查看更多>>摘要：In high-latitude regions, the cold hardiness of buds and canes of grapevine is important for budburst time and yield in the next season. The freezing resistance of buds and canes sampled from six wine grapes currently cultivated in Hokkaido, Japan, all of them grown from autumn to winter, was investigated. A significant difference between the cultivars in their freezing resistance was detected in the buds harvested in winter. In addition, outstanding differences in the lower temperature exotherms (LTE) related to the supercooling ability of tissue cells happened in the winter buds, and there is a close relationship between freezing resistance and LTE detected in the winter buds. This suggests that the supercooling ability of tissue cells in winter buds is strongly related to the freezing resistance. However, detailed electron microscopy exposed that the differences in freezing resistance among cultivars appeared in freezing behavior of leaf primordium rather than apical meristem. This indicated that as the water mobility from the bud apical meristem to the spaces around the cane phloem progressed, the slightly dehydrated cells improved the supercooling ability and increased the freezing resistance.
查看更多>>摘要：In high-latitude regions, the cold hardiness of buds and canes of grapevine is important for budburst time and yield in the next season. The freezing resistance of buds and canes sampled from six wine grapes currently cultivated in Hokkaido, Japan, all of them grown from autumn to winter, was investigated. A significant difference between the cultivars in their freezing resistance was detected in the buds harvested in winter. In addition, outstanding differences in the lower temperature exotherms (LTE) related to the supercooling ability of tissue cells happened in the winter buds, and there is a close relationship between freezing resistance and LTE detected in the winter buds. This suggests that the supercooling ability of tissue cells in winter buds is strongly related to the freezing resistance. However, detailed electron microscopy exposed that the differences in freezing resistance among cultivars appeared in freezing behavior of leaf primordium rather than apical meristem. This indicated that as the water mobility from the bud apical meristem to the spaces around the cane phloem progressed, the slightly dehydrated cells improved the supercooling ability and increased the freezing resistance.
查看更多>>摘要：Somatic resource banks play a crucial role in the conservation of genetic diversity, allowing for the preservation of biological samples from different populations. Puma somatic cells can be recovered from these banks and used in assisted techniques toward enhancing their multiplication and conservation. In response to the population reduction of this ecologically importance species, we aimed to evaluate the capacity of cryopreservation of somatic tissues on the maintenance of the integrity and quality of the cells recovered after culture, with the aim of establishing a somatic tissue bank that will allow for the safeguarding of a wide genetic sampling of pumas. Cryopreservation increased the thickness of the corneum layer in the tissues, and the number of perinuclear halos and empty gaps. Nevertheless, cryopreservation was able to maintain normal fibroblast patterns, even showing an increase in the percentage of collagen fibers. Cryopreservation maintained the proliferative potential of the tissues and the parameters evaluated during in vitro culture, mainly regarding the viability, proliferative activity, and apoptosis levels. Nevertheless, cells from cryopreserved tissues showed decreased metabolism and mitochondrial membrane potential when compared to cells from non-cryopreserved tissues. In summary, we demonstrated for the first time that puma somatic tissues subjected to cryopreservation are viable and maintain tissue integrity, featuring minimal changes after warming. Although viable somatic cells are obtained from these tissues, they undergo alterations in their metabolism and mitochondrial membrane potential. Improvements in the conservation conditions of somatic samples are needed to increase the quality of somatic tissue banks in this species.
de Oliveira Lira, Gabriela PereiraBorges, Alana Azevedodo Nascimento, Matheus BarbosaCosta de Aquino, Leonardo Vitorino...
9页
查看更多>>摘要：Somatic resource banks play a crucial role in the conservation of genetic diversity, allowing for the preservation of biological samples from different populations. Puma somatic cells can be recovered from these banks and used in assisted techniques toward enhancing their multiplication and conservation. In response to the population reduction of this ecologically importance species, we aimed to evaluate the capacity of cryopreservation of somatic tissues on the maintenance of the integrity and quality of the cells recovered after culture, with the aim of establishing a somatic tissue bank that will allow for the safeguarding of a wide genetic sampling of pumas. Cryopreservation increased the thickness of the corneum layer in the tissues, and the number of perinuclear halos and empty gaps. Nevertheless, cryopreservation was able to maintain normal fibroblast patterns, even showing an increase in the percentage of collagen fibers. Cryopreservation maintained the proliferative potential of the tissues and the parameters evaluated during in vitro culture, mainly regarding the viability, proliferative activity, and apoptosis levels. Nevertheless, cells from cryopreserved tissues showed decreased metabolism and mitochondrial membrane potential when compared to cells from non-cryopreserved tissues. In summary, we demonstrated for the first time that puma somatic tissues subjected to cryopreservation are viable and maintain tissue integrity, featuring minimal changes after warming. Although viable somatic cells are obtained from these tissues, they undergo alterations in their metabolism and mitochondrial membrane potential. Improvements in the conservation conditions of somatic samples are needed to increase the quality of somatic tissue banks in this species.
