- Email: [email protected]
chelator in bovine sperm desiccation
322
Abstracts / Cryobiology 57 (2008) 315–340
extracted from hen egg yolk. (Conflicts of interest: None declared. Source of funding: None declared.)
36. Study of the effect of intracellular sugars and antioxidant/chelator in bovine sperm desiccation. Ranjan Sitaula, Sankha Bhowmick, University of Masachusetts Dartmouth, North Dartmouth, MA, USA
doi:10.1016/j.cryobiol.2008.10.034
34. Development of a new plant-based extender for the cryopreservation of bovine semen. Mathieu Boilard, Chantal Brisson, Patrick Blondin, L’Alliance Boviteq Inc., Saint-Hyacinthe, QC, Canada Avian flu and bovine spongiform encephalopathy (mad cow disease) have increased worldwide awareness regarding the bio-security of exported bovine semen. The bovine artificial insemination industry generally uses animal proteins, like egg yolk (EY) or milk, as major components of cryoprotectant solutions (extender) used to freeze bovine semen. In order to avoid potential biosecurity problems, L’Alliance Boviteq (R&D division of The Semex Alliance) recently developed a proprietary animal protein-free extender based on plant lipids. Firstly, semen from four bulls was recovered, diluted in either EY or plant based extenders (PBE) and frozen following industrial procedures used in most AI centers. Semen was thawed and washed using a Tyrode modified media (sp-TALP). Sperm were analyzed using a computer-assisted semen analyzer (CASA) immediately (0 h) and after a 5 h incubation period in sp-TALP. Motility was higher in semen samples that had been cryopreserved with the PBE when compared to the EY extender (70.5 vs 45.8 p < 0.05 at 0 h, 39.5 vs 10.3 p < 0.02 at 5 h). Secondly, semen was used for in vitro fertilization. Semen from three bulls was frozen in the two different extenders. Equivalent fertilization rates and blastocyst rates were obtained with the PBE compared to the EY extender (82.6 vs 85.3 p = 0,5 and 22.2 vs 19.6 p = 0.4). Finally, semen from four different bulls was distributed commercially for artificial insemination upon freezing in PBE or EY extender. Cow pregnancy was evaluated by ultrasound 30 days post insemination. No significant difference was observed between the PBE and the EY extender (37.1% vs 46.0% p > 0.12). In conclusion, the new PBE developed at L’Alliance Boviteq is a good alternative to extenders containing animal proteins and eliminates any biosecurity concerns for semen exportation. We acknowledge the help of Dr. Mary Buhr (University of Guelph), Dr. Reuben Mapletoft (University of Saskatchewan). (Conflicts of interest: None declared. Source of funding: Bioniche Animal Health, the Ontario CanAdapt program, SEMEX Alliance.) doi:10.1016/j.cryobiol.2008.10.035
35. Cellular biophysics during freezing of rat and mouse sperm. Ramachandra Devireddy a, Jeung H. Choi b, Mie Hagiwara b, Wim Wolkers b, Ken P. Roberts b, John C. Bischof b, a Louisiana State University, Baton Rouge, LA, USA, b University of Minnesota, Minneapolis, MN, USA While cryopreservation of mouse sperm yields good survival and motility after thawing, rat sperm cryopreservation remains a challenge. This study measures the cellular biophysics during freezing of both sperm types to suggest improvements in rat cryopreservation approaches. A Differential Scanning Calorimeter(DSC) was used to study the state of water in the cellular and extracellular space and extract cellular water transport information during freezing. Data in eluted mouse and rat (eluted and epididymal) sperm in PBS and mBWW respectively was obtained at cooling rates of 5 and 20 °C/min and a model was fit to the data to obtain a set of combined best fit water-transport parameters (Lpg and ELp). Mouse sperm was modeled as a cylinder of length 122 lm and radius of 0.46 lm with an osmotically inactive cell volume of 0.61 Vo(Vo: isotonic cell volume). Rat sperm was modeled as a cylinder of length 188.7 lm and radius of 0.71 lm, with an osmotically inactive cell volume of 0.61 Vo. Optimal rates for mouse sperm yielded Lpg = 1.53 10 15m3/Ns (0.009 lm/ min-atm) and ELp = 