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)

Células fetais bovinas de cultivo primário submetidas a diferentes pressões negativas antes do congelamento em palhetas / Fetal bovine primary culture cells submitted to different negative pressures before straw freezing

O congelamento de células é uma importante ferramenta na preservação de espécies ameaçadas de extinção. Células fetais de cultivo primário obtidas de um bovino clone foram submetidas à pressão negativa (PN) de 200, 500 ou 800 mbar, imediatamente (PN0h) ou três horas antes (PN3h) do congelamento em palhetas finas, com 10% de DMSO como crioprotetor. Células frescas e congeladas sem submissão à PN foram utilizadas como controles. Avaliou-se a viabilidade pós-descongelamento, a curva de proliferação celular, assim como o tempo de duplicação da população (PDT) celular, a cada 24 horas, durante oito dias. Os dados obtidos foram submetidos ao teste de Tukey ou Qui quadrado (P≤0,05). A sobrevivência média dos grupos controle (89,8%) e PN500 0h (88,1%) foi superior aos outros grupos; o tempo de PDT foi semelhante nos grupos fresco (27,5 ± 0,35 h), controle congelado (30,1 ± 2,3 h) e PN500 0h (32,4 ± 1,6 h). O menor tempo foi observado no grupo PN800 0h (21,9 h). O congelamento de células fetais bovinas de cultivo primário, realizado em palhetas de 0,25 mL, com 10% de DMSO, possibilita elevadas taxas de sobrevivência após o descongelamento. A PN modifica a curva de crescimento de células criopreservadas, sendo que as intensidades de 200 ou 500 mbar, aplicadas imediatamente antes do congelamento das células, possibilitam curvas de proliferação semelhantes às obtidas com células frescas.

Cell cryopreservation is an important tool in the preservation of endangered species. Fetal bovine primary culture-obtained cells were subjected to negative pressure (NP) 200, 500 or 800 mbar, just before (NP0h) or 3 hours before (NP3h) freezing into fine (0.25 mL) straws, using 10% DMSO as cryoprotectant. Fresh and frozen fibroblasts non submitted to NP were used as controls. We evaluated cell viability after freezing, cell proliferation curve, and population doubling time (PDT) every 24 hours during 8 days. Data were submitted to Tukey or Chi square test (P≤ 0.05). The average survival of control group (89.8%) and NP500-0h (88.1%) was higher than other groups; the time of PDT was similar in the fresh (27.5 ± 0.35 h), frozen control (30.1 ± 2.3 h) and NP500-0h groups (32.4 ± 1.6 h). The lowest time was observed in the NP800-0h (21.9 h) group. Freezing of bovine fibroblasts into 0.25 mL straws with 10% DMSO enables high survival rates after thawing. The NP modifies the growth curve of cryopreserved cells and the intensities of 200 or 500 mbar, applied just before freezing the cells, allow proliferation curves similar to those obtained with fresh cells.

Células fetais bovinas de cultivo primário submetidas a diferentes pressões negativas antes do congelamento em palhetas / Fetal bovine primary culture cells submitted to different negative pressures before straw freezing

O congelamento de células é uma importante ferramenta na preservação de espécies ameaçadas de extinção. Células fetais de cultivo primário obtidas de um bovino clone foram submetidas à pressão negativa (PN) de 200, 500 ou 800 mbar, imediatamente (PN0h) ou três horas antes (PN3h) do congelamento em palhetas finas, com 10% de DMSO como crioprotetor. Células frescas e congeladas sem submissão à PN foram utilizadas como controles. Avaliou-se a viabilidade pós-descongelamento, a curva de proliferação celular, assim como o tempo de duplicação da população (PDT) celular, a cada 24 horas, durante oito dias. Os dados obtidos foram submetidos ao teste de Tukey ou Qui quadrado (P≤0,05). A sobrevivência média dos grupos controle (89,8%) e PN500 0h (88,1%) foi superior aos outros grupos; o tempo de PDT foi semelhante nos grupos fresco (27,5 ± 0,35 h), controle congelado (30,1 ± 2,3 h) e PN500 0h (32,4 ± 1,6 h). O menor tempo foi observado no grupo PN800 0h (21,9 h). O congelamento de células fetais bovinas de cultivo primário, realizado em palhetas de 0,25 mL, com 10% de DMSO, possibilita elevadas taxas de sobrevivência após o descongelamento. A PN modifica a curva de crescimento de células criopreservadas, sendo que as intensidades de 200 ou 500 mbar, aplicadas imediatamente antes do congelamento das células, possibilitam curvas de proliferação semelhantes às obtidas com células frescas.(AU)

