I trig".-

              

 

            

 

Yb
.. £14... .
Jawuvdzr .
fwd... I r .
.A vi; . 2 :
.~ L
Q. ‘
o.
I C. 0
T O
'2': v _ v... v.1:
c... 4 4r .
v4...» . v.» to! . A v. ..:
.2. . ~ 3: A . s. .93.. f.
, r 4 . .
. . .rte . ..e no...» . .
. . . g .2:. . . . . 512...... iti.§&vi .. NM. .. . 3..
A . .. a. Av. . x . . ‘ A
u x . .tlv é§u§.§2§1fih§“fi‘c§fi innwflvflmhfimfizé.fo I
g fiat-4 §:+filltb$‘lsz{.l flirs‘vql... ,séfifl‘t‘tt .7: vi. . .
0 hi. , étht .41.... ..ILi’ «Itoifrio’!» Sn £0.qu .zuw...1f1:.r~ .\ .
I gig . 1. ‘Nv‘MJ-fi. [1.5“st . Von-..:.ézlnfluflwffl (UMP-1.7».o! *tfi. L
t . Vt“ e . {5* . t.vou!§.§3fl.‘x ..IT‘ b\ t.§AtI&S1\.Su¢ID L45. it
n v ‘tfvirplfr 779i. . 1.57%: 0%! .tLaIK‘.¢vabcrtB\I-vl§
HE .ItrDVT-vtr‘ni. .L. Shi‘ée; 3:81.... Man-“14.; i. by! n. .
o . va§§9oku¢t E‘Es‘ig‘” grist?! iii-bu 5.1;...) ..:
u ‘ mtg-.33.: I .i... {i133 . ..:tv. Lanfifi. tittfésfi tbb}... 2
c s . 1 49% 12;... .1... yiétlfl‘infifi fr. .0...).r¢flt§fezlxhr9u+khv§ .
hug. . 4221...“...52. epfififikfiwczvzktt.ikt.roti: . s... it».
hit... ..fiviubfioztehug tact; ifkatfiuvdy . than“.
t i1}: hAibEfl-u .3
.fl It... 11¢

1 A
E:[{
{tn-I x. E
EVtrhv‘V . t E! 1.0%.: v0u|h$q}o! .ttifz51 it: . .
J ..J'mt v AA‘tl, {Pl-.144? \‘ik? lésflflb‘qfi'zlt .4 1 h A
It...“ 54.. {.943 A. N1 _ .rl .fii‘wmg.
.q A .3Lé.’
t2. 2
. Sta-tit...
it
I

 

‘
..:... \I-E
Sir , :ILAA.£$V%EO4>5 V00 v3.02?
A 1%‘v§.,£ O. ‘K {.fiutn‘e ..:rrrflhbbnn‘tg .MQwHAAV 7.. ,9.
3 it? 9!; {4.4L . Eéttiflh.
u . 09!... . {IQ £11 I. .oilA:.¢I.v«i .
.. L 991‘. §£un 5‘4““ .15???- . mill
.4. a, . .. .tkt‘fthFDétAi E$EL¥§§E§AWP§¢NN§ A .
. . D. I. . 1. . i‘ .
Eigxhfivfitefiriéfikmizz 1%.»?
. ‘1!!! \s‘kfin

390‘ ((50... $.(fwtfro...

     

 

 

                  

" i 0‘ 16-. It .1
i060“. .. vital! 1.1.
. . . if. 0! 1:09....» .1 1‘1 OU‘ML l
. . . 1%“. , ..:. .3?§Lfitzfi$~%¥$§ . “v.1? ..:.rt that Rift? ..
. fir»!!! A a. . .. .1 . 1‘35... 4 A 4 . .,.~ 2 .
uni.» til... . 4%.?) c i§.xflflflh§1, ii“... ‘3‘; taunt; 19» 0.04.; “Dog
:0 0‘5... 0 ..mi‘tfrxk. .lzlfixvv1f¥o«¢r dual u.tf%§.§)t£tf§eol
..: t... l!.4lt§l:-§li¢tfuq¥¥l.{. t.

 
  
 

flank Ar .
ii :4....§:§.§

.53..
E AF . to?» . 0.02.4.3.
«I... § Agthmflubfisbt'fl'fltfinié at ..r {luv-A.

 

 

 

 

  

 

 

  

        

   

 

    

 

3 $1.11; u: Eff?!
. n. 1.1.5.... :E i :
igfiédtdfllmwmbh «up... . r... J... 13% . fidvtrtvésodi L 1.4 344.3314»:
4. . 3." .Wéi: 01‘1"? E lb... ”Rut his... {#9. . “ugh—..:. L .In . 695...}..59}. £411). A...» , .
. . PM. ..: . Estgifivr 13:34:! 5+. ..3 ..ttulgmhfiA 1.4..ifiuvtweflrtsmh fin 4.313114%“.«1.
. . vi. , f a: (I. u. v 623.7% 2:02.15: 1.0.2va . . ,0 . like;
.1 r ..v 0 hrftoh}. vixuwcg. P l‘nufihfiufihrcxl‘athi V§h§ffii~ iniqlbflut.iu6¢?é ¥1§v¢tfiit§ t1) tf. fig. 5 I. 1...} upéauu.ifl
v a u .r 0 up. u a. J. 1" ..wtul.rto.IUP-l§vvh\|.vo‘hv»\pb:h to. 9.1.47!!! .13.}.4E . .lEK . ’L5VTtbni4L419: ‘dfn if]
.9- o... v HUI. 4.3; r .u o vvc R... .e Hts...9r.r.fl.‘¢..t86{|{. ‘4‘!» sly! . ISL... fit.‘ .Lrhcflfiffius. . . r . ”tuna .v 1 .nvi...‘ . .. u. 1.5;... L‘Anu... 3.1.9. o: . 1.... {5.0 . ..v. $.va
r u i . u v: a. c A . Kid. itaufitizli Ch}: .9. i. :14}. .9! .t‘. . . . 1%...ng. I..vb.l.u|§...v \Agé . .h!\§....¢ %¥§.2.f_u.
2.... we ..: . ..v .. E... .x . .u . :sum.0.n..wbvvtulitu.$.mr§. ivy/2?” . . it .gr‘eméivfxnfiiifivfigfiAfipiiii ..:. 19bit 4 v . 1. 454411.443
n a .a . v 11: .9 a .. .. ’E‘ttifi gigiat It . Tvv.\ .. Zigtxhvxi Etiétflég it)... icky.” 54.91...» «L45
. it‘ikv‘il... 1"}.(35 iTvErV‘ieaoflug . it”: E‘)3§i {fix . #7; I... flfififlrtfl":
. DI o erg... tvtllkt: Whit; . iivfi,“ 3. .A . Iv: . . . 5...... ”VA 3
A I .3. .t‘r‘. trinity). ;§t.‘u§ . in... . Riff}; 2 . 1.1123515: {gr-.314. .. . 595.11.! . ..1rIAHIAKtstkéilllLJ"H.411.
v.4 Lipi‘htout +4.4; . I .thfiJW-Fu ii'ifilwxé‘tn L \{If ci‘tafk..§»b\§\$‘i
gr; .tt :églclxt; 2.:. It. 12%;. 2.43... giiiiilfivztflvtii
‘3) . ti P992. EI‘VOV‘gi . "If; {twig .. A r. .131...
It}! \ $2.1oietiglfv'l‘cvfl g} g.- kixhb i 4......
.Yvit‘ .iié . \l A 5:. Iii. O)... gtéh‘1t1}
It}... 241%:— u fi.‘ . I .. IV .§.§1§r§fihufll§
. . git-Di it... . .h it! 5‘3): (fly. fit-0'4 .‘i A A};
; . 9 401701}?! «*4!!! t; . 0%? ALLAH“. 56in»! igftthflaofl to...
{.431 .%E.f§zt3{ltott%tk§tfiii téifiiitntgx irflbz.ittfi.}.i{3.§
.. Eéi {ktfiliv‘véi E11. nip-is"... A
t; %.io . :FEFNK“. .52 $.- A.§1kxii ‘hfin.fi&.i
EW3E%.£ i \ . . J L§XEH£4¥F§KQ¢W|££L1LLJ in“
E: i.“ .. .‘ta‘... 7. . .Jflkn-T. ._ v u ,. e “Margit-ht... Cm.&1ih..&a.:\1f$\|ir§
) At 1 . 3 .
...
A

 

you»! i
If E? . if»? . . . . I} .
toll-1.1;“ to. L1 A . . gig)... ..fi Sig {I
Y. éryfiii. . “Eu-11.09.. l. .E ) ‘fivfi . :i‘fw “WIMMM
\. VIE... . . ”E u r..r¢3c¢omrir§§;1ll tfifilfifi A . L98§fli$
$95.1 i)... . ..Ex.|rt.§.¥4~§;i . .fiv..\uo..9.... _ A fréifn . 4.44.... Ethyl-(v!
. A . Er . {it $51. ”1360*...it)
gu¥§i§|¥ffi92tLté§rk§tt13 our

         
                

   

. EEO) I ’Ttt‘hniizx 10531.
2%.}:5‘ £3.55LK7A‘; kettfxtbcfflfztl: L .. l. .90; .
but: A a 3:. in! , LgnwwumVViI
¢ . A . .
5

..§
\A‘
.9

   

. . .
A ntssgfiuix. .. - ...
4...: :fiiyxéi ..: A
s X 1.0.4.! giggiihitl "WW4“...

         

 

 

     

         

‘- I
r
o
. .3“... 1% $2.313... ..:.
9 I .1 '9 \. . .7
N
.
‘v u y .- .
o r. . c
v... .
. if
’ I A (v
. x v.5; ..
I . 1
‘ 'f \
v
0 O.
_.
.! r a
3,-
l 0.1
. A...
v \ v¢
..A. t.
T . i
. 1 . 1A.. . .a .
. h. .hflw. . Iu afixfik‘fliivflutv
. . . 1.. v4... nag..fl31fi..,
k v - 4“... H... .4
..: v n. I .Ythlhfibzrhi
I .1 .
. . .2. 354.04....“42 f r. .
.. 1.9.5.5413; ..x .
.‘Vbi‘i‘ . ~ J VI.“‘¥ . .II i
..::M v .v . .. tit . .
..: fr¢\.£.( tl n u . It!|ufl.!ilbtli&bll%jfitk..
$Vfi§hmsflifli§l.i. . . . c . ¢v . giving“! 4.031(1th
I A... . .‘Cxitxot‘rthxfifi 9 » Egli‘nh‘rv? . . 6 7 infilv .r-Dtfisktnk... ..4 I.
4.3%: ...1% . . i! trust... viii... AI. 3;!
, 9.. . 041‘ t0 tgythbIbfi‘)?
. . O . ‘§§E:LSQ|.‘L.J;~+A
. . hEUVdAu [Eras ‘3‘»...9? “.04.... h... blink thunk
.l )‘n.§¥.§.£}ififlit}”lfiwlfub. it?
A: bl.- tttulbk...?...1;\.. -

 

                      

. .2... .. ..:...ii. 4...... . :6.-. ..:... . . 3 .51 .
h v.. ... 4.... 4w.v,1mpw.%mm§m.Mwfiflvfimyllfit§ .X A 4.1!}: . .
.... , it...» 9.4131 Liv-L, .iA.lr9§;{tolsb bx. GO... A
H. . .- H.“ ..h m.-:...u...uxl.. ..A§.fi#%%.§§iktfiwb§ui
¢ 7., r .tyn‘fivbilix. _ vYMF.NQrYIl!...:WVZfi.€BZJk\If .Johni . .rrvvfixnunl . .5149}?! v .
. . . . . . .. L . . ,. hfiéiilrlifi?!»

"5.94:1 ..
- "L‘X‘ub |v‘9.f.lrt v.5: ....... .
.A hit}! {.11

 

      
    

§§PI¢L Iii tit! . .
. .gbt‘nll'i ‘01 7 . O b .

E‘irtl ._ Luv-fit. 5;" £55..“mele can.

”$3.5240llt . «tis’li

but»; Mfg; b'nlx‘tkhunu. tarnif’f
.. I. 1.71.134... i 2..

. . 4.
i :.....: .2. . 2... . .. .
, A . A , , .. I. .4... .1...) .. . ..:...;......r...... t... . . n
. r .2: . Iii} 2.4.".

 

in,
9.2
0

Date

 

Heme? _.
Michigan State
University

 

 

 

This is to certify that the

thesis entitled

TRIAL HEAT SHOCKING TO INDUCE TRIPLOIDY
IN COHO SALMON, CHINOOK SALMON,
AND COHO x CHINOOK SALMON RECIPROCAL HYBRIDS

presented by

Douglas J. Sweet

has been accepted towards fulfillment
Of the requirements for

 

 

M.S. degree in Fisheries & Wildlife

 

 

July 29, 1986

 

0-7639

MS U is an Affirmative Action/Equal Opportunity Institution

 

MSU
LIBRARIES
m

RETURNING MATERIALS:

 

 

Place in book drop to
remove this checkout from
your record. FINES will
be charged if book is
returned after the date
stamped below.

 

 

 

 

TRIAL HEAT SHOCKING TO INDUCE TRIPLOIDY
IN COHO SALMON, CHINOOK SALMON,
AND COHO x CHINOOK SALMON RECIPROCAL HYBRIDS

By

Douglas J. Sweet

A THESIS

Submitted to
Michigan State University
in partial fulfillment of the requirements
for the degree of

MASTER OF SCIENCE

Department of Fisheries and Wildlife

1986

 

 

ABSTRACT
TRIAL HEAT SHOCKING T0 INDUCE TRIPLOIDY

IN COHO SALMON, CHINOOK SALMON,
AND OOHO x CHINOOK SALMON RECIPROCAL HYBRIDS

By

Douglas J. Sweet

The life span and growth capabilities of Pacific salmonids
may be increased by sterilization via triploid induction. A heat
shock, at 36°C, applied for one minute starting ten minutes after
fertilization induced triploidy in 21% to 42% of the Chinook
salmon and between 0% and 14% chinook female x coho male salmon
hybrids. No triploids were found in coho salmon or coho female x
Chinook male salmon hybrids.

Heat shocking significantly (P g .001) decreased survival by
an average of 32% at swim up.

Chinook female x coho male salmon hybrids exhibited severe
malformations in 30% of the individuals. Survival of this hybrid
was 11% to 12% lower than pure chinook crosses.

Survival of coho female x Chinook male hybrids was not
significantly different from coho pure crosses.

This heat shock treatment was not optimal because triploid

induction rates near 100% are desirable.

LIL-u

ACKNOWLEDGEMENTS

First of all, I would like to thank Michigan State
University, Department of Fisheries and Wildlife, for this
research opportunity and the Michigan Sea Grant College Program
for funding my research assistantship. Additional funds were
also provided by the American Fishing Tackle Manufacturers
Association, Brownis Sporting Goods, Lowrance, Inc., and various
Chapters of Michigan Salmon and Steelheaders Fisherman's
Association.

I would also like to thank Dr. Donald Carling, Dr. Charles
Liston, and Dr. Robert Robbins, my committee members, for their
guidance in my course work and experimental design of the
research. Special thanks go to Dr. Donald Garling for his
efforts in supplying financial assistance through Michigan Sea
Grant for the duration of the study.

I would also like to specially thank Tony Ostrowski for his
help during the heat shocking process at the Little Manistee
Weir. His timing of the procedures was crucial to the outcome of
the experiments. I am also grateful for the help and comradeship
from Mark Sargent, Mike Thomas, Mark Ducharme and Ric Westerhoff.

I am also greatly appreciative to personnel from the

Michigan Department of Natural Resources, Fisheries Division, for

supplying the salmon used in this research and for the use of the
facilities at the Little Manistee Weir.

I would also like to express gratitude towards the Michigan
State University Genetics Laboratory personnel, and Paul
Scheerer, of Washington State University, for their suggestions
and advice on improving the solid tissue karyotyping.

Statistical advice was given by Dr. John Gill, Professor of
Biometry, which I appreciated immensely.

Finally, I express my most sincere appreciation and love for
my wife, Jennifer Sweet, for her support and patience, not to
mention her time and efforts in typing and editing the manu-
script, and helping conduct various aspects of data gathering.

Her name belongs on this degree as much as my own.

iv

 

TABLE OF CONTENTS

LIST OF TABLES. . . . . . . . . . . . . . . . .

LIST OF FIGURES . . . . . . . . . . . . . .

INTRODUCTION. . . . . . . . . . . . . . . . . .

MATERIALS AND METHODS . . . . . . . . . . . . .

I.

II.

III.

IV.

VI.

VII.

VIII.

IX.

X.

Specific Crosses Tested. . . . . . .
Spawning Procedures. . . . . . .

Triploid Induction . . . . . . . . .
Egg Incubation Conditions. . . . . .
Statistical Analysis of Survival . .
Solid Tissue Karyotyping . . . . . .
Graphs of Chromosome Counts. . . . .

Determination of Percentage of
Triploid Individuals. . . . . .

Karyograms . . . . . . . . . . . . .

Erythrocyte Nuclear Preparations . .

RESULTS AND DISCUSSION. . . . . . . . . . . . .

I.

II.

III.

IV.

