CRYOBIOLOGY
15, 24%248 ( 1978)
Viability
Assays for Mahmalian
Oval
D. G. WHITTINGHAM MRC Mammalian Development Unit, Wolfson House (CJnive&ty College London), 4 Stephenson Wug> London NWI 2HE, England
In a recent Ciba Foundation symposium on “The Freezing of Mammalian Embryos” (3) the feasibility of preserving the eggs and embryos of several mammalian species at subzero temperatures was clearly demonstrated. The ultimate test of viability for mammalian ova after subjection to in vitro manipulations such as low-temperature storage is their ability to develop into normal live offspring when introduced into the reproductive tracts of appropriately synchronised foster mothers. Obviously, this type: of assay is not always practicable since it is both expensive and time consuming especialIy in species with protracted gestation times, e.g., sheep and cow. To be effective alternative methods of assaying the survival of frozen embryos must be closely correlated with viability after transfer to foster mothers. Whenever possible, they shouId provide information on the type of damage that may occur in cells during freezing and thawing. With the large domestic animals, where the number of embryos is limited, even with superovulation, the ideal viability assay should not interfere with the subsequent development of the embryo. The following account summarises current and possible Received October 21, 1977; accepted December I, 1977. Synopsis paper presented at the 14th Annual Meeting of the Society for Cryobiology, August 1977, Minneapohs, Minnesota. 1 Presented at the 14th Annual Meeting, Society for Cryobiology, Minneapolis, Minnesota, August l-3, 1977.
future methods of assaying the survival of mammalian eggs and embryos after freezing and thawing and aIso the factors which affect embryonic viability after transfer, 1. NORMALITY
(a) Morphological
ON THAWING
Appearance
The early developmental stages in the mouse (one to eight cells) are easily observed at relatively low magnification, and lysis and swelling of blastomeres can be recognised. However, it is not possible to observe changes in the individual cells of later stages (morulae and blastocysts) unless they are examined histologically. In some species, e.g., horse, pig, ferret, and to some extent cattle and sheep, intracellular details are masked by large amounts of Iipid in the cytoplasm of the unfixed embryos at all preimplantation stages. However, in the mouse at least, it is possible to divide embryos after freezing and thawing into the following categories on the basis of their morphological appearance: (i) normal in comparison with unfrozen control embryos of the same developmental age; (ii) swollen with blastomeres apparently still intact; (iii ) damaged with one or more blastomeres lysed or degenerated; and (iv) totally degenerated. Loss of or damage to the zona pellucida is noted in all categories. This initial assessment of survival provides information on the extent of gross morphological changes that are immediately apparent after thawing. In the 245
OOll-2240/78/0152-0245$02.00/O Copyright Q 1978 by Academic Press, Inc. All rights of repwductionin any form reserved.
246
D. G. WHITTINGHAM
mouse we have found that the proportion of frozen eight-cell embryos scored as normal on recovery after thawing is simiIar to the proportion which develops to the blastocyst stage during culture ( 17, 18). Ultrastructural examination of frozen eightcell embryos which appear normal after thawing fails to reveal any submicroscopic disorganisation of protoplasmic components most susceptibIe to freezing injury, i.e., plasma membrane, nuclear envelope, GoIgi complex, mitochondria, and fibrous lamellar components ( 14). Swollen embryos usually recover and continue development in cuhure (17). Swelling may indicate the incomplete removal of the cryoprotective agent during dilution or that a certain amount of membrane or intracellular damage has occurred during freezing or thawing. Large proportions of totalIy degenerated embryos are generally the result of either suboptimal rates of coohng and/or thawing or faiIure of the additive to afford cryoprotection or incorrect addition and dilution of the cryoprotective agent.
