Effect of different concentrations of Trolox® in association with docosahexaenoic acid on equine semen freezing

The aim of this study was to evaluate the association of different concentrations of Trolox® and the addition of a fixed concentration of DHA in the freezing of semen of Mangalarga Marchador stallions. To that end, 16 ejaculates were frozen in the following extenders: E1) BotuCrio® (BC; Control); E2) BC + 50 ngml-1 DHA + 30 µM Trolox® (BCDHA30T); E3) BC + 50 ngml-1 DHA + 40 µM Trolox® (BCDHA40T); E4) BC + 50 ngml-1 DHA + 50 µM Trolox® (BCDHA50T). All the tested extenders were similar in preserving different kinematic parameters, cell functional integrity, compacted DNA, and high and intermediate mitochondrial activity (P>0.05). However, sperm cryopreserved in BCDHA40T showed higher velocities than sperm frozen in the control extender (P<0.05). The 30 µM concentration of Trolox® was worse for sperm motility and the 50 µM concentration of Trolox® did not adequately preserve the structural integrity of the membranes in an extender containing DHA when compared to the BotuCrio® (P<0.05) extender. The use of Trolox® in freezing extenders containing DHA did not maximize the effect of BotuCrio®, except for in the case of sperm velocity parameters when at a concentration of 40 µM.(AU)

Coenzyme Q-10 improves preservation of mitochondrial functionality and actin structure of cryopreserved stallion sperm

Abstract Coenzyme Q-10 (CoQ-10) is a cofactor for mitochondrial electron transport chain and may be an alternative to improve sperm quality of cryopreserved equine semen. This work aimed to improve stallion semen quality after freezing by adding CoQ-10 to the cryopreservation protocol. Seven saddle stallions were utilized. Each animal was submitted to five semen collections and freezing procedures. For cryopreservation, each ejaculate was divided in three treatments: 1) Botucrio® diluent (control); 2) 50 μmol CoQ-10 added to Botucrio® diluent; 3) 1 mmol CoQ-10 added to Botucrio® diluent. Semen batches were analyzed for sperm motility characteristics (CASA), plasma and acrosomal membranes integrity and mitochondrial membrane potential (by fluorescence probes propidium iodide, Hoechst 33342, FITC-PSA and JC-1, respectively), alterations in cytoskeletal actin (phalloidin-FITC) and mitochondrial function (diaminobenzidine; DAB). The 1 mmol CoQ-10 treatment presented higher (P<0.05) amount (66.8%) of sperm cells with fully stained midpiece (indicating high mitochondrial activity) and higher (P<0.05) amount (81.6%) of cells without actin reorganization to the post-acrosomal region compared to control group (60.8% and 76.0%, respectively). It was concluded that the addition of 1 mmol CoQ-10 to the freezing diluent was more effective in preserving mitochondria functionality and cytoskeleton of sperm cells submitted to cryopreservation process.

Coenzyme Q-10 improves preservation of mitochondrial functionality and actin structure of cryopreserved stallion sperm

Coenzyme Q-10 (CoQ-10) is a cofactor for mitochondrial electron transport chain and may be an alternative to improve sperm quality of cryopreserved equine semen. This work aimed to improve stallion semen quality after freezing by adding CoQ-10 to the cryopreservation protocol. Seven saddle stallions were utilized. Each animal was submitted to five semen collections and freezing procedures. For cryopreservation, each ejaculate was divided in three treatments: 1) Botucrio® diluent (control); 2) 50 μmol CoQ-10 added to Botucrio® diluent; 3) 1 mmol CoQ-10 added to Botucrio® diluent. Semen batches were analyzed for sperm motility characteristics (CASA), plasma and acrosomal membranes integrity and mitochondrial membrane potential (by fluorescence probes propidium iodide, Hoechst 33342, FITC-PSA and JC-1, respectively), alterations in cytoskeletal actin (phalloidin-FITC) and mitochondrial function (diaminobenzidine; DAB). The 1 mmol CoQ-10 treatment presented higher (P<0.05) amount (66.8%) of sperm cells with fully stained midpiece (indicating high mitochondrial activity) and higher (P<0.05) amount (81.6%) of cells without actin reorganization to the post-acrosomal region compared to control group (60.8% and 76.0%, respectively). It was concluded that the addition of 1 mmol CoQ-10 to the freezing diluent was more effective in preserving mitochondria functionality and cytoskeleton of sperm cells submitted to cryopreservation process.(AU)

The interference of ozone gas in kinects and mitochondrial potential of equine sperm submitted on cryopreservation

The objective of this study was to evaluate the effects of the addition of different concentrations of ozone to quarter horse semen submitted to cryopreservation. Six ejaculates from four stallions were collected and were divided in four experimental groups: a control group (BotuCRIO® extender) and three other groups with BotuCRIO® ozonized at concentrations of 6, 8 and 12 μg of O3/mL. The semen samples were diluted (200 x 106 spermatozoa/mL), filled in straws and frozen. After thawing (37 ºC, 30s), the samples were evaluated at 0, 30 and 60 minutes of incubation regarding sperm kinetics by a computer-assisted sperm analysis (CASA), and plasma membrane integrity (PMI), acrosome integrity (ACi) and mitochondrial membrane potential (MMP) by fluorescent probes. There was a reduction in the kinetic parameters total motility (TM), progressive motility (PM), curvilinear velocity (VCL), straight line velocity (VSL) and average path velocity (VAP) in all groups during the thermoresistance test (TT), a pattern also found in PMI and MMP analyses (p<0.05). There was no difference (p>0.05) between the control and treatment (6, 8, and 12 μg of O3/mL) groups, in any of the evaluated times for the kinetic parameters TM, linearity (LIN), straightness (STR), wobble index (WOB), amplitude of lateral head displacement (ALH) and beat cross frequency (BCF). Regarding the VCL, VSL and VAP parameters, the group treated with 6 μg did not differ from the control or from 8 μg, but was higher than 12 μg at 30 and 60 minutes. ACi and PMI did not differ between groups (p>0.05), but PMI was lower in groups 8 μg and 12 μg compared to the control and 6 μg (p<0.05). It was concluded that the addition of ozone does not present beneficial effects for cryopreservation of equine semen at the concentrations used and decreases important parameters of fertility.(AU)

