- Email: [email protected]
Volume changes of pig oocytes after exposure to different cryoprotectants
26th ANNUAL
ABSTRACTS,
penetration of zona-free hamster ova using citrateyolk extender, pH 6.5, fast freeze and large surface area. However, there was significant interaction between the protocol parameters and the single most successful freeze protocol was citrate-yolk extender at pH 6.5, fast freeze with large surface area. Studies with cheetah sperm are ongoing and additional freeze protocol parameters, as well as thawing, sperm capacitation, and evaluation methods, will be introduced into the experimental design in an attempt to identify an optimal cryopreservation technique for this species. (Support provided by the Zoological Society of San Diego and the Nixon Griffts Fund for Zoological Research.) 24. Optimized Volume Meter Method to Study Mammalian Embryo Permeability. N. D. BEZUGLY, F. I. OSTASHKO, AND A. V. MEDVEDOVSKY
(Research Institute of Animal Breeding of the Forest-Steppe and Woodlands, Kharkov, USSR).
543
MEETING
PB 1 + 20% FCS. The times (min) needed for recovery of the isotonic volume for each group of oocytes are reported in Table 1. The permeability rates are not affected by different concentrations of the three cryoprotectants used. There is no time difference to reach the isotonic volume when the propandiol solution is compared to the DMSO solution; however, the glycerol solution doubles the time to regain the isotonic volume. When mixtures of cryoprotectants are used, only the propandiol + DMSO solution equilibrates in 8 min while the other mixture solutions require at least 20 min. The oocytes were fixed and stained after 24 hr of incubation for morphological examination (zona pellucida, cytoplasmatic components, nuclear membrane, G.V.). Our preliminary data show that propandiol solutions (10 or 40%) may lead to less injury to the oocytes than to the other cryoprotectants. (Supported by MPI 40% and SEMENITALY .) SESSION
II---GENERAL
CRYOPRESERVATION-CONVERSAZIONE
25A. Mammalian
Embryo Permeability to Water and Cryoprotectants. F. I. OSTASHKO, N. D. BEZUGLY, N. A. GORDIENKO, AND E. G. VOLKOVA (Research Institute of Animal Breeding of
the Forest-Steppe and Woodlands, Kharkov, USSR). 293 Volume Changes of Pig Oocytes after Exposure to Different Cryoprotectants. A. ARAV AND M. L. BACCI (Instituto di Fisiologia Veterinaria, Bologna, Italy). For cryopreservation of pig gametes we studied the osmotic behavior of immature oocytes and their injury after exposure to various cryoprotective compounds. The volume changes of 45 germinal vesicle oocytes surrounded by their cumulus cells were measured after a one-step exposure to a low (lo%, v/v) or high (40%, v/v) concentration of diierent cryoprotectants (glycerol, propandiol, DMSO). A mixture of two different cryoprotectants (20%, v/v + 20%, v/v) of glycerol and propandiol, glycerol and DMSO, and DMSO and propandiol were also checked at 20°C under pa&in oil in PBl + 20% FCS. Pictures of the oocytes were taken at 1, 2, 3, 5, 8, 10, and 18 min and volumes of the cells were calculated using the cross section and assuming each oocyte has a spherical shape. The volumes were expressed as the percentage of the isotonic volume in
26. Comparison of Two Technics of Hypothermic Conservation of the Rats Heart: Immersion or Perfusion; Investigation with “P NMR. A. LARESE, J. AUSSEDAT, A. RAY, P. MARCSEK, N. ROUBI, A. ROSSI, AND G. DUREAU (Blood
Transfusion Center, Lyon, France). Although organ conservation by a perfusion technic seems to be a better process compared to a simple immersion, it is still the most used method in the human allografted hearts preservation. The NMR spectroscopy technic allows precise biochemical investigations into the energy state of the stored hearts. We compared three groups of conserved rats hearts: group I was stored by immersion during 6 hr, group II was immersed under the same conditions during 15 hr and group III was perfused by a low flow perfusate (0.3 ml/min) during 15 hr. In all cases, the cardioplegic solution was the St. Thomas perfusate (20 mM KC1 and 147 mM NaCl). The temperature for all technics was kept at + 4°C. At the end of the experiment, biochemical parameters were evaluated: NMR spectroscopy, quantitative biochemistry, and functional parameters of progressively rewarmed perfused hearts (37°C). Results are expressed in Table 1. Results show that (a) perfused hearts maintained better functional properties (LVP) and (b) the myocardium energy state is less altered by a perfusion process.