Cell cryopreservation is an important tool in the preservation of endangered species. Fetal bovine primary culture-obtained cells were subjected to negative pressure (NP) 200, 500 or 800 mbar, just before (NP0h) or 3 hours before (NP3h) freezing into fine (0.25 mL) straws, using 10% DMSO as cryoprotectant. Fresh and frozen fibroblasts non submitted to NP were used as controls. We evaluated cell viability after freezing, cell proliferation curve, and population doubling time (PDT) every 24 hours during 8 days. Data were submitted to Tukey or Chi square test (P≤ 0.05). The average survival of control group (89.8%) and NP500-0h (88.1%) was higher than other groups; the time of PDT was similar in the fresh (27.5 ± 0.35 h), frozen control (30.1 ± 2.3 h) and NP500-0h groups (32.4 ± 1.6 h). The lowest time was observed in the NP800-0h (21.9 h) group. Freezing of bovine fibroblasts into 0.25 mL straws with 10% DMSO enables high survival rates after thawing. The NP modifies the growth curve of cryopreserved cells and the intensities of 200 or 500 mbar, applied just before freezing the cells, allow proliferation curves similar to those obtained with fresh cells.(AU)

Intracytoplasmic sperm injection after vitrifcation of immature oocytes in follicular fluid increases bovine embryo production

Background: Despite the low efficiency caused by its harmful effects, vitrification is the technique of choice for oocyte cryopeservation, especially at the germinal vesicle (GV) stage. This enables the banking of female gametes without linkage to the male genotype. Follicular fluid (FF), in vivo, is known to provide an adequate environment to the immature oocyte. The intra-cytoplasmic sperm injection (ICSI), by the other hand, can be used to bypass any sperm penetration disorder, including the ones caused by cryopreservation. This study aimed to evaluate oocyte vitrification in FF based solution, and to asses ICSI efficiency in the fertilization of vitrified/warmed bovine GV oocytes. Material, Methods & Results: Follicles of 2-8 mm in diameter were aspirated from bovine ovaries obtained from a slaughterhouse, selected and maintained into FF from aspiration, until their allocation in the experimental groups. The FF used to prepare the vitrification solution was centrifuged, heat inactivated, filtered through a 0.22 mm pore and stored at -20°C. Oocyte vitrification was done into one of these three solutions: The standard solution TCM-Hepes (TH-Vitri) was compared to a totally FF based solution (FF-Vitri), and to a 50:50 (v/v) mix of both solutions (TH:FF-Vitri). Oocytes were submitted to in vitroembryo production in order to assess embryo production efficiency. A second set of experiments using the FF-Vitri solution compared IVF versus ICSI. With basis on cleaved structures, the morula + blastocyst rate obtained in the Fresh Control (43.9%) was similar to FF-Vitri (31.1%). Conversely, the TH-Vitri (15.7%) and the TH:FF-Vitri (20.4%) rates were significantly lower than the Fresh Control. ICSI showed a positive effect in comparison with IVF. The embryo development rate of Vitri-IVF (18.8%) was the lowest, whereas Vitri-ICSI (37.3%) was similar to the Fresh-IVF (43.9%), but lower than the Fresh-ICSI (57.8%).[...]

Intracytoplasmic sperm injection after vitrifcation of immature oocytes in follicular fluid increases bovine embryo production

Background: Despite the low efficiency caused by its harmful effects, vitrification is the technique of choice for oocyte cryopeservation, especially at the germinal vesicle (GV) stage. This enables the banking of female gametes without linkage to the male genotype. Follicular fluid (FF), in vivo, is known to provide an adequate environment to the immature oocyte. The intra-cytoplasmic sperm injection (ICSI), by the other hand, can be used to bypass any sperm penetration disorder, including the ones caused by cryopreservation. This study aimed to evaluate oocyte vitrification in FF based solution, and to asses ICSI efficiency in the fertilization of vitrified/warmed bovine GV oocytes. Material, Methods & Results: Follicles of 2-8 mm in diameter were aspirated from bovine ovaries obtained from a slaughterhouse, selected and maintained into FF from aspiration, until their allocation in the experimental groups. The FF used to prepare the vitrification solution was centrifuged, heat inactivated, filtered through a 0.22 mm pore and stored at -20°C. Oocyte vitrification was done into one of these three solutions: The standard solution TCM-Hepes (TH-Vitri) was compared to a totally FF based solution (FF-Vitri), and to a 50:50 (v/v) mix of both solutions (TH:FF-Vitri). Oocytes were submitted to in vitroembryo production in order to assess embryo production efficiency. A second set of experiments using the FF-Vitri solution compared IVF versus ICSI. With basis on cleaved structures, the morula + blastocyst rate obtained in the Fresh Control (43.9%) was similar to FF-Vitri (31.1%). Conversely, the TH-Vitri (15.7%) and the TH:FF-Vitri (20.4%) rates were significantly lower than the Fresh Control. ICSI showed a positive effect in comparison with IVF. The embryo development rate of Vitri-IVF (18.8%) was the lowest, whereas Vitri-ICSI (37.3%) was similar to the Fresh-IVF (43.9%), but lower than the Fresh-ICSI (57.8%).[...](AU)