The interference of ozone gas in kinects and mitochondrial potential of equine sperm submitted on cryopreservation

The objective of this study was to evaluate the effects of the addition of different concentrations of ozone to quarter horse semen submitted to cryopreservation. Six ejaculates from four stallions were collected and were divided in four experimental groups: a control group (BotuCRIO® extender) and three other groups with BotuCRIO® ozonized at concentrations of 6, 8 and 12 μg of O3/mL. The semen samples were diluted (200 x 106 spermatozoa/mL), filled in straws and frozen. After thawing (37 ºC, 30s), the samples were evaluated at 0, 30 and 60 minutes of incubation regarding sperm kinetics by a computer-assisted sperm analysis (CASA), and plasma membrane integrity (PMI), acrosome integrity (ACi) and mitochondrial membrane potential (MMP) by fluorescent probes. There was a reduction in the kinetic parameters total motility (TM), progressive motility (PM), curvilinear velocity (VCL), straight line velocity (VSL) and average path velocity (VAP) in all groups during the thermoresistance test (TT), a pattern also found in PMI and MMP analyses (p0.05) between the control and treatment (6, 8, and 12 μg of O3/mL) groups, in any of the evaluated times for the kinetic parameters TM, linearity (LIN), straightness (STR), wobble index (WOB), amplitude of lateral head displacement (ALH) and beat cross frequency (BCF). Regarding the VCL, VSL and VAP parameters, the group treated with 6 μg did not differ from the control or from 8 μg, but was higher than 12 μg at 30 and 60 minutes. ACi and PMI did not differ between groups (p>0.05), but PMI was lower in groups 8 μg and 12 μg compared to the control and 6 μg (p<0.05). It was concluded that the addition of ozone does not present beneficial effects for cryopreservation of equine semen at the concentrations used and decreases important parameters of fertility.

Características seminais pós-descongelamento em garanhões da raça crioula / Post-thaw seminal characteristics of Criollo breed stallions

Este trabalho teve como objetivo avaliar as características seminais de garanhões da raça Crioula após o processo de criopreservação utilizando o BotuCrio® . Foram utilizados 24 garanhões alojados nas proximidades de Porto Alegre, RS, Brasil. As análises do sêmen foram realizadas pré e pósdescongelamento através do sistema Computer Assisted Sperm Analysis AndroVision®. Somente foram utilizadas amostras com motilidade total ≥ 60 % e vigor ≥ 3, estas foram envasadas em palhetas de 0,5 ml, dispostas horizontalmente a 5ºC durante 20 minutos. Após colocadas a 6 cm acima do nível de nitrogênio líquido durante 20 minutos. Sendo finalmente imersas em nitrogênio líquido, após descongeladas em banho-maria a 37ºC durante 30 segundos. Os valores médios e desvios padrões das variáveis pós-descongelamento foram: motilidade total (52,85 % ± 7,27); motilidade progressiva (31,15 % ± 10,21); motilidade rápida (4,78 % ± 5,37); motilidade local (22,44 % ± 8,68); velocidade curvilinear (97,67 μm/s ± 35,25); velocidade em linha reta (35,33 μm/s ± 15,29); velocidade da trajetória média (44,99 μm/s ± 17,98); linearidade (0,3 % ± 0,15); BCF (2,5 Hz ± 1,7); ALH (0,35 μm ± 0,3); integridade funcional da membrana (42,73 % ± 7,06) e integridade estrutural da membrana (48,06 % ± 8,99). Concluímos que o protocolo utilizando diluente BotuCrio® é viável para criopreservação do sêmen de cavalos da raça Crioula, pois garante motilidade acima de 30 % pós-descongelamento que é recomendado pelo Colégio Brasileiro de Reprodução Animal.(AU)

The aim of this study was to evaluate seminal characteristics of Criollo breed stallions, after cryopreservation process using the BotuCrio®. One ejaculate from twenty-four stallions located near to Porto Alegre, RS, Brazil were used. Semen analysis were performed pre and post-freezing through the Computer Assisted Sperm Analysis AndroVision® system. Only samples with total motility ≥ 60 % and vigor ≥ 3 were used. The samples were placed in 0.5 ml straws, arranged horizontally at 5ºC for 20 minutes. Then, 6 cm above the level of liquid nitrogen in vapor for 20 minutes. Finally, being immersed in liquid nitrogen, the samples thawed in a water bath at 37ºC for 30 seconds. Mean values and standard deviations of post-freeze variables were: total motility (52.85 % ± 7.27); progressive motility (31.15 % ± 10.21); rapid motility (4.78 % ± 5.37); local motility (22.44 % ± 8.68); Curvilinear Velocity (97.67 μm/s ± 35.25); Straight Line Velocity (35.33 μm/s ± 15.29); Average Path Velocity (44.99 μm/s ± 17.98); Linearity (0.3 % ± 0.15); BCF (2.5 Hz ± 1.7); ALH (0.35 μm ± 0.3) functional integrity (42.73 % ± 7.06) and physical integrity (48.06 % ± 8.99). We conclude that the protocol used for semen cryopreservation is feasible for use in the Criollo breed, because it has motility above 30 % post-freezing semen parameters, wich is the recommended by the Brazilian College of Animal Reproduction.(AU)