查看更多>>摘要：Wider implementation of AI in sheep in the field condition has not been possible till date due to very poor conception rate after cervical insemination with cryopreserved semen. Poor cervical penetrability in ewe and diminished sperm functions in cryopreserved semen are considered responsible for it. In the present study, effect of carboxymethyl cellulose (CMC) on post-thaw qualities of ram semen was investigated. Ejaculates from eight adult Malpura rams were pooled and diluted (800 x 106 sperm mL-1) with TES-Tris-fructose-egg yolk extender having either 5 or 6% glycerol and supplemented with 0, 0.25, 0.5, 0.75 and 1.0% (w/v) CMC and packaged into 0.25 mL French mini straws. The straws were progressively cooled to 5 degrees C inside a cold cabinet (5 degrees C) and then equilibrated for 22 h inside a refrigerator (2-5 degrees C). Straws were frozen at -25 degrees C min-1 up to -125 degrees C using a programmable cell freezer (Planer Biomed R-204, UK) and finally plunged into liquid nitrogen. The post-thaw progressive motility was higher (P < 0.05) in 0.75% CMC-treated group compared to control. Overall, both pre-freeze and post-thaw sperm kinetics was comparable between CMC-treated and control groups. The postthaw sperm viability, acrosomal integrity and sperm with high mitochondrial membrane potential (hMMP) were relatively higher while sperm with high membrane cholesterol was significantly (P < 0.05) higher in presence of 0.25% CMC compared to the control. Both sperm having hMMP and non-capacitated sperm were significantly (P < 0.05) higher in presence of 5% glycerol than 6% glycerol. Similarly, functional membrane integrity (FMI) was higher in presence of 5% glycerol than 6% glycerol when CMC was added at 0.5% to extender. In conclusion, both 0.25% CMC and 5% glycerol resulted in improvement in several post-thaw sperm functions in cryopreserved ram semen. Thus CMC demonstrated cryoprotective effect on ram sperm in a synergistic manner with glycerol.
查看更多>>摘要：Wider implementation of AI in sheep in the field condition has not been possible till date due to very poor conception rate after cervical insemination with cryopreserved semen. Poor cervical penetrability in ewe and diminished sperm functions in cryopreserved semen are considered responsible for it. In the present study, effect of carboxymethyl cellulose (CMC) on post-thaw qualities of ram semen was investigated. Ejaculates from eight adult Malpura rams were pooled and diluted (800 x 106 sperm mL-1) with TES-Tris-fructose-egg yolk extender having either 5 or 6% glycerol and supplemented with 0, 0.25, 0.5, 0.75 and 1.0% (w/v) CMC and packaged into 0.25 mL French mini straws. The straws were progressively cooled to 5 degrees C inside a cold cabinet (5 degrees C) and then equilibrated for 22 h inside a refrigerator (2-5 degrees C). Straws were frozen at -25 degrees C min-1 up to -125 degrees C using a programmable cell freezer (Planer Biomed R-204, UK) and finally plunged into liquid nitrogen. The post-thaw progressive motility was higher (P < 0.05) in 0.75% CMC-treated group compared to control. Overall, both pre-freeze and post-thaw sperm kinetics was comparable between CMC-treated and control groups. The postthaw sperm viability, acrosomal integrity and sperm with high mitochondrial membrane potential (hMMP) were relatively higher while sperm with high membrane cholesterol was significantly (P < 0.05) higher in presence of 0.25% CMC compared to the control. Both sperm having hMMP and non-capacitated sperm were significantly (P < 0.05) higher in presence of 5% glycerol than 6% glycerol. Similarly, functional membrane integrity (FMI) was higher in presence of 5% glycerol than 6% glycerol when CMC was added at 0.5% to extender. In conclusion, both 0.25% CMC and 5% glycerol resulted in improvement in several post-thaw sperm functions in cryopreserved ram semen. Thus CMC demonstrated cryoprotective effect on ram sperm in a synergistic manner with glycerol.
查看更多>>摘要：Stallion sperm is typically cryopreserved using low cooling rates and low concentrations of cryoprotective agents (CPAs). The inevitable water-to-ice phase transition during cryopreservation is damaging and can be prevented using vitrification. Vitrification requires high cooling rates and high CPA concentrations. In this study, the feasibility of stallion sperm vitrification was investigated. A dual-syringe pump system was used to mix sperm equilibrated in a solution with a low concentration of CPAs, with a solution containing a high CPA concentration, and to generate droplets of a defined size (i.e., -20 mu L) that were subsequently cooled by depositing on an aluminum alloy block placed in liquid nitrogen. Mathematical modeling was performed to compute the heat transfer and rate of cooling. The minimum CPA concentration needed for vitrification was determined for various CPAs (glycerol, ethylene glycol, propylene glycol, dimethyl sulfoxide) and combinations thereof, while effects of droplet size and carrier solution were also identified. Sperm vitrification was eventually done using a glycerol/ propylene glycol (1/1) mixture at a final concentration of 45% in buffered saline supplemented with 3% albumin and polyvinylpyrrolidon, while warming was done in standard diluent supplemented with 100 mM sucrose. The sperm concentration was found to greatly affect sperm membrane integrity after vitrification-and-warming, i.e., was found to be 21 +/- 12% for 10 x 106 sperm mL-1 and 54 +/- 8% for 1 x 106 sperm mL-1. However, an almost complete loss of sperm motility was observed. In conclusion, successful sperm vitrification requires establishing the narrow balance between droplet size, sperm concentration, CPA type and concentration, and exposure time.