Pre-incubation of porcine semen reduces the incidence of polyspermy on embryos derived from low quality oocytes / Pré-incubação dos espermatozoides suínos diminui polispermia e aumenta a produção embrionária em oócitos de baixa qualidade

The main cause of low efficiency of in vitro produced porcine embryos is the high polyspermic penetration rates at fertilization, which is aggravated in low quality oocytes. Experiment 1 evaluated the embryo development in high and low quality oocytes. Experiment 2 evaluated the embryo development and quality of low quality oocytes fertilized with sperm pre-incubated during 0h (control), 0.5h, 1h and 1.5h. Experiment 3 investigated fertilization and monospermic rates of the same groups of Experiment 2. Experiment 4 evaluated embryo development, cell density, fertilization and monospermic rates of high quality oocytes using semen pre incubated during the best time observed in the previous experiments. Cleavage and blastocyst rates were analyzed by chi-square test, and remaining data by ANOVA and Tukey test (P0.05). The cleavage (74.8 vs 51.7%) and blastocyst (33.7 vs 9.8%) rates were greater in oocytes of high versus low quality, with no differences in cell density. Fertilization rates (65.6 to 79.5%) were not influenced by pre-incubation time. However, semen pre-incubation during 1.5h increased monospermic penetration (53.3%) and cleavage rates (92.5%) in low quality oocytes. Blastocyst rate was improved with 1.5h of semen pre incubation; however they were still lower than that observed with high quality control oocytes. Ultimately, pre-incubation did not influence fertilization, monospermic penetration, embryo development rates, nor cell density in oocytes of high quality. Low-quality porcine oocytes resulted in better rates of embryo development if in vitro fertilized with sperm pre-incubated for 1.5 hour.(AU)

A principal causa da baixa eficiência na PIV de embriões suínos é a elevada taxa de polispermia, que é exacerbada em oócitos de baixa qualidade. O experimento 1 avaliou o desenvolvimento embrionário de oócitos de baixa e alta qualidade. O experimento 2 avaliou a qualidade e o desenvolvimento embrionário de oócitos de baixa qualidade fecundados com sêmen pré-incubado por 0h (controle), 0,5h, 1h e 1,5h. O experimento 3 investigou a fecundação e as taxas de monospermia dos mesmos grupos do experimento 2. O experimento 4 avaliou o desenvolvimento embrionário, a densidade celular, a fecundação e as taxas de monospermia de oócitos de alta qualidade, fecundados com sêmen pre-incubado com o melhor tempo observado nos experimentos anteriores. As taxas de clivagem e de blastocistos foram submetidas ao teste de Qui-quadrado e os demais dados submetidos à ANOVA e teste de Tukey (P0,05). As taxas de clivagem (74,8 vs 51,7%) e de blastocistos (33,7 vs 9,8%) foram superiores nos oócitos de alta qualidade, comparados aos de baixa qualidade, não havendo diferenças na quantidade de células embrionárias. As taxas de fecundação (65,6 vs 79,5%) não foram influenciadas pelo tempo de pré-incubação. Todavia, a pré-incubação do sêmen por 1,5h aumentou a penetração monospérmica (53,3%) e a taxa de clivagem (92,5%), nos oócitos de baixa qualidade. As taxas de blastocisto aumentaram com sêmen pré-incubado por 1,5h, que foram ainda inferiores às obtidas dos oócitos de alta qualidade do grupo controle. Finalmente, a pré-incubação do sêmen não influencia na fecundação, na penetração monospérmica, no desenvolvimento embrionário, nem na quantidade de células embrionárias com oócitos de alta qualidade. Oócitos suínos de baixa qualidade produzem melhores taxas de desenvolvimento embrionário se fecundados in vitro com sêmen pré-incubado por 1,5 horas.(AU)

Criotolerância de oócitos e embriões bovinos maturados com líquido folicular e/ou -mercaptoetanol / Cryotolerance of bovine oocytes and embryos maturated with follicular fluid and/or -mercaptoethanol