Survival of Crosses. . . . . . .

Disembryogenesis of Hybrid Offspring .

Maternal Dominance in Hybrids. . .
Gynogenesis Versus Hybrid Production

Karyograms of Coho Salmon. . . . . .

12

12

13

13

14

15

17

l8

19

19

20

23

23

28

37

38

40

 

VI.

VII.

VIII.

IX.

XI.

XII.

XIII.

XIV.

XV.

XVI.

CONCLUSIONS

APPENDIX A.

APPENDIX B.

REFERENCES.

Karyograms of Chinook Salmon . . . . . .

Verification of Hybridization: Karyograms
of Reciprocal Hybrids Between Coho and
Chinook Salmon. . . . . . . . . . . . .

Karyograms of Triploid Chinook Salmon. .

Comparisons of Efficiency of Various
Triploidy Induction Techniques. . . . .

Improvement of Triploidy Induction
Techniques (Heat Shocking). . . . . .

Improvement of Triploidy Induction
(Hydrostatic Pressure). . . . . . . . .

Failure of Triploid Induction in Coho Salmon

Solid Tissue Karyotyping . . . . . . . . .

Lymphocyte and Tissue Culture Karyotyping
Techniques. . . . . . . . . . . . . . .

Erythrocyte Parameters Used for Triploid
Identification. O O O O O O O O O O O O

Other Methods of Triploid Indentification.

O O O O O O O O O O O O C O O O 0 O
O O O O O O O O O O O O O O O O O O O O

O O O O O O O O O O O O O O O O O O 0

vi

46

57

65

67

69

71

73

78

80

87

89

91

93

97

LIST OF TABLES

Number Page

1. Percent triploid individuals per treatment
group. O O O C O C O O O O O O O I I O O O O O O O 66

2. Nuclear parameters of erythrocytes from triploid
and diploid chinook salmon . . . . . . . . . . . . 85

Vii

 

LIST OF FIGURES

Number

1.

Survival of coho maternal side crosses from
fertilization to swim-up. Eyeing occurred at
approximately 285 thermal units Co. Hatching
ogcurred between 450 and 500 thermal units
C . . .

Survival of chinook maternal side crosses from
fertilization to swim-up. Eyeing occurred at
approximately 285 thermal units Co. Hatching
ogcurred between 500 and 550 thermal units
C

Varying degrees of disembryogenesis occurring in
swim-up chinook female x coho male hybrid
salmon. Most severe on bottom with decreasing
disembryogenesis toward top. A normal hybrid
is in each photo (top) for comparison . . . . . . .

Four month old chinook female x coho male hybrid
salmon showing moderate disembryogenesis.
(Above - side view, below, top view).

Six month old coho female x chinook male hybrid
salmon. . . . . . . . . . . . . . . . . . .

Six month old chinook female x coho male hybrid
salmon. . . . . . . . . . . . . . . . . . . . .

Six month old coho salmon. . . . . . . . . . . . . .

Chromosome number composition of metaphase spreads
sampled from coho salmon. . . . . . . . . . . . . .

Metaphase spread (above) and karyogram (below) of
coho salmon diploid with 60 chromosomes (44
metacentric-submetacentrics and 16 acrocentric-
telocentrics) . . . . . . . . . . . . . . . . . .

viii

24

25

30

31

33

33

34

41

42

Number Page

10. Metaphase spread (above) and karyogram (below) of
coho salmon diploid with 60 chromosomes (46
metacentric-submetacentrics, and 14 acrocentric-
telocentrics) . . . . . . . . . . . . . . . . . . . 43

11. Chromosome number compositions of metaphase spreads
sampled from chinook salmon . . . . . . . . . . . . 45

12. Metaphase spread (above) and karyogram (below of
chinook salmon diploid with 68 chromosomes (34
metacentric-submetacentrics and 34 acrocentric-
telocentrics) . . . . . . . . . . . . . . . . . . . 47

13. Metaphase spread (above) and karyogram (below) of
chinook salmon diploid with 68 chromosome (36
metacentric-submetacentrics and 32 acrocentric-
telocentrics) . . . . . . . . . . . . . . . . . . . 48

 

14. Chromosome number composition of metaphase spreads
sampled from coho female x chinook male hybrid
salmon. . . . . . . . . . . . . . . . . . . . . . . 49

15. Chromosome number composition of metaphase spreads
sampled from chinook female x coho male hybrid
salmon. . . . . . . . . . . . . . . . . . . . . . . 50

16. Metaphase spread (above) and karyogram (below) of
coho female x chinook male hybrid diploid salmon
with 64 chromosomes (40 metacentric-submetacentrics
and 24 acrocentric-telocentrics). . . . . . . . . . 52

17. Metaphase spread (above) and karyogram (below) of
coho female x chinook male hybrid diploid salmon
with 63 chromosomes (40 metacentric-submetacentrics
and 23 acrocentric-telocentrics). . . . . . . . . . 53

18. Metaphase spread (above) and karyogram (below) of
chinook female x coho male hybrid diploid salmon
with 64 chromosomes (40 metacentric-submetacentrics
and 24 acrocentric-telocentrics). . . . . . . . . . 54

19. Metaphase spread (above) and kayrogram (below) of
chinook female x coho male hybrid diploid salmon
with 65 chromosomes (40 metacentric-submetacentrics
and 25 acrocentric-telocentrics). . . . . . . . . . 55

20. Metaphase spread (above) and karyogram (below) Of
chinook female x coho male hybrid diploid salmon
with 65 chromosomes. (38 metacentric-submeta-
centrics and 27 acrocentric-telocentrics) . . . . . 56

ix

Number

21. Chromosome number composition of metaphase spreads
sampled from heat shocked chinook salmon. . . . .

22. Chromosome number composition of metaphase spreads
sampled from heat shocked chinook female x coho
male hybrid salmon. . . . . . . . . . . . . . . .

23. Chromosome number composition of metaphase spreads
sampled from heat shocked coho salmon . . . . .

24. Chromosome number composition of metaphase spreads
sampled from heat shocked coho female x chinook
male hybrid salmon. . . . . . . . . . . . . . . .

25. Metaphase spread (above) and karyogram (below) of
chinook salmon triploid with 96 chromosomes (50
metacentric-submetacentrics and 46 acrocentric-
telocentrics) . . . . . . . . . . . . . . . . . .

26. Metaphase spread (above) and karyogram (below) Of
chinook salmon triploid with 101 chromosomes
(50 metacentric-submetacentric, and 51
acrocentric-telocentrics) . . . . . .

27. Distribution of diploid and triploid erythrocytes
per length of long nuclear axis . . . . . . .

28. Distribution of diploid and triploid erythrocytes
per area of erythrocytic nuclei . . . . . . . . .

29. Distribution of diploid and triploid erythrocytes
per volume of erythrocytic nuclei . . . . . . . .

30. Distribution of diploid and triploid erythrocytes
per length of short nuclear axis. . . . . . . . .

59

60

61

63

64

81

82

83

84

INTRODUCTION

Salmonid aquaculture has benefitted from developments in
nutrition, husbandry, and genetics. However, a major unsolved
problem affecting production of Pacific salmonids is the reduced
growth rate, flesh degradation, and increased mortality that
occurs during maturation and spawning (Gjedrem, 1976; Lemoine and
Smith, 1980; McBride and van Overbeeke, 1971; Refstie et a1.,
1977). An increase in production is expected if a method could
be developed to inhibit the physiological changes associated with
maturation. One potential solution is sterilization of fish
intended for food or sport-fishery purposes.

Sterilization was previously tested on Pacific salmon in

order to increase their life span. Kokanee salmon (Oncorhynchus

 

nerka kennerlyi) were surgically castrated resulting in a longer

 

life span (Robertson, 1961). Gonadectomy performed on sockeye

salmon (Oncorhynchus nerka) also resulted in prolongation of life

 

with cessation of the tissue degeneration that occurs during
sexual maturation (van Overbeeke and McBride, 1971). The gonadal
steroids, 11-ketotestosterone, 17q-methyltestosterone, and
estradiol, are directly responsible for tissue degeneration
during spawning and they also initiate hyperadrenocorticism
causing further degeneration (Schreck and Fowler, 1982; van

Overbeeke and McBride, 1971). Therefore, an effective

sterilization technique should prevent degeneration of tissue and
mortalities associated with maturation in Pacific salmonids.
Since fish have indeterminant growth, (Beverton and Holt, 1957)
Older sterile fish should continue growing without putting energy
into reproduction.

Since surgical castration of large numbers of hatchery
produced salmonids is not economical, other methods of steriliza-
tion have been tested. Steroid hormones were used to sterilize
and alter the sex ratios of salmonids. This technique requires
the eggs to be immersed in and the swim up fry fed on steroid
hormones (Goetz, et a1., 1979).

Another, more simplified method of sterilizing salmonids is
the induction of triploidy. Triploidy, the condition of having
three haploid chromosome sets (3N) instead of two haploid chromo-
some sets (2N), can be easily induced in various fish and
amphibians. Triploidy was induced in various urodeles and
anurans with a heat shock of 35.000 - 37.0°C, applied to newly
fertilized eggs for 4 to 7.5 minutes (Briggs, 1947). Briggs
found the optimal time to administer the heat shock was 20
minutes post ferilization. This time corresponds to metaphase of
the second meiotic division of the egg nucleus, which suggests
that heat shock causes triploidy by preventing shortening of
spindle fibers during anaphase. The spindle fibers are probably
partially denatured by the heat (Briggs, 1947). Other
researchers agree that triploid induction occurs through

prevention of the second meiotic division. For example, marker

chromosomes were used to determine the origins of triploidy in

the newt (Pleurodeles waltlii), and these Observations proved

 

that the two sets of maternal chromosomes and one set of paternal
chromosomes that occured in a triploid individual arise from
suppression of the second meiotic division of the egg (Ferrier
and Jaylet, 1978L

Triploidy causes sterility in the adult organism because
gametogenesis has been arrested in the gonial stages. Gameto-
genesis is probably arrested due to the odd chromosome number and
resulting aneuploidy that occurs in the sex cells of triploids
(Cassoni et a1., 1984). In addition to possessing non-functional
gametocytes, the gonads of triploids are often severely retarded

in development. Gonads of triploid Atlantic salmon (Salmo salar)

 

were reduced in size by 48% for males and 92.3% for females
(Benfey and Sutterlin, 1984a). Gonad formation was also markedly
reduced in triploids of carp, catfish, plaice, and plaice x
flounder hybrids (Gervai, et a1., 1980; Lincoln, 1981; Wolters et
a1., 1981b 1982d). Triploid salmonids, such as the rainbow trout

(Salmo gairdneri), also show reduced gonad size and function but

 

not to the degree as other species. The testes of male triploid
rainbow trout develop normally except for nominal production of
milt. Also steroid levels in triploid male rainbow trout did
not differ significantly from diploids. However, female triploid
rainbow trout had markedly reduced gonad size with low levels of

gonadal steroids as compared to diploids (Lincoln and Scott,

1984; Thorgaard and Call, 1979). By most indicators, triploid
individuals are typically sterile.

There are exceptions to the rule of sterility in triploids
because reproducing populations of triploid gynogenetic
amphibians and fish have been discovered. All of these are
specialized cases which frequently have unusual mechanisms for
reproduction. lFor example, the silvery salamander (Ambystoma

platineum) and the Tremblays salamander (Ambystoma tremblayi) are

 

naturally occuring, all-female, triploid hybrids. These hybrids
were the result of Jefferson salamanders (Ambystoma

jeffersonianum) mating with blue-spotted salamanders (Ambystoma

 

laterale). The silvery and Tremblays salamanders have no
reductional meiotic divisions and sperm from one of the hybrid
parental species males only activates the egg to develop (Behler

and King, 1979L Triploid axolotl (Ambystoma mexicanum) females

 

also produce Offspring. No special meiotic mechanism to conserve
normal chromosome numbers is involved because surviving Offspring
have variable chromosome numbers. Obviously the development of
axolotls has some tolerance to aneuploidy (Frankhauser and

Humphrey, 1950). .A triploid fish species (Poeciliopsis spJ also

 

reproduces gynogenetically. In this case the triploid number of
chromosomes is believed to be increased to hexaploid by an
endomitotic division. The triploid number is then maintained by
a meiotic division (Schultz, 1967). Due to the exceptions of
sterility in these triploids, care must be taken when claiming

any man-made triploids to be completely sterile.

Triploids usually do not differ from diploids morpho-
logically. Triploid carp (Cyprinus carpio) were phenotypically
identical to diploids except for a minor disturbance in scale
pattern in the triploids (Gervai, et a1., 1980). Comparis0ns of
multiple morphological measurements, fin ray numbers and
pharyngeal-teeth arrangement yielded no significant differences
between diploid and triploid hybrid grass carp (Cassani et a1.
1984). Triploid frog embryos and larvae develop normally and are
identical to diploid larvae except for larger cell size and fewer
cells in triploids (Briggs, 1947X

Cytologically, triploids can be distinguished from diploids.
Karyological examination reveals triploids have an additional
haploid set of chromosomes over the normal diploid set. The cell
nuclei and cell size is usually larger for triploids than
diploids. This occurs because triploids have 1/3 more DNA than
diploids (Briggs, 1947).

Since triploids contain more DNA and have larger cells, it
has been hypothesized that triploids also should grow faster and
obtain larger sizes than diploids (Purdom, 1973). Growth in
triploids of various species seems to be highly variable. The
reported variation in triploid growth probably arises not only
from actual differences between species but also the variation
and inadequacies of experimental design. For example, triploid
rainbow trout had slower growth than diploids as reported by
Solar, et a1” (1984), whereas Atlantic salmon had no significant

differences in growth between diploids and triploids (Benfey and

 

Sutterlin, 1984a). Benfey and Sutterlin stated that although
triploid Atlantic salmon may not grow faster than diploids in
early growth stages, the triploids may outgrow the diploids
during sexual maturation. Neither of the above studies included
sexual maturation in their growth measurements. In other
species, triploids were considered to grow faster and reach
heavier weights compared to diploids. Triploid channel catfish
(Ictalurus punctatus) were significantly heavier than diploids at
8 months old and older (Wolters et a1., 1982d). This age and
growth period corresponds with sexual maturity of the catfish.
Grass carp x bighead carp hybrid triploids (Ctenopharyngodon
idella i x Hypothalmichthys nobilis d) grew faster than diploid
hybrids (Cassani et a1., 1984). Finally, triploid Tilapia 22323
were larger than diploids at 14 weeks old (Valenti, 1975). The
results of Valenti's study should be taken cautiously because of
limited sample size. Only one to six polyploid fish per
experimental group survived to the end of 14 weeks.

Another potential advantage of triploid induction is
enhanced survival of triploid hybrids. Triploidy may increase
survival of inter-generic or intra-generic hybrids by providing
one complete maternal set of chromosomes. In an ordinary diploid
hybrid, some vital hereditary material may be absent because only
a haploid set of chromosomes originates from each parental
species. In a triploid the extra set of maternal chromosomes
gives the hybrid at least one complete diploid set of genes from

one species. This compensates for any deficiencies caused by the

hybrid. For example Elinson and Briedis (1981) observed that

diploid hybrids of bullfrog (Rana catesbiana) x green frog (Rana

 

clamitans) died during gastrulation while the triploid hybrids
flourished. Scheerer and Thorgaard (1983) induced triploidy in

brown trout (Salmo trutta), brook trout (Salvelinus fontinalis),

 

 

and rainbow trout hybrids. The triploid hybrids had higher
survival rates than the diploid hybrids. In some cases the
triploid hybrids had lower survival to the eyed stage of develop-
ment but had better survival to the initiation of feeding. This
reduced survival was probably due to effects of heat shock rather
than triploidy.

Having discussed the advantages that inducing triploidy has
in fish, the management implications for this process become
clear. Triploid salmon and triploid hybrid salmon may benefit
sport fisheries as well as aquaculture. Benefits for anglers
include a potentially larger and different fish to capture, less
flesh degradation during the spawning season, and in the case of
hybrids, a combination of good traits such as faster growth with
good fighting capability. Having combined characteristics, these
triploid salmon hybrids may occupy a different ecological niche
than their purebred parents. This Offers the advantage of
potentially increasing the carrying capacity and stability Of the
aquatic ecosystem involved. .A good example of a successful
hybrid occupying a new niche is the splake, a hybrid between lake

trout (Salvelinus namaycush) and brook trout, that occupied a

 

different niche than the lake trout and successfully avoided

heavy predation by the sea lamprey (Pillay and Dill, 1976L
Triploid salmon can Offer easy management of the number of fish
stocked since no natural reproduction of these individuals should
occur. Finally, sterile triploids may be introduced as exotic
species to assess environmental impact before fertile fish are
stocked.

Triploids and various polyploids have been produced and
found to spontaneously occur in many salmonid species.
Spontaneous triploids were discovered by Thorgaard and Gall,
(1979), in the McCloud River rainbow trout strain. Utter et al.
(1983) also found spontaneous triploids in pink salmon

(Oncorhynchus‘gorbuscha).

