Viability of Toxoplasma gondii in cattle semen cryopreserved with different concentrations of cryoprotectant / Viabilidade do Toxoplasma gondii em sêmen de bovino criopreservado em diferentes concentrações de crioprotetores

ABSTRACT: Toxoplasma gondii can be eliminated in bovine semen. Cryopreserved semen is often used due to the fact that artificial insemination in dairy and beef cattle provides benefits in terms of production. However, little is known regarding the viability and infectivity of T. gondii tachyzoites in cryopreserved bovine semen. In the present study, cattle semen negative for T. gondii were contaminated with 1 x 106 tachyzoites (RH strain) and cryopreserved with and without different cryoprotectants, such as DMSO (concentrations of 2.5%, 5.0%, 7.5%, 8.0% and 10.0%) and glycerol (2.25%, 2.5%, 3.0%, 5.0%, 7.5% and 10.0%), followed by freezing in liquid nitrogen (-196°C). After 24 hours, the samples were thawed and inoculated in 10 mice per cryoprotectant concentration. The mice were evaluated for clinical signs of toxoplasmosis (rough coat, diarrhea, hypoactivity and sudden death) as well as serum titers of IgM and IgG and the presence of tachyzoites in the peritoneal lavage. The results revealed that T. gondii remained infective in all samples. Clinical signs of toxoplasmosis were observed in the mice beginning with the 6th day post-inoculation (DPI) and 100% lethality was found between the 7th and 9th DPI. Viable tachyzoites were recovered from peritoneal exudate of dead mice (except for the control group), with higher mean of tachyzoite counts in the intraperitoneal lavage for 5% DMSO (±3.32 x 106), 8% DMSO (±3.53 x 106), 3% glycerol (±4.75 x 106), 7.5% glycerol (±6.26 x 106) and the absence of cryoprotectant (±3.11 x 106). Seroconversion occurred in the treated groups, with titers of IgG from 1:16 to 1:128 and IgM from 1:16 to 1:512. T. gondii viability and infectivity were maintained in cattle semen during 24 hours of cryopreservation at -196°C with and without cryoprotectant. However, further studies are necessary to determine whether cryopreserved semen contributes to the spread of toxoplasmosis through artificial insemination.

RESUMO: Sabe-se que Toxoplasma gondii pode ser eliminado no sêmen bovino. A inseminação artificial em bovinos leiteiros e de corte proporcionou avanços e benefícios nas produções e para isso o sêmen criopreservado é frequentemente utilizado. No entanto, pouco se sabe sobre a viabilidade e infectividade dos taquizoítos de T. gondii em sêmen bovino criopreservado. Para isso o sêmen bovino, negativo para T. gondii, foi contaminado com 1x106 taquizoítos (cepa RH), criopreservados com ou sem diferentes crioprotetores como DMSO (2.5%, 5.0%, 7.5%, 8.0% e 10.0%) e Glicerol (2.25%, 2.5%, 3.0%, 5.0%, 7.5% e 10.0%) e congelados em nitrogênio líquido (-196°C). Após 24 horas, essas amostras foram descongeladas e inoculadas em 10 camundongos por diluente de concentração de crioprotetor. Os camundongos foram avaliados quanto a sinais clínicos de toxoplasmose (pele áspera, diarreia, hipoatividade e morte súbita), títulos séricos de IgM e IgG e presença de taquizoítos no lavado peritoneal. Os resultados mostraram que T. gondii se manteve infectante em todas as amostras, inclusive naquelas sem crioprotetor. Sinais clínicos de toxoplasmose foram observados nos camundongos a partir do 6º dia pós-inoculação (DPI) e 100% de letalidade foi verificada entre o 7º ao 9º DPI. Nos camundongos mortos, exceto no grupo controle, taquizoítos viáveis foram recuperados do exsudato peritoneal, com maior média de taquizoítos quantificados na lavagem intraperitoneal para DMSO a 5% (±3.32x106), 8% (±3.53x106) e glicerol 3% (±4.75x106), 7,5% (±6.26x106) e livre de crioprotetor (±3.11x106). A soroconversão ocorreu nos grupos tratados com títulos de IgG (1:16 a 1:128) e IgM (1:16 a 1:512). A viabilidade e infectividade do T. gondii no sêmen bovino durante as 24 horas de criopreservação a -196°C foram mantidas com ou sem crioprotetor. No entanto, mais estudos são necessários para verificar se o sêmen criopreservado contribui para a disseminação da toxoplasmose na inseminação artificial.

Kinetic vitrification: concepts and perspectives in animal sperm cryopreservation

Sperm cryopreservation is an important tool for genetic diversity management programs and the conservation of endangered breeds and species. The most widely used method of sperm conservation is slow freezing, however, during the process, sperm cells suffer from cryoinjury, which reduces their viability and fertility rates. One of the alternatives to slow freezing is vitrification, that consist on rapid freezing, in which viable cells undergo glass-like solidification. This technology requires large concentrations of permeable cryoprotectants (P- CPA's) which increase the viscosity of the medium to prevent intracellular ice formation during cooling and warming, obtaining successful results in vitrification of oocytes and embryos. Unfortunately, this technology failed when applied to vitrification of sperm due to its higher sensitivity to increasing concentrations of P-CPAs. Alternatively, a technique termed 'kinetic sperm vitrification' has been used and consists in a technique of permeant cryoprotectant-free cryopreservation by direct plunging of a sperm suspension into liquid nitrogen. Some of the advantages of kinetic vitrification are the speed of execution and no rate-controlled equipment required. This technique has been used successfully and with better results for motility in human (50-70% motility recovery), dog (42%), fish (82%) and donkey (21.7%). However, more studies are required to improve sperm viability after devitrification, especially when it comes to motility recovery. The objective of this review is to present the principles of kinetic vitrification, the main findings in the literature, and the perspectives for the utilization of this technique as a cryopreservation method.(AU)

Eficiência de três diluidores inespecíficos na refrigeração do sêmen de coelho (Oryctolagus cuniculus f. domesticus) / Efficiency of three unspecific diluents in refrigeration of rabbit (Oryctolagus cuniculus f. domesticus) semen / Eficiencia de tres diluyentes inespecíficos en la refrigeración de semen de conejo (Oryctolagus cuniculus f. domesticus)