Cryotolerance of bovine oocytes and embryos maturated with addition of follicular fluid (LF) and ?-mercaptoethanol (BM) was evaluated. After vitrification, oocytes were maturated in: TCM-199 (control); BM (24h TCM-199+100µM BM); LF (6h in LF+18h TCM-199), and LF+BM (6h LF+18h TCM-199+100µM BM). There was not difference (p>0.05) in blastocysts rate in TCM (6.4%), BM (4.0%) and LF (3.4%) treatments. The hatching rates and cell density of hatched embryos did not differ (p>0.05) among treatments. In Experiment 2, hatched blastocysts (Bx) obtained in D7 or D8 were vitrified and evaluated according to its expanding and hatching rates. The expanding rate was similar (p>0.05), being observed a distinct pathway in hatching rate in D7 and D8 Bx. Higher hatching rate was observed in D7 Bx from control (TCM-54.2%) compared to BM (40.32%) and LF+BM (33.89%) treatments. The D8 Bx showed lower hatching rate in control (TCM-199) compared with D7 Bx. In BM, LF and LF+BM treatments, the hatching rate was similar for D7 or D8 embryos. Maturation with addition of LF and/or BM does not increase the oocyte or IVP embryo cryotolerance. Expanded blastocysts (D7) have higher cryotolerance and show a distinct pathway when added with LF or BM, in comparison with D8 embryos.

Foi avaliada a criotolerância de oócitos e embriões bovinos maturados com adição de líquido folicular (LF) e/ou ?-mercaptoetanol (BM). Após vitrificação, os oócitos foram maturados em: TCM-199 (controle); BM (24h TCM-199+100µM BM); LF (6h em LF+18h TCM-199) e LF+BM (6h LF+18h TCM-199+100µM BM). Não houve diferença (p>0,05) nas taxas de blastocistos dos tratamentos TCM (6,4%), BM (4,0%) e LF (3,4%). A eclosão e densidade celular dos embriões eclodidos não diferiram (p>0,05) nos tratamentos. No Experimento 2 blastocistos expandidos (Bx) obtidos em D7 ou D8 foram vitrificados, avaliando-se sua reexpansão e eclosão. A reexpansão foi semelhante (p>0,05), sendo observado comportamento distinto na eclosão entre Bx D7 e D8. Nos Bx D7 houve maior eclosão no controle (TCM54,2%) em relação ao BM (40,32%) e LF+BM (33,89%). Os Bx D8 apresentaram menor eclosão no controle (TCM) em relação aos Bx D7. Nos tratamentos BM, LF e LF+BM a eclosão foi semelhante para Bx D7 ou D8. A maturação com adição de LF e/ou BM não melhora a criotolerância de oócitos imaturos e embriões PIV. Blastocistos expandidos precoces (D7) são mais criotolerantes e apresentam um comportamento distinto à adição de LF e BM, em relação aos tardios (D8).

Criotolerância de oócitos e embriões bovinos maturados com líquido folicular e/ou -mercaptoetanol / Cryotolerance of bovine oocytes and embryos maturated with follicular fluid and/or -mercaptoethanol

Cryotolerance of bovine oocytes and embryos maturated with addition of follicular fluid (LF) and ?-mercaptoethanol (BM) was evaluated. After vitrification, oocytes were maturated in: TCM-199 (control); BM (24h TCM-199+100µM BM); LF (6h in LF+18h TCM-199), and LF+BM (6h LF+18h TCM-199+100µM BM). There was not difference (p>0.05) in blastocysts rate in TCM (6.4%), BM (4.0%) and LF (3.4%) treatments. The hatching rates and cell density of hatched embryos did not differ (p>0.05) among treatments. In Experiment 2, hatched blastocysts (Bx) obtained in D7 or D8 were vitrified and evaluated according to its expanding and hatching rates. The expanding rate was similar (p>0.05), being observed a distinct pathway in hatching rate in D7 and D8 Bx. Higher hatching rate was observed in D7 Bx from control (TCM-54.2%) compared to BM (40.32%) and LF+BM (33.89%) treatments. The D8 Bx showed lower hatching rate in control (TCM-199) compared with D7 Bx. In BM, LF and LF+BM treatments, the hatching rate was similar for D7 or D8 embryos. Maturation with addition of LF and/or BM does not increase the oocyte or IVP embryo cryotolerance. Expanded blastocysts (D7) have higher cryotolerance and show a distinct pathway when added with LF or BM, in comparison with D8 embryos.(AU)