 

Other polyploids have been induced by a variety of treat-
ments with different species of salmonids. For example, the use
of mitotic-inhibiting chemicals, such as cytochalasin B and
colchicine, induced a variety of polyploids and chimeras (having
a combination of diploid and polyploid cells) in rainbow trout
(Refstie et a1., 1977), atlantic salmon (Allen and Stanley,
1979), and brook trout (Smith and Lemoine, 1979). These chemical
treatments, which were usually applied to the early embryo, did
not lead to consistent results. Some individuals were diploid,
triploid, tetraploid or mosaics combinations.

Cold shocking eggs and early embryos of salmonids have also
been found to induce various polyploids and tetraploids. This
technique has been used on brook trout, producing mosaic poly-

ploids (Lemoine and Smith, 1980), and on Atlantic salmon

(Lincoln, et a1., 1974). Tetraploids, which are theoretically
fertile, are only desirable if they are crossed to diploids to
produce all triploid offspring (Chourrout, 1984; Gjedrem, 1976X
The most effective means of inducing triploidy in salmonids
was by heat shock. Using this method, triploids or tetraploids
have been produced, with the specific result depending on the
time after fertilization the eggs are shocked. Eggs shocked
within the first hour of fertilization typically produce
triploids, whereas eggs shocked about the time of the first
mitotic division (about 5 hours post fertilization) typically
produce tetraploids (Thorgaard et a1., 1981). Heat shocks
ranging from 26°C to 36°C, and lasting from 1 to 20 minutes, have
been attributed to successful triploid induction (Benfey and
Sutterlin, 1984b; Chourrout, 1980; Johnstone, 1985; Lincoln and
Scott, 1983; Solar et a1” 1984; Utter et alu 1983). Higher
temperatures and longer heat shock periods increased the per-
centage of triploids but also decreased the number of survivors.
The lower temperatures and shorter shocking periods increased
fish survival but resulted in lower percentage of triploids. A
review of the literature suggests the optimal temperature, time,
and length of heat shock was between 26°C - 28°C, for 10-20
minutes, 20-25 minutes post fertilization (Solar et a1., 1984i
A heat shock of these parameters typically produced nearly 100%
triploids and minimized heat shock mortalities. Utter et al.
(1983) has previously induced triploidy in 58% to 84% of chinook

salmon, pink salmon, and pink x chinook salmon reciprocal

10

hybrids. They used a ten minute heat shock, at 280-3000, applied
ten minutes post fertilization.

The main objective of this research was to determine the
feasability of inducing triploidy in coho salmon (Oncorhynchus
kisutch), chinook salmon (9; tchawytscha), and coho salmon x
chinook salmon reciprocal hybrids. Coho and chinook salmon were
chosen because they are popular sport fish and are easily
obtained from the Great Lakes. Hybridization is feasible because
these species have overlapping spawning runs. The secondary
objective of this research was to determine the viabililty of the
hybrids, triploid hybrids, and triploid purebreds in relation to
the pure breds, by recording survivorship to swim up. Previous
reports of hybridization between these two species indicated poor
survival of offspring from chinook females crossed to coho males
and variable survival of offspring from coho females crossed to
chinook males (Blanc and Chevassus, 1979, 1982; Chevassus 1979A
Induction of triploidy in these hybrids was hoped to correct this
situation and create a viable sport fish. The triploid hybrid
would contain two haploid maternal sets of chromosomes (2N) and
one haploid paternal set of chromosomes (1N). Hopefully this
double set of maternal chromosomes, which is a complete chromo-
some set from the maternal species could compensate for any
deficiencies of hereditary material caused by hybridization.

Although the most optimal heat-shocking technique seems to
be at 26-28°C, for 10-20 minutes, at 20 to 25 minutes post

fertilization, this technique was not used in this trial. The

11

majority of salmonids on which the Optimal heat shocking
technique was developed were smaller species, with smaller eggs,
than the coho and chinook salnunn A hotter heat shock was
thought necessary for adequate heating of the larger coho and
chinoOk salmon eggs. Thorgaard et a1. (1981) produced triploid
rainbow trout at optimal numbers and survival with a heat shock
of 36°C for one minute, ten-minutes post fertilization. This

hotter heat-shock regiment was chosen for this trial.

MATERIALS AND METHODS

Specific Crosses Tested

 

Eight different crosses, run in triplicate, were used to
assess induction of triploidy in coho and chinook salmon. The
crosses were:

1) coho g x coho O,

2) coho S! x cohot<f'+- triploid induction

3) coho a; x chinook (f,

4) coho g; x chinook ny+ triploid induction

5) chinookgx chinook 0,

6) chinookgx chinook 0’4- triploid induction
7) chinookgx coho 0'

8) chinookgx coho (7+ triploid induction

Crosses 1 and 5 were used as references for egg and sperm quality
for the coho salmon and chinook salmon, respectively in the
experiment. Crosses 2 and 6 were to test triploid induction in
coho salmon and chinook salmon, respectively, while crosses 3 and
7 were designed to test diploid reciprocal hybrids of coho and
chinook salmon. Finally, crosses 4 and 8 were designed to test
triploid induction in reciprocal hybrids between coho and chinook

salmon.

12

ll‘

 

 

 

l3

Spawning Procedures

Eggs and milt of coho and chinook salmon were obtained from
Michigan Department of Natural Resources, Fish Division, at the
Little Manistee Weir on October 15, 1984. Complete hatchery
procedures are outlined in Appendix A. It was estimated that
approximately 500 eggs per test group would be needed to supply
enough fish for karyotype samples (based on expected survival of
chinook salmon in our fish laboratory, Carling and Masterson,
1985). To obtain 500 coho eggs per test group, the eggs of three
females were pooled and mixed prior to separation into test
groups and fertilization. The eggs of three chinook females were
similarly treated. Milt from six coho males was pooled together
because fully ripe coho males with adequate quantities of milt
were difficult to obtain. Milt from only three chinook males was
needed to supply adequate quantities for fertilization. Pooling
of gametes would also minimize the effects of any sterile gamete

donors.

Triploid Induction

To induce triploidy, the eggs were heat shocked in a 17-
gallon fiberglass rectangular tank at ten minutes post fertiliza-
tion. Water was circulated through this tank by an Autoflow
external box filter. The water was heated to 36°C by two Fisher
Automerse thermostatic immersion heaters. The eggs were sus-
pended in this tank during heat shock in 2 mm-mesh floating

screen boxes measuring 8" x 8" x 5". After application of the

 

14

heat treatment, eggs were immediately transferred to water at
ambient temperature. ,All non-heat-shocked eggs were sham-heat
shocked by moving and immersing the eggs for a similar period of

time in water at ambient incubation temperature.

Egg Incubation Conditions

 

After all treatments, the eggs were allowed to water harden
for 1 hour at ambient water temperature before transporting. The
eggs were transported in plastic rectangular buckets, on ice,
inside of coolers.

Upon arrival at MSU Fisheries Research Laboratory, the eggs
were unpacked and numbers were estimated by the California
Volumetric method (Leitritz and Lewis, 1980). Each test group
was split into three equal replicates. Each replicate was
randomly assigned a position in one of 24 vertical flow incubator
trays. Each stack Of eight trays was supplied with one gallon of
aerated well water per minute at approximately 12°C.

To assess the various treatments, survival of eggs and fry
was recorded weekly from fertilization to swinrwqx Time within
this developmental period was recorded as thermal units in
degrees Celsius. Thermal units were calculated by summing the
degrees Celsius that the incubation water was at for every suc-
cessive 24 hour period. IFor example, if the water temperature
was a constant 10°C for five days, then 50 thermal units would be
accumulated. Eggs were considered dead when they turned white or

fungused. .Anti-fungal treatments were not used because frequent

 

15

egg picking prevented the spread of fungal infections. Care was
also taken to prevent physical disruption of the eggs during the
sensitive period between 48 hours post fertilization and eyeing

approximately 25 days later (Leitritz and Lewis, 1980).

Statistical Analysis of Survival

 

Survival was compared statistically at two independent times
during development. These times are considered crucial for sal-
mon development and included the stages of (1) eyeing and (2)
swim up. .A three-way analysis of variance with orthogonal con-

trasts was used for these comparisons. The model is as follows:

Yijk1=u+ oci+Bj+(ocB)ij +Yk+ (aY)ik+ (BY)jk

'*(C*BY)ijk,+ E(13101
where u = mean survival

a i = average effects of heat shock

Bj = average effects of maternal side of cross
( a3)(ij) = interaction of <1 and B

Yk = average effects of paternal side of cross

interaction of B and Y

(BY)jk

(OLBY)ijk interaction of a, B, and Y
E(ijk)l = random effect of unspecified variables
(Gill, 1978).
Initial tests for homogeneous variance (f-max test) on

survival for these times revealed heterogeneous variances. Three

transformations were attempted on both time periods of data.

16

These transformations were:
1) y = Arcsin (P)

where P is proportion surviving at that time
2) y = loge P
1
3) y = l - P
P
(Gill, personal communication).

None of these transformations resulted in homogeneous variance

(P 5_.25) for time (2) swim up and only transformation No. 2,
above, achieved homogeneous variance for time (1) eyeing. Due to
this heterogeneous variance, survival at time (1) eyeing was com-
pared statistically after the data was transformed using trans-
formation No. 2% Interactions and main effects were considered
significant if a type I probability of error was .05 or less

(P g .05).

Since time (2) swim-up data could not be successfully trans-
formed to a homogeneous variance (P 5_.25), the transformation
which most closely achieved this was used. Transformation No. 2
was used for this purpose but since it was not as complete as in
time (1) eyeing the interactions and main effects were considered
significant only if a type I error was .01 or less (P S .01)

(Gill, personal communication)

17

Solid Tissue Karyotyping

 

To verify if heat treatment induced triploidy, karyotypes
were performed on samples from each test group. Initially,
karyotyping was attempted on swim up fry using Kligerman and
Bloom's (1977) solid-tissue technique. Failure of this technique
led to alternate trials with modifications on solid tissue karyo-
typing and some use of tissue culture. Modifications on the
Kligerman and Bloom methods eventually resulted in moderately
good results (See Appendix B). However, due to the amount of
time the procedure required, the moderate success rate, and the
age of the fish when sampling became possible, only twenty fish
from each non-hybrid cross and ten fish from each hybrid group
could be analyzed. These chromosome counts were completed when
the fish were between four and six months old.

Metaphase chromosome spreads were examined with a Microstar
American Optical Series One-Ten Laboratory Microscope. Slides
made by the modified Kligerman and Bloom (1977) solid-tissue
karyotyping technique were initially scanned at 100 power.
Special attention was paid to the circumference of the dried-cell
suspension rings. In this region the most acceptable metaphase
spreads were found. Once a suspected spread was located at 100
power (a very small scattering of dots), a 1,000 power oil
immersion lens was used while enumerating chromosomes. Chromo-
some contrast was optimal when stained with Giemsa and ammonium
hydroxide intensifier; and when viewed with near maximum light

power with diaphragms wide open. An eyepiece micrometer grid was

18

used as a position reference when counting chromosomes. This
grid was centered over the metaphase spread while chromosome
centromeres were counted in each grid square. The chromosomes
within each grid square were counted in order starting with the
upper left hand corner square, moving to each square to the
immediate right, then down one row and back to the left. This
pattern was followed as if each grid was counted as words that
are read in this sentence. The actual count was performed on a
Lion tally counter to minimize counting error and possible bias
from prior knowledge of expected chromosome counts. Only three
of the largest and most intact appearing metaphase spreads were

counted per slide.

Graphs of Chromosome Counts

All metaphase spreads counted per test group were bar
graphed according to chromosome count. These graphs essentially
show the distribution of chromosome counts in the population of
cells in that test group. Each individual fish is usually repre-
sented by three cells, or counts, on that graph. Expected
diploid chromosome counts for chinook and coho salmon were taken
from Simon (1963) and are indicated on the graphs. Calculated
expected chromosome counts for diploid hybrids, triploid hybrids,
and triploids are also indicated. The purpose of these graphs
was to indicate if triploid cells are in the population. Hypo-

thetically, if some triploids are present a bimodel distribution

19

peak is expected on the graph peaking at the expected diploid

(dotted line) and triploid (dashed line) chromosome numbers.

Determination of Percentage of Triploid Individuals

 

Various parameters were set in order to determine the per-
centage of triploid individuals in each test group. This was
deemed necessary because of the variable chromosome counts
obtained and the likelihood that two overlapping partial diploid
metaphase spreads may have appeared as a triploid. An individual
fish was determined triploid if at least two of the three largest
metaphase spreads had a chromosome count exceeding the median
number between the expected diploid and triploid counts. This
arbitrary limit was set in hopes of eliminating any artificially
high diploid counts (due to counting error) but was low enough to
include triploid spreads missing some chromosomes. Standard
error for percent triploids was calculated for each test group by
the binomial formula:

S.E. = w1(l - wl)
n

where wl = percent triploid individuals
n = total number of fish examined per test group

(Gill, personal communication).

Karyograms

Photomicrographs of the best metaphase spreads in each test
group were taken. An Olympus Photomicrographic System Camera,

model PM-lO-M with accompanying exposure meter, model EMM-7, was

 

20

used. Karyograms were made by projecting negatives with a
Beseler enlarger onto a piece of papen. Chromosomes were traced,
blackened in, and later cut out. The chromosome images were then
arranged into karyograms according to a Denver type arrangement
(Al-Sabti, 1985; Blaxhall, l983aL This technique has the meta-
centric chromosomes arranged in order of decreasing size followed
by acrocentric chromosomes also arranged in order of decreasing
size. Due to similiarity in size and shape of many chromosomes
exact homologous pairs might not have been placed together.
Initially chromosomes were classified into four categories as
described by Levan, et a1” (1965L These four categories were
determined by chromosome arm ratios and consisted of metacentric,
submetacentric, subtelocentric and acrocentric chromosomes. This
system was abandoned due to high variability of chromosome con-
densation and relatively poor resolution from the film (ASA 64)
used for karyograms. A simplified two category technique for
chromosome classification was used instead. 'Using this technique
chromosomes were categorized as having the centromere position
either near the center of the chromosome (metacentric-submeta-
centric) or near the ends of the chromosome (acrocentric-telocen-

tric).

Erythrocyte Nuclear Preparations

 

In addition to karyotyping, erythrocyte preparations were
made for each fish karyotyped. These preparations were taken as

an alternate system for determining triploidy if karyotyping

 

21

proved inadequate. Erythrocyte cell and nuclear size can be used
as an indicator of triploidy (Beck and Biggers, 1983; Benfey et
a1., 1984; Benfey and Sutterlin, 1984c; Cimino, 1973; Walters et
al., 1982c). Blood was taken in a heparinized capillary tube
from the severed anterior dorsal aorta of fish being prepared for
karyotyping. The blood was then expelled onto a clean slide,
smeared with the edge of another slide, and allowed to air dry.
Dried blood smears were fixed in methanol for two minutes,
allowed to air dry again, and stained in buffered Giemsa as
described in step 10 of Appendix B (Beck and Diggers, 1983).

Stained blood smears were observed under 1,000 X (using the
same microscope for viewing metaphase spreads). Individual ery-
throcytes were randomly selected and nuclei width and length were
measured using a micrometer scale. Any erythrocytes which
appeared malformed, ruptured, or otherwise disrupted were not
included in the measurements. Area and volume of the erythrocyte
nuclei were calculated using formulae taken from Lou and Purdom,
(1984). Area was calculated by the ellipse formula:

Area = (a)(b) /4
where a is the diameter of the short axis

b is the diameter of the long axis.

Volume was calculated by the formula:

Volume = azb
1.91

 

22

Since karyotyping provided sufficient data, only a limited
number of erythrocyte slides were measured. Erythrocytes from
six chinook salmon determined by karyotyping to be triploid, and
six chinook salmon determined by karyotyping to be diploid were
fully analyzed. This small test sample was analyzed to check the
validity of using erythrocyte dimensions as triploidy indicators
for future work. Bar graphs were made displaying the distri-
bution of triploid and diploid erythrocytes vs. length of long
nuclear axis, length of short nuclear axis, area and volume of
erythrocytic nuclei.

Disembryogenesis associated with the hybrid salmon was
examined histopathologically and described by the Animal Health

Diagnostic Laboratory at Michigan State University.

,_ .._a ...

 

RESULTS AND DISCUSSION

Survival of Crosses

 

Incubation conditions for the eight different crosses can be
considered favorable for optimal survival since survival of
chinook salmon controls at time of swim-up was 91%. Carling and
Masterson (1985), reported only 50% survival at swim up for
chinook salmon incubated at the same facilities under similar
conditions. Since survival of chinook salmon controls in this
study was exceptional, it can be assumed that conditions were
favorable for development Of hybrid and triploid-induced (heat-
shocked) crosses.

Three-way analysis of variance with orthogonal contrasts
indicated that heat shocking was the main factor decreasing
survival between fertilization to eyeing. Heat shocking
decreased survival by an average of 24% (Figures 1 and 2). This
difference is significant with a type I probability of error of
.001 or less (P S .001). The majority Of this decrease Occurred
in the few days post fertilization reflecting immediate effects
from the heat shock.

