EDMUND FAY GRAHAM (1924-1991)
CRYOBIOLOGY
29, 433442
(1992)
In Memoriam Edmund
Fay Graham
Ed Graham passed away on November 26, 1991, after a long and painful fight against cancer, We will remember him for his enthusiasm for cryobiology and his vision of the practical possibilities that cryopreservation offers, particularly of gametes. This enthusiasm and his fighting spirit rubbed off on many of the students, colleagues, and professionals within and outside the country who met him during his 40-year career as professor of reproductive physiology and cryobiology at the University of Minnesota. Ed Graham was born in Mesilla, New Mexico, on July 25, 1924, and grew up on a small dairy farm outside El Paso, Texas. He occasionally told about his childhood poverty and having to milk cows and deliver the milk before going to school and how this made him want to go to college and “learn all there is to know about the cow.” Before this could happen, however, he served in the Army’s 1Olst Airborne division which landed in France behind the enemy lines on the day before D-Day. It was nearly wiped out and Ed Graham was among the few survivors. After the war, Ed Graham earned a B.S. in dairy husbandry from Utah State University to “learn all about the cow,” but found that the Bachelor’s degree only gave him a taste for more knowledge. Studies for an M.S. degree, which he earned in 1952 at South Dakota State University, introduced him to reproductive physiology. He then proceeded to the University of Minnesota to study with the well-known dairy physiologist W. E. Petersen and was immediately put on the payroll. He remained at the University of Minnesota until his death. Ed
(1924-1991)
Graham started to teach courses in reproductive physiology in 1953 and his first M.S. advisee graduated in the same year that Ed Graham finished his Ph.D. Over the years more than one thousand students experienced his stimulating and enthusiastic classroom teaching. W. E. Peterson had attracted many talented graduate students, among them P. E. Dzuik, to Minnesota and lively discussions on how gamete preservation and manipulations could revolutionize animal breeding were common in the group. The very first attempt to artificially inseminate cows had been made at Minnesota in 1938 by C. L. Cole, a dairy geneticist and head of the department . County cooperatives for dairy cattle breeding were mushrooming in Minnesota and Wisconsin. In Cambridge a few years earlier, Chris Polge had devised the first technique for freeze preservation of bull semen. In this environment, Ed Graham was chosen by the department to stay and develop the promising “high tech” field of artificial insemination. He jumpstarted with his peers to work on various aspects of semen production and semen collection and last, but not least, to further develop freeze preservation of bull semen. It is no exaggeration to say that Ed Graham was very successful and instrumental in making it possible for these artificial insemination centers to convert to frozen semen. Many of his graduates went on to work in the centers and the technicians were initially trained at the University of Minnesota. The Minnesota/Wisconsin AI cooperatives supported his research until 1980. He was often an appreciated speaker at the National Association of Animal Breeders’
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Copyright 0 1992 by Academic Press. Inc. All rights of reproduction in any form reserved.
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technical conferences. One of his highlights was “The usefulness of useless data,” which points out how human bias affects the fertility in field trials. The 1960swere a very active period in Ed Graham’s professional life. The feasibility of “the other gene multiplication system,” embryo transfer, was explored. Its practicality was shown and the second calf in the world (the first one was born in Madison, WI) from ova transfer was produced. Ed Graham always liked to see discoveries from his laboratory translate into practical uses, and so with former graduate students Harry Rajamannan and Melvyn Fahning, he founded in 1973 the world’s first embryo transfer company, International CryoBiological Services, Inc. He attempted to improve semen diluents by finding which individual constituents of semen were positively correlated to fertility. The greatest success was achieved in the turkey, and Ed Graham’s turkey semen extender may well be the one most used in the world today. At the beginning of the decade, he visited Japan and made lifelong professional relationships with prominent researchers there. As an immediate result of the visit, Drs. Nagase and Niwa were invited to Minnesota, where they together furthered the socalled pellet freezing technique. Pellet freezing on dry ice proved to be very useful for freezing large volumes, for example, as required for boar semen. I had the pleasure to begin working in Ed Graham’s laboratory in the late 1960s. The work was often interrupted by fishing trips on which research was discussed. Foremost on his mind was how to freeze semen from other agriculturally important species, and the process of freeze-drying. Ed Graham felt rightly that glycerol would be the major detriment to sperm survival in freeze-dried semen. In developing a glycerol-free system suitable for freeze-drying spermatozoa, Ed Graham laid the foundation for the solution of freezing boar spermatozoa, since the discovery that glycerol
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increased the permeability of boar spermatozoa for intracellular enzymes implied that glycerol was toxic. This information was received with skepticism by cryobiologists at the time but it is now clear that lower concentrations of glycerol favor fertility for the semen of many species. In the late 196Os,Ed Graham began collaboration with a transplantation surgeon, the late Richard C. Lillehei, on a project aiming to freeze-preserve kidneys for transplantation. This project continued into the mid-1970s. Some success was achieved in that kidneys frozen to -20°C survived and were able to sustain the life of dogs. Work on freeze-drying continued and it was found that sperm containing only 15% water could be rehydrated and retain fertility. However, sperm with less water lost their fertilizing ability. Although grain containing 15% water will not spoil, spermatozoa with this water content had to be stored in the frozen stage. This finding eliminated its practical use. Frozen spermatozoa and embryos of endangered species-a “library of life”were on Ed Graham’s mind from the 1970s through the rest of his life. He further developed electroejaculation to accomplish collection from animals of all sizes. He worked on dogs, cats, sheep, chicken, turkeys, and zoological species ranging from rare cats and tigers to elephants as well as fish. At the time of his death he had frozen semen stored from 30 to 40 species. Assay of frozen semen was another of Ed Graham’s interests. His eyesight throughout his scientific career was poor and he could not use a microscope well. To overcome this, he hooked up a videocamera to the microscope and was able to see sperm motility on the TV screen. This technique for visualizing sperm motility was appreciated by personnel at artificial insemination centers, most of which now employ this technique. Other techniques investigated include electronic sizing of spermatozoa to assess their osmotic reactivity, enzyme
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leakage, and Sephadex filtration. The latter technique was developed after Ed Graham for some reason poured bull semen through a Sephadex column and discovered that all spermatozoa in the effluent were motile. It may prove to be one of his most important discoveries. Ed Graham never found the mechanism of action of the filter, but this work is continuing. Apparently, the surface structure of the spermatozoa is the discriminating factor. Assay results with Sephadex filters seem to be better correlated to the fertility of the semen than any other laboratory technique described to date. Ed Graham gained an international reputation in many ways. He worked on a Spanish-American bilateral research project on artificial insemination in swine and sheep. He received honorary degrees from the Veterinary College of Madrid, Spain, and the Italian “Instituto Sperimentale Italian0 ‘Lazzaro Spallanzani’ per Fecondazione Artificiale” and was an honorary member of the Japanese Society for artificial insemination. He participated in an official USDA trip to Russia in 1976 with six other U.S. scientists. Vern Pursel, a participant on the trip and one of Ed’s former graduate students, wrote “that it was evident that Dr. Graham was the most widely recognized U.S. scientist in the group and considered the equal by no less than Soviet Academy member Milovanov (who wrote the first modern book on artificial insemination in 1934). It was Dr. Graham who was accorded at the opening ceremony and most honored at the closing ceremony even if he was not the leader or the eldest in the group.” He was also recognized nationally with several awards, for example, the National Association of Animal Breeder’s Physiology Research Award, by Minnesota Turkey Growers, Midwest Breeders, and several honor societies. Ed Graham’s favorite meetings were the International Congresses of Animal Reproduction and Artificial Insemination and those of the Society for Cryobiology. Many
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may remember his enthusiasm when he chaired the 14th Cryobiology meeting in Minneapolis in 1977. Members and pioneers in the society, perhaps foremost Father Luyet and his co-workers Lou Rapatz and Alan MacKenzie, influenced his interest in cryobiology substantially. In the mid-1980s Ed suffered from a detached retina in his good eye and became practically blind. This naturally had a dampening effect on his ability to do science but did not decrease the hours he spent at work. Nor did he stay home when a back problem proved to be bone cancer. His fighting spirit kept him going to the last day despite severe pain. However, we like to remember him as the powerful man that he was at the height of his career, enthusiastically talking about the advantages and possibilities of freeze preservation of biological material. Few persons could remain unaffected by Ed Graham and many students and collaborators carry his mark. With the death of Ed Graham, we have lost a remarkable man and researcher. Bo G. Crabo University of Minnesota Department of Animal Science St. Paul, Minnesota 55108 PUBLICATIONS
OF EDMUND
F. GRAHAM
“The Mechanical and Hormonal Methods of Dilating the Bovine Cervix.” MS. thesis, South Dakota State University, 1952. “The Effects of Different Planes of Nutrition and Frequencies of Collection on Bovine Semen Production.” Ph.D. thesis, University of Minnesota, 1955. 1. Hirsch, M., Graham, E. F., and Dracy, A. E. A classification for the identification of bovine noseprints. J. Dairy Sci. 35, 31&319 (1952). 2. Graham, E. F., Dracy, A. E., and Hirsch, M. Some similarities of monozygotic cattle noseprints. J. Dairy Sci. 36, 1l-13 (1953). 3. Graham, E. F., and Dracy, A. E. The effects of relaxin and mechanical dilation of the bovine cervix. J. Dairy Sci. 36, 772-777 (1953). 4. Dziuk, P. J., Graham, E. F., Donker, J. D., Marion, G. B., and Petersen, W. E. Electroejaculation in farm animals. J. Anim. Sci. 12, 945-946 (1953).
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5. Dziuk, P. J., Graham, E. F., Donker, J. D., Marion, G. B., and Petersen, W. E. Some observations in collection of semen from bulls, goats, boars and rams by electrical stimulation. Vet. Med. 49, 455-458 (1954). 6. Dziuk, P. J., Graham, E. F., and Petersen, W. E. The technique of electroejaculation and its use in dairy bulls. J. Duiry Sci. 37, 10351041 (1954). 7. Graham, E. F., Erickson, W. E., and Bayley, N. D. Conception results with frozen semen. In “Proceedings, 7th Annual Conference of the National Association of Artificial Breeders” Vol. 7, pp. 198-199, 1954. 8. Graham, E. F., Erickson, W. E., and Bayley, N. D. The effect of glycerol equilibration on frozen bovine spermatozoa. J. Dairy Sci. 40, 51c.515 (1957). 9. Donker, J. D., Nichols, J. R., Graham, E. F., and Petersen, W. E. Controlled estrus in cattle. In “Reproduction and Infertility,” Part IV, pp. 171-178. Pergamon, New York, 1958. IO. Larson, G. L. and Graham, E. F. Effects of low temperatures in storage of bovine semen. A.I. Dig. 6(12), 6-8 (1958). Il. Graham, E. F., Vogt, D. W., and Fisher, G. R. Effect of method of glycerol addition on the fertility of frozen bovine spermatozoa. J. Dairy Sci. 41, 1553-1556 (1958). 12. Erickson, W. E., and Graham, E. F. Effect of storage on nonreturn rate of frozen spermatozoa. A.I. Dig. 7, 8-9 (1959). 13. Larson, G. L., and Graham, E. F. Effects of rapid freezing on progressive motility of semen. A.I. Dig. 7, C7 (1959). 14. Graham, E. F., and Erickson, W. E. Comparison of glass ampules and plastic vials based on the nonreturn rate of frozen bovine spermatozoa. J. Dairy Sci. 42, 365-367 (1959). 15. Erickson, W. E., and Graham, E. F. Factors affecting the fertility of frozen bovine spermatozoa. J. Dairy Sci. 42, 520-528 (1959). 16. Graham, E. F., and Erickson, W. E. The effect of long time storage of frozen bovine spermatozoa. A.I. Dig. 7, 5-8 (1959). 17. Graham, E. F. Artificial insemination in the United States. Jpn. J. Anim. Reprod. 5, 81-86 (1959). 18. Dukelow, W. R., Frederick, E. C., and Graham, E. F. Frequency of ejaculation in the bovine. J. Dairy Sci. 43, 1335-1339 (1960). 19. Graham, E. F. New ideas in freezing semen. A.Z. ’ Dig. 9(ll), 8 (1961). 20. Graham, E. F. New ideas in freezing semen. In “Proceedings, 14th Annual Conference of the National Association of Animal Breeders, Syracuse, NY,” pp. 59-62, 1961.
