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The aim of this study was to evaluate the effects of different selenium compounds on the sperm quality of cryopreserved ram semen. Ejaculates from four rams, collected using an artificial vagina heated to 38 °C, were individually evaluated. The approved ejaculates were pooled and diluted (1:1 v:v) in Tris-egg yolk extender (20%, v/v) and separated into two control groups, one cooled for 2 h and the other for 4 h. The pooled ejaculates at the two cooling periods were supplemented with two doses (0.5 and 1 µg/mL) of organic selenium (ORG), and inorganic selenium (SeNa), each. The samples were packed in 0.25 ml straws, at a concentration of 400 × 106 sperms/mL and stored in liquid nitrogen. The straws were thawed in a water bath at 37 °C for 20 s, and the samples were subjected to sperm kinetics evaluation by Computer Assisted Semen Analysis software. Sperm membrane integrity, acrosome morphology, and mitochondrial potential were assessed. In addition, oxidative stress markers reactive oxygen species (ROS), ferric reducing antioxidant power (FRAP), thiobarbituric acid reactive species (TBARS), and glutathione peroxidase (GPx) enzyme activity) were also evaluated. No significant improvement was observed in the ram semen quality at the two cooling times. Supplementation of the freezing extender with 0.5 µg/mL ORG, subjected to 4 h cooling period, increased the sperm motility when compared with the control group at the same cooling time. In addition, the 0.5 µg/mL SeNa group, under the 2 h cooling period, showed an increase in sperm motility when compared to the control group at the same cooling period. Considering the importance of sperm motility as a fertility parameter, our study indicates that supplementation with ORG and SeNa can help improve the total motility of the cryopreserved ram semen.

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While sperm migrate within the reproductive tract of cows experiencing negative energy balance (NEB), they come into contact with elevated concentrations of non-esterified fatty acids (NEFA). For this reason, this study aimed to investigate the effects of three different NEFA - palmitic acid (PA), stearic acid (SA), and oleic acid (OA) - on bovine sperm motility, kinetic parameters, oxidative status, and morphology. Frozen thawed semen samples from Bos taurus bulls were incubated with varying concentrations of each fatty acid, and the sperm's characteristics were analysed at different time points. Computer-Assisted Sperm Analysis (CASA) was employed to assess sperm motility and kinetic parameters. Concurrently, the production of the reactive oxygen species (ROS) and total antioxidant capacity were measured to determine the oxidative status. Additionally, sperm morphology was evaluated. In Experiment 1, different concentrations of PA did not show significant effects on total motility, progressive motility, or any kinetic parameters analysed. Similarly, PA did not have a significant impact on the oxidative status or sperm morphology. In Experiment 2, SA at various concentrations did not lead to significant changes in total motility, progressive motility, or any kinetic parameters evaluated. Furthermore, SA did not affect oxidative status or sperm morphology. In Experiment 3, the concentrations of OA used did not result in significant changes in total motility, progressive motility, or any kinetic parameters studied. Likewise, OA did not induce any alterations in oxidative status or sperm morphology. Overall, the results from all three experiments indicate that PA, SA and OA, at the in vitro conditions and tested concentrations, do not exert detrimental effects on bovine sperm function and morphology. These results provide insights that contribute to our understanding of how fatty acids can impact the reduction of fertility rates in cows facing NEB. This, in turn, lays the foundation for additional critical investigations in this area. Further studies are necessary to validate these findings in vivo.

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The aim of this study was to evaluate the morphology and superoxide dismutase enzyme (SOD) activity of bovine preantral follicles (PFs) preserved in TCM 199, saline solution or PBS at different conservation periods. Cow ovaries (n=6) were divided into 7 fragments. One small piece of each ovarian fragment was randomly removed to evaluate SOD activity, while the remainder was immediately fixed for morphological evaluation as a control group. The other 6 fragments were randomly distributed in tubes containing TCM 199, saline solution, or PBS and maintained at 4ºC for 6 or 24 h. For histological evaluation, the fragments were fixed in Carnoy and stained with PAS-hematoxylin, following being classified PFs in relation to their follicular morphology in normal or degenerated. Determination of SOD activity was based on the ability to inhibit autoxidation of adrenaline in adrenochrome. Evaluation of follicular morphology showed that follicles preserved in TCM 199 for 6 h did not differ from the control (P > 0.05). In contrast, preservation in saline solution and PBS for 6 or 24 h and TCM 199 for 24 h decreased normal PFs compared to the control (P < 0.05). SOD showed a lower activity in ovarian cortical tissue kept in TCM 199 for 6 h and saline solution for 24 h than in the other groups. Our study shows that incubation using TCM 199 at 4°C for 6 h can be used to efficiently conserve female bovine PFs in situ.(AU)

