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i
A CLINICAL CYTOGENETICAL STUDY OF
THE HORSE
A thesis presented in partial
fulfilment of the requirements for the degree
of Doctor of Philosophy in
Veterinary Science at
Massey University
Murray Garfield Blue
B. V. Se. ( Massey )
1976
ii
ABSTRACT
Chromosome preparations were derived from short-term
cultures of blood lymphocytes and from fibroblast-type cell
cultures of subc�taneous fascia obtained from
normal horses.
44
clinically
The quality of mitotic cells compared
favourably with similar studies in other species.
Some
characteristic morphological features were found which aid
in the recognition of certain chromosomes, but the identification of many homologous pairs by conventional techniques
was difficult.
The normal karyotype of the horse ( 2n= 64)
was characterized following Giemsa staining and C-banding.
The latter procedure was shown to be a valuable aid for the
identification of specific chromosomes, in particular the
sex chromosomes.
After centromere staining, the X chromo-
some of the horse
lS
characterized by the presence of
prominent C-bands on its long arms, while the Y chromosome
appears as an intensely-stained block.
Analysis of mitotic chromosomes prepared from ten
phenotypically normal but infertile mares revealed, in one
animal, the consistent lack of one sex chromosome in the four
tissues studied.
Centromere staining and sex chromatin
studies confirmed that this mare had the karyotype 63XO.
Examination of the genitalia of this XO mare disclosed the
presence of apparently normal
reproductive organs except
that macroscopic follicles were absent from both ovaries.
Histologically, a number of presumptive atretic Graafian
follicles and a discrete area of apparently functional
luteal tissue were discovered in the right ovary.
��
The sig-
nificance of these findings are discussed and compared with
iii
the features of the analogous syndromes associated with the
XO anomaly in other species.
It is suggested that disorders of the sex chromosomes
may occur commonly in horses and may contribute significantly
towards the low r ertility of the thoroughbred mare. Attention
is drawn to the important practical applications of cyto­
genetic studies in the investigation of infertility in
broodmares, and in the certification for sale of young horses
intended for use as breeding stock.
Cytogenetic studies of spontaneous abortions in women
have established that a large proportion of pregnancy wastage
in humans is due to chromosome aberrations.
The counterparts
of many of the chromosome errors initially described in
humans have now been reported among domestic animals.
In
particular, the XO anomaly seems remarkably common among
infertile mares.
These findings have indicated that chromo­
somal abnormalities may play a significant role in the
aetiology of spontaneous abortion and embryonic loss in
domestic animals.
To investigate an hypothesis that chromo­
some anomalies are an important cause of prenatal loss in
the mare, an attempt was made to analyze, cytogenetically,
a ser1es of equine abortions.
F rom a range of tissues
obtained from 26 aborted foals, cell cultures were prepared
but failed to grow, and chromosome analysis was therefore
not possible for any of these specimens.
It seems that the
equine foetus is retained in the uterus of the mare for a
considerable period after the foetus has died and usually
consists of partially-autolyzed non-vital tissues at the
time of its expulsion.
Consequently, a study was made of the metaphase
iv
chromosomes prepared from 22 equine embryos after their
surgical removal from mares' uteri.
found in each specimen.
A normal karyotype was
Although inconclusive on their own,
the results of this study form an important contribution
towards cumulatiye studies of embryonic loss in the horse.
The current findings are discussed in the light of similar
studies of induced abortions in women and embryonic loss in
animals, and the potential for further investigation
in
this
field is emphasized.
From the limited number of cytogenetic studies in
domestic animals, it appears that chromosome analyses,
particularly with the aid of more refined techniques such as
centromere staining, may be of considerable value in investi­
gations of infertility and embryonic loss in the horse.
V
ACKNOWLEDGEMENTS
This thesis is the record of a research project
involving three years of study and research within the
faculty of Veterinary S6ience at Massey University,
Palmerston North, New Zealand.