Objetivou-se com este trabalho avaliar a viabilidade seminal de coelhos após refrigeração a 5°C utilizando três diluentes comerciais, indicados para refrigeração de sêmen de outras espécies monogástricas. Foram realizadas dez colheitas de sêmen de cada reprodutor. Os ejaculados de cada coelho eram misturados, obtendo-se um pool de sêmen. Em seguida, o pool heterospérmico foi dividido em três tratamentos, de acordo com o diluente utilizado: BotuDogⓇ (cães); BotuSemen® (equinos); e Beltsville Thawing Solution - BTS (suínos). As amostras foram avaliadas quanto aos parâmetros macroscópicos, microscópicos e funcionais em quatro momentos: sêmen fresco (0 h) e refrigerado (16, 24 e 48 h). Os valores médios de motilidade, vigor e defeitos totais do sêmen fresco foram de 86,0%, 3,05 e 11,5%, respectivamente. Após o início do resfriamento, observou-se comportamento linear decrescente para a motilidade e vigor espermático do sêmen em função do tempo, diferindo (P < 0,05) entre os tratamentos. Destaca-se que o tratamento diluído em BTS apresentou redução na manutenção dos parâmetros avaliados de forma mais pronunciada (P < 0.05) ao longo do tempo, apresentando os menores valores para motilidade e vigor espermático dentro de cada tempo avaliado: 16h [20,6 ± 7,28 e 0,75 (0,375 1,5)], 24h [12,0 ± 6,80 e 0 (0 - 1,0)] e 48h [3,0 ± 2,13 e 0 (0 - 0,125)]. Após 48 h de resfriamento, o BotuDogⓇ foi o que apresentou maior valor para motilidade espermática (71,0 ± 2,08) e, juntamente, com o BotuSemenⓇ apresentaram maior valor para vigor espermático (2,5). De acordo com os dados obtidos os diluentes BotuDogⓇ e BotuSemenⓇ mostraram-se eficientes em manter parâmetros espermáticos adequados, apresentando boa viabilidade espermática em até 48h pós refrigeração a 5°C. Foram obtidos, resultados inéditos quanto a parâmetros espermáticos do sêmen de coelho refrigerado que podem ser utilizados como valores de referência para o manejo reprodutivo e processamento do semen desses animais, visto a inexistênca dessas recomendações técnicas.

The objective of this work was to evaluate the seminal viability of rabbits after refrigeration at 5°C using three commercial diluents, indicated for refrigeration of semen from other monogastric species. Ten semen collections were performed from each breeder. The ejaculates of each rabbit were mixed, obtaining a pool of semen. Then, the heterospermic pool was divided into three treatments, according to the diluent used: BotuDog (dogs); BotuSemenⓇ (horses); and Beltsville Thawing Solution - BTS (pigs). The samples were evaluated for macroscopic, microscopic, and functional parameters in four moments: fresh (0 h) and refrigerated (16, 24, and 48 h) semen. The average values of motility, vigor, and total defects of fresh semen were 86.0%, 3.05 and 11.5%, respectively. After the beginning of cooling, a decreasing linear behavior was observed for motility and sperm vigor of semen as a function of time, differing (P < 0.05) between treatments. It is noteworthy that the treatment diluted in BTS® showed a more pronounced reduction in the maintenance of the evaluated parameters (P < 0.05) over time, with the lowest values for sperm motility and vigor within each evaluated time: 16h [20.6 +7.28 and 0.75 (0.375 - 1.5)], 24h [12.0± 6.80 and 0 (0 - 1.0)] and 48h [3.0±2.13 and 0 (0 - 0.125)]. After 48 h of cooling, BotuDog presented the highest value for sperm motility (71.0 ± 2.08) and, together with BotuSemenⓇ, presented the highest value for sperm vigor (2.5). According to the data obtained, BotuDog and BotuSemenⓇ diluents were efficient in maintaining adequate sperm parameters, showing good sperm viability in up to 48 hours after refrigeration at 5°C. Through this work, unpublished results were obtained regarding spermatic parameters of refrigerated rabbit semen that can be used as reference values for reproductive management and semen processing of these animals, given the lack of these technical recommendations.

El objetivo de este trabajo fue evaluar la viabilidad seminal de conejos después de refrigeración a 5°C utilizando tres diluyentes comerciales, indicados para la refrigeración de semen de otras especies monogástricas. Se realizaron diez colectas de semen de cada reproductora. Se mezclaron los eyaculados de cada conejo, obteniendo un pool de semen. Luego, el pool heterospérmico se dividió en tres tratamientos, según el diluyente utilizado: BotuDogⓇ (perros); BotuSemenⓇ (caballos); y Beltsville Thawing Solution - BTS® (cerdos). Las muestras fueron evaluadas para parámetros macroscópicos, microscópicos y funcionales en cuatro momentos: semen fresco (0 h) y refrigerado (16, 24 y 48 h). Los valores promedio de motilidad, vigor y defectos totales del semen fresco fueron 86,0%, 3,05 y 11,5%, respectivamente. Después del inicio del enfriamiento, se observó un comportamiento lineal decreciente para la motilidad y el vigor espermático del semen en función del tiempo, difiriendo (P < 0.05) entre tratamientos. Cabe destacar que el tratamiento diluido en BTS® mostró una reducción más pronunciada en el mantenimiento de los parámetros evaluados (P<0,05) a lo largo del tiempo, con los valores más bajos de motilidad espermática y vigor dentro de cada tiempo evaluado: 16h [20,6 ± 7,28 y 0,75 (0,375 -1.5)], 24h [12,0 ± 6,80 y 0 (0 - 1,0)] y 48h [3,0±2,13 y 0 (0 - 0,125)]. Después de 48 h de enfriamiento, BotuDogⓇ presentó el valor más alto de motilidad espermática (71,0 +2,08) y, junto con BotuSemenⓇ, presentó el valor más alto de vigor espermático (2,5). De acuerdo con los datos obtenidos, los diluyentes BotuDogⓇ y BotuSemenⓇ fueron eficientes en mantener parámetros espermáticos adecuados, mostrando buena viabilidad espermática hasta 48 horas después de la refrigeración a 5°C. A través de este trabajo se obtuvieron resultados inéditos respecto a parámetros espermáticos de semen de conejo refrigerado que pueden ser utilizados como valores de referencia para el manejo reproductivo y procesamiento de semen de estos animales, dada la falta de estas recomendaciones técnicas.

