RESUMO
Sperm samples used on fertilization strongly influence the in vitro production (IVP) rates. However, sperm traits behind this effect are not stated consistently until now. This study aimed to evaluate the isolated and combined effect of some sperm traits (MB: total motility before Percoll® gradient, MA: total motility after Percoll® gradient, AI: acrosome integrity, MI: membrane integrity, MP: mitochondrial membrane potential, and CR: chromatin resistance) on IVP rates. This is the first study focusing on the isolated effect of distinct traits. For this purpose, the experiment was divided in three steps. In first step, to study behavior of traits sperm samples (n = 63 batches) were analyzed and ranked based on each trait. In second step, samples ranked were selected from target ranks regions and allocated in groups of four to five batches, creating Higher and Lower groups, according to two different approaches. One aimed to form groups that differed to all sperm traits simultaneously (effect of combined traits). The other aimed to form groups that differed only to a single sperm trait while no differences were observed for the remaining traits (effect of each isolated trait). In third step, for each group successfully formed in step 2, sperm samples were individually and prospectively used for IVP. Cleavage, embryo development and blastocyst rates were recorded and compared between Higher and Lower of respective trait groups. Surprisingly, evaluation of isolated effects revealed that lower levels of MB, AI and MP resulted in higher embryo development and blastocyst rates (p<0.05), which was not observed on cleavage rate. We conclude that sperm traits strongly influence embryo development after in vitro fertilization (IVF), affecting the zygote competence to achieve blastocyst stage. Individually, levels of MB, AI or MP could be some of the key traits that may define IVP efficiency on current systems of embryo production.
Assuntos
Bovinos/embriologia , Espermatozoides/fisiologia , Acrossomo/fisiologia , Animais , Blastocisto/fisiologia , Cromatina/metabolismo , Fase de Clivagem do Zigoto/fisiologia , Técnicas In Vitro , Masculino , Potencial da Membrana Mitocondrial , Povidona , Dióxido de Silício , Motilidade dos Espermatozoides , Zigoto/fisiologiaRESUMO
Sperm DNA fragmentation is considered one of the main causes of male infertility. The most accepted causes of sperm DNA damage are deleterious actions of reactive oxygen species (ROS), defects in protamination, and apoptosis. Ram sperm are highly prone to those damages due to the high susceptibility to ROS and to oxidative stress caused by heat stress. We aimed to evaluate the effects of heat stress on the chromatin of ejaculated and epididymal sperm and the activation of apoptotic pathways in different cell types in ram testis. We observed higher percentages of ejaculated sperm with increased chromatin fragmentation in the heat stress group; a fact that was unexpectedly not observed in epididymal sperm. Heat stress group presented a higher percentage of spermatozoa with DNA fragmentation and increased number of mRNA copies of transitional protein 1. Epididymal sperm presented greater gene expression of protamine 1 on the 30th day of the spermatic cycle; however, no differences in protamine protein levels were observed in ejaculated sperm and testis. Localization of proapoptotic protein BAX or BCL2 in testis was not different. In conclusion, testicular heat stress increases ram sperm DNA fragmentation without changes in protamination and apoptotic patterns.
Assuntos
DNA/efeitos dos fármacos , Motilidade dos Espermatozoides/efeitos dos fármacos , Espermatozoides/fisiologia , Testículo/fisiologia , Animais , Masculino , ProtaminasRESUMO
Higher temperatures lead to an increase of testicular metabolism that results in spermatic damage. Oxidative stress is the main factor responsible for testicular damage caused by heat stress. The aim of this study was to evaluate lasting effects of heat stress on ejaculated sperm and immediate or long-term effects of heat stress on epididymal sperm. We observed decrease in motility and mass motility of ejaculated sperm, as well as an increase in the percentages of sperm showing major and minor defects, damaged plasma and acrosome membranes, and a decrease in the percentage of sperm with high mitochondrial membrane potential in the treated group until one spermatic cycle. An increased enzymatic activity of glutathione peroxidase and an increase of stressed cells were observed in ejaculated sperm of the treated group. A decrease in the percentage of epididymal sperm with high mitochondrial membrane potential was observed in the treated group. However, when comparing immediate and long-term effects, we observed an increase in the percentage of sperm with low mitochondrial membrane potential. In conclusion, testicular heat stress induced oxidative stress that led to rescuable alterations after one spermatic cycle in ejaculated sperm and also after 30 days in epididymal sperm.
Assuntos
Epididimo/patologia , Estresse Oxidativo , Sêmen/metabolismo , Espermatozoides/fisiologia , Reação Acrossômica , Animais , Antioxidantes/metabolismo , Citometria de Fluxo , Radicais Livres , Glutationa Peroxidase/metabolismo , Temperatura Alta , Peroxidação de Lipídeos , Masculino , Potencial da Membrana Mitocondrial , Ovinos , Motilidade dos Espermatozoides , Temperatura , Substâncias Reativas com Ácido Tiobarbitúrico/metabolismoRESUMO
This study proposed a quantitative evaluation of oxidative status (OS) in bovine embryos. Sixteen-cell stage embryos, cultured under 5% O2, were treated with oxidative stress inducer menadione (0, 1, 2.5 and 5 µmol/l) for 24 h. Blastocyst rate (BLR) was recorded and expanded blastocysts were stained with CellROX®Green (CRG; OS evaluation) and evaluated under epifluorescence microscopy (ratio of pixel/blastomere). A significant effect of menadione was observed for BLR (P = 0.0039), number of blastomeres/embryo (P < 0.0001) and OS (P < 0.001). Strong negative correlations were found between BLR and the number of blastomeres with OS evaluation, demonstrating the efficacy of this analysis to evaluate OS levels of IVF bovine embryos.