Copyright is owned by the Author of the thesis. Permission is given for a copy to be downloaded by an individual for the purpose of research and private study only. The thesis may not be reproduced elsewhere without the permission of the Author.

Some mares are completely sterile; some conceive3 but cannot complete their pregnancy: ...

AN INVESTIGATION OF MOSAIC / CHIMAERISM IN THE DOMESTIC HORSE ( Eguus cabal lus caba l lu s )

A thesi s presented i n partial

f u l f i lment of the requi rements for the degree of

Doc tor of Phi losophy in

Cytogeneti c s at

Massey Unive r s i ty

Karen Shei la Walker

1980

. t• I• f • ' J- • •

ABSTRACT

Thi s study invo lved three aspect s of the field of Cytogeneti c s

app lied to the domestic horse (�. caba l lus cabal lus ) . The first aspect , the identific at ion and investigat ion of individua l s with

cytogenetic anomalies , is fundamental to Cytogenet ics . The individ

ual s di scovered , however , pointed to t he c lose interrel at ionshi p of

Cytogeneti c and Genetic research. Thirdly , questions of fert i lity ,

and in one mare the t en tative demonstrat ion of an app l ication of

cytogenetic s in veteri nary c l inic a l evaluati on , were concerned with

functions of C l inical Cytogeneti c s .

The pr imary hypothesi s of thi s study was that individual s with

abnormal sex chromosome con s t i tution s , the maj ority of whi ch are

either mosaics or chimaeras , form a significant proport ion of some

�. £· cabal lus breeds , in part icu lar the Thoroughbred Breed . Sub s idiary to that hypothes i s was that these mosai c /chimaeras as a group

were d i sproport ionately responsible for the sub - fert i l i ty of some

hor s e popu l atio ns , through inferti l ity , sub - ferti l i ty and generating

abnorma l off spring .

Five hors es were inves tigated . One , a XY /XYY /?XO mosaic / chimaeric

sta l l ion was ful ly ferti le . However , he sired an XO st i l l - born fi l ly . Limited studies of three of his relatives and three of hi s

o ther progeny , sugges ted the possib i l i ty of cytogenetic anoma l ies in

tha t kinshi p . He was the first reported sta l l ion with an XYY line ,

and his XO fi l ly was the first reported sti l l -born foal with a chromo

some abnorma l i ty .

Two 64 , XX / 65 ,XXY mares wi th no physical signs of intersexua l i ty

were studied . One was infert i l e , however , there was insufficient

information to assess the o ther mare ' s fert i l ity . An infert i l e mare

was di scovered and inves tigated in depth , whi ch had 65 , XXX/65 , XXY/

64 , XX/ 6 6 , XXY / 6 6 , XXXX mixopl oid mosaic/chimaeri sm , al though she was

primari ly a tri p l e-X mare . The fourth mare was primari ly thought to

ii

be a pro l i f e rative mosaic , whi c h exhibited mitotic ins tabi l i ty . Post

mortem evidence , however , confirmed that she had been cytogenetical ly

abnorma l and that she probably was a mosaic / chimaera.

Aside from one XX/ XXY mare , whi ch had been born wi th congenital

defec t s , the re was no thing ei ther in t he hi story or phenotype of these

f ive horses to dis tingui sh them from o ther Thoroughbreds. Only one

i i i

o f the mares was presented fo r ana l ys i s because of inferti l i ty , al though

two of the othe r mares might have been termed " shy breeders".

The discovery of al l the maj o r sex chromosome aneuploid conditions

infrequent l y f ound in humans , XO , XXY , XYY and XXX , in the mosai c / chi

maeric cond i t i o n among less than 200 Thoroughbreds evaluated , suggested

that chromo some anomal ies are preva l ent in the Thoroughbred population.

ACKNOWLEDGEMENTS

I am i ndebted to the Faculty of Veterinary Science , Massey

U niver s i ty , for providing the opportun i ty and faci l i t i es for thi s

i nvestigation .

In par t icular I woul d l i ke to thank my supervisor Prof . A . N . \

Bruere and acknowledge the patient assis tance and guidance of bo th

the academic and technical staff of the Depar tment of Veterinary

S c ience , wi th mention of : Prof . E . D . Fielden , Dr . B . E . Goulden ,

Dr . R . E . Harri s , Dr . R . J . Ho l mes , Dr . I . L . Anderson , Dr . H . G .

P earce , Mrs . R . B . Foo t e , Ms Karen Armitage , and the staff of the

Large Anima l Uni t .

Recogni tion i s al so due to the .Department of Veterinary Path

o logy and Pub l i c Heal t h s taff and fac u l ty , wi th speci a l mention of :

M s L. Frey , the H i s to l ogy Preparation Uni t , the Po st-mortem Examin

a t i on Fac i l i ty , Dr . A . C . Johns tone , Dr . M . R. Al ley , Dr . K . M .

Moriarty , Dr . A . J . Robinson an d P . Ramadas s , Ph.D. candidate ; and

P rof . • R . E . Munford and Mr . M . J . Birtles of the Depar tment of

Anatomy and Physiology .