The acetate groups of FDA arc cleaved by cellular esterases releasing free fluorescein intracelhrlarly and cells will maintain fluorescence when the plasma membranes are intact. This should be a usefu1 test for assessing the survival of uufertilized ova and the embryos of species which are refractory to culture with present techniques. (c) Histological
Examination
The appearance of the nuclei and the cell membranes of representative samples of embryos can be examined by conventional light microscopy of fixed material but details of other submicroscopic structures sensitive to freezing injury can only be observed at the ultrastructural Ievel. So far, such details are limited to an ultrastructural examination of eight-cell mouse embryos after thawing (14) (see above) and a freeze-substitution study of ice formation in one-cell mouse embryos cooIed at various rates (Wilmut and Hay, unpublished, cited in the Ciba Foundation Symposium No. 52, 1977). Freeze fracture and scanning eIectron microscopic studies of (b) Dye Tests frozen embryos, although not strictly conThese have been routineIy used to assay sidered to constitute a viability assay, membrane damage, as shown by changes should provide a means of determining in membrane permeabihty, in many ceI1 where embryos are most susceptible to types after freezing but, so far, there are damage during freezing. only two reports of their use in embryo storage. The ability of sheep embryos to (d) Metabolic Activity retain the vital dye neutral red after storThe metabolic activity of frozen sperm age at low temperatures above freezing provided a rough indication of their via- has been studied extensiveIy and in many bility (5). More recently, Jackowski (4) instances such activity has not been we11 developed a fluorescent dye technique pre- correlated with fertilizibility. There are no viously used by McGrath et d. (6) to simiIar studies with frozen embryos alassay the viability of mouse ova. There was though energy metabolism, nucleic acid, a high correlation (0.96) between the abil- and protein synthesis were studied in rabity of embryos to retain fluorescein after bit embryos after storage at 5 and 10°C thawing and their ability to develop in cul- ( 1, 2). Clearly, such parameters warrant ture. The principle of the technique is that investigation in frozen embryos especially normal embryos are impermeable to fluo- now that techniques are sufficiently re. rescein but permeable to the nonffuorescent fined to require only relatively small numderivative, ffuorescein diacetate ( FDA). bers of embryos.
VIABILITY 2. IA’ VITRO
(a) Fe&x&on
ASSAYS
CULTURE
of Oocytes in ViEro
At present this technique is only valuable in the few mammals where in U&O festilization can be accomplished routinely and where the fertilized oocytes are capable of developing to live offspring after transfer. Some of the important parameters which can be used to evaluate the normality of frozen-thawed oocytes are sperm penetration of the zona pellucida, fusion of sperm and egg, the release of cortical granules, completion of meiosis, and pronuclear formation in the egg. Oocytes of the mouse and hamster were fertilized in vitro after freezing (7, 9) but only the mouse oocytes developed to live offspring after transfer (12). So far, unfrozen hamster oocytes have failed to deveIop in ~i,it~o(15).
OF OVA
247
13). Apparently frozen mouse embryos experience a deIay in the resumption of embryonic development after thawing, 3. DEVELOPMENT IN THE OVIDUCTS OF OTHER SPECIES
This procedure is laborious involving surgical interference for transfer and collection of the embryos, and the ability of embryos to develop further within interspecific oviducts is not always comparable with development within intraspecific oviducts. Nevertheless, in species where the embryos are presently refractory to development an Vitro, e.g., sheep and cow, the development of stored embryos has been examined successfully after transfer to the rabbit oviduct (8). 4. TRANSFER