Sperm cryopreservation of Prochilodus brevis using different concentrations of non-permeable cryoprotectants

Abstract The action of substances with non-permeable cryoprotectant potential, besides glucose, has not yet been studied for the species Prochilodus brevis. The objective of this work was to evaluate the action of four non-permeable cryoprotectants on this species sperm cryopreservation. Five pools were cryopreserved in a solution of 5% glucose and 10% dimethyl sulfoxide (Me2SO) associated or not (control) with cryoprotectants egg yolk (5, 10 or 12%), soy lecithin (2.5, 7.5 or 10%), sucrose (5, 10 or 20%) and lactose (5, 8 or 15%). After thawing, samples were evaluated for sperm kinetics (total motility, motility duration, velocities, and wobble - WOB), morphology and membrane and DNA integrity. The treatments containing egg yolk improved significantly (P<0.05) results when compared the control for the membrane integrity parameter. When compared to other treatments, egg yolk, at any concentration, presented higher results (P<0.05) for membrane integrity, total motility, curvilinear velocity (VCL) and average path velocity (VAP) parameters. Egg yolk also showed the best results for WOB, but it did not differ from 5% and 8% lactose and 5% and 20% sucrose. Soy lecithin had the lowest percentages of morphologically normal sperm (P<0.05), while the other treatments did not differ from each other. There was no difference regarding DNA integrity data. Thus, 5% egg yolk is indicated as a non-permeable cryoprotectant for P. brevis, in association with 5% glucose and 10% Me2SO.

Sperm cryopreservation of Prochilodus brevis using different concentrations of non-permeable cryoprotectants

The action of substances with non-permeable cryoprotectant potential, besides glucose, has not yet been studied for the species Prochilodus brevis. The objective of this work was to evaluate the action of four non-permeable cryoprotectants on this species sperm cryopreservation. Five pools were cryopreserved in a solution of 5% glucose and 10% dimethyl sulfoxide (Me2SO) associated or not (control) with cryoprotectants egg yolk (5, 10 or 12%), soy lecithin (2.5, 7.5 or 10%), sucrose (5, 10 or 20%) and lactose (5, 8 or 15%). After thawing, samples were evaluated for sperm kinetics (total motility, motility duration, velocities, and wobble - WOB), morphology and membrane and DNA integrity. The treatments containing egg yolk improved significantly (P<0.05) results when compared the control for the membrane integrity parameter. When compared to other treatments, egg yolk, at any concentration, presented higher results (P<0.05) for membrane integrity, total motility, curvilinear velocity (VCL) and average path velocity (VAP) parameters. Egg yolk also showed the best results for WOB, but it did not differ from 5% and 8% lactose and 5% and 20% sucrose. Soy lecithin had the lowest percentages of morphologically normal sperm (P<0.05), while the other treatments did not differ from each other. There was no difference regarding DNA integrity data. Thus, 5% egg yolk is indicated as a non-permeable cryoprotectant for P. brevis, in association with 5% glucose and 10% Me2SO.(AU)

Assessment of coconut water added to Numida meleagris egg yolk as cryoprotectant for goat semen

Effects were assessed of the dilutants TRIS and ACP - 101c® with the addition of different guinea fowl (Numida meleagris) egg yolk concentrations. Fifteen ejaculates were collected from five goats of the Anglo Nubian breed. The ejaculates were pooled and then divided into 12 groups, two control groups (GC1 TRIS, with 2.5% Gallus gallus domesticus hen egg yolk GOGD), (GC2 Control Group ACP - 101c®, with the addition of 2.5% Gallus gallus domesticus hen egg yolk GOGD) and ten experimental groups (EG), containing TRIS and ACP added with different concentrations of egg yolk from guinea hen (Numida meleagris) (TRIS 2,5% GONM; TRIS 5% GONM; TRIS 10% GONM; TRIS 15% GONM; TRIS 20% GONM; ACP® 2,5% GONM; ACP® 5% GONM; ACP® 10% GONM; ACP® 15% GONM; ACP® 20% GONM). Then cryopreservation was carried out and the samples stored in liquid nitrogen (-196 °C). After seven days, the samples were thawed and assessed for spermatic kinetics, immunofluorescence and sperm morphology. Analysis of GOMN by the CASA system showed that the various parameters were similar to those of GOGD (P>0.05). The membrane integrity, mitochondrial potential and the acrosome were not influenced by the treatment (P>0.05) nor by the dilutant used for cryopreservation (P>0.05). The spermatic morphology was also preserved by the different GOGD and GONM concentrations in the ACP® and TRIS dilutants, with no statistically significant differences (P<0.05). It was concluded that Numida meleagris egg yolk, as external membrane cryoproctant added to the dilutants ACP-101c® and TRIS, improved goat semen quality.(AU)

Effect of cryoprotectant on the motility, viability, fertilization, and DNA integrity of naleh fish Barbonymus sp. (Cyprinidae) sperm / Efeito do crioprotetor na motilidade, viabilidade, fertilização e integridade do DNA no sêmen de peixes naleh Barbonymus sp. (Cyprinidae)

Naleh fish Barbonymus sp. is a commercial freshwater fish, which is indigenous to Aceh, Indonesia. The population of this species has declined over the years as a result of habitat perturbations and overfishing. Hence, the crucial need to develop a cryopreservation method to support breeding programs. This involved the use of a cryoprotectant as an important component. The objective of this study, therefore, was to explore the best cryoprotectant for naleh fish spermatozoa, and a total of five types were tested. These include the DMSO, Methanol, Ethanol, Glycerol, and Ethylene Glycol at a similar concentration of 10%, which were individually combined with 15% egg yolk, and every treatment was performed in three replications. Conversely, Ringer's solution was adopted as an extender, and the sperm was cryopreserved in liquid nitrogen for 15 days. The results showed significant influence on sperm motility and viability, as well as egg fertility of naleh fish (P <0.05), although the DMSO provided the best outcome, compared to others at 47.17%, 50.13%, and 45.67%, respectively. Furthermore, DNA fragmentation had not occurred in the fresh and cryopreserved sperm samples, indicating the protective effect of tested cryoprotectants. It is concluded that the 10% DMSO and 15% egg yolk is the best cryoprotectant for naleh fish spermatozoa.(AU)

O peixe naleh Barbonymus sp. é um peixe comercial de água doce, originário de Aceh, Indonésia. Durante vários anos, as perturbações provocadas no seu habitat e a pesca predatória determinaram o declínio da sua população, cuja preservação deve apoiar-se em um programa de reprodução controlada, com o emprego de espermatozoides criopreservados. O presente trabalho realizou um estudo comparativo de cinco crioprotetores: dimetilsultóxido, metanol, etanol, glicerol e etileno glicol. Todos os crioprotetores foram testados na concentração de 10%, combinados a 15% de gema de ovo. Cada tratamento foi efetuado em triplicatas. A solução de ringer foi utilizada como extensor e o esperma foi criopreservado em nitrogênio líquido por 15 dias. Os resultados obtidos revelaram a existência de influência significante (P<0,05) na viabilidade e motilidade espermática bem como na fertilidade dos ovos do peixe naleh, em que o dimetilsulfóxido apresentou o melhor resultado com os valores de 47,17%, 50,13% e 45,67%, respectivamente. Por outro lado, a fragmentação do DNA não ocorreu nas amostras de esperma fresco e criopreservado, indicando o efeito protetor dos crioprotetores testados. A conclusão obtida foi que o dimetilsulfóxido e 15% de gema de ovo foram o melhor crioprotetor para os espermatozoides do peixe naleh.(AU)

