Canine sperm vitrification with nonpermeable cryoprotectants and coconut water extender

This study was aimed to assess the efficiency of coconut water extender with addition of soy lecithin and sucrose as nonpermeable cryoprotectants for canine semen vitrification, using a simple method that yields a high survival rate of spermatozoa for clinical use. Twelve ejaculates from 12 adult normozoospermic dogs were collected separately by digital manipulation and only the second semen fraction was used in this study. After evaluation of volume, concentration, viability, total and progressive motility, velocity parameters and morphology, semen was diluted with a coconut water extender (50% (v/v(volume per volume)) coconut water, 25% (v/v) distilled water and 25% (v/v) 5% anhydrous monosodium citrate solution) with addition of soy lecithin and fructose at 1% and 0.25M sucrose until final concentration of 100x106 spermatozoa/ml. After equilibration at 5ºC for 60 minutes, semen was vitrified by "direct dropping method" into liquid nitrogen in spheres with a volume of 30 µl. After a week of storage the spheres were devitrified as three of them were dropped into 0.5 mL of CaniPlus AI medium (Minitüb, Germany), which was previously warmed in a water bath at 42ºC for 2 minutes and evaluated about the above mentioned parameters. It was found that vitrification resulted in a lower percentage of viable sperms, normal morphology, total and progressive motilities (p0.05) compared to fresh semen samples. In conclusion, our results demonstrate that vitrification with coconut water extender with addition of 1% soy lecithin and 0.25M sucrose as cryoprotectants, has an excellent potential for routine canine sperm cryopreservation.(AU)

Use of melatonin as an inhibitor of apoptotic process for cryopreservation of zebrafish (Danio rerio) embryos / Uso da melatonina como inibidor do processo apoptotico para a criopreservação de embriões de zebrafish (Danio rerio)

This study investigated the use of melatonin to arrest the effects of apoptosis in vitrified zebrafish (D. rerio) embryos. Dechorionated embryos at 22-24 somite-stage were divided (n = 60/treatment) into a non-vitrified (Control Group, 0 M melatonin) and vitrified treatments with 0 M (T1), 1 µM (T2) and 1 mM of melatonin (T3). For vitrified treatments, a solution methanol/propylene glycol based was used and the embryos stored in -196 °C for a week. After thaw, survival rate, scanning electron microscopy, expression of anti (bcl-2) and pro-apoptotic (bax/caspase-3) genes, reactive oxygen species (ROS) formation and DNA fragmentation analyses were performed. No live embryos were obtained from vitrified treatments, observing a rapid degeneration immediately after thawing, with the vitelline layer rupture and leakage of its content, followed by breakdown of epithelial cells and melanisation of the tissue. Regarding the apoptotic process, T3 had the highest relative gene expression, for the three genes (P 0.05). The inclusion of 1 µM of melatonin in the vitrifying solution, countered the effects of apoptotic process in post-thaw embryos, suggesting its utility in cryopreserving fish embryos.

Este estudo investigou o uso da melatonina para conter os efeitos da apoptose em embriões vitrificados de zebrafish (D. rerio). Embriões descorionados no estágio de 22-24 somitos foram divididos (n = 60 / tratamento) em tratamento não vitrificado (Grupo Controle, melatonina 0 M) e tratamentos vitrificados com 0 M (T1), 1 µM (T2) e 1 mM de melatonina (T3). Para os tratamentos vitrificados, utilizou-se uma solução à base de metanol/propilenoglicol e os embriões foram armazenados em -196 °C por uma semana. Após o descongelamento, foram realizadas análises de taxa de sobrevivência, microscopia eletrônica de varredura, expressão dos genes anti (bcl-2) e pró-apoptóticos (bax/caspase-3), formação de espécies reativas de oxigênio (EROS) e análises de fragmentação de DNA. Não foram obtidos embriões vivos a partir dos tratamentos vitrificados, observando uma rápida degeneração imediatamente após o descongelamento, com ruptura da camada vitelina e vazamento de seu conteúdo, seguida de quebra das células epiteliais e melanização do tecido. Em relação ao processo apoptótico. T3 apresentou expressão gênica relativa alta para os três genes (P 0,05). A inclusão de 1 µM de melatonina na solução de vitrificação, contrariou os efeitos do processo apoptótico em embriões pós-descongelamento, sugerindo sua utilidade na criopreservação de embriões de peixes.

Use of melatonin as an inhibitor of apoptotic process for cryopreservation of zebrafish (Danio rerio) embryos

Abstract This study investigated the use of melatonin to arrest the effects of apoptosis in vitrified zebrafish (D. rerio) embryos. Dechorionated embryos at 22-24 somite-stage were divided (n = 60/treatment) into a non-vitrified (Control Group, 0 M melatonin) and vitrified treatments with 0 M (T1), 1 µM (T2) and 1 mM of melatonin (T3). For vitrified treatments, a solution methanol/propylene glycol based was used and the embryos stored in -196 °C for a week. After thaw, survival rate, scanning electron microscopy, expression of anti (bcl-2) and pro-apoptotic (bax/caspase-3) genes, reactive oxygen species (ROS) formation and DNA fragmentation analyses were performed. No live embryos were obtained from vitrified treatments, observing a rapid degeneration immediately after thawing, with the vitelline layer rupture and leakage of its content, followed by breakdown of epithelial cells and melanisation of the tissue. Regarding the apoptotic process, T3 had the highest relative gene expression, for the three genes (P 0.05) furthermore, T2 had similar expression of pro-apoptotic genes to CG (P 0.05). ROS formation revealed that CG presented lower percentage of embryo surface area affected (3.80 ± 0.40%) (P 0.05), in contrast, no differences were found among the other groups. T1 was most significantly (P 0.05) damaged by DNA fragmentation. The vitrified groups with melatonin had similar damage levels of CG (P > 0.05). The inclusion of 1 µM of melatonin in the vitrifying solution, countered the effects of apoptotic process in post-thaw embryos, suggesting its utility in cryopreserving fish embryos.

