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Titration of vitrification solution in mouse embryo cryopreservation
ABSTRACTS, 25th ANNUAL optimal rates has no practical application, because of significantly lower survival in comparison to that obtained after freezing at optimal rates. REFERENCE 1. Fiser, P. S., Fairfidl, R. W., and Marcus, G. J. Cryobiology 23, 141-149 (1986). 169. Survival of Trichomonas preservation
of Human
Vaginalis during CryoSemen. J. K. SHER-
MAN, J. DALY, T. HOSTETLER, AND K. MCHENRY (University of Arkansas for Medical Sciences, Little Rock, Arkansas). Trichomoniasis is a sexual disease transmitted by the parasite Trichomonas vaginalis, often in semen of men who are asymptomatic. Undetected cryosurvival of T. vaginalis in the cryobanking of infected human semen for clinical inseminations, therefore, is a concern. Under optimal conditions of growth phase of organisms, composition of medium, rates of freezing, and concentration of DMSO, 65 to 90% of populations of T. vaginalis in their growth medium can survive freezing to and storage at - 196°C. However, recent experiments reveal no survival of T. vaginalis during cryopreservation of infected human semen for cryobanking. In our experiments, both semen and control (Diamond’s incomplete TYM) growth medium were inoculated with T. vaginalis (20,000-40,000/m1)in their late logarithmic growth phase. Aliquots (0.3 ml) of both infected controls and infected semen from nine ejaculates from five donors were processed for cryopreservation with 10% glycerol (v/v) after our two-step method, and crybanked at - 196°C.Cryosurvival of T. vaginalis, measured as colony growth in solid medium, averaged about 0.1% in TYM-glycerol, but averaged 29% (range of 8 to 34%) in semen-glycerol, at 0 hr as well as after 3 months at - 196°C. Results indicate not only that T. vaginalis can survive during successful cryobanking of human semen, but also that the extent of cryosurvival is greater in semen than in the parasite’s growth medium, with semen acting as an additional cryoprotectant. The special conditions for cryopreservation and for measurement of cryosurvival are discussed as possible explanations for results which contlict with reports of others. Cryosurvival, during cryobanking of human semen, of other microorganisms which cause sexually transmitted diseases, is noted. 170. Titration
of VitriJication Cryopreservation.
Solution in Mouse Em-
A. ARAV, L. GIANAROLI, AND P. SURIANO (Department of Physiopathology of Reproduction, University of Bologna, Bologna, Italy). bryo
Vitrification has been proposed for cryopreservation of preimplantation mouse embryos since intra and
MEETING
567
extra crystallization is avoided by rapid cooling of high concentrations of cryoprotectant solution. The purpose of this work was to try to reduce the toxicity of the vitrification solution by decreasing the time and temperature of embryo exposure to cryoprotectant solution. Eight-cell mouse embryos (174) were placed in 10 ml PBl + 10% FCS containing sucrose (0.25 ikf) at room temperature. After 3 min a titration was started with 10 ml of cooled (4°C) drops containing sucrose (0.25 M) + glycerol (12 M) in PBl + 10% FCS. After 12 min when the final solution was close to 6 M glycerol + 0.25 M sucrose, embryos were partially dehydrated and permeated by vitrification solution. After loading embryos in 0.25ml straws, the straws were cooled by placing them on a thick steel plate in the vapor phase of LN,. Two minutes later the straws were plunged directly in LN2 and stored for 1 week. The straws were warmed in a 37°C water bath for 10 sec. The contents of the straws were mixed with 10 ml PBl + 10% FCS containing sucrose (0.25 M) and glycerol (3 M) at 4°C. After 3 min, titration of 10 ml was started at room temperature with drops containing only PBl + 10% FCS; 12 min later embryos were washed in T, medium before culture. Out of 174 vitrified, stored, and warmed eight-cell mouse embryos, 145(83%) reached the blastocyst stage after culture. 41 blastocysts were transferred to pseudopregnant mice, and 7 normal fetuses were found on the 20th day of gestation. 171. Freezing Experiments with Normal and Bisected Mouse Embryos. A. LUCAS-HAHN AND H. NIEMANN (Institut fur Tierzucht und Tierverhalten (FAL), Neustadt, West Germany). Morulae and early blastocysts were collected from superovulated NMRI mice and were randomly assigned to five different groups of treatment. Embryos were either frozen as controls (group 1) or after removal of the zona pellucida by pronase (groups 2-5) and subsequent embedding in agar (group 3) or microsurgical bisection (group 4) and embedding in agar (group 5). All embryos were frozen by a method successfully used in bovine embryos; using 1.4 M glycerol as the cryoprotectant, 0.25-ml straws, and slow cooling to -35°C when they were plunged into liquid nitrogen. Embryos were thawed in a 30°C water bath and the cryoprotectant was removed using sucrose in two steps. After thawing, embryos were morphologically evaluated and then cultured for 48 hr in microdrops of Whitten’s medium under silicone oil. Postthaw morpholigical appearance was similar in all groups (86.8,70.6,90.9,85.9, and 84.2%, respectively) were considered to have survived after thawing. After 48 hr of culture, development in groups 2 and 4 was significantly (P < 0.05) reduced compared to groups 1 and 3. The respective rates of development were 61.8, 26.5, 59.1, 42.8 and 13.2%. Results of these prelimi-
