Survival rates of mouse blastocyst vitrified in dimethylformamide based solutions associated with ethylene glicol or 1-2 propanediol

The aim of this study was to determine the effect of dimethylformamide (DF) associated with ethylene glycol (EG) or 1-2 propanediol (PROH) during vitrification, on the in vitro development of mouse blastocysts. Cryoprotectant toxicity was evaluated exposing embryos into three different equilibrium solutions (ES) composed by DF, EG or PROH mixtures (10% v/v of each) in mPBS + 0.5% PVA at different interval times (1, 3 and 10min). In a second experiment, embryos were exposed to the same ES (either 1 or 3min), following for the three respectively vitrification solutions (VS) (20% v/v of each) for 30s. After 72 hours of in vitro culture, embryo hatching and expansion rates were similar for the ES1 and ES2 equilibration solutions during the time interval of 1 or 3min. However embryos exposed for 10 min to the DF equilibration solutions, had lower survival rates than EG-PROH solution (P 0.01). Furthermore, survival rates for embryos exposed to DF-PROH (ES+VS) were lower than embryos exposed to the other solutions (P 0.01). Blastocyst vitrification was performed with the three ES+VS (for 1min and 30s, respectively), using glass micropipettes (GMP). Survival rates were lower for blastocysts vitrified with DF solutions (3%-3/108 and 17.1%-19/111) (P 0.01) than with PROH+EG vitrification solutions (69%-73/105). In conclusion, DF as a cryoprotectant into vitrification solutions have deleterious effects on the in vitro developmental competence of vitrified mouse blastocysts.

O objetivo deste estudo foi determinar o efeito da dimetilformamida (DF) associada com etileno glycol (EG) ou 1-2 propanediol (PROH) durante a vitrificação, no desenvolvimento in vitro de blastocistos murinos. A toxicidade dos crioprotetores foi avaliada ao expor os embriões as três soluções de equilíbrio (ES) compostas pelas misturas de DF, EG ou PROH (10% v/v de cada) em mPBS + 0,5% PVA, em diferentes intervalos de tempo (1, 3 e 10min). Em um segundo experimento, os embriões foram expostos as mesmas ES (durante 1 e 3min), seguido da exposição as três respectivas soluções de vitrificação (VS) (20% v/v de cada) durante 30seg. Após 72 horas de cultivo in vitro, as taxas de expansão e eclosão dos embriões expostos durante os períodos de 1 e 3min às soluções de equilíbrio ES1 e ES2 foram semelhantes. No entanto, os embriões expostos durante 10min às soluções de equilíbrio com DF apresentaram taxas de sobrevivência inferiores à solução de EG-PROH (P 0,01). Além disso, as taxas de sobrevivência dos embriões expostos à DF-PROH (ES+VS) foram menores que as dos embriões expostos as outras soluções (P 0,01). A vitrificação dos blastocistos foi realizada após a exposição dos embriões nas três ES+VS (por 1min e 30seg, respectivamente), usando micropipetas de vidro (GMP). As taxas de sobrevivência foram menores nos blastocistos vitrificados nas soluções compostas por DF (3%-3/108 e 17,1%-19/111), em relação à solução EG-PROH (69%-73/105) (P 0,01). Em conclusão, a DF adicionada como crioprotetor às soluções de vitrificação apresenta efeitos deletérios na capacidade de desenvolvimento in vitro dos blastocistos murinos vitrificados.

Survival rates of mouse blastocyst vitrified in dimethylformamide based solutions associated with ethylene glicol or 1-2 propanediol

The aim of this study was to determine the effect of dimethylformamide (DF) associated with ethylene glycol (EG) or 1-2 propanediol (PROH) during vitrification, on the in vitro development of mouse blastocysts. Cryoprotectant toxicity was evaluated exposing embryos into three different equilibrium solutions (ES) composed by DF, EG or PROH mixtures (10% v/v of each) in mPBS + 0.5% PVA at different interval times (1, 3 and 10min). In a second experiment, embryos were exposed to the same ES (either 1 or 3min), following for the three respectively vitrification solutions (VS) (20% v/v of each) for 30s. After 72 hours of in vitro culture, embryo hatching and expansion rates were similar for the ES1 and ES2 equilibration solutions during the time interval of 1 or 3min. However embryos exposed for 10 min to the DF equilibration solutions, had lower survival rates than EG-PROH solution (P 0.01). Furthermore, survival rates for embryos exposed to DF-PROH (ES+VS) were lower than embryos exposed to the other solutions (P 0.01). Blastocyst vitrification was performed with the three ES+VS (for 1min and 30s, respectively), using glass micropipettes (GMP). Survival rates were lower for blastocysts vitrified with DF solutions (3%-3/108 and 17.1%-19/111) (P 0.01) than with PROH+EG vitrification solutions (69%-73/105). In conclusion, DF as a cryoprotectant into vitrification solutions have deleterious effects on the in vitro developmental competence of vitrified mouse blastocysts.