In the course of this work
I have had the cooperation of a large number of people, for
which I am most grateful.
I am especially indebted to my supervisor,
Professor A.N. Bruere, for providing me with the opportunity
to undertake this study.
His continual guidance and enthus­
iasm is greatly appreciated.
F or the availability of the technical facilities
required for this work and for assistance in their use, my
thanks are due to the Heads and personnel of the following
departments of Massey University:
Veterinary Clinical
Sciences, Veterinary Pathology and Public Health, and the
Library.
Although it is not possible to personally acknow­
ledge all of the people who assisted with this study, special
Dr. Helen Chapman
mention must be made of the following:
for her constant and invaluable advice;
with the preparation of photographs;
Mr. Torn Law for aid
Messrs. Carol Black,
Cedric Barnett, Linley Fray, Bert de Cleene and Priscilla
Jaine for their tireless technical assistance;
and
Mr. Mervyn Birtles for his patient tuition with the histolo­
gical preparations.
Great enthusiasm for this study was shown by
Mr. Harry Dewes;
behalf.
I with to thank him for his efforts on my
I am also grateful to Professor E. D. F ieldon and
Dr. B.E. Goulden who helped in proof-reading of the thesis,
vi
and to Mr. Martin Copland for his eternal comments and
criticism.
Finally, I must acknowledge the Mackenzie Education
Foundation and in particular Mr. R.A. Mackenzie, who
provided the financial grant to support this research
project.
vii
TABLE OF CONTENTS
P age
PART
I
PAR T
II
PRELUDE
2'1
MAT ER IA LS A ND METHODS
29
II. '1 . -. PRELIMINARY ST UDY OF ABORTED
FOETUSES
II. 2.
30
METHOD FOR OBTAINING EQUINE
EMBRYOS
30
i)
Exper imental an imals
30
Pregnancy diagnosis
3'1
i i)
-
II. 3.
i i i) Anaesthes ia
3'1
iv)
32
Surgery
IN V ITRO CULTURE OF BLOOD
LEUCOCYTES
34
i)
Hi story
34
i i)
Culture of cells
34
i i i) Metaphase arrest with
colch i c ine
iv)
35
Treatment with hypoton i c
flu id
35
v)
Fixation of cells
36
v i)
Sta ining techniques
37
a) General procedure for
m itotic c ells
b) C entromere sta in ing
II. 4.
37
37
FIBROBLAST CULTUR E
39
i)
Introduction
39·
i i)
Prep aration of cell monolayer
from t i s sue explant s
40
v iii
Page
a) Preparation of tis sue
40
explants
b) Meth od for o btaining plasma
41
c) Explant attachment for
p rlmary culture
41
d) Development of cell
monolayer
42
iii) Tryp s in disaggregation technique f o r cell culture
44
a) Treatment o f embryo s
44
b) Primary culture
45
iv)
Replen ishment o f gro wth medium
46
v)
A s ses sment of cell growth
47
v i)
Preparatio n o f cultures for
subculture or harvest
49
a) Subcultures
49
b) Harvest of f ibroblast
cultures
II . 5 .
v ii) Storage of cells
50
SEX C HROMATIN STUDIES
51
i)
Sex chromatin of nerve tis sue
ii)
Sex chromatin o f p olymorpho­
nuclear neutrophil s
II. 6 .
51
51
A NALYSIS OF RESULTS
52
i)
M icro s c opy
52
Photographic techniques
52
ii)
·
iii) C onstruction of karyotypes
PART III
50
53
GESTATION A ND A BORTION IN THE MARE
54
III . 1.
55
LITERATURE REVIEW
ix
Page
i)
Normal gestation
i i)
Cau ses of abortion in the
mare
PAR T IV
-55
59
III . 2.
RESULTS FROM A BORTED FOETUSES
66
III . 3.
DISCUSSION
70
CYTOGE NETIC STUDIES IN THE HORSE
IV . 1 .