Resumo Este estudo investigou o uso da melatonina para conter os efeitos da apoptose em embriões vitrificados de zebrafish (D. rerio). Embriões descorionados no estágio de 22-24 somitos foram divididos (n = 60 / tratamento) em tratamento não vitrificado (Grupo Controle, melatonina 0 M) e tratamentos vitrificados com 0 M (T1), 1 µM (T2) e 1 mM de melatonina (T3). Para os tratamentos vitrificados, utilizou-se uma solução à base de metanol/propilenoglicol e os embriões foram armazenados em -196 °C por uma semana. Após o descongelamento, foram realizadas análises de taxa de sobrevivência, microscopia eletrônica de varredura, expressão dos genes anti (bcl-2) e pró-apoptóticos (bax/caspase-3), formação de espécies reativas de oxigênio (EROS) e análises de fragmentação de DNA. Não foram obtidos embriões vivos a partir dos tratamentos vitrificados, observando uma rápida degeneração imediatamente após o descongelamento, com ruptura da camada vitelina e vazamento de seu conteúdo, seguida de quebra das células epiteliais e melanização do tecido. Em relação ao processo apoptótico. T3 apresentou expressão gênica relativa alta para os três genes (P 0,05), além disso, T2 apresentou expressão semelhante as dos genes pró-apoptóticos de GC (P 0,05). A formação de EROS revelou que GC apresentou menor percentual de área de superfície embrionária afetada (3,80 ± 0,40%) (P 0,05), ao contrário, não foram encontradas diferenças entre os outros grupos. T1 foi mais significativamente (P 0,05) danificado pela fragmentação do DNA. Os grupos vitrificados com melatonina apresentaram níveis de dano semelhantes ao do GC (P> 0,05). A inclusão de 1 µM de melatonina na solução de vitrificação, contrariou os efeitos do processo apoptótico em embriões pós-descongelamento, sugerindo sua utilidade na criopreservação de embriões de peixes.

Use of melatonin as an inhibitor of apoptotic process for cryopreservation of zebrafish (Danio rerio) embryos / Uso da melatonina como inibidor do processo apoptotico para a criopreservação de embriões de zebrafish (Danio rerio)

This study investigated the use of melatonin to arrest the effects of apoptosis in vitrified zebrafish (D. rerio) embryos. Dechorionated embryos at 22-24 somite-stage were divided (n = 60/treatment) into a non-vitrified (Control Group, 0 M melatonin) and vitrified treatments with 0 M (T1), 1 µM (T2) and 1 mM of melatonin (T3). For vitrified treatments, a solution methanol/propylene glycol based was used and the embryos stored in -196 °C for a week. After thaw, survival rate, scanning electron microscopy, expression of anti (bcl-2) and pro-apoptotic (bax/caspase-3) genes, reactive oxygen species (ROS) formation and DNA fragmentation analyses were performed. No live embryos were obtained from vitrified treatments, observing a rapid degeneration immediately after thawing, with the vitelline layer rupture and leakage of its content, followed by breakdown of epithelial cells and melanisation of the tissue. Regarding the apoptotic process, T3 had the highest relative gene expression, for the three genes (P < 0.05) furthermore, T2 had similar expression of pro-apoptotic genes to CG (P < 0.05). ROS formation revealed that CG presented lower percentage of embryo surface area affected (3.80 ± 0.40%) (P < 0.05), in contrast, no differences were found among the other groups. T1 was most significantly (P < 0.05) damaged by DNA fragmentation. The vitrified groups with melatonin had similar damage levels of CG (P > 0.05). The inclusion of 1 µM of melatonin in the vitrifying solution, countered the effects of apoptotic process in post-thaw embryos, suggesting its utility in cryopreserving fish embryos.

Este estudo investigou o uso da melatonina para conter os efeitos da apoptose em embriões vitrificados de zebrafish (D. rerio). Embriões descorionados no estágio de 22-24 somitos foram divididos (n = 60 / tratamento) em tratamento não vitrificado (Grupo Controle, melatonina 0 M) e tratamentos vitrificados com 0 M (T1), 1 µM (T2) e 1 mM de melatonina (T3). Para os tratamentos vitrificados, utilizou-se uma solução à base de metanol/propilenoglicol e os embriões foram armazenados em -196 °C por uma semana. Após o descongelamento, foram realizadas análises de taxa de sobrevivência, microscopia eletrônica de varredura, expressão dos genes anti (bcl-2) e pró-apoptóticos (bax/caspase-3), formação de espécies reativas de oxigênio (EROS) e análises de fragmentação de DNA. Não foram obtidos embriões vivos a partir dos tratamentos vitrificados, observando uma rápida degeneração imediatamente após o descongelamento, com ruptura da camada vitelina e vazamento de seu conteúdo, seguida de quebra das células epiteliais e melanização do tecido. Em relação ao processo apoptótico. T3 apresentou expressão gênica relativa alta para os três genes (P <0,05), além disso, T2 apresentou expressão semelhante as dos genes pró-apoptóticos de GC (P <0,05). A formação de EROS revelou que GC apresentou menor percentual de área de superfície embrionária afetada (3,80 ± 0,40%) (P <0,05), ao contrário, não foram encontradas diferenças entre os outros grupos. T1 foi mais significativamente (P <0,05) danificado pela fragmentação do DNA. Os grupos vitrificados com melatonina apresentaram níveis de dano semelhantes ao do GC (P> 0,05). A inclusão de 1 µM de melatonina na solução de vitrificação, contrariou os efeitos do processo apoptótico em embriões pós-descongelamento, sugerindo sua utilidade na criopreservação de embriões de peixes.