O objetivo deste estudo foi determinar o efeito da dimetilformamida (DF) associada com etileno glycol (EG) ou 1-2 propanediol (PROH) durante a vitrificação, no desenvolvimento in vitro de blastocistos murinos. A toxicidade dos crioprotetores foi avaliada ao expor os embriões as três soluções de equilíbrio (ES) compostas pelas misturas de DF, EG ou PROH (10% v/v de cada) em mPBS + 0,5% PVA, em diferentes intervalos de tempo (1, 3 e 10min). Em um segundo experimento, os embriões foram expostos as mesmas ES (durante 1 e 3min), seguido da exposição as três respectivas soluções de vitrificação (VS) (20% v/v de cada) durante 30seg. Após 72 horas de cultivo in vitro, as taxas de expansão e eclosão dos embriões expostos durante os períodos de 1 e 3min às soluções de equilíbrio ES1 e ES2 foram semelhantes. No entanto, os embriões expostos durante 10min às soluções de equilíbrio com DF apresentaram taxas de sobrevivência inferiores à solução de EG-PROH (P 0,01). Além disso, as taxas de sobrevivência dos embriões expostos à DF-PROH (ES+VS) foram menores que as dos embriões expostos as outras soluções (P 0,01). A vitrificação dos blastocistos foi realizada após a exposição dos embriões nas três ES+VS (por 1min e 30seg, respectivamente), usando micropipetas de vidro (GMP). As taxas de sobrevivência foram menores nos blastocistos vitrificados nas soluções compostas por DF (3%-3/108 e 17,1%-19/111), em relação à solução EG-PROH (69%-73/105) (P 0,01). Em conclusão, a DF adicionada como crioprotetor às soluções de vitrificação apresenta efeitos deletérios na capacidade de desenvolvimento in vitro dos blastocistos murinos vitrificados.

Survival rates of mouse blastocyst vitrified in dimethylformamide based solutions associated with ethylene glicol or 1-2 propanediol

The aim of this study was to determine the effect of dimethylformamide (DF) associated with ethylene glycol (EG) or 1-2 propanediol (PROH) during vitrification, on the in vitro development of mouse blastocysts. Cryoprotectant toxicity was evaluated exposing embryos into three different equilibrium solutions (ES) composed by DF, EG or PROH mixtures (10% v/v of each) in mPBS + 0.5% PVA at different interval times (1, 3 and 10min). In a second experiment, embryos were exposed to the same ES (either 1 or 3min), following for the three respectively vitrification solutions (VS) (20% v/v of each) for 30s. After 72 hours of in vitro culture, embryo hatching and expansion rates were similar for the ES1 and ES2 equilibration solutions during the time interval of 1 or 3min. However embryos exposed for 10 min to the DF equilibration solutions, had lower survival rates than EG-PROH solution (P 0.01). Furthermore, survival rates for embryos exposed to DF-PROH (ES+VS) were lower than embryos exposed to the other solutions (P 0.01). Blastocyst vitrification was performed with the three ES+VS (for 1min and 30s, respectively), using glass micropipettes (GMP). Survival rates were lower for blastocysts vitrified with DF solutions (3%-3/108 and 17.1%-19/111) (P 0.01) than with PROH+EG vitrification solutions (69%-73/105). In conclusion, DF as a cryoprotectant into vitrification solutions have deleterious effects on the in vitro developmental competence of vitrified mouse blastocysts.