72
THE KARYOTYPE OF THE HORSE
73
i)
73
L iterature review
a) Chromo s omes of the hor se,
Equus caballu s
73
b) Chromo s omes of the donkey ,
Equu s asinu s
i i)
78
Results and Discu s s i o n
78
a) Chromo s ome number
78
b) Chromo s ome morphology
86
c) Centromere stain ing of
horse chromo somes
IV . 2.
96
CYTOGENETIC STUDIES OF
INFERTILE HORSE S
99
i)
99
Literature review
a) Ster ility of equine hybrids 99
b) The chromo s omes of intersex h o rses
101
c) Sex chromo s ome anomalies
in infertile mares
ii)
Results:
Studies o n XO mare
103
107
a) H i story
110
b) Chromo s ome studies
110
c) Examinati o n of reproductive tract
116
X
d) Sex chromatin studies
iii) Dis cus sion
Origin of
xo
'140
anomaly
F requency of XO anomaly
Effect of
xo
'1 40
'1 4'1
anomaly on
'146
phenotype
Effect of
'1 30
xo
anomaly o n the
reproductive o rgans
'1 48
Inactivation o f X chromos omes
149
Triple-X females
'1 5 '1
Fertility and the lack of o ne
X chromo some
Part V
'1 5 2
CHROMOSOME STUDIES IN ABOR TION A ND
EMBRYONIC LOSS
'1 5 6
V. '1 .
'1 5 7
L ITERATURE R EVIEW
i)
C hromo somes and human
abo rtion
'1 5 7
a) Incidence of abo rtion
'1 5 7
b) Chromo s ome studies in
spo ntaneous abortions
'1 57
c) Types of c hromo s ome anomalies in abo rtions
'1 60
d) Tris omy and mono s omy in
spontaneous abortions
e) Polyploidy
'1 63
'1 64
..
f) Mis cellaneous chromo s ome
anomalies in spontaneous
abo rtions
'1 68
xi
Page
g ) Parental chromo s omes and
sp ontaneo u s abo rtion
h ) Polymorphisms
1 68
1 69
i ) Chromo s ome anomalies in
induced abo rtions
ii )
iii )
Chromo s omes and embryo nic
death in animals
1 71
The chicken
1 71
The rabbit
1 71
Domestic animals
172
Aetiology o f chromo s ome
dis order s in abortions
V. 2 .
RESULTS AND DISCUSSION
i)
ii )
1 69
1 74
1 76
Clinical details of equine
embryo s
1 76
Chromo some studies
1 81
Pr oblems with cell
culture s
1 84
Mo saicism
1 85
iii )
Po s sibility o f a sire effect
iv )
Chromo some anomalies and
embryonic lo s s in the horse
v)
1 86
1 87
The aetiology of chromo some
dis o rders in relation to
embryonic lo s s in the horse
Drugs
1 91
1 91
Heritable chromo s o me
aberrations
1 91
Maternal age
1 92
Aging o f gametes
1 93
xii
Page
v i) Conclus ion
1 95
APPENDICES
1 97
BIBLIOGRAPHY
21 1
x i ii
LIST OF TA BLE S
Page
T able
III. 1 .
Factors assoc iated with abo rtion in the
60
mare .
III. 2 .
IV. 1 .
IV. 2 .
Autopsy details of abo rtuses .
�
Establishment of the chromosome numbers
68
of the horse, donkey and mule .
76
Culture methods used for o btaining ho rse
karyotyp e .
IV. 3 .
D istr ibuti o n of chromosome counts from
horses with no rmal karyotypes .
IV. 4 .
77
82
Distr ibut i o n of chromosome c ounts from
ho rses with no rmal karyotypes and at least
30 cells c ounted .
IV. 5 .
Spec ies c o mpar ison of aneuploidy from
embryo c ell counts .
IV. 6 .
1 36
Posit i o n o f the sex c hromatin body within
the nuc leus .