106.7 kJ/mole (25.5 kcal/mole). This is similar to earlier published results in PBS+egg yolk for mouse sperm. Corresponding parameters for eluted rat sperm were Lpg = 1.7 10 15m3/Ns (0.01 lm/min-atm) and ELp = 77.8 kJ/mole (18.6 kcal/mole). As an interesting and potentially important comparison to eluted sperm additional DSC experiments were also conducted on rat epididymal tissue sperm (2 mg) in mBWW media (8 lL). The tissue(presumed mostly sperm) behaved somewhat differently than eluted sperm as shown in the permeability parameters obtained: Lpg = 2.9 10 15m3/Ns (0.017 lm/min-atm) and ELp = 74.1 kJ/mole (17.7 kcal/ mole). Parameters obtained in this study were finally used to predict optimal cooling rates of 23, 51 and 73 °C/min for eluted mouse, eluted rat and epididymal rat sperm, respectively. Post freeze-thaw motility experiments for mouse and rat sperm show maximum motility at similar cooling rates to those predicted from permeability measurements (data not shown). (Conflicts of interest: None declared. Source of funding: NIH R21 RR021698 and the Institute for Engineering in Medicine, University of Minnesota.) doi:10.1016/j.cryobiol.2008.10.036
The objective of the study was to investigate the protective effect of intracellular and extracellular disaccharides during bovine sperm desiccation, and the supplemental effect of the addition of an antioxidant (catalase) and a chelator (desferal, an iron chelator). The study was motivated by a goal to achieve partial to complete desiccation preservation of mammalian sperm, which holds promises as a simplified alternative to the cryopreservation technique. The addition of antioxidant and chelator was based on the hypothesis that an optimal amount of the additives helps reduce the oxidative stresses, thus minimizing membrane and mitochondrial damage. Sperm loaded with two different sugars, trehalose and sucrose, were dried with and without catalase/ desferal and evaluated for motility and membrane integrity immediately upon rehydration. Intracellular concentrations of the sugars were loaded by the method of ATP poration. Drying was performed through slow convective drying which was achieved by placing the sperm in desiccator boxes maintained at 11% RH. Motility was evaluated under a bright field microscope and membrane integrity was assayed in a fluorescent microscope using the dyes, Hoechst and Ethidium Homodimer. Results suggested an improved desiccation tolerance of sperm in the presence of intracellular trehalose or sucrose. Survival was further enhanced by the addition of 1 mM desferal in the desiccation buffer. Though the motility at lower dry basis water fractions (DBWF) did not show a significant improvement, there was a marked increase in the sperm membrane integrity at lower water contents (DBWF < 2) which correspond to a partially desiccated state. Moreover, the critical water content at which membrane integrity was completely lost was lowered to a DBWF value of around 0.2 for sperm with intracellular sugar and desferal as compared to around 0.6 for sperm with extracellular sugars only. (Conflicts of interest: None declared. Source of funding: None declared.) doi:10.1016/j.cryobiol.2008.10.037
37. Effects of raffinose concentration and methyl-beta-cyclodextrin on IVF with cryopreserved C57BL/6 sperm. Keiji Mochida a, Chihiro Koshimoto b, Keisuke Edashige c, Kentaro Aizawa d, Kyuichi Taguma a, Akihiko Ohta d, Atsuo Ogura a,e, a RIKEN Bioresource center, b University of Miyazaki, c Kochi University, d Meiji University, e Tsukuba University Spermatozoa from certain strains of mice, including C57BL/6, are very sensitive to freezing and thawing, and frequently fail to fertilize oocytes by conventional in vitro fertilization. We examined whether this problem could be overcome by optimizing the raffinose concentration and osmolality of freezing solutions. First, we prepared raffinose solutions at concentrations of 9, 12, 15, 18 and 21% (w/v). C57Bl/6Cr Slc epididymal spermatozoa from males aged 3–6 months were suspended in either of the freezing solutions. After equilibration for 1 min, sperm