Effect of cryoprotectant on the motility, viability, fertilization, and DNA integrity of naleh fish Barbonymus sp. (Cyprinidae) sperm / Efeito do crioprotetor na motilidade, viabilidade, fertilização e integridade do DNA no sêmen de peixes naleh Barbonymus sp. (Cyprinidae)

Naleh fish Barbonymus sp. is a commercial freshwater fish, which is indigenous to Aceh, Indonesia. The population of this species has declined over the years as a result of habitat perturbations and overfishing. Hence, the crucial need to develop a cryopreservation method to support breeding programs. This involved the use of a cryoprotectant as an important component. The objective of this study, therefore, was to explore the best cryoprotectant for naleh fish spermatozoa, and a total of five types were tested. These include the DMSO, Methanol, Ethanol, Glycerol, and Ethylene Glycol at a similar concentration of 10%, which were individually combined with 15% egg yolk, and every treatment was performed in three replications. Conversely, Ringer's solution was adopted as an extender, and the sperm was cryopreserved in liquid nitrogen for 15 days. The results showed significant influence on sperm motility and viability, as well as egg fertility of naleh fish (P <0.05), although the DMSO provided the best outcome, compared to others at 47.17%, 50.13%, and 45.67%, respectively. Furthermore, DNA fragmentation had not occurred in the fresh and cryopreserved sperm samples, indicating the protective effect of tested cryoprotectants. It is concluded that the 10% DMSO and 15% egg yolk is the best cryoprotectant for naleh fish spermatozoa.(AU)

O peixe naleh Barbonymus sp. é um peixe comercial de água doce, originário de Aceh, Indonésia. Durante vários anos, as perturbações provocadas no seu habitat e a pesca predatória determinaram o declínio da sua população, cuja preservação deve apoiar-se em um programa de reprodução controlada, com o emprego de espermatozoides criopreservados. O presente trabalho realizou um estudo comparativo de cinco crioprotetores: dimetilsultóxido, metanol, etanol, glicerol e etileno glicol. Todos os crioprotetores foram testados na concentração de 10%, combinados a 15% de gema de ovo. Cada tratamento foi efetuado em triplicatas. A solução de ringer foi utilizada como extensor e o esperma foi criopreservado em nitrogênio líquido por 15 dias. Os resultados obtidos revelaram a existência de influência significante (P<0,05) na viabilidade e motilidade espermática bem como na fertilidade dos ovos do peixe naleh, em que o dimetilsulfóxido apresentou o melhor resultado com os valores de 47,17%, 50,13% e 45,67%, respectivamente. Por outro lado, a fragmentação do DNA não ocorreu nas amostras de esperma fresco e criopreservado, indicando o efeito protetor dos crioprotetores testados. A conclusão obtida foi que o dimetilsulfóxido e 15% de gema de ovo foram o melhor crioprotetor para os espermatozoides do peixe naleh.(AU)

Uso da galactose na vitrificação de sêmen ovino em palhetas / Use of galactose to vitrify ram semen in straws

A vitrificação de espermatozoides é uma técnica que apresenta grande potencial para criopreservação de material genético, e sua eficácia tem sido superior aos métodos convencionais em algumas espécies. No entanto, existem poucos estudos sobre sua eficiência com sêmen ejaculado de carneiros e o uso da galactose como crioprotetor extracelular durante a vitrificação. Objetivou-se com este estudo avaliar o efeito da galactose (0,01 M), associada ou não ao glicerol (3% e 7%), em meio comercial (Steridyl® - controle), na criopreservação de espermatozoides de carneiros pelo método de palhetas, comparando o método clássico de congelação e a vitrificação. Ejaculados de seis carneiros da raça Dorper em idade reprodutiva foram coletados com vagina artificial, aliquotados, diluídos individualmente (100 × 106 espermatozoides/mL) nos meios testados, envasados em palhetas de 0,25 mL e submetidos à congelação clássica ou vitrificação. Foram analisadas a cinemática, morfologia, morfometria, viabilidade, integridade física e funcional da membrana espermática. A congelação clássica obteve melhores resultados de motilidade total e progressiva do que a vitrificação nos quatro extensores testados, uma vez que as amostras vitrificadas não apresentaram motilidade pós-reaquecimento (p < 0,05). A adição de galactose ou glicerol ao meio comercial não trouxe efeito benéfico tanto para a vitrificação quanto congelação clássica.

Sperm vitrification is a technique with great potential for cryopreservation of genetic material, with superior effectiveness compared to conventional methods in some species. However, few studies have shown its efficiency with ejaculated sperm of rams and the use of galactose as an extracellular cryoprotectant during vitrification. This study aimed to evaluate the effect of galactose (0.01 M), with or without glycerol addition (3% and 7%) in a commercial extender (Steridyl® - control) for ram sperm cryopreservation in straws, comparing the classic freezing method and vitrification. Ejaculates from six breeding soundness Dorper rams were collected with an artificial vagina, aliquoted, individually diluted (100 × 106 sperm/mL) on extenders tested, loaded into 0.25 mL straws, and subjected to a classic freezing method or vitrification. Sperm kinematics, morphology, morphometry, viability, and physical and functional integrity of the sperm membrane were evaluated. The classic freezing method resulted in higher total and progressive motility than vitrification, as no motility was detected in vitrified samples after rewarming (p<0.05). The addition of galactose or glycerol to the commercial medium did not benefit both vitrification and the classic freezing method.