O objetivo deste estudo foi determinar o efeito da dimetilformamida (DF) associada com etileno glycol (EG) ou 1-2 propanediol (PROH) durante a vitrificação, no desenvolvimento in vitro de blastocistos murinos. A toxicidade dos crioprotetores foi avaliada ao expor os embriões as três soluções de equilíbrio (ES) compostas pelas misturas de DF, EG ou PROH (10% v/v de cada) em mPBS + 0,5% PVA, em diferentes intervalos de tempo (1, 3 e 10min). Em um segundo experimento, os embriões foram expostos as mesmas ES (durante 1 e 3min), seguido da exposição as três respectivas soluções de vitrificação (VS) (20% v/v de cada) durante 30seg. Após 72 horas de cultivo in vitro, as taxas de expansão e eclosão dos embriões expostos durante os períodos de 1 e 3min às soluções de equilíbrio ES1 e ES2 foram semelhantes. No entanto, os embriões expostos durante 10min às soluções de equilíbrio com DF apresentaram taxas de sobrevivência inferiores à solução de EG-PROH (P 0,01). Além disso, as taxas de sobrevivência dos embriões expostos à DF-PROH (ES+VS) foram menores que as dos embriões expostos as outras soluções (P 0,01). A vitrificação dos blastocistos foi realizada após a exposição dos embriões nas três ES+VS (por 1min e 30seg, respectivamente), usando micropipetas de vidro (GMP). As taxas de sobrevivência foram menores nos blastocistos vitrificados nas soluções compostas por DF (3%-3/108 e 17,1%-19/111), em relação à solução EG-PROH (69%-73/105) (P 0,01). Em conclusão, a DF adicionada como crioprotetor às soluções de vitrificação apresenta efeitos deletérios na capacidade de desenvolvimento in vitro dos blastocistos murinos vitrificados.

Mice Embryos Cryopreservation: Vitrification or Ultra-Rapid Freezing?

The importance of mice as animal model for research has promoted the surge of many strains with important characteristics which need to be preserved. Embryo cryopreservation appears as the most suitable technique. However, until now there is not an effective methodology for this specie. This study aimed to evaluate three methods for mice embryos cryopreservation.

Mice Embryos Cryopreservation: Vitrification or Ultra-Rapid Freezing?

The importance of mice as animal model for research has promoted the surge of many strains with important characteristics which need to be preserved. Embryo cryopreservation appears as the most suitable technique. However, until now there is not an effective methodology for this specie. This study aimed to evaluate three methods for mice embryos cryopreservation.(AU)

Artificial feeding of Amblyomma cajennense (Acari: Ixodidae) fasting females through capillary tube technique

The present study aimed to adjust the artificial feeding technique through capillaries and to verify its influence over the biology of Amblyomma cajennense females. Five groups of 20 female ticks were formed. Females were starved for 45 days and then fed with citrated bovine blood using capillary tubes in different periods of time. Females were divided in five experimental groups with 20 individuals each and fed as follows: groups uninterruptedly fed for 12, 24, and 48 hours and groups fed 2 and 6 h a day, for a period of 8 days. Subsequently, ticks were exposed to rabbits for complementary feeding and their biological parameters were analyzed. TIcks were capable of feeding, showing rounded idiosoma, visible even to naked eyes, following the feeding period. The groups fed for 24 hours, 2 hours/day for eight consecutive days or 6h/day for eight consecutive days presented greater weight gain, without statistically significant differences. These results suggested that 24 hours of artificial feeding were enough for fasting females to increase weight by 2.43 mg. Artificial feeding through capillaries did not interfere with parasitic and non-parasitic phases of A. cajennense females.

O presente trabalho teve como objetivos adaptar a técnica de alimentação artificial por meio de tubos capilares para fêmeas de Amblyomma cajennense e verificar a sua influência sobre os parâmetros biológicos da espécie. Fêmeas em jejum por 45 dias foram alimentadas com sangue bovino citratado usando tubos capilares por diferentes períodos de tempo: 12, 24 e 48 horas, 2 horas/dia e 6 horas/dia por 8 dias consecutivos, sendo que cada grupo foi composto por 20 carrapatos. Em seguida, os carrapatos foram expostos a coelhos para completar a alimentação e seus parâmetros biológicos foram analisados. Após alimentação artificial, os carrapatos apresentaram idiossoma arredondado mesmo à vista desarmada. Os grupos 24 horas, 8 dias/2 horas/dia e 8 dias/6 horas/dia tiveram maior ganho de peso, não havendo diferença estatística entre eles. Desta forma, nas condições do presente trabalho, 24 horas de alimentação artificial foram suficientes para que fêmeas em jejum aumentassem seu peso em 2,43mg. A alimentação artificial utilizando tubos capilares não interferiu nos parâmetros relativos às fases parasitária e não-parasitária de fêmeas A. cajennense.

