Sperm function and oxidative status: Effect on fertility in Bos taurus and Bos indicus bulls when semen is used for fixed-time artificial insemination.

Semen quality is one of the criteria used for the selection of bulls with relatively greater fertility. In addition, bull fertility depends on the integrity and function of all sperm structures. The aim of this study, therefore, was to determine associations when there was conducting of conventional and functional techniques for the evaluation of sperm samples from bulls with known fertility history as determined when semen from these bulls was used for fixed-time artificial insemination programs. The study was designed in a 2 × 2 factorial arrangement, with one factor being breed (Angus x Nellore) and the other fertility (greater x lesser). Greater fertility groups were composed of ten Angus and 11 Nellore bulls, while lesser fertility groups were composed of ten Angus and seven Nellore bulls. Sperm were analyzed, in four cryopreserved distinct batches for each animal, for morphology, kinetics, plasma and acrosomal membrane integrity, mitochondrial activity and mitochondrial membrane potential, DNA integrity and oxidative status. There was no difference in characteristics commonly used in sperm quality conventional analysis. The results from functional analysis indicated an important association between mitochondrial dysfunctions, oxidative stress, and damage to sperm structures in lesser fertility bulls. Greater fertility bulls had greater sperm quality and indicators of functional cell structures. The associations, when there were evaluations using different techniques, indicate the importance of evaluation and correlation between different sperm functions to understand effects of distinct parameters on sperm fertilization capacity.

Induced lipid peroxidation in ram sperm: semen profile, DNA fragmentation and antioxidant status.

Action of reactive oxygen species, protamination failures and apoptosis are considered the most important etiologies of sperm DNA fragmentation. This study evaluated the effects of induced lipid peroxidation susceptibility on native semen profile and identified the mechanisms involved in sperm DNA fragmentation and testicular antioxidant defense on Santa Ines ram sperm samples. Semen was collected from 12 adult rams (Ovis aries) performed weekly over a 9-week period. Sperm analysis (motility, mass motility, abnormalities, membrane and acrosome status, mitochondrial potential, DNA fragmentation, lipid peroxidation and intracellular free radicals production); protamine deficiency; PRM1, TNP1 and TNP2 gene expression; and determination of glutathione peroxidase (GPx), glutathione reductase, catalase (CAT) and superoxide dismutase activity and immunodetection in seminal plasma were performed. Samples were distributed into four groups according to the sperm susceptibility to lipid peroxidation after induction with ascorbate and ferrous sulfate (low, medium, high and very high). The results were analyzed by GLM test and post hoc least significant difference. We observed an increase in native GPx activity and CAT immunodetection in groups with high susceptibility to induced lipid peroxidation. We also found an increase in total sperm defects, acrosome and membrane damages in the group with the highest susceptibility to induced lipid peroxidation. Additionally, the low mitochondrial membrane potential, susceptible to chromatin fragmentation and the PRM1 mRNA were increased in the group showing higher susceptibility to lipid peroxidation. Ram sperm susceptibility to lipid peroxidation may compromise sperm quality and interfere with the oxidative homeostasis by oxidative stress, which may be the main cause of chromatin damage in ram sperm.

Photobiological effect of low-level laser irradiation in bovine embryo production system.

The objectives of this study were to evaluate the effect of low-level laser irradiation (LLLI) on bovine oocyte and granulosa cells metabolism during in vitro maturation (IVM) and further embryo development. Cumulus-oocytes complexes (COCs) were subjected (experimental group) or not (control group) to irradiation with LLLI in a 633-nm wavelength and 1 J/cm2 fluency. The COCs were evaluated after 30 min, 8, 16, and 24 h of IVM. Cumulus cells were evaluated for cell cycle status, mitochondrial activity, and viability (flow cytometry). Oocytes were assessed for meiotic progression status (nuclear staining), cell cycle genes content [real-time polymerase chain reaction (PCR)], and signal transduction status (western blot). The COCs were also in vitro fertilized, and the cleavage and blastocyst rates were assessed. Comparisons among groups were statistically performed with 5% significance level. For cumulus cells, a significant increase in mitochondrial membrane potential and the number of cells progressing through the cycle could be observed. Significant increases on cyclin B and cyclin-dependent kinase (CDK4) levels were also observed. Concerning the oocytes, a significantly higher amount of total mitogen-activated protein kinase was found after 8 h of irradiation, followed by a decrease in all cell cycle genes transcripts, exception made for the CDK4. However, no differences were observed in meiotic progression or embryo production. In conclusion, LLLI is an efficient tool to modulate the granulosa cells and oocyte metabolism.

Assessment of post-thawed ram sperm viability after incubation with seminal plasma.

A suggested alternative to improve post-thawed ram semen quality is the addition of seminal plasma (SP). This is thought to be capable of improving sperm resistance to thermal shock, reverting cryocapacitation and helping sperm survival. The aim of this study was to evaluate the effect of frozen-thawed ram semen incubation with SP on mitochondrial activity, acrosomal membrane integrity, necrosis and apoptosis. Frozen/thawed semen was divided into two groups: the SP Group and the control group. After 0, 30 and 60 min, fluorescent probes were added to aliquots from each treatment group and evaluated using flow cytometry. There was no difference between treatment groups in almost all viability parameters evaluated, with exception of the apoptosis, which was found increased in SP group. The increase in incubation period resulted in a decreased percentage of sperm with high mitochondrial membrane potential and acrosomal membrane integrity and an increased percentage of necrotic and apoptotic sperm cells. In conclusion, the present study showed that addition of seminal plasma after thawing cryopreserved ram sperm had no identifiable beneficial effect on sperm quality.