Características seminais pós-descongelamento em garanhões da raça crioula / Post-thaw seminal characteristics of Criollo breed stallions

Este trabalho teve como objetivo avaliar as características seminais de garanhões da raça Crioula após o processo de criopreservação utilizando o BotuCrio® . Foram utilizados 24 garanhões alojados nas proximidades de Porto Alegre, RS, Brasil. As análises do sêmen foram realizadas pré e pósdescongelamento através do sistema Computer Assisted Sperm Analysis AndroVision®. Somente foram utilizadas amostras com motilidade total ≥ 60 % e vigor ≥ 3, estas foram envasadas em palhetas de 0,5 ml, dispostas horizontalmente a 5ºC durante 20 minutos. Após colocadas a 6 cm acima do nível de nitrogênio líquido durante 20 minutos. Sendo finalmente imersas em nitrogênio líquido, após descongeladas em banho-maria a 37ºC durante 30 segundos. Os valores médios e desvios padrões das variáveis pós-descongelamento foram: motilidade total (52,85 % ± 7,27); motilidade progressiva (31,15 % ± 10,21); motilidade rápida (4,78 % ± 5,37); motilidade local (22,44 % ± 8,68); velocidade curvilinear (97,67 μm/s ± 35,25); velocidade em linha reta (35,33 μm/s ± 15,29); velocidade da trajetória média (44,99 μm/s ± 17,98); linearidade (0,3 % ± 0,15); BCF (2,5 Hz ± 1,7); ALH (0,35 μm ± 0,3); integridade funcional da membrana (42,73 % ± 7,06) e integridade estrutural da membrana (48,06 % ± 8,99). Concluímos que o protocolo utilizando diluente BotuCrio® é viável para criopreservação do sêmen de cavalos da raça Crioula, pois garante motilidade acima de 30 % pós-descongelamento que é recomendado pelo Colégio Brasileiro de Reprodução Animal.

The aim of this study was to evaluate seminal characteristics of Criollo breed stallions, after cryopreservation process using the BotuCrio®. One ejaculate from twenty-four stallions located near to Porto Alegre, RS, Brazil were used. Semen analysis were performed pre and post-freezing through the Computer Assisted Sperm Analysis AndroVision® system. Only samples with total motility ≥ 60 % and vigor ≥ 3 were used. The samples were placed in 0.5 ml straws, arranged horizontally at 5ºC for 20 minutes. Then, 6 cm above the level of liquid nitrogen in vapor for 20 minutes. Finally, being immersed in liquid nitrogen, the samples thawed in a water bath at 37ºC for 30 seconds. Mean values and standard deviations of post-freeze variables were: total motility (52.85 % ± 7.27); progressive motility (31.15 % ± 10.21); rapid motility (4.78 % ± 5.37); local motility (22.44 % ± 8.68); Curvilinear Velocity (97.67 μm/s ± 35.25); Straight Line Velocity (35.33 μm/s ± 15.29); Average Path Velocity (44.99 μm/s ± 17.98); Linearity (0.3 % ± 0.15); BCF (2.5 Hz ± 1.7); ALH (0.35 μm ± 0.3) functional integrity (42.73 % ± 7.06) and physical integrity (48.06 % ± 8.99). We conclude that the protocol used for semen cryopreservation is feasible for use in the Criollo breed, because it has motility above 30 % post-freezing semen parameters, wich is the recommended by the Brazilian College of Animal Reproduction.

Padronização do Sperm Class Analyzer® (SCA®) para avaliação de espermatozoides equinos criopreservados / [Standardization of the Sperm Class Analyzer® (SCA®) for the evaluation of cryopreserved equine sperm]

Objetivou-se, no primeiro experimento, avaliar o efeito da velocidade de captura de imagens de 25Hz, 30Hz e 50Hz na cinética dos espermatozoides equinos criopreservados. Todas as velocidades mostraram-se adequadas para capturar o movimento espermático (P>0,05). No segundo experimento, objetivou-se avaliar o efeito da deposição de sêmen em lâmina sob lamínula, Leja®10 e 20, na cinética espermática. O uso de lâmina e lamínula foi superior às lejas para manter a LIN e o WOB (P<0,05). No terceiro experimento, objetivou-se avaliar o efeito das concentrações de 25, 50 e 100x106 na cinética espermática. As concentrações de 25 e 50 x106 foram superiores a 100x106 para preservar a LIN, a STR e a BCF e não afetar negativamente a motilidade (P<0,05). No quarto experimento, objetivou-se avaliar o efeito dos diluidores BotuCrio®, BotuSêmen®, TALP sperm e da solução fisiológica na cinética espermática. O BotuCrio® foi superior a todos os diluidores em preservar a BCF e os hiperativos (P<0,05). Conclui-se que o emprego da velocidade de captura entre 25 e 50Hz, a deposição do sêmen entre lâmina e lamínula e a rediluição em diluidor de congelação para atingir 25 a 50x106 de espermatozoides/mL são ideais para o SCA® avaliar, de forma fidedigna, o sêmen equino criopreservado.(AU)

