GLYCEROL-FREEZING TECHNIQUE AS AN AID
IN BLOOD TYPING OF WHALES*JOHN E. CUSHING**, KAZUO FUJINO AND
KOZO TAKAHASHI***This paper describes the application of the glycerol-freezing preservation
technique to blood type studies on whales. Following the lead ofrecent trends in anthropology (cf. Mourant, 1954), blood type antigens
are receiving increasing attention as " genetic tags " useful as aids inthe definition of intraspecific populations of marine animals (cf. Fujino,
1956; Ridgway et al., 1958; Suzuki et al., 1958). Method for preserving intact erythrocytes between the time of their collection and serological characterization are therefore of considerable interest. In contrast
to samples of human or animal bloods which often can be taken andpreserved under controlled conditions (Stormont et al., 1958 ; Strumia,1958), the erythrocytes of marine forms are usually collected where
sterile and other precautions are not practical. (Of note is the fact thatwhale erythrocytes can be preserved for one month or longer by theaddition of the antimicrobial, guanofuracin, 5-Nitro-2-furfurylidine-aminoguanidine Hydrochloride, Toyama Chemical Co., Tokyo).

Preliminary studies on fish (Cushing, 1956; Cushing et al., 1957) having shown that the glycerol-freezing technique offered considerable
promise, efforts were made to learn of its suitability for large scale
research. This technique has been in use in various laboratories concerned with human and domestic animal blood type studies since it was
first applied to blood bank research (cf. Kabat, 1956, p. 92 for a reviewof this subject). The general method employed here is based on consideration of the various researches noted above. The typing techniquesand methods used are the same as described in earlier papers on the
blood typing of marine animals (see above). (Of basic significance are thefacts that erythrocytes hemolyze completely if frozen and thawed in theabsence of glycerol (or related preservatives such as ethylene glycol),and that glycerol inhibits agglutination reactions very markedly in concentrations suitable for preservation.

* Supported in part by grants of the Japanese Fisheries Agency; the United States
Dept. of the Navy, Office of Naval Research; and the University of California.
** University of California, Santa Barbara, California, U.S. A., John Simon Guggenheim
Memorial Foundation Fellow, 1958-59, Dept. of Fisheries, Faculty of Agriculture, Tokyo
University, Tokyo.
*** Department of Fisheries, Faculty of Agriculture, Tokyo University, Tokyo.
89

90

J.E. CUSHING, K. FUJINO AND K. TAKAHASHI

The application of the glycerol-freezing technique will be taken up by
separate species.

Finback whales (Balaenoptera physalus).
Samples of blood from finback whales were first preserved by glycerolfreezing in the summer of 1957, when six were obtained through thecourtesy of Mr. Charles Caito, Whaling Station, Del Monte Fishing Co.,San Francisco, California. There were part of a collection made by Mr.
Caito from whales captured off San Francisco by his company. The
success of these efforts and of a small collection of finback samples
taken in 1957 (Fujino) led to the collection of 460 finback samples fromthe north Pacific during the months of June, July and August 1958.
This collection was made under the supervision of Mr. K. Nasu, Whales
Research Institute, Tokyo.
Samples from these whales were taken during the flensing of carcasses on the factory ship. While these whales had been dead for from ahalf to twenty six hours (seven and a half hours in average) at thetime of sampling, the biological log showed that this variability had
no effect on subsequent sample condition. 2.5 ml. or 125 ml. of whole
blood were mixed with an approximately equal volume of a solution consisting of four parts of glycerol and six parts of a five percent solutionof trisodium citrate. (As an additional precaution against accidental
warming guanofuracin had been added to this mixture at 200 mg. per
100 ml. However, the use of antimicrobials is not essential to the successof the glycerol-freezing method). The samples were kept at 5°C for
2 to 16 days on the factory ship until a box of 50 was accumulated.
This box was then transferred to a refrigerator ship and kept at - 20°C
until the eventual recovery of samples for study at Tokyo University.
Analysis started four months after collection of the last samples andincluded a set of fifteen 125 ml. samples taken eight months before in
the Antarctic. Intact erythrocytes were recovered by dialysis at room
temperatures for two hours against 1.5% saline, followed by several
times' washing. Cells prepared in this manner remained in good condition for several days at refrigeration temperatures, being rewashed at
the start of each day's work. It was found convenient to handle from
fifteen to twenty 10 ml. aliquots of samples per day, but this number
could have been greatly expanded with additional assistance. Aliquots
could be removed with a spatula without thawing samples, or obtainedby thawing the samples under running tap water. Several freezings andthawings did not appear to affect the fragility of the cells.
Varying degrees of initial hemolysis were found in almost all samples,but good yields of cells were obtained from all the large samples

