Correlation between insemination dose and bovine sperm traits on the in vitro embryo production outcomes / Correlação da dose inseminante e características do sêmen bovino no sucesso da produção in vitro de embriões

In the present study we aimed to test the best insemination dose for in vitro embryo production (IVEP) and to correlate sperm traits in bovine. In vitro matured oocytes were inseminated with three different sperm concentrations of the same bull: G1 (1*106), G2 (2*106) and G3 (4*106) sperm/mL. At 18 h post-insemination (hpi), presumptive zygotes [G1 (n=114), G2 (n=139) and G3 (n=136)] were stained to evaluate the pronuclei numbers, or continued to in vitro culture [G1 (n=102), G2 (n=111) and G3 (n=106)]. Sperm kinetics were analyzed using Computer-Assisted Semen Analysis (CASA). Sperm plasma membrane, acrosome integrity and mitochondrial activity were analyzed using fluorescent probes. In vitro fertilization (IVF) and IVEP data were compared using chi-square (P<0.05) and correlated with CASA and fluorescence data using Person Correlation (P<0.05). The IVF efficiency, cleavage and total blastocyst rates did not show any significant difference (P>0.05) among the groups. In G3, the polyspermy rate was the highest (7.4%; P<0.05) without difference (P>0.05) between G1 (0%) and G2 (0%). In G1, the early blastocyst rate was the highest (7.8%; P<0.05), without significant difference (P>0.05) between G2 (1.8%) and G3 (0.9%). The IVF efficiency and total blastocyst rates were positively correlated with curvilinear velocity (VCL) (r≃+1; P<0.05). We concluded that the reduction of insemination dose may negatively affect embryo development and VCL may be used as a parameter to improve the IVEP outcomes.

O objetivo deste estudo foi testar a melhor dose inseminante para a produção de embriões in vitro (IVEP) e sua correlação com as características espermáticas na espécie bovina. Oócitos maturados in vitro foram inseminados com três concentrações diferentes de espermatozoides de único touro: G1 (1*106), G2 (2*106) e G3 (4*106) espermatozoides/mL. Às 18h pós-inseminação (hpi), os presumíveis zigotos [G1 (114), G2 (139) e G3 (136)] foram corados para avaliar o número de pronúcleos, ou continuaram para o cultivo in vitro [G1 (102), G2 (111) e G3 (106)]. Os parâmetros da cinética espermática foram analisados usando o Computer-Assisted Semen Analysis (CASA). A integridade de membrana plasmática espermática, acrossomal e a atividade mitocondrial foram analisadas usando sondas fluorescentes. Os dados da fertilização in vitro (FIV) e IVEP foram comparadas com qui-quadrado (P=0,05) e correlacionados com dados de CASA e Fluorescência usando Correlação de Pearson (r=±1; P<0,05). A eficiência da FIV, taxas de clivagem e blastocisto total não mostraram diferença significativa (P>0,05) entre os grupos. Em G3, a taxa de polispermia foi a maior (7,4%; P<0,05), sem diferença (P>0,05) entre G1 (0%) e G2 (0%). Em G1, a taxa de blastocisto inicial foi a maior (7,8%; P<0,05), sem apresentar diferença significativa (P>0,05) com G2 (1,8%) e G3 (0,9%). A eficiência de FIV e a taxa de blastocisto total foram positivamente correlacionadas com velocidade curvilinear (VCL) (P<0,05). Concluímos que a dose inseminante reduzida pode negativamente afetar o desenvolvimento embrionário e VCL pode ser usada como parâmetro para melhorar os resultados da PEIV.

Correlation between insemination dose and bovine sperm traits on the in vitro embryo production outcomes / Correlação da dose inseminante e características do sêmen bovino no sucesso da produção in vitro de embriões

In the present study we aimed to test the best insemination dose for in vitro embryo production (IVEP) and to correlate sperm traits in bovine. In vitro matured oocytes were inseminated with three different sperm concentrations of the same bull: G1 (1*106 ), G2 (2*106 ) and G3 (4*106 ) sperm/mL. At 18 h post-insemination (hpi), presumptive zygotes [G1 (n=114), G2 (n=139) and G3 (n=136)] were stained to evaluate the pronuclei numbers, or continued to in vitro culture [G1 (n=102), G2 (n=111) and G3 (n=106)]. Sperm kinetics were analyzed using Computer-Assisted Semen Analysis (CASA). Sperm plasma membrane, acrosome integrity and mitochondrial activity were analyzed using fluorescent probes. In vitro fertilization (IVF) and IVEP data were compared using chisquare (P0.05) among the groups. In G3, the polyspermy rate was the highest (7.4%; P0.05) between G1 (0%) and G2 (0%). In G1, the early blastocyst rate was the highest (7.8%; P0.05) between G2 (1.8%) and G3 (0.9%). The IVF efficiency and total blastocyst rates were positively correlated with curvilinear velocity (VCL) (r≃+1; P<0,05). We concluded that the reduction of insemination dose may negatively affect embryo development and VCL may be used as a parameter to improve the IVEP outcomes.(AU)