Uso da galactose na vitrificação de sêmen ovino em palhetas / Use of galactose to vitrify ram semen in straws

A vitrificação de espermatozoides é uma técnica que apresenta grande potencial para criopreservação de material genético, e sua eficácia tem sido superior aos métodos convencionais em algumas espécies. No entanto, existem poucos estudos sobre sua eficiência com sêmen ejaculado de carneiros e o uso da galactose como crioprotetor extracelular durante a vitrificação. Objetivou-se com este estudo avaliar o efeito da galactose (0,01 M), associada ou não ao glicerol (3% e 7%), em meio comercial (Steridyl® - controle), na criopreservação de espermatozoides de carneiros pelo método de palhetas, comparando o método clássico de congelação e a vitrificação. Ejaculados de seis carneiros da raça Dorper em idade reprodutiva foram coletados com vagina artificial, aliquotados, diluídos individualmente (100 × 106 espermatozoides/mL) nos meios testados, envasados em palhetas de 0,25 mL e submetidos à congelação clássica ou vitrificação. Foram analisadas a cinemática, morfologia, morfometria, viabilidade, integridade física e funcional da membrana espermática. A congelação clássica obteve melhores resultados de motilidade total e progressiva do que a vitrificação nos quatro extensores testados, uma vez que as amostras vitrificadas não apresentaram motilidade pós-reaquecimento (p < 0,05). A adição de galactose ou glicerol ao meio comercial não trouxe efeito benéfico tanto para a vitrificação quanto congelação clássica.

Sperm vitrification is a technique with great potential for cryopreservation of genetic material, with superior effectiveness compared to conventional methods in some species. However, few studies have shown its efficiency with ejaculated sperm of rams and the use of galactose as an extracellular cryoprotectant during vitrification. This study aimed to evaluate the effect of galactose (0.01 M), with or without glycerol addition (3% and 7%) in a commercial extender (Steridyl® - control) for ram sperm cryopreservation in straws, comparing the classic freezing method and vitrification. Ejaculates from six breeding soundness Dorper rams were collected with an artificial vagina, aliquoted, individually diluted (100 × 106 sperm/mL) on extenders tested, loaded into 0.25 mL straws, and subjected to a classic freezing method or vitrification. Sperm kinematics, morphology, morphometry, viability, and physical and functional integrity of the sperm membrane were evaluated. The classic freezing method resulted in higher total and progressive motility than vitrification, as no motility was detected in vitrified samples after rewarming (p<0.05). The addition of galactose or glycerol to the commercial medium did not benefit both vitrification and the classic freezing method.

Adição de colesterol carregado com ciclodextrina ao sêmen ovino na criopreservação / Addition of cholesterol loaded with cyclodextrin to sheep semen in cryopreservation

Objetivou-se avaliar o efeito da adição de diferentes concentrações do colesterol carregado por ciclodextrina (CCC) sobre os espermatozoides congelados de ovinos. Foram coletados dois ejaculados de 10 carneiros (n=20) e diluídos em Tris-Gema de ovo até a concentração final de 200 x106 sptz/mL e mantidos em banho maria a 32 °C. O CCC foi adicionado: controle (0,0mg), 1,5mg, 3,0mg e 6,0mg de CCC/120 x106 sptz/mL. Após adição, o sêmen foi resfriado a 5 °C por duas horas, após esse período, envasado em palhetas de 0,5mL e então acondicionado sob vapor de nitrogênio líquido (N2L), a 8 cm da lâmina líquida/15 minutos e depois imersos no N2L. As amostras foram analisadas quanto à motilidade espermática, integridade da membrana plasmática e da membrana acrossomal, atividade mitocondrial e teste de ligação. As variáveis foram submetidas à análise de variância e médias comparadas pelo teste de Tukey a 5% de probabilidade. O maior percentual de integridade da membrana plasmática, acrossomal e a maior atividade mitocondrial foram obtidos utilizando 6,0mg de CCC. A adição de3,0mg de CCC manteve o percentual de motilidade espermática após a criopreservação, quando comparado aos demais tratamentos e controle. A adição de 1,5 e 3,0mg de CCC mantiveram o percentual de viabilidade espermática após a criopreservação acima de 65%. O número de espermatozoides com capacidade de ligação a membrana perivitelina da gema de ovo foi maior (p<0,05) no tratamento com 3,0mg de CCC. Concluiu se que a adição de CCC ao sêmen diluído, nas concentrações avaliadas, melhora a qualidade espermática após descongelação.

The objective was to evaluate the effect of adding different concentrations of cholesterol carried by cyclodextrin (CCC) on frozen ovine sperm. Two ejaculates were collected from 10 rams (n=20) and diluted in Tris-Yolk until the final concentration of 200 x 106 sptz/mL was reached and kept in a water bath at 32 °C. The CCC was added: control (0,0mg), 1.5mg, 3.0mg, and 6.0mg of CCC/120 x 106 sptz/mL. After the addition, the semen was cooled at 5 °C for two hours, after that period, filled in 0.5 mL straws, and then conditioned under liquid nitrogen vapor (N2L), at 8 cm of the liquid/15 minutes, and then immersed in N2L. The samples were analyzed for sperm motility, plasma membrane and acrosomal membrane integrity, mitochondrial activity, and binding test. The variables were subjected to analysis of variance and means compared by Tukey's test at 5% probability. The highest percentage of plasma membrane integrity and the highest mitochondrial activity were obtained using 6.0mg of CCC. The addition of 3.0mg of CCC maintained the percentage of sperm motility after cryopreservation, when compared to other treatments and control. The addition of 1.5 and 3.0mg of CCC maintained the percentage of sperm viability after cryopreservation, above 65%. The count of sperm with ability to bind to the egg yolk perivitelline membrane was higher (p<0.05) with 3.0mg of CCC. It is concluded that the addition of CCC to the diluted semen, in the evaluated concentrations, improves the sperm quality after thawing.

Adição de colesterol carregado com ciclodextrina ao sêmen ovino na criopreservação / Addition of cholesterol loaded with cyclodextrin to sheep semen in cryopreservation

Objetivou-se avaliar o efeito da adição de diferentes concentrações do colesterol carregado por ciclodextrina (CCC) sobre os espermatozoides congelados de ovinos. Foram coletados dois ejaculados de 10 carneiros (n=20) e diluídos em Tris-Gema de ovo até a concentração final de 200 x106 sptz/mL e mantidos em banho maria a 32 °C. O CCC foi adicionado: controle (0,0mg), 1,5mg, 3,0mg e 6,0mg de CCC/120 x106 sptz/mL. Após adição, o sêmen foi resfriado a 5 °C por duas horas, após esse período, envasado em palhetas de 0,5mL e então acondicionado sob vapor de nitrogênio líquido (N2L), a 8 cm da lâmina líquida/15 minutos e depois imersos no N2L. As amostras foram analisadas quanto à motilidade espermática, integridade da membrana plasmática e da membrana acrossomal, atividade mitocondrial e teste de ligação. As variáveis foram submetidas à análise de variância e médias comparadas pelo teste de Tukey a 5% de probabilidade. O maior percentual de integridade da membrana plasmática, acrossomal e a maior atividade mitocondrial foram obtidos utilizando 6,0mg de CCC. A adição de3,0mg de CCC manteve o percentual de motilidade espermática após a criopreservação, quando comparado aos demais tratamentos e controle. A adição de 1,5 e 3,0mg de CCC mantiveram o percentual de viabilidade espermática após a criopreservação acima de 65%. O número de espermatozoides com capacidade de ligação a membrana perivitelina da gema de ovo foi maior (p<0,05) no tratamento com 3,0mg de CCC. Concluiu se que a adição de CCC ao sêmen diluído, nas concentrações avaliadas, melhora a qualidade espermática após descongelação.(AU)