91

GLYCEROL-FREEZING TECHNIQUE IN BLOOD TYPING(total=15 bottles), and from all but 30 of the small samples (total=450
bottles). Of these 19 came from a single box of fifty which apparently
had not been refrigerated properly. The reasons for hemolysis have
not yet been studies, but it was found that smaller samples (e.g. 5 ml.)showed more tendency to hemolyze than larger ones (e.g. 250 ml). In
addition to hemolysis, the yield of cells also is influenced by internal
sedimentation and clotting in the body of the whale following death, sothat care should be taken to collect from blood vessels that appear tocontain relatively high concentrations of erythrocytes.
The Ju system of antigens (Fujino, 1956, 1958) was selected for
detailed study. The authors have found no reason to believe thateither the specificities or titers of the serological reactions of this system were altered during the period of freezing. Various absorptions
also showed that the cells were as useful in this work as unfrozen
ones. Table 1 shows that the frequencies of antigens as distributed
TABLE 1.

COMPARISON OF FREQUENCIES OF FINBACK Ju TYPES EXAMINEDBY FRESH AND GLYCEROL PRESERVED CELLS

Area

East China Sea

Ju blood
type
Fresh
cells

I

a)

Frozencells b)off Navarin

off Kamchatka

1

1.2

2

Sum

1

1.2

2

Sum

1

1.2

2

Sum

95

13

21

129
c)

329

27

73

429
d)

-

-

-

no
data

-

-

-

no
data

113

1

5

119
e)

71

I

I

1

1.2

2

Sum

1

1.2

2

-

-

-

no
data

29

1

2

13

167

4

6

13

0

0

1.2

2

Sum

32 11401

9

17

1427

177

0

1

1

Sum

74

I
a)
b)
c)
d)
e)

76

3

North of east
AleutianSouth of east
Aleutian

off Oleutorskie

2

75
f)

East China Sea examined in 1956 and 1957, other areas in 1954 and 1955.
Examined in 1958 only.
Fujino, unpublished data.
Yearly and seasonal fluctuations have been observed.
May be distinguished into two separate operating seasons in 1958 in which
seasonal fluctuations were seen.
f) May be distinguished into two operating seasons in 1958 in which no seasonal
fluctuation was seen.

92

J.E. CUSHING, K. FUJINO AND K. TAKAHASHI

in the north Pacific finbacks are comparable with those observed inearlier studies.
Various observations suggest that cells with Ju 2 specificities may
tend to slightly more fragile than those of Ju 1 type. These include
field observations on fresh cells, and laboratory observations on recovered
cells. This possibility has not yet been confirmed, but it is importantto keep in mind in general, as the rejection of badly hemolyzed samples
without typing might conceivable bias frequency counts in future studies.
In this particular study, observations on the agglutination of ghosts andresidual cells, as well as inhibition tests (Table 2), made it possible totype all the badly hemolysed samples.
TABLE 2. EXAMPLES OF THE AGGLUTINATION.INHIBITION TESTS OF
FINBACK Ju 2 TYPE CELLS (NO. 14) BY SUCCESSIVE DILUTIONS OF THE
DIALYSED SUPERNATANT FROM Ju 2 TYPE WHOLE BLOODS (NO. 14)
Dilutions of no. 14
supernatant:
2
4
8
16
32
64
Saline control

Dilution Of horse serum
32

+
+
+

**

**

64

+
+

***

128

256

512

±

+

**

+

*

+

Each test was made by mixing one drop of supernatant with one drop of serum
diluted, and fifteen minutes later, adding one drop of cell suspension. Reaction
were read thirty minutes later.