suspension was enclosed inside a 0.25 mL plastic straw and cooled in the vapor of liquid nitrogen (LN2) for 10 min. Then the straw was immersed in LN2 and stored for more than 5 days. For thawing, the straw was immersed in a water bath at 37 °C for 15 min. The thawed sperm suspension was added to HTF medium and incubated for 1 h at 37 °C. The motility and the progressive movement were assessed using a Hamilton Thorn IVOS computerized semen analyzer. Normalized motilities were 21%, 32%, 36%, 41%, and 47%, respectively, in raffinose solutions described above. Next, 0/4–4/4 X strength SD-PBS(supplemented Dulbecco’s phosphate-buffered saline; Koshimoto et al., 2000) contained raffinose at a concentration of 15%, 18%, or 21% were used for sperm freezing. When the thawed sperm were assessed for their motilities, practicable motilities were obtained with medium including 18% or 21% raffinose and 400– 525 mOsm/kg. The highest motility (56%) was obtained with 18% raffinose in 1/4 SD-PBS. The frozen sperm in this medium were preincubated in HTF supplemented with BSA and/or methyl-beta-cyclodextrin (after Choi et al.) for 1 h and inseminated into oocyte-containing HTF medium. The fertilization rates were 45% and 70%, respectively. These results indicated that C57BL/6 sperm cryopreservation could be improved by optimizing raffinose concentration and osmolality of the freezing solution. Furthermore, the supplementation of methyl-beta-cyclodextrin to preincubation medium was effective for increase of in vitro fertilization rate. (Conflicts of interest: None declared. Source of funding: None declared.) doi:10.1016/j.cryobiol.2008.10.038
Contributed papers 5. Cryobiology of oocytes
38. The pathway for the movement of water and cryoprotectants in bovine oocytes and embryos. Keisuke Edashige, Takao Hara, Yasunori Kawai, Masahiro Yoshimura, Bo Jin, Delgado Valdez Jr., Magosaburo Kasai, Kochi University, Nankoku, Kochi, Japan We have shown that the permeability of mouse oocytes and embryos at early cleavage stages to water and cryoprotectants is low, whereas that of morulae and
Abstracts / Cryobiology 57 (2008) 315–340
extracted from hen egg yolk. (Conflicts of interest: None declared. Source of funding: None declared.)
36. Study of the effect of intracellular sugars and antioxidant/chelator in bovine sperm desiccation. Ranjan Sitaula, Sankha Bhowmick, University of Masachusetts Dartmouth, North Dartmouth, MA, USA
doi:10.1016/j.cryobiol.2008.10.034
34. Development of a new plant-based extender for the cryopreservation of bovine semen. Mathieu Boilard, Chantal Brisson, Patrick Blondin, L’Alliance Boviteq Inc., Saint-Hyacinthe, QC, Canada Avian flu and bovine spongiform encephalopathy (mad cow disease) have increased worldwide awareness regarding the bio-security of exported bovine semen. The bovine artificial insemination industry generally uses animal proteins, like egg yolk (EY) or milk, as major components of cryoprotectant solutions (extender) used to freeze bovine semen. In order to avoid potential biosecurity problems, L’Alliance Boviteq (R&D division of The Semex Alliance) recently developed a proprietary animal protein-free extender based on plant lipids. Firstly, semen from four bulls was recovered, diluted in either EY or plant based extenders (PBE) and frozen following industrial procedures used in most AI centers. Semen was thawed and washed using a Tyrode modified media (sp-TALP). Sperm were analyzed using a computer-assisted semen analyzer (CASA) immediately (0 h) and after a 5 h incubation period in sp-TALP. Motility was higher in semen samples that had been cryopreserved with the PBE when compared to the EY extender (70.5 vs 45.8 p < 0.05 at 0 h, 39.5 vs 10.3 p < 0.02 at 5 h). Secondly, semen was used for in vitro fertilization. Semen from three bulls was frozen in the two different extenders. Equivalent fertilization rates and blastocyst rates were obtained with the PBE compared to the EY extender (82.6 vs 85.3 p = 0,5 and 22.2 vs 19.6 p = 0.4). Finally, semen from four different bulls was distributed commercially for artificial insemination upon