Use of melatonin as an inhibitor of apoptotic process for cryopreservation of zebrafish (Danio rerio) embryos / Uso da melatonina como inibidor do processo apoptotico para a criopreservação de embriões de zebrafish (Danio rerio)

This study investigated the use of melatonin to arrest the effects of apoptosis in vitrified zebrafish (D. rerio) embryos. Dechorionated embryos at 22-24 somite-stage were divided (n = 60/treatment) into a non-vitrified (Control Group, 0 M melatonin) and vitrified treatments with 0 M (T1), 1 µM (T2) and 1 mM of melatonin (T3). For vitrified treatments, a solution methanol/propylene glycol based was used and the embryos stored in -196 °C for a week. After thaw, survival rate, scanning electron microscopy, expression of anti (bcl-2) and pro-apoptotic (bax/caspase-3) genes, reactive oxygen species (ROS) formation and DNA fragmentation analyses were performed. No live embryos were obtained from vitrified treatments, observing a rapid degeneration immediately after thawing, with the vitelline layer rupture and leakage of its content, followed by breakdown of epithelial cells and melanisation of the tissue. Regarding the apoptotic process, T3 had the highest relative gene expression, for the three genes (P < 0.05) furthermore, T2 had similar expression of pro-apoptotic genes to CG (P < 0.05). ROS formation revealed that CG presented lower percentage of embryo surface area affected (3.80 ± 0.40%) (P < 0.05), in contrast, no differences were found among the other groups. T1 was most significantly (P < 0.05) damaged by DNA fragmentation. The vitrified groups with melatonin had similar damage levels of CG (P > 0.05). The inclusion of 1 µM of melatonin in the vitrifying solution, countered the effects of apoptotic process in post-thaw embryos, suggesting its utility in cryopreserving fish embryos.(AU)

Este estudo investigou o uso da melatonina para conter os efeitos da apoptose em embriões vitrificados de zebrafish (D. rerio). Embriões descorionados no estágio de 22-24 somitos foram divididos (n = 60 / tratamento) em tratamento não vitrificado (Grupo Controle, melatonina 0 M) e tratamentos vitrificados com 0 M (T1), 1 µM (T2) e 1 mM de melatonina (T3). Para os tratamentos vitrificados, utilizou-se uma solução à base de metanol/propilenoglicol e os embriões foram armazenados em -196 °C por uma semana. Após o descongelamento, foram realizadas análises de taxa de sobrevivência, microscopia eletrônica de varredura, expressão dos genes anti (bcl-2) e pró-apoptóticos (bax/caspase-3), formação de espécies reativas de oxigênio (EROS) e análises de fragmentação de DNA. Não foram obtidos embriões vivos a partir dos tratamentos vitrificados, observando uma rápida degeneração imediatamente após o descongelamento, com ruptura da camada vitelina e vazamento de seu conteúdo, seguida de quebra das células epiteliais e melanização do tecido. Em relação ao processo apoptótico. T3 apresentou expressão gênica relativa alta para os três genes (P <0,05), além disso, T2 apresentou expressão semelhante as dos genes pró-apoptóticos de GC (P <0,05). A formação de EROS revelou que GC apresentou menor percentual de área de superfície embrionária afetada (3,80 ± 0,40%) (P <0,05), ao contrário, não foram encontradas diferenças entre os outros grupos. T1 foi mais significativamente (P <0,05) danificado pela fragmentação do DNA. Os grupos vitrificados com melatonina apresentaram níveis de dano semelhantes ao do GC (P> 0,05). A inclusão de 1 µM de melatonina na solução de vitrificação, contrariou os efeitos do processo apoptótico em embriões pós-descongelamento, sugerindo sua utilidade na criopreservação de embriões de peixes.(AU)

Efeitos da vitrificação na preservação morfológica do tecido testicular de gato doméstico / Vitrification effects during morphological preservation of testicular tissue from domestic cat

This study aimed to evaluate the effect of domestic cats (Felis Catus) testicular parenchyma vitrification by Ovarian Tissue Cryosystem and conventional straw. Three (n=3) adult cats were submitted to routine orchiectomy. For the vitrification, the samples were exposed to equilibrium solution composed by RPMI, containing 20% of ethylene glycol (EG) and 0,1M of sucrose at 20 °C, for 3 minutes. Subsequently, exposed to vitrification solution, containing RPMI added to 40% of EG and 0,1 M of sucrose at 20 °C, for 2 minutes. After devitrification, 10 seminiferous tubules of each treatment were analyzed by histology assay. The viability of spermatic and Sertolicells were analysed with light histology, as well as the seminiferous tubules morphometry. The vitrified groups were inferior to the control group in the morphologically integral cells analysis. However, the OTC was superior to straw in terms of morphological preservation of the germinative cells. Nevertheless, in morphometric analysis there was no statistical difference between the treatments (control, OTC and straw). Therefore, the vitrification in OTC method showed better results than vitrification in straw based on histological evaluation of germ and Sertoli cells of domestic cats.