O objetivo deste estudo foi testar a melhor dose inseminante para a produção de embriões in vitro (IVEP) e sua correlação com as características espermáticas na espécie bovina. Oócitos maturados in vitro foram inseminados com três concentrações diferentes de espermatozoides de único touro: G1 (1*106 ), G2 (2*106 ) e G3 (4*106 ) espermatozoides/mL. Às 18 h pós-inseminação (hpi), os presumíveis zigotos [G1 (114), G2 (139) e G3 (136)] foram corados para avaliar o número de pronúcleos, ou continuaram para o cultivo in vitro [G1 (102), G2 (111) e G3 (106)]. Os parâmetros da cinética espermática foram analisados usando o ComputerAssisted Semen Analysis (CASA). A integridade de membrana plasmática espermática, acrossomal e a atividade mitocondrial foram analisadas usando sondas fluorescentes. Os dados da fertilização in vitro (FIV) e IVEP foram comparadas com qui-quadrado (P=0,05) e correlacionados com dados de CASA e Fluorescência usando Correlação de Pearson (r=±1; P0,05) entre os grupos. Em G3, a taxa de polispermia foi a maior (7,4%; P0,05) entre G1 (0%) e G2 (0%). Em G1, a taxa de blastocisto inicial foi a maior (7,8%; P0,05) com G2 (1,8%) e G3 (0,9%). A eficiência de FIV e a taxa de blastocisto total foram positivamente correlacionadas com velocidade curvilinear (VCL) (P<0,05). Concluímos que a dose inseminante reduzida pode negativamente afetar o desenvolvimento embrionário e VCL pode ser usada como parâmetro para melhorar os resultados da PEIV.(AU)

Correlation between insemination dose and bovine sperm traits on the in vitro embryo production outcomes / Correlação da dose inseminante e características do sêmen bovino no sucesso da produção in vitro de embriões

In the present study we aimed to test the best insemination dose for in vitro embryo production (IVEP) and to correlate sperm traits in bovine. In vitro matured oocytes were inseminated with three different sperm concentrations of the same bull: G1 (1*106 ), G2 (2*106 ) and G3 (4*106 ) sperm/mL. At 18 h post-insemination (hpi), presumptive zygotes [G1 (n=114), G2 (n=139) and G3 (n=136)] were stained to evaluate the pronuclei numbers, or continued to in vitro culture [G1 (n=102), G2 (n=111) and G3 (n=106)]. Sperm kinetics were analyzed using Computer-Assisted Semen Analysis (CASA). Sperm plasma membrane, acrosome integrity and mitochondrial activity were analyzed using fluorescent probes. In vitro fertilization (IVF) and IVEP data were compared using chisquare (P0.05) among the groups. In G3, the polyspermy rate was the highest (7.4%; P0.05) between G1 (0%) and G2 (0%). In G1, the early blastocyst rate was the highest (7.8%; P0.05) between G2 (1.8%) and G3 (0.9%). The IVF efficiency and total blastocyst rates were positively correlated with curvilinear velocity (VCL) (r≃+1; P<0,05). We concluded that the reduction of insemination dose may negatively affect embryo development and VCL may be used as a parameter to improve the IVEP outcomes.

O objetivo deste estudo foi testar a melhor dose inseminante para a produção de embriões in vitro (IVEP) e sua correlação com as características espermáticas na espécie bovina. Oócitos maturados in vitro foram inseminados com três concentrações diferentes de espermatozoides de único touro: G1 (1*106 ), G2 (2*106 ) e G3 (4*106 ) espermatozoides/mL. Às 18 h pós-inseminação (hpi), os presumíveis zigotos [G1 (114), G2 (139) e G3 (136)] foram corados para avaliar o número de pronúcleos, ou continuaram para o cultivo in vitro [G1 (102), G2 (111) e G3 (106)]. Os parâmetros da cinética espermática foram analisados usando o ComputerAssisted Semen Analysis (CASA). A integridade de membrana plasmática espermática, acrossomal e a atividade mitocondrial foram analisadas usando sondas fluorescentes. Os dados da fertilização in vitro (FIV) e IVEP foram comparadas com qui-quadrado (P=0,05) e correlacionados com dados de CASA e Fluorescência usando Correlação de Pearson (r=±1; P0,05) entre os grupos. Em G3, a taxa de polispermia foi a maior (7,4%; P0,05) entre G1 (0%) e G2 (0%). Em G1, a taxa de blastocisto inicial foi a maior (7,8%; P0,05) com G2 (1,8%) e G3 (0,9%). A eficiência de FIV e a taxa de blastocisto total foram positivamente correlacionadas com velocidade curvilinear (VCL) (P<0,05). Concluímos que a dose inseminante reduzida pode negativamente afetar o desenvolvimento embrionário e VCL pode ser usada como parâmetro para melhorar os resultados da PEIV.