Thi s study woul d not have been pos sible wi thout the hel p and

cooperation of horse owners and breeders in Ne\v Zeal and , to whom the

author expresses gratitude . In addi t ion , the active partici pation of

members of the veterinary profes sion was an invaluabl e hel p , espec i a l ly

Mr . H . Dewes , M r . J . Hope , Ms G . Verkerk , M s J . Franci s , Mr . P . Wi l l iams

and Mr . C . Kel ly . I shou l d al s o l i ke to acknowledge the f inanci a l

assistance of The New Zealand Equine Research Foundation .

I woul d l i ke to express my appreci ation to the Librarians and

M r . T . Law (Photographic Uni t ) for their tolerance and professiona l i sm,

a s wel l as to the Department of Bo tany and Zoo logy for thei r support

and sufferance .

For their hel p , encouragement and endur ing faith , a very specia l

thankyou to my paren t s .

Many peo pl e , known and unknown , have made this s tudy possib l e .

To them, et.��. C"CL ouJ

TABLE OF CONTENTS

Page

Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . iv

Conten t s . . . . . . • . . . . . . . . . . . . . • . . . . . . . . . . . . . . . . . . . . . . • • . . . . . . . . . . v

L i s t of Figures • . . . • . . . . . . . . . . . . . . . • . • . . . . . . . . . . . . . • . . • • . . . . . . • xi i

Lis t o f Tab l es . . . . . . • • . . . . . . . . • . . . • • . . . . . . . . . . . . . . . . . . . . . . . . . . . xv

CHAPTER I - Introduc tion 1

1.1. Infer t i l i ty in the domes t i c hor se ( E . c abal lus cabal lus): the aims of thi s study • . . . . . . . . . . . . . . . • • • . . . . . . . . . . . . . . . . 3

1.2. Mosaic i sm and chimaerism : defini tions and pos s i b l e relation to inferti l ity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5

1.2.1. Mosai c i sm , chimaeri sm and " mo sai c / chimaeri sm" : def i n i tions . . . . . . . . . . • . . . . . . . . . . . . . . . . . . . . . . . . . . . 5

1.2.2. Chimaeri sm: types , po ssible relation to fert i l -i ty and po ssible pred i s posing fac tors . . . . . . . . .. . . 6

1.2.2.1. Haemopoietic chimaer i sm . . . . . . . . . . . . . . . . 6

1. 2. 2. 2. Whole body chimaeri sm . . . . . . . . . . . . . . . . . . 7

1.2.3. Mosai c i sm: possible impl i cations in infert i l i ty and pos s i b l e pred i s po s i ng fac tors . . • . . . . . . . . . . . . . 7

1.2.3.1. Factors which might pred i spo s e to format ion of mosaic i sm . . . . . . . . . . • . . ·. . • . 8

1.2.3.2. The formation of mo saic i sm . . . . . . . . . . . . . 9

1.3. Factor s affec ting mosaic /chimaer i c compos i tion and t hei r interpretation • . • • . . . . . . • • • • . . . . . . . . . • • • • • . . . . . . . . • • . . . . . 10

1 . 4 . Aspec t s o f the cytogenet i c evaluation of mosaic /chimaeri sm • . . 1 0

1 . 4 . 1 . Chromosome preparations from cul tured cel l s in relation to the in- vivo mosai c /chimaeric condi tion 1 0

1 . 4 . 1 . 1 . Representat iveness o f the sample . . . . . . . . . . . 1 1

1 . 4 . 1 . 2 . Di fferential viabi l i ty and growth of c e l l l ines in c u l ture . . . . • . . . . . . . . . . . . . . . . . 1 2

1 . 4 . 1 . 3 . Natura l , ar tifac tual and symptomati c aneu p l o idy and hyperploidy . . . . . . . . . . . . . . . . . 1 2

1 . 4 . 1 . 4 . Mi togenic s timu l ation and mi toic i nhibi t ion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 3

1 . 4 . 2 . Mosai c /chimaeri s m and sex-chromatin s tudies . . . • . . • . . . 1 3

1 . 5 . Atypical development and cytogenetic abnorma l i ties . . • . . . . . . . . 1 5

1 • 6 . The fami ly Egui dae . . . . . • . . . . . . . . . • . • • . • . . . . . . . . . . . • . . . . . . . . . 1 6

1 . 6 . 1 . The karyotypes o f Equidae . . . . . . . • . . . . . . • . • • . • • . . . . . . • 1 6

1 . 6 . 2 . E quine hybri d s • . . . . • • . . . . . . • • • . • . . . . . • . • • • . . . . . . . • • . . 1 7

1 . 7 . Cytogenetics of�· caba l l u s caba l l u s . . . . • . . . . . . . . • • • • � • . . . . • . 1 9

1 . 7 . 1 . The normal karyotyp e of�· £· cabal lus • • • • . . . . . . . . • • . 1 9