The objective of the first experiment was to evaluate the effect of 25, 30 and 50Hz frame acquisition rate on equine cryopreserved sperm. All frame acquisition rates tested were adequate to capture the sperm movement (P>0.05). The aim of the second experiment was to evaluate the effect of chambers, slide-coverslip, Leja®10 and 20 on sperm movement. The use of slide-coverslip was superior to maintain LIN and WOB (P<0.05). The aim of the third experiment was to evaluate the effect of 25, 50 and 100x106 sperm/mL concentration on sperm movement. Concentrations of 25 and 50x106 sperm/mL were greater than 100x106 to preserve LIN, STR and BCF and did not adversely affect motility (P<0.05). The aim of the fourth experiment was to evaluate the effect of BotuCrio®, BotuSêmen®, TALP sperm and physiological solution on sperm movement. BotuCrio® was superior among other extenders in preserving BCF and hyperactive (P<0.05). It is concluded that the use of the frame acquisition rate between 25 and 50 Hz; the deposition of semen between slide and coverslip and new dilution in the freezing extender to 25-50x106 of sperm/mL is ideal to reliably evaluate cryopreserved equine semen by SCA®.(AU)

Padronização do Sperm Class Analyzer® (SCA®) para avaliação de espermatozoides equinos criopreservados / [Standardization of the Sperm Class Analyzer® (SCA®) for the evaluation of cryopreserved equine sperm]

Objetivou-se, no primeiro experimento, avaliar o efeito da velocidade de captura de imagens de 25Hz, 30Hz e 50Hz na cinética dos espermatozoides equinos criopreservados. Todas as velocidades mostraram-se adequadas para capturar o movimento espermático (P>0,05). No segundo experimento, objetivou-se avaliar o efeito da deposição de sêmen em lâmina sob lamínula, Leja®10 e 20, na cinética espermática. O uso de lâmina e lamínula foi superior às lejas para manter a LIN e o WOB (P<0,05). No terceiro experimento, objetivou-se avaliar o efeito das concentrações de 25, 50 e 100x106 na cinética espermática. As concentrações de 25 e 50 x106 foram superiores a 100x106 para preservar a LIN, a STR e a BCF e não afetar negativamente a motilidade (P<0,05). No quarto experimento, objetivou-se avaliar o efeito dos diluidores BotuCrio®, BotuSêmen®, TALP sperm e da solução fisiológica na cinética espermática. O BotuCrio® foi superior a todos os diluidores em preservar a BCF e os hiperativos (P<0,05). Conclui-se que o emprego da velocidade de captura entre 25 e 50Hz, a deposição do sêmen entre lâmina e lamínula e a rediluição em diluidor de congelação para atingir 25 a 50x106 de espermatozoides/mL são ideais para o SCA® avaliar, de forma fidedigna, o sêmen equino criopreservado.(AU)

The objective of the first experiment was to evaluate the effect of 25, 30 and 50Hz frame acquisition rate on equine cryopreserved sperm. All frame acquisition rates tested were adequate to capture the sperm movement (P>0.05). The aim of the second experiment was to evaluate the effect of chambers, slide-coverslip, Leja®10 and 20 on sperm movement. The use of slide-coverslip was superior to maintain LIN and WOB (P<0.05). The aim of the third experiment was to evaluate the effect of 25, 50 and 100x106 sperm/mL concentration on sperm movement. Concentrations of 25 and 50x106 sperm/mL were greater than 100x106 to preserve LIN, STR and BCF and did not adversely affect motility (P<0.05). The aim of the fourth experiment was to evaluate the effect of BotuCrio®, BotuSêmen®, TALP sperm and physiological solution on sperm movement. BotuCrio® was superior among other extenders in preserving BCF and hyperactive (P<0.05). It is concluded that the use of the frame acquisition rate between 25 and 50 Hz; the deposition of semen between slide and coverslip and new dilution in the freezing extender to 25-50x106 of sperm/mL is ideal to reliably evaluate cryopreserved equine semen by SCA®.(AU)

Can we use LDL instead of egg yolk in BotuCrio® extender to cryopreserve sperm from the Mangalarga Marchador stallion?

The objective of this study was to compare the BotuCrio® extender with the Merk - egg yolk and the INRA 82 modified by the inclusion of acetamide, methyl cellulose and trehalose in substitution of glycerol for freezing equine semen. The semen was diluted after centrifugation to obtain 100 x 106 of sperm/ml in: BotuCrio® (control); Merk - egg yolk or INRA 82 modified (Experiment 1). The extended semen was packaged in 0.5 ml straws, cooled and frozen in a freezing machine. The control extender was superior in preserving the motility, VCL, VSL, VAP, LIN, STR and the BCF when compared to the Merk - egg yolk and INRA 82 modified (P < 0.05). The BotuCrio® preserved more effectively the equine sperm viability characteristics evaluated in Experiment 1 and was used as a control extender in Experiment 2 to test the effectiveness of using LDL in replacement of egg yolk. BotuCrio® was superior to preserve progressive motility, VCL, VSL, VAP, LIN, STR and the percentage of functional integrity of sperm membranes compared to BotuCrio LDL (P < 0.05). However, both extenders preserved similarly the total motility, ALH, BCF and the structural integrity of the membranes (P > 0.05). The fertility rate after AI with frozen semen in BotuCrio LDL was 37.5%.(AU)

Can we use LDL instead of egg yolk in BotuCrio® extender to cryopreserve sperm from the Mangalarga Marchador stallion?