Influência do meio de conservação à base de água de coco em pó (ACP-102c) na manutenção da atividade mitocondrial de espermatozoides ovinos criopreservados / Influence of coconut powder water-based conservation medium (APC-102c) for maintaining mitochondrial activity of cryopreserved ram sperm

Background: Semen extenders are required to protect and preserve semen, and the development of suitable extenders iskey for artificial insemination. Although the use of Tris-based diluent is widespread, new diluents such as powdered coconut water have been developed for better sperm protection. One way to evaluate the effectiveness of diluents is throughmicroscopic analyses that evaluate sperm motility, vigor, and concentration. However, these analyses are limited, and maynot provide accurate results. New evaluation techniques have been studied, and one of the tests that can be used to addreliability to these analyses is mitochondrial activity evaluation, which can sum all the parameters, and provide a moreaccurate evaluation. Thus, the present study aimed to evaluate the efficacy of ACP-102c in cryopreserved ram semen.Materials, Methods & Results: Five semen samples were collected from two ram breeders using artificial vagina (n = 10).Each ejaculate was divided into the following two treatments: T1 - ACP-102c + 20% egg yolk + 7% glycerol and T2 - TRIS+ 20% egg yolk + 7% glycerol. Extended semen samples were then packed in 0.5 mL plastic straws, subjected through therefrigeration curve up to 4°C (0.35°C/min), and equilibrated for 2 h at 4°C. Subsequently, the straws were placed at 4 cmabove liquid nitrogen level (-60°C) for 15 min, immersed, and then finally stored in the liquid nitrogen at -196°C. Bothfresh and thawed samples were evaluated for total and progressive sperm motility using conventional microscopy (40x),and the same evaluator on each occasion. For plasma membrane integrity (IMP), the smear staining technique with theEosin-Nigrosin staining was used; 200 sperms were counted and classified as whole (unstained) and unhealthy (stained).Mitochondrial activity was evaluated using a cytochemical technique based on the oxidation of 3,3’-diaminobenzidine(DAB); 200 sperms were counted, and classified into four...

Influência do meio de conservação à base de água de coco em pó (ACP-102c) na manutenção da atividade mitocondrial de espermatozoides ovinos criopreservados / Influence of coconut powder water-based conservation medium (APC-102c) for maintaining mitochondrial activity of cryopreserved ram sperm

Background: Semen extenders are required to protect and preserve semen, and the development of suitable extenders iskey for artificial insemination. Although the use of Tris-based diluent is widespread, new diluents such as powdered coconut water have been developed for better sperm protection. One way to evaluate the effectiveness of diluents is throughmicroscopic analyses that evaluate sperm motility, vigor, and concentration. However, these analyses are limited, and maynot provide accurate results. New evaluation techniques have been studied, and one of the tests that can be used to addreliability to these analyses is mitochondrial activity evaluation, which can sum all the parameters, and provide a moreaccurate evaluation. Thus, the present study aimed to evaluate the efficacy of ACP-102c in cryopreserved ram semen.Materials, Methods & Results: Five semen samples were collected from two ram breeders using artificial vagina (n = 10).Each ejaculate was divided into the following two treatments: T1 - ACP-102c + 20% egg yolk + 7% glycerol and T2 - TRIS+ 20% egg yolk + 7% glycerol. Extended semen samples were then packed in 0.5 mL plastic straws, subjected through therefrigeration curve up to 4°C (0.35°C/min), and equilibrated for 2 h at 4°C. Subsequently, the straws were placed at 4 cmabove liquid nitrogen level (-60°C) for 15 min, immersed, and then finally stored in the liquid nitrogen at -196°C. Bothfresh and thawed samples were evaluated for total and progressive sperm motility using conventional microscopy (40x),and the same evaluator on each occasion. For plasma membrane integrity (IMP), the smear staining technique with theEosin-Nigrosin staining was used; 200 sperms were counted and classified as whole (unstained) and unhealthy (stained).Mitochondrial activity was evaluated using a cytochemical technique based on the oxidation of 3,3-diaminobenzidine(DAB); 200 sperms were counted, and classified into four...(AU)

Comparação da qualidade seminal de caprinos das raças Canindé e Alpina Britânica no Nordeste brasileiro / Comparison of the seminal quality of Canindé and British Alpine goats in the Brazilian Northeast