The availability of frozen cells also made it possible to discover antibodies in the normal serums of the horse, pig and sheep (only one
individual each was studied) that reacted specifically with cells carrying
the Ju2 factor. The use of horse and pig serum, combined with rabbit
immune serum, has revealed variations in the agglutination titer of Ju 2
cells of different types that resemble similar variations in other species.
Examples of these differences are shown in Table 3. At present it is notknown whether these are due to quantitative or qualitative variations
among the factors involved. The specificity and relative avidity ofhorse and pig serums for different Ju types were found to parallel
exactly the reactions of immune serums with these types. All theseserums were used in this series of typings and the relative frequencies
of homozygotes and heterozygotes were found to be same as in previous
years.
Inspection of these observed relative frequencies (Table 1) shows thatthey deviate from those that would be expected for a two allele system

GLYCEROL-FREEZING TECHNIQUE IN BLOOD TYPINGTABLE 3.

93

VARIATIONS IN THE AGGLUTINATION TITERS OF CELLS WITH
DIFFERENT FINBACK Ju SPECIFICITIESa) Examples of reactions with normal animal serums
Dilution of animal serums
Finback whale cells:2{No.
No. 14116
Ju1Ju2type {No.
No. 184{No. 12Ju 1 type
No. 13
Ju 2 type

Horse

Pig

16

32

64

128 256 512

4

8

16

32

64

128

+H+H+H-

+H+H-

+H+H-

+H+H-

+

+H+H-

+H+H-

+H+H-

+H+H-

+H+H-

** ++

+

*+ +

*

+
+

** +±

256

b) Examples of reactions with immune serums
Dilution of anti-Ju 2 immune no. 34 serum
Finback whale cells:

Ju2 type

{No.
No.

2
14

Ju1Ju2 type {No. 116
No. 184
12
Jul type
No. 13to.

Unabsorbed
160 320 640 1280 2560 5120 10240+H+H+H+H-+H+H+H+H-**+++H+H+H-{No. 2
No. 14
J lJ
jNo. 116
u u2 type iNo. 184{No. 12
Jul type
No. 13

+H+H-

+H+H-

* **
+
+H-

80 160 320 640 1280*- **- +++++H- +H+H- +H- +H+H- ++H- ±*± *+ +

- - -- - -Dilution of anti-Ju 1 immune no. 47 serum

Finback whale cells:

Ju2 type

Absorbed by Ju 1 cells

80

Unabsorbed
80

160

320

+H+H+H+H+H+H-+H+H+H+H+H+H-+H+H+H+H-

-/ff

+H-

Absorbed by Ju 2 cells

640

1280

5

10

20

**
**
**

+
+
+
+
+
+

+H+H+H+H-

**
**

+
+
+
+

40

of antigens in populations in Hardy-Weinberg equilibria. It is too early
to state which of the several possible factors that might be expected
to cause this deviation are actually the responsible ones. (cf. Srb andOwen, 1952 for an introductory discussion of the Hardy-Weinberg equilibrium).