Efeitos da vitrificação na preservação morfológica do tecido testicular de gato doméstico / Vitrification effects during morphological preservation of testicular tissue from domestic cat

This study aimed to evaluate the effect of domestic cats (Felis Catus) testicular parenchyma vitrification by Ovarian Tissue Cryosystem and conventional straw. Three (n=3) adult cats were submitted to routine orchiectomy. For the vitrification, the samples were exposed to equilibrium solution composed by RPMI, containing 20% of ethylene glycol (EG) and 0,1M of sucrose at 20 °C, for 3 minutes. Subsequently, exposed to vitrification solution, containing RPMI added to 40% of EG and 0,1 M of sucrose at 20 °C, for 2 minutes. After devitrification, 10 seminiferous tubules of each treatment were analyzed by histology assay. The viability of spermatic and Sertolicells were analysed with light histology, as well as the seminiferous tubules morphometry. The vitrified groups were inferior to the control group in the morphologically integral cells analysis. However, the OTC was superior to straw in terms of morphological preservation of the germinative cells. Nevertheless, in morphometric analysis there was no statistical difference between the treatments (control, OTC and straw). Therefore, the vitrification in OTC method showed better results than vitrification in straw based on histological evaluation of germ and Sertoli cells of domestic cats.(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)

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.

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.

Avaliação da técnica de vitrificação de ovários de camundongos da linhagem B6D2F1, pertencentes ao Instituto de Ciência e Tecnologia em Biomodelos - ICTB/Fiocruz-RJ / Evaluation of ovary vitrification technique from B6D2F1 strain belonging to the Institute of Biomodels Science and Technology ICTB / Fiocruz-RJ

Objetivou-se testar a vitrificação de ovários de camundongos do ICTB/Fiocruz. Inicialmente, fez-se coleta e maturação in vitro dos oócitos de ovários a fresco e vitrificados, bem como avaliação de estruturas no cultivo embrionário, pós-fertilização in vitro. Fêmeas B6D2F1 foram eutanasiadas para remoção dos ovários (n=60) e divididas em três grupos: grupo 1 (n=30 animais) - oócito de ovários vitrificados, maturados e fertilizados in vitro (120 fragmentos); grupo 2 (n=15) (controle 1) - oócitos coletados a fresco, maturados e fertilizados in vitro; e grupo 3 (n=15) (controle 2) - oócitos maturados in vivo e fertilizados in vitro. A técnica foi verificada no desenvolvimento embrionário in vitro, que foi avaliado pelo teste de qui-quadrado (BioStat 5.0). Recuperaram-se 123, 224 e 328 oócitos nos G1, G2 e G3, respectivamente. Observaram-se diferenças significativas nas taxas de clivagem às 24 horas (embriões ≥ 2 células) entre G1 (8%) e G2 (32%) (P<0,1) e G1 e G3 (49%) (P<0,05), mas não entre G2 e G3 (P>0,05). Para blastocistos, às 96 horas, os grupos G1, G2 e G3 apresentaram, respectivamente, 6%, 11% e 46%, diferindo significativamente entre eles (P<0,05). A vitrificação de ovários, a maturação oocitária e a fertilização in vitro são alternativas para a produção de embriões de camundongos in vitro.(AU)

This work aimed test ovarian vitrification of hybrid mouse from ICTB/Fiocruz. Protocol collection and oocyte in vitro maturation from fresh and vitrified ovaries was established and embryos were evaluated after fertilization. B6D2F1 females were euthanized for ovarian removal (n= 60) and divided into 3 groups: G1 (n= 30) - ovaries fragmented (n= 120), vitrified, matured and fertilized; G2 (n= 15) - in vitro fertilization of oocytes matured in vitro from fresh ovaries; G3 (n= 15) - ampulla region oocytes in vitro fertilizated. Viability was verified by thawing, oocyte in vitro maturation and fertilization. In vitro embryo development of each group was evaluated by Chi-square test (BioStat 5.0). 123, 224 and 328 oocytes were recovered from G1, G2 and G3, respectively. Significant differences were observed in cleavage rates at 24 hours (embryos with 2 cells or more) between G1 (8%) and G2 (32%) (P< 0.1) and G1 and G3 (49%) (P< 0.05) but not between G2 and G3 (P> 0.05). Blastocysts at 96 hours presented 6%, 11% and 46%, respectively for G1, G2 and G3, differing significantly (P< 0.05). Ovary vitrification, oocyte in vitro maturation and in vitro fertilization were available for the production of in vitro mouse embryos.(AU)

Avaliação da técnica de vitrificação de ovários de camundongos da linhagem B6D2F1, pertencentes ao Instituto de Ciência e Tecnologia em Biomodelos - ICTB/Fiocruz-RJ / Evaluation of ovary vitrification technique from B6D2F1 strain belonging to the Institute of Biomodels Science and Technology ICTB / Fiocruz-RJ

Objetivou-se testar a vitrificação de ovários de camundongos do ICTB/Fiocruz. Inicialmente, fez-se coleta e maturação in vitro dos oócitos de ovários a fresco e vitrificados, bem como avaliação de estruturas no cultivo embrionário, pós-fertilização in vitro. Fêmeas B6D2F1 foram eutanasiadas para remoção dos ovários (n=60) e divididas em três grupos: grupo 1 (n=30 animais) - oócito de ovários vitrificados, maturados e fertilizados in vitro (120 fragmentos); grupo 2 (n=15) (controle 1) - oócitos coletados a fresco, maturados e fertilizados in vitro; e grupo 3 (n=15) (controle 2) - oócitos maturados in vivo e fertilizados in vitro. A técnica foi verificada no desenvolvimento embrionário in vitro, que foi avaliado pelo teste de qui-quadrado (BioStat 5.0). Recuperaram-se 123, 224 e 328 oócitos nos G1, G2 e G3, respectivamente. Observaram-se diferenças significativas nas taxas de clivagem às 24 horas (embriões ≥ 2 células) entre G1 (8%) e G2 (32%) (P<0,1) e G1 e G3 (49%) (P<0,05), mas não entre G2 e G3 (P>0,05). Para blastocistos, às 96 horas, os grupos G1, G2 e G3 apresentaram, respectivamente, 6%, 11% e 46%, diferindo significativamente entre eles (P<0,05). A vitrificação de ovários, a maturação oocitária e a fertilização in vitro são alternativas para a produção de embriões de camundongos in vitro.(AU)