O objetivo desse estudo foi avaliar a morfologia e atividade da enzima superóxido dismutase (SOD) de folículos pré-antrais bovinos (PFs) preservados em TCM 199, solução salina ou PBS por diferentes períodos de conservação. Ovários de vacas (n=6) foram divididos em 7 fragmentos. Um pequeno pedaço de cada fragmento ovariano foi removido para avaliar a atividade da SOD enquanto que o restante foi imediatamente fixado para avaliação morfológica como grupo controle. Os outros 6 fragmentos foram distribuídos aleatoriamente em tubos contendo TCM 199, solução salina ou PBS e mantidos a 4ºC por 6 ou 24 h. Para avaliação histológica, os fragmentos foram fixados em Carnoy e corados com PAShematoxilina, sendo classificados em seguida os PFs em relação à sua morfologia folicular em normal ou degenerados. A determinação da atividade da SOD foi baseada na capacidade de inibir a autooxidação da adrenalina no adrenocromo. A avaliação da morfologia folicular mostrou que os folículos preservados em TCM199 por 6 h não diferiram do controle (P > 0,05). Em contraste, a preservação em solução salina e PBS por 6 ou 24 h e TCM 199 por 24 h diminuiu os PFs normais em comparação com o controle (P < 0,05). A SOD mostrou uma menor atividade no tecido cortical ovariano mantido em TCM 199 por 6 h e solução salina por 24 h do que nos outros grupos. Nosso estudo mostra que a incubação usando TCM 199 a 4° C por 6 h pode ser usada eficientemente para conservar PFs de fêmeas bovinas in situ.(AU)

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Over the last several years human sperm quality was found to be significantly reduced and the role environmental contaminants play in this phenomenon remain to be determined. Mercury (Hg) is one of the most widespread contaminants; however the correlation between metal exposure and adverse consequences on human and animals fertility are not completely established. The aim of this study was to determine the effects of direct exposure to inorganic Hg on male gametes using spermatozoa (bovine sperm) which characteristically resemble human sperm. Sperm were divided and incubated for 0.5, 1 or 2 h at low levels of Hg: i) Control: without exposure; ii) Hg8 nM: mercury chloride (HgCl2) at 8 nM and iii) Hg8 µM: HgCl2 at 8 µM. Sperm kinetics, morphology, sperm membrane integrity, and in vitro fertilization were assessed. In addition the levels of reactive oxygen species (ROS), lipid peroxidation and total antioxidant capacity were measured. Hg exposure for 2 h impaired sperm morphology and membrane integrity as well as kinetic parameters including curvilinear velocity and straight-line velocity, which are needed for fertilization as evidenced by the reduced fertilization rate in 8 µM Hg-treated gametes. Hg enhanced oxidative stress in male sperm as reflected by elevated levels of ROS and lipid peroxidation and decreased antioxidant capacity. Data demonstrated that low levels of Hg when incubated with spermatozoa are sufficient to increase oxidative stress, adversely affect sperm quality parameters, subsequently impairing sperm fertility capacity.

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The present study assessed the effects of daily supplementation with 33 mg/metabolic weight (MW) of γ-oryzanol on testicular degeneration induced by scrotal insulation in rams. Eight animals were divided into two groups: Control (subjected to scrotal insulation without treatment) and Gamma (subjected to scrotal insulation and γ-oryzanol treatment). The rams were subjected to scrotal insulation by covering the scrotum with a thermal bag for 72 h. Animals in the Gamma group received 33 mg/MW oral γ-oryzanol once-daily, beginning 7 days before insulation and continuing during insulation and for 20 days afterward, for a total treatment period of 30 days. Samples of semen and blood were collected during the experiment to perform biochemical evaluations of oxidative stress, seminal kinetics and morphology, and plasma testosterone concentrations. Ultrasound examinations of the testicular parenchyma and clinical evaluations of its consistency and the scrotal perimeter were also performed at weekly intervals. Testicular tissue was collected for biochemical analyses of oxidative stress parameters at the end of the experiment by orchiectomy. The results showed that testicular degeneration was induced by scrotal insulation, as was demonstrated by the reduced scrotal perimeter and increased in testicular flaccidity immediately after insulation. Moreover, a delayed increase in the number of hyperechoic points in the parenchyma and a delayed reduction in sperm motility were observed at 10 weeks after insulation by ultrasonography. Treatment with γ-oryzanol reduced levels of reactive oxygen species (ROS) levels in the testes, and increased the total antioxidant potential (assessed based on the ferric reducing ability (FRAP)) in week 10 and levels of lipid peroxidation (TBARS). It also increased the number of intact spermatozoa in week 3, but increased the total number of sperm defects from week 5 onwards. Although γ-oryzanol protected the semen and testes by reducing the levels of the parameters of oxidative stress evaluated herein, the other parameters studied were not improved by the treatment. In addition, supplementation with γ-oryzanol led to more morphological abnormalities in the sperm. This study presented new information on the oral administration of γ-oryzanol to rams with testicular degeneration, and described potential therapies for this pathology, which currently has no established treatment and has important impacts on reproductive health.