Hegermann, JanWolkers, Willem F.Sieme, HaraldOldenhof, Harriette...
11页
查看更多>>摘要：Stallion sperm is typically cryopreserved using low cooling rates and low concentrations of cryoprotective agents (CPAs). The inevitable water-to-ice phase transition during cryopreservation is damaging and can be prevented using vitrification. Vitrification requires high cooling rates and high CPA concentrations. In this study, the feasibility of stallion sperm vitrification was investigated. A dual-syringe pump system was used to mix sperm equilibrated in a solution with a low concentration of CPAs, with a solution containing a high CPA concentration, and to generate droplets of a defined size (i.e., -20 mu L) that were subsequently cooled by depositing on an aluminum alloy block placed in liquid nitrogen. Mathematical modeling was performed to compute the heat transfer and rate of cooling. The minimum CPA concentration needed for vitrification was determined for various CPAs (glycerol, ethylene glycol, propylene glycol, dimethyl sulfoxide) and combinations thereof, while effects of droplet size and carrier solution were also identified. Sperm vitrification was eventually done using a glycerol/ propylene glycol (1/1) mixture at a final concentration of 45% in buffered saline supplemented with 3% albumin and polyvinylpyrrolidon, while warming was done in standard diluent supplemented with 100 mM sucrose. The sperm concentration was found to greatly affect sperm membrane integrity after vitrification-and-warming, i.e., was found to be 21 +/- 12% for 10 x 106 sperm mL-1 and 54 +/- 8% for 1 x 106 sperm mL-1. However, an almost complete loss of sperm motility was observed. In conclusion, successful sperm vitrification requires establishing the narrow balance between droplet size, sperm concentration, CPA type and concentration, and exposure time.
Macoretta, Christian LeandroMiranda, Leandro Andres
9页
查看更多>>摘要：The Siamese fighting fish (Betta splendens) has great importance as an ornamental aquarium fish as well as laboratory model species. Due to its rapid development, a cooling-embryo protocol could provide some advantages in their transportation, embryonic synchronization, and optimization of hatcheries. In this context, this work aimed to develop a protocol to storage B. splendens embryos at two temperatures (5 and 14 degrees C), testing three cryoprotective solutions (S1: 0.5 M sucrose, 1.5 M methanol; S2: 0.25 M sucrose, 0.75 M methanol; and S3: 0.125 M sucrose, 0.375 M methanol) and evaluating the quality of the larvae obtained. Moreover, a method to isolate the embryos from the bubble nest constructed by the male and to incubate them without parental care was applied in this study. The cooling assays were done using embryos of 24-h-post-fertilization at 26 degrees C and the results demonstrated that it is possible to store these embryos deprived of cryoprotectants at 5 degrees C for at least 6-h without negative effects. Meanwhile, S2 and S3 were the most suitable solutions for its storage for 9-h at 5 degrees C or 24-h at 14 degrees C, obtaining 77% hatching and 52% normal larvae in the first case or 88% hatching and 81% larvae with mild abnormalities in the second one. Indeed, type and frequency of larval abnormalities were evaluated and, remarkably, a partial recovery was described on malformed larvae from embryo cooled at 14 degrees C. Finally, this work is the first report about the cooling of B. splendens embryos and establishes the conditions for further studies on this field with this species.
Macoretta, Christian LeandroMiranda, Leandro Andres
9页
查看更多>>摘要：The Siamese fighting fish (Betta splendens) has great importance as an ornamental aquarium fish as well as laboratory model species. Due to its rapid development, a cooling-embryo protocol could provide some advantages in their transportation, embryonic synchronization, and optimization of hatcheries. In this context, this work aimed to develop a protocol to storage B. splendens embryos at two temperatures (5 and 14 degrees C), testing three cryoprotective solutions (S1: 0.5 M sucrose, 1.5 M methanol; S2: 0.25 M sucrose, 0.75 M methanol; and S3: 0.125 M sucrose, 0.375 M methanol) and evaluating the quality of the larvae obtained. Moreover, a method to isolate the embryos from the bubble nest constructed by the male and to incubate them without parental care was applied in this study. The cooling assays were done using embryos of 24-h-post-fertilization at 26 degrees C and the results demonstrated that it is possible to store these embryos deprived of cryoprotectants at 5 degrees C for at least 6-h without negative effects. Meanwhile, S2 and S3 were the most suitable solutions for its storage for 9-h at 5 degrees C or 24-h at 14 degrees C, obtaining 77% hatching and 52% normal larvae in the first case or 88% hatching and 81% larvae with mild abnormalities in the second one. Indeed, type and frequency of larval abnormalities were evaluated and, remarkably, a partial recovery was described on malformed larvae from embryo cooled at 14 degrees C. Finally, this work is the first report about the cooling of B. splendens embryos and establishes the conditions for further studies on this field with this species.
NSTL
Elsevier