RESUMO
Cloning procedures often interfere with conceptus growth and life ex utero, in a set of symptoms known as abnormal offspring syndrome (AOS). The aim of the present study was to compare the developmental pattern of in vivo-derived (IVD), IVF-derived and handmade cloning-derived (NT-HMC) Day 225 bovine concepti using established procedures. Pregnancy diagnosis was performed on Day 30 following blastocyst transfer on Day 7. Conceptus morphometry was assessed by ultrasonography on Day 51, and on Day 225 pregnant cows were killed for morphological examination of concepti. Pregnancy outcome was similar between groups, with greater pregnancy losses in the first trimester (70.6%) and smaller fetuses on Day 51 in the NT-HMC group than in the IVD (14.3%) and IVF (20.0%) groups. However, NT-HMC-derived concepti were twofold larger on Day 225 of gestation than controls. A higher frequency (63.5%) of placentomes larger than the largest in the IVD group was observed in the NT-HMC group, which may be relevant to placental function. Conceptus traits in the IVF group were similar to the IVD controls, with only slight changes in placentome types. Morphological changes in cloned concepti likely affected placental function and metabolism, disrupting the placental constraining mechanism on fetal growth in mid- to late pregnancy.
Assuntos
Clonagem de Organismos , Desenvolvimento Embrionário/fisiologia , Animais , Bovinos , Transferência Embrionária/veterinária , Feminino , Fertilização in vitro/veterinária , Técnicas de Transferência Nuclear/veterinária , Gravidez , Resultado da Gravidez , Ultrassonografia Pré-Natal/veterináriaRESUMO
Embryo sexing is a powerful tool for livestock producers because it allows them to manage their breeding stocks more effectively. However, the cost of supplies and reagents, and the need for trained professionals to biopsy embryos by micromanipulation restrict the worldwide use of the technology to a limited number of specialized groups. The aim of this study was to couple a fast and inexpensive DNA extraction protocol with a practical biopsy approach to create a simple, quick, effective, and dependable embryo sexing procedure. From a total of 1847 sheep and cattle whole embryos or embryo biopsies, the sexing efficiency was 100% for embryo biopsies, 98% for sheep embryos, and 90.2% for cattle embryos. We used a primer pair that was common to both species and only 10% of the total extracted DNA. The whole protocol takes only 2 h to perform, which suggests that the proposed procedure can be readily applied to field conditions. Moreover, in addition to embryo sexing, the procedure can be used for further analyses, such as genotyping and molecular diagnosis in preimplantation embryos.
Assuntos
Embrião de Mamíferos/metabolismo , Gado , Reação em Cadeia da Polimerase/métodos , Análise para Determinação do Sexo/métodos , Animais , BlastocistoRESUMO
The recombinant production of therapeutic proteins for human diseases is currently the largest source of innovation in the pharmaceutical industry. The market growth has been the driving force on efforts for the development of new therapeutic proteins, in which transgenesis emerges as key component. The use of the transgenic animal platform offers attractive possibilities, residing on the low production costs allied to high productivity and quality of the recombinant proteins. Although many strategies have evolved over the past decades for the generation of transgenic founders, transgenesis in livestock animals generally faces some challenges, mainly due to random transgene integration and control over transgene copy number. But new developments in gene editing with CRISPR/Cas system promises to revolutionize the field for its simplicity and high efficiency. In addition, for the final approval of any given recombinant protein for animal or human use, the production and characterization of bioreactor founders and expression patterns and functionality of the proteins are technical part of the process, which also requires regulatory and administrative decisions, with a large emphasis on biosafety. The approval of two mammary gland-derived recombinant proteins for commercial and clinical use has boosted the interest for more efficient, safer and economic ways to generate transgenic founders to meet the increasing demand for biomedical proteins worldwide.
Assuntos
Animais Geneticamente Modificados/genética , Biofarmácia/tendências , Gado/genética , Proteínas Recombinantes/genética , Animais , Reatores Biológicos , Técnicas de Transferência de Genes/tendências , Humanos , Proteínas Recombinantes/biossínteseRESUMO
The presence of the zona pellucida has been perceived as a requirement for the oviductal transfer of cloned embryos at early stages of development while protecting the embryo from an immune system response. We hypothesized that steroid hormone therapy could reduce a potential cellular immune response after the transfer of zona-free cloned embryos into the oviduct of recipient female goats. In Experiment 1, seven does were used to study the systemic immunosuppressant effect of the methylprednisolone administration (for 3 days) on blood cell counts. Whole blood was collected prior to treatment with methyprednisolone and then on Days 1, 2, 3, 4, 7, 14, 21 and 28 after the first dose of methylprednisolone for the analysis of haematological parameters. Methylprednisolone treatment significantly reduced circulating white blood cells and neutrophils in comparison with pre-treatment levels, demonstrating a systemic immunosuppressant effect. In Experiment 2, a group of 58 does were used as recipient females to study the effect of administration of methylprednisolone for 3 days on the establishment of pregnancies after the transfer of zona-free cloned embryos into the oviducts. No effects on pregnancy rates on Day 30 were observed regarding the distinct treatment groups (control vs. methylprednisolone), the source of oocytes (in vivo- vs in vitro-matured) or the presence or absence of the zona pellucida in embryos. In summary, methylprednisolone was effective at inducing a systemic immunosuppressed state in goats, but the treatment prior to embryo transfer did not affect pregnancy rates. Moreover, pregnancy rates were similar between zona-free and zona-intact goat cloned embryos.