Sperm-mediated gene transfer: effect on bovine in vitro embryo production.

The technique of sperm-mediated gene transfer (SMGT) can be used to delivery exogenous DNA into the oocyte. However, it has low repeatability and produces inconsistent results. In order to optimize this technique, it is necessary to study the mechanism by which DNA enters the sperm cell and integrates in the sperm genome. Furthermore, studies must focus in the maintenance of sperm cell viability and function. The aim of this study was to evaluate different SMGT protocols of sperm electroporation or capacitation (CaI) aiming to maintain sperm viability in the production of bovine embryos in vitro. Frozen-thawed semen was divided in two experimental groups (electroporation or CaI) and one control group (non-treated cells). For the electroporation method, five different voltages (100, 500, 750, 1000 or 1500 V) with 25 µF capacitance were used. For CaI treatment, combinations of two CaI concentrations (250 nM or 500 nM), two incubation periods of sperm cells with CaI (1 or 5 min) and two incubation periods that mimicked time of sperm cell interaction with exogenous DNA molecules (1 or 2 h) were evaluated. According to our data, electroporation and CaI treatments do not prevent sperm penetration and oocyte fertilization and can be an alternative method to achieve satisfactory DNA delivery in SMGT protocols.

Bovine oocyte vitrification: effect of ethylene glycol concentrations and meiotic stages.

Success in oocyte cryopreservation is limited and several factors as cryoprotectant type or concentration and stage of oocyte meiotic maturation are involved. The aim of the present study was to evaluate the effect of maturation stage and ethylene glycol (EG) concentration on survival of bovine oocytes after vitrification. In experiment 1, kinetics of oocyte in vitro maturation (IVM) was evaluated. Germinal vesicle (GV), germinal vesicle breakdown (GVBD), metaphase I (MI), and metaphase II (MII) oocytes were found predominantly at 0, 0-10, 10-14, and 18-24h of IVM, respectively. In experiment 2, in vitro embryo development after in vitro fertilization (IVF) of oocytes exposed to equilibrium (ES) and vitrification solution VS-1 (EG 30%), or VS-2 (EG 40%) at 0, 12 or 18 h of IVM was evaluated. Only blastocyst rate from oocytes vitrified in SV-2 after 18 h of IVM was different from control oocytes. Hatched blastocyst rates from oocytes vitrified in VS-1 after 12 and 18 h, and SV-2 after 18 h of IVM were different from unvitrified oocytes. In experiment 3, embryo development was examined after IVF of oocytes vitrified using VS-1 or VS-2 at 0, 12 or 18 h of IVM. Rates of blastocyst development after vitrification of oocytes in VS-1 at each time interval were similar. However, after vitrification in VS-2, blastocyst rates were less at 18 h than 0 h. Both cleavage rates and blastocyst rates were significantly less in all vitrification groups when compared to control group and only control oocytes hatched. In conclusion, both EG concentration and stage of meiotic maturation affect the developmental potential of oocytes after vitrification.

Cryopresevation of mouse morulae in glycerol, sucrose and honeybee royal jelly

Compacted mouse morulae were frozen at 0.3§C/min. or 0.5§C/min. from -6§C to -24§C or -32§C in 10 por cento of glycerol plus different sucrose concentrations with or without 0.1 por cento of honeybee royal jelly. Embryos were thawed in water bath at 22§C for 20 seconds and cryoprotectant dilution was done in three steps. Embryos were cultured in Whitten's medium for 24, 48 and 72 hours at 37§C, 5 por cento of CO2 and 100por cento of humidity. The in vitro development ranged from 56.6 por cento to 100 por cento after 72 hours. Expanded blastocysts were transferred to pseudopregnant recipients on the third day of the estrous cycle. Viable fetuses rates for embryos frozen to -24 or -32§C at 0.3§C/minute in 10 por cento glycerol + 10 por cento sucrose, 10 por cento glycerol + 10 por cento sucrose + 0.1 por cento honeybee royal jelly, 10 por cento glycerol + 0.1 por cento honeybee royal jelly or 10 por cento glycerol were respectively: 28.1 por cento and 13.6 por cento, 48.7 por cento and 31.9 por cento, 28.6 por cento and 13.2 por cento, 20.0 por cento and 42.4 por cento. Viable fetuses for embryos frozen to -24§C or -32§C at 0.5§C/minute in 10 por cento glycerol + 10 por cento sucrose or 10 por cento glycerol + 10 por cento sucrose + 0.1 por cento honeybee royal jelly were respectively 29.0 por cento and 15.3 por cento, 48.8 por cento and 32.0 por cento. We can conclude that addition of 10 por cento sucrose to 10 por cento glycerol was efficient for embryo freezing at 0.3 or 0.5§C/minute and plunged in liquid nitrogen at -24§C. The honeybee royal jelly addition provided higher viable fetuses rates when embryos were cooled at 0.3 or 0.5§C/minute and plunged in liquid nitrogen at -24§C