O estudo objetivou avaliar a qualidade seminal de caprinos das raças Canindé (autóctone) e Alpina Britânica (exótica) no Nordeste brasileiro. O experimento foi realizado nos períodos de julho a setembro dos anos de 2015 e 2016. As coletas seminais foram realizadas com auxílio de vagina artificial, de machos das raças Canindé (n = 4) e Alpina Britânica (n = 7). Após a coleta, o sêmen foi avaliado quanto a: volume (ml), concentração (sptz/ml), motilidade (%) e vigor (1-5). Em seguida, diluído em ACP-101c e criopreservado em máquina TK3000TM. Posteriormente, foram analisados os parâmetros cinéticos, através do software SCA®, e a atividade mitocondrial. No sêmen fresco, as duas raças apresentaram valores de motilidade e concentração dentro do preconizado para a espécie. Entretanto, os valores de volume e circunferência escrotal foram superiores na raça Alpina Britânica (0,73 ml ± 0,32; 27,35 cm ± 2,09) do que na raça Canindé (0,36 ml ± 0,07; 23,25 cm ± 0,95) (p < 0,05). Já no sêmen descongelado, os parâmetros motilidade total - MT (36,96% ± 11,16 vs. 20,50% ± 14,15), motilidade progressiva - MP (26,67% ± 11,13 vs. 11,29% ± 9,42), vigor (2,91 ± 0,90 vs. 1,88 ± 0,67), velocidade curvilinear - VCL (78,12 µm/s ± 12,41 vs. 59,28 µm/s ± 15,57), velocidade linear - VSL (49,23 µm/s ± 9,87 vs. 29,9 µm/s ± 9,48), velocidade média da trajetória - VAP (66,08 µm/s ± 12,77 vs. 45,70 µm/s ± 12,20), linearidade - LIN (62,97% ± 6,96 vs. 49,54%±9,50), retilinearidade - STR (74,63% ± 6,44 vs. 65,77% ± 8,92), e oscilação - WOB (84,32% ± 5,94 vs. 74,42% ± 7,31) foram superiores para a raça Canindé em detrimento à Alpina Britânica (p < 0,05). Portanto, o sêmen fresco das duas raças podem ser utilizados em biotécnicas reprodutivas. Já o sêmen pós-descongelação da raça Canindé apresentou melhor qualidade, provavelmente pelos animais estarem mais adaptados às condições adversas da região Nordeste, sendo recomendado para programas de inseminação artificial.(AU)

The objective of this study was to evaluate the sperm quality of Canindé (native) and British Alpine (exotic) goats in the Northeast of Brazil. The experiment was carried out from July to September of the years 2015 and 2016. Sperm collections of Canindé (n = 4) and British Alpine (n = 7) males were performed using artificial vagina. After collection, the sperm was evaluated for volume (ml), concentration (sptz/ml), motility (%), and vigor (1-5). Then diluted in ACP-101c and cryopreserved in TK3000TM machine. Subsequently, kinetic parameters were analyzed through SCA TM software and mitochondrial activity. In fresh sperm, the two races presented values of motility and concentration within the recommended for the specie. However, volume and scrotal circumference values were higher in the British Alpine breed (0.73 mL ± 0.32; 27.35 cm ± 2.09) than in the Canindé breed (0.36 mL ± 0.07; 23 , 25 cm ± 0.95) (p <0.05). In the thawed sperm, the parameters total motility - TM (36.96% ± 11.16 vs. 20.50% ± 14.15), progressive motility - PM (26.67% ± 11.13 vs. 11.29 % ± 9.42), vigor (2.91 ± 0.90 vs. 1.88 ± 0.67), curvilinear velocity - VCL (78.12 µm/s ± 12.41 vs. 59.28 µm/s ± 15.57), linear velocity - VSL (49.23 µm/s ± 9.87 vs. 29.9 µm/s ± 9.48), mean velocity of the trajectory - VAP (66.08 µm/s ± 12.77 vs. 45.70 µm/s ± 12.20), linearity - LIN (62.97% ± 6.96 vs. 49.54% ± 9.50), rectilinearity - STR (74.63% ± 6.44 vs. 65.77% ± 8.92), and oscillation -WOB (84.32% ± 5.94 vs. 74.42% ± 7.31) were higher for Canindé breed than for British Alpine ( p < 0.05). Therefore, fresh sperm from both breeds can be used in reproductive biotechniques. On the other hand, the post-thawed sperm of the Canindé breed showed better quality, probably because the animals were more adapted to the adverse conditions of the Northeast region and are recommended for artificial insemination programs.(AU)

Comparação da qualidade seminal de caprinos das raças Canindé e Alpina Britânica no Nordeste brasileiro / Comparison of the seminal quality of Canindé and British Alpine goats in the Brazilian Northeast

O estudo objetivou avaliar a qualidade seminal de caprinos das raças Canindé (autóctone) e Alpina Britânica (exótica) no Nordeste brasileiro. O experimento foi realizado nos períodos de julho a setembro dos anos de 2015 e 2016. As coletas seminais foram realizadas com auxílio de vagina artificial, de machos das raças Canindé (n = 4) e Alpina Britânica (n = 7). Após a coleta, o sêmen foi avaliado quanto a: volume (ml), concentração (sptz/ml), motilidade (%) e vigor (1-5). Em seguida, diluído em ACP-101c e criopreservado em máquina TK3000TM. Posteriormente, foram analisados os parâmetros cinéticos, através do software SCA®, e a atividade mitocondrial. No sêmen fresco, as duas raças apresentaram valores de motilidade e concentração dentro do preconizado para a espécie. Entretanto, os valores de volume e circunferência escrotal foram superiores na raça Alpina Britânica (0,73 ml ± 0,32; 27,35 cm ± 2,09) do que na raça Canindé (0,36 ml ± 0,07; 23,25 cm ± 0,95) (p < 0,05). Já no sêmen descongelado, os parâmetros motilidade total - MT (36,96% ± 11,16 vs. 20,50% ± 14,15), motilidade progressiva - MP (26,67% ± 11,13 vs. 11,29% ± 9,42), vigor (2,91 ± 0,90 vs. 1,88 ± 0,67), velocidade curvilinear - VCL (78,12 µm/s ± 12,41 vs. 59,28 µm/s ± 15,57), velocidade linear - VSL (49,23 µm/s ± 9,87 vs. 29,9 µm/s ± 9,48), velocidade média da trajetória - VAP (66,08 µm/s ± 12,77 vs. 45,70 µm/s ± 12,20), linearidade - LIN (62,97% ± 6,96 vs. 49,54%±9,50), retilinearidade - STR (74,63% ± 6,44 vs. 65,77% ± 8,92), e oscilação - WOB (84,32% ± 5,94 vs. 74,42% ± 7,31) foram superiores para a raça Canindé em detrimento à Alpina Britânica (p < 0,05). Portanto, o sêmen fresco das duas raças podem ser utilizados em biotécnicas reprodutivas. Já o sêmen pós-descongelação da raça Canindé apresentou melhor qualidade, provavelmente pelos animais estarem mais adaptados às condições adversas da região Nordeste, sendo recomendado para programas de inseminação artificial.(AU)