IV. 1 2.
1 13
Sex chromatin in nerve cells of a normal
stalli o n, normal mare and XO mare .
IV. 1 1 .
1 09
C hromosome counts of metaphase spreads from
XO mare .
IV. 1 0 .
1 08
C hromosome c ounts fro m infertile mares
shown to have normal karyotypes .
IV. 9 .
1 06
Clini c al details of infert ile mares
referred for chromosome analysis .
IV. 8 .
84
Clinical details o f mares with sex chromo­
some disorders .
IV. 7 .
83
1 36
C o mparison of sex chromosome disorders
among i nfertile mares and women .
1 42
xiv
Page
Table
IV . 13 .
Spec ies character i st i c s a s s o c iated with
sex chromo s ome dis orders .
V.
1.
Chromo s o me anomalies among human spontan­
eous abo rt i o ns .
V.
2.
1 50
1 60
Summary of chromo some studies in human
;
spontaneous abo rti ons.
1 62
V.
3.
Chromo s ome studies in induced abo rtions .
1 70
V.
4.
Clini cal details of equine embryo s .
1 78
V.
5.
Chromo s ome studies of equine embryo s .
1 82
XV
LIST OF F IGURES
Page
PART I I .
1.
Surgical removal of an e quine embryo
from the ut erus of a mare .
2.
F low diagram of leuco cyt e cu lture
t e chnique from horse b lood .
3.
43
F ibrob last c e l l culture which has
f o rmed a comp l e t e mono layer .
5.
38
Primary c e l l culture showing out growth of c e l l s from a t i s sue exp lant .
4.
33
48
Mono layer c e l l culture aft er 16 hours
expo sure to c o lchic ine .
48
69
PART I I I .
1.
G e st at ional age s of abortu se s .
PART IV .
1.
Karyotype of a t etrap l o id c e l l in a
c e ll culture from an e quine embryo .
85
2.
Karyotype of a normal stal l i on.
88
3.
Karyotype of a normal mare.
89
4.
Karyotype o f a normal stall ion showing
characterist ic s e condary c onstrict ions
on acroc entric chromo s ome s .
5.
91
Pre sumed s e condary c onstrict ions on the
s hort arms of a large met ac e ntric
chromosome .
6.
92
Pre sumptive chromo somal translo c at ion
invo lving a large met acentric chromo­
s ome from a normal mare .
7.
94
Karyotype of a c e l l from an e qu ine
embryo showing pre sumpt ive premature
c hromat id · separat ion and a s s o c iat ion
o f t wo homo logous met acentric chromo­
s o me s .
95
xvi
Page
8.
Karyotype from a normal stall ion
aft e r c entromere st aining .
9.
97
Charact eris t i c app earance of the sex
chromo s ome s of the horse after
c e ntromere staining .
�0.
98
Phenotype o f thoroughbred mare
shown t o have XO anomaly .
�� -
Karyo type of mare shown to have XO
anomaly .
�2.
��4
Karyotype of 63XO mare aft er centromere st aining .
��5
�3 -
Int ernal genit alia of
xo
mare .
�4.
Int ernal ge n it alia of
xo
mare ; vagina ,
cervix and ut erus op ened .
�5.
Left ovary o f XO mare .
�6.
H i s t o logical s e ct ion o f endom etrium
from XO mare .
� 7-
��7
��8
� 2�
Pre sump t ive at ret ic Graaf ian f o l l i­
c l e s ( a and b ) and lut eal t i s sue ( c )
in the right ovary o f the XO mare .
�8.
� 22
Pre sumpt ive Graafian f o l l i c le s in
the right ovary of the XO mare .
� 22
�9.
Wall o f pre sump t ive Graaf ian f o llic le� 23
20 .
Atre t i c or degenerat ing Graaf ian
f o l l ic l e .
2� .
D i s c r e t e are a of lut e a l t i s sue in the
right ovary of the XO mare .
22 .