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21. Pursel, V. G., and Graham, E. F. Induced estrus in anestrous ewes by use of progestogens and follicle stimulating hormone. J. Anim. Sci. 21, 132-136 (1962). 22. Avery, T. L., Cole, C. L., and Graham, E. F. Investigations associated with the transplantation of bovine ova. I. Synchronization of oestrus. J. Reprod. Fertil. 3, 206211 (1962). 23. Avery, T. L., Fahning, M. L., and Graham, E. F. Investigations associated with the transplantation of bovine ova. II. Superovulation. J. Reprod. Fertil. 3, 212-217 (1962). 24. Avery, T. L., and Graham, E. F. Investigations associated with the transplantation of bovine ova. III. Recovery and fertilization. J. Reprod. Fe&/. 3, 218-228 (1962). 25. Avery, T. L., Fahning, M. L., Pursel, V. G., and Graham, E. F. Investigations associated with the transplantation of bovine ova. IV. Transplantation of ova. J. Reprod. Fertil. 3, 229-238 (1962). 26. Graham, E. F. Controlling estrus in farm animals. In “Proceedings, Minnesota Nutrition Conference for Feed Manufacturers and Dealers,” pp. 13-19, 1962. 27. Graham, E. F. Factors affecting the freezing of bovine spermatozoa. Jpn. J. Anim. Reprod. 17, 591-597 (1963). 28. Graham, E. F. Biochemistry of semen. Jpn. J. Anim. Reprod. 17, 715-720 (1963). 29. Nagase, H., and Graham, E. F. Pelleted semen: Comparison of different extenders and processes on fertility of bovine spermatozoa. In “Proceedings, 5th Int. Cong. Anim. Reprod. AI, Trento.” Vol. 4, pp. 387-391, 1964. 30. Rajamannan, A. H. J., Graham, E. F., and Smith, F. The effect of a new extender, incorporating dried egg yolk on the fertility of bovine spermatozoa. In “Proceedings, 5th Int. Cong. Anim. Reprod. AI, Trento,” Vol. 4, pp. 392-397, 1964. 31. Nagase, H., Graham, E. F., and Niwa, T. Pelleted semen: The effect of glycerol level and composition of thawing solution on fertility of bovine spermatozoa. In “Proceedings, 5th Int. Cong. Anim. Reprod. AI, Trento,” Vol. 4, pp. 404-409, 1964. 32. Graham, E. F., Schultz, R. H., Johnson, L. A., and Fahning, M. L. A comparison of the free amino acids in seminal plasma of the bull, boar, cock, turkey and uterine fluids of the cow during estrus. In “Proceedings, 5th Int. Cong. Anim. Reprod. AI, Trento,” Vol. 4, pp. 381-386, 1964. 33. Rajamannan, A. H. J., Linnerud, A., and Graham, E. F. Lactation studies. IX. Relationship of milk flow rate and complementary milk to
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milk yields in a herd of Holstein cattle. J. Dairy Sci. 49, 31-35.
34. Graham, E. F. Comments on freezing spermatozoa. In “Proceedings, 1st Technical Conference, National Association of Animal Breeders, Chicago,” pp. 61-63, 1966. 35. Graham, E. F. The use of a dye indicator for testing, training and evaluating technicians in artificial insemination. In “Proceedings, 1st Technical Conference, National Association of Animal Breeders, Chicago,” pp. 57-60, 1966. 36. Schultz, R. H., Fahning, M. L., and Graham, E. F. A technique for exposure of the bovine internal genetalia. Vet. Rec. 78, 91-93 (1966). 37. Ahluwalia, B. S., and Graham, E. F. Free amino acids in the semen of the fowl and turkey. J. Reprod. Fertil.
12, 365-368 (1966).
38. Ahluwalia, B. S., and Graham, E. F. Free carbohydrates in seminal plasma and sperm cells of semen of fowl. J. Reprod. Fertil. 12, 359361 (1966). 39. Fahning, M. L., Schultz, R. H., and Graham, E. F. A technique for collection of uterine fluid from the live cow. Vet. Rec. 79, 23&233 (1966). 40. Fahning, M. L., Schultz, R. H., Graham, E. F., Donker, J. D., and Mohrenweiser, H. W. Synchronization of oestrus in dairy heifers with 6-methyl-17-acetoxyprogesterone and its effect on conception rate. J. Reprod. Ferril. 12, 569-571 (1966). 41. Clark, J. B. K., Graham, E. F., Lewis, B. A., and Smith, F. o-Mannitol, erythritol and glycerol in bovine semen. J. Reprod. Fertil. 13, 189-197 (1967). 42. Fahning, M. L., Schultz, R. H., and Graham, E. F. The free amino acid content of uterine fluids and blood serum in the cow. J. Reprod. Fertil. 13, 229-236 (1967). 43. Graham, E. F., and Pace, M. M. Some biochemical changes in spermatozoa due to freezing. Cryobiology
4, 75-84 (1967).
44. Pursel, V. G., and Graham, E. F. Phospholipids of bovine spermatozoa and seminal plasma. J. Reprod. Ferril. 14, 203-211 (1967). 45. Rajamannan, A. H. J., and Graham, E. F. Spin freezing of semen. In “Proceedings, 6th Int. Cong. Anim. Reprod. AI, Paris,” Vol. 2, pp. 1145-1146, 1968. 46. Rajamannan, A. H. J., and Graham, E. F. Film freezing of semen. In “Proceedings, 6th Int. Cong. Anim. Reprod. AI, Paris,” Vol. 2, pp. 1141-1143, 1968. 47. Graham, E. F., Thompson, L., and Neville, W. Physical, physiochemical and chemical characteristics of boar semen. In “Proceedings,
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2nd Technical Conference on Artificial Insemination and Reproduction, National Association of Animal Breeders,” pp. 55-63, 1968. 48. Graham, E. F. The usefulness of useless data field tests and responsibilities. In “Proceedings, 2nd Technical Conference on Artificial Insemination and Reproduction, National Association of Animal Breeders,” pp. 18-22, 1968. 49. Beisang, A. A., Dietzman, R. H., Mayo, C. H., Graham, E. F., and Lillehei, R. C. Damage during perfusion procedures used to prepare whole organs for freezing. Cryobiology 5, 273277 (1969). 50. Carter, J. E., Beisang, A. A., Ahlgren, F. F.,
Dorman, D. F., and Graham, E. F. An automatic temperature control device. Cryobiology 5, 343-346(1969). 51. Schultz, R. H., Burcalow,
H. B., Fahning, M. L., Graham, E. F., and Weber, A. F. Karyometric study of epithelial cells lining the glands of the bovine endometrium. J. Reprod. Fertil. 19, 169-171 (1969). 52. Gibson, C. D., and Graham, E. F. The relationship between fertility and post-freeze motility of bull spermatozoa (by pellet freezing) without glycerol. J. Reprod. Fertil. 20, 155-157 (1969). 53. Beisang, A. A., Lillehei,
R. C., and Graham, E. F. Cryoprotective techniques in the freezing of whole organs. Cryobiology 5, 223-229 (1969). 54. Beisang, A. A., Graham, E. F.. Lillehei, E. C., Dietzman, R. H., and Carter, J. E. Enzyme levels in the effluent perfusates from frozen canine kidneys. Transplant. hoc. 1, 862-881
(1969). 55. Al-Hakim, M. K.. Graham, E. F., and Schmehl,
M. L. Free amino acids and amino compounds in bovine seminal plasma. J. Dairy Sci. 53, 84-88 (1970). 56. Graham, E. F.. Schmehl, M. K. L., and MakiLaurila, M. Some physical and chemical methods of evaluating semen. In “Proceedings, 3rd Technical Conference on Artificial Insemination, National Association of Animal Breeders,” pp. 44-48, 1970. 57. Carter, J. E., Schellpfeffer, D. A., Beisang, A. A., Graham, E. F., and Lillehei, R. C. The effect of storage temperature and media on the activity of lactic dehydrogenase and glutamic oxalacetic transaminase in perfusates from frozen canine kidneys. Cryobiology 6,533-541 (1970). 58. Beisang, A. A., Feemster, J., Dietzman, R. H., Uchida, H., Carter, J. E., Graham, E. F., and Lillehei, R. C. Damage assay of kidneys fro-
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zen by intraarterial perfusion with a fluorocarbon. Fed. Proc. 29, 1782-1788 (1970). Pace, M. M., and Graham, E. F. The release of glutamic oxaloacetic transaminase from bovine spermatozoa as a test method of assessing semen quality and fertility. Rio/. Reprod. 2, 140-146 (1970). Crabo, B. G., Bower, R. E., Brown, K. I., Graham, E. F., and Pace, M. M. Extracellular glutamic-oxaloacetic transaminase as a measure on membrane injury in spermatozoa during treatment. In “Current Problems in Fertility” (A. Ingelman-Sundberg and N.-O. Lunell, Eds.), pp. 33-38. Plenum, New York, 1971. Graham, E. F., Rajamannan, A. H. J., Schmehl, M. K. L., Maki-Laurila, M., and Bower, R. E. Preliminary report on procedure and rationale for freezing boar semen. A.I. Dig. 19, 12-14 (1971). Brown, K. I., and Graham, E. F. Effect of semen quality on fertility in turkeys. Poult. Sci. 50, 295-297 (1971). Brown, K. I., and Graham, E. F. Effect of some cryophylactic agents on turkey spermatozoa. Poult. SC;. 50, 832-835 (1971). Graham, E. F., and Brown, K. I. Effect of osmotic pressure of semen extenders on the fertility and hatchability of turkey eggs. Poult. Sci. 50, 836-838 (1971). Rajamannan, A. H. J., Graham, E. F., and Schmehl, M. K. L. Effect of holding on bovine semen. A.I. Dig. 19, 6 (1971). Graham, E. F., Rajamannan, A. H. J., Schmehl, M. K. L., Maki-Lauria, M., and Bower, R. E. Fertility studies with frozen boar spermatozoa. A.I. Dig. 19, 6-16 (1971). Brown, K. I., Crabo, B. G., Graham, E. F., and Pace, M. M. Some factors affecting loss of intracellular enzymes from spermatozoa. Cryobiology 8, 220-224 (1971). Graham, E. F., Schmehl, M. L., Brown, K. I., Crabo, B. G., and Ertel, G. Some chemical studies of the female reproductive tract and seminal plasma of the male turkey and their relationship to fertility. Poult. Sci. 50, 11701181 (1971). Schultz, R. D., Fahning, M. L., and Graham, E. F. A chemical study of uterine fluid and blood serum of normal cows during the oestrous cycle. J. Reprod. Fertil. 27, 335-367 (1971). Alexander, F. C. M., Zemjanis, R., Graham, E. F., and Schmehl, M. L. Semen characteristics and chemistry from bulls before and after seminal vesiculectomy and after vasectomy. J. Dairy Sci. 54, 1530-1535(1971). Ellery, J. C., Graham, E. F., and Zemjanis, R.