The objective of this study was to evaluate the sperm quality of Canindé (native) and British Alpine (exotic) goats in the Northeast of Brazil. The experiment was carried out from July to September of the years 2015 and 2016. Sperm collections of Canindé (n = 4) and British Alpine (n = 7) males were performed using artificial vagina. After collection, the sperm was evaluated for volume (ml), concentration (sptz/ml), motility (%), and vigor (1-5). Then diluted in ACP-101c and cryopreserved in TK3000TM machine. Subsequently, kinetic parameters were analyzed through SCA TM software and mitochondrial activity. In fresh sperm, the two races presented values of motility and concentration within the recommended for the specie. However, volume and scrotal circumference values were higher in the British Alpine breed (0.73 mL ± 0.32; 27.35 cm ± 2.09) than in the Canindé breed (0.36 mL ± 0.07; 23 , 25 cm ± 0.95) (p <0.05). In the thawed sperm, the parameters total motility - TM (36.96% ± 11.16 vs. 20.50% ± 14.15), progressive motility - PM (26.67% ± 11.13 vs. 11.29 % ± 9.42), vigor (2.91 ± 0.90 vs. 1.88 ± 0.67), curvilinear velocity - VCL (78.12 µm/s ± 12.41 vs. 59.28 µm/s ± 15.57), linear velocity - VSL (49.23 µm/s ± 9.87 vs. 29.9 µm/s ± 9.48), mean velocity of the trajectory - VAP (66.08 µm/s ± 12.77 vs. 45.70 µm/s ± 12.20), linearity - LIN (62.97% ± 6.96 vs. 49.54% ± 9.50), rectilinearity - STR (74.63% ± 6.44 vs. 65.77% ± 8.92), and oscillation -WOB (84.32% ± 5.94 vs. 74.42% ± 7.31) were higher for Canindé breed than for British Alpine ( p < 0.05). Therefore, fresh sperm from both breeds can be used in reproductive biotechniques. On the other hand, the post-thawed sperm of the Canindé breed showed better quality, probably because the animals were more adapted to the adverse conditions of the Northeast region and are recommended for artificial insemination programs.(AU)

Identification of ultrastructural and functional damages in sperm from six-banded armadillos (Euphractus sexcinctus) due to cryopreservation / Identificação de danos ultraestruturais e funcionais em espermatozoides de tatus-peba (Euphractus sexcinctus) oriundos da criopreservação

The aim of the study was to cryopreserve the semen of six-banded armadillos (Euphractus sexcinctus) in Tris-yolk and glycerol diluent, and to determine the damage caused by the freezing-thawing process, using fluorescent markers and ultrastructural analysis. Semen samples (n=11) collected from 4 adult six-banded armadillos by electroejaculation were cryopreserved in Tris diluent plus 20% egg yolk and 3% glycerol, in a fast freezing curve. Classical analysis of samples was performed after dilution, refrigeration and thawing, followed by fluorescence analysis, using a combination of fluorescent probes to assess membrane integrity (propidium iodide - PI and Hoechst - H342), and mitochondrial activity (CMXRos - Mito Tracker Red®). We also used the ultrastructural analysis to verify possible morphological alterations caused by cryoinjuries. When compared with fresh samples, we verified a significant decline in all the armadillos' semen parameters after thawing, in which only 6.1% motile sperm were found. However, the percentage of sperm which remained with viable (13%) and functional (24.7%) membranes after thawing suggests that some cells could be live but immotile. Analysis using fluorescent markers revealed that the mitochondria of armadillos' sperm is highly sensible to the freezing protocol and the findings through ultrastructure analysis proved this statement. Additionally, the images obtained by transmission electron microscopy revealed that frozen-thawed sperm presented damaged plasma membrane, nuclear modifications as changes in chromatin and acrossomal changes relative to sperm capacitation. In conclusion, this study is the first attempt to cryopreserve the semen of an armadillo species, and to help us to identify critical points on the freezing-thawing procedure in order to improve the protocol.(AU)