TO FOSTER
MOTHERS
As mentioned earIier, this is the finite test of the success of freezing mammalian This is a convenient and direct method embryos. While many of the previous asfor making an initial assessment of sur- says can indicate the early manifestation viva1 of frozen embryos. The practical ad- of freezing damage, it is only by studying vantage of this method is that surviving the developmental potential of the frozenembryos will continue development when thawed embryo after transfer that the fulI transferred to foster mothers after culture, extent of such damage can be evaluated. and only embryos surviving the in vitro In the mouse, there are two major sources culture test need be transferred, thus econ- of embryonic loss after embryo storage; omising on time and the animals required the first is inherent in the freezing techfor the ultimate viability test. At present, nique itself and results in a proportion of this assay only applies to those species compIetely degenerated embryos on thawwhere embryo culture techniques are ade- ing; the second occurs mainly during the quate for continued normal development early postimplantation period. In a recent (10). It has been extensively used in the study, we found that the early postimplanmouse where development in vitro is highly tation loss of frozen-thawed embryos did correlated with embryonic development in not differ significantIy from unfrozen conuivo after transfer (16-18). Moreover, a trols (17). This success was achieved by period in culture is beneficial to the sur- carefully controlling the synchrony bevival of frozen-thawed eight-cell mouse tween embryo and recipient at transfer, the embryos and, blastocysts to live offspring use of naturally mated recipients, and after transfer since significantly higher per- limiting the culture period for the assesscentages of frozen-thawed embryos de- ment of initial survival to no more than velop to live offspring if cultured for 24 24 hr. In conclusion, for the mouse there are hr. before transfer when compared to the direct transfer of embryos on thawing (11, several effective methods of estimating em(b) Development
of Embryos in Vitro
D. G. WRITTINGHAM
248
bryonic survival after freezing, namely, morphoIogica1 appearance on thawing and development in culture, and these are closely correlated with the ultimate surviva1 of the frozen embryos to live offspring. However, in species where morphological examination and culture techniques are unreliable, other indirect methods are of value, i.e., transfer to interspecific oviducts and histological
examination.
The develop-
ment of the fluorescent dye test for the examination of these latter embryos would make assessmentof viabiIity much simpler. Consideration should aIso be given to the development of metabolic tests for survival as well as more detailed uItrastructura1
studies of frozen embryos to ascertain the type and extent of damage after freezing and thawing. REFERENCES 1, Anderson, G. B., and Foote, R. H. Effects of low temperature storage upon subsequent energy metabolism of rabbit embryos. Exp. CeZERes. 87, 302306 ( 1974). 2. Anderson, G, B., and Foote, R. H. Effects of low temperature upon subsequent nucleic acid and protein synthesis of rabbit embryos. Exp. CeEERes. 90, 73-78 ( 1975). 3. Elliot, K., and Whelan, J, (Eds.) “The Freezing of Mammalian Embryos,” Ciba Foundation Symposium 52 (new series). Elsevier/ Excerpta Medics/North-HoIland, Amsterdam, 1977. 4. Jackowski, S. C. “Physiological Differences between Fertilized and Unfertilized Mouse Ova: Glycerol Permeability and Freezing Sensitivity.” Ph.D. Dissertation, University of Tennessee, 1977. 5. Kardymowicz, 0. A method of vital staining for determining the viabi&y of fertilized sheep ova stored in vitro. In “‘Proceedings, Proc. 7th International Congress on Animal Reporduction & A.I., Munich,” Z, Vol. 1, pp. 503-506 ( 1972). 6, McGrath, J. J., Cravalho, E. G., and Huggins, C. E., An experimental comparison of intracelhdar ice formation and freeze-thaw surviva.l of Hela S-3 cells. Cryobiology 12, 540-550 (1975). 7. Parkening, T. A., Tsunoda, Y., and Chang, M. C. Effects of various low temperatures, cryoprotective agents and cooling rates on
