RESUMO
Horse cloning by somatic cell nuclear transfer (SCNT) is an attractive scientific and commercial endeavor. Moreover, SCNT allows generating genetically identical animals from elite, aged, castrated, or deceased equine donors. Several variations in the horse SCNT method have been described, which may be useful for specific applications. This chapter describes a detailed protocol for horse cloning, thus including SCNT protocols using zona pellucida (ZP)-enclosed or ZP-free oocytes for enucleation. These SCNT protocols are under routine use for commercial equine cloning.
Assuntos
Técnicas de Transferência Nuclear , Zona Pelúcida , Cavalos , Animais , Técnicas de Transferência Nuclear/veterinária , Oócitos , Clonagem de Organismos/métodosRESUMO
Field collection of oocytes in mares using transvaginal follicular aspiration (TVA) for embryo production has the potential to revolutionate the equine industry. Protocols for TVA in specialized laboratory settings have been described in the scientific literature since the early 1980s. The objective of this study was to determine the success rate of TVA oocytes recovery under ambulatory conditions. A secondary goal of this study was to determine if TVA is associated with any health complications when performed by recently trained practitioners in the field. Follicles (n = 296) from 66 adult clinically healthy mares were aspirated over a period of 6 days. TVAs were performed by 22 veterinarians with 5-20 years of experience in equine and bovine reproductive medicine, but no previous experience in TVA. Oocytes (n = 145) were recovered. No short- or long-term systemic or local complications were observed following TVA in any of the mares used in this study. Fifty-six out of 66 mares became pregnant within 3 months following TVA. This study shows that with proper training, TVA can be successfully used to obtain equine oocytes with no health complications under field conditions in nonspecialized laboratory settings.
Assuntos
Recuperação de Oócitos , Oócitos , Animais , Bovinos , Embrião de Mamíferos , Feminino , Cavalos , Recuperação de Oócitos/veterinária , GravidezRESUMO
There is currently no objective, real-time and non-invasive method for evaluating the quality of mammalian embryos. In this study, we processed images of in vitro produced bovine blastocysts to obtain a deeper comprehension of the embryonic morphological aspects that are related to the standard evaluation of blastocysts. Information was extracted from 482 digital images of blastocysts. The resulting imaging data were individually evaluated by three experienced embryologists who graded their quality. To avoid evaluation bias, each image was related to the modal value of the evaluations. Automated image processing produced 36 quantitative variables for each image. The images, the modal and individual quality grades, and the variables extracted could potentially be used in the development of artificial intelligence techniques (e.g., evolutionary algorithms and artificial neural networks), multivariate modelling and the study of defined structures of the whole blastocyst.
Assuntos
Blastocisto , Animais , Bovinos , Feminino , Processamento de Imagem Assistida por Computador , GravidezRESUMO
Morphological analysis is the standard method of assessing embryo quality; however, its inherent subjectivity tends to generate discrepancies among evaluators. Using genetic algorithms and artificial neural networks (ANNs), we developed a new method for embryo analysis that is more robust and reliable than standard methods. Bovine blastocysts produced in vitro were classified as grade 1 (excellent or good), 2 (fair), or 3 (poor) by three experienced embryologists according to the International Embryo Technology Society (IETS) standard. The images (n = 482) were subjected to automatic feature extraction, and the results were used as input for a supervised learning process. One part of the dataset (15%) was used for a blind test posterior to the fitting, for which the system had an accuracy of 76.4%. Interestingly, when the same embryologists evaluated a sub-sample (10%) of the dataset, there was only 54.0% agreement with the standard (mode for grades). However, when using the ANN to assess this sub-sample, there was 87.5% agreement with the modal values obtained by the evaluators. The presented methodology is covered by National Institute of Industrial Property (INPI) and World Intellectual Property Organization (WIPO) patents and is currently undergoing a commercial evaluation of its feasibility.
Assuntos
Inteligência Artificial , Automação Laboratorial , Blastocisto/citologia , Processamento de Imagem Assistida por Computador , Microscopia , Algoritmos , Animais , Automação Laboratorial/métodos , Bovinos , Transferência Embrionária , Feminino , Processamento de Imagem Assistida por Computador/métodos , Microscopia/métodos , Redes Neurais de Computação , Curva ROCRESUMO
Interest in equine somatic cell nuclear transfer technology has increased significantly since the first equid clones were produced in 2003. This is demonstrated by the multiple commercial equine cloning companies having produced numerous cloned equids to date; worldwide, more than 370 cloned horses have been produced in at least six different countries. Equine cloning can be performed using several different approaches, each with different rates of success. In this review we cover the history and applications of equine cloning and summarise the major scientific advances in the development of this technology in horses. We explain the advantages and disadvantages of different procedures to produce cloned equine embryos and describe the current status of equine clone commercialisation, along with observations of differences in regional breed association registration regulations.
Assuntos
Clonagem de Organismos , Cavalos , Técnicas de Transferência Nuclear/veterinária , Animais , Clonagem de Organismos/história , Clonagem de Organismos/métodos , Clonagem de Organismos/veterinária , Embrião de Mamíferos , História do Século XXI , Cavalos/embriologia , Técnicas de Transferência Nuclear/história , Técnicas de Transferência Nuclear/tendênciasRESUMO
Morphological embryo classification is of great importance for many laboratory techniques, from basic research to the ones applied to assisted reproductive technology. However, the standard classification method for both human and cattle embryos, is based on quality parameters that reflect the overall morphological quality of the embryo in cattle, or the quality of the individual embryonic structures, more relevant in human embryo classification. This assessment method is biased by the subjectivity of the evaluator and even though several guidelines exist to standardize the classification, it is not a method capable of giving reliable and trustworthy results. Latest approaches for the improvement of quality assessment include the use of data from cellular metabolism, a new morphological grading system, development kinetics and cleavage symmetry, embryo cell biopsy followed by pre-implantation genetic diagnosis, zona pellucida birefringence, ion release by the embryo cells and so forth. Nowadays there exists a great need for evaluation methods that are practical and non-invasive while being accurate and objective. A method along these lines would be of great importance to embryo evaluation by embryologists, clinicians and other professionals who work with assisted reproductive technology. Several techniques shows promising results in this sense, one being the use of digital images of the embryo as basis for features extraction and classification by means of artificial intelligence techniques (as genetic algorithms and artificial neural networks). This process has the potential to become an accurate and objective standard for embryo quality assessment.