O objetivo deste estudo foi criopreservar o sêmen de tatus-peba (Euphractus sexcinctus) em diluente Tris-gema e glicerol, e determinar os danos causados pelo processo de congelação-descongelação, utilizando marcadores fluorescentes e análise ultraestrutural. As amostras de sêmen (n=11) coletadas de 4 tatus-peba adultos por eletroejaculação foram criopreservadas em diluente Tris acrescido de 20% de gema de ovo e 3% de glicerol, em curva rápida de congelação. A análise clássica das amostras foi realizada após a diluição, refrigeração e descongelação, seguida por análise de fluorescência, utilizando uma combinação de sondas fluorescentes para avaliar a integridade da membrana (Iodeto de Propídio - PI e Hoechst - H342), e a atividade mitocondrial (CMXRos - Mito Tracker RED®). Foi também utilizada a análise ultraestrutural para verificar possíveis alterações morfológicas causadas pela crioinjúria. Quando comparadas com as amostras a fresco, verificou-se uma queda significativa em todos os parâmetros seminais dos tatus após a descongelação, em que apenas 6,1% de espermatozoides móveis foram encontrados. No entanto, o percentual de espermatozoides que permaneceu com membrana viável (13%) e funcional (24,7%) após a descongelação sugere que algumas células podem estar vivas, mas imóveis. Análises utilizando marcadores fluorescentes revelaram que as mitocôndrias dos espermatozoides de tatus são altamente sensíveis ao protocolo de congelação e os achados através da análise ultraestrutural comprovaram esta afirmação. Além disso, as imagens obtidas por microscopia eletrônica de transmissão revelaram que espermatozoides congelados-descongelados apresentaram membranas plasmáticas danificadas, modificações nucleares como alterações na cromatina, e alterações acrossomais relativas à capacitação espermática. Em conclusão, este estudo é a primeira tentativa de criopreservação de sêmen em uma espécie de tatu, e nos auxiliou a identificar pontos críticos no processo de congelação-descongelação, a fim de melhorar o protocolo.(AU)

Identification of ultrastructural and functional damages in sperm from six-banded armadillos (Euphractus sexcinctus) due to cryopreservation / Identificação de danos ultraestruturais e funcionais em espermatozoides de tatus-peba (Euphractus sexcinctus) oriundos da criopreservação

The aim of the study was to cryopreserve the semen of six-banded armadillos (Euphractus sexcinctus) in Tris-yolk and glycerol diluent, and to determine the damage caused by the freezing-thawing process, using fluorescent markers and ultrastructural analysis. Semen samples (n=11) collected from 4 adult six-banded armadillos by electroejaculation were cryopreserved in Tris diluent plus 20% egg yolk and 3% glycerol, in a fast freezing curve. Classical analysis of samples was performed after dilution, refrigeration and thawing, followed by fluorescence analysis, using a combination of fluorescent probes to assess membrane integrity (propidium iodide - PI and Hoechst - H342), and mitochondrial activity (CMXRos - Mito Tracker Red®). We also used the ultrastructural analysis to verify possible morphological alterations caused by cryoinjuries. When compared with fresh samples, we verified a significant decline in all the armadillos' semen parameters after thawing, in which only 6.1% motile sperm were found. However, the percentage of sperm which remained with viable (13%) and functional (24.7%) membranes after thawing suggests that some cells could be live but immotile. Analysis using fluorescent markers revealed that the mitochondria of armadillos' sperm is highly sensible to the freezing protocol and the findings through ultrastructure analysis proved this statement. Additionally, the images obtained by transmission electron microscopy revealed that frozen-thawed sperm presented damaged plasma membrane, nuclear modifications as changes in chromatin and acrossomal changes relative to sperm capacitation. In conclusion, this study is the first attempt to cryopreserve the semen of an armadillo species, and to help us to identify critical points on the freezing-thawing procedure in order to improve the protocol.(AU)

O objetivo deste estudo foi criopreservar o sêmen de tatus-peba (Euphractus sexcinctus) em diluente Tris-gema e glicerol, e determinar os danos causados pelo processo de congelação-descongelação, utilizando marcadores fluorescentes e análise ultraestrutural. As amostras de sêmen (n=11) coletadas de 4 tatus-peba adultos por eletroejaculação foram criopreservadas em diluente Tris acrescido de 20% de gema de ovo e 3% de glicerol, em curva rápida de congelação. A análise clássica das amostras foi realizada após a diluição, refrigeração e descongelação, seguida por análise de fluorescência, utilizando uma combinação de sondas fluorescentes para avaliar a integridade da membrana (Iodeto de Propídio - PI e Hoechst - H342), e a atividade mitocondrial (CMXRos - Mito Tracker RED®). Foi também utilizada a análise ultraestrutural para verificar possíveis alterações morfológicas causadas pela crioinjúria. Quando comparadas com as amostras a fresco, verificou-se uma queda significativa em todos os parâmetros seminais dos tatus após a descongelação, em que apenas 6,1% de espermatozoides móveis foram encontrados. No entanto, o percentual de espermatozoides que permaneceu com membrana viável (13%) e funcional (24,7%) após a descongelação sugere que algumas células podem estar vivas, mas imóveis. Análises utilizando marcadores fluorescentes revelaram que as mitocôndrias dos espermatozoides de tatus são altamente sensíveis ao protocolo de congelação e os achados através da análise ultraestrutural comprovaram esta afirmação.[...] (AU)