The objective was to evaluate the effect of adding different concentrations of cholesterol carried by cyclodextrin (CCC) on frozen ovine sperm. Two ejaculates were collected from 10 rams (n=20) and diluted in Tris-Yolk until the final concentration of 200 x 106 sptz/mL was reached and kept in a water bath at 32 °C. The CCC was added: control (0,0mg), 1.5mg, 3.0mg, and 6.0mg of CCC/120 x 106 sptz/mL. After the addition, the semen was cooled at 5 °C for two hours, after that period, filled in 0.5 mL straws, and then conditioned under liquid nitrogen vapor (N2L), at 8 cm of the liquid/15 minutes, and then immersed in N2L. The samples were analyzed for sperm motility, plasma membrane and acrosomal membrane integrity, mitochondrial activity, and binding test. The variables were subjected to analysis of variance and means compared by Tukey's test at 5% probability. The highest percentage of plasma membrane integrity and the highest mitochondrial activity were obtained using 6.0mg of CCC. The addition of 3.0mg of CCC maintained the percentage of sperm motility after cryopreservation, when compared to other treatments and control. The addition of 1.5 and 3.0mg of CCC maintained the percentage of sperm viability after cryopreservation, above 65%. The count of sperm with ability to bind to the egg yolk perivitelline membrane was higher (p<0.05) with 3.0mg of CCC. It is concluded that the addition of CCC to the diluted semen, in the evaluated concentrations, improves the sperm quality after thawing.(AU)

Ultraestrutura e criopreservação de sêmen de jundiá amazônico (Leiarius marmoratus) em cativeiro / Ultrastructure and sperm cryopreservation of the amazon catfish (Leiarius marmoratus) in captivity

Os objetivos do presente estudo foram analisar a ultraestrutura do espermatozoide do jundiá amazônico e avaliar a sua criopreservação com três agentes crioprotetores (metanol 10%, DMSO 10% e etilenoglicol 10%) e duas soluções ativadoras (NaCl 0,29% e NaHCO3 1%). Como diluente, foi utilizada uma solução de glicose a 5%, sendo o sêmen envasado em palhetas de 0,25mL e congelado em vapor de nitrogênio (botijão dry shipper). No sêmen fresco, o espermatozoide apresentou comprimento de 25,46±2,54µm, cabeça esférica (1,51±0,18µm), ausência de acrossoma, peça intermediária com formato cônico (0,93±0,17µm), ligeiramente assimétrica, com presença de vesículas, e flagelo único (21,48±2,45µm). O sêmen descongelado apresentou valores mais altos (P<0,05) para duração, vigor e taxa de motilidade espermática com os crioprotetores metanol 10% e DMSO 10%. A duração da motilidade espermática foi maior (P<0,05) com o ativador NaHCO3 1% (21-96 s). O sêmen de Leiarius marmoratus criopreservado com DMSO e metanol apresentou, respectivamente, 7,32±4,21% e 8,94±6,69% de taxa de motilidade. No entanto, os resultados não foram satisfatórios para estabelecer um protocolo para a espécie.(AU)

The aims of this study were to describe the spermatozoon ultrastructure and to evaluate the sperm cryopreservation of the amazon catfish with three cryoprotectant agents (10% methanol, 10% DMSO, and 10% ethylene glycol) and two activator agents (0.29% NaCl and 1% NaHCO3). Glucose 5% extender was used as a diluent solution and sperm loaded in 0.25 straws was frozen in nitrogen vapor (dry shipper). Fresh spermatozoon was 25.46±2.54µm long, the head was spherical (1.51±0.18µm) with no acrosome, the midpiece was cone shaped (0.93±0.17µm) with presence of vesicles, slightly asymmetric, and the flagellum was single (21.48±2.45µm). Post-thawed semen presented higher values (P< 0.05) for duration, vigor and sperm motility rate with cryoprotectants 10% methanol and 10% DMSO. The duration of sperm motility was longer (P< 0,05) when triggered in 1% NaHCO3 (96-21 s). Leiarius marmoratus semen cryopreserved with DMSO and methanol, presented respectively 7.32±4.21% and 8.94±6.69% of motility. However, the results were not satisfactory to establish a protocol for the specie.(AU)

Ultraestrutura e criopreservação de sêmen de jundiá amazônico (Leiarius marmoratus) em cativeiro / Ultrastructure and sperm cryopreservation of the amazon catfish (Leiarius marmoratus) in captivity

Os objetivos do presente estudo foram analisar a ultraestrutura do espermatozoide do jundiá amazônico e avaliar a sua criopreservação com três agentes crioprotetores (metanol 10%, DMSO 10% e etilenoglicol 10%) e duas soluções ativadoras (NaCl 0,29% e NaHCO3 1%). Como diluente, foi utilizada uma solução de glicose a 5%, sendo o sêmen envasado em palhetas de 0,25mL e congelado em vapor de nitrogênio (botijão dry shipper). No sêmen fresco, o espermatozoide apresentou comprimento de 25,46±2,54µm, cabeça esférica (1,51±0,18µm), ausência de acrossoma, peça intermediária com formato cônico (0,93±0,17µm), ligeiramente assimétrica, com presença de vesículas, e flagelo único (21,48±2,45µm). O sêmen descongelado apresentou valores mais altos (P<0,05) para duração, vigor e taxa de motilidade espermática com os crioprotetores metanol 10% e DMSO 10%. A duração da motilidade espermática foi maior (P<0,05) com o ativador NaHCO3 1% (21-96 s). O sêmen de Leiarius marmoratus criopreservado com DMSO e metanol apresentou, respectivamente, 7,32±4,21% e 8,94±6,69% de taxa de motilidade. No entanto, os resultados não foram satisfatórios para estabelecer um protocolo para a espécie.(AU)