the survival, fertilizabiIity and development of frozen-thawed mouse eggs. J. Exp. zooz. 197, 369-374 ( 1976). 8. Trounson, A. O., Willadsen, S. M., Rowson, L. E. A., and Newcomb, R. The storage of cow eggs at room temperature and at Iow temperatures. J. Rqwod. FM. 46, 173178 (1976). 9. Tsunoda, Y., Parkening, T. A., and Chang, M. C. In U&O fertilization of mouse and hamster eggs after freezing and thawing. Experientiu 32, 223-224 ( 1976). 10. Whittingham, D. G, FertiIization, early development rind storage of mammalian ova in u&o. In “The Early Development of Mammals” (M. Balls and A. E. Wild, Eds. ), pp. l-24. Cambkidge University Press, London and New York, 1975. 11. Whittingham, D. G. Low temperature storage of mammalian embryos. In “Basic Aspects of Freeze Preservation of Mouse Strains” (0. MuhIbock, Ed,), pp. 4555. 0. Muhlbock Gustav-Fischer Verlag, Strittgart, 1976. 12. Whittingham, D. G. Fertilization in L&O and development to term of unfertilized mouse oocytes previously stored at -196°C. J. Reprod. Fert. 49, 89-94 (1977). 13. Whittingham, D. G. Some factors affecting embryo storage in laboratory animals. In “The Freezing of Mammalian Embryos” ( K. ElIiott and J. Whelan, Eds. ), Ciba Foundation Symposium 52 (new series), pp. 97-108. EIsevier/Excerpta Medica/ North-Holland, Amsterdam, 1977. 14. Whittingham, D. G., and Andersen, E. WItrastructural studies of frozen-thawed S-celI mouse embryos. 1. Reprod. Fert. 48, 137140 ( 1978) * 15. Whittingham, D. G., and Bavister, B. D. Development of hamster eggs fertilized in vitro or in viva. 1. Reprod. FM. 38, 489492 (1974). 16. Wbittingham, D. G., Leibo, S. P., and Mazur, P. Survival of mouse embryos frozen to 196°C and -269°C. Science 178, 411-414 (1972). 17. Whittingham, D. G., Lyon, M. F., and GIenister, P. H. Long-term storage of mouse embryos at -196%: The effect of background radiation. Gkmt Res. Cambridge 29, 171-181 1977. 18. Whittingham, D. G., Lyon, M. F., and GIenister, P. H. Re-establishment of breeding stocks of mutant and inbred strains of mice from embryos stored at -196°C for prolonged periods. Genet. Res. Cambridge 30, 287-299 (1977).
15, 24%248 ( 1978)
Viability
Assays for Mahmalian
Oval
D. G. WHITTINGHAM MRC Mammalian Development Unit, Wolfson House (CJnive&ty College London), 4 Stephenson Wug> London NWI 2HE, England
In a recent Ciba Foundation symposium on “The Freezing of Mammalian Embryos” (3) the feasibility of preserving the eggs and embryos of several mammalian species at subzero temperatures was clearly demonstrated. The ultimate test of viability for mammalian ova after subjection to in vitro manipulations such as low-temperature storage is their ability to develop into normal live offspring when introduced into the reproductive tracts of appropriately synchronised foster mothers. Obviously, this type: of assay is not always practicable since it is both expensive and time consuming especialIy in species with protracted gestation times, e.g., sheep and cow. To be effective alternative methods of assaying the survival of frozen embryos must be closely correlated with viability after transfer to foster mothers. Whenever possible, they shouId provide information on the type of damage that may occur in cells during freezing and thawing. With the large domestic animals, where the number of embryos is limited, even with superovulation, the ideal viability assay should not interfere with the subsequent development of the embryo. The following account summarises current and possible Received October 21, 1977; accepted December I, 1977. Synopsis paper presented at the 14th Annual Meeting of the Society for Cryobiology, August 1977, Minneapohs, Minnesota. 1 Presented at the 14th Annual Meeting, Society for Cryobiology, Minneapolis, Minnesota, August l-3, 1977.