TABLE l-Abstract Glycerol 10% 18’
40% >20’
DMSO 10% 8’
40% 10’
Propandiol 10% 8’
40% 12’
25B Gly-Prop
Gly-DMSO
Prop-DMSO
20% + 20% 20’-30’
20% + 20% b30’
20% + 20% 8’
ABSTRACTS,
penetration of zona-free hamster ova using citrateyolk extender, pH 6.5, fast freeze and large surface area. However, there was significant interaction between the protocol parameters and the single most successful freeze protocol was citrate-yolk extender at pH 6.5, fast freeze with large surface area. Studies with cheetah sperm are ongoing and additional freeze protocol parameters, as well as thawing, sperm capacitation, and evaluation methods, will be introduced into the experimental design in an attempt to identify an optimal cryopreservation technique for this species. (Support provided by the Zoological Society of San Diego and the Nixon Griffts Fund for Zoological Research.) 24. Optimized Volume Meter Method to Study Mammalian Embryo Permeability. N. D. BEZUGLY, F. I. OSTASHKO, AND A. V. MEDVEDOVSKY
(Research Institute of Animal Breeding of the Forest-Steppe and Woodlands, Kharkov, USSR).
543
MEETING
PB 1 + 20% FCS. The times (min) needed for recovery of the isotonic volume for each group of oocytes are reported in Table 1. The permeability rates are not affected by different concentrations of the three cryoprotectants used. There is no time difference to reach the isotonic volume when the propandiol solution is compared to the DMSO solution; however, the glycerol solution doubles the time to regain the isotonic volume. When mixtures of cryoprotectants are used, only the propandiol + DMSO solution equilibrates in 8 min while the other mixture solutions require at least 20 min. The oocytes were fixed and stained after 24 hr of incubation for morphological examination (zona pellucida, cytoplasmatic components, nuclear membrane, G.V.). Our preliminary data show that propandiol solutions (10 or 40%) may lead to less injury to the oocytes than to the other cryoprotectants. (Supported by MPI 40% and SEMENITALY .) SESSION
II---GENERAL
CRYOPRESERVATION-CONVERSAZIONE
25A. Mammalian
Embryo Permeability to Water and Cryoprotectants. F. I. OSTASHKO, N. D. BEZUGLY, N. A. GORDIENKO, AND E. G. VOLKOVA (Research Institute of Animal Breeding of
the Forest-Steppe and Woodlands, Kharkov, USSR). 293 Volume Changes of Pig Oocytes after Exposure to Different Cryoprotectants. A. ARAV AND M. L. BACCI (Instituto di Fisiologia Veterinaria, Bologna, Italy). For cryopreservation of pig gametes we studied the osmotic behavior of immature oocytes and their injury after exposure to various cryoprotective compounds. The volume changes of 45 germinal vesicle oocytes surrounded by their cumulus cells were measured after a one-step exposure to a low (lo%, v/v) or high (40%, v/v) concentration of diierent cryoprotectants (glycerol, propandiol, DMSO). A mixture of two different cryoprotectants (20%, v/v + 20%, v/v) of glycerol and propandiol, glycerol and DMSO, and DMSO and propandiol were also checked at 20°C under pa&in oil in PBl + 20% FCS. Pictures of the oocytes were taken at 1, 2, 3, 5, 8, 10, and 18 min and volumes of the cells were calculated using the cross section and assuming each oocyte has a spherical shape. The volumes were expressed as the percentage of the isotonic volume in
26. Comparison of Two Technics of Hypothermic Conservation of the Rats Heart: Immersion or Perfusion; Investigation with “P NMR. A. LARESE, J. AUSSEDAT, A. RAY, P. MARCSEK, N. ROUBI, A. ROSSI, AND G. DUREAU (Blood
Transfusion Center, Lyon, France). Although organ conservation by a perfusion technic seems to be a better process compared to a simple immersion, it is still the most used method in the human allografted hearts preservation. The NMR spectroscopy technic allows precise biochemical investigations into the energy state of the stored hearts. We compared three groups of conserved rats hearts: group I was stored by immersion during 6 hr, group II was immersed under the same conditions during 15 hr and group III was perfused by a low flow perfusate (0.3 ml/min) during 15 hr. In all cases, the cardioplegic solution was the St. Thomas perfusate (20 mM KC1 and 147 mM NaCl). The temperature for all technics was kept at + 4°C. At the end of the experiment, biochemical parameters were evaluated: NMR spectroscopy, quantitative biochemistry, and functional parameters of progressively rewarmed perfused hearts (37°C). Results are expressed in Table 1. Results show that (a) perfused hearts maintained better functional properties (LVP) and (b) the myocardium energy state is less altered by a perfusion process.
TABLE l-Abstract Glycerol 10% 18’
40% >20’
DMSO 10% 8’
40% 10’
Propandiol 10% 8’
40% 12’
25B Gly-Prop
Gly-DMSO
Prop-DMSO
20% + 20% 20’-30’
20% + 20% b30’
20% + 20% 8’