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This study aimed to evaluate the effect of the force and duration of centrifugation and the impact of cushioned centrifugation on sperm selection by Percoll gradient, on sperm quality and development kinetics of in vitro produced bovine embryos. Two experiments were performed. In Experiment I, a pool of semen was selected by Percoll gradients and the pellet was divided into four groups and distributed in a 2 × 2 factorial, with two forces (2200 × g or 9000 × g) and two durations (1 min or 3 min) of centrifugation. In Experiment II, semen was divided into two groups and selected by Percoll gradient with Cushion Fluid (CF) or without CF (Control) in the second centrifugation. The morphofunctionality, biochemical characteristics and fertilizing capacity of the selected sperms were evaluated. In addition, the development of the resulting bovine embryos was monitored for 48 h post-insemination. Duncan and Chi-square tests (P < 0.05) were used to compare the means. In Experiment I, there was a significant increase in sperm vigor (P < 0.05) after sperm selection in all treatments. The force and duration of centrifugation did not have any effect on sperm motility, vigor, and recovery rate among the different treatments (P > 0.05). In Experiment II, the recovery rate and reactive oxygen species (ROS) production in semen were similar among treatments (P > 0.05) although a higher ROS production was observed in the CF fertilization medium. Total fertilization rate was superior in the CF group (65.4 ± 5.3%) compared to that in Control (39.6 ± 4.9%). However, the normal fertilization and cleavage rate did not differ between the Control (94 ± 6.3% and 58.3 ± 8.3%) and CF (89 ± 7.1% and 75.0 ± 7.3%) groups. The reduction in the force and duration of centrifugation did not decrease the sperm recovery during selection by the Percoll gradient and the use of CF in the second centrifugation did not affect the normal fertilization and development of bovine IVF embryos up to 48 h.(AU)

Esse estudo objetivou avaliar o efeito da força e duração da centrifugação, e o impacto da centrifugação amortecida na seleção espermática por gradientes de Percoll, na qualidade espermática e cinética do desenvolvimento de embriões bovinos produzidos in vitro. Dois experimentos foram realizados. No experimento I, um pool de sêmen foi selecionado por gradientes de Percoll e o pellet dividido em quatro grupos e distribuído em um fatorial 2 x 2, com duas forças (2200 e 9000 X g) e dois tempos (1 e 3 min) de centrifugação. No Experimento II, o sêmen foi dividido em dois grupos e selecionado com (CF) ou sem CushionFluid (Controle) na segunda centrifugação. A morfofuncionalidade, características bioquímicas e capacidade fecundante dos espermatozoides selecionados foram avaliadas. Além disso, o desenvolvimento dos embriões bovinos resultantes foi monitorado por 48 horas pós-inseminação. Os testes Duncan e Qui-quadrado (P<0,05) foram usados para comparar as médias. No Experimento I, houve um aumento significativo no vigor após a seleção espermática para todos os tratamentos. A força e a duração da centrifugação não tiveram nenhum efeito na motilidade, vigor e recuperação espermática entre os diferentes tratamentos (P > 0.05). No Experimento II, a taxa de recuperação e a produção de espécies reativas de oxigênio (EROs) no sêmen foram similares entre os tratamentos (P>0,05), embora a maior produção de EROs foi observada no meio de fecundação do grupo CF. A taxa de fecundação total, foi superior no grupo CF (65.4±5.3%) comparada com o Controle (39.6±4.9%). Entretanto a fecundação normal e a taxa de clivagem não diferiram entre os grupos Controle (94±6.3% e 58.3±8.3%) e CF (89±7.1% and 75.0±7.3%). A redução na força e duração da centrifugação não diminuiu a recuperação espermática durante a seleção por gradientes de Percoll e o uso de CF na segunda centrifugação não influenciou a fecundação normal e o desenvolvimento de embriões bovinos PIV até 48 horas.(AU)