1 . 7 . 1 . 1 . Chromo some identifi ca tion . • . • . . . . . . . . • . . . . . 1 9

1 . 7 . 1 . 2 . Chrorao some po lymorphi sms . . . . . • • . . . . . . . . • . . . 20

1 . 7 . 1 . 3 . The karyo type of the domestic horse . . . . . . . . 20

1 . 7 . 1 . 4 . Arrangement of the karyotype o f the dome s t i c horse . . . . . . . . . . . . . . . . . . . . . . . . . . • . . 2 1

1 . 7 . 2 . Abnormal karyo types in the domestic hor se . . . . . . . . . . . . 2 1

1 . 7 . 3 . Infert i l i ty and chromosome anomalies in the domestic horse . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 22

1 . 7 . 3 . 1 . Inferti l i ty: comments on termino logy • . . . • . . 2 2

1 . 7 . 3 . 2 . Chromosome anomalies i n the domestic hor se . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 26

CHAPTER II - Materials and Methods 28

2 . 1 . Subj ec t s • . . . . . • . . . . . . . • • • • • . . . . . . . . . . . . . . . . . . . . . . . . • • . . . . . . . . 29

2 . 2 . Cytogenetic techniques • . . . . . . • • . • . . . . • • • • • . . • . . . . . . . . . . . . . • . . 29

2 . 2 . 1 . Leucocyte cul tures . . . . . . . . . . . . . • . • • • • . . . . . . . . . . . . . . . • 30

vi

2 . 2 . 2 . Cel l cultu res other than l eucocyte cultures • • • • • • • . • 30

2 . 2 . 3 . Harvesting cel l cultures and s l ide preparation . . . . . . 30

2 . 2 . 4 . Staining . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 30

2 . 2 . 5 . Sex- chromatin 30

2 . 2 . 6 . Evaluation of cul tured cel l s . . . . . . . • . . . . . . . . . . . . . . . . 3 1

CHAPTER Ill - H- 1 1 , A fert i l e XY/XYY/?XO Mo saic/Chimaer;c S tallion 3 2

3 . 1 . Introduction . . . . . . . . . . . • . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 33

3 . 2 . Materials and methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3 6

3 . 2 . 1 . Sources o f information . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 36

3 . 2 . 2 . H - 1 1 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . • . . . 36

3 . 2 . 2 . 1 . Description . . . . . . . . . . . . . . . . • . . • • . . . . . . . . . . 3 6

3 . 2 . 2 . 2 . General his tory . . . . . . . . . . . . . • • • • • � • . . . . • • . 36

3 . 2 . 2 . 3 . Reproduc t ive performance . . . . . . : • . . . . . . . . • • 37 3 . 2 . 2 . 4 . H- l l ' s s i re and dam • . . . • • • • • . • . . . • • • • • • • • . 38

3 . 2 . 3 . Relatives and progeny investigated • • . . . . . . • • • • • • • . • • 38

3 . 2 . 4 . Cytogenetic materials and methods • • • • • • • . • . • . . • • . . • . 41

3 . 3 . Resu l ts of cytogenetic analysi s of H- 1 1 • • . . . • • . . . . . . . . • • • • . . 44

3 . 4 . Extended cytogenetic s tudy of H- 1 1 : hi s relatives and progeny . . . . . . . . . . . . . . . . • . • . . . . . • • . . . . . . . . . . . . . . . . . . . . . . . . . . . 50

3 . 5 . Di scussion . . . . . • . . . . . . . . . • . . . . . . . . . . . • . . . . . . . . . . . . • . . . . . . . . . 58

3 . 5 . 1 . H - 1 1 ' s ferti l i ty . • . . . . . . . • . . . . . . . . • . . . . . . . . . . . . . . . . . 5 8

3 . 5 . 2 . H - l l ' s XO f i l ly . . . . . . . . . . . . . . . . . . . . . . . . • . . . . . . . . . . . . 59

3 . 5 . 3 . The abnormal chromosome arrangements in cul tured c e l l s of H- 1 1 ' s fami ly . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 60

3 . 5 . 4 . H- l l ' s size and behaviour in view of hi s XYY line . . . • . 6 1

3 . 5 . 4 . 1 . H- l l ' s s1.ze . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6 1

3 . 5 . 4 . 2 . H- l l ' s behaviour . . . . . . . . . . . . . . • . . . . . . . . • • . . 63

3 . 5 . 5 . Possible manners of formation of H- l l ' s mosai c /chimaeri sm . . . . . • . . . . . . . • • . . . . • • • • . . . . . . . . . . . . 64

3. 5 . 6 . Summary . . . • . . . . . • . . . . . . • . . . . . . . • . . . . . . . • • • • . . . . . . . . . 65

vii

CHAPTER IV - H-52, A Mare of Dis tinction 66

4 . 1 . Introduc tion • • • • • . . . . . • • . . . . . • . • • • • . . . • . . . . . . . . . . . • . . . . . • . . • 67

4 . 2 . Material s and methods . • . . . • . . . • . • • . . . . • . • . . . . . • • • . . . . . . . . . . . 69