Blue white dolphins (Stenella caeruleo-albus).
Blood samples were taken from thirty blue white dolphins captured
January 1, 1959 on the east side of the Izu Peninsula, Shizuoka Pref.,Japan. These samples, 25 ml. of whole blood each, were collected into
glycerol solution, frozen with dry ice and returned to Tokyo. A parallel set of samples from the same individuals was collected into citrateguanofuracin solution, as described above, and returned to Tokyo unfrozen. Almost all samples gave good yields of cells after one month,

J.E. CUSHING, K. FUJINO AND K. TAKAHASHI

94

and the frozen samples gave good yields of cells after three months.
The agglutinin titers and specificities of cells obtained at this time did
not differ materially from those of freshly collected, unfrozen and frozen
cells as determined in January. These dolphin cells were used in various
studies, including the work on radioactive antibodies reported in thisvolume (Fujino et al., 1959).

Humpback whale (Megaptera nodosa).
Cells from twenty five humpback whales were among the samples
obtained from Mr. Charles Caito at San Francisco. These were preserved by freezing (50 ml. of whole blood per sample) and almost all
gave sufficient cells for study, although like the finback whales, varying
degrees of hemolysis occurred.
A second collection of one hundred and five humpback whale bloods(25 ml. of whole blood per sample) were obtained from shore stations
on Okinawa, Ryukyu Islands. These were collected under the supervision of Dr. M. Nishiwaki, Whales Research Institute, whose special efforts in this regard are greatly appreciated (see Nishiwaki, this volume).
These samples were stored at -20°C and were returned to Tokyo by
air where they were examined two months after collection. Sufficient
cells were obtained from all samples excepting 5. Of these, 3 were left
in the room temperatures (20,....,22°C) before freezing for 8 hours or
longer in which decomposition had started.
Table 4 shows the reactions of anti-finback Ju 2 no. 34 serum (sent to
California by Fujino) with finback and humpback erythrocytes collected
TABLE 4. THE REACTIONS OF ANTI-FINBACK Ju 2 No. 34 SERUM WITH
FINBACK AND HUMPBACK ERYTHROCYTES COLLECTED OFF
SAN FRANCISCO, U.S.A.
Unabsorbed
Finback cells:
Ju 1 (4)Ju 1 (10)Ju 1 (25)
Ju 2 (31)
Humpback cells:
8
19
24
27
33
34

200
+H-

Absorbed by finback Ju 1 cells 4:50+H+H+H+H-

100
+H-

+H-

+H-

+H-

+H-

+H-

+H-

*

** *+
+

*
* *+

+H+H-

+H-

400

50

100

200

400

+H-

*

+
+

+H-

+H-

*

±

+

±
+H+H+
This table presents evidence that the humpback varies individually with respect
to an antigen with specificities related to the Ju 2 antigen of the finback. Additional
evidence for a Ju-like system in humpbacks is shown Tables 5 and 6.

95

GLYCEROL-FREEZING TECHNIQUE IN BLOOD TYPING

off San Francisco. As the reactions show, humpback cells vary individually with respect to an antigen with Ju2-like specificities (cf. Fujino,1958). Further studies on humpbacks collected off Okinawa have led toa tentative gr.ouping of individuals into four types with respect to theJu-like antigens. The first three of these types react positively tovarying degrees with Ju 2 specific serums, while the fourth does not.
The individuals in the different types are as follows: Type 1, K-9; Type2, K-24, K-61, N-12 and R-82; Type 3, R-10, R-29, R-48, R-66, K-12
and K-81; Type 4, K-4 and all other individuals excepting R-45. This
individual reacted positively with Ju2 serums, but could not be studied
sufficiently to decide whether it belonged to type 2 or type 3.
Table 5 illustrates typical reactions of each cell type with respect toanti-finback Ju serums. These show that a reciprocal relationship exists
among the types that strengthens the argument that humpbacks varywith respect to Ju-like antigens.
TABLE 5. AGGLUTININ TITERS OF IMMUNE ANTI-FINBACK Ju SERUMSAGAINST DIFFERENT TYPES OF HUMPBACK ERYTHROCYTESFROM OKINAWA
Anti-finback Ju 1 serum
Humpback
Absorbed by K9 cells
whale cells:
Unabsorbed
Type no.
80
160
40 80 160 320 640 1280 2560 5120
40
320
640
1 K 9
+
2 K24
+
+
3 R29
+
+
4 K4
+
+
+
Humpback
whale cells:
Type no.