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This study aimed to evaluate the effect of the force and duration of centrifugation and the impact of cushioned centrifugation on sperm selection by Percoll gradient, on sperm quality and development kinetics of in vitro produced bovine embryos. Two experiments were performed. In Experiment I, a pool of semen was selected by Percoll gradients and the pellet was divided into four groups and distributed in a 2 × 2 factorial, with two forces (2200 × g or 9000 × g) and two durations (1 min or 3 min) of centrifugation. In Experiment II, semen was divided into two groups and selected by Percoll gradient with Cushion Fluid (CF) or without CF (Control) in the second centrifugation. The morphofunctionality, biochemical characteristics and fertilizing capacity of the selected sperms were evaluated. In addition, the development of the resulting bovine embryos was monitored for 48 h post-insemination. Duncan and Chi-square tests (P 0.05). In Experiment II, the recovery rate and reactive oxygen species (ROS) production in semen were similar among treatments (P > 0.05) although a higher ROS production was observed in the CF fertilization medium. Total fertilization rate was superior in the CF group (65.4 ± 5.3%) compared to that in Control (39.6 ± 4.9%). However, the normal fertilization and cleavage rate did not differ between the Control (94 ± 6.3% and 58.3 ± 8.3%) and CF (89 ± 7.1% and 75.0 ± 7.3%) groups. The reduction in the force and duration of centrifugation did not decrease the sperm recovery during selection by the Percoll gradient and the use of CF in the second centrifugation did not affect the normal fertilization and development of bovine IVF embryos up to 48 h.

Esse estudo objetivou avaliar o efeito da força e duração da centrifugação, e o impacto da centrifugação amortecida na seleção espermática por gradientes de Percoll, na qualidade espermática e cinética do desenvolvimento de embriões bovinos produzidos in vitro. Dois experimentos foram realizados. No experimento I, um pool de sêmen foi selecionado por gradientes de Percoll e o pellet dividido em quatro grupos e distribuído em um fatorial 2 x 2, com duas forças (2200 e 9000 X g) e dois tempos (1 e 3 min) de centrifugação. No Experimento II, o sêmen foi dividido em dois grupos e selecionado com (CF) ou sem CushionFluid (Controle) na segunda centrifugação. A morfofuncionalidade, características bioquímicas e capacidade fecundante dos espermatozoides selecionados foram avaliadas. Além disso, o desenvolvimento dos embriões bovinos resultantes foi monitorado por 48 horas pós-inseminação. Os testes Duncan e Qui-quadrado (P 0.05). No Experimento II, a taxa de recuperação e a produção de espécies reativas de oxigênio (EROs) no sêmen foram similares entre os tratamentos (P>0,05), embora a maior produção de EROs foi observada no meio de fecundação do grupo CF. A taxa de fecundação total, foi superior no grupo CF (65.4±5.3%) comparada com o Controle (39.6±4.9%). Entretanto a fecundação normal e a taxa de clivagem não diferiram entre os grupos Controle (94±6.3% e 58.3±8.3%) e CF (89±7.1% and 75.0±7.3%). A redução na força e duração da centrifugação não diminuiu a recuperação espermática durante a seleção por gradientes de Percoll e o uso de CF na segunda centrifugação não influenciou a fecundação normal e o desenvolvimento de embriões bovinos PIV até 48 horas.