All eggs originating from chinook salmon had better survival

than eggs originating from coho salmon by time of eyeing. Mean

23

 

  
 
 
  
  

.3
O
O

9 0.,

sum
PF

0)
O
I

96 SURVIVAL ( Average of 3 Replicates )
0:
o
I

24

Coho x Coho

 

 

 

\ A
Wk fl.
A A AA -

A
f v fl

Coho X Chinook

Coho X Chinook +
Heat Shock

 

 

40-- Coho XCoho +
30.. Heat Shock
20..
10...
O ‘ l r 'I l I
160 ' 360 ' 500 700
TIME IN THERMAL UNITS c"
Figure 1. Survival of coho maternal side crosses from fertili-

zation to swim-up. Eyeing occurred at approximately
285 thermal units C . gatching occurred between 450
and 500 thermal units C .

 

 
    
 
 

S

90.
so.
70.
so,
so.
40.
so.
.01

10..

96 SURVIVAL ( Average of 3 Replicates )

O

 

Figure 2.

25
Chinook X Chinook

A

Chinook X Coho

Chinook .X Chinook +
Heat Shock

Chinook X Coho +
Heat Shock

1 -‘”” I I l I ' I

166 300 500 700
TIME IN'THERMAL UNITS c'

Survival of chinook maternal side crosses from fertili-
zation to swim-up. Eyeing occurred at approximately

285 thermal units C . Hatching occurred between 500
and 550 thermal units C .

 

26

survival of chinook eggs was 7% higher than coho eggs and this
maternal-effect was highly significant (P 5_.001L

At time of swimrup, heat shock was also found to signifi-
cantly (P 5 .001) decrease survival by an average of 32%. A
large proportion of these mortalities originated in the period
prior to eyeing. ‘This is evident from the similarity of sur-
vival curves after eyeing. The only difference between heat-
shocked and non-heat-shocked curves is the displaced position of
heat-shocked curves due to high mortalities prior to eyeing
(Figures 1 and 2). This indicates, as would be expected, that
heat shock was most damaging to development during the heat shock
or in the stages immediately thereafter.

In contrast to significant maternal effects occurring
between fertilization to eyeing, the period from fertilization to
swimrup had significant main effects caused by the paternal
source of the cross. During this period survival averaged 10%
lower for eggs fertilized by coho males than for eggs fertilized
by chinook males. This difference was significant with a type I
probability of error of .001 or less (P g .001).

Hybrids between brown trout females and brook trout males
were shown to have a similar pattern of maternal dominance of
survival characteristics occurring before the eyeing stage (Blanc
and Poisson, 1983L Paternal effects only become important after
yolk sac absorption and in the case of many hybrids seems to be
the source of blue sac disease (Blanc and Poisson, 1983). One

potential explanation of the decreased survival of coho maternal

 

27

and paternal sides of the crosses is the observation that coho
salmon were not at peak ripeness and peak of the spawning run
when eggs and milt were taken. Ripe female and male cohos were
difficult to Obtain at egg-taking time, indicating a possible
condition of underripe or overripe eggs. This could easily
explain the lower survival Of coho maternal-side crosses.

Further evidence for the low quality of eggs Obtained from coho
salmon is seen in the sudden drop of survival of coho eggs within
the first few days post fertilization (Figure 1%

Survival of all hybrid groups, except coho female x chinook
male plus heat shock, was lower than their respective control
groups at time of swim up. Survival of chinook female x coho
male hybrids was 12-13% lower for both heat-shocked and non-heat-
shocked groups as compared to pure chinook crosses. Survival of
coho female x chinook male hybrids (84%) was lower but similar to
survival of coho pure matings (87%). The exception to this trend
was the higher survival of heat-shocked coho female x chinook
male hybrids (61%) than heat-shocked coho pure matings (43%).
This extremely low survival of heat-shocked coho pure matings may
be explained by the compounded effects of low survival character-
istics of coho eggs, the low survival characteristics occurring
from coho paternal side crosses, and low survival caused by the
heat-shocked treatment. Lower survival of hybrids is expected.
Lower survival of heat-shocked hybrids over diploid hybrids con-
tradicts what other researchers have found and what is expected

Of triploids. Using female brook trout x male brown trout and

28

female rainbow trout x male brown trout Scheerer and Thorgaard
(1983) listed a hierarchy of survival. Diploids of either
species had the highest survival, followed by triploids, triploid
hybrids, and diploid hybrids having the lowest survival. In this
case triploid hybrids had lower survival to eyeing (probably from
detrimental effects of heat shock) but better survival to initia-
tion of feeding. Induced triploidy also increased the survival

of Tilapia nilotica x Tilapia aurea hybrids (Chourrout and

 

 

Itskovich, 1982). Heat shocked hybrids may not have better
survival in this research because of relatively low triploid
induction rates of this specific heat-shock treatment. Coho
female x chinook male hybrid heat-shocked groups contained no
triploids. Heat-shocked chinook female x coho male hybrid groups
contained 0 to 14.3% triploids (Table 1). This low number of
triploids may not have been adequate to overcome the negative
survival stress of hybridization and heat shocking. If triploid
induction was more successful and in the range of at least 80-90%

of the individuals, survival might have been considerably higher.

Disembryoggnesis of Hybrid Offspring

 

Interesting cases of disembryogenesis were observed in
hybrid offspring of chinook and coho salmon. Marked malforma-
tions of chinook female x coho male hybrids was noted shortly
after hatching. These malformations were present in as many as
30% of the hybrid individuals at any given time. The gross

appearance of these fish suggested a degeneration of the cervical

29

and caudal areas (Figures 3 and 4). Scoliosis, lordosis, and
sometimes both were present. Fish continued to developed these
traits over a four month period of time, with cervical degenera-
tion appearing first, followed by caudal degeneration and dis-
coloration. The degeneration made swimming and feeding difficult
for the fish, eventually causing death.

A very small percentage (less than 1%) of fish in the other
types of crosses also developed these problems. Specifically,
coho female x chinook male hybrids had a few individuals with
slight degeneration located only in the cervical area and no
degeneration in the caudal areas. Some of these fish also had
shortened gill opercula.

Normal and malformed individuals at four weeks post hatching
were sacrificed, preserved, and sent to the Animal Health
Diagnostic Laboratory at Michigan State University for diagnosis.
Radiographic and histopathological examinations revealed the
following results. ‘Vertebrae were poorly ossified and malformed
into triangular shapes. Areas of deformed vertebrae coincided
with scoliosis and lordosis. In areas where degeneration
occurred there was unequal development of segmental myotomes with
muscle tissue missing in certain areas and loosely organized in
other areas. Epithelium in the degenerated areas was lacking
scales but was pigmented and contained mucus glands. NO inflam-
matory cells were located in these areas indicating a non-infec-
tious etiology; 'The final diagnosis from the Animal Health

Laboratory was musculo-skeletal disembryogenesis of unknown

 

30

 

Figure 3.

Varying degrees of disembryogenesis occurring in
swim-up chinook female x coho male hybrid salmon.
Most severe on bottom with decreasing disembryogenesis

toward top. A normal hybrid is in each photo (top)
for comparison.

31

 

I. H mm" IMTW‘ LA?
IPWWW "I: ‘5,

,9”! 2 3

G 7 t 9 l

   
 

 

Figure 4. Four month old chinook female x coho male hybrid salmon
showing moderate disembryogenesis (Above - side View,
below, top view).

32

origin (Figures 3 through 7 for comparisons of normal and
abnormal individualsL A genetic cause may be suggested since
only chinook female x coho male hybrids had disembryogenesis in
large percentages.

Other workers have found various forms of disembryogenesis
or "cripples" associated with hybrids. Hybrids between Atlantic
salmon and pink salmon produced non-viable fry with severe
morphological anomalies. Some Of these fry apparently had only
one eye directed forward on the head and were described as
"cyclops like" (Loginova and Krasnoperova, 1982). Simon and
Noble, (1968) also reported variable survival of reciprocal

crosses of pink salmon and chum salmon (Oncorhynchus ketaL. Chum

 

salmon males x pink salmon females produced relatively normal
appearing Offspring with few mortalities. The reciprocal cross,
with chum salmon females x pink salmon.males suffered total
mortalities from weak deformed offspring. All hybrids between
chum and pink salmon had less robust caudal peduncles similar to
the results in chinook female x coho male hybrids from this
study. In brown trout female x brook trout male hybrids, the
percentage of cripples depended on which individual female brown
trout was used in the cross (Buss and Wright, 1956). This
occurrence may also hold true for this study. There is the
possibility that one of the three chinook females used in the
chinook female x coho male cross was genetically responsible for
the 30% disembryogenesis Observed. Buss and Wright, (1956) also

reported high variation in viability of fry in splake x brook

 

33

 

Figure 5. Six month old coho female x chinook male hybrid
salmon.

 

 

Figure 6. Six month old chinook female x coho male hybrid
salmon.

34

 

Figure 7. Six month old coho salmon.

35

trout hybrids and high percentages of "caudal cripples" in brook
trout females x lake trout males.

Previous trials of chinook salmon x coho salmon hybrids had
mixed results. Coho females x chinook males were rated as a
potentially useful hybrid (Chevassus, 1979 and Refstie et a1”
1982). This hybrid was shown to have a hatching rate of 40-88%
of control. (Chevassus, 1979 and Refstie et a1., 1982), and the
growth rate of this hybrid was reported to be five times the
control rate at 230 days of age (Refstie et a1., 1982). My study
agrees with these findings since coho female x chinook males had
minimal disembryogenesis with survival slightly less then control
at time of swim up (84% compared to 87%). Chevassus (1979)
reported lower success for chinook females x coho males because
only a few individuals survived to one year Old. ‘My study again
agrees with these findings since this cross contained severe
disembryogenesis and lower survival compared to control (12-l3%
lower). Blanc and Chevassus (1979 and 1982) also reported good
survival of coho female x chinook male hybrids. Their study
reported survival as high as 50% of control up to the fourth
‘month post hatching. These hybrids were noted to have slower
growth and achieved a smaller size compared to coho controls. In
contrast, using Foerster%3(l935) work as an example, Chevassus
(1979), stated that hybrids using coho salmon were typically non-
viable. Specifically, coho females x chinook males did not even

survive to the eyed stage.

36

The cause of non-viability in hybrids is Often difficult to
ascertain. Blue sac disease CLe. the inability to absorb the
yolk sac properly), has been implicated in many hybrid problems
(Blanc and Poisson, 1983; Buss and Wright, 1956). Other
potential explanations for hybrid mortalities include failure at
fertilization or embryogenesis, failure at hatching, differences
in incubation times, egg sizes, or chromosome counts causing
incompatibilities between the two species (Buss and Wright,

1956). Crippling and disembryogenesis is obviously reSponsible
for many mortalities and is probably caused by some genetic
inbalance of the hybrid genome during hybrid development. In the
present study the majority of hybrid mortality occurred between
eyeing and hatching (Figure l and 2). In fact, many chinook
female x coho male hybrid alevins were observed to have died in
the process of hatching. Disembryogenesis definitely contributed
to mortalities of the hybrids, especially after the period of
swim up when these individuals were not able to swiuinormally and
compete for feed. In Atlantic salmon, high mortalities at time
of hatching have been associated with defective enzymes (Battle,
1944). Hybrids with defective hatching enzymes would have
problems during hatching, whereas hybrids with severe disembryo-
genesis may not have the physical capability to hatch. It would
be interesting to discern what genetic and developmental
mechanism is disrupted to cause the disembryogenesis in hybrid

salmonids.

37

Maternal-Dominance in Hybrids

 

Another interesting phenomenon observed in coho x chinook
salmon hybrids was maternal dominance in the characteristics of
the dorsal and anal fin. All hybrids originating from coho eggs
retained the coho salnunfls elongated and white colored leading
fin rays of the anal and dorsal fin. In contrast, all hybrids
originating from chinook eggs lacked this white leading edge and
retained the appearance of chinook salmon. Other authors have
reported various forms of maternal and paternal dominance in fish
species. Hybrids between chum salmon males and pink salmon
females may show characteristics of one parent, the other parent,
unlike both parents, or a combination from both parents (Simon
and Noble, 1968L The F1 generation of chum salmon females x
pink salmon males developed a dorsal hump which was intermediate
in size to the large hump Observed on pink salmon males. Tail
spots, which are very evident on pink salmon, are present but are
reduced in size on the hybrid. Another characteristic which is
expressed in the hybrid in an intermediate manner is tooth size.
Pink salmon have small teeth while chum salmon have large teeth.
The hybrids have intermediate sized teeth. In four different
characteristics the hybrid chum x pink salmon resembles either
one parent species or the other. The pink salnunfs white belly
and white background coloration is retained over the dark belly
and yellow background coloration of the chum salmon. IParr marks
of the chum salmon, which are absent in pink salmon, remain

evident in the hybrid. Gill raker counts in the hybrid are

38

closer to pink salmon counts than to chum salmon counts.
Finally, scale counts of the first row Of scales above the
lateral line resembles chum salmon scale counts more closely than
pink salmon scale counts. Simon and Noble, (1968) explain the
parr mark characteristics as a simple Mendelian recessive. The
other characteristics are considered to be inherited in a
multiple gene system.

Reciprocal hybrids between channel catfish (Ictalurus

punctatus) and blue catfish (Ictalurus furcatus) exhibited

 

paternal predominance (Dunham, et a1., 1982). The male parent Of
these two hybrids was more responsible for determining the
external appearance, swim bladder shape, and fin ray number of
the hybrid offspringn In addition, growth and morphometric
uniformity also exhibited paternal predominance. Finally, even
behavior was effected by paternal predominance. Susceptibility
to capture by seine was influenced by the male parent more than

the female parent (Dunham, et a1., 1982L

Gynogenesis Versus Hybrid Production

 

Maternal dominance seen in the coho salmon x chinook salmon
hybrids actually may be an indicator Of gynogenetic production of
the maternal species. Gynogenesis has been induced in various
salmonids with several treatments. Gynogenetic rainbow trout
were produced by fertilization with irradiated sperm coupled with
heat shock (Chourout and Quillet, 1982; Purdom et a1., 1985L

Gynogenesis has also occurred in rainbow trout through the use of

 

39

pressure shocks of 7,000 psi applied for 4 minutes, 40 minutes
post fertilization in conjunction with irradiated sperm
(Chourrout, 1984). Finally, cold shock was also used to induce
gynogenesis in coho salmon (Refstie et a1., 1982). In my study,
gynogenetic production may have occurred in the hybrids by
activation of the maternal genome by sperm from the paternal
species. The sperm would contribute no genetic material to the
zygote. Most offspring activated in this matter would be haploid
and probably would be inviable (Refstie et a1., 1982). However,
if fertilization via a hybrid sperm was associated with a
disruption of the second meiotic division of the egg, then a
diploid gynogenetic may have been produced. A diploid gyno-
genetic is produced if the sperm pro-nucleus is excluded from
fusion, while a triploid hybrid is produced if the sperm pro-
nucleus is included (Chevassus, 1983). Spontaneous prevention of
the second meiotic division by fertilization with another species
sperm has occurred in grass carp females crossed to big head carp
males. In this case retention of the sperm pronucleus creates a
triploid hybrid (Allen and Stanley, 1983; Cassani, et a1., 1984)

If gynogenesis occurred with the coho salmon x chinook
salmon hybrids then the similar appearance of the offspring to
the maternal species could not be contributed to maternal
dominance. These offspring would appear identical to the
maternal side of the cross because they are gynogenetics of the
maternal side. The possibility of such an occurrence is

increased dramatically in the heat shocked hybrid groups because

40

the heat shock applied to induce triploidy may also induce
diploid gynogenesis if the sperm pro-nucleus fails to unite with
the egg pro-nucleus. Care must be taken when a hybrid is claimed
to be produced because of the above mentioned possibilities.
These pseudo-hybrids (gynogenetics) may be differentiated from
true hybrids by morphological characteristics, physiological
characteristics, biochemical characteristics, or karyological
characteristics (Chevassus, 1983). Since hybrids do not always
have intermediate characteristics of the two species involved,
morphological, physiological, and biochemical characteristics are
not always reliable at determining validity of a hybridfia
genomes. The study of karyotypes is the only reliable means of
determining the validity of an individualis genetic structure
(Chevassus, 1983). Gynogenesis was not found in my study but
before I discuss the evidence for this the various character-
istics of karyograms from coho and chinook salmon must be

discussed.

Karyograms of Coho Salmon

 

Chromosome counts of all metaphase spreads from coho salmon
indicated a peak occurrance and probable diploid number of 60
(Figure 8). This number agrees with the findings Of Gold et a1.
(1980) and Simon (1963). Karyograms of coho salmon indicate a
range of 44 to 46 metacentrics-submetacentrics and 14 to 16 acro-
centric-telocentric chromosomes (Figures 9 and 10). Simon (1963)

describes coho salmon having 52 metacentrics-submetacentrics and

 

 

41

.coEHmm osoo

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Eoum mOHmEmm monoumm ommammuoe mo noHuHmanoo nomads oEOmoEouno .w onswwm
..ODE:Z mEomoEth—U
o: 8. cm 8 2. 8 cm 3 on
_ _ . .p A _ O
_ . u .|.l %
mm o _ mm o
_ _ _ n M"
_ . ,

_ mad lo. M.

_ e

. cu

m

_ . m

_ m m. l on n.