4 . 2 . 1 . Description . . . . • • • • . . . . . . • • . . . . . • . • • • • • . . . . . . . . . • . . . 69

4 . 2 . 2 . History • • • . . . . . . . . . . . . • • . . • . . . . . . . . • • • • . . . . . . . . . . • . . 69

4 . 2 . 3 . Cytogenet i c material s and methods . . . . . . . . • . . . . . . . • . . 69

4 . 2 . 3 . 1 . Ce l l cultures . . . . . . . • • . • . . . . . . . . . . . . . . . . . . 69

4 . 2 . 3 . 2 . Sex- chromatin investigations . . . . . . . . • . . . . . 7 1

4 . 2 . 4 . Examinations and observations . . . . . . . . . . . . . . . . . . . . . . . 7 1

4 . 3 . Resu lts . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 73

4 . 3 . 1 . Resu l t s of cytogenetic investigations . . . . . . . . . . . . . . . 73

4 . 3 . 1 . 1 . Lymphocyte cu ltures . . . . . . . . . . . . . . . . . . . . . . . 86

4 . 3 . 1 . 2 . Cu l tured cel l s other than lymphocytes . . . . . 90

4 . 3 . 1 . 3 . S ex- chromatin s tudies . . . . . . . . . . . . . . . . . . . . 96

4 . 3 . 2 . Reports of examinations and observations • . . • • • . . . . . . 99

4 . 3 . 2 . 1 . Physical examinations of reproductive organs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 99

4 . 3 . 2 . 2 . General medical hi s tory at Massey Universi ty . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 99

4 . 3 . 2 . 3 . S erum hormone analysis • . . . • • • • . . . • • • • . . . • 101

4 . 3 . 3 . Post-mortem findings . . . • . . . • • . . . • • . • . . . . • . • • • • • • • • . 102

4 . 3 . 4 . Observations on H- 52' s behaviour • • • • . . . . . • • • • . • . • • . 1 1 1

4 . 4 . Di scussion . . . . . . . . . . . . . . . • . . . . . . . . . . . . . . . • . . . . . . . . . . . . . . . . . 1 1 2

4 . 4 . 1 . Cytogenetic aspec t s of H-5 2 ' s mixoploidy 1 1 2

4 . 4 . 1 . 1 . The chromosome cons ti tution of H-52 • . • . . . 1 1 2 .

4 . 4 . 1 . 2 . The po ssible zygote/ embryonic chromosome consti tution of H-52 . . . . . . . . . . . . . . . . . . . . . 1 13

4 . 4 . 1 . 3 . Po ss ible predi sposing fac tors to H-5 2 ' s mixoploidy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 1 7

4 . 4 . 2 . Phys ical and behavioural aspect s o f H-53 ' s mixoploidy . . . . . . . . . . • . . . . . . . . . . . . . . . . . . . . . . . . . . • • . . 1 1 7

4 . 4 . 3 . Summary . . . . . . . . . . . . . . . . . . . . • . . . . . . . . . . . . . . . . . . . . . . . 1 21

vi ii

CHAPTER V - Two 64,XX/ 65,XXY Mosaic / Chimaeric Mares 1 22

5 . 1 . Introduction . . . • • . . . . . . • • • . . . . . • . • • • . • • . . . . . . • • . . • • • . • • 1 23

5 . 2 . Materials and methods • • • • . • . • • • • • • • . . . . . • • • • . • . . . . . . . • . 1 24

5 . 2.1 . Sources of information . . . . . . . . . . . • . . . . . . . . • . . . • . 1 24

5 . 2 . 2 • H- 46 • . . . . . . . . • . . . . . . . . . . . . . . • . • . . . . . . • . . . • . . . . . . 1 2 4

5 .2 . 2.1 . Description . . . • . . . . . . . . . . . . . . . . . . . . . . . 1 24

5 . 2 . 2 . 2 . Feminine structures . . .. . . . . .. . . . .. .. . . 1 24

5.2 . 2 . 3 . Reproduc tive hi s tory .. . ..... . . . . . . . . . . 1 24

5 .2 . 2 . 4 . Behaviour • . . . • . . . . . . . . . . . . . . • . . . . . . . . . 1 27

5 .2.3. H - 47 ... . . . . . . . . . . .. . . . . .. . . .. . . . . . . .. .... . . .. . . . 1 27

5 . 2 . 3 . 1 . Description • . . . . . . . . . . . . . . . . . • • . . . . . . . 1 27

5.2 . 3 . 2.

5 . 2.3 . 3 .

5 . 2.3.4 .