Foi avaliada a criotolerância de oócitos e embriões bovinos maturados com adição de líquido folicular (LF) e/ou ?-mercaptoetanol (BM). Após vitrificação, os oócitos foram maturados em: TCM-199 (controle); BM (24h TCM-199+100µM BM); LF (6h em LF+18h TCM-199) e LF+BM (6h LF+18h TCM-199+100µM BM). Não houve diferença (p>0,05) nas taxas de blastocistos dos tratamentos TCM (6,4%), BM (4,0%) e LF (3,4%). A eclosão e densidade celular dos embriões eclodidos não diferiram (p>0,05) nos tratamentos. No Experimento 2 blastocistos expandidos (Bx) obtidos em D7 ou D8 foram vitrificados, avaliando-se sua reexpansão e eclosão. A reexpansão foi semelhante (p>0,05), sendo observado comportamento distinto na eclosão entre Bx D7 e D8. Nos Bx D7 houve maior eclosão no controle (TCM54,2%) em relação ao BM (40,32%) e LF+BM (33,89%). Os Bx D8 apresentaram menor eclosão no controle (TCM) em relação aos Bx D7. Nos tratamentos BM, LF e LF+BM a eclosão foi semelhante para Bx D7 ou D8. A maturação com adição de LF e/ou BM não melhora a criotolerância de oócitos imaturos e embriões PIV. Blastocistos expandidos precoces (D7) são mais criotolerantes e apresentam um comportamento distinto à adição de LF e BM, em relação aos tardios (D8).(AU)

Effects of exposure to halothane, isoflurane, and sevoflurane on embryo viability and gestation in female mice / Efeitos da exposição ao halotano, isofluorano, e sevofluorano na viabilidade embrionária e gestação em fêmeas de camundongos

Although the negative effects of inhalation anaesthetics on fertility have been known for a while, the stages during the reproductive cycle at which these effects occur and the mechanisms of action are largely unknown. This study aimed to evaluate the effects of acute exposure of female mice to halothane, isoflurane, and sevoflurane prior to mating. BALB/c female mice (n=160) were allocated in groups of 20 to halothane (HG), isoflurane (IG), sevoflurane (SG), and oxygen/sham (SH) treatment groups and their respective control groups (CGs). The mice were exposed to 1 minimum alveolar concentration (MAC) of the corresponding anaesthetic or oxygen for 4 h/day over 5 consecutive days. Two days after exposure, females were mated with males (ratio 2:1 female/male) for five consecutive days. Every morning, females were checked for the presence of vaginal plugs. Half of the females that exhibited plugs were euthanised 72 h later for embryo evaluation. The remaining females were euthanised on the 14th day of pregnancy for foetal evaluation. A low number of morulae and total embryos (morulae + blastocysts) were observed in the HG (P 0.05). The number of implantations was lower in the HG (6.0) compared with the IG (11.8) and SG (12.4) (P 0.05). Exposure to halothane is not recommended for use in female mice prior to mating procedures because it leads to decreased embryo production...(AU)

Embora os efeitos negativos dos anestésicos inalatórios na fertilidade já foram descritos há algum tempo, os estágios durante o ciclo reprodutivo em que estes efeitos ocorrem bem como os mecanismos de ação ainda permanecem desconhecidos. Os objetivos deste estudo foram avaliar os efeitos da exposição aguda em fêmeas de camundongos ao halotano, isofluorano, e sevofluorano prévio ao acasalamento. Fêmeas de camundongos BALB/c (n=160) foram alocadas em grupos de 20 aos tratamentos halotano (HG), isofluorano (IG), sevofluorano (SG), oxigênio (SH), e seus respectivos grupos controle (CGs). As fêmeas de camundongos foram expostas a uma concentração alveolar mínima (CAM) do anestésico correspondente ou a oxigênio por 4 horas diárias durante 5 dias consecutivos. Após dois dias do fim da exposição às fêmeas foram acasaladas com os machos (proporção 2:1 fêmea/macho) durante 5 dias consecutivos. A cada manhã, as fêmeas foram avaliadas para a observação da presença de plug vaginal. Metade das fêmeas que exibiam plug foram submetidas à eutanásia após 72 horas para avaliação embrionária. As fêmeas restantes foram eutanasiadas no 14º dia de gestação para avaliação fetal. No HG foi observado um menor número de mórulas e embriões totais (mórulas + blastocistos) (P 0,05). O número de implantações foi menor no HG (6,0) comparado ao IG (11,8) e SG (12,4) (P 0,05). A exposição ao halotano não...(AU)

Effect of the cryopreservation method used, the embryonic stage and the use of conjugated linoleic acid isomers on the cryotolerance of in vitro-produced bovine embryos / Efeito do método de criopreservação utilizado, estágio de desenvolvimento embrionário e uso de isômeros do ácido linoleico conjugado na criotolerância de embriões bovinos produzidos in vitro