The objective of this study was to compare the BotuCrio® extender with the Merk - egg yolk and the INRA 82 modified by the inclusion of acetamide, methyl cellulose and trehalose in substitution of glycerol for freezing equine semen. The semen was diluted after centrifugation to obtain 100 x 106 of sperm/ml in: BotuCrio® (control); Merk - egg yolk or INRA 82 modified (Experiment 1). The extended semen was packaged in 0.5 ml straws, cooled and frozen in a freezing machine. The control extender was superior in preserving the motility, VCL, VSL, VAP, LIN, STR and the BCF when compared to the Merk - egg yolk and INRA 82 modified (P 0.05). The fertility rate after AI with frozen semen in BotuCrio LDL was 37.5%.

Functional assessment of diluent choice for semen cryopreservation from stallions with high and low freezability

Background: Fertility using horse frozen-thawed semen remains lower than in other livestock species. This fact suggeststhat horse semen hold intrinsic sensitivity to cryoinjury that must be investigated. Moreover, there is substantial evidenceof genetic factors upon horse cryopreservation outcome. Nonetheless, diluent and cryoprotectant choice for horse semencryopreservation are under intense research. Thus these factors could be explored to identify conditions that may increasesemen viability after thawing. The aim of this work was to evaluate the effect of diluents Botu-Crio®,Lactose-EDTA®, andINRA-82® on cryopreserved semen from stallions with high (HFA) and low freezability (LFA).Materials, Methods & Results: Frozen-thawed semen was evaluated for motility and membrane integrity using computerassisted semen analysis (CASA), and also inferred for sperm DNA fragmentation by sperm chromatin structure assay during the thermoresistance test (TRT). Comparisons for each parameter were done in a pair-wise fashion between HFA andLFA semen at one-hour intervals during the TRT (0 h - 4 h). Sperm motility in HFA, regardless of the diluent, was larger(P < 0.05) than LFA, both on 0h and 1h. In the 2h evaluation, sperm motility using Botu-Crio® and Lactose-EDTA® wasgreater (P < 0.05) for HFA. Analysis of sperm membrane integrity was similar between HFA and LFA semen (P > 0.05)at 0 h and 3 h. Sperm DNA fragmentation was lower (P < 0.05) in HFA semen at 0 h and 1 h.Discussion: Artificial insemination in horses using frozen-thawed semen is gaining wider acceptance under commercialsettings, although its current limited outreach due to low semen viability after thawing. Therefore, several efforts weremade toward ... (AU)

Cat epididymal semen cryopreserved with and without vitamin E: effect on sperm parameters and lipid peroxidation

The aims of this study were to investigate: 1) if the addition of α-tocopherol (vitamin E) in three concentrations (0.3, 0.6 and 0.9 mM) is able to preserve spermatozoa integrity after thawing and 2) the effect of α-tocopherol supplementation on lipid peroxidation. Fifty four domestic cats were used in this study constituting 18 pools (3 cats per pool). Each pool was submitted at four experimental groups: group 0 (control) – epididymal sperm were frozen with a commercial Botucrio® extender; group 0.3, group 0.6 and group 0.9 – the extender was supplemented with 0.3, 0.6 and 0.9 mM of α-tocopherol, respectively. Each semen sample was evaluated for motility, progressive forward motility, morphology, sperm viability (plasma membrane integrity-PMI), hypo-osmotic swelling test (HOST), before and after thawing. The evaluation of lipid peroxidation reaction by Thiobarbituric Acid Reactive Substances (TBARS) test was performed on thawed semen only. Results demonstrated that there was no significant difference between control and the three α-tocopherol groups with regards to motility and progressive motility after thawing (P > 0.05). As expected, in fresh samples viability was significantly higher than in all the cryopreserved groups in which there was no positive influence of any of the αtocopherol concentration used. Lipid peroxidation was higher in the supplemented groups 0.6 and 0.9 mM of α-tocopherol than in control and in 0.3 mM group. In conclusion, the addition of α-tocopherol to the commercial extender had no positive influence on reduction of lipid peroxidation. This topic deserves further investigations to better understand the effect of cryopreservation procedures on epididymal spermatozoa and to establish adequate strategies to counteract sperm cryodamages.

Cat epididymal semen cryopreserved with and without vitamin E: effect on sperm parameters and lipid peroxidation

The aims of this study were to investigate: 1) if the addition of α-tocopherol (vitamin E) in three concentrations (0.3, 0.6 and 0.9 mM) is able to preserve spermatozoa integrity after thawing and 2) the effect of α-tocopherol supplementation on lipid peroxidation. Fifty four domestic cats were used in this study constituting 18 pools (3 cats per pool). Each pool was submitted at four experimental groups: group 0 (control) epididymal sperm were frozen with a commercial Botucrio® extender; group 0.3, group 0.6 and group 0.9 the extender was supplemented with 0.3, 0.6 and 0.9 mM of α-tocopherol, respectively. Each semen sample was evaluated for motility, progressive forward motility, morphology, sperm viability (plasma membrane integrity-PMI), hypo-osmotic swelling test (HOST), before and after thawing. The evaluation of lipid peroxidation reaction by Thiobarbituric Acid Reactive Substances (TBARS) test was performed on thawed semen only. Results demonstrated that there was no significant difference between control and the three α-tocopherol groups with regards to motility and progressive motility after thawing (P > 0.05). As expected, in fresh samples viability was significantly higher than in all the cryopreserved groups in which there was no positive influence of any of the αtocopherol concentration used. Lipid peroxidation was higher in the supplemented groups 0.6 and 0.9 mM of α-tocopherol than in control and in 0.3 mM group. In conclusion, the addition of α-tocopherol to the commercial extender had no positive influence on reduction of lipid peroxidation. This topic deserves further investigations to better understand the effect of cryopreservation procedures on epididymal spermatozoa and to establish adequate strategies to counteract sperm cryodamages.(AU)