General hi story . . . . . . . • • • . . . . • • . • • . . . . 1 27

Reproductive his tory • . . • • • . . . . . . . • • • • • 1 29

Behaviour . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 3 1

5 .2.4. Cytogeneti c material s and methods • • . . • • • • • • • • • • • 1 3 1

5 . 2 . 4 . 1 . H- 46 . . . . . . . . • . . . . . . . . . . . . . . . . . . . . . . . . . 1 3 1

5.2.4 . 2. H- 47 • • • • • . . . . • • • • . . . . . • • • • • • . . • . • • . . • • 13 1

5 .2 . 4 . 3 . Evaluation of cel l s • • • • • • • . • . • • . • . . . . . 1 3 1

5 .3 . Results • • . . . . . . . • • . . . . . . . . • . . . . . . . . . • . . . . . . • . . . • . • . . . . • 1 3 2

5 . 4 .

5 . 3 . 1 . H-46

5 .3 . 2 . H-47

Discussion .... . . . ..... . ...... . . . .... ...... . . .. . ... . . . .. .

1 3 2

136

140

5 .4 . 1 . Errors of development and H-46 and H- 47 . ........ 144

5 . . 4.2. Summary . . . . . . . • . . . . . . . . . • . . . . . . . . . . . . . . . . . . . . . . . 1 47

CHAPTER VI - H- 27, An Enigma 1 48

6 . 1 . Introduction • . • . . • . . . . . . • . . . • . • . • • • . . . . . • . . . . . . . . • . . . . . 1 49

6.2 . Materials and methods • . . • • . • • . . . . . . • • • . . • . . . . . • • . . . . . . . 1 5 1

6.2.1 . H- 27 . . ... . ... .. ... .... . . . . . . . ...... .. . . ... .. ... . 1 5 1

6 . 2 . 1 . 1 . Descri p tion . • . . • • • • • • • . . . . . • • • • . . • . . . • 1 5 1

6 . 2 . 1 . 2 . General information • . . • • • • • • • . • • • . . • . • 1 5 1

i x

6 . 3 .

6 . 2 . 1 . 3 . Pre- admi ssion history • . . . . • • . • . . • • . . . . . . 1 5 1

6 . 2 . 1 . 4 . History at Massey University . . . . . . . . . . • . . 1 5 1

6 . 2 . 1 . 5 . Behavi our . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 5 2

6 . 2 . 1 . 6 . Pos t-mortem report . . . . • . • . • . . . . . . . . . • . . . 1 5 2

6 . 2 . 2 . Cytogeneti c material s and methods • . . • • • . . . . . . . . . • . 1 55

6 . 2 . 2 . 1 . Cu ltured cel ls . . . . . • . • • . • . . . . . . . . • • • . . . . 1 55

6 . 2 . 2 . 2 . S ex- chromatin investigations . . . • . • • • • • . • 1 5 6

6 . 2 . 2 , 3 . Lymphocyte transformation test . • . . • • • . • . 1 5 6

Resu l t s 1 5 7

6 . 3 . 1 . Resu lts of cytogeneti c investigations -.... . . . . . . . . . . 1 5 7

6 . 3 . 1 . 1 . Cultured cel l s • • • . • • . • • . . . • • • . • . . . . • . . . . 1 5 7

6 . 3 . 1 . 2 . Sex- chromatin invest igations . .. . . . . .... . . 1 67

6 . 3 . 2 . Lymphocyte pro l iferation test . • . . . . . . . . . • . . . . . • • • . 1 69

6 . 4 . Di scussion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 7 2

6 . 4 . 1 . Cytogeneti c abnormality . . . .. . . . . . . .. . . . . . . . . . . . . . . . 1 7 2

6 . 4 . 2 . Some impli cations of the fai lure of lymphocyte cu ltures . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 73

6.4.3 . Po s s ible impli cations of mitotic instabi lity in c e l l cu l tures . . . • . . . . . . . . . . • . • . . . . . . • • . . . . . . . . • • . • . 1 74

6 . 4 . 4 . Inferti l i ty

6 . 4 . 5 . Summary . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

CHAPTER VII - General Discussion

7 . 1 . Preval ence of the mo saic /chimaeric condi tion in

1 7 6

177

179

�- .£.· cabal lus . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 180

7 . 2 . Possibl e predi spo sing factors to the mosaic / chimaeri c condi tion and the horses in this study • • • . • • • • • • . • • • • • • • . . • • 1 8 2

7 . 2 . 1 . Parental age effect 1 8 2

7 . 2 . 2 . Birth order and dam' s obstetric hi s tory • . • • • • • • • . • • 183

7 . 2 . 3 . Possible inf luences of metabol i sm and physio logy • • • . 184

7 . 2 . 4 . Inbreeding and relationships between the mosai c / chimaeras i n thi s study . . . • . . . • • . • • • • • . . . • • . . . • . . . . 185

X

7 . 2 . 5 . Genetic predi sposi tion to masked karyotype change . . . . . . . . . . . . . . . . . . . . . . . . . . . . . • . . . . . . . . . . . . . 186

7 . 3 . Mosaic/ chimaeric individual s and breed sub-ferti lity . . . . . . 188

7 . 3 . 1 . Mosaic/ chimaerism and fert i l i ty . . . . . . . . . . . . • . . • . • 188

7 .4 . Cytogenetics and the Domestic Horse . . . . . . . . . . . . . . . . . . . . . . 1 9 1