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Background: Despite the low efficiency caused by its harmful effects, vitrification is the technique of choice for oocyte cryopeservation, especially at the germinal vesicle (GV) stage. This enables the banking of female gametes without linkage to the male genotype. Follicular fluid (FF), in vivo, is known to provide an adequate environment to the immature oocyte. The intra-cytoplasmic sperm injection (ICSI), by the other hand, can be used to bypass any sperm penetration disorder, including the ones caused by cryopreservation. This study aimed to evaluate oocyte vitrification in FF based solution, and to asses ICSI efficiency in the fertilization of vitrified/warmed bovine GV oocytes. Material, Methods & Results: Follicles of 2-8 mm in diameter were aspirated from bovine ovaries obtained from a slaughterhouse, selected and maintained into FF from aspiration, until their allocation in the experimental groups. The FF used to prepare the vitrification solution was centrifuged, heat inactivated, filtered through a 0.22 mm pore and stored at -20°C. Oocyte vitrification was done into one of these three solutions: The standard solution TCM-Hepes (TH-Vitri) was compared to a totally FF based solution (FF-Vitri), and to a 50:50 (v/v) mix of both solutions (TH:FF-Vitri). Oocytes were submitted to in vitroembryo production in order to assess embryo production efficiency. A second set of experiments using the FF-Vitri solution compared IVF versus ICSI. With basis on cleaved structures, the morula + blastocyst rate obtained in the Fresh Control (43.9%) was similar to FF-Vitri (31.1%). Conversely, the TH-Vitri (15.7%) and the TH:FF-Vitri (20.4%) rates were significantly lower than the Fresh Control. ICSI showed a positive effect in comparison with IVF. The embryo development rate of Vitri-IVF (18.8%) was the lowest, whereas Vitri-ICSI (37.3%) was similar to the Fresh-IVF (43.9%), but lower than the Fresh-ICSI (57.8%).[...]

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Background: Despite the low efficiency caused by its harmful effects, vitrification is the technique of choice for oocyte cryopeservation, especially at the germinal vesicle (GV) stage. This enables the banking of female gametes without linkage to the male genotype. Follicular fluid (FF), in vivo, is known to provide an adequate environment to the immature oocyte. The intra-cytoplasmic sperm injection (ICSI), by the other hand, can be used to bypass any sperm penetration disorder, including the ones caused by cryopreservation. This study aimed to evaluate oocyte vitrification in FF based solution, and to asses ICSI efficiency in the fertilization of vitrified/warmed bovine GV oocytes. Material, Methods & Results: Follicles of 2-8 mm in diameter were aspirated from bovine ovaries obtained from a slaughterhouse, selected and maintained into FF from aspiration, until their allocation in the experimental groups. The FF used to prepare the vitrification solution was centrifuged, heat inactivated, filtered through a 0.22 mm pore and stored at -20°C. Oocyte vitrification was done into one of these three solutions: The standard solution TCM-Hepes (TH-Vitri) was compared to a totally FF based solution (FF-Vitri), and to a 50:50 (v/v) mix of both solutions (TH:FF-Vitri). Oocytes were submitted to in vitroembryo production in order to assess embryo production efficiency. A second set of experiments using the FF-Vitri solution compared IVF versus ICSI. With basis on cleaved structures, the morula + blastocyst rate obtained in the Fresh Control (43.9%) was similar to FF-Vitri (31.1%). Conversely, the TH-Vitri (15.7%) and the TH:FF-Vitri (20.4%) rates were significantly lower than the Fresh Control. ICSI showed a positive effect in comparison with IVF. The embryo development rate of Vitri-IVF (18.8%) was the lowest, whereas Vitri-ICSI (37.3%) was similar to the Fresh-IVF (43.9%), but lower than the Fresh-ICSI (57.8%).[...](AU)

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Green tea presents catechins as its major components and it has a potential antioxidant activity. Cyclophosmamide (CP) is an antineoplastic and immunosuppressive agent, known to reduce fertility. In the present study, we evaluated the effect of green tea infusion on cyclophosphamide-induced damage in male mice reproductive system. Mice received green tea infusion (250 mg/kg) or vehicle by gavage for 14 days. Saline or CP were injected intraperitoneally at a single dose (100 mg/kg) at the 14th day. Animals were euthanized 24 h after CP administration and testes and epididymis were removed for biochemical analysis and sperm evaluation. Catechins concentration in green tea infusion was evaluated by HPLC. CP increased lipid peroxidation, DNA damage and superoxide dismutase activity whereas sperm concentration, glutathione peroxidase (GPx), glutathione S-transferase (GST) and 17ß-hydroxysteroid (17ß-HSD) dehydrogenase activities were reduced in both tissues tested. Catalase activity and protein carbonyl levels were changed only in testes, after CP administration. Green tea pre-treatment reduced significantly lipid peroxidation, protein carbonylation, DNA damage and restored GPx and GST activity in testes. In epididymis, therapy significantly increased sperm concentration and restored GPx and 17ß-HSD activity. Green tea improves CP-induced damage on reproductive system, probably due to their high catechins content.