1
2
3
4

K 9
K24
R29
K 4

* * * *** * * * ** *** * * * ** ** * * * * *Anti-finback Ju 2 serum* * *Absorbed by K4 cells

Unabsorbed

40 80 160 320 640 1280 2560** * * * ** *+ * +

*

+ -

+

20

40

80

160

320

640

* * *+ *±
*+ *

+

+

That the relations among the Ju 2 positive types (1, 2 and 3) arecomplex is illustrated by Table 6 which summarizes the results of reactions of different cell types with anti-finback Ju2 no. 34 serum following absorption with different type cells.
Isoagglutinins, similar to those described by Fujino (1952), were easily
recovered in the supernatants of dialysed samples at approximate dilutions of one in two from whole blood. These could be used directly, or
freed from excessive hemoglobin through salting-out by one third saturation with ammonium sulfate. The relationship of isoagglutinins to antigenic types is shown in Table 7. These parallel the results of Ju 2

J. E. CUSHING, K. FUJINO AND K. TAKAHASHI

96
TABLE 6.

THE REACTIONS OF DIFFERENT CELL TYPES WITH ANTI-FINBACKJu 2 No. 34 SERUM ABSORBED IN VARIOUS WAYSa) Cross-absorption reactions, serum dilution one in ten
Test cells:

Absorbing cells:
Type no.
1
2
2
2
3
3
3
4

K 9
N12
K24
K61
RlO
R29
R48
K 4

K9

N12

K24

K61

-tit
-tit
-tit
-tit
-tit
-tit
-tit

***
-tit

***
-tit

***
-tit

RlO

R29

R48

*

*

*

b) Agglutinin titers after progressive absorptions
Dilution of serum
Test cells:
Type no.
320
160
10
20
40
80
1 K 9 -tit
-tit
-tit
-tit
+
2 Nl2
±
-tit
-tit
+
2 K24 -tit±
-tit+
Absorbed by
2 K61
±
-tit
-tit
+
K4 cells
3 RlO
+
3 R29
+
4 K 4
1 K 9 -tit
-tit
+
2 Nl2
+
2 K24
+
Absorbed by
2 K61
K4 and R29
+
cells
3 RlO
3 R29
4 K 4
1 K 9 -tit
-tit
+
2 N12
2 K24
Absorbed by
2 K61
K4, R29 andK61 cells
3 RlO
3 R29
4 K 4

**
*

**
**
*

*

640

K4

1280

+

*
*

TABLE 7.

THE RELATIONS OF HUMPBACK ISOAGGLUTININS TO CELL TYPES
Type of test cells
Isoagglutinins
in serum from
1
4
4
2
3
3
supernatant:
RlO
R29
R38
K24
K9
K4
K 9
K24
-tit
RlO
-tit
+
R29
-tit
K 4
-tit
+
+
R38
-tit
+
The isoagglutinins in the undiluted supernatants of dialysed samples. Additional
data were obtained from the other Ju 2 positive types and eight negative individuals
that conformed with the observations shown above,*

97

GLYCEROL-FREEZING TECHNIQUE IN BLOOD TYPING

serum absorptions, excepting for the individual serum variations within
types as shown. (One explanation for this variation simply could be
differences in titers of the antibody fractions concerned).
As in the finback whale, natural antibodies were found in the serumsof various animals that reacted positively with cells showing Ju 2-likespecificities (Table 8). As far as studied the reactions of these serums
appears to be similar to that of the immune serums and isoagglutinins
already described, and shows that normal serums will provide a usefulsource of antibody for further research.
TABLE 8.