Appendix I - The 1978 Pi lot Survey

Appendix ll - Unpubl i shed XO Mares Di s covered in New Zealand and Other Horses Inves tigated for which there

193

was a Sugges ti on of a Cytogenetic Anomaly • • • • • • • • 200

Appendix Ill - Anci l lary Information to Chapter IV

A . H - 5 2 1s Dimen sions

B . H - 5 2 Pos t - Mortem • . . . • . . • • . . . . . . • • • • · � . . . . 204

C . Estimates of Pr imordia l Ce l l Pool -Size . . . 207

Appendix IV - H- 27 Post Mortem . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 209

Appendix V - Materials

A. Supp l i es 2 1 1

B . Tissue cul ture materia l s . • . . . . . . . . . . . . . . . 2 1 2

c. D .

Cel l cu lture media

Staining solutions

2 1 4

2 1 4

E . E qui pment . . . . . . . . . . . • • . . . . . . . . . . . . . . . . . . . 2 1 5

Appendix V I - Methods

I . Leucocyte culture system • . . . . . . • • • . • . . • . . • . . 21 6

1 1 . Cel l culture techni ques • • . • . . • • • . . . • • . . . • . . • 217

I II . Slide preparation • • . • • • • . . . . • . . • • • • . . . • . . . . . 2 1 9

IV . Sex- chromatin • • • • • . • . . • • • • . • . . . . . . . • . . • • • . . • 220

V . Differential staining . • • • . • . . • • • • • • • . . . . • • . . 220

VI . Photographic methods . • . . • . • • • . . . . . • • • • • • • . • • 2 2 1

VII . Lymphocyte transfo r ma. tion test . • • . • . • • • • . • . • 2 2 2

-

Bibliography • • • . • . . . . • • • • . • . • . . . . • • • • • . . . . . . . . . . . . • • . • • • . • • . • • • 2 24

xi

1 11 . 1

1 11 . 2

III . 3 a

3b

LIST OF FIGURES

H- 1 1 as a four year old

Mare "B" wi th three day old 1 979 f i l ly by H- 1 1 . . • • • • •

H- 1 1 , the sire o f H- 58 • • • • • . • . • • . . • • • • • • • • • . . . • • • • • • •

H- 58 age nine months . . . . . . . . . . . . • . . . . . . . . . . . . . . . . . . . .

Page

35

40

4 2

42

111 . 4 A norma l 64 , XY metaphase from H- 1 1 . . . . . . . . . . . . . . . . . . . 46

111 . 5 A metaphase wi th 65 chromos omes from H- l l ' s cul tured s kin (designated XYY ) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 47

111 . 6 A metaphase wit h 65 chromosomes from H- ll ' s cu l tured lymphocytes ( designated XYY ) . . . . . . . . . . . . . . . . . . . . . . . . . 48

111 . 7 An example of a 63 , XO cel l from H- 1 1 . . . . . . . . . . • . . . . . . 49

111 . 8 Metaphase with 6 5 chromosomes and t r i- radial (non-homo l ogou s chromatid interchange ) invo lving chromosome 1 and possibly 9 ( from H- l l ' s sire ) . . .. . . . 5 2

111 . 9 Examples o f abnormal chromosome arrangemen ts found in one of H - l l ' s ha lf brother s and H- l l ' s 1978 co l t . . 53

1 1 1 . 10 C- banded cel l from H- 68 , an XO fi l ly sired by H- 1 1 . . . 55

111 . 1 1 G - banded cel l f rom H- 68 , an XO f i l ly sired by H- 1 1 . . . 56

IV . l

IV . 2

I V . 3

IV. 4

IV. 5

Left lateral view o f H- 5 2 • • • • • . • • • • . • . . . . . • . • . . . . . . . •

Metaphase containing 65 chromosomes ( designated XXY ) ,

G -banded metaphase containing 65 chromosomes , confirming the 65 ,XXY cel l line • • • • . • . • • • . • . • . • • • • • • .

A metaphase wi th 65 chromosomes ( designated XXX ) • • • • •

G- banded metaphase containing 65 chromosomes , confi rming the 65 ,XXX cel l line • • • . . • • • . • • . . . • • • • • • • •

68

75

76

77

78

IV. 6 A metaphase \vi th 64 chromosomes ( 28 non-acrocentric ) containing two X- chromosomes . . . . . . . . . . . . . . . . . . . . . . . . . 79

IV . 7

IV . 8

Another exampl e of a 64 ,XX cel l from H-52 • • • • . . • • • . • •

One of the me taphases with 66 chromosomes ( designated 6 6 , XXXY) . . . . • • . . . . . . . • • • • . . . . . • • . . . . . . . • .

IV. 9 Another metapha·se wi th 6 6 chromo somes designated

80

8 1

66 , XXXY . • • . . . . • . • . . . • . . . . . . . • . • • • . . • . . . . . . . . . . . . . . . . . 8 2

IV. lO

IV. l l

One of three metaphases wi th 66 chromosomes ( 30 non acrocentric ) �esignated 66 , XXXX ) • . . . . . . . . . . . . . . . . . . .