Assuntos
Clonagem de Organismos/veterinária , Transferência Embrionária/veterinária , Cabras , Terapia de Imunossupressão/veterinária , Metilprednisolona/administração & dosagem , Animais , Transferência Embrionária/métodos , Feminino , Terapia de Imunossupressão/métodos , Contagem de Leucócitos/veterinária , Neutrófilos , Técnicas de Transferência Nuclear/veterinária , Gravidez , Taxa de Gravidez , Zona Pelúcida/imunologiaRESUMO
Lipid-rich and energy-dense diets can have significant effects on the reproductive physiology, including the ovarian function and fertility. The aim of this study was to assess the effect of cashew nut bran supplementation as a lipid source on follicle development, plasma and intrafollicular concentrations of cholesterol, and developmental competence of in vitro-matured goat oocytes. The inclusion of cashew nut bran as 24% of the goats' diet for 28 days increased the percentage and number of degenerated oocytes compared with the control (P < 0.05), and also the plasma cholesterol levels and the proportion of grade IV oocytes compared with all other treatments (P < 0.05). Moreover, a significant reduction was observed in the proportion of viable oocytes compared with the control and in the percentage of grade II oocytes compared with all other treatments (P < 0.05). Oocyte maturation, cleavage, and blastocyst rates after parthenogenetic activation of viable oocytes were not affected by the type of diet. In conclusion, the inclusion of cashew nut bran as 24% of the diet of adult goats for 28 days changed plasma cholesterol levels and reduced the proportion of viable immature oocytes; however, the 12% and 24% diet supplementations with cashew nut bran did not interfere with competence of resulting viable oocytes to reach the metaphase II stage after IVM, and to develop after parthenogenetic activation.
Assuntos
Anacardium , Cabras/fisiologia , Metabolismo dos Lipídeos , Oócitos/citologia , Ração Animal , Animais , Colesterol/sangue , Feminino , Cabras/metabolismo , Técnicas de Maturação in Vitro de Oócitos , ReproduçãoRESUMO
Re-suspension of frozen-thawed ram sperm with ovine and bovine whole seminal plasma (SP) is beneficial for its post-thawing viability. However, neither the influence of SP incubation duration nor the re-suspension with equine SP has been tested. In the first experiment, frozen-thawed ram sperm were incubated with SP from bovine (BSP), equine (ESP) or ovine (OSP) or without SP (-SP) for short (5 min) or long (6 h) periods. Viability parameters such as sperm progressive motility, percentage of live cells and membrane functionality were assessed every 2 h for 6 h. All SP treatments showed higher spermatozoa viability than the -SP treatment in most evaluations. Incubation time did not affect sperm viability for BSP treatment, however, ESP and OSP induced a transitory benefic effect in the short incubation period and detrimental effect in the longer period. In the second experiment, frozen-thawed sperm, with or without short incubation with SP, were selected by swim-up, and their DNA fragmentation rate was assessed using comet assay immediately after swim-up completion and after 5 h of incubation. BSP, ESP and OSP protein profiles were determined by SDS-PAGE. Only ESP was associated with sperm DNA stabilization capacity and SP from rams and bulls showed protein profiles different from that of stallions. These experiments indicate that equine or ovine, but not bovine whole SP supplementation to post-thawing incubation medium of frozen-thawed ram sperm affects its viability in a time dependent manner. The beneficial effect of ESP on stabilizing DNA integrity, even after sperm washing with swim-up method and incubation for 5 h, can be determined by SPP or by antioxidant components from SP.(AU)
Assuntos
Animais , Sêmen/citologia , Espermatozoides/citologia , Criopreservação , Cavalos/classificação , OvinosRESUMO
Re-suspension of frozen-thawed ram sperm with ovine and bovine whole seminal plasma (SP) is beneficial for its post-thawing viability. However, neither the influence of SP incubation duration nor the re-suspension with equine SP has been tested. In the first experiment, frozen-thawed ram sperm were incubated with SP from bovine (BSP), equine (ESP) or ovine (OSP) or without SP (-SP) for short (5 min) or long (6 h) periods. Viability parameters such as sperm progressive motility, percentage of live cells and membrane functionality were assessed every 2 h for 6 h. All SP treatments showed higher spermatozoa viability than the -SP treatment in most evaluations. Incubation time did not affect sperm viability for BSP treatment, however, ESP and OSP induced a transitory benefic effect in the short incubation period and detrimental effect in the longer period. In the second experiment, frozen-thawed sperm, with or without short incubation with SP, were selected by swim-up, and their DNA fragmentation rate was assessed using comet assay immediately after swim-up completion and after 5 h of incubation. BSP, ESP and OSP protein profiles were determined by SDS-PAGE. Only ESP was associated with sperm DNA stabilization capacity and SP from rams and bulls showed protein profiles different from that of stallions. These experiments indicate that equine or ovine, but not bovine whole SP supplementation to post-thawing incubation medium of frozen-thawed ram sperm affects its viability in a time dependent manner. The beneficial effect of ESP on stabilizing DNA integrity, even after sperm washing with swim-up method and incubation for 5 h, can be determined by SPP or by antioxidant components from SP.