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F. GRAHAM Artificial insemination of pony mares with semen frozen and stored in liquid nitrogen. Am. J. Vet. Res. 32, 1693-1698 (1971). Carter, J. E., Graham, E. F., Lillehei, R. C., and Blackshear, P. L. The effect of high hydrostatic pressure and low temperature on lactate dehydrogenase and glutamic oxalacetic transaminase. Cryobiology 8, 524-534 (1971). Graham, E. F., Crabo, B. G., and Brown, K. I. Effect of some zwitter ion buffers on the freezing and storage of spermatozoa. I. Bull. .I. Dairy Sci. 55, 372-378 (1972). Brown, K. I., Graham, E. F., and Crabo, B. G. Effect of some hydrogen-ion buffers on storage and freezing of turkey spermatozoa. Poult. Sci. 51, 840-849 (1972). Crabo, B. G., Brown, K. I., and Graham, E. F. Effect of some buffers on the storage and freezing of boar spermatozoa. 1. Anim. Sci. 35, 377-382 (1972). Dziuk, P. J., Lewis, J. M., Graham, E. F., and Moyer, R. H. Comparison between natural service and artificial insemination with fresh or frozen sperm at an appointed time in the ewe. .I. Anim. Sci. 35, 572-582 (1972). Crabo, B. G., and Graham, E. F. Correlation between some laboratory methods for evaluation of boar semen after freezing. In “Proceedings, 7th Int. Cong. Anim. Reprod. AI, Munich,” Vol. 2, pp. 1639-1644, 1972. Graham, E. F., and Crabo, B. G. Some factors influencing the freezing of boar semen. In “Proceedings, 7th Int. Cong. Anim. Reprod. AI, Munich,” Vol. 2, pp. 1627-1632, 1972. Smith, J. D., and Graham, E. F. Free amino acids in ram seminal plasma. J. Anim. Sci. 34, 601604 (1972). Graham, E. F. The state of the art in cryobiology. In “Application of Cryogenic Technology,” Vol. 5, pp. 182-192. Scholium Intemational, Inc., Whitestone, NY, 1972. Carter, J. E., Risch, S. J., Graham, E. F., and Lillehei, R. C. The effect of pressure and cooling rate on spermatozoa, kidney tissue and the whole kidney. Cryobiology 10, 263-270 (1973). Graham, E. F., Crabo, B. G., and Schmehl, M. K. L. Utilization of enzyme assay in developing techniques for freezing semen. In “atti dell’ Simposio Internazional di Zootecina, 15-17 April, Milan. Italy,” pp. 95111, 1973. Bower, R. E., Jr., Crabo, B. G., Pace, M. M., and Graham, E. F. Effects of dilution and glycerol on the release of glutamic oxaloacetic transaminase (GOT) from boar spermatozoa. J. Anim. Sci. 36, 319-324 (1973). Bower, R. E., Jr., Graham, E. F., and Crabo, B.
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A technique for collecting epididymal spermatozoa and fluids from the boar virtually free from accessory sex gland secretions. .I. Reprod. Fertil. 33, 319-322 (1973). Dietzman, R. H., Rebelo, A. E., Graham, E. F., Crabo, B. G., and Lillehei, R. C. Long-term functional success following freezing of canine kidneys. Surgery 74, 181-189 (1973). Pace, M. M., and Graham, E. F. Components in egg yolk which protect bovine spermatozoa during freezing. J. Anim. Sci. 39, 1144-1149 (1974). Rebelo, A. E., Graham, E. F., Lillehei, R. C., and Dietzman, R. H. Freeze-thaw parameters in successful renal cryopreservation. J. Surg. Res. 16, XI-57 (1974). Rebelo, A. E., Graham, E. F., Crabo, B. G., Lillehei, R. C., and Dietzman, R. H. Surgical preparation, perfusion techniques and cryoprotectants used in successful freezing of the kidney. Surgery 75, 319-331 (1974). Graham, E. F. Ova transfer. In “Proceedings, 6th Technical Conference on Artificial Insemination, National Association of Animal Breeders, Chicago,” pp. 14-20, 1974. Graham, E. F., Larson, E. V., and Crabo, B. G. Freezing and freeze-drying bovine spermatozoa. In “Proceedings, 6th Technical Conference on Artificial Insemination, National Association of Animal Breeders, Chicago,” 1974. Pace, M. M., and Graham, E. F. Components in egg yolk which protect bovine spermatozoa during freezing. .I. Anim. Sci. 39, 1144-l 149 (1974). Dietzman, R. H., Kubota, S., Rebelo, A. E., Graham, E. F., Kim, C. H., and Lillehei, R. C. The effect of freezing the kidney on lifesupporting function after contralateral nephrectomy. Transplant. Proced. 6, 267-273 (1974). Kubota, S., Graham, E. F., Crabo, B. G., Lillehei, R. C., and Dietzman, R. H. Freeze-thaw parameters in successful renal cryopreservation. J. Surg. Res. 16, XI-57 (1974). Crabo, B. G., Zimmerman, K. J., Gustafsson, B., Holtman, M., Koh, T. J. P., and Graham, E. F. Effect of low doses of a-chlorohydrin on fertility and semen characterization and binding of the drug to spermatozoa in swine. Int. J. Fertil. 20, 87-94 (1975). Koh, T. J., Crabo, B. G., Tsou, H. L., and Graham, E. F. Fertility of liquid boar semen as influenced by breed and season. J. Anim. Sci. 42, 138-144 (1976). Graham, E. F., Vazquez, I. A., Schmehl, M. K. L., and Evensen, B. K. An assay of semen quality by use of Sephadex filtration. In
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“Proceedings, 8th Int. Cong. Anim. Reprod. AI, Cracow,” Vol. 4, pp. 896-899, 1976. 97. Smith, J. E., and Graham, E. F. High resolution size distributions of bull spermatozoa. In “Proceedings, 8th Int. Cong. Anim. Reprod. AI, Cracow,” Vol. 4, pp. 943-945, 1976. 98. Larson, E. V., and Graham, E. F. Fertility of freeze-dried bovine spermatozoa. In “Proceedings, 8th Int. Cong. Anim. Reprod. AI, Cracow,” Vol. 4, pp. 822-825, 1976. 99. Deibel, F. C., Jr., Smith, J. E., Crabo, B. G., and Graham, E. F. Evaluation of six assays of sperm quality by means of their accuracy, precision, and sensitivity in separating known induced levels of damage. In “Proceedings, 8th Int. Cong. Anim. Reprod. AI, Cracow,” Vol. 4, pp. 888-891, 1976. 100. Kubota, S., Graham, E. F., Crabo, B. G., Lillehei, R. C., and Dietzman, R. H. The effect of freeze rate, duration of phase transition and warming rate on survival of frozen canine kidneys. Cryobiology 13, 455-462 (1976). 101. Larson, E. V., and Graham, E. F. Freeze-drying of spermatozoa. International Symposium on Freeze-Drying. Develop. Biol. Stud. 36, 343348 (1977). 102. Graham, E. F., Schmehl, M. K. L., Evensen, B. K. and Nelson, D. S. An overview of research advances in A.I. and semen preservation and its application to the breeding industry. In “Proceedings, Annual Meeting of the Society for Theriogenology,” pp. 11l-l 19, 1977. 103. Graham, E. F., Schmehl, M. K. L., Evenson, B. K., and Nelson, D. S. Semen preservation in non-domestic animals. In “Proceedings of the Symposium of the London Zoological Society” Vol. 43, pp. 153-173, 1978. 104. Graham, E. F. Fundamentals of the preservation of spermatozoa. In “The Integrity of Frozen Spermatozoa,” pp. 4-44. Natl. Acad. Sci.. , Washington, DC, 1978. 105. Graham, E. F., and Crabo, B. G. Some methods of freezing and evaluating human spermatozoa. In “The Integrity of Frozen Spermatozoa,” pp. 274-304. Natl. Acad. Sci., Washington, DC, 1978. 106. Graham, E. F., Schmehl, M. K. L., Evensen, B. K., and Nelson, D. S. Viability assays for frozen semen. Cryobiology 15,242-244 (1978). 107. Erdahl, D. A., and Graham, E. F. Cryopreservation of Salmonid spermatozoa. Cryobiology 15, 362-364 (1978). 108. Graham, E. F., Schmehl, M. K. L., and Evensen, B. K. An overview of column separation of spermatozoa. In “Proceedings, 7th Technical Congress on Artificial Insemination
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and Reproduction, National Association Animal Breeders,” pp. 69-73, 1978. 109. Deibel, F. C., Jr., Graham, E. F., and Evensen, B. K. Technique and application of electronic counting and sizing of spermatozoa. In “Proceedings, 7th Technical Congress on Artificial Insemination and Reproduction, National Association Animal Breeders,” pp. 4.5-52, 1978. 110. Graham, E. F., Crabo, B. G., and Pace, M. M. Current status of semen preservation in the ram, boar and stallion. J. Anim. Sci. 47(Suppl. II), 80-l 18 (1978). 111. Crabo, B. G., and Graham, E. F. Freezing of spermatozoa. In “Animal Models for Research on Contraception and Fertility” (N. J. Alexander, Ed.), Hagerstown, MD, pp. 521527. Harper & Rowe, 1979. 112. Crabo, B. G., Zimmerman, K. J., Hunter, A. G., Moore, R., and Graham, E. F. Effect of a-chlorhydrin on epididymal sperm and plasma in swine. Arch. Androl. 3, 79-87 (1979). 113. Zimmerman, K. J., Crabo, B. G., Moore, R., and Graham, E. F. Movements of sodium and potassium into epididymal boar spermatozoa. Biol. Reprod. 21, 173-179 (1979). 114. Fayemi, E. O., Crabo, B. G., and Graham, E. F. Assay of frozen boar semen with Sephadex filtration. Theriogenology 12, 13-17 (1979). 115. Vazquez, I. A., and Graham, E. F. Effect of dialysis and glycerol on extender for freezing boar spermatozoa. In “Proceedings, 9th Int. Cong. Anim. Reprod. AI, Madrid” Vol. 5, pp. 442444, 1980. 116. Schmehl, M. K. L., Graham, E. F., and Nelson, D. S. Effects of cell concentration and added dead cells on fertility of frozen bovine spermatozoa. In “Proceedings, 9th Int. Cong. Anim. Reprod. AI, Madrid,” Vol. 5, pp. 393-395, 1980. 117. Nelson, D. S., Graham, E. F., and Schmehl, M. K. L. Fertility after dialyzing frozen turkey semen. In “Proceedings, 9th Int. Cong. Anim. Reprod. AI, Madrid,” Vol. 5, pp. 490492, 1980. 118. Erdahl, D. A., and Graham, E. F. Preservation of spermatozoa of fresh water fishes. In “Proceedings, 9th Int. Cong. Anim. Reprod. AI, Madrid,” Vol. 2, pp. 317-326, 1980. 119. Erdahl, D. A., and Graham, E. F. Preservation of spermatozoa of fish. Cryo-Left. 1, 203-208 (1980). 120. Graham, E. F., Schmehl, M. K. L., and Nelson, D. S. Problems with laboratory assays. In “Proceedings, 8th Technical Conference on Reproduction and Artificial Insemination, Na-
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tional Association Animal Breeders, Milwaukee,” pp. 67-70, 1980. 121. Jeyendran, R. S., Graham, E. F., and Schmehl, M. K. L. Fertility of dehydrated bovine spermatozoa. Cryobiology 18, 292-300 (1980). 122. Jeyendran, R. S., and Graham, E. F. An evaluation of cryoprotective compounds on bovine spermatozoa. Cryobiology 17, 458-464 (1980). 123. Graham, E. F. Sinopsis of problems in artificial insemination. In “Proceedings, 9th Int. Cong. Anim. Reprod. AI, Madrid,” Vol. I, pp. 9598, 1980. 124. Memon, M. A., Gustafsson, B. K., Graham, E. F., and Crabo, B. G. Influence of prostaglandins on acrosome morphology of ram spermatozoa. In “Proceedings, 9th Int. Cong. Anim. Reprod. AI, Madrid,” Vol. 4, pp. 458461, 1980. 125. Zavos, P. M., and Graham, E. F. Preservation of turkey spermatozoa by use of emulsion and supercooling. Cryobiology 18, 497-505 (1981). 126. Bower, R. E., Jr., Crabo, B. G., and Graham, E. F. The in vitro effect of boar semen on the contractility of rat uteri. Theriogenology 16, 553-563 (1981). 127. Pace, M. M., Sullivan, J. J., Elliott, F. I., Graham, E. F., and Coulter, G. H. Effects of thawing temperature, number of spermatozoa and spermatozoa1 quality on fertility of bovine spermatozoa packaged in .5 ml French straws. J. Anim. Sci. 53, 693-701 (1981). 128. Graham, E. F., Nelson, D. S., and Schmehl, M. K. L. Development of extender and techniques for frozen turkey semen. 1. Development. Poult. Sci. 61, 550-557 (1982). 129. Graham, E. F., Nelson, D. S., and Schmehl, M. K. L. Development of extender and techniques for frozen turkey semen. Il. Fertility trials. Poult. Sci. 61, 558-563 (1982). 130. Jeyendran, R. S., and Graham, E. F. pH changes of buffers during freezing. Cryobiology 19, 16-19 (1982). 131. Zavos, P. M., and Graham, E. F. Effects of various degrees of supercooling on motility and fertility of turkey spermatozoa. Cryo-Letf. 3, 71-77 (1982). 132. Zavos, P., and Graham, E. F. Effects of various degrees of supercooling and nucleation temperature on fertility of turkey spermatozoa. Cryobiology 20, 553-559 (1983). 133. Jeyendran, R. S., Hunter, A. G., and Graham, E. F. Alteration of seminal plasma proteins during freeze-drying. J. Dairy Sci. 66, 887-891 (1983). 134. Erdahl, A. E., Erdahl, D. E., and Graham, E. F. Cryopreservation of Salmonid spermatozoa. Aquaculture 43, 341-350 (1984).
