Sêmen refrigerado ­ estado da arte em diferentes espécies / Chilled semen ­ state of the art in different species

Na atual conjuntura da criação artificial de bovinos e bubalinos, o material genético masculino de qualidade superior de reprodutores é explorado ao máximo possível através da inseminação artificial em tempo fixo de um grande número de fêmeas com apenas um único ejaculado. Para isso, é necessário um sêmen de boa qualidade que desempenhe um papel indispensável na melhoria das taxas de fertilidade, independente de qual tipo seja utilizado (fresco, refrigerado e congelado). Porém, o processo de congelação/descongelação causa uma série de injúrias aos espermatozoides, ocasionando resultados inferiores para percentuais de viabilidade espermática, motilidade, membrana plasmática e integridade acrossomal, potencial de membrana mitocondrial, cinemática do esperma, quando comparado ao sêmen refrigerado. Assim, o objetivo desta revisão é disseminar o conhecimento sobre o uso sêmen refrigerado na preservação de germoplasma de reprodutores bovinos e bubalinos para melhorar as taxas de concepção em propriedades. Para isso, serão abordados comentários sobre o armazenamento do sêmen refrigerado, com ênfase nas diferenças entre curvas de refrigeração, suas vantagens e desvantagens relativas para procedimentos de uso na IATF, identificando o método mais indicado por diversos autores, o estado atual da biotécnica, seus méritos e possibilidades futuras.(AU)

In the current conjuncture of the artificial creation of bovines and buffaloes, the male genetic material of superior quality of sires is exploited to the maximum possible through the fixed-time artificial insemination of a large number of females with only a single ejaculate. For this, a good quality semen is needed that plays an indispensable role in improving fertility rates, regardless of which type is used (fresh, chilled and frozen). However, the freezing/thawing process causes a series of injuries to spermatozoa, causing lower results for percentages of sperm viability, motility, plasma membrane and acrosomal integrity, mitochondrial membrane potential, sperm kinematics, when compared to refrigerated semen. Thus, the objective of this review is to disseminate knowledge about the use of chilled semen in the preservation of germplasm of bovine and buffalo breeders to improve conception rates in properties. For this, comments on the storage of refrigerated semen will be addressed, with emphasis on the differences between refrigeration curves, their relative advantages and disadvantages for procedures for use in FTAI, identifying the method most indicated by several authors, the current state of biotechnics, its future merits and possibilities.(AU)

Equine semen cooled with chemically defined extender containing soy lecithin / Sêmen equino refrigerado com diluidor quimicamente definido contendo lecitina de soja

Equine semen has historically been chilled using milk-based media. However, the use of animal-based components presents several potential concerns, such as variability in formulations, microbial contamination and regulatory issues. We aimed to evaluate the potential of including different concentrations of soy lecithin (LS) in chemically defined Biggers, Whitten and Whittingham (BWW) medium for cooling equine semen to 15°C. Ejaculates were diluted as six different experimental groups: 1) BotuSêmen® (control); 2) BWW; 3) BWW + 1% LS; 4) BWW + 2% LS; 5) BWW + 4% LS and 6) BWW + 6% LS. BWW medium, did not preserve motility, velocity, straightness (STR), linearity (LIN), amplitude of lateral sperm head displacement (ALH), cross flagellar beat frequency (BCF), functional and structural integrity of equine spermatozoa during 24 h of refrigeration when compared to BotuSêmen® (P <0.05). The use of BWW for cooling equine semen was only possible with the addition of LS, being the concentrations equal or higher than 2% better, because they preserved total motility, curvilinear velocity (VCL) and LIN with the same potential of BotuSêmen® (P >0.05). Nevertheless, BotuSêmen® showed superiority in preserving the percentage of sperm progressive motility, average path velocity (VAP), linear progressive velocity (VSL) and BCF during cooling compared to the other extenders (P <0.05). The inclusion of soy lecithin, from 2 to 6% in the BWW medium, allowed maintaining the viability of equine semen cooled at 15ºC for up to 24 hours.

O sêmen equino tem sido historicamente refrigerado usando meios à base de leite. No entanto, o uso de componentes de origem animal causa várias preocupações potenciais, como variabilidade nas formulações, contaminação microbiana e questões regulatórias. Objetivou-se avaliar o potencial de inclusão de diferentes concentrações de lecitina de soja (LS) no meio quimicamente definido BWW - Biggers, Whitten e Whittingham para refrigeração de sêmen equino e armazenamento na temperatura de 15°C. Os ejaculados foram diluídos em seis diferentes grupos experimentais: 1) BotuSêmen® (controle); 2) BWW; 3) BWW + 1% lecitina de soja (LS); 4) BWW + 2% LS; 5) BWW + 4% LS e 6) BWW + 6% LS. O meio BWW, não preservou a motilidade, a velocidade, a retilinearidade (STR), a linearidade (LIN), a amplitude do deslocamento lateral da cabeça (ALH), a frequência de batimento flagelar cruzado (BCF), a integridade funcional e estrutural dos espermatozoides equino durante 24 h de refrigeração quando comparado ao BotuSêmen® (P <0,05). O uso de BWW para refrigeração de sêmen equino só foi possível com adição de lecitina de soja, sendo as concentrações igual ou superior a 2% melhores, pois preservaram a motilidade total, a velocidade curvilinear (VCL) e LIN com mesmo potencial do BotuSêmen® (P >0,05). Ainda assim, o diluidor comercial BotuSêmen® apresentou superioridade em preservar o percentual de espermatozoides progressivamente móveis, a velocidade média da trajetória (VAP), a velocidade linear progressiva (VSL) e a frequência do batimento flagelar cruzado (BCF) durante a refrigeração comparado aos demais diluidores (P <0,05). A inclusão de lecitina de soja, de 2 a 6% no meio BWW, permitiu a manutenção da viabilidade do sêmen equino refrigerado a 15ºC por até 24 horas.