_ s

cm u .5 3.8 w

.1

no

l.on mm

0,

w

IO

8

s . m

modm ice 9

3:03am”. ..:. H m
. H w

. "H

C O . e

.358 x m ..o . is.

 

owncw

 

 

 

 

   

«94% .4

   
 
  
 
 

9
\u I! u u M at n n u 1r,

ii! it at in xx xx xx xx xx

  
 

I! 88 U! M M M «A n M;

 

 

Figure 9. Metaphase spread (above) and karyogram (below)
of coho salmon diploid with 60 chromosomes
(44 metacentric-submetacentrics and 16 acro-
centric-telocentrics).

 

 

10 .
ii xi llil It. xx in: in NW

mm a u u ’xx in. u: u

  
 
 
    

   

’8 ll 3; at an 4.4 A. Mi.

 

Figure 10. Metaphase spread (above) and karyogram (below)
of coho salmon diploid with 60 chromosomes
(46 metacentric-submetacentrics, and 14 acro-
centric-telocentrics).

 

44

eight acrocentrics. This dissimiliarity reflects differing
nomenclature systems rather than differences in chromosome
morphology. Ikzobserved and made drawings from chromosomes in
anaphase in contrast to my use of metaphase chromosomes. Simonfis
drawings, made from camera lucida and direct microsc0pe Observa-
tions, appear to have much more detail than my own, hence, his
nomenclature system is different. Simon breaks up the chromosome
morphology into two groups. The first group consists of V-shaped
(metacentric) and J-shaped (submetacentric) chromosomes grouped
together. The second group consists of only rod shaped
(acrocentric) chromosomes. Chromosomes in my study were grouped
into the acrocentric-telocentric category when the centromeres
were at the end, or nearer to the end of than the middle of the
chromosome. In contrast, because of finer resolution, Simon
(1963) only categorized chromosomes as acrocentric when the
centromere was located at the end of the chromosome. When the
nomenclature system employed in this study is used on Simonks
coho salmon.karyogram, 46 meta-centric-submetacentrics with 14
acrocentric-telocentric Chromosomes is Observed. This completely

agrees with the findings from karyograms in my study.

Karyograms of Chinook Salmon

 

Chromosome counts for chinook salmon metaphase spreads
peaked at and indicate a probable diploid number of 68 (Figure
11). .A diploid number Of 68 chromosomes for chinook salmon

agrees with the findings of Gold et a1., (1980) and Simon (1963).

.coEHmm xooawso
Eouw moHQEMm mmmouam ommsmmuoe mo cofiuwmomfioo Humans oEomoEouso .HH madman

 

 

45

39.52 oEomoEoEo

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

o a F 09 on on 2. on on .9. on
P .b p P _ J— _ O %
_ _ ..
_ - . H ...|.. 1.
_ mg m mm m .m
_ l3 m.
. _ m mos. _ o
_ . S
_ .r .m
mo. n 5 H I 2 m
1 9
H d
. s
U ad
m I on m
. m
in mode a
k 8
r o
u .... 3 w
Eaammfl .:< W0..N.v m
n a
m ..855 x w ..855 . .....
. . A 1 cm
mm H :N

 

 

 

46

Simon (1963) also classified the chinook's chromosomes as 36
metacentric-submetacentric with 32 acrocentrics. This is similar
to the findings of my study where 34 to 36 metacentric-submeta-
centrics and 32 to 34 acrocentric-telocentrics were observed

(Figures 12 and 13%

Verification of Hybridization: Karyoggams of Reciprocal Hybrids

 

Between Coho and Chinook Salmon

 

Since standard chromosome complements of chinook and coho
salmon are known, the expected hybrid complement may be deter-
mined. 'This expected complement may then be compared with the
actual hybrid karyograms to determine if true hybridization or if
gynogenetic production has occurred. Taking the haploid chromo-
some number for chinook salmon (34) and adding it to the haploid
chromosome number of coho salmon (30) results in an expected
diploid hybrid number of 64. The peak chromosome number found in
chinook female x coho male and coho female x chinook male bar
graphs was 64 (Figure 14 and 15). This indicates that true
hybridization, rather than gynogenetic production, occurred. The
peak chromosome number would have been equal to the maternal
diploid number if gynogenetic production had occurred. Further
evidence for true hybridization can be found upon examination of
hybrid karyograms. Since chinook salmon have 34 to 36 metacen-
tric-submetacentrics and 32 to 34 acrocentric-telocentrics, then
a haploid number of 17 to 18 metacentric-submetacentrics and 16

to 17 acrocentric-telocentrics would be expected to be

 

 

.xx 3» :15: xx u an: Jug

in! ”i 81' L! 8818 M

Jr. 3* AA AA an AA 59!

[an AA 05 5454.»...05

  

64 ‘1‘ “" ‘5 “if;

 

Figure 12. Metaphase spread (above) and karyograms (below)
of chinook salmon diploid with 68 chromosomes
(34 metacentric-submetacentrics and 34 acro-
centric-telocentrics).

48

,a .. "Xf‘ "
5
‘ 1'; ‘ 25"
C‘ 7 a )
1‘ ‘ 7 [, $1.“. - :‘0
1' " ’J’
:,r' J ‘b 3‘ it‘:
1 A \ ' 5’ f A
" t! ,. a“, ’

 

2

l

*_M u n u n x: xx xx xx

 

1

’45” n I! u H Ii! M. u 1"-

 

)5. an as u m M u. up.“

 
  

  
 

. 14;.“ us M 6913

Figure 13. Metaphase spread (above) and karyogram (below)
of chinook salmon diploid with 68 chromosomes
(36 metacentric-submetacentrics and 32 acro-
centric-telocentrics).

49

.noEHMm pawn»: mama xooawfio x onEow onoo

 

 

 

 

 

Scum moHaEmm mmmouam ommnmmuoa mo aofluflmomaoo nomads oEomoEopno .¢H osswflm
500532 OEOmOEOhr—U
cow 0m on ON cm on JW on
p P . .

_ _ .1 - L m o a.
_ . - .

m. EPA m

_ .lllh mw

_ . m6 .m.

_ _ H lop w

_ m... a

_ . S

_ H .d

. m

_ nv

ill 9

mm H :0 . d

H _9

o la

n nu

. lion “H

J

. O

.l. I... i m

H 4 mm s

m

nice Av

mm

3.563%:— .34.. _. _ m

3.3. o.

. 9

1

.e .856 x m 2.8

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

50

Eoum moHaEmm mmmoumm o

.moEHmm mwunha mama 0500 x onEom xoocHno
mmsmwuofi mo cowufimomfioo “snaps oEOmoEouno

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

.m. asaw.a
.3532 06305030
0: 00w 8 8 Os 8 Om 3 on

_ L _ L _ p o
_ T u u an
“ tau." m we m
u m... m 12 m
_ M so u
. 9
_ u cu
. m
_ H .

H I.ON
mm n .5 H w
H d
. 9
. Id
m I... m
0
. m
. O
H .I... w
H ,mKM l 9. w
to... a
322:: :< . m
. m.
.e 2.8 x 3855 w is m

VGHCN

 

 

 

 

 

 

51

contributed to the hybrid. Likewise, coho salmon were determined
to have 44 to 46 metacentric-submetacentrics and 14 to 16
acrocentric-telocentrics. The coho salnunfs haploid contribution
to the hybrid would be 22 to 23 metacentric-submetacentrics and 7
to 8 acrocentric-telocentrics. Combining the chinook and coho
haploid chromosome complements gives an expected hybrid with 39
to 41 metacentric-submetacentric and 23 to 25 acrocentric-
telocentric chromosomes which was Observed in hybrid karyograms
shown in Figures 16 through 19. Figures 16 and 18 show karyo-
grams of individuals with 40 metacentric-submetacentric and 24
acrocentric-telocentric chromosomes. Figure 17 shows the karyo-
gram Of an individual with 40 metacentric-submetacentric and 23
acrocentric-telocentric chromosomes. Figure 19 shows an
individual with 40 metacentric-submetacentric and 25 acrocentric-
telocentric chromosomes. Figure 20 shows the karyogram of an
individual that deviates the furthest from the expected hybrid
number with 38 metacentric-submetacentric and 27 acrocentric-
telocentric chromosomes. This last example is much closer to an
expected hybrid karyogram than either parental species karyogram
indicating it is a true hybrid. This is true because when the
differences in chromosome number between the deviant hybrid and
coho salmon, the deviant hybrid and chinook salmon, and the
deviant hybrid and expected hybrid are compared; the difference
is least between the deviant hybrid and the expected hybrid
chromosome counts. Specifically, the deviant hybrid has two to

four extra metacentric-submetacentric chromosomes and five to

 

. ———w———v

Figure 16.

u is it 18 u an «a {g

    

 

 

Metaphase spread (above) and karyogram (below)
of coho female x chinook male hybrid diploid
salmon with 64 chromosomes (40 metacentric-
submetacentrics and 24 acrocentric-telocentrics).

53

 

  
 

'1 14
Mix ii xx it n mm;-
119mm» xa . "

  

 

Figure 17. Metaphase spread (above) and karyogram (below)
of coho female x chinook male hybrid diploid
salmon with 63 chromosomes (40 metacentric-sub-
metacentric and 23 acrocentric-telocentrics).

 

 

,,

 

Figure 18. Metaphase spread (above) and karyogram (below)
of chinook female x coho male hybrid diploid
salmon with 64 chromosomes (40 metacentric-

submetacentrics and 24 acrocentric-telocen-
trics).

 

55

 

 
 
  

12
gum ii iii n n n n n

2; 3:: es in am x:- xn

   
  

x.,‘

.9

4 '3 L2!!— “L4 0 A .62 4;?"

Figure 19. Metaphase spread (above) and karyogram (below)
of chinook female x coho male hybrid diploid
salmon with 65 chromosomes (40 metacentric-
submetacentrics and 25 acrocentric-telocen-
trics).

56

 

 

Figure 20. Metaphase spread (above) and karyogram (below)

of chinook female x coho male hybrid diploid
salmon with 65 chromosomes (38 metacentric-

submetacentrics and 27 acrocentric-telocen-
trics).

 

57

seven fewer acrocentric-telocentric chromosomes when compared to
the chinook salmon karyograms. The deviant hybrid also has six
to eight fewer metacentric-submetacentric Chromosomes and 11-13
extra acrocentric-telocentric chromosomes when compared to coho
salmon karyograms. The smallest difference occurs when the
deviant hybrid is compared to the expected hybrid chromosome
counts. The deviant hybrid has only one to three fewer
metacentric-submetacentric chromosomes and two to four extra
acrocentric-telocentric chromosomes when compared to the expected

hybrid counts.

Karyograms of Triploid Chinook Salmon

 

Karyogram and metaphase spread chromosome count distribu-
tions also indicate triploidy was induced in heat-shocked chinook
salmon and chinook female x coho male salmon hybrids. Bimodal
peaks centered around the expected chinook salmon diploid (2N =
68) and triploid (3N = 102) number indicate triploid and diploid
individuals were contained within the heat-shocked chinook salmon
pure crosses (Figure 21). Likewise, bimodal peaks centered at
the diploid chinook female x coho male hybrid number (2N = 64)
and triploid number (3N = 98) also indicate the presence Of
diploid and triploid individuals (Figure 22). The absence of
such bimodal peaks from heat-shocked coho pure matings and coho
female x chinook male crosses indicated no triploid induction
occurred in coho maternal side crosses (Figures 23 and 24). The

absence of bimodal distributions from all other non-heated

 

 

58

.noEHmm xooawno moxoonm umon Scum
monEmm mmwouam ommsmmuoe wo cofiuwmomaoo gonads oEOm080pco

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

.HN spawns
conEaz oEomoEoEo
0w p 0% r 0_m 0.0 on 00 0.0 0.0 00
121:2... . T. ...
a... I I “ s3 . 43 m
m.m_ . _ Oww H h 0 mm.
. I109
_ _ . .2 e
_ mm... m mo a
. n0
" m 9...: m
N _ . ION m
09 u an .
. _ .0
9mm m
H O
u i.00 mm
. O
H w
H w
3:05.. 32 H is. w
. 9
“u : um
v.85 so: +. s a m
a. .3850 x a .3820 s m

 

 

 

 

 

 

.noEHmm panama mama 0500 x onEom xooawno moxoonm “no:
Eouw mofiafimm mmmopam ommnmwuofi mo :oHuflmanoo amass: oEOmoEOMSU .NN ouswflm

 

59

.3532 080880.30

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

0 w p 00 r 00 00 Oh 00 on 0* on
. l. I A “T . I . I so a.
E. . 58w... m m m m .lmmmu w
_ star... is M mud n w
_ 0h.¢ mm V . . . F _ B
. . la. .a
A m m 2 . -2 a
_ m I a
_ H 0.9 S
_ . m
H a u

. i.0
wmwnucmu H mw
H .0
L198 m
.H , Ion m
. 0
mm 5 m
. O
u 9
H m
3.50:0: .:< 13 m
H n
xuocm «no: + «an cu m.
.e 2.8 x a .388 . ..s ..

 

 

 

6O

.SOEHmm OSOU UGMOOSm ummfi

Eoum monEmm mmmouam ummaamuoe mo cofluwmoasoo popes: oEomoEouso

.mm spawns

 

338:2 oanoEoEO

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

o.” 2.: cm o.o a.» on on c... on
We. 8.83m; m .n
_ I. I,
_ Juno
. la.
n l -83
cone»
8de , Jon
c‘na
no»
woman
85038.. ._._< A 13.
.625 .8: + 8H5
w. 28 X m 2.8 To...

 

 

Jeqmnu owosowoaqa .Iod spends essudezew a,

 

 

   

.coEHmm mflunzs onE xoocmso N onEom oaoo moxoonm use:
Scum monEMm mvmoumm ommnamuofi mo coauflmomaoo Humans OEOmEounu .qm ouswwm

 

61

500-252 0809080550

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

. on.
0w _. ow. 0.m 0.0 0x. 0 m m 0% on m
a. min .n L A IN... a.
_ WNW .560 0nd m.
u 363 L n
_ iwl e. «pm 10' u.
_ . w
_ UH“ n.
_ AH“ s
VON...” ,lllli JON .0
ill m
,nHWIIIIi nu

ii I . m .
W Hon Jon 0
m
A - o
T 555 w
mMP<03nmm 44¢ . 10¢ w
emucu N
xoonw “mm...— + m
. . a.
% xooch X. w 0200 Tom m

 

 

 

 

62

shocked crosses indicated that no natural or hybrid produced
triploid individuals were encountered (Figures 8, 11, 14, and
15). It is important to determine that no natural triploids are
in the population because natural triploids have been found in
rainbow trout (Thorgaard and Call, 1979) and pink salmon.(Utter
et a1” 1983).

Further evidence Of triploid induction comes from examina-
tion of nearly complete karyograms of triploid chinook salmon. A
triploid chinook salmon would be expected to have one diploid
(2N) plus one haploid (1N) set of chromosomes. Using this guide-
line, plus the standard karyological composition of chinook
salmon, a triploid chinook salmon would be expected to have a
total of 51 to 54 metacentric-submetacentric chromosomes from 34
to 36 diploid plus 17 to 18 haploid metacentric-submetacentric
chromosomes. iLikewise, 48 to 51 acrocentric-telocentric chromo-
somes would be expected in a triploid from a diploid count of 32
to 34 plus a haploid number of 16 to 17 acrocentric-telocentric
chromosonmm. 'When analyzed, the two nearly complete triploid
karyograms (Figures 25 and 26) come very close to the expected
counts. The karyogram represented in Figure 25 contained 96
chromosomes (expected 3N = 102) with 50 metacentric-submetacen-
tric (expected 51 to 54) and 46 acrocentric-telocentric chromo-
somes (expected 48 to 51). The karyogram in Figure 26 is more
similar to the expected counts with 101 chromosomes (expected 3N
= 102%. Fifty of these chromosomes were metacentric-submetacen-

tric (expected 51 to 43) and 51 acrocentric-telocentric (expected

63

 

      

”x kw: xxx 1“”
1013i xxx XH“ it“! K55
Xxx 2W ”x “'5‘" xxx

xix an: an

I

’ ’\ I‘lN. 0'» lflcnv IN I\ '4‘ .g .\\
I -— - fiik p» hil.a 5",‘.i

-"“"' 6“" ""‘ “can

 

 

Figure 25. Metaphase spread (above) and karyogram (below)
of chinook salmon triploid with 96 chromosomes
(50 metacentric-submetacentrics and 46 acrocentric-

telocentrics).

Figure 26.

64

 

 

,' i" .'.'
. , ‘,
'1 ~ I
:1 - I
.
4A
’ .
’
‘3’

7 h f”!!!
vl’kx ”(xx xxx Md

mm xxx xxx Xxx
Metaphase spread
(above) and karyo- .
gram (below) of xxx 2*“ “I!” x XX \.
chinook salmon
triploid with 101
chromosomes (50 ‘ 0‘ i x xx N ”X . *V’
metacentric-sub-
metacentrics, and
51 acrocentric-

telocentrics). 04 A AA VIN-“h I‘m“

AAA “on “Ga-q 650

  
     

 

‘~~“‘.A“L~A.z
a!” "l . i ‘37,?” V ., .