Assuntos
Animais , Criopreservação , Espermatozoides/citologia , Sêmen/citologia , Cavalos/classificação , OvinosRESUMO
Bull breeding soundness evaluation (BBSE) usually neglects the libido and mating ability evaluation. The internal artificial vagina (IAV) permits semen sampling, as well as mating ability evaluation. Few studies have been performed using IAV with young bulls and there are none with Bos indicus bulls. The present study evaluated sexual behavior, mating ability and semen quality in young Bos taurus (Devon) and B. indicus (Nellore) bulls using the IAV device. In the first experiment, 52 Devon bulls, 18-25 months old were observed, and the behavior and mating ability recorded over a 10-min period within a restrained mount-cow with an IAV inserted. In the second experiment, 20 Nellore bulls, 20-30 months old were evaluated over a 20 min period. Of the 52 Devon bulls, 45 (86.5%) had semen recovered with the IAV, 31 (69.0%) were considered satisfactory. Nellore bulls exhibited a different sexual behavior, with 10 bulls not showing any interest in the females. Four bulls demonstrated sexual interest only once, e.g., sniffing, two showed interest on more than one occasion, and four had more than two mounts or mounting attempts. None out of the Nellore bulls was collected with IAV. The IAV was an effective and welfare-promoting animal technology for the evaluation of semen quality and mating ability of B. taurus bulls. However, the IAV was not adequate for young Nellore bulls, probably due to their quiescent sexual behavior and delayed sexual maturity. Further studies are needed to evaluate the performance of the IAV for older Nellore bulls.
Assuntos
Cruzamento/métodos , Bovinos/fisiologia , Animais , Feminino , Masculino , Sêmen/fisiologia , Comportamento Sexual Animal/fisiologia , VaginaRESUMO
Cat scratch disease (CSD) is an infectious illness caused by a Gram-negative rod named Bartonella henselae. Typical CSD is characterized by a small skin lesion at the site of a scratch or a bite, followed by regional lymphadenopathy, one to two weeks later. Atypical forms may present as ocular manifestations, neurological manifestations, hepatosplenic involvement and vertebral osteomyelitis. Among neurological complications, encephalopathy is by far the most common. Other neurological manifestations are very rare. We report a case of an 11-year-old boy, with a posterior cervical lymphadenopathy and fever. Cat scratch disease was diagnosed and treated after a positive "Whartin-Starry" stain on lymph node biopsy. Two weeks after treatment, the patient was readmitted presenting an acute episode of left hemiplegia. A brain MRI demonstrated a right subcortical fronto-parietal lesion with no contrast enhancement. Complete recovery was observed after corticosteroid treatment.
Assuntos
Bartonella henselae/isolamento & purificação , Doença da Arranhadura de Gato/complicações , Hemiplegia/etiologia , Doença Aguda , Corticosteroides/uso terapêutico , Animais , Antibacterianos/uso terapêutico , Doença da Arranhadura de Gato/diagnóstico , Doença da Arranhadura de Gato/tratamento farmacológico , Gatos , Criança , Claritromicina/uso terapêutico , Humanos , MasculinoRESUMO
Cat scratch disease (CSD) is an infectious illness caused by a Gram-negative rod named Bartonella henselae. Typical CSD is characterized by a small skin lesion at the site of a scratch or a bite, followed by regional lymphadenopathy, one to two weeks later. Atypical forms may present as ocular manifestations, neurological manifestations, hepatosplenic involvement and vertebral osteomyelitis. Among neurological complications, encephalopathy is by far the most common. Other neurological manifestations are very rare. We report a case of an 11-year-old boy, with a posterior cervical lymphadenopathy and fever. Cat scratch disease was diagnosed and treated after a positive Whartin-Starry stain on lymph node biopsy. Two weeks after treatment, the patient was readmitted presenting an acute episode of left hemiplegia. A brain MRI demonstrated a right subcortical fronto-parietal lesion with no contrast enhancement. Complete recovery was observed after corticosteroid treatment.