freezing in PBE or EY extender. Cow pregnancy was evaluated by ultrasound 30 days post insemination. No significant difference was observed between the PBE and the EY extender (37.1% vs 46.0% p > 0.12). In conclusion, the new PBE developed at L’Alliance Boviteq is a good alternative to extenders containing animal proteins and eliminates any biosecurity concerns for semen exportation. We acknowledge the help of Dr. Mary Buhr (University of Guelph), Dr. Reuben Mapletoft (University of Saskatchewan). (Conflicts of interest: None declared. Source of funding: Bioniche Animal Health, the Ontario CanAdapt program, SEMEX Alliance.) doi:10.1016/j.cryobiol.2008.10.035
35. Cellular biophysics during freezing of rat and mouse sperm. Ramachandra Devireddy a, Jeung H. Choi b, Mie Hagiwara b, Wim Wolkers b, Ken P. Roberts b, John C. Bischof b, a Louisiana State University, Baton Rouge, LA, USA, b University of Minnesota, Minneapolis, MN, USA While cryopreservation of mouse sperm yields good survival and motility after thawing, rat sperm cryopreservation remains a challenge. This study measures the cellular biophysics during freezing of both sperm types to suggest improvements in rat cryopreservation approaches. A Differential Scanning Calorimeter(DSC) was used to study the state of water in the cellular and extracellular space and extract cellular water transport information during freezing. Data in eluted mouse and rat (eluted and epididymal) sperm in PBS and mBWW respectively was obtained at cooling rates of 5 and 20 °C/min and a model was fit to the data to obtain a set of combined best fit water-transport parameters (Lpg and ELp). Mouse sperm was modeled as a cylinder of length 122 lm and radius of 0.46 lm with an osmotically inactive cell volume of 0.61 Vo(Vo: isotonic cell volume). Rat sperm was modeled as a cylinder of length 188.7 lm and radius of 0.71 lm, with an osmotically inactive cell volume of 0.61 Vo. Optimal rates for mouse sperm yielded Lpg = 1.53 10 15m3/Ns (0.009 lm/ min-atm) and ELp = 106.7 kJ/mole (25.5 kcal/mole). This is similar to earlier published results in PBS+egg yolk for mouse sperm. Corresponding parameters for eluted rat sperm were Lpg = 1.7 10 15m3/Ns (0.01 lm/min-atm) and ELp = 77.8 kJ/mole (18.6 kcal/mole). As an interesting and potentially important comparison to eluted sperm additional DSC experiments were also conducted on rat epididymal tissue sperm (2 mg) in mBWW media (8 lL). The tissue(presumed mostly sperm) behaved somewhat differently than eluted sperm as shown in the permeability parameters obtained: Lpg = 2.9 10 15m3/Ns (0.017 lm/min-atm) and ELp = 74.1 kJ/mole (17.7 kcal/ mole). Parameters obtained in this study were finally used to predict optimal cooling rates of 23, 51 and 73 °C/min for eluted mouse, eluted rat and epididymal rat sperm, respectively. Post freeze-thaw motility experiments for mouse and rat sperm show maximum motility at similar cooling rates to those predicted from permeability measurements (data not shown). (Conflicts of interest: None declared. Source of funding: NIH R21 RR021698 and the Institute for Engineering in Medicine, University of Minnesota.) doi:10.1016/j.cryobiol.2008.10.036
The objective of the study was to investigate the protective effect of intracellular and extracellular disaccharides during bovine sperm desiccation, and the supplemental effect of the addition of an antioxidant (catalase) and a chelator (desferal, an iron chelator). The study was motivated by a goal to achieve partial to complete desiccation preservation of mammalian sperm, which holds promises as a simplified alternative to the cryopreservation technique. The addition of antioxidant and chelator was based on the hypothesis that an optimal amount of the additives helps reduce the oxidative stresses, thus minimizing membrane and mitochondrial damage. Sperm loaded with two different sugars, trehalose and sucrose, were dried with and without catalase/ desferal and evaluated for motility and membrane integrity immediately upon rehydration. Intracellular concentrations of the sugars were loaded by the method of ATP