Cell viability of bovine spermatozoa subjected to DNA electroporation and DNAse I treatment.

Many mechanisms involved in sperm-mediated gene transfer (SMGT) are still unknown. It is still a matter of debate whether exogenous DNA fragments incorporated by the embryo are originated from those bound to the sperm membrane or by those that penetrated the intracellular compartment. In an attempt to elucidate the transmission mechanism of exogenous DNA molecules by sperm, some authors suggested a treatment with DNAse I to remove DNA molecules outside the sperm. But little is known regarding the effects of DNAse I treatment on sperm viability and its impact on sperm organelles. An important aspect of the SMGT technique is the amount of exogenous DNA incubated with sperm, which may influence the internalization rate. Due to the inconsistencies found in literature, this work aimed to contribute to bovine sperm physiology knowledge evaluating the effects of different DNA concentrations, electroporation, and DNAse I treatments on sperm viability characteristics, DNA uptake, and IVF. For that, the effects of different concentrations of exogenous DNA (250, 500 and 1000 ng/10(6) cells) and incubation or electroporation were tested on sperm functional characteristics and in vitro embryo production. No effect of DNA concentration was observed on uptake, plasma membrane integrity, and mitochondrial membrane potential. The addition of exogenous DNA induced a decrease on acrosomal lesion in the 500-ng group when compared to the control. Cells incubated with DNA, electroporated, and treated with DNAse I presented a deleterious influence on mitochondrial membrane potential. In vitro fertilization was made with 1000 ng of DNA, sperm cells incubated or electroporated followed by DNAse I treatment. No significant difference was found in cleavage rate. Blastocyst rates were 24.36% for the control; 19.65% for incubated; 3.5% for electroporated control; and 17.40% for electroporated. There is a significant difference in blastocyst rate between the control and electroporated control groups. The incubated group yielded five and electroporated two positive blastocysts evaluated by epifluorescence microscopy. Polymerase chain reaction screening shows 17% of positive embryos for incubation and 11% for electroporation. Fluorescence in situ hybridization showed the presence of exogenous gene in embryos. These results show that exogenous DNA molecules can be conducted by an intracellular mechanism. The SMGT protocol using electroporation and DNAse I treatment reduces sperm mitochondrial function, in vitro embryo production and increases sperm DNA fragmentation.

Tratamento de folículo anovulatório persistente em égua: relato de caso / Treatment of persistent anovulatory follicle in mare: a case report

O presente estudo busca apresentar o processo diagnóstico, evolução e tratamento, bem como registrar um caso de folículo anovulatório persistente na espécie equina. Para tanto, realizou-se atendimento clínico em uma égua da raça Quarto de Milha de nove anos de idade, no Hospital Veterinário da Universidade Paulista de São José dos Campos, com histórico de infertilidade. Foi realizada a avaliação clínica do paciente e exame ultrassonográfico do sistema reprodutivo, utilizando o equipamento Ultrassom Chison D600 com probe linear 7,5MHz no qual foi identificada estrutura compatível com folículo anovulatório no ovário direito. O tratamento proposto seria a utilização 5000 UI de gonadotropina coriônica humana (hCG - Vetecor - Hertape Calier) por via intravenosa e 250 mg de Cloprostenol (Prolise® -Tecnopec), por via intramuscular, no nono dia após a aplicação do hCG. No entanto por apresentar quadro de desconforto abdominal 24 horas após a aplicação do hCG, o tratamento foi suspenso a pedido do proprietário, não sendo realizada a indução da luteólise. O animal foi reavaliado com novo exame ultrassonográfico após sete dias da aplicação do hCG, onde foram observadas imagens ultrassonográficas compatíveis com a ovulação do folículo. A terapia proposta foi eficiente para resolução do folículo anovulatório, no entanto, a origem do quadro de desconforto abdominal pode ser evidenciada, sugerindo que maiores esforços sejam necessários para investigar o efeito do tratamento na saúde do animal...

This study seeks to make the diagnostic process, evolution and treatment, as well as to register a case of persistent anovulatory follicle in the equine species. Therefore, a mare of the Quarter Horse race of nine-year-old with a history of infertility was examined at the Veterinary Hospital of Universidade Paulista in São José dos Campos. It was performed a clinical assessment of the patient and ultrasound examination of the reproductive system, using ultrasound equipment Chison D600 with 7.5 MHz linear probe. It was identified a structure compatible with anovulatory follicle in the right ovary. The proposed treatment would be to use 5000 IU IV human chorionic gonadotropin (hCG - Vetecor - Hertape Calier) and 250 mg Cloprostenol IM (Prolise® - Tecnopec) on the ninth day after application of hCG. However, the animal showed abdominal discomfort 24 hours after application of hCG, so the treatment was suspended at the request of the owner, has not been accomplished to induce luteolysis. The animal was reassessed with new ultrasound seven days after application of hCG, which were seen ultrasound images compatible with ovulation follicle. The proposed therapy was effective for solving the anovulatory follicle, however, it was evident abdominal discomfort, suggesting that further efforts are necessary to investigate the treatment effect on the health of the animal...