The addition of the salmon oil in the freezing of equine semen / A adição do óleo de salmão no congelamento do sêmen equino

The aim of the present study was to verify the effect of salmon oil addition on cryopreservation of equine semen. The experiment consisted of two treatments. Treatment 1 (T1) (control diluent), BotuCrio® was used without addition of salmon oil and treatment 2 (T2) (experimental diluent) BotuCrio® plus (with) 2% salmon oil. Three ejaculates of four stallions were used, totalizing 12 collections (n=12). Overall motility and progressive motility were evaluated by the Hamilton Thorn Research (HTR) Ceros 10.8 program, as well as the plasma membrane functionality through the hyposmotic test. Both treatments did not present statistical differences in relation to motility (T1 25,2±1,7 a, T2 29,7±1,9 a) and progressive motility (T1 11,0±1,1 a, T2 14,1±1,3 a). With respect to the hyposmotic test, the treatment 2 plus 2% of Salmon oil, presented better protection of sperm membrane functionality in relation to the control treatment (T2 77,3±1,0 a, T1 68,0±1,0 b). It can be concluded that salmon oil, although not altering the total and progressive motility, confers a better efficiency of sperm membrane functionality after thawing in equine semen.(AU)

O objetivo com este trabalho foi verificar o efeito da adição do óleo de salmão no congelamento do sêmen equino. O experimento constituiu de dois tratamentos. O tratamento 1 (T1) (diluente controle), utilizou-se o BotuCrio® sem a adição de óleo de salmão e o tratamento 2 (T2) (diluente experimental) BotuCrio® adicionado de 2% de óleo de salmão. Foram utilizados três ejaculados de quatro garanhões, totalizando doze coletas (n=12). Avaliou-se a motilidade total e motilidade progressiva pelo programa Ceros 10.8 da Hamilton Thorn Research (HTR), além da funcionalidade de membrana plasmática através do teste hiposmótico. Ambos os tratamentos não apresentaram diferenças estatísticas em relação a motilidade total (T1 25,2±1,7 a, T2 29,7±1,9 a) e motilidade progressiva (T1 11,0±1,1 a, T2 14,1±1,3 a). Com relação ao teste hiposmótico, o tratamento 2 acrescido de 2 % de óleo de Salmão, apresentou melhor proteção da funcionalidade de membrana espermática em relação ao tratamento controle (T2 77,3±1,0 a, T1 68,0±1,0 b). Pode-se concluir que o óleo de salmão, apesar de não alterar a motilidade total e progressiva, confere uma melhor eficiencia da funcionalidade de membrana espermática pós descongelamento em sêmen de equinos.(AU)

The addition of the salmon oil in the freezing of equine semen / A adição do óleo de salmão no congelamento do sêmen equino

The aim of the present study was to verify the effect of salmon oil addition on cryopreservation of equine semen. The experiment consisted of two treatments. Treatment 1 (T1) (control diluent), BotuCrio® was used without addition of salmon oil and treatment 2 (T2) (experimental diluent) BotuCrio® plus (with) 2% salmon oil. Three ejaculates of four stallions were used, totalizing 12 collections (n=12). Overall motility and progressive motility were evaluated by the Hamilton Thorn Research (HTR) Ceros 10.8 program, as well as the plasma membrane functionality through the hyposmotic test. Both treatments did not present statistical differences in relation to motility (T1 25,2±1,7 a, T2 29,7±1,9 a) and progressive motility (T1 11,0±1,1 a, T2 14,1±1,3 a). With respect to the hyposmotic test, the treatment 2 plus 2% of Salmon oil, presented better protection of sperm membrane functionality in relation to the control treatment (T2 77,3±1,0 a, T1 68,0±1,0 b). It can be concluded that salmon oil, although not altering the total and progressive motility, confers a better efficiency of sperm membrane functionality after thawing in equine semen.

O objetivo com este trabalho foi verificar o efeito da adição do óleo de salmão no congelamento do sêmen equino. O experimento constituiu de dois tratamentos. O tratamento 1 (T1) (diluente controle), utilizou-se o BotuCrio® sem a adição de óleo de salmão e o tratamento 2 (T2) (diluente experimental) BotuCrio® adicionado de 2% de óleo de salmão. Foram utilizados três ejaculados de quatro garanhões, totalizando doze coletas (n=12). Avaliou-se a motilidade total e motilidade progressiva pelo programa Ceros 10.8 da Hamilton Thorn Research (HTR), além da funcionalidade de membrana plasmática através do teste hiposmótico. Ambos os tratamentos não apresentaram diferenças estatísticas em relação a motilidade total (T1 25,2±1,7 a, T2 29,7±1,9 a) e motilidade progressiva (T1 11,0±1,1 a, T2 14,1±1,3 a). Com relação ao teste hiposmótico, o tratamento 2 acrescido de 2 % de óleo de Salmão, apresentou melhor proteção da funcionalidade de membrana espermática em relação ao tratamento controle (T2 77,3±1,0 a, T1 68,0±1,0 b). Pode-se concluir que o óleo de salmão, apesar de não alterar a motilidade total e progressiva, confere uma melhor eficiencia da funcionalidade de membrana espermática pós descongelamento em sêmen de equinos.