Conjugated linoleic acid (CLA) might be able to improve the cryotolerance of in vitro-produced (IVP) embryos. The effect of two CLA isomers on the cryotolerance of bovine IVP embryos, as well as that of the stage of embryonic development and the method used for cryopreservation was evaluated by three experiments. In Experiment 1, oocytes (n = 3,917) were fertilized in vitro and cultured with 0, 50, 100, or 200 μM trans-10, cis-12 (t10, c12 CLA). In Experiment 2, fertilized oocytes (n = 2,131) were cultured with 100 μM t10, c12 or cis-9, trans-11 (c9, t11 CLA), or a combination of both isomers. The embryos were vitrified at the blastocyst (BL) or the expanded blastocyst (EB) stage. In Experiment 3, oocytes (n = 1,720) were fertilized and cultured with or without 100 μM t10, c12 CLA, and the blastocysts were vitrified or frozen. Blastocyst development rate as well as the rates of re-expansion and hatching after thawing was recorded. Moreover, the mean cell number and mRNA expression of acetyl-CoA carboxylase (ACC1) and stearoyl-CoA desaturase (SCD1) as well as fatty acid synthase (FASN) multienzyme complex were determined. In Experiment 1, the highest concentration of t10, c12 CLA that did not reduce blastocyst development rate was 100 μM. In Experiment 2, the rates of re-expansion and hatching among the EBs obtained through IVP after supplementation with t10, c12 CLA (73.1% and 57.7%), with c9, t11 CLA (80.0% and 68.6%), with the combination (78.3% and 52.2%), and with the control group (85.4% and 58.3%) were similar. At the BL stage, the rates of re-expansion and hatching were lower than those at the EB stage, and CLA combination allowed a hatching rate (8.0%) lower than that observed in the control group (40.0%). In Experiment 3, the hatching rates for vitrified EBs (vitrified control; 67.4%) and vitrified CLA EBs (65.8%) were higher than those obtained for frozen EBs, exposed (13.3%) or not exposed (28.6%) to CLA.(AU)

Existem evidências de que o ácido linoleico conjugado (CLA) pode aumentar a criotolerância de embriões produzidos in vitro (PIV). O efeito de dois isômeros do CLA na criotolerância de embriões bovinos PIV, assim como o estágio de desenvolvimento e o efeito do método de criopreservação, foi avaliado através de três experimentos. No Experimento 1, oócitos (n = 3.917) foram fecundados in vitro e embriões foram cultivados com 0, 50, 100 ou 200 μM de trans-10, cis-12 (t10, c12 CLA). No Experimento 2, oócitos fecundados (n = 2.131) foram cultivados com 100 μM de t10, c12 ou cis-9, trans-11 (c9, t11 CLA), ou ainda com uma associação de ambos. Os embriões foram vitrificados nos estágios de blastocisto (BL) ou blastocisto expandido (BE). No Experimento 3, oócitos (n = 1.720) foram fecundados e cultivados com ou sem 100 μM de t10, c12 CLA e os blastocistos foram vitrificados ou congelados. As taxas de desenvolvimento dos blastocistos, bem como de re-expansão e eclosão após o reaquecimento foram observadas. Adicionalmente, o número médio de células e a expressão de mRNA das enzimas acetil-CoA carboxilase (ACC1) e estearoil-CoA dessaturase (SCD1) e do complexo enzimático ácido graxo sintase (FASN) foram avaliados. No Experimento 1, a maior concentração de t10, c12 CLA que não reduziu a taxa de blastocisto foi 100 μM. No Experimento 2, as taxas de re-expansão e eclosão obtidas entre EB obtidos por PIV após suplementação com t10, c12 CLA (73,1 e 57,7%), com c9, t11 CLA (80,0 e 68,6%), com a associação de ambos (78,3 e 52,2%) e com o grupo Controle (85,4 e 58,3%) foram similares. As taxas de re-expansão e eclosão foram mais baixas no estágio de BL do que no estágio de BE, e a associação dos isômeros apresentou taxa de eclosão (8,0%) mais baixa do que a do grupo controle (40,0%).(AU)

CRIOTOLERÂNCIA DE OÓCITOS E EMBRIÕES BOVINOS MATURADOS COM LÍQUIDO FOLICULAR E/OU -MERCAPTOETANOL

Cryotolerance of bovine oocytes and embryos maturated with addition of follicular fluid (LF) and -mercaptoethanol (BM) was evaluated. After vitrification, oocytes were maturated in: TCM-199 (control); BM (24h TCM-199+100µM BM); LF (6h in LF+18h TCM-199), and LF+BM (6h LF+18h TCM-199+100µM BM). There was not difference (p>0.05) in blastocysts rate in TCM (6.4%), BM (4.0%) and LF (3.4%) treatments. The hatching rates and cell density of hatched embryos did not differ (p>0.05) among treatments. In Experiment 2, hatched blastocysts (Bx) obtained in D7 or D8 were vitrified and evaluated according to its expanding and hatching rates. The expanding rate was similar (p>0.05), being observed a distinct pathway in hatching rate in D7 and D8 Bx. Higher hatching rate was observed in D7 Bx from control (TCM-54.2%) compared to BM (40.32%) and LF+BM (33.89%) treatments. The D8 Bx showed lower hatching rate in control (TCM-199) compared with D7 Bx. In BM, LF and LF+BM treatments, the hatching rate was similar for D7 or D8 embryos. Maturation with addition of LF and/or BM does not increase the oocyte or IVP embryo cryotolerance. Expanded blastocysts (D7) have higher cryotolerance and show a distinct pathway when added with LF or BM, in comparison with D8 embryos.