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135. Heath, E., Jeyendran, R. S., and Graham, E. F. Ultrastructure of spermatozoa of the Asiatic elephant. Zentralbl. Veterinaermed. Reihe C 12, 245-252 (1984). 136. Smith, F. O., and Graham, E. F. Cryopreservation of canine semen: Technique and performance. In “Proceedings, 10th Int. Cong. Anim. Reprod. AI, Urbana,” Vol. I, Abstract 216, 1984. 137. Memon, M. A., Gustafsson, B. R., Graham, E. F., and Crabo, B. G. Effect of prostaglandin supplementation on frozen-thawed ram spermatozoa. In “Proceedings, 10th Int. Cong. Anim. Reprod. AI, Urbana,” Vol. 1, Abstract 201, 1984. 138. Graham, E. F. Semen quality and its preservation. Turkey Research 30, 32-38 (I 984). 139. Graham, E. F., Schmehl, M. L., and Deyo. R. C. M. Cryopreservation and fertility of fish, poultry and mammalian spermatozoa. In “Proceedings, 10th Technical Conference on Artificial Insemination and Reproduction, National Association Animal Breeders, Milwaukee,” pp. 4-31, 1984. 140. Burton, M., and Graham, E. F. A new method for thawing straw-frozen bovine spermatozoa. Theriogenology 4, 179-185 (1985). 141. Schmehl, M. K., and Graham, E. F. Changes in elemental composition of fertilized and unfertilized northern pike (Esox lucis) eggs incubated in buffer with and without dimethyl sulfoxide: An x-ray microanalysis study. Gamete Res. 14, 92-106 (1986). 142. Schmehl, M. K., Anderson, S. P., Vazquez, I. A., and Graham, E. F. The effect of dialysis on extended ram semen prior to freezing on post-thaw survival fertility. Cryobiology 23, 406-414 (1986). 143. Schmehl, M. K., Vazquez, I. A., and Graham, E. F. The effects of non-penetrating cryoprotectants added to TEST-yolk-glycerol extender on post thaw motility of ram spermatozoa. Cryobiology 23, 512-517 (1986). 144. Martin, R. S., Vazquez, I. A., Pursel, V., and Graham, E. F. Fertility results with dialyzed and refrigerated porcine semen transported between continents. In “Proceedings, World Pork Congress, Barcelona, Spain,” Vol. 2, pp. 5763, 1986. 145. Garcia, M. A., Fahning, M. L., and Graham, E. F. In vitro culture, freezing, thawing, and transfer of bovine embryos versus transfer of fresh embryos from the same collection: Preliminary results. Theriogenoiogy 26, 803-812 (1986). 146. Schmehl, M. K., Graham, E. F., and Erdahl, D. A. Chemical constituents of trout seminal
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plasma after minimal and maximal cell damage treatments with possible applications to semen evaluation assays. Aquaculture 62, 31 l-318 (1987). 147. Erdahl, A. W., Cloud, J. C., and Graham, E. F. Fertility of rainbow trout (Salmo gairdneri) gametes: Gamete viability in artificial media. Aquaculture 60, 323-332 (1987). 148. Garcia, M. A., and Graham, E. F. Effects of low molecular-weight fractions (LMWF) from milk, egg yolk, and seminal plasma on freezability of bovine spermatozoa. Cryobiology 24, 429-436 (1987). 149. Garcia, M. A., and Graham, E. F. Factors affecting the removal of low molecular weight fractions (LMWF) from egg yolk and seminal plasma in extended semen by dialysis: Effect on post-thaw sperm survival. Cryobiology 24, 437-445 (1987). 150. Garcia, M. A., and Graham, E. F. Dialysis of bovine semen and its effect on fresh and freezethawed spermatozoa. Cryobiology 24, 44&454 (1987). 151. Schmehl, M. K., and Graham, E. F. Comparative ultrastructure of the zona radiata from eggs of six species of Salmonids. Ce// Tissue Res. 250(3). 513-519 (1987).
152. Erdahl, A. W., and Graham, E. F. Fertility of teleost semen as affected by dilution and storage in a seminal plasma-mimicking medium. Aquaculture 60, 311-321 (1987). 153. Vazquez, I., Graham, E. F., Martinez, F., Alcaide, M., and Soriano, 1. Fertility with frozen ram semen in Manchego sheep. In “Proceedings, 11th Int. Congr. Anim. Reprod. A.I., Dublin,” Vol. 1, No. 3, p. 305, 1988. 154. Li, S. R., and Graham, E. F. Effect of dialysis, air, semen ratio, osmotic pressure, glycerol and glycerol removal on stored fowl spermatozoa. In “Proceedings, 11th Int. Congr. Anim. Reprod. AI., Dublin,” Vol. 3, p. 270, 1988. 155. Cloud, J. G., Erdahl, A. L., and Graham, E. F. Survival and continued normal development of fish embryos following incubation at reduced temperatures. Trans. Am. Fish. Sot. 117, 503-506 (1988). 156. Tajima, A., Graham, E. F., and Hawkins, D. M. Estimation of the relative fertilizing ability of frozen chicken spermatozoa using a heterospermic competition method. J. Reprod. Ferfil. 85, 1-5 (1989). 157. Schmehl, M. K. L., and Graham, E. F. Ultrastructure of the domestic tom cat (Fe/is domesrica) and Siberian tiger (Panrhea tigris alfica) spermatozoa. Theriogenology 31, 861874 (1989).
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158. Garcia, M. A., and Graham, E. F. Development of a buffer system for dialysis of bovine spermatozoa before freezing. I. Effect of zwitter ion buffers. Theriogenology 31, 1021-1028 (1989). 159. Garcia, M. A., and Graham, E. F. Development of a buffer system for dialysis of bovine spermatozoa before freezing. II. Effect of sugars and sugar alcohols on post-thaw motility. Theriogenology 31, 1029-1037 (1989). 160. Garcia, M. A., and Graham, E. F. Development of a buffer system for dialysis of bovine spermatozoa before freezing. III. Effect of different inorganic and organic salts on fresh and 31, frozen thawed semen. ‘fheriogenology 103%1048 (1989). 161. Ijaz, A., Hunter, A. G., and Graham, E. F. Identification of the capacitating agent for bovine sperm in egg yolk-TEST semen extender. J. Dairy Sci. 72, 2700-2706 (1989). 162. Byers, A. P., Hunter, A. G., Seal, U. S., Binczik, G. A., Graham, E. F., Reindl, N. J., and Tilson, R. L. In vitro induction of capacitation of fresh and frozen spermatozoa of the Siberian tiger (Panthera tigris). J. Reprod. Ferril. 86, W-607 (1989). 163. Graham, E. F., and Graham, J. K. The effect of whole ejaculate filtration on the morphology and fertility of bovine semen. J. Dairy Sci. 73, 91-97 (1989). 164. Tajima, A., Graham, E. F., Shoffner, R. N., Otis, J. S., and Hawkins, D. M. Cryopreservation of semen from unique lines of chicken germ plasm. Poulr. Sci. 69, 999-1002 (1990). 165. Abdelhakeam, A. A., Graham, E. F., and Vazquez, I. A. Studies on the presence and
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absence of glycerol in unfrozen and frozen ram semen: Fertility trials and the effect of dilution methods on freezing ram semen in the absence of glycerol. Cryobiology 28, 3W2 (1991). 166. Abdelhakeam, A. A., Graham, E. F., Vazquez, I. A., and Chaloner, K. M. Studies on the absence of glycerol in unfrozen and frozen ram semen. Development of an extender for freezing: Effects of osmotic pressure, egg yolk levels, type of sugars, and method of dilution. Cryobiology 28, 43-49 (1991). 167. Schmehl, M. K., Graham, E. F., and Kilkowski, S. M. Thermographic studies of phantom and canine kidneys thawed by microwave radiation. Cryobiology 27, 311-318 (1990). 168. Tibary, A., Graham, E. F., Asri, A., Boukhliq, R., and Deyo, R. Effect of dialysis or centrifugation on post-thaw motility and fertility of Santa Gertrudis bull semen collected by electroejaculation. Theriogenology 33, 733-739 (1990). 169. Schmehl, M. L., Graham, E. F., Byers, A. P., and Hunter, A. G. Characteristics of fresh and cryopreserved Siberian tiger (Panthera tigris altaica) semen and its seminal plasma chemical constituents. Zoo Biol. 9, 431436 (1990). 170. Anzar, M., Hassan, M. M., Graham, E. F., Deyo, R. C. M., and Singh, G. Usefulness of Hamilton Thorn Motility Analyzer (HTM2030)for the evaluation of bovine semen. Theriogenology 36, 307-317 (1991). 171. Tajima, A., Graham, E. F., and Hawkins, D. M. The effect of glycerol addition and reduction on the morphology of frozen and unfrozen 13, 6-14 chicken spermatozoa. Cryo-Lett. (1992).