CONGELAÇÃO DE ESPERMATOZOIDES EQUINOS COM A ADIÇÃO DE EXTRATO DE Eugenia uniflora L (PITANGUEIRA) AO MEIO DILUIDOR COMERCIAL

As biotecnologias da reprodução utilizadas em equinos estão em constante aprimoramento e a criopreservação espermática é uma das mais utilizadas, por permitir o melhor aproveitamento do ejaculado, otimizando a disseminação de material genético de animais geneticamente superiores. Porém, esta biotécnica induz a alterações às células espermáticas, decorrente da produção exacerbada de espécies reativas do oxigênio (ERO). As ERO quando em excesso, promovem lesão a membrana plasmática do espermatozoide, diminuindo a sua viabilidade. Neste sentido, antioxidantes atuam no combate aos efeitos nocivos provocados pelos radicais livres, proporcionando melhor qualidade do sêmen fresco, refrigerado e congelado. A Eugenia uniflora L, popularmente conhecida como (pitangueira), apresenta compostos fenólicos com ação antioxidante, inibindo a peroxidação lipídica e a lipoperoxidação in vitro. Estes compostos desempenham um papel importante, agindo tanto na etapa de iniciação como na propagação do processo oxidativo. Entretanto, não existem relatos na literatura sobre a utilização do extrato de pitanga como agente antioxidante adicionado ao meio diluidor para sêmen equino. Assim, este trabalho teve como objetivo estudar os efeitos da fração solúvel em etanol das folhas da E. uniflora adicionado ao meio diluidor comercial para melhorar a cinética e a viabilidade do sêmen equino criopreservado. Foram utilizados quatro garanhões da raça quarto de milha, entre 5 e 15 anos de idade selecionados de acordo com o escore de condição corporal e exame andrológico. Foram colhidas 3 amostras de ejaculados, utilizando vagina artificial, com análise imediata quanto a motilidade, vigor espermático, morfologia espermática e integridade de membrana plasmática, avaliação subjetiva utilizando microscopio. Após avaliação, os ejaculados foram diluídos conforme os grupos experimentais: 1) grupo controle: diluidor BotuCrio®; 2) grupo 1: diluidor BotuCrio®, com a adição de extrato de E. uniflora na concentração de 1 mg para cada 120 x 106 espermatozoides por mL e 3) grupo 2: diluidor BotuCrio®, com a adição de extrato de E. uniflora na concentração de 2 mg para cada 120 x 106 espermatozoides por mL. Depois de diluido, as amostras foram submetidas a congelação. Após a descongelação em banho-maria a 46ºC por 12 segundos, as amostras foram analisadas em sistema computadorizado (CASA) para avaliação da cinética espermática. De acordo com as análises realizadas, apenas o parâmetro de porcentagem de espermatozoides com movimento médio diferenciou entre os grupos, com o controle apresentando maior porcentagem em relação aos grupos 1 e 2. Pôde-se concluir então, que a adição da fração solúvel em etanol das folhas da E. uniflora ao ejaculado equino, não altera a cinética espermática pós-descongelação.

The biotechnologies of reproduction used in horses are constantly improving and sperm cryopreservation is one of the most used, as it allows the best use of ejaculate, optimizing the dissemination of genetic material from genetically superior animals. However, this biotechnology induces alterations to cells, leading to the exacerbated production of reactive oxygen species (ROS). In excess, ROS damage the sperm's plasma membrane, decreasing its viability. In this sense, antioxidants act to combat the harmful effects caused by free radicals, providing better quality of fresh, chilled and frozen semen. Eugenia uniflora L (pitanga) has phenolic compounds with antioxidant action, inhibiting lipid peroxidation and lipoxygenase in vitro. These compounds play an important role, acting both in the initiation stage and in the propagation of the oxidative process. However, there are no reports in the literature on the use of pitanga extract as an antioxidant agent added to the diluting medium for equine semen. Thus, this work aimed to study the effect of E. uniflora L (pitanga) added to the commercial diluting medium to improve the kinetics and viability of cryopreserved equine semen. Four stallions of different breeds were used, selected according to the body condition score and andrological examination. Five samples of ejaculates were collected, using an artificial vagina, with immediate analysis for motility, sperm vigor, sperm morphology and plasma membrane integrity. Then, the ejaculates were diluted according to the experimental groups: 1) control group: BotuCrio® Diluidor; 2) group 1: BotuCrio® Diluter, with the addition of Eugenia uniflora L extract at a concentration of 1 mg for each 120 x 106 sperm per mL and 3) group 2: BotuCrio® Diluter, with the addition of E. uniflora L extract at a concentration of 2 mg for each 120 x 106 sperm per ml. Then, the samples were subjected to freezing. After thawing, the samples were analyzed in a computerized system (CASA) to evaluate sperm kinetics and to evaluate cell viability by flow cytometry. According to the analyzes performed, only the parameter of percentage of sperm with medium movement differed between the groups, with the control group presenting a higher percentage (P0.05) in relation to groups 1 and 2. It was concluded that the addition of the ethanol-soluble fraction of the leaves of E. uniflora L (pitanga) to the equine ejaculate does not alter the kinetics and sperm viability after thawing.