Foi avaliada a criotolerância de oócitos e embriões bovinos maturados com adição de líquido folicular (LF) e/ou -mercaptoetanol (BM). Após vitrificação, os oócitos foram maturados em: TCM-199 (controle); BM (24h TCM-199+100µM BM); LF (6h em LF+18h TCM-199) e LF+BM (6h LF+18h TCM-199+100µM BM). Não houve diferença (p>0,05) nas taxas de blastocistos dos tratamentos TCM (6,4%), BM (4,0%) e LF (3,4%). A eclosão e densidade celular dos embriões eclodidos não diferiram (p>0,05) nos tratamentos. No Experimento 2 blastocistos expandidos (Bx) obtidos em D7 ou D8 foram vitrificados, avaliando-se sua reexpansão e eclosão. A reexpansão foi semelhante (p>0,05), sendo observado comportamento distinto na eclosão entre Bx D7 e D8. Nos Bx D7 houve maior eclosão no controle (TCM54,2%) em relação ao BM (40,32%) e LF+BM (33,89%). Os Bx D8 apresentaram menor eclosão no controle (TCM) em relação aos Bx D7. Nos tratamentos BM, LF e LF+BM a eclosão foi semelhante para Bx D7 ou D8. A maturação com adição de LF e/ou BM não melhora a criotolerância de oócitos imaturos e embriões PIV. Blastocistos expandidos precoces (D7) são mais criotolerantes e apresentam um comportamento distinto à adição de LF e BM, em relação aos tardios (D8).

Effect of the cryopreservation method used, the embryonic stage and the use of conjugated linoleic acid isomers on the cryotolerance of in vitro-produced bovine embryos / Efeito do método de criopreservação utilizado, estágio de desenvolvimento embrionário e uso de isômeros do ácido linoleico conjugado na criotolerância de embriões bovinos produzidos in vitro

Conjugated linoleic acid (CLA) might be able to improve the cryotolerance of in vitro-produced (IVP) embryos. The effect of two CLA isomers on the cryotolerance of bovine IVP embryos, as well as that of the stage of embryonic development and the method used for cryopreservation was evaluated by three experiments. In Experiment 1, oocytes (n = 3,917) were fertilized in vitro and cultured with 0, 50, 100, or 200 μM trans-10, cis-12 (t10, c12 CLA). In Experiment 2, fertilized oocytes (n = 2,131) were cultured with 100 μM t10, c12 or cis-9, trans-11 (c9, t11 CLA), or a combination of both isomers. The embryos were vitrified at the blastocyst (BL) or the expanded blastocyst (EB) stage. In Experiment 3, oocytes (n = 1,720) were fertilized and cultured with or without 100 μM t10, c12 CLA, and the blastocysts were vitrified or frozen. Blastocyst development rate as well as the rates of re-expansion and hatching after thawing was recorded. Moreover, the mean cell number and mRNA expression of acetyl-CoA carboxylase (ACC1) and stearoyl-CoA desaturase (SCD1) as well as fatty acid synthase (FASN) multienzyme complex were determined. In Experiment 1, the highest concentration of t10, c12 CLA that did not reduce blastocyst development rate was 100 μM. In Experiment 2, the rates of re-expansion and hatching among the EBs obtained through IVP after supplementation with t10, c12 CLA (73.1% and 57.7%), with c9, t11 CLA (80.0% and 68.6%), with the combination (78.3% and 52.2%), and with the control group (85.4% and 58.3%) were similar. At the BL stage, the rates of re-expansion and hatching were lower than those at the EB stage, and CLA combination allowed a hatching rate (8.0%) lower than that observed in the control group (40.0%). In Experiment 3, the hatching rates for vitrified EBs (vitrified control; 67.4%) and vitrified CLA EBs (65.8%) were higher than those obtained for frozen EBs, exposed (13.3%) or not exposed (28.6%) to CLA.