This work aimed test ovarian vitrification of hybrid mouse from ICTB/Fiocruz. Protocol collection and oocyte in vitro maturation from fresh and vitrified ovaries was established and embryos were evaluated after fertilization. B6D2F1 females were euthanized for ovarian removal (n= 60) and divided into 3 groups: G1 (n= 30) - ovaries fragmented (n= 120), vitrified, matured and fertilized; G2 (n= 15) - in vitro fertilization of oocytes matured in vitro from fresh ovaries; G3 (n= 15) - ampulla region oocytes in vitro fertilizated. Viability was verified by thawing, oocyte in vitro maturation and fertilization. In vitro embryo development of each group was evaluated by Chi-square test (BioStat 5.0). 123, 224 and 328 oocytes were recovered from G1, G2 and G3, respectively. Significant differences were observed in cleavage rates at 24 hours (embryos with 2 cells or more) between G1 (8%) and G2 (32%) (P< 0.1) and G1 and G3 (49%) (P< 0.05) but not between G2 and G3 (P> 0.05). Blastocysts at 96 hours presented 6%, 11% and 46%, respectively for G1, G2 and G3, differing significantly (P< 0.05). Ovary vitrification, oocyte in vitro maturation and in vitro fertilization were available for the production of in vitro mouse embryos.(AU)

Vitrified embryo transfer in Merino sheep under extensive conditions

The aim was to evaluate pregnancy success after transfer of embryos vitrified in micropipette tips in Merino sheep under extensive conditions. A second objective was to evaluate the influence of embryo stage in such pregnancy rate. One hundred and twenty-seven embryos were rewarmed and transferred into recipient ewes. On rewarming, the embryos were placed into three-step cryoprotectant dilutions. Finally, prior to transfer to recipient females, embryos were maintained in Basic Medium for 5 min at 25ºC and were reevaluated by morphological criteria; all degenerated embryos were eliminated. Recipient ewes (n = 150) were treated for estrus with sponges placed for 14 days and 300 IU of eCG. At embryo transfer, three experimental groups were defined: morulae transferred on Day 7, blastocysts transferred on Day 7 and blastocysts transferred on Day 8 after sponge removal. In all groups, semi-laparoscopic transfer of one rewarmed embryo per recipient was performed. Pregnancy was diagnosed by ultrasonography on day 28 after embryo transfer. The embryo selection rate after rewarming was higher for blastocysts (89.3% - 67/75) compared to morulae (65.9% - 60/91) (P < 0.05). Pregnancy diagnosis showed a 38.3% (23/60) of success after morula transfer on Day 7 post progestagen removal. The day of transfer showed a significant influence on pregnancy rate after blastocyst transfer (Day 8, 55.9% - 19/34 vs Day 7, 21.2% - 7/33) (P < 0.05). Blastocysts transfer on Day 8 showed the highest global efficiency (pregnancies/total embryos after rewarming) (47.5% - 19/40) (P < 0.05). In conclusion, reproductive efficiency obtained by vitrified embryo transfer allows its recommendation for embryo transfer programs under extensive conditions. The importance of considering the synchrony between the embryo age and the recipient uterus stage is emphasized.

Vitrification of in vitro produced porcine blastocysts: influence of cryoprotectants toxicity and embryo age

Background: Porcine embryos are sensible to all assisted reproduction manipulations, especially the ones that involvecryopreservation. Despite the high cryoprotectant concentrations routinely applied, vitrification is the most effective technique to date. These substances toxicity can also play a negative role in embryo viability. During in vitro porcine embryoproduction, the speed of development is often unevenly distributed. It is possible that their development speed, affectsembryo tolerance to cryoprotectants. This study aimed to evaluate the toxicity of porcine embryos of days 5 or 6 of cultureto cryoprotectant agents; as well as to assess embryo survival to vitrification.Materials, Methods & Results: Parthenogenetic porcine blastocysts and expanded blastocysts of days 5 and 6 of culturewere exposed to toxicity tests (experiments 1 and 2) and vitrification (experiment 3) using different protocols. In the firstexperiment, three different cryoprotectants were used (Dimethyl sulfoxide - DMSO, Ethylene glycol - EG, and Sucrose- SUC), combined in three different associations (G1: 15% EG + 15% DMSO with 0.5 M SUC; G2: 16% EG + 16%DMSO with 0.4 M SUC; G3: 18% EG + 18% DMSO with 0.5 M SUC). In the fresh Control, embryos of day 6 are moresensible than the ones of day 5, whom showed a lower hatching rate (39.7 vs. 60.8%). After the toxicity (Experiment 1)test, the G1 showed better expansion rates in day 6 (50.0 vs 31.0 and 3.6% for G2 and G3) and higher hatching of day 6compared to G2 and G3 (23.2, vs. 8.6 and 0.0% for G2 and G3). The fresh non hatched embryos at day 8, derived at day6, had a lower percentage of cells with cleaved caspase-3 (20.2%) compared with the G1 (30.5%), G2 (31.4%) and G3(30.5%). The hatched embryos of day 5 from G2 had lower total cell number (TCN) compared with the day 6 hatchedembryos, whereas in G1 the TCN was not affected. The second experiment compared EG combined to one of these threeextracellular...