A preliminary study on the effects of E-Z Mixin® and EquiPlus® extenders supplemented with Edible Birds Nest on the quality of chilled Arabian stallion semen

The aim of this study was to evaluate the effects of adding different concentrations of edible birds nest (EBN) which is secreted by swiftlet birds (Aerodramus fuciphagus), into EquiPlus® and E-Z Mixin® extenders on the quality of chilled Arabian stallion semen at various storage times (0, 24 and 48 h). Ten ejaculates were collected from five stallions, and diluted using the two extenders containing 0% (control), 0.12%, 0.24% and 0.24% of EBN + seminal plasma (SP). All the diluted semen samples were then cooled and stored at 5 °C, and examined at 0, 24 and 48 h. Sperm kinetic parameters were assessed using computer assisted sperm analysis (CASA) and viability were assessed using Hoechst33342/PI stain. In both extenders, total motility (TM) and progressive motility (PM) were significantly higher at 0.12% and 0.24% compared to 0.24% + SP at 24 and 48 h. At 0.12%, EZ mixin® treated semen had significantly higher TM and PM than EquiPlus® at 24 and 48 h. At 0.12% and 0.24%, average path velocity (VAP), straight-line velocity (VSL) and curvilinear velocity (VCL) were significantly higher in E-Z mixin® treated semen compared to EquiPlus® at 24 and 48 h. Comparisons between the two extender types at different concentrations of EBN showed no significant difference in lateral head amplitude (ALH), linearity (LIN), straightness (STR), beat cross frequency (BCF) and viability, irrespective of the storage time. The percentage of viable was significantly higher in E-Z mixin® than EquiPlus® at 0 and 48 h in control and 0.12%. Supplementation of the E-Z mixin® extender with 0.12% and 0.24% EBN concentrations in the absence of SP provided better CASA parameters such as TM, PM, VAP, VSL, and VCL at 24 and 48 h storage time. In conclusion, the results of this study indicated that chilled semen from Arabian stallion that was extended using E-Z mixin® and supplemented with 0.12% and 0.24% EBN concentrations performed better and yielded superior results in sperm kinetic parameters and % viable compared to EquiPlus® at 24 and 48 h storage time.(AU)

A preliminary study on the effects of E-Z Mixin® and EquiPlus® extenders supplemented with Edible Bird's Nest on the quality of chilled Arabian stallion semen

Abstract The aim of this study was to evaluate the effects of adding different concentrations of edible bird's nest (EBN) which is secreted by swiftlet birds (Aerodramus fuciphagus), into EquiPlus® and E-Z Mixin® extenders on the quality of chilled Arabian stallion semen at various storage times (0, 24 and 48 h). Ten ejaculates were collected from five stallions, and diluted using the two extenders containing 0% (control), 0.12%, 0.24% and 0.24% of EBN + seminal plasma (SP). All the diluted semen samples were then cooled and stored at 5 °C, and examined at 0, 24 and 48 h. Sperm kinetic parameters were assessed using computer assisted sperm analysis (CASA) and viability were assessed using Hoechst33342/PI stain. In both extenders, total motility (TM) and progressive motility (PM) were significantly higher at 0.12% and 0.24% compared to 0.24% + SP at 24 and 48 h. At 0.12%, E-Z mixin® treated semen had significantly higher TM and PM than EquiPlus® at 24 and 48 h. At 0.12% and 0.24%, average path velocity (VAP), straight-line velocity (VSL) and curvilinear velocity (VCL) were significantly higher in E-Z mixin® treated semen compared to EquiPlus® at 24 and 48 h. Comparisons between the two extender types at different concentrations of EBN showed no significant difference in lateral head amplitude (ALH), linearity (LIN), straightness (STR), beat cross frequency (BCF) and viability, irrespective of the storage time. The percentage of viable was significantly higher in E-Z mixin® than EquiPlus® at 0 and 48 h in control and 0.12%. Supplementation of the E-Z mixin® extender with 0.12% and 0.24% EBN concentrations in the absence of SP provided better CASA parameters such as TM, PM, VAP, VSL, and VCL at 24 and 48 h storage time. In conclusion, the results of this study indicated that chilled semen from Arabian stallion that was extended using E-Z mixin® and supplemented with 0.12% and 0.24% EBN concentrations performed better and yielded superior results in sperm kinetic parameters and % viable compared to EquiPlus® at 24 and 48 h storage time.

Reduction of seminal plasma concentration can decrease detrimental effects of seminal plasma on chilled ram spermatozoa