Existem evidências de que o ácido linoleico conjugado (CLA) pode aumentar a criotolerância de embriões produzidos in vitro (PIV). O efeito de dois isômeros do CLA na criotolerância de embriões bovinos PIV, assim como o estágio de desenvolvimento e o efeito do método de criopreservação, foi avaliado através de três experimentos. No Experimento 1, oócitos (n = 3.917) foram fecundados in vitro e embriões foram cultivados com 0, 50, 100 ou 200 μM de trans-10, cis-12 (t10, c12 CLA). No Experimento 2, oócitos fecundados (n = 2.131) foram cultivados com 100 μM de t10, c12 ou cis-9, trans-11 (c9, t11 CLA), ou ainda com uma associação de ambos. Os embriões foram vitrificados nos estágios de blastocisto (BL) ou blastocisto expandido (BE). No Experimento 3, oócitos (n = 1.720) foram fecundados e cultivados com ou sem 100 μM de t10, c12 CLA e os blastocistos foram vitrificados ou congelados. As taxas de desenvolvimento dos blastocistos, bem como de re-expansão e eclosão após o reaquecimento foram observadas. Adicionalmente, o número médio de células e a expressão de mRNA das enzimas acetil-CoA carboxilase (ACC1) e estearoil-CoA dessaturase (SCD1) e do complexo enzimático ácido graxo sintase (FASN) foram avaliados. No Experimento 1, a maior concentração de t10, c12 CLA que não reduziu a taxa de blastocisto foi 100 μM. No Experimento 2, as taxas de re-expansão e eclosão obtidas entre EB obtidos por PIV após suplementação com t10, c12 CLA (73,1 e 57,7%), com c9, t11 CLA (80,0 e 68,6%), com a associação de ambos (78,3 e 52,2%) e com o grupo Controle (85,4 e 58,3%) foram similares. As taxas de re-expansão e eclosão foram mais baixas no estágio de BL do que no estágio de BE, e a associação dos isômeros apresentou taxa de eclosão (8,0%) mais baixa do que a do grupo controle (40,0%).

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)

Effects of exposure to halothane, isoflurane, and sevoflurane on embryo viability and gestation in female mice / Efeitos da exposição ao halotano, isofluorano, e sevofluorano na viabilidade embrionária e gestação em fêmeas de camundongos

Although the negative effects of inhalation anaesthetics on fertility have been known for a while, the stages during the reproductive cycle at which these effects occur and the mechanisms of action are largely unknown. This study aimed to evaluate the effects of acute exposure of female mice to halothane, isoflurane, and sevoflurane prior to mating. BALB/c female mice (n=160) were allocated in groups of 20 to halothane (HG), isoflurane (IG), sevoflurane (SG), and oxygen/sham (SH) treatment groups and their respective control groups (CGs). The mice were exposed to 1 minimum alveolar concentration (MAC) of the corresponding anaesthetic or oxygen for 4 h/day over 5 consecutive days. Two days after exposure, females were mated with males (ratio 2:1 female/male) for five consecutive days. Every morning, females were checked for the presence of vaginal plugs. Half of the females that exhibited plugs were euthanised 72 h later for embryo evaluation. The remaining females were euthanised on the 14th day of pregnancy for foetal evaluation. A low number of morulae and total embryos (morulae + blastocysts) were observed in the HG (P 0.05). The number of implantations was lower in the HG (6.0) compared with the IG (11.8) and SG (12.4) (P 0.05). Exposure to halothane is not recommended for use in female mice prior to mating procedures because it leads to decreased embryo production...

Embora os efeitos negativos dos anestésicos inalatórios na fertilidade já foram descritos há algum tempo, os estágios durante o ciclo reprodutivo em que estes efeitos ocorrem bem como os mecanismos de ação ainda permanecem desconhecidos. Os objetivos deste estudo foram avaliar os efeitos da exposição aguda em fêmeas de camundongos ao halotano, isofluorano, e sevofluorano prévio ao acasalamento. Fêmeas de camundongos BALB/c (n=160) foram alocadas em grupos de 20 aos tratamentos halotano (HG), isofluorano (IG), sevofluorano (SG), oxigênio (SH), e seus respectivos grupos controle (CGs). As fêmeas de camundongos foram expostas a uma concentração alveolar mínima (CAM) do anestésico correspondente ou a oxigênio por 4 horas diárias durante 5 dias consecutivos. Após dois dias do fim da exposição às fêmeas foram acasaladas com os machos (proporção 2:1 fêmea/macho) durante 5 dias consecutivos. A cada manhã, as fêmeas foram avaliadas para a observação da presença de plug vaginal. Metade das fêmeas que exibiam plug foram submetidas à eutanásia após 72 horas para avaliação embrionária. As fêmeas restantes foram eutanasiadas no 14º dia de gestação para avaliação fetal. No HG foi observado um menor número de mórulas e embriões totais (mórulas + blastocistos) (P 0,05). O número de implantações foi menor no HG (6,0) comparado ao IG (11,8) e SG (12,4) (P 0,05). A exposição ao halotano não...