Evaluation of sperm quality in successive regular collections from captive black-and-gold howler monkeys (Alouatta caraya)

The black-and-gold howler monkey serves as a suitable model for development of assisted reproductive techniques (ARTs), which may later be applied in threatened species of the same genus. The objectives of this study were to evaluate semen characteristics of black-and-gold howler monkeys (Alouatta caraya), with emphasis on mitochondrial activity and acrosome integrity, using staining methods that are suitable for use under field conditions. Twenty six semen samples were collected by rectal probe electroejaculation from six adult captive males from the National Primate Center (CENP), Ananindeua, PA, Brazil. The following parameters were evaluated in each semen sample: volume, pH, concentration, plasma membrane integrity, acrosome integrity, and mitochondrial activity. Both Spermac stain and the Simple Staining Method were suitable for acrosome evaluation. Diaminobenzidine needs further investigation to improve its application for this species. Overall semen quality improved from the first collection to the fifth collection. This highlights the importance of repeating semen collections when evaluating a male for use in ARTs. This research brought novel information on semen characteristics of Alouatta caraya which will aid further studies on the application of ARTs in this and other primate species.

Evaluation of sperm quality in successive regular collections from captive black-and-gold howler monkeys (Alouatta caraya)

The black-and-gold howler monkey serves as a suitable model for development of assisted reproductive techniques (ARTs), which may later be applied in threatened species of the same genus. The objectives of this study were to evaluate semen characteristics of black-and-gold howler monkeys (Alouatta caraya), with emphasis on mitochondrial activity and acrosome integrity, using staining methods that are suitable for use under field conditions. Twenty six semen samples were collected by rectal probe electroejaculation from six adult captive males from the National Primate Center (CENP), Ananindeua, PA, Brazil. The following parameters were evaluated in each semen sample: volume, pH, concentration, plasma membrane integrity, acrosome integrity, and mitochondrial activity. Both Spermac stain and the Simple Staining Method were suitable for acrosome evaluation. Diaminobenzidine needs further investigation to improve its application for this species. Overall semen quality improved from the first collection to the fifth collection. This highlights the importance of repeating semen collections when evaluating a male for use in ARTs. This research brought novel information on semen characteristics of Alouatta caraya which will aid further studies on the application of ARTs in this and other primate species.(AU)

EFEITO DA MELATONINA NA MATURAÇÃO IN VITRO SOBRE A EXPRESSÃO GÊNICA E A QUALIDADE DE OÓCITOS BOVINOS SOB CHOQUE TÉRMICO

Conduziu-se este trabalho, com o objetivo de avaliar o efeito da adição de melatonina ao meio de maturação in vitro (MIV) de oócitos bovinos submetidos ao choque térmico sobre sua qualidade e expressão de genes específicos. Foram aspirados folículos ovarianos, provenientes de ovários de abatedouro, com 3 a 8 mm de diâmetro. Os oócitos foram maturados sob choque térmico em meio MIV acrescido de melatonina nas concentrações 0; 10-12 ;10-9 ; 10-6 e 10-3 M. No controle positivo, os oócitos foram maturados na ausência de melatonina (NS) sob condição convencional. Foram realizadas de três a seis baterias por análise. Foi considerado o delineamento em blocos ao acaso, sendo os blocos formados pelas baterias. As taxas de maturação e de apoptose, a produção de espécies reativas de oxigênio (EROS) e a atividade mitocondrial foram analisadas por modelos lineares generalizados pelo procedimento GLIMMIX (SAS®). Na análise de expressão gênica, foi utilizado como calibrador o tratamento NS e a eficiência do primer foi calculada pelo programa LinRegPCR, com os resultados analisados pelo software REST®. A taxa de maturação não diferiu entre oócitos maturados sob choque térmico com diferentes concentrações de melatonina e não diferiu entre oócitos dos tratamentos 0 M de melatonina sob choque térmico e 0 M de melatonina sob condição convencional. As concentrações 10-3 e 10-9 M de melatonina reduziram a taxa de maturação em relação ao NS. A melatonina não influenciou a taxa de apoptose. A taxa de apoptose do grupo NS foi menor do que a observada no grupo 0 M, mas não diferiu da observada no tratamento 10-3 M. A produção de EROS foi menor nos tratamentos 10-9 , 10-6 e 10-3 M e maior nos tratamentos 0 e 10-12 M. A atividade mitocondrial foi menor no tratamento 10-3 M em comparação aos demais tratamentos sob choque térmico. A atividade mitocondrial dos oócitos maturados com 10-12, 10-9 e 10-6 M de melatonina não diferiu da observada nos oócitos do tratamento NS. Oócitos do grupo 0 M apresentaram maior atividade mitocondrial comparado ao grupo NS. Houve sub expressão dos genes C-MOS nos tratamentos 10-9 e 10-12 M de melatonina e de MAPK no tratamento 10-12M. Os tratamentos 10-3 ; 10-6 e 10-9 M de melatonina apresentaram superexpressão do gene GDF9. Os genes ZAR1 e HSP40 não foram afetados pela adição de melatonina ao meio MIV ou pelo choque térmico. Conclui-se que a melatonina é capaz de proteger os oócitos contra prejuízos causados pelo choque térmico, sendo a concentração 10-6 M a mais eficiente.