poration. Drying was performed through slow convective drying which was achieved by placing the sperm in desiccator boxes maintained at 11% RH. Motility was evaluated under a bright field microscope and membrane integrity was assayed in a fluorescent microscope using the dyes, Hoechst and Ethidium Homodimer. Results suggested an improved desiccation tolerance of sperm in the presence of intracellular trehalose or sucrose. Survival was further enhanced by the addition of 1 mM desferal in the desiccation buffer. Though the motility at lower dry basis water fractions (DBWF) did not show a significant improvement, there was a marked increase in the sperm membrane integrity at lower water contents (DBWF < 2) which correspond to a partially desiccated state. Moreover, the critical water content at which membrane integrity was completely lost was lowered to a DBWF value of around 0.2 for sperm with intracellular sugar and desferal as compared to around 0.6 for sperm with extracellular sugars only. (Conflicts of interest: None declared. Source of funding: None declared.) doi:10.1016/j.cryobiol.2008.10.037
37. Effects of raffinose concentration and methyl-beta-cyclodextrin on IVF with cryopreserved C57BL/6 sperm. Keiji Mochida a, Chihiro Koshimoto b, Keisuke Edashige c, Kentaro Aizawa d, Kyuichi Taguma a, Akihiko Ohta d, Atsuo Ogura a,e, a RIKEN Bioresource center, b University of Miyazaki, c Kochi University, d Meiji University, e Tsukuba University Spermatozoa from certain strains of mice, including C57BL/6, are very sensitive to freezing and thawing, and frequently fail to fertilize oocytes by conventional in vitro fertilization. We examined whether this problem could be overcome by optimizing the raffinose concentration and osmolality of freezing solutions. First, we prepared raffinose solutions at concentrations of 9, 12, 15, 18 and 21% (w/v). C57Bl/6Cr Slc epididymal spermatozoa from males aged 3–6 months were suspended in either of the freezing solutions. After equilibration for 1 min, sperm suspension was enclosed inside a 0.25 mL plastic straw and cooled in the vapor of liquid nitrogen (LN2) for 10 min. Then the straw was immersed in LN2 and stored for more than 5 days. For thawing, the straw was immersed in a water bath at 37 °C for 15 min. The thawed sperm suspension was added to HTF medium and incubated for 1 h at 37 °C. The motility and the progressive movement were assessed using a Hamilton Thorn IVOS computerized semen analyzer. Normalized motilities were 21%, 32%, 36%, 41%, and 47%, respectively, in raffinose solutions described above. Next, 0/4–4/4 X strength SD-PBS(supplemented Dulbecco’s phosphate-buffered saline; Koshimoto et al., 2000) contained raffinose at a concentration of 15%, 18%, or 21% were used for sperm freezing. When the thawed sperm were assessed for their motilities, practicable motilities were obtained with medium including 18% or 21% raffinose and 400– 525 mOsm/kg. The highest motility (56%) was obtained with 18% raffinose in 1/4 SD-PBS. The frozen sperm in this medium were preincubated in HTF supplemented with BSA and/or methyl-beta-cyclodextrin (after Choi et al.) for 1 h and inseminated into oocyte-containing HTF medium. The fertilization rates were 45% and 70%, respectively. These results indicated that C57BL/6 sperm cryopreservation could be improved by optimizing raffinose concentration and osmolality of the freezing solution. Furthermore, the supplementation of methyl-beta-cyclodextrin to preincubation medium was effective for increase of in vitro fertilization rate. (Conflicts of interest: None declared. Source of funding: None declared.) doi:10.1016/j.cryobiol.2008.10.038
Contributed papers 5. Cryobiology of oocytes
38. The pathway for the movement of water and cryoprotectants in bovine oocytes and embryos. Keisuke Edashige, Takao Hara, Yasunori Kawai, Masahiro Yoshimura, Bo Jin, Delgado Valdez Jr., Magosaburo Kasai, Kochi University, Nankoku, Kochi, Japan We have shown that the permeability of mouse oocytes and embryos at early cleavage stages to water and cryoprotectants is low, whereas that of morulae and