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In Memoriam Edmund
Fay Graham
Ed Graham passed away on November 26, 1991, after a long and painful fight against cancer, We will remember him for his enthusiasm for cryobiology and his vision of the practical possibilities that cryopreservation offers, particularly of gametes. This enthusiasm and his fighting spirit rubbed off on many of the students, colleagues, and professionals within and outside the country who met him during his 40-year career as professor of reproductive physiology and cryobiology at the University of Minnesota. Ed Graham was born in Mesilla, New Mexico, on July 25, 1924, and grew up on a small dairy farm outside El Paso, Texas. He occasionally told about his childhood poverty and having to milk cows and deliver the milk before going to school and how this made him want to go to college and “learn all there is to know about the cow.” Before this could happen, however, he served in the Army’s 1Olst Airborne division which landed in France behind the enemy lines on the day before D-Day. It was nearly wiped out and Ed Graham was among the few survivors. After the war, Ed Graham earned a B.S. in dairy husbandry from Utah State University to “learn all about the cow,” but found that the Bachelor’s degree only gave him a taste for more knowledge. Studies for an M.S. degree, which he earned in 1952 at South Dakota State University, introduced him to reproductive physiology. He then proceeded to the University of Minnesota to study with the well-known dairy physiologist W. E. Petersen and was immediately put on the payroll. He remained at the University of Minnesota until his death. Ed
(1924-1991)
Graham started to teach courses in reproductive physiology in 1953 and his first M.S. advisee graduated in the same year that Ed Graham finished his Ph.D. Over the years more than one thousand students experienced his stimulating and enthusiastic classroom teaching. W. E. Peterson had attracted many talented graduate students, among them P. E. Dzuik, to Minnesota and lively discussions on how gamete preservation and manipulations could revolutionize animal breeding were common in the group. The very first attempt to artificially inseminate cows had been made at Minnesota in 1938 by C. L. Cole, a dairy geneticist and head of the department . County cooperatives for dairy cattle breeding were mushrooming in Minnesota and Wisconsin. In Cambridge a few years earlier, Chris Polge had devised the first technique for freeze preservation of bull semen. In this environment, Ed Graham was chosen by the department to stay and develop the promising “high tech” field of artificial insemination. He jumpstarted with his peers to work on various aspects of semen production and semen collection and last, but not least, to further develop freeze preservation of bull semen. It is no exaggeration to say that Ed Graham was very successful and instrumental in making it possible for these artificial insemination centers to convert to frozen semen. Many of his graduates went on to work in the centers and the technicians were initially trained at the University of Minnesota. The Minnesota/Wisconsin AI cooperatives supported his research until 1980. He was often an appreciated speaker at the National Association of Animal Breeders’
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$5.00
Copyright 0 1992 by Academic Press. Inc. All rights of reproduction in any form reserved.
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technical conferences. One of his highlights was “The usefulness of useless data,” which points out how human bias affects the fertility in field trials. The 1960swere a very active period in Ed Graham’s professional life. The feasibility of “the other gene multiplication system,” embryo transfer, was explored. Its practicality was shown and the second calf in the world (the first one was born in Madison, WI) from ova transfer was produced. Ed Graham always liked to see discoveries from his laboratory translate into practical uses, and so with former graduate students Harry Rajamannan and Melvyn Fahning, he founded in 1973 the world’s first embryo transfer company, International CryoBiological Services, Inc. He attempted to improve semen diluents by finding which individual constituents of semen were positively correlated to fertility. The greatest success was achieved in the turkey, and Ed Graham’s turkey semen extender may well be the one most used in the world today. At the beginning of the decade, he visited Japan and made lifelong professional relationships with prominent researchers there. As an immediate result of the visit, Drs. Nagase and Niwa were invited to Minnesota, where they together furthered the socalled pellet freezing technique. Pellet freezing on dry ice proved to be very useful for freezing large volumes, for example, as required for boar semen. I had the pleasure to begin working in Ed Graham’s laboratory in the late 1960s. The work was often interrupted by fishing trips on which research was discussed. Foremost on his mind was how to freeze semen from other agriculturally important species, and the process of freeze-drying. Ed Graham felt rightly that glycerol would be the major detriment to sperm survival in freeze-dried semen. In developing a glycerol-free system suitable for freeze-drying spermatozoa, Ed Graham laid the foundation for the solution of freezing boar spermatozoa, since the discovery that glycerol
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increased the permeability of boar spermatozoa for intracellular enzymes implied that glycerol was toxic. This information was received with skepticism by cryobiologists at the time but it is now clear that lower concentrations of glycerol favor fertility for the semen of many species. In the late 196Os,Ed Graham began collaboration with a transplantation surgeon, the late Richard C. Lillehei, on a project aiming to freeze-preserve kidneys for transplantation. This project continued into the mid-1970s. Some success was achieved in that kidneys frozen to -20°C survived and were able to sustain the life of dogs. Work on freeze-drying continued and it was found that sperm containing only 15% water could be rehydrated and retain fertility. However, sperm with less water lost their fertilizing ability. Although grain containing 15% water will not spoil, spermatozoa with this water content had to be stored in the frozen stage. This finding eliminated its practical use. Frozen spermatozoa and embryos of endangered species-a “library of life”were on Ed Graham’s mind from the 1970s through the rest of his life. He further developed electroejaculation to accomplish collection from animals of all sizes. He worked on dogs, cats, sheep, chicken, turkeys, and zoological species ranging from rare cats and tigers to elephants as well as fish. At the time of his death he had frozen semen stored from 30 to 40 species. Assay of frozen semen was another of Ed Graham’s interests. His eyesight throughout his scientific career was poor and he could not use a microscope well. To overcome this, he hooked up a videocamera to the microscope and was able to see sperm motility on the TV screen. This technique for visualizing sperm motility was appreciated by personnel at artificial insemination centers, most of which now employ this technique. Other techniques investigated include electronic sizing of spermatozoa to assess their osmotic reactivity, enzyme
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leakage, and Sephadex filtration. The latter technique was developed after Ed Graham for some reason poured bull semen through a Sephadex column and discovered that all spermatozoa in the effluent were motile. It may prove to be one of his most important discoveries. Ed Graham never found the mechanism of action of the filter, but this work is continuing. Apparently, the surface structure of the spermatozoa is the discriminating factor. Assay results with Sephadex filters seem to be better correlated to the fertility of the semen than any other laboratory technique described to date. Ed Graham gained an international reputation in many ways. He worked on a Spanish-American bilateral research project on artificial insemination in swine and sheep. He received honorary degrees from the Veterinary College of Madrid, Spain, and the Italian “Instituto Sperimentale Italian0 ‘Lazzaro Spallanzani’ per Fecondazione Artificiale” and was an honorary member of the Japanese Society for artificial insemination. He participated in an official USDA trip to Russia in 1976 with six other U.S. scientists. Vern Pursel, a participant on the trip and one of Ed’s former graduate students, wrote “that it was evident that Dr. Graham was the most widely recognized U.S. scientist in the group and considered the equal by no less than Soviet Academy member Milovanov (who wrote the first modern book on artificial insemination in 1934). It was Dr. Graham who was accorded at the opening ceremony and most honored at the closing ceremony even if he was not the leader or the eldest in the group.” He was also recognized nationally with several awards, for example, the National Association of Animal Breeder’s Physiology Research Award, by Minnesota Turkey Growers, Midwest Breeders, and several honor societies. Ed Graham’s favorite meetings were the International Congresses of Animal Reproduction and Artificial Insemination and those of the Society for Cryobiology. Many
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may remember his enthusiasm when he chaired the 14th Cryobiology meeting in Minneapolis in 1977. Members and pioneers in the society, perhaps foremost Father Luyet and his co-workers Lou Rapatz and Alan MacKenzie, influenced his interest in cryobiology substantially. In the mid-1980s Ed suffered from a detached retina in his good eye and became practically blind. This naturally had a dampening effect on his ability to do science but did not decrease the hours he spent at work. Nor did he stay home when a back problem proved to be bone cancer. His fighting spirit kept him going to the last day despite severe pain. However, we like to remember him as the powerful man that he was at the height of his career, enthusiastically talking about the advantages and possibilities of freeze preservation of biological material. Few persons could remain unaffected by Ed Graham and many students and collaborators carry his mark. With the death of Ed Graham, we have lost a remarkable man and researcher. Bo G. Crabo University of Minnesota Department of Animal Science St. Paul, Minnesota 55108 PUBLICATIONS
OF EDMUND
F. GRAHAM
“The Mechanical and Hormonal Methods of Dilating the Bovine Cervix.” MS. thesis, South Dakota State University, 1952. “The Effects of Different Planes of Nutrition and Frequencies of Collection on Bovine Semen Production.” Ph.D. thesis, University of Minnesota, 1955. 1. Hirsch, M., Graham, E. F., and Dracy, A. E. A classification for the identification of bovine noseprints. J. Dairy Sci. 35, 31&319 (1952). 2. Graham, E. F., Dracy, A. E., and Hirsch, M. Some similarities of monozygotic cattle noseprints. J. Dairy Sci. 36, 1l-13 (1953). 3. Graham, E. F., and Dracy, A. E. The effects of relaxin and mechanical dilation of the bovine cervix. J. Dairy Sci. 36, 772-777 (1953). 4. Dziuk, P. J., Graham, E. F., Donker, J. D., Marion, G. B., and Petersen, W. E. Electroejaculation in farm animals. J. Anim. Sci. 12, 945-946 (1953).
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5. Dziuk, P. J., Graham, E. F., Donker, J. D., Marion, G. B., and Petersen, W. E. Some observations in collection of semen from bulls, goats, boars and rams by electrical stimulation. Vet. Med. 49, 455-458 (1954). 6. Dziuk, P. J., Graham, E. F., and Petersen, W. E. The technique of electroejaculation and its use in dairy bulls. J. Duiry Sci. 37, 10351041 (1954). 7. Graham, E. F., Erickson, W. E., and Bayley, N. D. Conception results with frozen semen. In “Proceedings, 7th Annual Conference of the National Association of Artificial Breeders” Vol. 7, pp. 198-199, 1954. 8. Graham, E. F., Erickson, W. E., and Bayley, N. D. The effect of glycerol equilibration on frozen bovine spermatozoa. J. Dairy Sci. 40, 51c.515 (1957). 9. Donker, J. D., Nichols, J. R., Graham, E. F., and Petersen, W. E. Controlled estrus in cattle. In “Reproduction and Infertility,” Part IV, pp. 171-178. Pergamon, New York, 1958. IO. Larson, G. L. and Graham, E. F. Effects of low temperatures in storage of bovine semen. A.I. Dig. 6(12), 6-8 (1958). Il. Graham, E. F., Vogt, D. W., and Fisher, G. R. Effect of method of glycerol addition on the fertility of frozen bovine spermatozoa. J. Dairy Sci. 41, 1553-1556 (1958). 12. Erickson, W. E., and Graham, E. F. Effect of storage on nonreturn rate of frozen spermatozoa. A.I. Dig. 7, 8-9 (1959). 13. Larson, G. L., and Graham, E. F. Effects of rapid freezing on progressive motility of semen. A.I. Dig. 7, C7 (1959). 14. Graham, E. F., and Erickson, W. E. Comparison of glass ampules and plastic vials based on the nonreturn rate of frozen bovine spermatozoa. J. Dairy Sci. 42, 365-367 (1959). 15. Erickson, W. E., and Graham, E. F. Factors affecting the fertility of frozen bovine spermatozoa. J. Dairy Sci. 42, 520-528 (1959). 16. Graham, E. F., and Erickson, W. E. The effect of long time storage of frozen bovine spermatozoa. A.I. Dig. 7, 5-8 (1959). 17. Graham, E. F. Artificial insemination in the United States. Jpn. J. Anim. Reprod. 5, 81-86 (1959). 18. Dukelow, W. R., Frederick, E. C., and Graham, E. F. Frequency of ejaculation in the bovine. J. Dairy Sci. 43, 1335-1339 (1960). 19. Graham, E. F. New ideas in freezing semen. A.Z. ’ Dig. 9(ll), 8 (1961). 20. Graham, E. F. New ideas in freezing semen. In “Proceedings, 14th Annual Conference of the National Association of Animal Breeders, Syracuse, NY,” pp. 59-62, 1961.