Another of the three metaphases designated 6 6 , XXXX . . .

83

84

IV . 1 2a An exampl e of a neuron wi th two sex- chromatin bodies , confi rming the presence of ei ther XXX or XXXY cel l s . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 85

1 2b An exampl e of a neuron �ith one sex- chromatin body , whi ch cou ld. repr�se�t.�tth�r an XX or XXY

IV . 13

IV . 14

IV . 15

IV . 16

IV . 17

IV . 18

IV . 19a

cel l . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 85

Hi stogram of cul tured lymphocytes analysed s howing the proportions ( %) of XX , XXX and XXY cel l s . . . . . . • . . . • 89

Propo ttions· ( %) of the cel l s evaluated from sol i d t i ssues wi th 64 , XX , 6 �,XXX . and_65 ,XXY chromosome constitu t ions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 95

A section of uterine tiss�e from H-52 showing endometri a l hypoplasia • • • • • • • . • • • • • • • • • • • • • • • • • • • • • • • • • 100

External (ventral ) view of H- 5 2 ' s genital organs • • • • • • • 104

External view of the . �terine horns , ovaries , and body of the u te rus . • • • • . • • . . • • . • • • . • . • • . . . • • • • • • • • • . . . • 105

Internal view of the uterus • • • • . . . . • • • • . . . • • • • • • • • • • • • • 106

Lef t cornu uteri and ovary • . . • . • • • • • . . . • • . . • • • • • • • • • • • • 1 07

1 9 b Sagittal bi section through_ the_ l ef t ovary ' s ovulation

IV . 20

IV . 2la

2 lb

IV . 22

IV . 23

V . 1

V . 2

V . 3

V . 4a

4b

V . 5

fossa . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 107

Drawings of Pear l ' s I ron and Sudan Black posi tive areas of left ovary sections and two photographs of a region of the area ( probabl e corpus albi cans ) . . . . . . • . . . . . . . . . . 108

Right cornu uteri and ovary . . • . . . . • • • • . . . . . . . . . • . . . . . . . 109

Sagi ttal bi section through the right ovary 's ovu lation fossa . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 109

Drawings of Pear l ' s Iron and Sudan Bl ack posi tive areas of right ovary sections and two photographs of a region of the area ( probab le corpus albicans ) . . . . . . . . . . . . . . . . . 1 10

Diagram of the possible mosaic manner of formation of H- 5 2's mixoploidy • . . . . . . • • • • . . . • • . • • . • . . . • . . . . . . • • • • . • . 1 1 6

H-46 as a �ine year o ld • . • • • . • • • • • • • . • . . . • . • . • . • • • • • • . • 1 25

Vulvar c l ef t and anus of H-46 • • • • • • • . . • . • • • • • • • • • • • • . . • 1 26

H- 47 as a two year o l d maiden mare • • • • • • • • . • • . • • • • . • • • • 1 28

Left lateral view of H-47 ' .s rump • • • • • • • • • • • • • • • • • • • • • • • 130

The appearance of H- 47 ' s artifica l ly formed continuous rectum and " c loaca" when she was two years o ld • • • • • • • • • 130

An exampl e of a . �el l from H-46 which was designated 65 , XXY. • • • • • • • • • • • • • . . • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • . • • 134

V . 6 A normal 64 , XX cel l from H-46 • • • • • • • • • • • • • • • • • • • • • • . • • • 135

V. 7 An exampl e o � _ �_ce��.�r9�.H747 which was designated 65 , XX.Y • • • • • • • • • • • • • • • . • . • • • • . • . • • • • • • • • . . • • • • • • • • • • • • • • 138

V . 8 A normal 64 ,XX �etaphase f rom H - 47 • . . • . • • • • • • . • . . . . . . . • 1 3 9

xii i

VI . la

lb

VI. 2

VI . 3a

Dor sal view o f H- 27 ' s internal geni talia . • • • . . • • • • • • • • 153

Ventr al view.-of H-2 7' s internal gen i talia. . . . . . . . . . . . . 153 Internal view o� H- 2 7�s cervix , uter�� �nd +ig�t u terine horn . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 154

Exampl e of a neuron with two sex- chromatin bodies from H- 27 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 159

3b Example of a neuron wi th one sex- chromatin body

VI . 4

VI . 5

VI . 6

VI . 7

VI . 8

VI . 9

VI • .!_

from H- 27 • . . . . . . . . . • . . . . . . . . . . . • . . . . . . . . . . . . . . . . . . . . . 159

The o nly 63 , XO cel l found in H- 27 ' s cul tured cel ls . . . . 160

One of the three cel ls which sugges ted , ini tial ly , that H- 27 might have a 66 ,XXXX cel l line • • • . . . . • . • . • • 1 6 2

One o f the subsequent metaphases wi th 66 chromosomes , sugges ting that H- 27 ' s cel l s were mi toti cal ly·unstable in-vi tro . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 63