This study was conducted to investigate the effect of different levels of seminal plasma (SP) and cold-shock on ram spermatozoa during 36 h storage at 5°C. In both ejaculated spermatozoa coated with egg yolk (second ejaculate; coated spermatozoa) and epididymal spermatozoa, samples were treated with 0, 50 and 100% seminal plasma. Different levels of seminal plasma were added on the basis of ram spermatocrit (32%). Then half of aliquots were suddenly put on ice water (cold-shock) and other half were gradually (0.25°C/min) chilled (non- cold shock). Sperm motility, viability and functional membrane integrity were determined in both aliquots at 0, 12, 24 and 36 h storage at 5°C. Under non- cold shock and cold-shock conditions, coated spermatozoa treated with 0% SP showed the highest motility compared to ejaculated spermatozoa (first ejaculate; uncoated spermatozoa) after 12, 24 and 36 h of storage at 5°C (P<0.05). Under non- cold shock and cold-shock conditions, viability and functional membrane integrity was higher in the coated spermatozoa treated with 0% SP than in the uncoated spermatozoa during 36 h storage (P<0.05). There was no significant difference between coated spermatozoa treated with 0 and 50% SP in the percentage of motility and viability after 24 and 36 h of storage (P>0.05). Under non- cold shock and cold-shock conditions, the percentage of motility of epididymal spermatozoa treated with 0% SP was significantly (P<0.05) higher than those treated with 100% SP after 36 h of storage at 5°C. In conclusion, removal of seminal plasma and/or reduction (up to 50%) of its concentration can decrease detrimental effects of seminal plasma on chilled ram spermatozoa.(AU)

Inseminação artificial e tecnologia de sêmen em jumentos / Artificial insemination and semen technology in donkey

A presente revisão enfoca a inseminação artificial e as tecnologias de sêmen de jumento (Equus asinus) que servem de apoio à implantação de programas de melhoramento genético, preservação da espécie ou na implantação de programas produtivos como a produção de muares, produção de rebanhos de jumentas leiteiras ou na produção de carne e derivados. O uso destas técnicas, se justificam pelo fato de que pode ser usado o potencial zootécnico das mesmas, como também por ter apresentado uma redução do efetivo mundial nos últimos 10 anos, fato este que pode colocar a espécie em perigo de extinção ou de comprometimento de diversos ambientes ao redor do mundo. Os jumentos têm prestado grande ajuda em diversas sociedades onde os mesmos servem ainda como meio de transporte ou tração em regiões com dificuldades topográficas e de menor poder aquisitivo. É apresentada uma relação histórica da inseminação artificial nos equídeos, com ênfase ao uso dos jumentos em nível mundial e no Brasil. Apresenta-se a técnica da inseminação artificial de forma simples e nos seus diversos processos técnicos, na execução, preparativos, avanços no uso de sêmen refrigerado, congelado e vitrificado.

This review focuses on artificial insemination and donkey semen technologies (Equus asinus) that support the implementation of genetic improvement programs, preservation of the species or in the implementation of productive programs such as the production of mules, production of dairy herds or in the production of meat and derivatives. The use of these techniques is justified by the fact that their zootechnical potential can be used, as well as by the fact that they have presented a reduction in the world herd in the last 10 years, a fact that can put the species in danger of extinction or compromise of several environments around the world. The donkeys have provided great help in several societies where they still serve as a means of transportation or traction in regions with topographical difficulties and less purchasing power. A historical relation of artificial insemination in horses is presented, with emphasis on the use of donkeys worldwide and in Brazil. The technique of artificial insemination is presented in a simple way and in its various technical processes, in the execution, preparations, advances in the use of chilled, frozen and vitrified semen.

Conservação da capacidade ligante de espermatozoides caninos refrigerados em diluente tris-gema / Conservation of the binding ability of chilled canine sperm in Tris-egg yolk extender

The aim was to evaluate the efficiency of the Tris-egg yolk extender in conserving the binding capability of canine sperm chilled for up to 48 h. The semen of four dogs was collected by digital manipulation and evaluated. The sperm fractions were diluted in Tris-egg yolk and stored at 4 °C. The sperm parameters of motility, vigor, morphology and osmotic response were evaluated in fresh semen, immediately after dilution (0h), at 24h and at48h. The sperm binding test using the chicken egg's perivitelline membrane was performed on fresh and 48 h-chilled samples. As a result, fresh semen showed motility of 99.2+/-0.8%, vigor 5+/-0,1 with 84.2+/-1.8% morphologically normal sperm, 95.0+/-0.8% osmotic response and 302.3+/-27.0 membrane-bound sperm. After refrigeration for 48 h, a significant reduction (p<0.05) in the sperm parameters was observed however, values were within the ideal range for the use of semen, such as 90.8+/-1.5% mobile sperm, with vigor 4.3+/-0.2, normal morphology of 71.5+/-1.9%, osmotic response of 77.0+/-4.1%, and 205.5+/-27.7 bound sperm. In conclusion, the hen egg perivitelline membrane binding test proved the efficiency of the Tris-egg yolk extender in conserving the binding capability of canine sperm chilled for 48h.

Conservação da capacidade ligante de espermatozoides caninos refrigerados em diluente tris-gema / Conservation of the binding ability of chilled canine sperm in Tris-egg yolk extender

The aim was to evaluate the efficiency of the Tris-egg yolk extender in conserving the binding capability of canine sperm chilled for up to 48 h. The semen of four dogs was collected by digital manipulation and evaluated. The sperm fractions were diluted in Tris-egg yolk and stored at 4 °C. The sperm parameters of motility, vigor, morphology and osmotic response were evaluated in fresh semen, immediately after dilution (0h), at 24h and at48h. The sperm binding test using the chicken egg's perivitelline membrane was performed on fresh and 48 h-chilled samples. As a result, fresh semen showed motility of 99.2+/-0.8%, vigor 5+/-0,1 with 84.2+/-1.8% morphologically normal sperm, 95.0+/-0.8% osmotic response and 302.3+/-27.0 membrane-bound sperm. After refrigeration for 48 h, a significant reduction (p<0.05) in the sperm parameters was observed however, values were within the ideal range for the use of semen, such as 90.8+/-1.5% mobile sperm, with vigor 4.3+/-0.2, normal morphology of 71.5+/-1.9%, osmotic response of 77.0+/-4.1%, and 205.5+/-27.7 bound sperm. In conclusion, the hen egg perivitelline membrane binding test proved the efficiency of the Tris-egg yolk extender in conserving the binding capability of canine sperm chilled for 48h.(AU)