The objective of this study was to evaluate the effect of melatonin addition to the in vitro maturation (IVM) medium of bovine oocytes subjected to heat shock on quality and expression of specific genes. Ovarian follicles from 3 to 8 mm of diameter, were aspirated from ovaries obtained in a slaughterhouse. Oocytes were matured under heat shock in IVM medium with melatonin, in the following concentrations: 0; 1012; 10-9 ; 10-6 e 10-3 M. In the positive control, oocytes were matured in the absence of melatonin (NS) on conventional condition. Three to six batteries were performed for each analysis. The randomized block design was utilized, being the blocks formed by the batteries. Maturation and apoptosis rate, reactive oxygen species (ROS) and mitochondrial activity were analyzed by generalized linear models through the GLIMIMIX procedure (SAS® ). To the gene expression analysis, the treatment NS was used as a calibrator and the primer efficiency was calculated through the program LinRegPCR, with the results being analyzed by the REST® software. Maturation rate did not differ between oocytes matured under heat shock with different melatonin concentration and did not differ between oocytes from treatments 0 M of melatonin under heat shock and 0 M of melatonin under conventional conditions. The concentrations 10-3 and 10-9 M of melatonin reduced the maturation rate in comparison to NS. Melatonin did not influence the apoptosis rate. Apoptosis rate from NS group was lower than observed on group 0 M, but it did not differ from the rate observed in the treatment 10-3 . ROS production was lower in the treatments 10-9 , 10-6 , and 10-3 M and greater in the treatments 0 and 10-12. Mitochondrial activity of oocytes matured at 10-12, 10- 9 , and 10-6 M of melatonin did not differ from the one observed in oocytes of treatment NS. Oocytes from group 0 M presented greater mitochondrial activity compared to oocytes from group NS. There was subexpression of genes c-MOS in the treatments 10-9 and 10-12 M of melatonin and of MAPK in the treatment 10-12 M. The treatments 10-3 ; 10-6 and 10-9 M of melatonin presented subexpression of the gene GDF-9. Genes ZAR1 and HSP40 were not affected by the melatonin addition to the IVM medium or by the heat shock. Concluding, melatonin is able to protect oocytes from the damage caused by heat shock, being the concentration 10-6 the most efficient.

Comparison of CapriPure® and Percoll® density gradients for sperm separation of frozen-thawed goat spermatozoa

Frozen - thawed semen from six Boer bucks was pooled and used to compare the recently available commercial product CapriPure ® with Percoll ® for sperm quality . Sperm quality parameters such as sperm motility, concentration, membrane integrity (PI/CFDA), acrosome integrity (FITC - PNA) and mitochondrial activity (JC - 1) were evaluated before and after sperm processing using CapriPure ® and Percoll ® density gradient s . There was a significant r eduction (P < 0.05) in the percentage of spermatozoa with intact acrosomes following Percoll ® gradient centrifugation. However , t here was a significant increase in the percentage of spermatozoa with high mitochondrial membrane potential in both CapriPure ® (P < 0.01) and Percoll ® (P < 0.05) density gradient centrifugation methods . The samples selected using the CapriPure ® gradient achieved higher numerical values of membrane integrity, acrosome integrity as well as high mitochondrial membrane potential and s howed a tendency for improvement of sperm parameters compared with the Percoll ® gradient, suggesting that CapriPure ® is a good alternative to Percoll ® for goat sperm separation . However, further studies are needed to assess the fertilizing capacity of sper matozoa following the use of the CapriPure ® gradient.

Comparison of CapriPure® and Percoll® density gradients for sperm separation of frozen-thawed goat spermatozoa

Frozen - thawed semen from six Boer bucks was pooled and used to compare the recently available commercial product CapriPure ® with Percoll ® for sperm quality . Sperm quality parameters such as sperm motility, concentration, membrane integrity (PI/CFDA), acrosome integrity (FITC - PNA) and mitochondrial activity (JC - 1) were evaluated before and after sperm processing using CapriPure ® and Percoll ® density gradient s . There was a significant r eduction (P < 0.05) in the percentage of spermatozoa with intact acrosomes following Percoll ® gradient centrifugation. However , t here was a significant increase in the percentage of spermatozoa with high mitochondrial membrane potential in both CapriPure ® (P < 0.01) and Percoll ® (P < 0.05) density gradient centrifugation methods . The samples selected using the CapriPure ® gradient achieved higher numerical values of membrane integrity, acrosome integrity as well as high mitochondrial membrane potential and s howed a tendency for improvement of sperm parameters compared with the Percoll ® gradient, suggesting that CapriPure ® is a good alternative to Percoll ® for goat sperm separation . However, further studies are needed to assess the fertilizing capacity of sper matozoa following the use of the CapriPure ® gradient.(AU)