future methods of assaying the survival of mammalian eggs and embryos after freezing and thawing and aIso the factors which affect embryonic viability after transfer, 1. NORMALITY
(a) Morphological
ON THAWING
Appearance
The early developmental stages in the mouse (one to eight cells) are easily observed at relatively low magnification, and lysis and swelling of blastomeres can be recognised. However, it is not possible to observe changes in the individual cells of later stages (morulae and blastocysts) unless they are examined histologically. In some species, e.g., horse, pig, ferret, and to some extent cattle and sheep, intracellular details are masked by large amounts of Iipid in the cytoplasm of the unfixed embryos at all preimplantation stages. However, in the mouse at least, it is possible to divide embryos after freezing and thawing into the following categories on the basis of their morphological appearance: (i) normal in comparison with unfrozen control embryos of the same developmental age; (ii) swollen with blastomeres apparently still intact; (iii ) damaged with one or more blastomeres lysed or degenerated; and (iv) totally degenerated. Loss of or damage to the zona pellucida is noted in all categories. This initial assessment of survival provides information on the extent of gross morphological changes that are immediately apparent after thawing. In the 245
OOll-2240/78/0152-0245$02.00/O Copyright Q 1978 by Academic Press, Inc. All rights of repwductionin any form reserved.
246
D. G. WHITTINGHAM
mouse we have found that the proportion of frozen eight-cell embryos scored as normal on recovery after thawing is simiIar to the proportion which develops to the blastocyst stage during culture ( 17, 18). Ultrastructural examination of frozen eightcell embryos which appear normal after thawing fails to reveal any submicroscopic disorganisation of protoplasmic components most susceptibIe to freezing injury, i.e., plasma membrane, nuclear envelope, GoIgi complex, mitochondria, and fibrous lamellar components ( 14). Swollen embryos usually recover and continue development in cuhure (17). Swelling may indicate the incomplete removal of the cryoprotective agent during dilution or that a certain amount of membrane or intracellular damage has occurred during freezing or thawing. Large proportions of totalIy degenerated embryos are generally the result of either suboptimal rates of coohng and/or thawing or faiIure of the additive to afford cryoprotection or incorrect addition and dilution of the cryoprotective agent.
The acetate groups of FDA arc cleaved by cellular esterases releasing free fluorescein intracelhrlarly and cells will maintain fluorescence when the plasma membranes are intact. This should be a usefu1 test for assessing the survival of uufertilized ova and the embryos of species which are refractory to culture with present techniques. (c) Histological
Examination
The appearance of the nuclei and the cell membranes of representative samples of embryos can be examined by conventional light microscopy of fixed material but details of other submicroscopic structures sensitive to freezing injury can only be observed at the ultrastructural Ievel. So far, such details are limited to an ultrastructural examination of eight-cell mouse embryos after thawing (14) (see above) and a freeze-substitution study of ice formation in one-cell mouse embryos cooIed at various rates (Wilmut and Hay, unpublished, cited in the Ciba Foundation Symposium No. 52, 1977). Freeze fracture and scanning eIectron microscopic studies of (b) Dye Tests frozen embryos, although not strictly conThese have been routineIy used to assay sidered to constitute a viability assay, membrane damage, as shown by changes should provide a means of determining in membrane permeabihty, in many ceI1 where embryos are most susceptible to types after freezing but, so far, there are damage during freezing. only two reports of their use in embryo storage. The ability of sheep embryos to (d) Metabolic Activity retain the vital dye neutral red after storThe metabolic activity of frozen sperm age at low temperatures above freezing provided a rough indication of their via- has been studied extensiveIy and in many bility (5). More recently, Jackowski (4) instances such activity has not been we11 developed a fluorescent dye technique pre- correlated with fertilizibility. There are no viously used by McGrath et d. (6) to simiIar studies with frozen embryos alassay the viability of mouse ova. There was though energy metabolism, nucleic acid, a high correlation (0.96) between the abil- and protein synthesis were studied in rabity of embryos to retain fluorescein after bit embryos after storage at 5 and 10°C thawing and their ability to develop in cul- ( 1, 2). Clearly, such parameters warrant ture. The principle of the technique is that investigation in frozen embryos especially normal embryos are impermeable to fluo- now that techniques are sufficiently re. rescein but permeable to the nonffuorescent fined to require only relatively small numderivative, ffuorescein diacetate ( FDA). bers of embryos.
