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Freezing, an alternative to discarding excess bovine embryos
602
ABSTRACTS,
16th
37°C. Tritiated serotonin was added (0.5 to 2.0 PmoV liter substrate concentration) and the uptake stopped after 10 set with ice-cold EDTA-saline to ensure that only the initial rate of uptake was measured. The activity in the platelets was determined by liquid scintillation counting and the rate of uptake expressed as picomoles per 10” cells per 10 sec. The Michaelis constant, K,, , and the theoretical maximum rate of uptake, V,,,, were determined from a double reciprocal plot of the rate of uptake against substrate concentration. After 2 min of incubation with saline at 37”C, the K,,, was 1.2 ? 0.2 pmoiliter and the V,,, 32.1 2 3.0 pmol/lO* cells/l0 sec. These values were unaffected by 30 min of incubation. When platelets were incubated for 2 min with glycerol, V,,, was reduced to 17.5 2 2.6 pmol/lO* cells/l0 set but K,,, was unchanged at 1.2 + 0.2 WmoVliter. This implied that the uptake of serotonin was inhibited noncompetitively. The inhibition lessened with time so that after 30 min of incubation with glycerol, V,,, had reached 29.2 2 1.4 pmol/lO* cells/l0 set, implying that the effect was osmotic and that it was nullified by the movement of glycerol into the platelets. WORKSHOP
3.
GAMETE
AND
EMBRYO
PRESERVATION
61. Comparati~~e
Aspects
of Freezing
Spermatozoa.
B. G. CRABO (Department of Animal Science, University of Minnesota, St. Paul, Minnesota 55108). In successful freezing of spermatozoa, cryoprotective systems, freeze rate, and method of thawing play an important role and have to be adjusted for each species. Differences in response are likely to depend on variations in sperm membrane composition. The importance and behavior of seminal proteins in freezing and for fertilization will be described, using swine as an example. Fertility trials are the only reliable assay of the outcome of freezing. This is complicated by specifics in the reproductive anatomy and physiology of particular species. Laboratory assays must evaluate many different functions of the fertilizing mechanisms in the spermatozoa to be of value for assessing the fertilizing capacity in some cases. Attempts to evaluate membrane function in the laboratory are discussed. 62. Status
of Rrseurch in the Cryopresenwtion Spermutozou. J. K. SHERMAN
of
(Department of Anatomy, University of Arkansas for Medical Sciences, Little Rock, Arkansas 72201). H1rt77un
A historical synopsis of research in the cryobiology of human spermatozoa is presented, to illustrate their contribution to basic and applied aspects of the discipline, especially in cryobanking. The type, extent, and results of recent and current investigations in this labo-
ANNUAL
MEETING
ratory and in others are discussed and evaluated. Contemporary methods of cryopreservation used in cryobanks here in the United States and abroad are described and compared. Finally, the future of cryobiology of human spermatozoa is discussed in terms of basic and applied research avenues and efforts. 63. The Importance tion in Farm
of Embryo or Oocyte PreservaAnimal Production and Research.
K. J. BETTERIDGE (Animal Pathology Directorate, Health of Animals Branch, Agriculture Canada, Animal Diseases Research Institute, P.O. Box 11300, Station H, Nepean, Ontario K2H 8P9, Canada). The current status of general embryo transfer procedures is briefly reviewed in order to give perspective to the need for preserving embryos and/or oocytes. The development of short- and long-term preservation methods to date is summarized with indications of their efficacy but without methodological details. Present and potential applications of cryopreservation of embryos and oocytes to farm animal production (e.g., in “banking” embryos; moving livestock internationally; holding embryos pending the results of sexing or other analyses) and research (e.g., comparison of foundation and improved stock in genetic work) are introduced. It is anticipated that subsequent reports will describe details of freezing and thawing methods and amplify selected examples of how preserved embryos are being used in transfer programmes. 64. Freezing, Bovine
an Alternative to Discarding Embryos. ROBERT D. BAKER
Embryos, Inc., Middleville,
Excess
(American Michigan 49333).
Effective methods have been developed to superovulate cattle and to recover embryos under commercial conditions. However, one out of every seven recoveries results in an excess of embryos, that is, more embryos than should or could be transferred into synchronized recipients. Procedures used to freeze the excess embryos for long-term storage or export are under investigation. Acceptable conception rates using frozen-thawed embryos have not yet been achieved. Rates over 40% will likely be required for extensive commercial use of frozen-thawed bovine embryos. 65. The Application cial Bovine
of Cryopreservation Embryo Transfer.
to Commer-
HARLEY J. SCHNEIDER, JR. (Rio Vista International, Inc., San Antonio, Texas).