Comparison of extenders and storage temperature in chilling canine semen / Diferentes diluentes e temperaturas de armazenamento para a refrigeração de sêmen canino

This study aimed to compare the effects a commercial milk-based extender and a self-made egg yolk extender had on the quality of canine semen stored at two different temperatures, 5ºC or 15ºC. The ejaculate obtained was split into two aliquots of equal volume and diluted with the milk or egg yolk extender. The final concentration was 100×106 spermatozoa/mL. Diluted semen was placed in transport containers and maintained at final storage temperatures of 5ºC and 15ºC. The quality of the chilled semen was assessed 12, 24, and 36 hours after storage. Semen diluted with the milk extender had higher motility, vigour, and plasma membrane integrity (p<0.05) of the spermatozoa than that diluted with the egg yolk extender. No difference in the semen quality was observed between the stored temperatures in both the groups. The difference observed between the extenders could be due to the standard formulation of the commercial milk extender and the presence of glucose in the mixture. In conclusion, the milk extender was better than the egg yolk extender at preserving the motility, viability, and membrane integrity of chilled canine semen for up to 36 hours. The storage temperature did not seem to affect the semen quality, suggesting that canine semen can be refrigerated at 15ºC.

O objetivo desse estudo foi avaliar a influência de um diluente comercial à base de leite, comparado à um diluente preparado com de gema de ovo na qualidade do sêmen de cão, armazenado em duas diferentes temperaturas (5º C ou 15º C). O ejaculado obtido foi dividido em duas alíquotas de volumes iguais, que foram diluídas com o diluente leite ou o diluente gema de ovo, apresentando uma concentração final de 100x106 espermatozoides/mL. O sêmen diluído foi colocado em caixas de transporte, mantendo temperaturas finais de refrigeração de 5º C e 15º C. A qualidade do sêmen refrigerado foi avaliada após 12, 24 e 36h de armazenamento. O sêmen diluído com o diluente leite resultou em maior motilidade, vigor e integridade de membrana plasmática (p<0,05) dos espermatozoides que o diluído com o diluente gema de ovo. Dentro de cada grupo, não foi encontrada nenhuma influencia da temperatura de armazenamento em relação à qualidade do sêmen. A diferença observada entre os diluentes, pode ter ocorrido devido à formulação padrão do diluente comercial à base de leite, além da presença de glicose em sua composição. Em conclusão, o diluente leite apresentou superior preservação da motilidade, viabilidade e integridade de membrana plasmática do sêmen de cão refrigerado, por até 36h de armazenamento, que o diluente gema de ovo, e a temperatura de armazenamento não interferiu na qualidade seminal, sugerindo que 15º C também pode ser utilizado para a refrigeração do sêmen canino.

Comparison of extenders and storage temperature in chilling canine semen / Diferentes diluentes e temperaturas de armazenamento para a refrigeração de sêmen canino

This study aimed to compare the effects a commercial milk-based extender and a self-made egg yolk extender had on the quality of canine semen stored at two different temperatures, 5ºC or 15ºC. The ejaculate obtained was split into two aliquots of equal volume and diluted with the milk or egg yolk extender. The final concentration was 100×106 spermatozoa/mL. Diluted semen was placed in transport containers and maintained at final storage temperatures of 5ºC and 15ºC. The quality of the chilled semen was assessed 12, 24, and 36 hours after storage. Semen diluted with the milk extender had higher motility, vigour, and plasma membrane integrity (p<0.05) of the spermatozoa than that diluted with the egg yolk extender. No difference in the semen quality was observed between the stored temperatures in both the groups. The difference observed between the extenders could be due to the standard formulation of the commercial milk extender and the presence of glucose in the mixture. In conclusion, the milk extender was better than the egg yolk extender at preserving the motility, viability, and membrane integrity of chilled canine semen for up to 36 hours. The storage temperature did not seem to affect the semen quality, suggesting that canine semen can be refrigerated at 15ºC.(AU)

O objetivo desse estudo foi avaliar a influência de um diluente comercial à base de leite, comparado à um diluente preparado com de gema de ovo na qualidade do sêmen de cão, armazenado em duas diferentes temperaturas (5º C ou 15º C). O ejaculado obtido foi dividido em duas alíquotas de volumes iguais, que foram diluídas com o diluente leite ou o diluente gema de ovo, apresentando uma concentração final de 100x106 espermatozoides/mL. O sêmen diluído foi colocado em caixas de transporte, mantendo temperaturas finais de refrigeração de 5º C e 15º C. A qualidade do sêmen refrigerado foi avaliada após 12, 24 e 36h de armazenamento. O sêmen diluído com o diluente leite resultou em maior motilidade, vigor e integridade de membrana plasmática (p<0,05) dos espermatozoides que o diluído com o diluente gema de ovo. Dentro de cada grupo, não foi encontrada nenhuma influencia da temperatura de armazenamento em relação à qualidade do sêmen. A diferença observada entre os diluentes, pode ter ocorrido devido à formulação padrão do diluente comercial à base de leite, além da presença de glicose em sua composição. Em conclusão, o diluente leite apresentou superior preservação da motilidade, viabilidade e integridade de membrana plasmática do sêmen de cão refrigerado, por até 36h de armazenamento, que o diluente gema de ovo, e a temperatura de armazenamento não interferiu na qualidade seminal, sugerindo que 15º C também pode ser utilizado para a refrigeração do sêmen canino.(AU)