65

48 to 51). In addition to the total chromosome counts indicating
triploidy, the natural grouping of homologous trios of chromo-
somes seems to occur (Figures 25 and 26%. The chance of two or
more partial diploid metaphase spreads combining to produce a
pseudo-triploid metaphase Spread seems remote due to the close-
ness of the expected counts with the actual counts and the

homologous trios evident in Figures 25 and 26.

Comparisons of Efficiency of Various Triploidy Induction

 

Techniques

 

Although triploidy was induced by heat shock of 36°C for one
minute 10 minutes post fertilization in chinook salmon and
chinook female x coho male salmon hybrids, the percentage of
triploids in each test group is not high enough for production.
Triploidy was induced in 42.1 i 11% and 21.1 i 9% of the
individuals in replicate one and two of heat-shocked chinook
salmon (Table 1). Heat shock also induced triploidy in 0%, 10 :_
9.5%, and 14.3 i 11% of the individuals in replicates one, two
and three of heat shocked chinook female x coho male salmon
hybrids (Table 1). This heat shock apparently was not effective
at inducing triploidy in coho salmon and coho female x chinook
male salmon hybrids since none were observed (Table 1%

Close to 100% triploid induction is desirable because
triploid individuals can only be detected from diploids by exten-
sive karyological or cytological techniques. This process would

be uneconomical for large hatchery operations. Using the same

66

 

Table 1. Percent triploid individuals per treatment group.
Cross and Treatment
Replicate Percent Triploid
Number Maternal x Paternal : Standard Error
1 Coho x Coho 0
2 Coho x Coho 0
3 Coho Coho 0
l Coho Coho + Heat Shock 0
2 Coho x Coho + Heat Shock 0
3 Coho Coho + Heat Shock 0
l Coho Chinook 0
2 Coho Chinook 0
1 Coho Chinook + Heat Shock 0
2 Coho Chinook + Heat Shock 0
3 Coho Chinook + Heat Shock 0
1 Chinook x Chinook 0
2 Chinook x Chinook 0
3 Chinook x Chinook 0
l Chinook x Chinook + Heat Shock 42.L:11
2 Chinook x Chinook + Heat Shock 21.1: 9
l Chinook x Coho 0
2 Chinook x Coho 0
3 Chinook x Coho 0
1 Chinook x Coho + Heat Shock 10.0:9.5
2 Chinook x Coho + Heat Shock 0
3 Chinook x Coho + Heat Shock 14.3111

 

 

67

heat shock as in this study, Thorgaard et a1” (1981) produced
an average of 40% triploid rainbow trout with 30% survival twenty
days post fertilization. When compared to this study's average
of 18% triploid induction with survival of heat shocked groups
ranging from 45-60% at swim up, a large improvement in triploid
induction rate with some sacrificing of survival can be accepted.
The lower success rate of the chinook salmon and chinook female x
coho male salmon hybrids triploid induction compared to rainbow
trout triploid induction rate may possibly be attributed to
larger egg size of the salmon. ILarger eggs may require high
temperatures, longer heating times, or a more effective heating
technique to be effective. Ways of improving the triploid induc-

tion rate must concentrate on these fHCtOI‘S.

Improvement of Triploidy Induction Techniques (Heat Shocking)

 

One way to improve triploid induction rate would be to
create a flow-through heat shocking apparatus rather than stati-
cally heat shocking the eggs. When a cluster of eggs are heat
shocked, eggs located toward the center of the egg mass may not
be heated as thoroughly. These eggs may not be successfully
triploid induced. .A flow through system, in which heated water
is pumped through the egg mass, would help in this situation by
moving warm.water to the center of the egg mass. This would lead
to more even and thorough heat shocking with presumably higher

triploid induction rates.

68

Other ways to improve triploid induction rate may be to
increase heat-shocking temperatures or duration. Both duration
and temperature probably should not be increased dramatically
together or excessive mortalities may result. One method may be
to lower heat-shock temperatures somewhat (temperatures in high
20°C range instead of 30°C range) while increasing the duration
of the shock. For example, rainbow trout have had triploid-
induction rates of 50-100% (depending on the strain of trout)
with heat shocks of 27°C to 28°C, for 10-15 minutes, at 40
minutes post fertilization. Survival for the heat shocked groups
was 30% at hatching compared to the control at 59%.(Lincoln and
Scott, 1983). Utter et a1., (1983) also induced high percentages
of triploids (58-84%) in Pacific salmon. This was accomplished
with a 28-30°C heat shock, for 10 minutes, ten minutes post
fertilization. Several authors induced 100% triploidy in various
salmonids. A 26°C heat shock, for 10 minutes, 1 minute post
fertilization produced 100% triploid rainbow trout (Solar et a1”
1984). Heat shocking of Atlantic salmon eggs resulted in 100%
triploids in two different cases. The first case used a heat
shock of 32°C, for 5 minute duration within 20 minutes post
fertilization (Benfey and Sutterlin, 1984b); and the second case
a heat shock of 30°C, for 10-14 minute duration, at 10 to 20
minutes post fertilization (Johnstone, 1985). Johnstone (1985)
prefers to use triploid yield (triploid induction rate x percent
survival at hatch) as a successful triploid induction scale.

Using this technique, Johnstone reports that 100% triploid

69

induction rates may not be optimal because of lower survival
rates that decrease yield. He states that a heat shock of 26°C,
for 20 minutes, starting immediately after fertilization produces
94% (instead of 100%) triploid individuals but with highest
possible yield (70JD of triploid individuals because of higher
survival. However, if losses due to egg mortalities may be
compensated for by slightly larger egg takes, a premium.can be
placed on production of 100% triploid individuals. Recent
communications received by Michigan State University from Hill et
a1., (1985) also supports medium temperature (28.5°C) heat shocks
of medium duration (10 to 15 minutes) to induce triploidy in 100%
of the chinook salmon.shocked. These workers also found that a
slow cooling period rather than fast cooling, after the heat
shock enhanced the production of triploid chinook. Preliminary
work at Michigan State University supports this supposition.

Heat shocks at 28.5°C, for 10 or 15 minutes starting ten minutes
after fertilization, followed by a 30 minute slow cooling period
induced triploidy in 100% chinook salmon (Westerhof, personal

communication).

Improvement of Triploidy Induction (Hydrostatic Pressure)

 

Another method to induce triploidy in salmonids which may
actually prove to be better than heat shocking, is the use of
hydrostatic pressure. IPressure shocks work on the same
principles as heat shocking, in that the second meiotic division

is disrupted or prevented during the period immediately post

70

fertilization. Pressure shocks of 10,000 psi, for eight minutes,
starting 60 minutes from oviposition, induced 97% triploid
efficiency (percent triploidy x percent survival) in the axolotl
salamander (Gillespie and Armstrong, 1979). The great advantage
in pressure shocks over heat shock is higher percent survival
with comparatively high triploid induction rates. IPressure
shocks have successfully induced triploidy in some salmonids.
Rainbow trout eggs, subjected to 7,000 psi of pressure for four
minutes, 40 minutes post fertilization had 100% triploid induc-
tion rates with over 70% survival 50 days after hatching
(Chourrout, 1984). Triploidy was also induced in 100% of
Atlantic salmon, using 10,150 psi of pressure applied for three
to six minutes within 20 minutes of fertilization. ‘These
Atlantic salmon had 70-90% survival compared to only 18% survival
of identically heat shocked groups (Benfey and Sutterlin, 1984b).
Benfey and Sutterlin stressed that pressure shocks above 10,150
psi, or for longer durations than six minutes, or shocks applied
after 20 minutes post fertlization caused 100% mortalities.
Obviously extreme care must be taken when using pressure shocks.
The high incidence of triploidy and high survival of pressure-
shocked eggs makes this a viable technique to be attempted on
coho and chinook salmon. 'The primary disadvantage to pressure
shocking is the cost associated with acquiring the specialized

equipment suitable to the task.

71

Failure of Triploid Induction in Coho Salmon

 

Failure of triploid induction in coho maternal side crosses
in this trial is difficult to explain. Coho salmon are liable to
triploid induction because a heat shock of 28-30°C for 10
minutes, ten minutes post fertilization induced triploidy in 50%
of the coho individuals (Utter et a1., 1983). Utter et a1.
(1983) also had some heat shocking trials which failed to produce
triploids in coho salmon. These trials were conducted at
temperatures of 33, 34 and 35°C for one minute at ten minutes
post fertilization which is almost identical to my heat shocking
regiment. One explanation arises from the observation that coho
were not at peak ripeness, or at the peak of their spawning run
when eggs were taken for this experiment. It is possible that
this overripe or underripe condition may be responsible for
triploid-induction failure. Other authors have stated the
susceptibility to triploid induction may be related to the degree
of egg ripeness and timing of ovulation (Lou and Purdom, 1984;
Thorgaard and Call, 1979). I hypothesize that eggs taken late in
the spawning season may have progressed into stages of the second
meiotic division which cannot be prevented by heat shock. ldke-
wise, eggs taken before they are ripe may not have progressed
into a stage which makes them liable to heat shock. This
hypothesis may explain why coho salmon, which were not at peak
ripeness, were not successfully triploid induced. This
hypothesis is further supported by observations on heat-shocking

trials performed on chinook salmon, by Michigan State University,

 

 

 

72

in the fall of 1985. In these trials, a heat-shocking technique
which produced 100% triploid individuals in the middle of the
spawning run, produced no triploids at the very end of the
spawning run. The only other difference in technique between
these two groups was the immediate fertilization of eggs after
oviposition in the early spawning run and delayed fertilization
of eggs in the late season spawning run. The delayed fertilized
eggs were stripped and packed dry for a several hour trip to Wolf
Lake State Fish Hatchery for subsequent fertilization and heat
shocking. It is also possible that eggs transported in this
manner prior to fertilization may undergo the second meiotic
division rendering a heat shock useless. It is doubtful that
this is true because Utter et a1., (1983) and Hill et a1., (1985)
had successful triploid induction using delayed fertilization.

If this were true, the question of "What stimuli induces the
second meiotic division?" would have to be answered. Could the
second meiotic division be stimulated by time after oviposition,
physical shock from eggs being transported, or is it fertiliza-
tion? Another question which needs to be answered is, "What
effect does early, mid, or late season egg taking have on
triploid induction rates?" If there are correlated differences
in triploid induction rate with egg ripeness the reason why these
differences exist and what relation does this have to the
formation and function of the spindle apparatus of the second
meiotic division must be answered before optimal triploid produc-

tion can occur.

73

Solid Tissue Karyotyping

 

Not only are improvements needed in triploid-induction tech-
niques, but triploid-detecting methods have many flaws and need
some improvement. Solid-tissue karyotyping, the primary method
of ploidy determination used in this study, proved to be very
time consuming and labor intensive. Appendix B lists the steps
involved with solid-tissue karyotyping and reviews the stages
which are critical to the procedure. Karyotyping of teleost
fishes in general is more difficult than other vertebrates due to
extremely small chromosome size and frequently high chromosome
numbers (Blaxhall, 1983a). In addition to this, much time is
required in perfecting karyotyping techniques for each species so
that optimal colchicine concentrations and exposure times must be
determined. If colchicine concentrations are too high or expo-
sure is too long, chromosome morphology is destroyed by condensa-
tion (Denton, 1973; Roberts, 1968L The longest (and most iden-
tifiable) chromosomes are Obtained by the shortest colchicine
treatments (Van Tuinen and Valentine, 1982). On the other hand,
if inadequate quantities of colchicine, or inadequate periods of
colchicine exposure are used, metaphase spreads are rarely
obtained. In order to obtain sufficient metaphase spreads for
this study, a colchicine exposure time of six to eight hours was
needed. This length of treatment, however, caused considerable
chromosome condensation resulting in poor chromosome morphology.
Identification and classification of metacentric-submetacentric

and acrocentric-telocentric chromosomes was difficult because of

 

74

condensation. This probably accounts for the differences seen in
chromosome classification between, for example, Figures 9 and 10.
Both the metaphase spreads in Figures 9 and 10 are of diploid
coho salmon with 60 chromosomes. However, Figure 9, which
contains chromosomes more condensed than Figure 10, has 44
metacentric-submetacentric chromosomeS‘with 16 acrocentric-
telocentric chromosomes. Figure 10, in contrast, has 46 metacen-
tric-submetacentric with 14 acrocentric-telocentric chromosomes.
Another good example of the differences encountered in karyograms
due to chromosome condensation can be seen in the comparison of
chromosome length and detail between Figures 16 and 17. 'The
differences in chromosome condensation probably accounts for the
majority of the variation of chromosome classification observed.
Another crucial factor in obtaining an adequate quantity of
metaphase spreads is the stage of development and growth of which
the individuals are sampled. Since actively dividing cells are
required to obtain metaphase spreads, fish in an active stage of
growth are required. Salmonids at the blastodisc stage are ideal
for good karyograms because chromosomes are larger at this time
and cell divisions are frequent (Roberts, 1967). In contrast,
eyed embryos of salmonids are undesirable for good karyogram
results (Roberts, 1967; Thorgaard, et a1., 1982). Similar
results were Obtained in this study with fingerling salmon.
Earlier samples of fingerling salmon which measured approximately
six centimeters or less provided more and better quality meta-

phase spreads than salmon sampled later which measured more than

 

75

six centimeters. This was the main reason sample size was
reduced from twenty to ten fish per replicate later in the study.
Considerably more time was required to process the larger fish
because fewer metaphase spreads could be found per preparation
and more slide preparations were needed for an adequate number of
metaphase spreads to be counted.

Highly variable chromosome counts were another problem.asso-
ciated with solid-tissue karyotyping. This variation is easily
seen in the bar graphs in Figures 8, 11, 14, 15, 21, 22, 23 and
24. Chromosome number usually peaks at the expected diploid or
triploid chromosome number. The majority of the other counts
fall below the expected number and a few usually fall above the
expected counts. These variations in counts may be attributed to
a variety of causes. First, the high predominance of metaphase
spreads containing fewer than the expected chromosome count may
be explained by loss of chromosomes during fixation and
processing. Chromosomes lost in this manner may not have been
fixed adequately to the slide and washed off in later stages of
staining and mounting. In addition, in some slides, which were
kept in hypotonic KCl solution for slightly longer periods than
30 minutes, or when tissues were not adequately fixed, seemed to
contain degraded, fuzzy appearing chromosomes. In these slides
it is possible some chromosomes may have been completely degraded
and lost. Some chromosomes may also have been hidden from view

beneath other chromosomes and cell debris.

 

76

The occasional higher than expected chromosome counts must
also be explained. Counting errors due to "false centromeres"
may explain some of these counts. ‘The procedure of counting
chromosomes used the centromere as an indicator of chromosome
position within the grid system as explained in Materials and
Methods Section. When a metaphase spread was compact, or the
chromosomes close together,:many chromosome arms overlapped.
These overlaps looked like centromeres especially when two darkly
stained chromosome arms were involved. 'These "false centromeres"
could have been counted as additional chromosome centromers.

Another possible explanation for higher than expected chro-
mosome counts is that some chromosomes from adjacent or nearby
metaphase spreads may have been dislodged from one spread and
fixed within another metaphase spread. I do not consider this
explanation as very viable because evidence does not support it.
Every metaphase spread seemed to be fixed at a different degree
of contraction with a characteristic stain color. If chromosomes
from different cells mixed, differently stained chromosomes would
have been present in one metaphase spread. Only one occurrence
of differently stained chromosomes contained within one metaphase
spread was observed in my study. Roberts (1967) also observed
variations Of chromosome number in Atlantic salmon karyotypes.
Chromosome counts may also deviate from.expected values due to
natural causes rather than methodological causes.

Naturally occurring intraspecific variation in chromosome

number has been reported in many species. Grammeltredt (1974) and

 

77

Fukuoka (1972) found diploid chromosome counts ranging from 58 to
65 in rainbow trout. Different populations of green sunfish

(Lepomis cyanellus) show chromosome polymorphism with some popu-

 

lations containing 46 chromosomes and other populations with 48
chromosomes (Becak et a1., 1966). Robertsonian translocations,
fissions and fusions have been implicated as the cause of these
variations in green sunfish and rainbow trout (Becak et a1”
1966; Thorgaard, 1976). 'Thorgaard (1976) also states that many
other salmonids, including the coho and sockeye salmon, have been
shown to contain Robertsonian polymorphisms. The possibility
that some of the chromosome variation found in this study may be
do to polymorphisms within coho salmon, chinook salmon, and their
hybrids can not be overlooked.