The use of embryo cryopreservation on a commercial basis is discussed. Selected topics for discussion are: selection of a freezing program, i.e., long vs short
ABSTRACTS,
16th
37°C. Tritiated serotonin was added (0.5 to 2.0 PmoV liter substrate concentration) and the uptake stopped after 10 set with ice-cold EDTA-saline to ensure that only the initial rate of uptake was measured. The activity in the platelets was determined by liquid scintillation counting and the rate of uptake expressed as picomoles per 10” cells per 10 sec. The Michaelis constant, K,, , and the theoretical maximum rate of uptake, V,,,, were determined from a double reciprocal plot of the rate of uptake against substrate concentration. After 2 min of incubation with saline at 37”C, the K,,, was 1.2 ? 0.2 pmoiliter and the V,,, 32.1 2 3.0 pmol/lO* cells/l0 sec. These values were unaffected by 30 min of incubation. When platelets were incubated for 2 min with glycerol, V,,, was reduced to 17.5 2 2.6 pmol/lO* cells/l0 set but K,,, was unchanged at 1.2 + 0.2 WmoVliter. This implied that the uptake of serotonin was inhibited noncompetitively. The inhibition lessened with time so that after 30 min of incubation with glycerol, V,,, had reached 29.2 2 1.4 pmol/lO* cells/l0 set, implying that the effect was osmotic and that it was nullified by the movement of glycerol into the platelets. WORKSHOP
3.
GAMETE
AND
EMBRYO
PRESERVATION
61. Comparati~~e
Aspects
of Freezing
Spermatozoa.
B. G. CRABO (Department of Animal Science, University of Minnesota, St. Paul, Minnesota 55108). In successful freezing of spermatozoa, cryoprotective systems, freeze rate, and method of thawing play an important role and have to be adjusted for each species. Differences in response are likely to depend on variations in sperm membrane composition. The importance and behavior of seminal proteins in freezing and for fertilization will be described, using swine as an example. Fertility trials are the only reliable assay of the outcome of freezing. This is complicated by specifics in the reproductive anatomy and physiology of particular species. Laboratory assays must evaluate many different functions of the fertilizing mechanisms in the spermatozoa to be of value for assessing the fertilizing capacity in some cases. Attempts to evaluate membrane function in the laboratory are discussed. 62. Status
of Rrseurch in the Cryopresenwtion Spermutozou. J. K. SHERMAN
of
(Department of Anatomy, University of Arkansas for Medical Sciences, Little Rock, Arkansas 72201). H1rt77un
A historical synopsis of research in the cryobiology of human spermatozoa is presented, to illustrate their contribution to basic and applied aspects of the discipline, especially in cryobanking. The type, extent, and results of recent and current investigations in this labo-
ANNUAL
MEETING
ratory and in others are discussed and evaluated. Contemporary methods of cryopreservation used in cryobanks here in the United States and abroad are described and compared. Finally, the future of cryobiology of human spermatozoa is discussed in terms of basic and applied research avenues and efforts. 63. The Importance tion in Farm
of Embryo or Oocyte PreservaAnimal Production and Research.
K. J. BETTERIDGE (Animal Pathology Directorate, Health of Animals Branch, Agriculture Canada, Animal Diseases Research Institute, P.O. Box 11300, Station H, Nepean, Ontario K2H 8P9, Canada). The current status of general embryo transfer procedures is briefly reviewed in order to give perspective to the need for preserving embryos and/or oocytes. The development of short- and long-term preservation methods to date is summarized with indications of their efficacy but without methodological details. Present and potential applications of cryopreservation of embryos and oocytes to farm animal production (e.g., in “banking” embryos; moving livestock internationally; holding embryos pending the results of sexing or other analyses) and research (e.g., comparison of foundation and improved stock in genetic work) are introduced. It is anticipated that subsequent reports will describe details of freezing and thawing methods and amplify selected examples of how preserved embryos are being used in transfer programmes. 64. Freezing, Bovine
an Alternative to Discarding Embryos. ROBERT D. BAKER
Embryos, Inc., Middleville,
Excess
(American Michigan 49333).
Effective methods have been developed to superovulate cattle and to recover embryos under commercial conditions. However, one out of every seven recoveries results in an excess of embryos, that is, more embryos than should or could be transferred into synchronized recipients. Procedures used to freeze the excess embryos for long-term storage or export are under investigation. Acceptable conception rates using frozen-thawed embryos have not yet been achieved. Rates over 40% will likely be required for extensive commercial use of frozen-thawed bovine embryos. 65. The Application cial Bovine
of Cryopreservation Embryo Transfer.
to Commer-
HARLEY J. SCHNEIDER, JR. (Rio Vista International, Inc., San Antonio, Texas).
The use of embryo cryopreservation on a commercial basis is discussed. Selected topics for discussion are: selection of a freezing program, i.e., long vs short