of Human
Vaginalis during CryoSemen. J. K. SHER-
MAN, J. DALY, T. HOSTETLER, AND K. MCHENRY (University of Arkansas for Medical Sciences, Little Rock, Arkansas). Trichomoniasis is a sexual disease transmitted by the parasite Trichomonas vaginalis, often in semen of men who are asymptomatic. Undetected cryosurvival of T. vaginalis in the cryobanking of infected human semen for clinical inseminations, therefore, is a concern. Under optimal conditions of growth phase of organisms, composition of medium, rates of freezing, and concentration of DMSO, 65 to 90% of populations of T. vaginalis in their growth medium can survive freezing to and storage at - 196°C. However, recent experiments reveal no survival of T. vaginalis during cryopreservation of infected human semen for cryobanking. In our experiments, both semen and control (Diamond’s incomplete TYM) growth medium were inoculated with T. vaginalis (20,000-40,000/m1)in their late logarithmic growth phase. Aliquots (0.3 ml) of both infected controls and infected semen from nine ejaculates from five donors were processed for cryopreservation with 10% glycerol (v/v) after our two-step method, and crybanked at - 196°C.Cryosurvival of T. vaginalis, measured as colony growth in solid medium, averaged about 0.1% in TYM-glycerol, but averaged 29% (range of 8 to 34%) in semen-glycerol, at 0 hr as well as after 3 months at - 196°C. Results indicate not only that T. vaginalis can survive during successful cryobanking of human semen, but also that the extent of cryosurvival is greater in semen than in the parasite’s growth medium, with semen acting as an additional cryoprotectant. The special conditions for cryopreservation and for measurement of cryosurvival are discussed as possible explanations for results which contlict with reports of others. Cryosurvival, during cryobanking of human semen, of other microorganisms which cause sexually transmitted diseases, is noted. 170. Titration
of VitriJication Cryopreservation.
Solution in Mouse Em-
A. ARAV, L. GIANAROLI, AND P. SURIANO (Department of Physiopathology of Reproduction, University of Bologna, Bologna, Italy). bryo
Vitrification has been proposed for cryopreservation of preimplantation mouse embryos since intra and
MEETING
567
extra crystallization is avoided by rapid cooling of high concentrations of cryoprotectant solution. The purpose of this work was to try to reduce the toxicity of the vitrification solution by decreasing the time and temperature of embryo exposure to cryoprotectant solution. Eight-cell mouse embryos (174) were placed in 10 ml PBl + 10% FCS containing sucrose (0.25 ikf) at room temperature. After 3 min a titration was started with 10 ml of cooled (4°C) drops containing sucrose (0.25 M) + glycerol (12 M) in PBl + 10% FCS. After 12 min when the final solution was close to 6 M glycerol + 0.25 M sucrose, embryos were partially dehydrated and permeated by vitrification solution. After loading embryos in 0.25ml straws, the straws were cooled by placing them on a thick steel plate in the vapor phase of LN,. Two minutes later the straws were plunged directly in LN2 and stored for 1 week. The straws were warmed in a 37°C water bath for 10 sec. The contents of the straws were mixed with 10 ml PBl + 10% FCS containing sucrose (0.25 M) and glycerol (3 M) at 4°C. After 3 min, titration of 10 ml was started at room temperature with drops containing only PBl + 10% FCS; 12 min later embryos were washed in T, medium before culture. Out of 174 vitrified, stored, and warmed eight-cell mouse embryos, 145(83%) reached the blastocyst stage after culture. 41 blastocysts were transferred to pseudopregnant mice, and 7 normal fetuses were found on the 20th day of gestation. 171. Freezing Experiments with Normal and Bisected Mouse Embryos. A. LUCAS-HAHN AND H. NIEMANN (Institut fur Tierzucht und Tierverhalten (FAL), Neustadt, West Germany). Morulae and early blastocysts were collected from superovulated NMRI mice and were randomly assigned to five different groups of treatment. Embryos were either frozen as controls (group 1) or after removal of the zona pellucida by pronase (groups 2-5) and subsequent embedding in agar (group 3) or microsurgical bisection (group 4) and embedding in agar (group 5). All embryos were frozen by a method successfully used in bovine embryos; using 1.4 M glycerol as the cryoprotectant, 0.25-ml straws, and slow cooling to -35°C when they were plunged into liquid nitrogen. Embryos were thawed in a 30°C water bath and the cryoprotectant was removed using sucrose in two steps. After thawing, embryos were morphologically evaluated and then cultured for 48 hr in microdrops of Whitten’s medium under silicone oil. Postthaw morpholigical appearance was similar in all groups (86.8,70.6,90.9,85.9, and 84.2%, respectively) were considered to have survived after thawing. After 48 hr of culture, development in groups 2 and 4 was significantly (P < 0.05) reduced compared to groups 1 and 3. The respective rates of development were 61.8, 26.5, 59.1, 42.8 and 13.2%. Results of these prelimi-