Humpback
erythrocytes:
Type no.
1 K 9
2 Nl2
3 R48
4 R14

INDIVIDUAL VARIATIONS IN THE REACTIONS OF HUMPBACKERYTHROCYTES WITH NORMAL ANIMAL SERUMS
Okinawa sample
Sheep-1

Pig
4-ttl-ttl-

16

8

32

4

* *+ *
*

8

16

8

16

32

64

4

128 25681632* *+ * **Horse (absorbed with R-45 cells)
8

512

16

32

64

128 256 512* * *± * * * +* * * *+ * ** ** *+ *** * * ± San Francisco samples+

Sheep (absorbed with 1 cell)

Sheep (unabsorbed)
No.
1
8
15
28
29

32

*+ *± * *Horse (unabsorbed)
Type no.
1 K9
2
K24
3
R48
4
K 4
2or3 R45

Sheep-2

2

4

8

16

+

±

±

32

64

2

4

8

16

32

64

*
±
* * *+ *± + ± *+ *+ * +
* *
±
±
±
* * *+ + + ± *+ * ±* shows* the potential usefulness of normal animal serums in population
This tablestudies on humpbacks.

While not enough work has been done to establish a genetic hypothesis explaining the interrelations of humpback blood types, it isobvious that more than two Ju-like alleles are involved, and probablethat these can exhibit both quantitative and qualitative differences withrespect to each other.
Table 9 shows that even in the present state of our knowledge the
humpback Ju antigens provide us with genetic markers that will bevery useful in population research.

J. E. CUSHING, K. FUJINO AND K. TAKAHASHI

98

TABLE 9. THE RELATIVE FREQUENCIES OF OCCURRENCE OF DIFFERENTCELL TYPES IN THE POPULATION OF HUMPBACK SAMPLESFROM OKINAWA
Type
~~-~

No. of samples~of each type J

1

2

3

4

1

4

7

87

unclassified

not tested(hemolysed)

total no.
of samples

1

5

105

TABLE 10. SUMMARY OF TEMPORARY CLASSIFICATION OF HUMPBACKBLOOD TYPES AS DETERMINED BY FINBACK Ju SPECIFICITIES
1Type of absorbing cells:

1
2
3
4

Type of test cells:
2
3

+
+
+

+
+

4

+

Additional species.
Two samples of sperm whale (Physeter catodon) blood from San
Francisco, and one of the True's Porpoise (Phocaena dallii truei) from
northern Japan have been collected and successfully recovered. Studies
on the preservation of fur seal (Callorhinus ursinus) blood also areproving successful. Recently Suzuki et al. (1958) have reported successful use of the glycerol-freezing technique in the investigation of
antigenic differences in tunas. They have evidence, from cells collectedin this manner, that shows population differences between the blood
types of albacore from the Pacific and Indian Oceans.
DISCUSSION AND SUMMARY

The above survey shows that it is possible to adapt on a large scale
the glycerol-freezing method of erythrocyte preservation as an aid inwhale blood type studies. The method is simple and well suited to therough conditions of field collection, providing refrigeration and freezing
equipment is available, and reasonable care is taken by the collector.
While the technique has proven adequate in its present form, further
research is desirable in order to control hemolysis, determine optimum
sample size, examine the effectiveness of ethylene glycol and related
compounds etc. (cf. Strumia et al., 1958; Stone, 1957). Attention iscalled in this regard to the facts that some species of fish vary withrespect to the relative fragility of their erythrocytes (Cushing, Ridgway, Suzuki, unpublished data), that marine teleosts are variable withrespect to the isotonicity of their blood (Green, 1953) and that the erythrocytes of some fishes are remarkably sensitive to traces of silver ion
in sodium chloride solutions (Ball, 1933).