Other problems with solid-tissue karyotyping related to the
multiple steps required for the process. If even minor devia-
tions were made in the sequence of steps outlined in Appendix B
the quality Of metaphase spreads was greatly affected. 'Notes of
the most critical steps used in this technique are also listed in
Appendix:B. The two most important of these steps appeared to be
the use of fresh fixative, and the proper application of the cell
suspension to the slide. The use of old fixative was probably
the major factor leading to failure of initial karyotyping
attempts. Cell suspensions made from inadequately fixed tissues
produced very "fuzzy" appearing images of cell particles. In
contrast, properly fixed tissues carefully applied to slides

produced distinct and sharp images of cell particles and the best

78

metaphase spreads. Application of the cell suspension to the
slide was also critical. Kligerman and Bloom (1977) state,
"expel" the cell suspension onto a clean slide. I interpreted
this as spreading the cell suspension with some force since most
karyotyping techniques require "dropping" cell suspensions onto a
slide from various heights (Gold, 1974; Hollenbeck and Chrisman,
1981). In fact, catfish karyotyping was completed by dropping
the cell suspension onto slides from a height of three meters
(Hollenbeck and Chrisman, 1981). Actually, no force is required
in this process and better terminology would be to "flow" the
cell suspension onto a hot slide. 'When cell suspensions were
applied with force during the Kligerman and Bloom (1977) pro-
cedure, very few cell particles and no chromosome spreads were

fixed to the slides.

Lymphocyte and Tissue Culture Karyotyping Techniques

 

Other karyotyping techniques may give better results than
the solid tissue method. ‘Very high quality metaphase spreads
with chromosomes of similar size and parallel chromatids are
frequently obtained with lymphocyte culturing techniques. .A
mitotic stimulant, phytohemagglutinin (PHA) used to synchronize
cell division followed by a colchicine treatment to stop cells at
the same time in metaphase has been used (Walters et a1., 1981a).
Tissue culture, using trypsin to emulsify tissue into individual
cells, may also achieve good metaphase spreads when treated with

PHA and colchicine (Blaxhall, 1975). The main disadvantage with

 

79

these two techniques is they require more sophisticated equipment
than a microscope, slides, and fixative such as culture media,
centrifuges, incubating containers, and incubators. Another
disadvantage is that culturing techniques require considerably
large amounts of tissue or blood making work on small specimens
impossible (Blaxhall, 1981; Denton, 1973). ‘Finally, even though
karyotypes may be Of better quality with cell culturing tech-
niques, results may still vary depending on the individual
person's technique (Grammeltvedt, 1975; Hartley and Horn, 1983).
The main advantages of solid-tissue karyotyping is that it
does not require highly specialized equipment or training and is
relatively inexpensive (Thorgaard, et a1., 1982). Karyotyping is
also a direct way of determining triploidy'(by counting chromo-
somes) and it is the only consistent way to confirm if hybridiza-
tion or gynogenesis has occurred in experimental hybrid crosses.
The main disadvantages of solid-tissue karyotyping are that it is
labor intensive and time consuming, resulting in relatively small
sample sizes to determine triploid induction rates. Due to the
small sample size, large standard errors are associated with the
percentage of triploid individuals induced. For example, in
Table 1 in the first replicate of chinook females x coho males +
heat shock, the percentage of triploid individuals is 10% ;i-_ 9.5%.
In order to reduce this large standard error, larger samples must
be taken. If larger samples are needed, a better triploid iden-

tification technique is required.

 

 

80

Erythrogyte Parameters Used for Triploid Identification

 

All other triploid-identification methods indirectly measure
the amount of DNA contained within the cell nuclei. iErythrocyte
parameters can be used for triploid identification. This system
works on the premise that triploids, having an additional haploid
(1N) set of chromosomes with more DNA, will have larger nuclei
and cells than diploids. In fishes nuclear measurements are
usually performed on erythrocytes since fish erythrocytes are
nucleated and are readily available by venous puncture. The
simplest and most inexpensive method of determining triploidy is
measuring parameters of erythrocytes and erythrocyte nuclei.
Erythrocyte parameters have previously been used to identify
triploids and diploids of hybrid grass x bighead carp (Beck and
Biggers, 1983), Atlantic salmon (Benfey et a1., 1984),
Poeciliopsis (Cimino, 1973), and channel catfish (Walters, et
a1., 1982c). In this study a comparison of diploid and triploid
erythrocyte distributions in relation to various nuclei para-
meters reveals a difference in nuclei size between diploid and
triploid chinook salmon (see the bar graphs of Figures 27-30).
The means, standard errors, and triploid/diploid ratios of the
various parameters are listed in Table 2. Ideally, the ratio of
triploid nuclei volume to diploid nuclei volume should be 1.5:1.
In this study, the actual ratio observed was 1.96:1 and probably
indicates that the nuclei are probably not true ellipses so the
formula overestimates the volume. Based on the means and the

sizes of the standard error, the best indicator of triploidy was

 

Numbers of Erythrocytes

81

 

19"
17“
15"
13‘

11-

 

D Diploid

I Triploid

 

 

 

Hi

 

 

 

 

 

1
5.32 5.99 6.65 7.32

Length of Long Nuclear Axis from Erythrocytes (:1)

Figure 27. Distribution of diploid and triploid

erythrocytes per length of long nuclear
axis.

 

 

82

.onosc owu>00p£umuo mo mono
pom mouxoou£ukuo mwofimwpu mam mHOHQHo mo :ofiusnflnumwm .wm ouswwm

AN: 0 .22.: ozaoocgtcw .0 o2<

50 m0 V” N” 00 cu nu ma an. _.N 0.. or 09 VF NP Pr

 

222:. I

2035 B

NF

sexKooaqMJa 10 Jeqwnu

 

83

.HoHosa oauhoou£uhuo
mo oEsHo> mom mouhoopnuhno oHoHaHuu mam oHonflm mo moflusnauumam .

Am: . .2032 2:82:35 *0 oE:.o>

m: mm, tion

{J

 

{I

  

 

 

maiou

 

 

i

 

 

 

222; I
2033 D

 

 

 

 

 

 

 

mm spawns

muivw

- av
.TN
.iv
.im
.Im
[gap
:3
|«.w
have

 

9915001043513 10 .quuinN

Number of Erythrocytes

84

 

20' E] Diploid

I Triploid

T

10_

 
 
  

 

 

 

 

 

 

 

 

2.66 3.33 3.99 4.66

Length of Short Nuclear Axis from Erythrocytes (u)

Figure 30. Distribution of diploid and triploid
erythrocytes per length of short nuclear
axis.

 

85

 

ZN

 

 

 

34 $4 2.4 mm; @333.
I I I I 23
on.ma+o~.om mm.m+ao.ma mm.+ma.m mo.+¢m.o vHoHan
I l I l A23
ma.oa+nn.am oo.m+mn.nm N¢.+mw.m mm.+om.a pHonHHH
Amsv osaao> Amsv mou< Asv nuwdoq Asv suwaog
wwx< uuonm mwx< waoq

.coaamw xoocwno owofimwv one mwoflnwuu Scum

moumoou£u%uo mo muouoamumm umofiosz .N manna

 

86

length of nuclear long axis. That is to say, the smallest amount
of overlap between diploid and triploid measurements occurred in
length of the long nuclear axis from the erythrocytes. Ranked in
increasing amount of overlap and decreasing ability to differen-
tiate triploidy is area of erythrocytic nuclei, followed by
volume of erythrocytic nuclei, and finally length of short
nuclear axis. These results are very similar to the findings of
Wolters et al. (1982c). Wolters et al. (1982c) states that the
best method for determining triploidy in channel catfish was to
consider all nuclear variables together. Measuring the length of
nuclear major axis was the second best method for determining
triploidy. Finally, volume of erythrocytic nuclei and length of
nuclear minor axis were the least effective means for determining
triploidy due to high percentages of improper classifications.
Wolters et al. (1982c) stated that although weighted mean values
and confidence intervals of erythrocyte measurements may be
distinct, the range of measurements still overlap and result in
some mis-classification. The reason for the poor discriminating
powers of the minor axis is a change in shape of erythrocytes
when diploids and triploids are compared. The major axis of the
triploid nuclei is increased at a greater rate than the minor
axis. Wolters et al. (1982c) also suggested that care must be
used when erythrocyte parameters are used to determine triploidy
because of the misclassification involved. They suggested that
only length of nuclear major axis needs to be used as a discrimi-

nating variable for channel catfish triploids.

 

87

Erythrocyte nuclear and cellular length was shown to
correctly identify triploid and diploid grass carp x bighead carp
hybrids with 98% accuracy (Beck and Biggers, 1983). Studies of
haematology of triploid landlocked Atlantic salmon also indicated
that triploids have increased erythrocyte nuclear and cellular
size (Benfey et al. 1984). 'Five of six erythrocyte parameters,
including cell major axis, cell surface area, nuclear major axis,
nuclear minor axis and nucleus volume could be used to success-
fully classify 100% of the individuals as triploids or diploids.
Measurement of erythrocyte minor axis was the only parameter
which could not determine triploidy with 100% accuracy. Even
this measurement was at least capable of 95% accuracy (Benfey et
al. 1984). Due to these results, I feel that triploidy may be
accurately detected in chinook salmon by measurements of the long
nuclear axis from erythrocytes. Mean nuclear lengths of eight

microns or greater would indicate a triploid.

Other Methods of Triploid Identification

 

Other methods have also been used to detect triploidy
including coulter counter channelyzer, microspectrophotometry,
and flow cytometry. The coulter counter channelyzer works on the
principle that triploids have larger erythroycytes than diploids,
with the machine sorting erythrocytes according to size. Flow
cytometry works by detecting laser excited fluorescence of the
stained nuclear DNA.(Allen, 1983). Flow cytometry offers the

advantages of being very accurate and relatively fast so large

 

88

samples can be determined. Other advantages are (a), Specimens
remain alive (only a small quantity of blood or tissue is
required), (b), any tissue is usable, and (c), almost any age of
a specimen may be analyzed (Allen, 1983; Thorgaard et al. 1982).
The disadvantages of flow cytometry are the prohibitively high
cost of the equipment and the high degree of technological
training required. Flow cytometry and various erythrocyte size
determination techniques are more suitable for triploid deter-
mination than solid-tissue karyotyping. These techniques are
less time consuming allowing more time to be spent analyzing
larger numbers of samples thus reducing the associated standard

error .

 

CONCLUSIONS

Heat shocks of 36°C for one minute, initiated at ten minutes
post fertilization, induced triploidy in chinook salmon and
chinook female x coho male salmon hybrids. Although
triploidy was induced, this heat-shock treatment was not
considered successful since only 0 to 42% triploid
individuals were produced. A triploid induction rate of

100% is desired for practical production uses.

The heat-shock treatment used failed to induce triploidy in

coho salmon and coho female x chinook male salmon hybrids.

The effects of degree of ripeness of eggs on triploid pro-
duction must be determined before optimal production of

triploids is achieved.

The effects of immediate fertilization versus delayed ferti-
lization on triploid induction rates must be determined

before optimal production of triploids is achieved.

The use of chinook female x coho male salmon hybrids is not
recommended due to high incidence of malformations which

occurred in the fry.

89

 

90

In contrast, coho female x chinook male salmon hybrids have
very good survival rates with low incidence of disembryo-

genesis. This may be a viable hybrid to use for production.

In order to determine the effects of triploidy on chinook
salmon x coho salmon reciprocal hybrids, a method of
producing near 100% triploids needs to be used to achieve

adequate numbers of triploid hybrids.

Solid-tissue karyotyping is a slow and inefficient means for
triploid determination. Faster, more accurate methods which
are capable of larger sample sizes should be used. Such
methods might include flow cytometry, coulter counter chan-

nelizer and erythrocytic nuclei size determinations.

Based on initial experiments, triploid induction rates of
100% can be achieved by heat shocks of approximately 28°C
applied for ten minutes, ten minutes post fertilization if

eggs are cooled slowly.

 

APPENDIX A

 

Appendix A

Heat-shocking procedures used at Little Manistee Weir,

October 15, 1984, to induce triploidy in coho salmon, chinook

salmon, and coho x chinook salmon reciprocal hybrids.

A.

Set up heat shocking container with river water and stabi-
lize temperature of water at 36°C (approximately 4 hours).
Prepare containers to receive eggs (clean, dry and insulated
from rapid temperature changes).
Strip eggs from three coho females into pan.
1. Mix eggs well
2. Split these in half (one half to be fertilized
immediately, the other half saved dry for step C-2-b.
Eggs not fertilized immediately but kept dry and cool
are viable for at least 4 hours) (Piper et. al., 1983).
a) The first half to be fertilized immediately by six
coho males (cOho males were not very ripe, (pro-
ducing less milt) so additional males were
needed). (Save some coho sperm for later
crosses).
1) Split these in half again.
Half to be false heat shocked ten minutes
post fertilization (Cross 1).
Half to be heat shocked ten minutes post

fertilization (Cross 2).

91

 

92

b) The second half to be fertilized by three chinook
males (save some chinook sperm for later crossesL
1) Split these in half again.
Half to be false heat shocked ten minutes
post fertilization (Cross 3).
Half to be heat shocked ten minutes post
fertilization (Cross 4).
Strip three chinook females into pan.
1. Mix eggs well.
2. Split these eggs in half (one half to be fertilized
immediately, the other half saved dry for step D-2-bL
a. The first half to be fertilized by three chinook.
1) Split these in half again.
Half to be false heat shocked (Cross 5%
Half to be heat shocked (Cross 6).
b. The second half to be fertilized by six coho
males.
1) Split these in half again.
Half to be false heat shocked (Cross 7).

Half to be heat shocked (Cross 8).

Each separate group of eggs was fertilized consecutively at

two-minute intervals. This time allotment was adequate to move

the various groups in and out of heat shocking and false heat

shocking. All eggs were fertilized in the same ten-minute period

to minimize time differences between collection of gametes and

fertilization for the different crosses.

 

APPENDIX B

 

 

Appendix B

Solid-tissue karyotyping technique for four month old

chinook salmon and coho salmon. Modified and adapted from

Kligerman & Bloom, (1977) and from Paul Scheerer, (Washington

State University, personal communicationL

Step

Step

Step

Step

Step

Step

Prepare colchicine solution in .85% sterile saline.
Inject fish, intermuscular on dorsal surface just
anterior to dorsal fin or interperitoneal with 25 ug
colchicine/g fish weight in .85% sterile saline
solution.

Allow injected fish to swim in aerated water for six to
eight hours post injection.

Sacrifice fish and remove kidneys and spleen. (Other
tissue types may also be removed such as gill arches,
intestine,eth

Place tissues in approximately 10 times their volume of
hypotonic .4% KCl solution for 20 £2 39 minutes.
Remove ALL hypotonic KCl solution and exchange with
FRESH 3:1 ethanol (or methanol): acetic acid. Allow
tissue to fix for 30 minutes, then exchange original
a1cohol:acetic acid fixative with fresh fixative.
Allow tissues to fix for another 30 minutes, then
exchange once again with fresh fixative. (Tissues may

be stored at 4°C at this time).

93

 

 

94

Step 7: Remove tissue from alcohol:acetic acid fixative and blot
off excess fixative on filter paper.

Step 8: Place tissue in four to six drops 50% acetic acid in
depression slide or small porcelain watch glass. Mince
tissue gently with bottom of test tube for one minute
until completely emulsified.

Step 9: Aspirate suspension in a micro-capillary tube and
transfer to a hot glass microscope slide (40-45°C) in a
ring of about 1 cm in diameter.

Wait four to five seconds, then draw off excess fluid not
dried to slide.

Repeat this process several times to produce several rings
at different locations on the slide.

(It is critical not to get too high of a concentration of
cells in the suspension. If too many cells are in the suspension
then too many cells will dry on the slide and impede spreading of
chromosomes and single cell resolutionL
Step 10: Stain slides in 4% Giemsa in..01 M phosphate buffer at

pH 7; or in 100% Giemsa for six minutes with.l.5 N NH4OH
used as intensifier for two minutes.

Step 11: Rinse slides in tap water or distilled water. Allow
slides to air dry.

Step 12: Fix slides for 10 minutes in Xylene.

Step 13: Mount slides in Apochromount, Eukitt, or other suitable

mounting media.

 

   

Step

Step

95

14: Scan slides at 100 X along periphery of rings. Spreads
will appear as small dots among cells.

15: View spreads at 1,000 X.

Notes of critical procedures found in this technique:

Step

Step

Step

Step

Step

2 - Various authors report fish may be allowed to swim in
colchicine solution. This method never produced good
results in my study.

5 - Amount and length of treatment Of tissue in hypotonic
solution seems moderately critical. If tissue is placed in
too large a volume of hypotonic solution or for too long a
time (> 30 minutes), cells seem to swell excessively,
possibly rupturing and losing chromosomes.

6 - Removing ALL hypotonic solution and using FRESH fixative
seems crucial to proper fixation of tissues. Fresh
alcohol:acetic acid fixation leads to good chromosome pre-
parations later. The use of old fixative was probably the
single most importance factor for initial failure of karyo-
typing. Methanol:acetic acid fixative begins to degrade to
methyl acetate within 30 minutes after mixing. IMethyl
acetate is not an effective fixative (Denton, 1973).

8 - It is important not to mince tissue excessively.
Experience may be your only guide here.

9 - Several critical phases are involved in this step.
Kligerman and Bloom (1977) state, "expel" the cell suSpen-

sion onto a clean slide. I interpreted this as spreading

 

 

96

the cell suspension with some force since most karyotyping
techniques require "dropping" cell suspensions from various
heights (Gold, 1974; Hollenbeck and Chrisman, 1981X
Actually, no force is required in this process and better
terminology would be to "flow" the cell suspension onto a
hot slide. Applying the suspension with any force may

impede the formation of good chromosome preparations.