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21. Pursel, V. G., and Graham, E. F. Induced estrus in anestrous ewes by use of progestogens and follicle stimulating hormone. J. Anim. Sci. 21, 132-136 (1962). 22. Avery, T. L., Cole, C. L., and Graham, E. F. Investigations associated with the transplantation of bovine ova. I. Synchronization of oestrus. J. Reprod. Fertil. 3, 206211 (1962). 23. Avery, T. L., Fahning, M. L., and Graham, E. F. Investigations associated with the transplantation of bovine ova. II. Superovulation. J. Reprod. Fertil. 3, 212-217 (1962). 24. Avery, T. L., and Graham, E. F. Investigations associated with the transplantation of bovine ova. III. Recovery and fertilization. J. Reprod. Fe&/. 3, 218-228 (1962). 25. Avery, T. L., Fahning, M. L., Pursel, V. G., and Graham, E. F. Investigations associated with the transplantation of bovine ova. IV. Transplantation of ova. J. Reprod. Fertil. 3, 229-238 (1962). 26. Graham, E. F. Controlling estrus in farm animals. In “Proceedings, Minnesota Nutrition Conference for Feed Manufacturers and Dealers,” pp. 13-19, 1962. 27. Graham, E. F. Factors affecting the freezing of bovine spermatozoa. Jpn. J. Anim. Reprod. 17, 591-597 (1963). 28. Graham, E. F. Biochemistry of semen. Jpn. J. Anim. Reprod. 17, 715-720 (1963). 29. Nagase, H., and Graham, E. F. Pelleted semen: Comparison of different extenders and processes on fertility of bovine spermatozoa. In “Proceedings, 5th Int. Cong. Anim. Reprod. AI, Trento.” Vol. 4, pp. 387-391, 1964. 30. Rajamannan, A. H. J., Graham, E. F., and Smith, F. The effect of a new extender, incorporating dried egg yolk on the fertility of bovine spermatozoa. In “Proceedings, 5th Int. Cong. Anim. Reprod. AI, Trento,” Vol. 4, pp. 392-397, 1964. 31. Nagase, H., Graham, E. F., and Niwa, T. Pelleted semen: The effect of glycerol level and composition of thawing solution on fertility of bovine spermatozoa. In “Proceedings, 5th Int. Cong. Anim. Reprod. AI, Trento,” Vol. 4, pp. 404-409, 1964. 32. Graham, E. F., Schultz, R. H., Johnson, L. A., and Fahning, M. L. A comparison of the free amino acids in seminal plasma of the bull, boar, cock, turkey and uterine fluids of the cow during estrus. In “Proceedings, 5th Int. Cong. Anim. Reprod. AI, Trento,” Vol. 4, pp. 381-386, 1964. 33. Rajamannan, A. H. J., Linnerud, A., and Graham, E. F. Lactation studies. IX. Relationship of milk flow rate and complementary milk to
IN MEMORIAM:
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milk yields in a herd of Holstein cattle. J. Dairy Sci. 49, 31-35.
34. Graham, E. F. Comments on freezing spermatozoa. In “Proceedings, 1st Technical Conference, National Association of Animal Breeders, Chicago,” pp. 61-63, 1966. 35. Graham, E. F. The use of a dye indicator for testing, training and evaluating technicians in artificial insemination. In “Proceedings, 1st Technical Conference, National Association of Animal Breeders, Chicago,” pp. 57-60, 1966. 36. Schultz, R. H., Fahning, M. L., and Graham, E. F. A technique for exposure of the bovine internal genetalia. Vet. Rec. 78, 91-93 (1966). 37. Ahluwalia, B. S., and Graham, E. F. Free amino acids in the semen of the fowl and turkey. J. Reprod. Fertil.
12, 365-368 (1966).
38. Ahluwalia, B. S., and Graham, E. F. Free carbohydrates in seminal plasma and sperm cells of semen of fowl. J. Reprod. Fertil. 12, 359361 (1966). 39. Fahning, M. L., Schultz, R. H., and Graham, E. F. A technique for collection of uterine fluid from the live cow. Vet. Rec. 79, 23&233 (1966). 40. Fahning, M. L., Schultz, R. H., Graham, E. F., Donker, J. D., and Mohrenweiser, H. W. Synchronization of oestrus in dairy heifers with 6-methyl-17-acetoxyprogesterone and its effect on conception rate. J. Reprod. Ferril. 12, 569-571 (1966). 41. Clark, J. B. K., Graham, E. F., Lewis, B. A., and Smith, F. o-Mannitol, erythritol and glycerol in bovine semen. J. Reprod. Fertil. 13, 189-197 (1967). 42. Fahning, M. L., Schultz, R. H., and Graham, E. F. The free amino acid content of uterine fluids and blood serum in the cow. J. Reprod. Fertil. 13, 229-236 (1967). 43. Graham, E. F., and Pace, M. M. Some biochemical changes in spermatozoa due to freezing. Cryobiology
4, 75-84 (1967).
44. Pursel, V. G., and Graham, E. F. Phospholipids of bovine spermatozoa and seminal plasma. J. Reprod. Ferril. 14, 203-211 (1967). 45. Rajamannan, A. H. J., and Graham, E. F. Spin freezing of semen. In “Proceedings, 6th Int. Cong. Anim. Reprod. AI, Paris,” Vol. 2, pp. 1145-1146, 1968. 46. Rajamannan, A. H. J., and Graham, E. F. Film freezing of semen. In “Proceedings, 6th Int. Cong. Anim. Reprod. AI, Paris,” Vol. 2, pp. 1141-1143, 1968. 47. Graham, E. F., Thompson, L., and Neville, W. Physical, physiochemical and chemical characteristics of boar semen. In “Proceedings,
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2nd Technical Conference on Artificial Insemination and Reproduction, National Association of Animal Breeders,” pp. 55-63, 1968. 48. Graham, E. F. The usefulness of useless data field tests and responsibilities. In “Proceedings, 2nd Technical Conference on Artificial Insemination and Reproduction, National Association of Animal Breeders,” pp. 18-22, 1968. 49. Beisang, A. A., Dietzman, R. H., Mayo, C. H., Graham, E. F., and Lillehei, R. C. Damage during perfusion procedures used to prepare whole organs for freezing. Cryobiology 5, 273277 (1969). 50. Carter, J. E., Beisang, A. A., Ahlgren, F. F.,
Dorman, D. F., and Graham, E. F. An automatic temperature control device. Cryobiology 5, 343-346(1969). 51. Schultz, R. H., Burcalow,
H. B., Fahning, M. L., Graham, E. F., and Weber, A. F. Karyometric study of epithelial cells lining the glands of the bovine endometrium. J. Reprod. Fertil. 19, 169-171 (1969). 52. Gibson, C. D., and Graham, E. F. The relationship between fertility and post-freeze motility of bull spermatozoa (by pellet freezing) without glycerol. J. Reprod. Fertil. 20, 155-157 (1969). 53. Beisang, A. A., Lillehei,
R. C., and Graham, E. F. Cryoprotective techniques in the freezing of whole organs. Cryobiology 5, 223-229 (1969). 54. Beisang, A. A., Graham, E. F.. Lillehei, E. C., Dietzman, R. H., and Carter, J. E. Enzyme levels in the effluent perfusates from frozen canine kidneys. Transplant. hoc. 1, 862-881
(1969). 55. Al-Hakim, M. K.. Graham, E. F., and Schmehl,
M. L. Free amino acids and amino compounds in bovine seminal plasma. J. Dairy Sci. 53, 84-88 (1970). 56. Graham, E. F.. Schmehl, M. K. L., and MakiLaurila, M. Some physical and chemical methods of evaluating semen. In “Proceedings, 3rd Technical Conference on Artificial Insemination, National Association of Animal Breeders,” pp. 44-48, 1970. 57. Carter, J. E., Schellpfeffer, D. A., Beisang, A. A., Graham, E. F., and Lillehei, R. C. The effect of storage temperature and media on the activity of lactic dehydrogenase and glutamic oxalacetic transaminase in perfusates from frozen canine kidneys. Cryobiology 6,533-541 (1970). 58. Beisang, A. A., Feemster, J., Dietzman, R. H., Uchida, H., Carter, J. E., Graham, E. F., and Lillehei, R. C. Damage assay of kidneys fro-
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zen by intraarterial perfusion with a fluorocarbon. Fed. Proc. 29, 1782-1788 (1970). Pace, M. M., and Graham, E. F. The release of glutamic oxaloacetic transaminase from bovine spermatozoa as a test method of assessing semen quality and fertility. Rio/. Reprod. 2, 140-146 (1970). Crabo, B. G., Bower, R. E., Brown, K. I., Graham, E. F., and Pace, M. M. Extracellular glutamic-oxaloacetic transaminase as a measure on membrane injury in spermatozoa during treatment. In “Current Problems in Fertility” (A. Ingelman-Sundberg and N.-O. Lunell, Eds.), pp. 33-38. Plenum, New York, 1971. Graham, E. F., Rajamannan, A. H. J., Schmehl, M. K. L., Maki-Laurila, M., and Bower, R. E. Preliminary report on procedure and rationale for freezing boar semen. A.I. Dig. 19, 12-14 (1971). Brown, K. I., and Graham, E. F. Effect of semen quality on fertility in turkeys. Poult. Sci. 50, 295-297 (1971). Brown, K. I., and Graham, E. F. Effect of some cryophylactic agents on turkey spermatozoa. Poult. SC;. 50, 832-835 (1971). Graham, E. F., and Brown, K. I. Effect of osmotic pressure of semen extenders on the fertility and hatchability of turkey eggs. Poult. Sci. 50, 836-838 (1971). Rajamannan, A. H. J., Graham, E. F., and Schmehl, M. K. L. Effect of holding on bovine semen. A.I. Dig. 19, 6 (1971). Graham, E. F., Rajamannan, A. H. J., Schmehl, M. K. L., Maki-Lauria, M., and Bower, R. E. Fertility studies with frozen boar spermatozoa. A.I. Dig. 19, 6-16 (1971). Brown, K. I., Crabo, B. G., Graham, E. F., and Pace, M. M. Some factors affecting loss of intracellular enzymes from spermatozoa. Cryobiology 8, 220-224 (1971). Graham, E. F., Schmehl, M. L., Brown, K. I., Crabo, B. G., and Ertel, G. Some chemical studies of the female reproductive tract and seminal plasma of the male turkey and their relationship to fertility. Poult. Sci. 50, 11701181 (1971). Schultz, R. D., Fahning, M. L., and Graham, E. F. A chemical study of uterine fluid and blood serum of normal cows during the oestrous cycle. J. Reprod. Fertil. 27, 335-367 (1971). Alexander, F. C. M., Zemjanis, R., Graham, E. F., and Schmehl, M. L. Semen characteristics and chemistry from bulls before and after seminal vesiculectomy and after vasectomy. J. Dairy Sci. 54, 1530-1535(1971). Ellery, J. C., Graham, E. F., and Zemjanis, R.