Effect of plasma separation techniques and sperm selection on sperm recovery and viability of cooled pony stallion semen for 48h at 5°C / [Efeito das técnicas de separação de plasma e de seleção de esperma na recuperação e viabilidade do sêmen de garanhão pônei resfriado por 48h a 5°C]

Separation techniques of seminal plasma [centrifugation (SC) and Sperm Filter® (SF)] and sperm selection [Androcoll-E (SCA) and filtration glass wool (GW)] were used in 24 ejaculates from 6 stallions. In experiment 1, the ejaculates were allocated into control (no spin), centrifugation at 600 g x 10min, SF and GW. In experiment 2, semen was submitted to SC, SGA and filtered through GW. Following the treatments in both experiments, samples were kept chilled at 5°C to 50 x 106 sperm/ml for 48h. The variables measured on fresh and cooling semen were pH, motility, membrane viability function by 6-carboxyfluorescein diacetate and propidium iodide (CFDA / PI), viability or vitality (eosin / nigrosine) and mitochondrial activity. In experiment 1, centrifugation to remove seminal plasma resulted in greater damage to sperm than separation by sperm filter, and selection by glass wool was more efficient in separating viable cells and maintaining viability during cooling. In experiment 2 Androcoll-E and glass wool treatments resulted in higher (P <0.0001) motility, membrane function, mitochondrial activity, and viability than centrifuged semen. Both selection by Androcoll- E and glass wool improved the quality of semen pony stallions for preservation for up to 48h to 5ºC.(AU)

As técnicas de separação do plasma seminal (centrifugação, SpermFilter) e de seleção espermática (Androcoll-E e filtração por lã de vidro) foram aplicadas em 24 ejaculados de seis garanhões da raça Pônei Brasileiro. Após coleta e separação da fração gel, os ejaculados foram diluídos 1:1 com diluente à base de leite em pó. No experimento 1, os ejaculados foram distribuídos em controle (sem centrifugação), centrifugação a 600g x 10min, SpermFilter e filtração por lã de vidro. No experimento 2, o sêmen foi submetido aos procedimentos: centrifugado (SC), centrifugado com Androcoll-E e filtrado por lã de vidro. Após os procedimentos de ambos os experimentos, as amostras foram mantidas refrigeradas a 5ºC, com 50 x 106 espermatozoides/mL, por 48h. As variáveis mensuradas a fresco, 24h e 48h foram: pH, motilidade, funcionalidade de membrana, viabilidade por diacetato de carboxifluoresceína e iodeto de propídio (CFDA/PI, vitalidade (eosina/nigrosina) e atividade mitocondrial. Já osmolaridade e morfologia espermática foram avaliadas somente imediatamente após a coleta. No experimento 1, a centrifugação para retirada do plasma seminal resultou em maiores danos aos espermatozoides do que a separação por SpermFilter. A filtração por lã de vidro mostrou-se mais eficiente em separar células viáveis e manter a viabilidade durante o resfriamento. No experimento 2, os tratamentos com Androcoll-E e filtrado por lã de vidro foram superiores (P<0,0001) ao sêmen centrifugado quanto à motilidade, à funcionalidade de membrana, à atividade mitocondrial e à viabilidade, tanto nas amostras de sêmen fresco como de sêmen refrigerado. O Androcoll-E e a lã de vidro permitiram manter por 48h, a 5ºC, o sêmen de garanhões pôneis utilizando-se diluente à base de leite.(AU)

Impact of in vitro fertilization by refrigerated versus frozen buffalo semen on developmental competence of buffalo embryos

The objective of this study was to evaluate the fertility of buffalo semen for in vitro embryo production (IVEP) by comparing the effectiveness of refrigerated versus frozen semen. Three OPU sessions were held at 30-day intervals. For oocyte fertilization three buffalo bulls were used, one per session. At each OPU-IVEP session, one ejaculate was collected and divided into two equal aliquots. Each aliquot was either refrigerated at 5ºC/24 hours or frozen. A TRIS extender containing 10% low density lipoproteins, 0.5% lecithin and 10 mM acetylcysteine was used adding 7% glycerol for freezing. Sperm motility/kinetic was evaluated by CASA and sperm membrane integrity by the hypoosmotic swelling test. The evaluations were performed at 0 h (post final dilution at 37ºC), at 4 and 24 hs post-incubation at 5ºC and post-thaw. At 24 hs incubation and immediately post thaw sperm cells were used for in vitro fertilization of buffalo oocytes equally distributed between both groups. Cleavage rates and embryo development were followed. The embryo/matured and embryo/cultured rates were 25.4 x 14.0% and 29.4 x 18.5% (P 0.05), for chilled and frozen semen, respectively. It is concluded that cooled semen can be used for in vitro embryo production in buffalo and that a better efficiency may be expected for cooled compared to frozen semen.