The aims of this study were to describe the spermatozoon ultrastructure and to evaluate the sperm cryopreservation of the amazon catfish with three cryoprotectant agents (10% methanol, 10% DMSO, and 10% ethylene glycol) and two activator agents (0.29% NaCl and 1% NaHCO3). Glucose 5% extender was used as a diluent solution and sperm loaded in 0.25 straws was frozen in nitrogen vapor (dry shipper). Fresh spermatozoon was 25.46±2.54µm long, the head was spherical (1.51±0.18µm) with no acrosome, the midpiece was cone shaped (0.93±0.17µm) with presence of vesicles, slightly asymmetric, and the flagellum was single (21.48±2.45µm). Post-thawed semen presented higher values (P< 0.05) for duration, vigor and sperm motility rate with cryoprotectants 10% methanol and 10% DMSO. The duration of sperm motility was longer (P< 0,05) when triggered in 1% NaHCO3 (96-21 s). Leiarius marmoratus semen cryopreserved with DMSO and methanol, presented respectively 7.32±4.21% and 8.94±6.69% of motility. However, the results were not satisfactory to establish a protocol for the specie.(AU)

Uso de gema de ovo em diferentes concentrações na congelação espermática de Prochilodus brevis e sua influência em parâmetros cinéticos / Use of egg yolk on Prochilodus brevis sperm freezing and its influence on kinect parameters

Prochilodus brevis is a fish species with an important ecological and economic role. Therefore, its captive breeding, together with sperm cryopreservation, come as an alternative to improve its availability. To perform sperm cryopreservation with quality results, it is necessary to develop studies on cryomedia composition, such as non-permeable cryoprotectants. The aim of the study was to test the efficacy of two non-permeable cryoprotectants on post-thawed kinetic parameters of Prochilodus brevis sperm. Twenty reproductive mature P. brevis males were selected and induced to sperm with carp pituitary extract. The sperm was collected, and five pools were made. Pools were diluted in 5% glucose and 10% DMSO added or not with three different concentrations of egg yolk (5; 10 or 12%). The samples were placed on French straws, frozen in dry shipper and stored in liquid nitrogen. After thawing, the kinetic analyses of computer assisted sperm analysis (CASA) were performed. There were no significant differences between the total motility of the control (60.48%) and treatments (49.0; 47.8 and 51.68%, respectively). The other kinetic parameters also showed no statistical difference. Thus, there is no need to use egg yolk in the freezing of P. brevis sperm when using a glucose and DMSO extender.

Avaliação morfológica do concentrado autólogo de plaquetas em cães sob refrigeração e criopreservação em DMSO 6% / [Morphological evaluation of the autologous platelet concentrate in dogs under refrigeration and criopreservation in 6% DMSO]

The morphological characteristics of the autologous platelet concentrate (APC) of 31 dogs were evaluated after cooling and freezing in 6% DMSO. Blood from the jugular vein of each patient was collected and centrifuged at 191g for six minutes to obtain APC. In the fresh sample, the platelet count, MPV, PDW and cell morphology were evaluated. Four samples of each animal were sent for storage, one refrigerated at 4°C for seven days, another for 30 days and two more stored in a freezer at -80°C in the same time interval, using 6% DMSO as cryoprotectant. The conserved samples were submitted to the same laboratory analysis as the fresh sample. There was a difference between fresh and preserved samples for platelet count, cell concentration, MPV and PDW (P<0.05), except in the 30-day refrigerated group, which showed severe morphological changes. In the frozen group for seven days, no difference was observed in the percentage of activation (P>0.05). The results obtained lead to the conclusion that cryopreservation with 6% DMSO at -80°C for seven days is a favorable option for the maintenance of platelet concentrations and the morphological characteristics of APC in dogs.(AU)

Uso de gema de ovo em diferentes concentrações na congelação espermática de Prochilodus brevis e sua influência em parâmetros cinéticos / Use of egg yolk on Prochilodus brevis sperm freezing and its influence on kinect parameters

Prochilodus brevis is a fish species with an important ecological and economic role. Therefore, its captive breeding, together with sperm cryopreservation, come as an alternative to improve its availability. To perform sperm cryopreservation with quality results, it is necessary to develop studies on cryomedia composition, such as non-permeable cryoprotectants. The aim of the study was to test the efficacy of two non-permeable cryoprotectants on post-thawed kinetic parameters of Prochilodus brevis sperm. Twenty reproductive mature P. brevis males were selected and induced to sperm with carp pituitary extract. The sperm was collected, and five pools were made. Pools were diluted in 5% glucose and 10% DMSO added or not with three different concentrations of egg yolk (5; 10 or 12%). The samples were placed on French straws, frozen in dry shipper and stored in liquid nitrogen. After thawing, the kinetic analyses of computer assisted sperm analysis (CASA) were performed. There were no significant differences between the total motility of the control (60.48%) and treatments (49.0; 47.8 and 51.68%, respectively). The other kinetic parameters also showed no statistical difference. Thus, there is no need to use egg yolk in the freezing of P. brevis sperm when using a glucose and DMSO extender.(AU)

Seminal characteristics and sensitivity of Astyanax lacustris (Characiformes: Characidae) sperm to cryoprotective solutions based on dimethylsufoxide and methylglicol

This study aimed to determine the semen characteristics of Astyanax lacustris after hormonal induction and to evaluate the sensitivity of the species sperm to cryoprotective solutions based on the cryoprotectants dimethyl sulfoxide and methyl glycol. Volume, color, sperm concentration, total motility and aspects of sperm movement were analyzed using "Integrated Semen Analysis System". Three different extenders were tested: A) glucose 5%+egg yolk 10%, B) BTS®5% and C) glucose 5% and two permeable cryoprotectants: dimethyl sulfoxide (Me2SO) and methyl glycol (MTG). Fresh A. lacustris semen presented total motility of 76.6±11.2%, motility duration of 33.0±2.2s, sperm concentration of 7.22±3.2×109sptz/mL and seminal osmolality of 219±0.03mOsm/kg-1. The toxicity test showed the highest total motility values at the MTG15%+A, Me2SO15%+B and Me2SO10%+C dilutions, and the Me2SO10%+C and Me2SO15%+C dilutions presented the highest values for curvilinear velocity, linear velocity and average velocity. The tested protocol was not effective at maintaining the viability of A. lacustris semen after freezing because no motility was observed in any of the dilutions. However, the Comet Assay demonstrated that cryoprotectant solutions were effective in protecting the genetic material of cells, as DNA damage levels were low, with no difference between control and Me2SO10% + A, dilutions MTG10%+C, Me2SO10%+B and Me2SO15%+B.(AU)