72.
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F. GRAHAM Artificial insemination of pony mares with semen frozen and stored in liquid nitrogen. Am. J. Vet. Res. 32, 1693-1698 (1971). Carter, J. E., Graham, E. F., Lillehei, R. C., and Blackshear, P. L. The effect of high hydrostatic pressure and low temperature on lactate dehydrogenase and glutamic oxalacetic transaminase. Cryobiology 8, 524-534 (1971). Graham, E. F., Crabo, B. G., and Brown, K. I. Effect of some zwitter ion buffers on the freezing and storage of spermatozoa. I. Bull. .I. Dairy Sci. 55, 372-378 (1972). Brown, K. I., Graham, E. F., and Crabo, B. G. Effect of some hydrogen-ion buffers on storage and freezing of turkey spermatozoa. Poult. Sci. 51, 840-849 (1972). Crabo, B. G., Brown, K. I., and Graham, E. F. Effect of some buffers on the storage and freezing of boar spermatozoa. 1. Anim. Sci. 35, 377-382 (1972). Dziuk, P. J., Lewis, J. M., Graham, E. F., and Moyer, R. H. Comparison between natural service and artificial insemination with fresh or frozen sperm at an appointed time in the ewe. .I. Anim. Sci. 35, 572-582 (1972). Crabo, B. G., and Graham, E. F. Correlation between some laboratory methods for evaluation of boar semen after freezing. In “Proceedings, 7th Int. Cong. Anim. Reprod. AI, Munich,” Vol. 2, pp. 1639-1644, 1972. Graham, E. F., and Crabo, B. G. Some factors influencing the freezing of boar semen. In “Proceedings, 7th Int. Cong. Anim. Reprod. AI, Munich,” Vol. 2, pp. 1627-1632, 1972. Smith, J. D., and Graham, E. F. Free amino acids in ram seminal plasma. J. Anim. Sci. 34, 601604 (1972). Graham, E. F. The state of the art in cryobiology. In “Application of Cryogenic Technology,” Vol. 5, pp. 182-192. Scholium Intemational, Inc., Whitestone, NY, 1972. Carter, J. E., Risch, S. J., Graham, E. F., and Lillehei, R. C. The effect of pressure and cooling rate on spermatozoa, kidney tissue and the whole kidney. Cryobiology 10, 263-270 (1973). Graham, E. F., Crabo, B. G., and Schmehl, M. K. L. Utilization of enzyme assay in developing techniques for freezing semen. In “atti dell’ Simposio Internazional di Zootecina, 15-17 April, Milan. Italy,” pp. 95111, 1973. Bower, R. E., Jr., Crabo, B. G., Pace, M. M., and Graham, E. F. Effects of dilution and glycerol on the release of glutamic oxaloacetic transaminase (GOT) from boar spermatozoa. J. Anim. Sci. 36, 319-324 (1973). Bower, R. E., Jr., Graham, E. F., and Crabo, B.
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A technique for collecting epididymal spermatozoa and fluids from the boar virtually free from accessory sex gland secretions. .I. Reprod. Fertil. 33, 319-322 (1973). Dietzman, R. H., Rebelo, A. E., Graham, E. F., Crabo, B. G., and Lillehei, R. C. Long-term functional success following freezing of canine kidneys. Surgery 74, 181-189 (1973). Pace, M. M., and Graham, E. F. Components in egg yolk which protect bovine spermatozoa during freezing. J. Anim. Sci. 39, 1144-1149 (1974). Rebelo, A. E., Graham, E. F., Lillehei, R. C., and Dietzman, R. H. Freeze-thaw parameters in successful renal cryopreservation. J. Surg. Res. 16, XI-57 (1974). Rebelo, A. E., Graham, E. F., Crabo, B. G., Lillehei, R. C., and Dietzman, R. H. Surgical preparation, perfusion techniques and cryoprotectants used in successful freezing of the kidney. Surgery 75, 319-331 (1974). Graham, E. F. Ova transfer. In “Proceedings, 6th Technical Conference on Artificial Insemination, National Association of Animal Breeders, Chicago,” pp. 14-20, 1974. Graham, E. F., Larson, E. V., and Crabo, B. G. Freezing and freeze-drying bovine spermatozoa. In “Proceedings, 6th Technical Conference on Artificial Insemination, National Association of Animal Breeders, Chicago,” 1974. Pace, M. M., and Graham, E. F. Components in egg yolk which protect bovine spermatozoa during freezing. .I. Anim. Sci. 39, 1144-l 149 (1974). Dietzman, R. H., Kubota, S., Rebelo, A. E., Graham, E. F., Kim, C. H., and Lillehei, R. C. The effect of freezing the kidney on lifesupporting function after contralateral nephrectomy. Transplant. Proced. 6, 267-273 (1974). Kubota, S., Graham, E. F., Crabo, B. G., Lillehei, R. C., and Dietzman, R. H. Freeze-thaw parameters in successful renal cryopreservation. J. Surg. Res. 16, XI-57 (1974). Crabo, B. G., Zimmerman, K. J., Gustafsson, B., Holtman, M., Koh, T. J. P., and Graham, E. F. Effect of low doses of a-chlorohydrin on fertility and semen characterization and binding of the drug to spermatozoa in swine. Int. J. Fertil. 20, 87-94 (1975). Koh, T. J., Crabo, B. G., Tsou, H. L., and Graham, E. F. Fertility of liquid boar semen as influenced by breed and season. J. Anim. Sci. 42, 138-144 (1976). Graham, E. F., Vazquez, I. A., Schmehl, M. K. L., and Evensen, B. K. An assay of semen quality by use of Sephadex filtration. In
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“Proceedings, 8th Int. Cong. Anim. Reprod. AI, Cracow,” Vol. 4, pp. 896-899, 1976. 97. Smith, J. E., and Graham, E. F. High resolution size distributions of bull spermatozoa. In “Proceedings, 8th Int. Cong. Anim. Reprod. AI, Cracow,” Vol. 4, pp. 943-945, 1976. 98. Larson, E. V., and Graham, E. F. Fertility of freeze-dried bovine spermatozoa. In “Proceedings, 8th Int. Cong. Anim. Reprod. AI, Cracow,” Vol. 4, pp. 822-825, 1976. 99. Deibel, F. C., Jr., Smith, J. E., Crabo, B. G., and Graham, E. F. Evaluation of six assays of sperm quality by means of their accuracy, precision, and sensitivity in separating known induced levels of damage. In “Proceedings, 8th Int. Cong. Anim. Reprod. AI, Cracow,” Vol. 4, pp. 888-891, 1976. 100. Kubota, S., Graham, E. F., Crabo, B. G., Lillehei, R. C., and Dietzman, R. H. The effect of freeze rate, duration of phase transition and warming rate on survival of frozen canine kidneys. Cryobiology 13, 455-462 (1976). 101. Larson, E. V., and Graham, E. F. Freeze-drying of spermatozoa. International Symposium on Freeze-Drying. Develop. Biol. Stud. 36, 343348 (1977). 102. Graham, E. F., Schmehl, M. K. L., Evensen, B. K. and Nelson, D. S. An overview of research advances in A.I. and semen preservation and its application to the breeding industry. In “Proceedings, Annual Meeting of the Society for Theriogenology,” pp. 11l-l 19, 1977. 103. Graham, E. F., Schmehl, M. K. L., Evenson, B. K., and Nelson, D. S. Semen preservation in non-domestic animals. In “Proceedings of the Symposium of the London Zoological Society” Vol. 43, pp. 153-173, 1978. 104. Graham, E. F. Fundamentals of the preservation of spermatozoa. In “The Integrity of Frozen Spermatozoa,” pp. 4-44. Natl. Acad. Sci.. , Washington, DC, 1978. 105. Graham, E. F., and Crabo, B. G. Some methods of freezing and evaluating human spermatozoa. In “The Integrity of Frozen Spermatozoa,” pp. 274-304. Natl. Acad. Sci., Washington, DC, 1978. 106. Graham, E. F., Schmehl, M. K. L., Evensen, B. K., and Nelson, D. S. Viability assays for frozen semen. Cryobiology 15,242-244 (1978). 107. Erdahl, D. A., and Graham, E. F. Cryopreservation of Salmonid spermatozoa. Cryobiology 15, 362-364 (1978). 108. Graham, E. F., Schmehl, M. K. L., and Evensen, B. K. An overview of column separation of spermatozoa. In “Proceedings, 7th Technical Congress on Artificial Insemination
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and Reproduction, National Association Animal Breeders,” pp. 69-73, 1978. 109. Deibel, F. C., Jr., Graham, E. F., and Evensen, B. K. Technique and application of electronic counting and sizing of spermatozoa. In “Proceedings, 7th Technical Congress on Artificial Insemination and Reproduction, National Association Animal Breeders,” pp. 4.5-52, 1978. 110. Graham, E. F., Crabo, B. G., and Pace, M. M. Current status of semen preservation in the ram, boar and stallion. J. Anim. Sci. 47(Suppl. II), 80-l 18 (1978). 111. Crabo, B. G., and Graham, E. F. Freezing of spermatozoa. In “Animal Models for Research on Contraception and Fertility” (N. J. Alexander, Ed.), Hagerstown, MD, pp. 521527. Harper & Rowe, 1979. 112. Crabo, B. G., Zimmerman, K. J., Hunter, A. G., Moore, R., and Graham, E. F. Effect of a-chlorhydrin on epididymal sperm and plasma in swine. Arch. Androl. 3, 79-87 (1979). 113. Zimmerman, K. J., Crabo, B. G., Moore, R., and Graham, E. F. Movements of sodium and potassium into epididymal boar spermatozoa. Biol. Reprod. 21, 173-179 (1979). 114. Fayemi, E. O., Crabo, B. G., and Graham, E. F. Assay of frozen boar semen with Sephadex filtration. Theriogenology 12, 13-17 (1979). 115. Vazquez, I. A., and Graham, E. F. Effect of dialysis and glycerol on extender for freezing boar spermatozoa. In “Proceedings, 9th Int. Cong. Anim. Reprod. AI, Madrid” Vol. 5, pp. 442444, 1980. 116. Schmehl, M. K. L., Graham, E. F., and Nelson, D. S. Effects of cell concentration and added dead cells on fertility of frozen bovine spermatozoa. In “Proceedings, 9th Int. Cong. Anim. Reprod. AI, Madrid,” Vol. 5, pp. 393-395, 1980. 117. Nelson, D. S., Graham, E. F., and Schmehl, M. K. L. Fertility after dialyzing frozen turkey semen. In “Proceedings, 9th Int. Cong. Anim. Reprod. AI, Madrid,” Vol. 5, pp. 490492, 1980. 118. Erdahl, D. A., and Graham, E. F. Preservation of spermatozoa of fresh water fishes. In “Proceedings, 9th Int. Cong. Anim. Reprod. AI, Madrid,” Vol. 2, pp. 317-326, 1980. 119. Erdahl, D. A., and Graham, E. F. Preservation of spermatozoa of fish. Cryo-Left. 1, 203-208 (1980). 120. Graham, E. F., Schmehl, M. K. L., and Nelson, D. S. Problems with laboratory assays. In “Proceedings, 8th Technical Conference on Reproduction and Artificial Insemination, Na-
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tional Association Animal Breeders, Milwaukee,” pp. 67-70, 1980. 121. Jeyendran, R. S., Graham, E. F., and Schmehl, M. K. L. Fertility of dehydrated bovine spermatozoa. Cryobiology 18, 292-300 (1980). 122. Jeyendran, R. S., and Graham, E. F. An evaluation of cryoprotective compounds on bovine spermatozoa. Cryobiology 17, 458-464 (1980). 123. Graham, E. F. Sinopsis of problems in artificial insemination. In “Proceedings, 9th Int. Cong. Anim. Reprod. AI, Madrid,” Vol. I, pp. 9598, 1980. 124. Memon, M. A., Gustafsson, B. K., Graham, E. F., and Crabo, B. G. Influence of prostaglandins on acrosome morphology of ram spermatozoa. In “Proceedings, 9th Int. Cong. Anim. Reprod. AI, Madrid,” Vol. 4, pp. 458461, 1980. 125. Zavos, P. M., and Graham, E. F. Preservation of turkey spermatozoa by use of emulsion and supercooling. Cryobiology 18, 497-505 (1981). 126. Bower, R. E., Jr., Crabo, B. G., and Graham, E. F. The in vitro effect of boar semen on the contractility of rat uteri. Theriogenology 16, 553-563 (1981). 127. Pace, M. M., Sullivan, J. J., Elliott, F. I., Graham, E. F., and Coulter, G. H. Effects of thawing temperature, number of spermatozoa and spermatozoa1 quality on fertility of bovine spermatozoa packaged in .5 ml French straws. J. Anim. Sci. 53, 693-701 (1981). 128. Graham, E. F., Nelson, D. S., and Schmehl, M. K. L. Development of extender and techniques for frozen turkey semen. 1. Development. Poult. Sci. 61, 550-557 (1982). 129. Graham, E. F., Nelson, D. S., and Schmehl, M. K. L. Development of extender and techniques for frozen turkey semen. Il. Fertility trials. Poult. Sci. 61, 558-563 (1982). 130. Jeyendran, R. S., and Graham, E. F. pH changes of buffers during freezing. Cryobiology 19, 16-19 (1982). 131. Zavos, P. M., and Graham, E. F. Effects of various degrees of supercooling on motility and fertility of turkey spermatozoa. Cryo-Letf. 3, 71-77 (1982). 132. Zavos, P., and Graham, E. F. Effects of various degrees of supercooling and nucleation temperature on fertility of turkey spermatozoa. Cryobiology 20, 553-559 (1983). 133. Jeyendran, R. S., Hunter, A. G., and Graham, E. F. Alteration of seminal plasma proteins during freeze-drying. J. Dairy Sci. 66, 887-891 (1983). 134. Erdahl, A. E., Erdahl, D. E., and Graham, E. F. Cryopreservation of Salmonid spermatozoa. Aquaculture 43, 341-350 (1984).