Vitrification of in vitro produced porcine blastocysts: influence of cryoprotectants toxicity and embryo age

Background: Porcine embryos are sensible to all assisted reproduction manipulations, especially the ones that involvecryopreservation. Despite the high cryoprotectant concentrations routinely applied, vitrification is the most effective technique to date. These substances toxicity can also play a negative role in embryo viability. During in vitro porcine embryoproduction, the speed of development is often unevenly distributed. It is possible that their development speed, affectsembryo tolerance to cryoprotectants. This study aimed to evaluate the toxicity of porcine embryos of days 5 or 6 of cultureto cryoprotectant agents; as well as to assess embryo survival to vitrification.Materials, Methods & Results: Parthenogenetic porcine blastocysts and expanded blastocysts of days 5 and 6 of culturewere exposed to toxicity tests (experiments 1 and 2) and vitrification (experiment 3) using different protocols. In the firstexperiment, three different cryoprotectants were used (Dimethyl sulfoxide - DMSO, Ethylene glycol - EG, and Sucrose- SUC), combined in three different associations (G1: 15% EG + 15% DMSO with 0.5 M SUC; G2: 16% EG + 16%DMSO with 0.4 M SUC; G3: 18% EG + 18% DMSO with 0.5 M SUC). In the fresh Control, embryos of day 6 are moresensible than the ones of day 5, whom showed a lower hatching rate (39.7 vs. 60.8%). After the toxicity (Experiment 1)test, the G1 showed better expansion rates in day 6 (50.0 vs 31.0 and 3.6% for G2 and G3) and higher hatching of day 6compared to G2 and G3 (23.2, vs. 8.6 and 0.0% for G2 and G3). The fresh non hatched embryos at day 8, derived at day6, had a lower percentage of cells with cleaved caspase-3 (20.2%) compared with the G1 (30.5%), G2 (31.4%) and G3(30.5%). The hatched embryos of day 5 from G2 had lower total cell number (TCN) compared with the day 6 hatchedembryos, whereas in G1 the TCN was not affected. The second experiment compared EG combined to one of these threeextracellular...(AU)

Vitrified embryo transfer in Merino sheep under extensive conditions

The aim was to evaluate pregnancy success after transfer of embryos vitrified in micropipette tips in Merino sheep under extensive conditions. A second objective was to evaluate the influence of embryo stage in such pregnancy rate. One hundred and twenty-seven embryos were rewarmed and transferred into recipient ewes. On rewarming, the embryos were placed into three-step cryoprotectant dilutions. Finally, prior to transfer to recipient females, embryos were maintained in Basic Medium for 5 min at 25ºC and were reevaluated by morphological criteria; all degenerated embryos were eliminated. Recipient ewes (n = 150) were treated for estrus with sponges placed for 14 days and 300 IU of eCG. At embryo transfer, three experimental groups were defined: morulae transferred on Day 7, blastocysts transferred on Day 7 and blastocysts transferred on Day 8 after sponge removal. In all groups, semi-laparoscopic transfer of one rewarmed embryo per recipient was performed. Pregnancy was diagnosed by ultrasonography on day 28 after embryo transfer. The embryo selection rate after rewarming was higher for blastocysts (89.3% - 67/75) compared to morulae (65.9% - 60/91) (P < 0.05). Pregnancy diagnosis showed a 38.3% (23/60) of success after morula transfer on Day 7 post progestagen removal. The day of transfer showed a significant influence on pregnancy rate after blastocyst transfer (Day 8, 55.9% - 19/34 vs Day 7, 21.2% - 7/33) (P < 0.05). Blastocysts transfer on Day 8 showed the highest global efficiency (pregnancies/total embryos after rewarming) (47.5% - 19/40) (P < 0.05). In conclusion, reproductive efficiency obtained by vitrified embryo transfer allows its recommendation for embryo transfer programs under extensive conditions. The importance of considering the synchrony between the embryo age and the recipient uterus stage is emphasized.(AU)

Combination of ethylene glycol with sucrose increases survival rate after vitrification of somatic tissue of collared peccaries (Pecari tajacu Linnaeus, 1758) / Combinação de etilenoglicol com sacarose aumenta a taxa de sobrevivência após a vitrificação de tecido somático de catetos (Pecari tajacu Linnaeus, 1758)

The cryopreservation of somatic tissue in collared peccaries promotes an alternative source of genetic material of this specie. The solid-surface vitrification (SSV) is a great option for tissue conservation; nevertheless, the optimization of SSV requirements is necessary, especially when referred to cryoprotectants that will compose the vitrification solution. Therefore, the aim was to evaluate the effect of the presence of 0.25 M sucrose in addition to different combinations (only or association) and concentrations (1.5 M or 3.0 M) of ethylene glycol (EG) and/or dimethyl sulfoxide (DMSO) in the somatic tissue vitrification of collared peccaries. Subsequently, we tested six combinations of cryoprotectants with or without sucrose in Dulbecco modified Eagle medium (DMEM) plus 10% fetal bovine serum (FBS). Thus, 3.0 M EG with sucrose was able to maintain normal tissue characteristics compared with non-vitrified (control), especially for the volumetric ratio of epidermis (61.2 vs. 58.7%) and dermis (34.5 vs. 36.6%), number of fibroblast (90.3 vs. 127.0), argyrophilic nucleolar organizer region (AgNOR) ratio (0.09 vs. 0.17%) and nucleus area (15.4 vs. 14.5 µm2) respectively. In conclusion, 3.0 M EG with 0.25 M sucrose and 10% FBS resulted in a better cryoprotectant composition in the SSV for somatic tissue of collared peccaries.(AU)

