or 0.4% lipid-rich BSA (Albumax I) in CJ1 was studied in Experiment 3. In Experiment 4, pregnancy/calving rates following the post-thaw transfer of in vitro produced embryos cryopreserved in the standard freezing medium were compared with those of in vitro and in vivo produced embryos cryopreserved in the improved medium (Albumax I in CJ1). Supplementation of the cryopreservation medium with 0.1M sucrose resulted in higher post-thaw survival rates at 24 h (71.3% versus 53.5 and 51.7%; P<0.05), 48 h (51.1% versus 45.3 and 40.2%), and 72 h (34.0% versus 24.4 and 23.0%) than 0.1M xylose or no supplement, respectively, in Experiment 1. Experiment 2 showed that embryos cryopreserved in the standard medium had poorer survival rates at 24 h (72.8% versus 86.5%; P<0.05), 48 h (53.1% versus 66.3%) or 72 h (28.4% versus 44.9%) than those frozen in CJ1. The post-thaw survival rate of embryos frozen in medium supplemented with Albumax I was better than that for the FBS or BSA supplements at 24h (92.0% versus 90.7 and 87.3%), 48 h (87.3% versus 76.9 and 70.9%; P<0.05), and 72 h (70.4% versus 49.1 and 46 4%; P<0.05; Experiment 3). In Experiment 4, in vitro produced embryos cryopreserved in CJ1 medium supplemented with Albumax I resulted in higher pregnancy rates at Day 35 (31.9% versus 22.9%) and Day 60 (24.1% versus 14.3%) of gestation, and calving rates (22.6% versus 10.0%; P<0.05) than similar embryos frozen in the standard medium. However, in vivo produced embryos cryopreserved in Albumax I in CJ1 resulted in higher pregnancy rates at Day 35 (50.7%; P<0.05) and Day 60 (45.1%; P<0.05) of gestation, and calving rate (43.7%; P<0.05). It was concluded that modification of the freezing medium by addition of lipid-rich BSA and replacing sodium chloride with choline chloride improves the post-thaw survival of in vitro produced embryos, and their viability post-transfer.
Asunto(s)
Blastocisto/fisiología , Criopreservación/veterinaria , Animales , Tasa de Natalidad , Bovinos , Supervivencia Celular , Colina/metabolismo , Medios de Cultivo/química , Técnicas de Cultivo de Embriones , Transferencia de Embrión , Femenino , Fertilización In Vitro , Masculino , Embarazo , Albúmina Sérica Bovina/metabolismo , Sacarosa/metabolismo , Factores de Tiempo , Xilosa/metabolismoRESUMEN
Over the world, canine species, including the gray wolf, have been gradually endangered or extinct. Many efforts have been made to recover and conserve these canids. The aim of this study was to produce the endangered gray wolf with somatic cell nuclear transfer (SCNT) for conservation. Adult ear fibroblasts from a female gray wolf (Canis lupus) were isolated and cultured in vitro as donor cells. Because of limitations in obtaining gray wolf matured oocytes, in vivo matured canine oocytes obtained by flushing the oviducts from the isthmus to the infundibulum were used. After removing the cumulus cells, the oocyte was enucleated, microinjected, fused with a donor cell, and activated. The reconstructed cloned wolf embryos were transferred into the oviducts of the naturally synchronized surrogate mothers. Two pregnancies were detected by ultrasonography at 23 days of gestation in recipient dogs. In each surrogate dog, two fetal sacs were confirmed by early pregnancy diagnosis at 23 days, but only two cloned wolves were delivered. The first cloned wolf was delivered by cesarean section on October 18, 2005, 60 days after embryo transfer. The second cloned wolf was delivered on October 26, 2005, at 61 days postembryo transfer. Microsatellite analysis was performed with genomic DNA from the donor wolf, the two cloned wolves, and the two surrogate female recipients to confirm the genetic identity of the cloned wolves. Analysis of 19 microsatellite loci confirmed that the cloned wolves were genetically identical to the donor wolf. In conclusion, we demonstrated live birth of two cloned gray wolves by nuclear transfer of wolf somatic cells into enucleated canine oocyte, indicating that SCNT is a practical approach for conserving endangered canids.