Impact of in vitro fertilization by refrigerated versus frozen buffalo semen on developmental competence of buffalo embryos

The objective of this study was to evaluate the fertility of buffalo semen for in vitro embryo production (IVEP) by comparing the effectiveness of refrigerated versus frozen semen. Three OPU sessions were held at 30-day intervals. For oocyte fertilization three buffalo bulls were used, one per session. At each OPU-IVEP session, one ejaculate was collected and divided into two equal aliquots. Each aliquot was either refrigerated at 5ºC/24 hours or frozen. A TRIS extender containing 10% low density lipoproteins, 0.5% lecithin and 10 mM acetylcysteine was used adding 7% glycerol for freezing. Sperm motility/kinetic was evaluated by CASA and sperm membrane integrity by the hypoosmotic swelling test. The evaluations were performed at 0 h (post final dilution at 37ºC), at 4 and 24 hs post-incubation at 5ºC and post-thaw. At 24 hs incubation and immediately post thaw sperm cells were used for in vitro fertilization of buffalo oocytes equally distributed between both groups. Cleavage rates and embryo development were followed. The embryo/matured and embryo/cultured rates were 25.4 x 14.0% and 29.4 x 18.5% (P 0.05), for chilled and frozen semen, respectively. It is concluded that cooled semen can be used for in vitro embryo production in buffalo and that a better efficiency may be expected for cooled compared to frozen semen.(AU)

Use of the ACP® and BTS extenders for cooling at 15°C white-lipped peccary (Tayassu pecari) semen / Uso dos diluidores ACP® e BTS para refrigeração a 15°C de sêmen de queixada (Tayassu pecari)

Knowledge about reproduction of white-lipped peccary is of great importance to assist with the conservation of this species and enable its rational use in captivity. This study aimed to evaluate the effect of ACP-103®, ACP-116® and BTS semen extenders on sperm viability during cooling of Tayassu pecari semen. Five ejaculates from four adult males were chilled. The animals were submitted to the protocols of sedation and anesthesia for semen collection by the electroejaculation method. After collection, the semen was macro- and microscopically assessed and diluted to reach 35x106 spermatozoa/mL in each of the three different extenders tested. The fresh-extended semen was packed in a BotuFLEX® thermal box to keep samples at 15°C for 24 hours. After cooling, the following semen parameters were analyzed: sperm motility, functional and structural integrity of sperm membranes, mitochondrial activity, chromatin condensation, and the thermoresistance test was performed. The parameters sperm motility, structural and functional integrity of sperm membranes, mitochondrial activity, and chromatin condensation were preserved after use of the extenders tested, and were similar to those of in natura semen (p>0.05). Curvilinear velocity (VCL) (p<0.05) was the only parameter with reduced values after cooling regardless of the extender used. The percentage of sperm with normal morphology was greater in samples cooled using the BTS extender (p<0.05). The ACP-103®, ACP-116® and BTS extenders can be used for the cooling and preservation of white-lipped peccary semen at 15°C for 24 hours.(AU)

Para auxiliar na conservação da espécie e permitir o uso racional do queixada em cativeiro é de grande importância o conhecimento sobre a reprodução da espécie. Objetivou-se avaliar o efeito dos diluidores de sêmen ACP-103®, ACP-116® e BTS na viabilidade espermática durante a refrigeração do sêmen do Tayassu pecari. Foram refrigerados cinco ejaculados provenientes de quatro machos adultos. Os animais foram contidos com auxílio de puçá e submetidos ao protocolo de sedação e anestesia para realização da coleta de sêmen pelo método da eletroejaculação. Depois da coleta, o sêmen foi avaliado macro e microscopicamente e diluído para atingir 35x106 espermatozoides/mL em cada um dos três diferentes diluidores testados. O sêmen diluído foi acondicionado em caixa térmica BotuFLEX® para manter as amostras a 15°C por um período de 24 horas. Depois da refrigeração, os espermatozoides foram avaliados quanto aos parâmetros de movimento espermático, integridade funcional e estrutural das membranas espermáticas, atividade mitocondrial, condensação da cromatina e teste de termorresistência. Os diluidores testados preservaram as características cinéticas, a integridade estrutural e funcional das membranas espermáticas, a atividade mitocondrial e a condensação da cromatina semelhante ao sêmen in natura (P>0,05). O único parâmetro que reduziu com o processo de refrigeração independente do diluidor utilizado foi a Velocidade Curvilinear (VCL) (P<0,05). Foi observado aumento do percentual de espermatozoides morfologicamente normais nas amostras refrigeradas em BTS (P<0,05). Os diluidores ACP-103®, ACP-116® e BTS podem refrigerar e conservar o sêmen de queixada a 15°C por 24 horas.(AU)

Use of the ACP® and BTS extenders for cooling at 15°C white-lipped peccary (Tayassu pecari) semen / Uso dos diluidores ACP® e BTS para refrigeração a 15°C de sêmen de queixada (Tayassu pecari)

Knowledge about reproduction of white-lipped peccary is of great importance to assist with the conservation of this species and enable its rational use in captivity. This study aimed to evaluate the effect of ACP-103®, ACP-116® and BTS semen extenders on sperm viability during cooling of Tayassu pecari semen. Five ejaculates from four adult males were chilled. The animals were submitted to the protocols of sedation and anesthesia for semen collection by the electroejaculation method. After collection, the semen was macro- and microscopically assessed and diluted to reach 35x106 spermatozoa/mL in each of the three different extenders tested. The fresh-extended semen was packed in a BotuFLEX® thermal box to keep samples at 15°C for 24 hours. After cooling, the following semen parameters were analyzed: sperm motility, functional and structural integrity of sperm membranes, mitochondrial activity, chromatin condensation, and the thermoresistance test was performed. The parameters sperm motility, structural and functional integrity of sperm membranes, mitochondrial activity, and chromatin condensation were preserved after use of the extenders tested, and were similar to those of in natura semen (p>0.05). Curvilinear velocity (VCL) (p<0.05) was the only parameter with reduced values after cooling regardless of the extender used. The percentage of sperm with normal morphology was greater in samples cooled using the BTS extender (p<0.05). The ACP-103®, ACP-116® and BTS extenders can be used for the cooling and preservation of white-lipped peccary semen at 15°C for 24 hours.(AU)