VIABILITY 2. IA’ VITRO
(a) Fe&x&on
ASSAYS
CULTURE
of Oocytes in ViEro
At present this technique is only valuable in the few mammals where in U&O festilization can be accomplished routinely and where the fertilized oocytes are capable of developing to live offspring after transfer. Some of the important parameters which can be used to evaluate the normality of frozen-thawed oocytes are sperm penetration of the zona pellucida, fusion of sperm and egg, the release of cortical granules, completion of meiosis, and pronuclear formation in the egg. Oocytes of the mouse and hamster were fertilized in vitro after freezing (7, 9) but only the mouse oocytes developed to live offspring after transfer (12). So far, unfrozen hamster oocytes have failed to deveIop in ~i,it~o(15).
OF OVA
247
13). Apparently frozen mouse embryos experience a deIay in the resumption of embryonic development after thawing, 3. DEVELOPMENT IN THE OVIDUCTS OF OTHER SPECIES
This procedure is laborious involving surgical interference for transfer and collection of the embryos, and the ability of embryos to develop further within interspecific oviducts is not always comparable with development within intraspecific oviducts. Nevertheless, in species where the embryos are presently refractory to development an Vitro, e.g., sheep and cow, the development of stored embryos has been examined successfully after transfer to the rabbit oviduct (8). 4. TRANSFER
TO FOSTER
MOTHERS
As mentioned earIier, this is the finite test of the success of freezing mammalian This is a convenient and direct method embryos. While many of the previous asfor making an initial assessment of sur- says can indicate the early manifestation viva1 of frozen embryos. The practical ad- of freezing damage, it is only by studying vantage of this method is that surviving the developmental potential of the frozenembryos will continue development when thawed embryo after transfer that the fulI transferred to foster mothers after culture, extent of such damage can be evaluated. and only embryos surviving the in vitro In the mouse, there are two major sources culture test need be transferred, thus econ- of embryonic loss after embryo storage; omising on time and the animals required the first is inherent in the freezing techfor the ultimate viability test. At present, nique itself and results in a proportion of this assay only applies to those species compIetely degenerated embryos on thawwhere embryo culture techniques are ade- ing; the second occurs mainly during the quate for continued normal development early postimplantation period. In a recent (10). It has been extensively used in the study, we found that the early postimplanmouse where development in vitro is highly tation loss of frozen-thawed embryos did correlated with embryonic development in not differ significantIy from unfrozen conuivo after transfer (16-18). Moreover, a trols (17). This success was achieved by period in culture is beneficial to the sur- carefully controlling the synchrony bevival of frozen-thawed eight-cell mouse tween embryo and recipient at transfer, the embryos and, blastocysts to live offspring use of naturally mated recipients, and after transfer since significantly higher per- limiting the culture period for the assesscentages of frozen-thawed embryos de- ment of initial survival to no more than velop to live offspring if cultured for 24 24 hr. In conclusion, for the mouse there are hr. before transfer when compared to the direct transfer of embryos on thawing (11, several effective methods of estimating em(b) Development
of Embryos in Vitro
D. G. WRITTINGHAM
248
bryonic survival after freezing, namely, morphoIogica1 appearance on thawing and development in culture, and these are closely correlated with the ultimate surviva1 of the frozen embryos to live offspring. However, in species where morphological examination and culture techniques are unreliable, other indirect methods are of value, i.e., transfer to interspecific oviducts and histological
examination.