It is also critical not to get too high of a concentration
of cells in the suspension. If excessive cells are in the
suspension and dry on the slide, then single cell resolution and

chromosome spreading may be impeded.

 

REFERENCES

Allen, Jr., S.K. 1983. Flow cytometry: assaying experimental
polyploid fish and shellfish. Aquaculture 33:317-328.

 

Allen, Jr., S. and J. Stanley. 1979. Polyploid mosaics induced
by cytochalasin B in landlocked Atlantic salmon (Salmo
salar). Trans. Am. Fish. Soc. 108:462-466.

Allen, Jr., S. and J. Stanley. 1983. Ploidy of hybrid grass
carp x bighead carp determined by flow cytometry. Trans.
Am. Fish. Soc. 112:431-435.

Al-Sabti, K. 1985. Chromosomal studies by blood leukocyte
culture technique on three salmonids from Yugoslavian
waters. g. Fish. Biol. 26:5-12.

 

Battle, H.I. 1944. The embryology of the Atlantic salmon (Salmo
salar). Can. i. 2E Res. 22(5):105-125.

Becak, W., M.L. Becak and S. Ohno. 1966. Intraindividual
chromosomal polymorphism in green sunfish (Lepomis
cyanellus) as evidence of somatic segregation.
Cytggenetics. 5:313-320.

 

Beck, M.L. and C.J. Biggers. 1983. Erythrocyte measurements of
diploid and triploid Ctenopharyngodon idella 9 x
Hypothalmichthys nobilis o>hybrids. .1. Fish. Biol.
22:497-502.

 

 

Behler, J.L. and F.W. King. 1979. The Audubon Society Field
Guide to North American Reptiles and Amphibians.
Chanticleer Press, Inc., New York. 719 pp.

Benfey, T.J. and A.M. Sutterlin. 1984a. Growth and gonadal
development in triploid landlocked Atlantic salmon (Salmo
salar). Can. J. Fish. Aquat. Sci. 41:1387-1392.

 

Benfey, T.J. and A.M. Sutterlin. 1984c. The haematology of
triploid Atlantic salmon (Salmo salar). .i- Fish. Biol.
24:333-338.

 

 

Benfey, T.J., A.M. Sutterlin and R.J. Thompson. 1984. Use of
erythrocyte measurements to identify triploid salmonids.
Can. i. Fish. Aquat. Sci. 41:980-984.

 

 

97

 

 

98

Beverton, R.J.H. and S.J. Holt. 1957. On the dynamics of
exploited fish populations. lLK. Min. Agric. Fish., Fish.
Invest. (Ser. 2). 19:533.

Blanc, J. and B. Chevassus. 1979. Interspecific hybridization
of salmonid fish I. Hatching and survival up to the 15th
day after hatching in F-l generation hybrids. Aquaculture.
18:21-34.

 

Blanc, J. and B. Chevassus. 1982. Interspecific hybridization
of salmonid fish II. Survival and growth up to the 4th
month after hatching in F-l generation hybrids.
Aquaculture. 9:383-387.

 

Blane, :LM. and H. Poisson. 1983. Parental sources of variation
and hatching and early survival rates of Salmo trutta x
Salvelinus fontinalis hybrid. Aquaculture. 32:115-122.

 

 

 

Blaxhall, PJL 1975. Fish chromosome techniques a review of
selected literature. g. Fish. Biol. 7:315-320.

 

Blaxhall, PAL 1981. A comparison of methods used for the
separation of fish lymphocytes. “J. Fish. Biol. 18:177-181.

 

Blaxhall, PJL 1983a. Chromosome karyotyping of fish using con-
ventional and G-banding methods. J. Fish. Biol. 22:417-
424.

 

Blaxhall, PJL 1983b. Factors affecting lymphocyte culture for
chromosome studies. g. Fish. Biol. 22:61-76.

 

Briggs, R. 1947. The experimental production and development of
triploid frog embryos. g, Exp. Zool. 106:237-265.

Buss, K. and.;LE. Wright. 1956. Results of species hybridi-
zation within family Salmonidae. Prog. Fish. Cult.
October, 1956, p. 149-156.

 

 

Cassani, J.R., W.E. Caton and B. Clark. 1984. Morphological
comparisons of diploid and triploid hybrid grass carp
Ctenopharynggdon idella 9 x Hypothalmichthys nobilis 0?.
J. Fish. Biol. 25:269-278.

 

 

 

Chevassus, B. 1979. Hybridization in salmonids - results and
perspectives. Aquaculture. 17:113-128.

 

Chevassus, B. 1983. Hybridization in fish. Aquaculture.
33:245-262.

 

   

99

Chourrout, D. 1980. Thermal induction of diploid gynogenesis
and triploidy in the eggs of the rainbow trout (Salmo
gairdneri Richardson). Reprod. Nutr. Develop. 20:727-733.

 

 

 

Chourrout, D. 1984. Pressure induced retention of second polar
body and suppression of first cleavage in rainbow trout:
production of all triploids, all tetraploids, and hetero-
zygous and homozygous diploid gynogenetics. Aquaculture.
36:111-126.

 

Chourrout, D. and J. Itskovich. 1982. Three manipulations per-
mitted by artificial insemination in Tilapia: induced
diploid gynogenesis, production of all triploid populations
and intergeneric hybridization. ‘International Symposium 23
Tilapia Proceedingg, Nazareth, Israel, May 8-13.

 

 

Chourrout, D. and E. Quillet. 1982. Induced gynogenesis in the
rainbow trout: Sex and survival of the pregenies production
of all triploid populations. Theor. Appl. Genet. 63:201-
205.

 

Cimino, ILC. 1973. Karyotypes and erythrocyte sizes of some
diploid and triploid fishes of the genus Poeciliopsis. J.
Fish. Res. 1351. Can. 30:1736-1737.

 

Denton, T.E. 1973. Fish Chromosome Methodology. C.C. Thomas
Publ., Illinois.

Dunham, R.A., R.O. Smitherman, M.J. Brooks, M. Benchakan and
.LA. Chappell. 1982. Paternal predominance in reciprocal
channel-blue hybrid catfish. Aquaculture. 29:389-396.

 

Elinson, RJL and A. Briedis. 1981. Triploidy permits survival
of an inviable amphibian hybrid. Developmental Genetics.
2:357-367.

 

Ferrier, V. and A. Jaylet. 1978. Induction of triploidy in the
newt Pleurodeles waltlii by heat shock or hydrostatic
pressure. Chromosoma. 69:47-63.

 

 

Frankhauser, G. and R.R. Humphrey. 1950. Chromosome number and
development of progeny of triploid axolotl females mated
with diploid males. J. Exper. 2001. 115(2):207-241.

 

Fukuoka, H. 1972. Chromosome number variations in the rainbow
trout (Salmo gairdneri irideus (Gibbons)). Japan. J.
Genet ics. 47(6):455-458.

 

Carling, Jr., D.L. and M. Masterson. 1985. Survival of Lake
Michigan chinook salmon eggs and fry incubated at three tem-
peratures. Prog.Fish.Cult. 47(Lh63-66.

 

 

100

Gervai, J., 3. Peter, A. Nagy, L. Horvarth and V. Csanyi. 1980.
Induced triploidy in carp, Cyprinus carpio L. J. Fish.
Biol. 17:667-671.

 

Gillespie, L.L. and J.B. Armstrong. 1979. Induction of triploid
and gynogenetic diploid axolotls (Ambystoma mexicanum) by
hydrostatic pressure. J. Exp. 2001. 210:117-122.

 

Gill, J.L. 1978. Design and Analysis of Experiments in the
Animal and Medical Sciences. Volume I. Iowa State
University Press, Ames, Iowa.

Gjedrem, T. 1976. Possibilities of genetic improvements in
salmonids. J. Fish. Res. Board. Com. 33:1094-1099.

 

 

Coetz, F.W., E.M. Donaldson, C.A. Hunter and E.M. Dye. 1979.
Effects of estradiol-17B and l7-OL methyltestosterone on
gonadal differentiation in the coho salmon (Oncorhynchus
kisutch). Aquaculture. 17:267-278.

 

 

Cold, J.R. 1974. A fast and easy method for chromosome karyo-
typing in adult teleosts. Prog. Fish. Cult. 36(3):169-171.

 

 

Cold, J.R., W.J. Karel and M.R. Strand. 1980. Chromosome
formulae of North American fishes. Prog. Fish. Cult.
42(1):10-23.

 

Grammeltvedt, A. 1974. A method of obtaining chromosome
preparations from rainbow trout (Salmo gairdneri) by
leucocyte culture. Norw. J. 2001. 22:129-134.

 

Grammeltvedt, A. 1975. Chromosomes of salmon (Salmo salar) by
leukocyte culture. Aquaculture. 5:205-209.

 

 

Hartley, S.E. and M.T. Horne. 1983. A method for obtaining
mitotic figures from blood leucocyte cultures of rainbow
trout, Salmo gairdneri. J. Fish. Biol. 22:77-82.

 

 

Hill, J., A. Hickerson, D. Sheldon, and K. Warren. 1985.
Production of triploid chinook salmon (Q. tshawytscha) using
flow through heat shock and subsequent gradual cooling.
Clatsop Economic Development Comm. Fisheries Proj. Astoria,
Oregon (unpublished paper).

 

Hollenbeck, P.J. and C.L. Chrisman. 1981. Kidney preparations
for chromosomal analysis of Ictaluridae. Copeia. 1:216-
217.

Johnstone, R. 1985. Induction of triploidy in Atlantic salmon
by heat shock. Aquaculture. 49:133-139.

 

101

Kligerman, A.D. and S.E. Bloom. 1977. Rapid chromosome prepara-
tions from solid tissues of fishes. J. Fish. Res. Board.
Can. 34:266-269.

 

Leitritz, E. and R.C. Lewis. 1980. Trout and Salmon Culture
(Hatchery Methods). California Fish Bulletin, Number 164.

Lemoine, Jr., H.L. and L.T. Smith. 1980. Polyploidy induced in
brook trout by cold shock. Trans. A_m. Fish. Soc. 109:626-
631.

Levan, A., K. Fredga and A.A. Sandberg. 1965. Nomenclature for
centromeric position on chromosomes. Hereditas. 52:201-
220.

Lincoln, R.F. 1981. Sexual maturation in triploid male plaice
(Pleuronectes platessa) and plaice x flounder (Platichthyes
flesus) hybrids. J. Fish. Biol. 19:415-426.

 

 

 

Lincoln, R.F., D. Aulstad, and A. Grammeltvedt. 1974. Attempted
triploid induction in Atlantic salmon (Salmo salar) using
cold shocks. Aquaculture. 4:287-297.

 

 

Lincoln, R.F. and A.P. Scott. 1983. Production of all female
triploid rainbow trout. Aquaculture. 30:375-380.

 

Lincoln, R.F. and A.P. Scott. 1984. Sexual maturation in

triploid rainbow trout, Salmo gairdneri Richardson. J.
Fish. Biol. 25:385-392.

 

 

Loginova, C.A. and S.V. Krasnoperova. 1982. An attempt at
crossbreeding Atlantic salmon and pink salmon (preliminary
report). Aquaculture. 27:329-337.

 

Lou, Y.D. and C.E. Purdom. 1984. Polyploidy induced by hydro-
static pressure in rainbow trout, Salmo gairdneri
Richardson. J. Fish. Biol. 25:345-351.

 

 

McBride, J.R. and A.P. van Overbeeke. 1971. Effects of
androgens, estrogens, and cortisol on the skin, stomach,
liver, pancreas and kidney in gonadectomized adult sockeye
salmon (Oncorhynchus nerka). J. Fish 1333. pg. 339. 28:485-
490.

 

Pillay, T.V.R. (Editor), and W.A. Dill (Editor). 1976. Chapter
X genetics and genetic improvement in fish by R. Moaz.
Advances in Aquaculture. Fishing News Books Ltd., Farnham,
Surrey, England, p. 610-621.

 

 

   

102

Piper, R.C., LB. McElwain, L.E. Orme, J.P. McCraren, L.G. Fowler
and J.R. Leonard. 1983. Fish Hatchery Management. United
States Department of the Interior, Fish and Wildlife
Service, Washington, D.C.

Purdom, C.E. 1973. Induced polyploidy in plaice (Pleuronectes
platessa) and its hybrid with the flounder (Platichthyes
flesus). Heredity. 29:11-24.

Purdom, C.E., D. Thompson and Y.D. Lou. 1985. Genetic
engineering in rainbow trout, Salmo gairdnerii Richardson,
by the suppression of meiotic and mitotic metaphase. J.
Fish. Biol. 27:73-79.

 

Refstie, T., J. Stoss, E.M. Donaldson. 1982. Production of all
female coho salmon (Oncorhynchus kisutch) by diploid gyno-

genesis using irradiated sperm and cold shock. Aguaculture.
29:67-82.

Refstie, T., V. Vassvik and T. Gjedrem. 1977. Induction of

polyploidy in salmonids by cytochalasin B. Aguaculture.
10:65-74.

Roberts, F.L. 1967. Chromosome cytology of the Osteichthyes.
Prog. Fish Cult. April, 1967, p. 75-83.

Roberts, F.L. 1968. Chromosome polymorphism in North American
landlocked Salmo salar. Can. J. Genet. Cytol. 10:865-875.

Robertson, O.H. 1961. Prolongation of the life span of kokanee
salmon (Oncorhynchus nerka kennerlyi) by castration before
beginning of gonad development. Zoology. 47:609-621.

 

Scheerer, P.D. and C.H. Thorgaard. 1983. Increased survival in

salmonid hybrids by induced triploidy. Can. J. Fish. Aguat.
Sci. 40:2040-2044.

Schreck, C.B. and L.G. Fowler. 1982. Growth and reproductive
development in fall chinook salmon: Effects of sex hormones
and their antagonists. Aguaculture. 26:253-263.

Schultz, R.J. 1967. Gynogenesis and triploidy in the viviparous
fish Poeciliopsis. Science. 157:1564-1567.

Simon, R.C. 1963. Chromosome morphology and species evolution
in the five North American species of Pacific salmon
(Oncorhynchus). J. Morphol. 11277-97.

Simon, R.C. and R.F.. Noble. 1968. Hybridization in Oncorhynchus
(Salmonidae) I. Viability and inheritance in; artificial
crosses of chum and pink salmon. Trans. fl. Fish. §o_c.
97(2):109-118.

103

Smith, lnT. and lLL. Lemoine. 1979. Colchicine induced poly-
ploidy in brook trout. Prog. Fish Cult. 41(2):86-88.

Solar,I.I” E.M.Donaldson and(LA.Hunter. 1984. Induction of
triploidy in rainbow trout (Salmo gairdneri Richardson) by
heat shock and investigation of early growth. Aquaculture.
42:57-67.

 

Thorgaard, GJL 1976. Robertsonian polymorphism and consti-
tutive heterochromatin distribution of chromosomes of the
rainbow trout (Salmo gairdneri). Cytogen. Cell. Genet.
17:174-184.

Thorgaard, G.H. and C.A.E. Call. 1979. Adult triploids in a
rainbow trout family. Genetics. 93:961-973.

Thorgaard,G.H” M.E.Jazwin and A.Stier. 1981. Polyploidy
induced by heat shock in rainbow trout. Trans. Am. Fish
Soc. 110:546-550.

Thorgaard, G.H., P.S. Rabinovitch, M.W. Shen, C.A.E. Call, J.
Propp and F.M. Utter. 1982. Triploid rainbow trout
identified by flow cytometry. Aguaculture. 29:305-309.

Utter, F.M.,O.W.Johnson, GJL Thorgaard and P.S.Rabinovitch.
1983. Measurement and potential applications of induced
triploidy in Pacific salmon. Aquaculture. 35:125-135.

Valenti, RuL 1975. Induced polyploidy in Tilapia aurea
(Steindachner) by means of temperature shock treatment. J.
Fish.Biol. 7:519-528.

 

van Overbeeke, A.P. and J.R. McBride. 1971. Histological
effects of ll-ketotestosterone, l74xmethy1testosterone,
estradiol, estradiol cypionate, and cortisol on the
interrenal tissue, thyroid gland, and pituitary gland
of gonadectomized sockeye salmon (Oncorhynchus nerkaL
J.Fish.§g§.Lg.ggA. 28:477-484.

Van Tuinen, P. and M. Valentine. 1982. Mammalian chromosome
newsletter. 23(4):182-185.

Wolters, W.R” (LL. Chrisman and (LS. Libey. 1981a. Lymphocyte
culture for chromosomal analysis of channel catfish
(Ictalurus pppctatus). Copeia, p. 503-504.

 

Wolters, W.R” C.S.Libey and OJ“ Chrisman. 1981b. Induction
of triploidy in channel catfish. Trans. Ag. Fish. Soc.
110:310-312.

 

 

104

Wolters, W.R., C.L. Chrisman and G.S. Libey. 1982c. Erythrocyte
nuclear measurements of diploid and triploid channel eat-
fish, (Ictalurus punctatus). g. Fish. Biol. 20:253-258.

Wolters, W.R., G.S. Libey and C.L. Chrisman. l982d. Effect of
triploidy on growth and gonad development of channel cat-
fish. Trans. fl. Fish. Soc. 111:102-105.