Para auxiliar na conservação da espécie e permitir o uso racional do queixada em cativeiro é de grande importância o conhecimento sobre a reprodução da espécie. Objetivou-se avaliar o efeito dos diluidores de sêmen ACP-103®, ACP-116® e BTS na viabilidade espermática durante a refrigeração do sêmen do Tayassu pecari. Foram refrigerados cinco ejaculados provenientes de quatro machos adultos. Os animais foram contidos com auxílio de puçá e submetidos ao protocolo de sedação e anestesia para realização da coleta de sêmen pelo método da eletroejaculação. Depois da coleta, o sêmen foi avaliado macro e microscopicamente e diluído para atingir 35x106 espermatozoides/mL em cada um dos três diferentes diluidores testados. O sêmen diluído foi acondicionado em caixa térmica BotuFLEX® para manter as amostras a 15°C por um período de 24 horas. Depois da refrigeração, os espermatozoides foram avaliados quanto aos parâmetros de movimento espermático, integridade funcional e estrutural das membranas espermáticas, atividade mitocondrial, condensação da cromatina e teste de termorresistência. Os diluidores testados preservaram as características cinéticas, a integridade estrutural e funcional das membranas espermáticas, a atividade mitocondrial e a condensação da cromatina semelhante ao sêmen in natura (P>0,05). O único parâmetro que reduziu com o processo de refrigeração independente do diluidor utilizado foi a Velocidade Curvilinear (VCL) (P<0,05). Foi observado aumento do percentual de espermatozoides morfologicamente normais nas amostras refrigeradas em BTS (P<0,05). Os diluidores ACP-103®, ACP-116® e BTS podem refrigerar e conservar o sêmen de queixada a 15°C por 24 horas.(AU)

Suplementação de selênio na dieta e qualidade espermática do sêmen de touros Brangus / Dietary selenium supplementation and sperm quality in Brangus bulls semen

Objetivou-se avaliar a qualidade espermática do sêmen de touros suplementados com selênio (Se) na dieta. Foram utilizados 16 touros Brangus, igualmente distribuídos em grupo controle (GC) e grupo Se (GSe − 0,1mg de Se/kg de MS de dieta). O experimento teve duração de 75 dias, e os animais foram suplementados por 60 dias. Foram realizadas quatro coletas de sêmen durante o período (0, 30, 60 e 75 dias) por animal. As amostras foram avaliadas quanto a motilidade e vigor espermáticos, integridade e funcionalidade da membrana plasmática (teste de expansão hiposmótico - HIPO) e viabilidade espermática e reação acrossomal (coloração tripla - TRI). Após avaliação, estas foram diluídas em meio Tris-gema com 5% de glicerol, envasadas (40x106 espermatozoides/palheta), resfriadas, congeladas e armazenadas em nitrogênio líquido até a análise. Após descongelação, foram submetidas às mesmas avaliações descritas para o sêmen fresco. Não houve interferência da suplementação com Se nas variáveis vigor espermático, HIPO e TRI do sêmen fresco e descongelado. Porém, constatou-se queda na motilidade espermática do GSe comparativamente ao GC no sêmen fresco (P=0,0035) e descongelado (P=0,0067) após 60 dias de suplementação. Portanto, a suplementação de Se na dieta não foi efetiva na promoção de melhorias dos parâmetros espermáticos de touros Brangus.

The aim of the present study was to evaluate sperm quality of bulls supplemented with selenium (Se) in the diet. Sixteen Brangus bulls were randomly divided in two groups: control (GC) and Se (GSe - 0.1mg Se/kg dietary DM). The experiment lasted 75 days and the animals were supplemented for 60 days. Four semen collections (0, 30, 60 and 75 days) per animal, were performed during the experimental period. Sperm motility and vigor, plasma membrane integrity and functionality (hyposmotic swelling test - HOST) and sperm viability and acrosome reaction (triple staining -TRI) were assessed. After immediate analysis, samples were diluted in Tris-egg yolk extender with 5% glycerol, packed (40x106 spermatozoa / straw), chilled, frozen and stored in liquid nitrogen until analysis. After thawing, sperm motility and vigor, HOST and TRI were performed. No significance was noticed on sperm vigor, HOST and TRI of fresh and post-thawed semen after dietary selenium supplementation. However, sperm motility decreased in GSe compared to GC in fresh (P = 0.0035) and post- thawed (P = 0.0067) semen samples after 60 days of supplementation. Therefore, dietary selenium supplementation was ineffective to improve semen parameters of fresh and post-thawed semen of Brangus bulls.

Suplementação de selênio na dieta e qualidade espermática do sêmen de touros Brangus / Dietary selenium supplementation and sperm quality in Brangus bulls semen

Objetivou-se avaliar a qualidade espermática do sêmen de touros suplementados com selênio (Se) na dieta. Foram utilizados 16 touros Brangus, igualmente distribuídos em grupo controle (GC) e grupo Se (GSe − 0,1mg de Se/kg de MS de dieta). O experimento teve duração de 75 dias, e os animais foram suplementados por 60 dias. Foram realizadas quatro coletas de sêmen durante o período (0, 30, 60 e 75 dias) por animal. As amostras foram avaliadas quanto a motilidade e vigor espermáticos, integridade e funcionalidade da membrana plasmática (teste de expansão hiposmótico - HIPO) e viabilidade espermática e reação acrossomal (coloração tripla - TRI). Após avaliação, estas foram diluídas em meio Tris-gema com 5% de glicerol, envasadas (40x106 espermatozoides/palheta), resfriadas, congeladas e armazenadas em nitrogênio líquido até a análise. Após descongelação, foram submetidas às mesmas avaliações descritas para o sêmen fresco. Não houve interferência da suplementação com Se nas variáveis vigor espermático, HIPO e TRI do sêmen fresco e descongelado. Porém, constatou-se queda na motilidade espermática do GSe comparativamente ao GC no sêmen fresco (P=0,0035) e descongelado (P=0,0067) após 60 dias de suplementação. Portanto, a suplementação de Se na dieta não foi efetiva na promoção de melhorias dos parâmetros espermáticos de touros Brangus.(AU)