GLYCEROL-FREEZING TECHNIQUE IN BLOOD TYPING99

The usefulness of the glycerol freezing technique is illustrated inseveral specific ways in the present study. These include the accumulation of additional data on the frequencies of Ju blood types in finback
whales, the discovery of antigens in humpback whales that are closely
related to the Ju antigens of finback whales, and the demonstration of
antibodies in the serums of various domestic animals that are highly
specific for the Ju 2 type antigens in both whale species.
ACKNOWLEDGEMENTS

The authors acknowledge with thanks the arrangements made by Dr. H.
Omura (Director, The Whales Research Institute) and Prof. Y. Suyehiro(Chief, Dept. of Fisheries, Faculty of Agriculture, Tokyo University)that made this cooperative research possible.
The assistance of Miss Nora Plecha and Mr. Gary Archer, Universityof California, Santa Barbara, is also gratefully acknowledged, as is thatof the persons whose help is specifically referred to in the text.
REFERENCES
BALL, E. G. (1933). Hemolytic action of silver occurring as an impurity in chemically puresodium chloride. Biol. Bull., 64: 277-88.
CUSHING, J. E. (1956). Observations on serology of tuna_ U.S. Dept. Interior, Fish andWildlife Serv., Sp. Sci. Rept. Fish., no. 183: 1-14.
CUSHING, J. E., RIDGWAY, G. J. & DURALL, G. L. (1957). The preservation of intact erythrocytes of marine vertebrates for blood group research. Biol. Bull., 113 (2): 343-4.
CUSHING, J. E., RIDGWAY, G. J. & SUZUKI, A. Unpublished data.
FUJINO, K. (1953). On the Serological Constitution of the Sei-, Fin-, Blue- and HumpbackWhales (1). Sci. Rep. Whales Res. Inst., no. 8: 103-24.
- - - (1956). On the Serological Constitution of the Fin Whales II. Further Studies on
Blood Groups. Sci. Rep. Whales Res. Inst., no. 11: 85-98.
- - - (1958). On the Serological Constitution of Fin Whale III. Human B Blood Group
Substances in Erythrocytes and Some Notes on Anti-fin Ju Specific Antibodies. Sci. Rep.
Whales Res. Inst., no. 13: 171-84.
FUJINO, K. & CUSHING, J. E. (1959). Blood Typing of Dried Whale Erythrocytes with J131
Labelled Antibody. Sci. Rep. Whales Res. Inst., no. 14: 101-106.
GREEN, J. & HOFFMAN, J. F. (1953). A study of isotonic solutions for the erythrocytes ofsome marine teleosts and elasmobranchs. Biol. Bull., 105: 289-295.
KABAT, E. A. (1956). Blood group substanaes. New York, Academic Press Inc., 330 p.
MOURANT, A. E. (1954). The distribution of the human blood groups., C. C. Thomas
Pub!., 438 p.
RIDGWAY, G. J., CUSHING, J. E. & DURALL, G. L. (1958). Serological differentiation of
populations of sockeye salmon, Onaorhynchus nerka. U. S. Dept. Interior, Fish andWildlife Serv., Sp. Sci. Rept., Fish., no. 257: 1-9.
SRB, A. M. & OWEN, R. D. (1952). General Genetias. San Francisco, California, W. H.
Freeman and Co., 561 p.

100

J. E. CUSHING, K. FUJINO AND K. TAKAHASHISTONE, W. H., Univ. of Wisconsin. Personal communication.
STORMONT, C. & SUZUKI, Y. (1958). The distribution of forssman blood factors in individuals of various Artiodactyl species. J. Immunol., 81: 276-84.
STRUMIA, M. M., COLWELL, L. c. & STRUMIA, P. v. (1958). Preservation of whole bloodin frozen state for transfusion. Science, 128 (3330): 1002-3.
SUZUKI, A., SHIMIZU, Y. & MORIO, T. (1958). Serological studies of the races of tuna. I.
The fundamental investigations and the blood groups of albacore. Rept. Nankai Reg.
Fish. Res. Lab., Kochi, Japan., no. 8: 104-16.