A criopreservação de tecido somático em catetos promove uma fonte alternativa de material genético nesta espécie. A vitrificação em superfície sólida (VSS) é uma ótima opção para a conservação do tecido; contudo, a otimização dos requerimentos da VSS é necessária, especialmente quanto aos crioprotetores que irão compor a solução de vitrificação. Portanto, o objetivo foi avaliar o efeito da presença de 0,25 M de sacarose em adição com diferentes combinações (individual ou associação) e concentrações (1,5 M ou 3,0 M) de etilenoglicol (EG) e/ou dimetilsulfóxido (DMSO) na vitrificação de tecido somático de catetos. Subsequentemente, nós testamos seis combinações de crioprotetores com ou sem sacarose em meio de Eagle modificado por Dulbecco (DMEM) acrescido de 10% de soro fetal bovino (SFB). Assim, 3,0 M de EG com sacarose foi capaz de manter as características normais do tecido comparado com o não vitrificado (controle), especialmente para a proporção volumétrica da epiderme (61,2 vs. 58,7%) e derme (34,5 vs. 36,6%), número de fibroblastos (90,3 vs. 127,0), razão da região argirófila organizadora de nucléolo (AgNOR) (0,09 vs. 0,17%) e área do núcleo (15,4vs.14,5 µm2), respectivamente. Em conclusão, 3,0 M de EG com 0,25 M de sacarose e 10% de SFB resultaram na melhor composição de crioprotetores na VSS para tecido somático de catetos.(AU)

Combination of ethylene glycol with sucrose increases survival rate after vitrification of somatic tissue of collared peccaries (Pecari tajacu Linnaeus, 1758) / Combinação de etilenoglicol com sacarose aumenta a taxa de sobrevivência após a vitrificação de tecido somático de catetos (Pecari tajacu Linnaeus, 1758)

The cryopreservation of somatic tissue in collared peccaries promotes an alternative source of genetic material of this specie. The solid-surface vitrification (SSV) is a great option for tissue conservation; nevertheless, the optimization of SSV requirements is necessary, especially when referred to cryoprotectants that will compose the vitrification solution. Therefore, the aim was to evaluate the effect of the presence of 0.25 M sucrose in addition to different combinations (only or association) and concentrations (1.5 M or 3.0 M) of ethylene glycol (EG) and/or dimethyl sulfoxide (DMSO) in the somatic tissue vitrification of collared peccaries. Subsequently, we tested six combinations of cryoprotectants with or without sucrose in Dulbecco modified Eagle medium (DMEM) plus 10% fetal bovine serum (FBS). Thus, 3.0 M EG with sucrose was able to maintain normal tissue characteristics compared with non-vitrified (control), especially for the volumetric ratio of epidermis (61.2 vs. 58.7%) and dermis (34.5 vs. 36.6%), number of fibroblast (90.3 vs. 127.0), argyrophilic nucleolar organizer region (AgNOR) ratio (0.09 vs. 0.17%) and nucleus area (15.4 vs. 14.5 µm2) respectively. In conclusion, 3.0 M EG with 0.25 M sucrose and 10% FBS resulted in a better cryoprotectant composition in the SSV for somatic tissue of collared peccaries.(AU)

A criopreservação de tecido somático em catetos promove uma fonte alternativa de material genético nesta espécie. A vitrificação em superfície sólida (VSS) é uma ótima opção para a conservação do tecido; contudo, a otimização dos requerimentos da VSS é necessária, especialmente quanto aos crioprotetores que irão compor a solução de vitrificação. Portanto, o objetivo foi avaliar o efeito da presença de 0,25 M de sacarose em adição com diferentes combinações (individual ou associação) e concentrações (1,5 M ou 3,0 M) de etilenoglicol (EG) e/ou dimetilsulfóxido (DMSO) na vitrificação de tecido somático de catetos. Subsequentemente, nós testamos seis combinações de crioprotetores com ou sem sacarose em meio de Eagle modificado por Dulbecco (DMEM) acrescido de 10% de soro fetal bovino (SFB). Assim, 3,0 M de EG com sacarose foi capaz de manter as características normais do tecido comparado com o não vitrificado (controle), especialmente para a proporção volumétrica da epiderme (61,2 vs. 58,7%) e derme (34,5 vs. 36,6%), número de fibroblastos (90,3 vs. 127,0), razão da região argirófila organizadora de nucléolo (AgNOR) (0,09 vs. 0,17%) e área do núcleo (15,4vs.14,5 µm2), respectivamente. Em conclusão, 3,0 M de EG com 0,25 M de sacarose e 10% de SFB resultaram na melhor composição de crioprotetores na VSS para tecido somático de catetos.(AU)

Combination of ethylene glycol with sucrose increases survival rate after vitrification of somatic tissue of collared peccaries (Pecari tajacu Linnaeus, 1758)