The aim of the present study was to evaluate sperm quality of bulls supplemented with selenium (Se) in the diet. Sixteen Brangus bulls were randomly divided in two groups: control (GC) and Se (GSe - 0.1mg Se/kg dietary DM). The experiment lasted 75 days and the animals were supplemented for 60 days. Four semen collections (0, 30, 60 and 75 days) per animal, were performed during the experimental period. Sperm motility and vigor, plasma membrane integrity and functionality (hyposmotic swelling test - HOST) and sperm viability and acrosome reaction (triple staining -TRI) were assessed. After immediate analysis, samples were diluted in Tris-egg yolk extender with 5% glycerol, packed (40x106 spermatozoa / straw), chilled, frozen and stored in liquid nitrogen until analysis. After thawing, sperm motility and vigor, HOST and TRI were performed. No significance was noticed on sperm vigor, HOST and TRI of fresh and post-thawed semen after dietary selenium supplementation. However, sperm motility decreased in GSe compared to GC in fresh (P = 0.0035) and post- thawed (P = 0.0067) semen samples after 60 days of supplementation. Therefore, dietary selenium supplementation was ineffective to improve semen parameters of fresh and post-thawed semen of Brangus bulls.(AU)

Technical recommendations for artificial insemination in sheep

Artificial insemination (AI) was the first important biotechnology applied to improve the genetics of farm animals. It allows the rapid and massive diffusion of desirable characteristics of males with high productive potential. We describe the different types of estrus induction and synchronization techniques and the use of the AI with fresh, chilled or frozen semen. Through the adequacy of the protocols of estrus synchronization and AI to the different production systems, the efficient use of reproductive techniques is possible, reaching acceptable pregnancy rates. Summary of reproductive results obtained using cervical and laparoscopic AI are presented.(AU)

Technical recommendations for artificial insemination in sheep

Artificial insemination (AI) was the first important biotechnology applied to improve the genetics of farm animals. It allows the rapid and massive diffusion of desirable characteristics of males with high productive potential. We describe the different types of estrus induction and synchronization techniques and the use of the AI with fresh, chilled or frozen semen. Through the adequacy of the protocols of estrus synchronization and AI to the different production systems, the efficient use of reproductive techniques is possible, reaching acceptable pregnancy rates. Summary of reproductive results obtained using cervical and laparoscopic AI are presented.

The use of reduced glutathione (GSH) as antioxidant for cryopreserved sperm in dogs / O uso da glutationa reduzida (GSH) como antioxidante na criopreservação seminal em cães

The aim of this study was to evaluate the effect of supplementation with different concentrations of reduced glutathione GSH (0; 5; 7.5; 10mM) in the extender for cryopreservation in dogs with evaluations performed after glycerolization (chilled) and thawing (thawed). For this purpose, we used 8 dogs and two semen collections were performed in a weekly interval, totaling 16 semen samples. The sperm were analyzed by automatic sperm motility (CASA) and flow cytometry analysis of mitochondrial potential (JC1 dye) and membrane/acrosome integrity (FITC-PI dyes). We evaluated subjectively the membrane and acrosome integrity, mitochondrial activity and DNA integrity. Seminal plasma was evaluated for lipid peroxidation (TBARS concentration). Chilled and thawed samples supplemented with 7.5 and 10mM of GSH had lower percentage of sperm with high (DAB - Class I) and medium (DAB - Class II) mitochondrial activity. And 10mM of GSH had higher percentage of low mitochondrial activity (DAB - Class III). Moreover, thawed samples of 10mM of GSH had high DNA fragmentation rates. Probably by a reductive stress effect on mitochondria which lead to an increase in reactive oxygen species, and a mitochondrial malfunction.(AU)

O objetivo deste estudo foi avaliar o efeito da suplementação com diferentes concentrações de glutationa reduzida (GSH - 0; 5; 7,5; 10mM) para criopreservação em cães com avaliações realizadas após glicerolização (refrigeração) e descongelação. Para tal, foram utilizados oito cães e foram realizadas duas coletas de sêmen em intervalo semanal, totalizando 16 amostras de sêmen. Foram avaliadas a motilidade espermática computadorizada (CASA) e a análise de citometria de fluxo do potencial mitocondrial (sonda JC-1) e integridade da membrana/acrossomal (sonda FITC-PI). Subjetivamente foi avaliada a integridade da membrana plasmática e do acrossomal, atividade mitocondrial e integridade do DNA. O plasma seminal foi avaliado quanto à peroxidação lipídica (concentração de TBARS). As amostras refrigeradas e descongeladas suplementadas com 7,5 e 10mM de GSH apresentaram menor porcentagem de espermatozoides com alta atividade mitocondrial (DAB - Classe I) e média (DAB - Classe II). Na concentração de 10mM de GSH, apresentaram maior porcentagem de baixa atividade mitocondrial (DAB - Classe III). Além disso, amostras descongeladas de 10mM de GSH apresentaram taxas de fragmentação de DNA elevadas, provavelmente por efeito de estresse redutivo sobre as mitocôndrias que elevam as espécies reativas de oxigênio e disfunção mitocondrial.(AU)