O objetivo deste estudo foi determinar as características do sêmen de Astyanax lacustris após indução hormonal e avaliar a sensibilidade dos espermatozoides da espécie a soluções crioprotetoras baseadas nos crioprotetores dimetilsulfóxido e metilglicol. Volume, cor, concentração espermática, motilidade total e aspectos do movimento espermático foram analisados usando o "Sistema Integrado de Análise de Sêmen (ISAS®CASA)". Três extensores diferentes foram testados: A) glicose 5%+gema de ovo 10%, B) BTS® 5% e C) glicose 5% e dois crioprotetores permeáveis: dimetilsulfóxido (Me2SO) e metilglicol (MTG). O sêmen fresco de A. lacustris apresentou motilidade total 76,6±11,2%, duração da motilidade 33,0±2,2s, concentração de espermatozoides 7,22±3,2×109sptz/mL e osmolalidade seminal 219±0,03mOsm/kg-1. O teste de toxicidade apresentou maiores valores de motilidade total nas diluições MTG15%+A, Me2SO15%+B e Me2SO10%+C, e as diluições Me2SO10%+C e Me2SO15%+C apresentaram maiores valores de velocidade curvilínea, velocidade linear e velocidade média. O protocolo testado não foi eficaz em manter a viabilidade do sêmen de A. lacustris pós-congelamento, pois não foi observada motilidade em nenhuma das diluições. No entanto, o Ensaio Cometa demonstrou que as soluções crioprotetoras eram eficazes na proteção do material genético das células, pois os níveis de dano ao DNA eram baixos, sem diferença entre controle e Me2SO10%+A, MTG10%+C, Me2SO10%+B e Me2SO15%+B.(AU)

Congelação de espermatozoides recuperados do epidídimo de cão (Canis familiares) / Spermatozoa freezing recovered from dog epididymis (Canis familiares)

O presente estudo tem como objetivo avaliar o espermatozoide recuperado do epidídimo congelado/descongelado em diferentes diluidores. O experimento foi realizado no Laboratório de Reprodução Animal do Setor Palotina no período de julho de 2017 a julho de 2019. Um mínimo de 40 CTE (complexo testículo epidídimo) de cães foi obtido de animais orquiectomizados. A recuperação dos espermatozoides da cauda do epidídimo foi realizada pela técnica de flutuação e submetida ao teste de swin-up para remoção dos resíduos celulares e espermatozoides inviáveis. Após a avaliação da concentração e motilidade, os espermatozoides foram diluidos pelo método “One Step” em Tris gema e/ou citrato gema e logo envazados em palhetas de 0,25mL numa concentração de 25 x 106 de espermatozoides viáveis. Logo as mesmas permaneceram em estabilização por 2 horas a temperatura de 5°C. Após foram colocadas em vapor de nitrogênio por 15 minutos, e por fim, mergulhadas no nitrogênio líquido e armazenadas em botijão criobiológico. A descongelação foi realizada em banho maria a 37°C por um minuto, e após avaliou-se a motilidade e vigor em microscopia de Contraste de Fase 100x. Para avaliação da viabilidade espermática, foi utilizada a coloração vital Azul de Tripan/Giemsa. Na análise do sêmen descongelado, o diluente Tris-gema foi estatisticamente superior ao Citratogema nas variáveis motilidade, vigor e morfologia de espermatozoides epididimarios de cão. O presente estudo, mostra que é possível recuperar e preservar espermatozoides provenientes do epidídimo de canídeos utilizando meio crioprotetor a base de tris-gema.(AU)

The present study aims to evaluate sperm recovered from frozen / thawed epididymis in different extenders. The experiment was carried out in Animal Reproduction Laboratory at the Veterinary Hospital at the Palotina Sector from July 2017 to July 2019. A minimum of 40 ETC (epididymis testis complex) from dogs was obtained from orchiectomized animals. Epididymis tail sperm retrieval was performed by flotation technique and then submitted to the swin-up test to remove the unfeasible sperm and cellular waste. After evaluation of concentration and motility, the sperm were diluted by the “One Step” method in Tris yolk and / or citrate yolk and then packed in previously identified 0.25mL straws at a concentration of 25 x 106 viable sperm. Soon they remained in stabilization for 2 hours at 5 ° C. After that they were placed in nitrogen vapor for 15 minutes, and finally, dipped in liquid nitrogen and stored in cryogenic cylinder. The thawing was performed in a 37 ° C water bath for one minute, and then motility and vigor were evaluated by x100 magnification Phase Contrast microscopy. For sperm viability evaluation, the Tripan Blue/Giemsa vital stain was used. In the analysis of thawed semen, Tris-yolk extender was statistically superior to Yolk Citrate in the motility, vigor and morphology of dog epididymis sperm variables. The present study shows that it is possible to recovery and preserve sperm from the canid epididymis using tris-yolk cryoprotectant extender.(AU)

Vitrificação de embriões equinos: alternativas para melhorar as taxas de sobrevivência embrionária / Vitrification of equine embryos: alternatives to improve embryonic survival rates

A criopreservação de embriões permite a criação de um banco genético de alto valor zootécnico, aproveitando-se do melhor momento reprodutivo das doadoras, facilitando a comercialização de material genético. Os crioprotetores utilizados durante o processo de vitrificação, afetam o equilíbrio osmótico, evitando a formação de cristais de gelo durante a diminuição de temperatura. Além da escolha adequada dos crioprotetores, o sistema de envase utilizado e a característica de manipulação prévia a vitrificação, impactam a viabilidade embrionária pós aquecimento. Apesar dos protocolos de vitrificação para embriões pequenos (300 µm), que apresentam maior taxa de recuperação, os mesmos protocolos não apresentam sucesso. A eficiência do processo de vitrificação em estruturas maiores está relacionada a redução do tamanho da blastocele, exigindo equipamentos de maior custo. O objetivo dessa revisão é descrever o “estado da arte” da vitrificação de embriões equinos, descrevendo diferentes protocolos de vitrificação, manipulação prévia e sistema de envase.(AU)

Embryo cryopreservation allows the formation of a genetic bank from high genetical value animals, taking advantage of the best reproductive moment of the mares, facilitating its genetic material commercialization. Cryoprotectants used during the vitrification process act in osmotic equilibrium, preventing ice crystals formation during the temperature decrease. In addition to the appropriate choice of cryoprotectants, the characteristic of support/packaging used and the type of manipulation prior to vitrification impact the embryonic viability after heating. Although vitrification protocols for small embryos ( 300 µm) have higher recovery rates but low survival rates when the same protocols are used. Vitrification efficiency in larger structures is related to the reduction of blastocele size, requiring equipment with higher cost. The purpose of this review is to describe the state of the art of equine embryo vitrification by describing different vitrification protocols, previous manipulation and different storage systems.(AU)