ABSTRACT: The cryopreservation of somatic tissue in collared peccaries promotes an alternative source of genetic material of this specie. The solid-surface vitrification (SSV) is a great option for tissue conservation; nevertheless, the optimization of SSV requirements is necessary, especially when referred to cryoprotectants that will compose the vitrification solution. Therefore, the aim was to evaluate the effect of the presence of 0.25 M sucrose in addition to different combinations (only or association) and concentrations (1.5 M or 3.0 M) of ethylene glycol (EG) and/or dimethyl sulfoxide (DMSO) in the somatic tissue vitrification of collared peccaries. Subsequently, we tested six combinations of cryoprotectants with or without sucrose in Dulbecco modified Eagle medium (DMEM) plus 10% fetal bovine serum (FBS). Thus, 3.0 M EG with sucrose was able to maintain normal tissue characteristics compared with non-vitrified (control), especially for the volumetric ratio of epidermis (61.2 vs. 58.7%) and dermis (34.5 vs. 36.6%), number of fibroblast (90.3 vs. 127.0), argyrophilic nucleolar organizer region (AgNOR) ratio (0.09 vs. 0.17%) and nucleus area (15.4 vs. 14.5 m2) respectively. In conclusion, 3.0 M EG with 0.25 M sucrose and 10% FBS resulted in a better cryoprotectant composition in the SSV for somatic tissue of collared peccaries.

RESUMO: A criopreservação de tecido somático em catetos promove uma fonte alternativa de material genético nesta espécie. A vitrificação em superfície sólida (VSS) é uma ótima opção para a conservação do tecido; contudo, a otimização dos requerimentos da VSS é necessária, especialmente quanto aos crioprotetores que irão compor a solução de vitrificação. Portanto, o objetivo foi avaliar o efeito da presença de 0,25 M de sacarose em adição com diferentes combinações (individual ou associação) e concentrações (1,5 M ou 3,0 M) de etilenoglicol (EG) e/ou dimetilsulfóxido (DMSO) na vitrificação de tecido somático de catetos. Subsequentemente, nós testamos seis combinações de crioprotetores com ou sem sacarose em meio de Eagle modificado por Dulbecco (DMEM) acrescido de 10% de soro fetal bovino (SFB). Assim, 3,0 M de EG com sacarose foi capaz de manter as características normais do tecido comparado com o não vitrificado (controle), especialmente para a proporção volumétrica da epiderme (61,2 vs. 58,7%) e derme (34,5 vs. 36,6%), número de fibroblastos (90,3 vs. 127,0), razão da região argirófila organizadora de nucléolo (AgNOR) (0,09 vs. 0,17%) e área do núcleo (15,4vs.14,5 m2), respectivamente. Em conclusão, 3,0 M de EG com 0,25 M de sacarose e 10% de SFB resultaram na melhor composição de crioprotetores na VSS para tecido somático de catetos.

A new simple and reliable vitrification device based on Hollow Fiber Vitrification (HFV) method evaluated using IVP bovine embryos

A new vitrification device based on hollow fiber vitrification (HFV) was constructed using a glass capillary, which lead to simplified construction process and increased practicality of the device. The hollow fiber was attached to heat-pulled tip of the glass capillary using forceps. A protective sheath fitted on the capillary provided protection for the cellulose triacetate hollow fiber with loaded embryos and allowed safe storage in liquid nitrogen for long periods of time (2-12 month), transfer between tanks with liquid nitrogen and transportation within these tanks. No embryos were lost in the process. The device was tested using seven-day-old and eight-day-old IVP bovine blastocysts and expanded blastocysts as a model. Obtained survival (90% at 24 h post warming) and hatching rates (62% at 72 h post warming) of day 7 blastocysts and expanded blastocysts were comparable to those gained using various vitrification carriers. Vitrified embryos did not show an increase in the number of cells with damaged membrane or a decrease in total cell number per embryos in comparison to their non-vitrified counterparts. Day 7 and 8 expanded blastocysts did not differ significantly in terms of survival at 24 (97.01 vs. 97.50%) and 48 h post warming (95.52 vs. 95%), but showed significantly higher survival and hatching rates than day 7 and 8 blastocysts. These results indicated that high and repeatable survival rates can be obtained by selection of IVP bovine embryos at the developmental stage of expanded blastocyst for HFV. Further modification of the method may be required to achieve high and stable results with different developmental stages of IVP bovine embryo. The vitrification device presented in the current article may contribute to wider application of HFV method in livestock production.

A new simple and reliable vitrification device based on Hollow Fiber Vitrification (HFV) method evaluated using IVP bovine embryos

A new vitrification device based on hollow fiber vitrification (HFV) was constructed using a glass capillary, which lead to simplified construction process and increased practicality of the device. The hollow fiber was attached to heat-pulled tip of the glass capillary using forceps. A protective sheath fitted on the capillary provided protection for the cellulose triacetate hollow fiber with loaded embryos and allowed safe storage in liquid nitrogen for long periods of time (2-12 month), transfer between tanks with liquid nitrogen and transportation within these tanks. No embryos were lost in the process. The device was tested using seven-day-old and eight-day-old IVP bovine blastocysts and expanded blastocysts as a model. Obtained survival (90% at 24 h post warming) and hatching rates (62% at 72 h post warming) of day 7 blastocysts and expanded blastocysts were comparable to those gained using various vitrification carriers. Vitrified embryos did not show an increase in the number of cells with damaged membrane or a decrease in total cell number per embryos in comparison to their non-vitrified counterparts. Day 7 and 8 expanded blastocysts did not differ significantly in terms of survival at 24 (97.01 vs. 97.50%) and 48 h post warming (95.52 vs. 95%), but showed significantly higher survival and hatching rates than day 7 and 8 blastocysts. These results indicated that high and repeatable survival rates can be obtained by selection of IVP bovine embryos at the developmental stage of expanded blastocyst for HFV. Further modification of the method may be required to achieve high and stable results with different developmental stages of IVP bovine embryo. The vitrification device presented in the current article may contribute to wider application of HFV method in livestock production.(AU)