IN MEMORIAM:
EDMUND
135. Heath, E., Jeyendran, R. S., and Graham, E. F. Ultrastructure of spermatozoa of the Asiatic elephant. Zentralbl. Veterinaermed. Reihe C 12, 245-252 (1984). 136. Smith, F. O., and Graham, E. F. Cryopreservation of canine semen: Technique and performance. In “Proceedings, 10th Int. Cong. Anim. Reprod. AI, Urbana,” Vol. I, Abstract 216, 1984. 137. Memon, M. A., Gustafsson, B. R., Graham, E. F., and Crabo, B. G. Effect of prostaglandin supplementation on frozen-thawed ram spermatozoa. In “Proceedings, 10th Int. Cong. Anim. Reprod. AI, Urbana,” Vol. 1, Abstract 201, 1984. 138. Graham, E. F. Semen quality and its preservation. Turkey Research 30, 32-38 (I 984). 139. Graham, E. F., Schmehl, M. L., and Deyo. R. C. M. Cryopreservation and fertility of fish, poultry and mammalian spermatozoa. In “Proceedings, 10th Technical Conference on Artificial Insemination and Reproduction, National Association Animal Breeders, Milwaukee,” pp. 4-31, 1984. 140. Burton, M., and Graham, E. F. A new method for thawing straw-frozen bovine spermatozoa. Theriogenology 4, 179-185 (1985). 141. Schmehl, M. K., and Graham, E. F. Changes in elemental composition of fertilized and unfertilized northern pike (Esox lucis) eggs incubated in buffer with and without dimethyl sulfoxide: An x-ray microanalysis study. Gamete Res. 14, 92-106 (1986). 142. Schmehl, M. K., Anderson, S. P., Vazquez, I. A., and Graham, E. F. The effect of dialysis on extended ram semen prior to freezing on post-thaw survival fertility. Cryobiology 23, 406-414 (1986). 143. Schmehl, M. K., Vazquez, I. A., and Graham, E. F. The effects of non-penetrating cryoprotectants added to TEST-yolk-glycerol extender on post thaw motility of ram spermatozoa. Cryobiology 23, 512-517 (1986). 144. Martin, R. S., Vazquez, I. A., Pursel, V., and Graham, E. F. Fertility results with dialyzed and refrigerated porcine semen transported between continents. In “Proceedings, World Pork Congress, Barcelona, Spain,” Vol. 2, pp. 5763, 1986. 145. Garcia, M. A., Fahning, M. L., and Graham, E. F. In vitro culture, freezing, thawing, and transfer of bovine embryos versus transfer of fresh embryos from the same collection: Preliminary results. Theriogenoiogy 26, 803-812 (1986). 146. Schmehl, M. K., Graham, E. F., and Erdahl, D. A. Chemical constituents of trout seminal
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plasma after minimal and maximal cell damage treatments with possible applications to semen evaluation assays. Aquaculture 62, 31 l-318 (1987). 147. Erdahl, A. W., Cloud, J. C., and Graham, E. F. Fertility of rainbow trout (Salmo gairdneri) gametes: Gamete viability in artificial media. Aquaculture 60, 323-332 (1987). 148. Garcia, M. A., and Graham, E. F. Effects of low molecular-weight fractions (LMWF) from milk, egg yolk, and seminal plasma on freezability of bovine spermatozoa. Cryobiology 24, 429-436 (1987). 149. Garcia, M. A., and Graham, E. F. Factors affecting the removal of low molecular weight fractions (LMWF) from egg yolk and seminal plasma in extended semen by dialysis: Effect on post-thaw sperm survival. Cryobiology 24, 437-445 (1987). 150. Garcia, M. A., and Graham, E. F. Dialysis of bovine semen and its effect on fresh and freezethawed spermatozoa. Cryobiology 24, 44&454 (1987). 151. Schmehl, M. K., and Graham, E. F. Comparative ultrastructure of the zona radiata from eggs of six species of Salmonids. Ce// Tissue Res. 250(3). 513-519 (1987).
152. Erdahl, A. W., and Graham, E. F. Fertility of teleost semen as affected by dilution and storage in a seminal plasma-mimicking medium. Aquaculture 60, 311-321 (1987). 153. Vazquez, I., Graham, E. F., Martinez, F., Alcaide, M., and Soriano, 1. Fertility with frozen ram semen in Manchego sheep. In “Proceedings, 11th Int. Congr. Anim. Reprod. A.I., Dublin,” Vol. 1, No. 3, p. 305, 1988. 154. Li, S. R., and Graham, E. F. Effect of dialysis, air, semen ratio, osmotic pressure, glycerol and glycerol removal on stored fowl spermatozoa. In “Proceedings, 11th Int. Congr. Anim. Reprod. AI., Dublin,” Vol. 3, p. 270, 1988. 155. Cloud, J. G., Erdahl, A. L., and Graham, E. F. Survival and continued normal development of fish embryos following incubation at reduced temperatures. Trans. Am. Fish. Sot. 117, 503-506 (1988). 156. Tajima, A., Graham, E. F., and Hawkins, D. M. Estimation of the relative fertilizing ability of frozen chicken spermatozoa using a heterospermic competition method. J. Reprod. Ferfil. 85, 1-5 (1989). 157. Schmehl, M. K. L., and Graham, E. F. Ultrastructure of the domestic tom cat (Fe/is domesrica) and Siberian tiger (Panrhea tigris alfica) spermatozoa. Theriogenology 31, 861874 (1989).
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IN MEMORIAM:
EDMUND
158. Garcia, M. A., and Graham, E. F. Development of a buffer system for dialysis of bovine spermatozoa before freezing. I. Effect of zwitter ion buffers. Theriogenology 31, 1021-1028 (1989). 159. Garcia, M. A., and Graham, E. F. Development of a buffer system for dialysis of bovine spermatozoa before freezing. II. Effect of sugars and sugar alcohols on post-thaw motility. Theriogenology 31, 1029-1037 (1989). 160. Garcia, M. A., and Graham, E. F. Development of a buffer system for dialysis of bovine spermatozoa before freezing. III. Effect of different inorganic and organic salts on fresh and 31, frozen thawed semen. ‘fheriogenology 103%1048 (1989). 161. Ijaz, A., Hunter, A. G., and Graham, E. F. Identification of the capacitating agent for bovine sperm in egg yolk-TEST semen extender. J. Dairy Sci. 72, 2700-2706 (1989). 162. Byers, A. P., Hunter, A. G., Seal, U. S., Binczik, G. A., Graham, E. F., Reindl, N. J., and Tilson, R. L. In vitro induction of capacitation of fresh and frozen spermatozoa of the Siberian tiger (Panthera tigris). J. Reprod. Ferril. 86, W-607 (1989). 163. Graham, E. F., and Graham, J. K. The effect of whole ejaculate filtration on the morphology and fertility of bovine semen. J. Dairy Sci. 73, 91-97 (1989). 164. Tajima, A., Graham, E. F., Shoffner, R. N., Otis, J. S., and Hawkins, D. M. Cryopreservation of semen from unique lines of chicken germ plasm. Poulr. Sci. 69, 999-1002 (1990). 165. Abdelhakeam, A. A., Graham, E. F., and Vazquez, I. A. Studies on the presence and
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absence of glycerol in unfrozen and frozen ram semen: Fertility trials and the effect of dilution methods on freezing ram semen in the absence of glycerol. Cryobiology 28, 3W2 (1991). 166. Abdelhakeam, A. A., Graham, E. F., Vazquez, I. A., and Chaloner, K. M. Studies on the absence of glycerol in unfrozen and frozen ram semen. Development of an extender for freezing: Effects of osmotic pressure, egg yolk levels, type of sugars, and method of dilution. Cryobiology 28, 43-49 (1991). 167. Schmehl, M. K., Graham, E. F., and Kilkowski, S. M. Thermographic studies of phantom and canine kidneys thawed by microwave radiation. Cryobiology 27, 311-318 (1990). 168. Tibary, A., Graham, E. F., Asri, A., Boukhliq, R., and Deyo, R. Effect of dialysis or centrifugation on post-thaw motility and fertility of Santa Gertrudis bull semen collected by electroejaculation. Theriogenology 33, 733-739 (1990). 169. Schmehl, M. L., Graham, E. F., Byers, A. P., and Hunter, A. G. Characteristics of fresh and cryopreserved Siberian tiger (Panthera tigris altaica) semen and its seminal plasma chemical constituents. Zoo Biol. 9, 431436 (1990). 170. Anzar, M., Hassan, M. M., Graham, E. F., Deyo, R. C. M., and Singh, G. Usefulness of Hamilton Thorn Motility Analyzer (HTM2030)for the evaluation of bovine semen. Theriogenology 36, 307-317 (1991). 171. Tajima, A., Graham, E. F., and Hawkins, D. M. The effect of glycerol addition and reduction on the morphology of frozen and unfrozen 13, 6-14 chicken spermatozoa. Cryo-Lett. (1992).