Ação do extrato etanólico de Mufumbo sobre a criopreservação do sêmen de garanhão / Action of the ethanol extract of Mufumbo on cryopreservation of stallion sêmen

Various natural antioxidants are being added to the freezing diluents in order to minimize deleteriouseffects to the membrane of spermatozoa during the cryopreservation process. Avaliou if stallions semencharacteristics cryopreserved amid thinner Botu-crio plus ethanol extract from the bark of mufumbo(Combretum leprosum) at different concentrations (30 mg, 60 mg and 120 mg). Samples of each treatment weresubmitted to hypoosmotic test membrane integrity tests and mitochondrial function (fluorescent tubes); Therewas no significant difference (P > 0.05) and the percentage of spermatozoa reactive to hiposmotic test betweenthe groups tested. The percentage of sperm with intact plasma membrane, as well as the mitochondrial potentialwere not statistically different (P > 0.05) between the control and treatment.(AU)

Ação do extrato etanólico de Mufumbo sobre a criopreservação do sêmen de garanhão / Action of the ethanol extract of Mufumbo on cryopreservation of stallion sêmen

Various natural antioxidants are being added to the freezing diluents in order to minimize deleteriouseffects to the membrane of spermatozoa during the cryopreservation process. Avaliou if stallions semencharacteristics cryopreserved amid thinner Botu-crio plus ethanol extract from the bark of mufumbo(Combretum leprosum) at different concentrations (30 mg, 60 mg and 120 mg). Samples of each treatment weresubmitted to hypoosmotic test membrane integrity tests and mitochondrial function (fluorescent tubes); Therewas no significant difference (P > 0.05) and the percentage of spermatozoa reactive to hiposmotic test betweenthe groups tested. The percentage of sperm with intact plasma membrane, as well as the mitochondrial potentialwere not statistically different (P > 0.05) between the control and treatment.

UTILIZAÇÃO DE DIFERENTES DILUENTES NA CRIOPRESERVAÇÃO DE ESPERMATOZOIDES DE GARANHÕES MANGALARGA MARCHADOR

title>Abstract /title> p>The techniques applied to animal reproduction such as artificial insemination, transfer and italic>in vitro /italic> production of embryos, heat synchronization and induction, and gametes cryopreservation have been more utilized in veterinary practice each day. Nevertheless, some techniques have not achieved their full technical capacity within the equine reproduction field, such as semen cryopreservation. The aim of this study was to evaluate the characteristics of post-thawing semen (total motility, strength, plasmatic and acrosomal membrane integrity) of Mangalarga Marchador breed stallions, using three different extenders (Botucrio, FR5 and FR6). Ejaculates from five stallions were collected and the gel-free semen was diluted in a 1:1 dilution in skim milk extender, and centrifuged at 600 g for 10 minutes. After the centrifugation the supernatant was removed and sperm pellet was divided and re-suspended using three different extenders to a concentration of 200 x 10 sup>6 /sup> cells/mL. The samples were packed into 0.5 mL straws, placed in a stainless steel support and kept inside the refrigerator (5 ºC) for one hour. Subsequently, these straws were kept at a height of 6 cm from liquid nitrogen for 15 minutes in an isotherm box and, after that, plunged into liquid nitrogen (-196 ºC) and stored in a liquid nitrogen holding tank. There were no differences in the parameters evaluated when extenders using mixed amides and glycerol (Botucrio and FR6) were used (P > 0.05). All parameters evaluated were lower for the extender containing only glycerol (P 0.05). The use of cryoprotectants (methylformamide and dimethylformamide) in association with glycerol concentrations around 1 to 2% is an alternative for semen cryopreservation of Mangalarga Marchador breed stallions. /p>

title>Resumo /title> p>As biotécnicas aplicadas à reprodução animal como inseminação artificial, transferência e produção italic>in vitro /italic> de embriões, indução e sincronização de cio e congelamento de gametas vêm sendo cada vez mais utilizadas na prática veterinária. No entanto, algumas biotécnicas ainda não alcançaram o seu total aperfeiçoamento técnico na reprodução equina, como a criopreservação de sêmen. Objetivou-se avaliar as características pós-descongelamento (motilidade total, vigor e integridade de membrana plasmática e acrosomal) dos espermatozoides de garanhões da raça Mangalarga Marchador (n=5), empregando-se três diluentes de criopreservação. Após a colheita, o sêmen foi diluído na proporção de 1:1 em meio à base de leite em pó desnatado e centrifugado a 600 G por 10 minutos. Após a centrifugação, o sobrenadante foi desprezado e o italic>pellet /italic> obtido dividido e ressuspendido com Botucrio, FR5 ou FR6. As amostras foram envasadas em palhetas de 0,5 mL sendo a concentração ajustada para 200x10 sup>6 /sup>espermatozoides/mL. As palhetas foram distribuídas em uma plataforma-suporte e estabilizadas a 5 ºC/60 min., em refrigerador comercial. Para o congelamento, as palhetas, posicionadas horizontalmente, foram expostas por 15 minutos ao vapor de nitrogênio líquido, em uma caixa de isopor, a 6 cm acima do nível de nitrogênio líquido. Logo em seguida, as palhetas foram imersas no nitrogênio líquido, acondicionadas em raques e estocadas em botijão criogênico a -196 ºC, para posterior avaliação. Não houve diferença para as variáveis motilidade, vigor e integridade das membranas plasmática e acrossomais quando se utilizaram diluentes que contêm a associação de amidas e glicerol (Botucrio e FR6; P>0,05). As variáveis seminais no diluente contendo apenas glicerol foram inferiores em todas as avaliações (P 0,05). A utilização de crioprotetores como a metilformamida, em associação com concentrações de 1 ou 2% de glicerol é uma alternativa para a criopreservação do sêmen de garanhões da raça Mangalarga Marchador. /p>