The develop-
ment of the fluorescent dye test for the examination of these latter embryos would make assessmentof viabiIity much simpler. Consideration should aIso be given to the development of metabolic tests for survival as well as more detailed uItrastructura1
studies of frozen embryos to ascertain the type and extent of damage after freezing and thawing. REFERENCES 1, Anderson, G. B., and Foote, R. H. Effects of low temperature storage upon subsequent energy metabolism of rabbit embryos. Exp. CeZERes. 87, 302306 ( 1974). 2. Anderson, G, B., and Foote, R. H. Effects of low temperature upon subsequent nucleic acid and protein synthesis of rabbit embryos. Exp. CeEERes. 90, 73-78 ( 1975). 3. Elliot, K., and Whelan, J, (Eds.) “The Freezing of Mammalian Embryos,” Ciba Foundation Symposium 52 (new series). Elsevier/ Excerpta Medics/North-HoIland, Amsterdam, 1977. 4. Jackowski, S. C. “Physiological Differences between Fertilized and Unfertilized Mouse Ova: Glycerol Permeability and Freezing Sensitivity.” Ph.D. Dissertation, University of Tennessee, 1977. 5. Kardymowicz, 0. A method of vital staining for determining the viabi&y of fertilized sheep ova stored in vitro. In “‘Proceedings, Proc. 7th International Congress on Animal Reporduction & A.I., Munich,” Z, Vol. 1, pp. 503-506 ( 1972). 6, McGrath, J. J., Cravalho, E. G., and Huggins, C. E., An experimental comparison of intracelhdar ice formation and freeze-thaw surviva.l of Hela S-3 cells. Cryobiology 12, 540-550 (1975). 7. Parkening, T. A., Tsunoda, Y., and Chang, M. C. Effects of various low temperatures, cryoprotective agents and cooling rates on
the survival, fertilizabiIity and development of frozen-thawed mouse eggs. J. Exp. zooz. 197, 369-374 ( 1976). 8. Trounson, A. O., Willadsen, S. M., Rowson, L. E. A., and Newcomb, R. The storage of cow eggs at room temperature and at Iow temperatures. J. Rqwod. FM. 46, 173178 (1976). 9. Tsunoda, Y., Parkening, T. A., and Chang, M. C. In U&O fertilization of mouse and hamster eggs after freezing and thawing. Experientiu 32, 223-224 ( 1976). 10. Whittingham, D. G, FertiIization, early development rind storage of mammalian ova in u&o. In “The Early Development of Mammals” (M. Balls and A. E. Wild, Eds. ), pp. l-24. Cambkidge University Press, London and New York, 1975. 11. Whittingham, D. G. Low temperature storage of mammalian embryos. In “Basic Aspects of Freeze Preservation of Mouse Strains” (0. MuhIbock, Ed,), pp. 4555. 0. Muhlbock Gustav-Fischer Verlag, Strittgart, 1976. 12. Whittingham, D. G. Fertilization in L&O and development to term of unfertilized mouse oocytes previously stored at -196°C. J. Reprod. Fert. 49, 89-94 (1977). 13. Whittingham, D. G. Some factors affecting embryo storage in laboratory animals. In “The Freezing of Mammalian Embryos” ( K. ElIiott and J. Whelan, Eds. ), Ciba Foundation Symposium 52 (new series), pp. 97-108. EIsevier/Excerpta Medica/ North-Holland, Amsterdam, 1977. 14. Whittingham, D. G., and Andersen, E. WItrastructural studies of frozen-thawed S-celI mouse embryos. 1. Reprod. Fert. 48, 137140 ( 1978) * 15. Whittingham, D. G., and Bavister, B. D. Development of hamster eggs fertilized in vitro or in viva. 1. Reprod. FM. 38, 489492 (1974). 16. Wbittingham, D. G., Leibo, S. P., and Mazur, P. Survival of mouse embryos frozen to 196°C and -269°C. Science 178, 411-414 (1972). 17. Whittingham, D. G., Lyon, M. F., and GIenister, P. H. Long-term storage of mouse embryos at -196%: The effect of background radiation. Gkmt Res. Cambridge 29, 171-181 1977. 18. Whittingham, D. G., Lyon, M. F., and GIenister, P. H. Re-establishment of breeding stocks of mutant and inbred strains of mice from embryos stored at -196°C for prolonged periods. Genet. Res. Cambridge 30, 287-299 (1977).