An exampl e of a 6 2 , XX . ce l l • . • • • • • • • • . • . • • • • • • • • • • • • • • • 1 64 An exampl e of an abnormal chromosome arrangement seen in t\vo of H- 27 1 s cultured cel l s . . . . . . •. . . • • • • • • • • • • • • • 1 65

An exampl e of endoredupl i cation i n H - 27 ' s cu l tured c el ls . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 66

Lymphocyte transformation tes t experimental design • • • 156

xiv

LIST OF T ABLES

Page

1 - 1 Extant members of•the fami ly Eguidae • . . • • • • • • • • • • • . . • . . . 1 6

1 - 2 Equine hybrids 18

1 - 3 Summary o f pub l ished reports of XO mares • • • • • • • • • • • . • . • • 23

1 - 4 Summary o f pu&fi shed reports of horses with non-mosai c / chimaeri �.���. ��+9�Qsome abnormal i tie s , other than XO mares

1 - 5 Summary of p������e� reports of horses wi th mosai c / ,

24

· c himaeri sm . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 25

3 - 1 Reproductive performance of H- 1 1 ( 1977- 1 980)

3 - 2 The di stri bu t i Qn� . 9�.c �romosome counts from H- l l ' s

3 9

cu l tured c el l s .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 45

3 - 3 Results of cytogenetic investigations o f H- l l 's relatives and progeny . . . . . . . . . . . . . . . . . . . . . . . • . . . . . . . . , . . . 5 1

4- 1 Summary of cel l cu l ture techniques and cul ture periods of non- lymphocyte cul tured cel l s . . . . . . . . . . . . . . . . . . . . . . . . . 70

4- 2 The di stri but ion of gross chromosome counts from H- 5 2's cu l tured c el l s . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 74

4 - 3 The di stribution o f gross chromosome counts in H- 52's cu l tured lymphocytes . . • . , • . . . • . . . . . . . . • • . . . . . . . . . . . . . . . . . 87

4 - 4 Condensed di stributions of differential chromosome counts from H- 5 2 ' s cul tured lymphocytes . . . . . � . • . . . . . . • . . • 88

4 - 5 The di s tributions of gro ss chromosome counts i n cu l tured cel l s other than lymphocytes • . • • . . . • . . • . • . . • • . . • . . . .' . . . . . 9 1

4 - 6 The di str ibutions o f differential chromosome counts in cul tured cel l s other than lymphocytes . • • • • . . . • • . • • • • . . . . • 9 2

4- 7 Percentages of artifactual aneuploidy and cel l s wi th 64/ 28 non- acrocentric chromosome counts • • • • • • • • • . • • • • . • . . 93

4- 8 The numbers and di stribu tion of sex- chromatin bodi es in neurons of H - 5 2 ' s c entral nervous system • • . . . . . . . . . . . • 97

4- 9 The proportions of cel l s with sex- chromatin bodies and their distr ibut ion in H-52 ' s central nervouS' system • • ·• • • • 98

4- 1 0 Results of serum hormone analysi s ' I and II • • • • • • • • • • • • • • 102

5 - l The di stributions of chromosome counts from H- 46 ' s cul tured cel l s • • • . • • • • • • • • • • • • . • • • . • • . • • • • • • • • . • • • . • . . • • • 133

5 - 2 The distributions of chromosome counts from H-47 ' s cu l tured ce l l s • • • • • • • • • • • • • • • • • • • • • • • • • • • • • I• • • • • • • • • • • • • • 1 3 7

6 - 1 The dis tributions of chromo some count s from H- 27 ' s c � l tured cel l s . • . . • . . • • • • • • . • • . • • • . . • • • . • • • • . • • • • . . • • • • 158

6- 2 Summary of recorded abnormal differential chromo some count s from cu l tured cel l s from H- 27 • • • . . . • . . • • . • . • • • • • 1 6 1

6 - 3 The numbers and di stributions of sex- chromatin bodies in the central nervous system of H - 27 . . • . . . . • . • . . . . • . . . 1 68

6 - 4 Thymidine incorporation of Eguine peripheral blood lymphocytes cul tured wi th PHA for three days • . . . . • • . • • • 1 70

7- 1 Compari son of the ages at conception of parents of XO mares and the ' mo saic/ chimaeras in this s tudy • . . • • . • . 183

7 - 2 Approximate coefficient of relationship • . • • • • . . • . . • . . . • • 1 85

A - I - 1 The distributions of chromo some counts in the 1 978 pi lot survey . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . • . • . . . . . 1 95

A- Il- l Summary of unpubli shed cases of XO mares in New Zealand. 201

A - II- 2 Summary of cytogenetic inves tigations of hor ses .whi ch had a suggestion of an anoma ly , but for which there was insuffi cient information to ve rify • • • • • • • • • • • • • • • • • • 202

A- lll- 1 Estimated primordial cel l pool si zes • • • • • • • • • • • • • • • • • • • • 207

xvi