� 24
� 27
Typ ic a l hist o l ogical appe aranc e of
funct ional lut eal t i s sue from right
ovary o f XO mare .
� 27
xvi i
Page
23 .
Charact erist ic act ive lut e a l endoc r ine c e lls .
24 .
1 29
Sex chromat in body ad jacent to the
nuc l e o lus in a neurone from a normal
mare .
25 .
1 37
Sex chromat in body free in the
nuc l e op lasm of a neurone from a
normal mare .
26 .
1 38
Sex chromat in body adj ac ent t o the
nuc l e ar membrane in a neurone from
a normal mare .
27 .
1 38
Charact eri s t i c nu c l e ar app endage
( " drumst ick " ) on a po lymorphonuc l e ar neutrophil from a normal mare .
28 .
1 39
Nuc l e ar app endage on a po lymo rpho nuc l e ar neutrophil from a nonua l
s t a l l ion .
PART V .
1.
Po s s ib l e me chani sms for the origin
of t r ip lo idy .
2.
1 80
E qu ine embryo with we l l deve loped
amnion and umbi l ical art eries .
6.
1 79
N o rmal horse embryo with membrane s
removed .
5.
1 79
Normal horse embryo showing remnant of
yo l k sac and the s inu s t e rminal i s .
4.
1 67
Norm a l horse embryo ( E . cabal lus )
with in intact all ant o chori o n .
3.
1 39
1 80
E qu ine twin embryo o f fewer than
20 d ay s ge s t at ion .
1 83
xvi ii
LIST OF APPENDICES
App endix
I.
Page
Paras ite c ontro l o f experiment al animal s .
II .
· Drugs used for anae sthe sia and surgery .
III .
Mat erial s re quired for blood leuco cyt e
1 98
1 98
culture .
1 99
a)
Preparat ion o f culture .
1 99
b)
Harve st ing of cultures .
200
c)
To mount slide s .
200
IV .
Preparat ion o f G iemsa stain .
201
V.
Procedure for cent romere staining .
201
VI .
Preparat ion o f glas sware .
202
VII .
Mat erial s re quired for fibroblast
culture :
V II I .
Plasma c lot emb e dding
t e chn ique .
202
a)
To obt ain t is su e from l ive horse s .
202
b)
Growth medium .
202
c)
To prepare tric ine buffer .
203
d)
To prepare pla sma c lot .
203
e)
A . T . V . ( Ant ib io t i c -tryp s in-versene
solut ion ) .
203
f)
PBS . ( Pho sphat e -buffered sal ine ) .
204
g)
General e quipment .
204
Mat erials re quired for f ibroblast
culture:
Tryp sin disaggregat io n
t echnique .
205
a)
Transport medium .
205
b)
Tryp s in s o lut ion .
205
c)
A nt ib iot ic s o lutio n .
205
d)
Co lchicine .
205
e)
Long t erm storage of c e l l s .
205
XlX
AJ2J2endix
Page
f)
IX .
X.
206
General equipment .
Analysis of re sult s .
206
..
Examinat ion of s lide s .
b)
Deve lopment o f f ilm .
206
c)
Deve l opment of print s .
206
Chromo s o me c ount s of hors e s prep ared
from leuc o cyt e cultures .
XI .
207
Chromo some c ount s of hors e s prep ared - by
f ibroblast culture s of t i s sue e xp lant s .
XII .
206
a)
208
Chromo some c ount s prepared by leucocyt e
culture f rom hors e s with normal karyo ­
type s .
C as e s with fewer than 30 c e l l s
are exc luded .
XIII .
209
Chrom o s ome count s from fibroblast c e l l
culture s , exc luding c a s e s with f eweE
than 30 c e l l s c ount e d .
a)
210
Chrom o s ome c ount s from hors e s with
norma l karyotyp e s prepared by c e l l
culture s o f t i s sue exp lant s .
b)
210
Chromo some c ount s f rom e quine embryo s
with normal karyotype s .
210