Bovine in vitro embryo production protocol: does it really influence embryo cryotolerance?

The protocol of  in vitro production (IVP) of bovine embryos is one of the critical factors determining embryo viability after cryopreservation. In this study were used two differents protocols to produce IVP bovine embryos, with variations in protein source, oocyte/zygote density per media volume, with the aim to determine the in vitro and in vivo embryo survival after vitrification using hand-pulled glass micropipettes. Expanded blastocysts (D7) were morphologically selected by size (?180 ?m) and osmotic behavior before they were randomly allocated to sub-groups by protocol: non-vitrified embryos (control; C) and vitrified embryos (V). For the evaluation of the in vitro survival, control embryos and a group of warmed vitrified embryos were in vitro-cultured (IVC) for 72 h. Re-expansion rates of warmed embryos at 24 h of IVC were 94.8% and 93.2% for Protocols 1 and 2, respectively. Hatching rates at 72 h of IVC of embryos from Protocol 1 (C=80% and V=75.8%) tended to be higher (P=0.0561, Chi2 test) than those from Protocol 2 (C=67.2% and V=59.3%). For the evaluation of in vivo survival, 21 vitrified embryos per protocol were singly non-surgically transferred to synchronized recipients (n=42) after the in-straw cryoprotectant dilution, resulting in 4 (19%) pregnancies per group on Day 60 of gestation. In conclusion, despite a lower variation on in vitro embryo development betwe

Bovine in vitro embryo production protocol: does it really influence embryo cryotolerance?

The protocol of  in vitro production (IVP) of bovine embryos is one of the critical factors determining embryo viability after cryopreservation. In this study were used two differents protocols to produce IVP bovine embryos, with variations in protein source, oocyte/zygote density per media volume, with the aim to determine the in vitro and in vivo embryo survival after vitrification using hand-pulled glass micropipettes. Expanded blastocysts (D7) were morphologically selected by size (?180 ?m) and osmotic behavior before they were randomly allocated to sub-groups by protocol: non-vitrified embryos (control; C) and vitrified embryos (V). For the evaluation of the in vitro survival, control embryos and a group of warmed vitrified embryos were in vitro-cultured (IVC) for 72 h. Re-expansion rates of warmed embryos at 24 h of IVC were 94.8% and 93.2% for Protocols 1 and 2, respectively. Hatching rates at 72 h of IVC of embryos from Protocol 1 (C=80% and V=75.8%) tended to be higher (P=0.0561, Chi2 test) than those from Protocol 2 (C=67.2% and V=59.3%). For the evaluation of in vivo survival, 21 vitrified embryos per protocol were singly non-surgically transferred to synchronized recipients (n=42) after the in-straw cryoprotectant dilution, resulting in 4 (19%) pregnancies per group on Day 60 of gestation. In conclusion, despite a lower variation on in vitro embryo development betwe

Bovine in vitro embryo production protocol: does it really influence embryo cryotolerance?

The protocol of  in vitro production (IVP) of bovine embryos is one of the critical factors determining embryo viability after cryopreservation. In this study were used two differents protocols to produce IVP bovine embryos, with variations in protein source, oocyte/zygote density per media volume, with the aim to determine the in vitro and in vivo embryo survival after vitrification using hand-pulled glass micropipettes. Expanded blastocysts (D7) were morphologically selected by size (?180 ?m) and osmotic behavior before they were randomly allocated to sub-groups by protocol: non-vitrified embryos (control; C) and vitrified embryos (V). For the evaluation of the in vitro survival, control embryos and a group of warmed vitrified embryos were in vitro-cultured (IVC) for 72 h. Re-expansion rates of warmed embryos at 24 h of IVC were 94.8% and 93.2% for Protocols 1 and 2, respectively. Hatching rates at 72 h of IVC of embryos from Protocol 1 (C=80% and V=75.8%) tended to be higher (P=0.0561, Chi2 test) than those from Protocol 2 (C=67.2% and V=59.3%). For the evaluation of in vivo survival, 21 vitrified embryos per protocol were singly non-surgically transferred to synchronized recipients (n=42) after the in-straw cryoprotectant dilution, resulting in 4 (19%) pregnancies per group on Day 60 of gestation. In conclusion, despite a lower variation on in vitro embryo development betwe