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Title Studies in Equine Reproduction
Name Sandra Ann Wilsher
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STUDIES IN
EQUINE REPRODUCTION
by
SANDRA ANN WILSHER
A report submitted to the University of Bedfordshire in partial fulfilment of the
requirements for the degree of Doctor of Philosophy by Publication
April 2009
I
Preface
All the experimental work and research described in this dissertation was
performed at the University of Cambridge, Department of Veterinary Medicine
Equine Fertility Unit, Mertoun Paddocks, Woodditton Road, Newmarket, Suffolk
CB8 9BH and the Paul Mellon Laboratory of Equine Reproduction, Cheveley Park,
Newmarket, Suffolk CB8 9DE under the supervision of Professor WR (Twink) Allen.
It was submitted for publication between 1999 and 2008. Supervision for the
preparation of this Report was kindly provided by Professor David Rawson,
University of Bedfordshire.
The studies represent original work carried out by the author and, where
assistance has been given, due acknowledgment has been made. The contribution of
the author to each paper is detailed in Appendix 2. The work has not been submitted
in part or full to any other University.
The papers submitted arose from work carried out on topics related to equine
reproduction over a 9-year period. They divide naturally into three research areas: -
1. Epidemiological surveys of the efficiency of Thoroughbred racing and breeding
2. The morphology and functions of the equine placenta
3. Embryo transfer in the horse
………………………….
Sandra Ann Wilsher
II
Acknowledgements
The research described in this report, and in the submitted publications was
undertaken during full-time employment at The Equine Fertility Unit in Newmarket. I
am very grateful for the guidance, support, advice and camaraderie that were ‘part-
and-parcel’ of being an employee there.
The animals used in the studies were part of the Equine Fertility Unit
experimental herd of horses and ponies maintained and managed at Mertoun
Paddocks by Professor WR (Twink) Allen. I am grateful to the many staff members
and students who assisted with the daily examinations of the mares and who helped to
collect blood and tissue samples. Worthy of special mention in this regard are the
Clinical Veterinary Researchers, Dr Lee Morris BVSc DVSc DipACT MRCVS, Miss
Anne Cecilè Lefranc DVM MRCVS, Miss Michaela Kölling VetMed MRCVS and Miss
Amber Clutton-Brock MA VetMB MRCVS.
A debt of gratitude is owed to the Thoroughbred studfarms situated in the
Newmarket area that kindly provided placentae for my studies from their foaling
mares. Likewise, to the many Thoroughbred studfarms and training yards throughout
England that prospectively recorded data for the two large epidemiological studies on
breeding efficiency and racing wastage. The two specialist equine veterinary practices
in Newmarket, Greenwood, Ellis and Partners, and Rossdales and Partners, also get
my wholehearted thanks for their marvellous cooperation with the collection of tissue
and data.
III
I am also grateful to Mrs Sue Gower, expert histology technician, for her help
and advice on all matters histological. She cheerfully processed the mounting backlog
of tissue samples when she arrived at the Equine Fertility Unit with far more skill and
efficiency than I could ever have achieved.
My co-authors on published papers have not only given me scientific food-
for-thought, but also provided the necessary expertise and advice to ensure that
submitted manuscripts were scientifically sound. In this respect, I thank, in particular,
Professor Abbey Fowden of the Physiological Laboratory of Cambridge University.
In addition, numerous scientific colleagues have guided, inspired, encouraged and
helped me at every stage of my career. To name but a few, Dr Lee Morris, Dr Bob
Moor FRS, Professor Ron Hunter, Dr Lulu Skidmore, Dr Jenny Ousey, Dr Tahera
Ansari and Professor Paul Sibbons.
From my first tentative investigations into the possibility of undertaking a
PhD by Publication at my old alma mata, to the final act of submission, my
supervisor, Professor David Rawson, and Professor Tiantian Zhang, have been
extremely encouraging and most helpful. They have made the whole undertaking
relatively stress free and I thank them most sincerely.
Throughout the course of my employment at the Equine Fertility Unit my
family have been a constant source of support and love. They have always allowed
encouraged me to follow my own path in life and I hope these scientific endeavours
in later years give them some pride and pleasure.
I owe the biggest debt of gratitude to Professor Twink Allen who, from the
day of my arrival at the Equine Fertility Unit in 1998, has guided and nurtured me
IV
scientifically and socially. He says things other people only dare to think, he refuses
to bow to bureaucratic authority and has a gusto for science and life that is infectious.
He always encouraged scientific discussion and never discouraged questions, no
matter how stupid, and he would unhesitatingly allocate research animals and
equipment to me whenever I came up with a worthwhile experiment. He continues to
inspire me and I am truly grateful to him for having made my research into the
science of equine reproduction such good fun.
______________
V
Abstract
The papers put forward by the candidate represent a significant contribution to
three main areas within the body of knowledge of equine reproduction. Namely, i)
epidemiological surveys of the efficiency of Thoroughbred racing and breeding, ii)
the morphology and functions of the equine placenta and, iii) embryo transfer in the
horse.
Two extensive surveys on reproductive efficiency of Thoroughbred mares and
stallions at stud and factors associated with the failure of Thoroughbred horses to
train and race demonstrated that increasing mare age is the greatest limiting factor to
an otherwise high rate of fertility in English Thoroughbreds although a high incidence
of early embryonic death remains a significant loss to the breeding industry. The
racing wastage survey showed little change over the past 20 years in the percentage
of 2- and 3-year-old horses that fail to run, the percentage that are never placed in a
race and the number that suffer significant injury or illness during their racing
careers. Radical and innovative changes to training methods are needed to overcome
these problems.
The morphology of the equine placenta was examined using gross
measurements, stereological-techniques, vascular casting and immunohistochemistry
and the findings related to fetal development and postnatal growth. Stereological
measurements applied to term placentae established reference parameters such as
surface area per unit volume of placental microcotyledons, the total microscopic area
of contact between mother and fetus at the placental interface, and placental
VI
efficiency. Maternal age, parity, size, genotype and nutrition were all shown to alter
placental morphology and, hence, pre- and postnatal fetal development.
A novel pair of cervical forceps were designed and marketed to provide a
simple and practical method for undertaking transcervical embryo transfer in the
horse which enables inexperienced operators to transfer horse embryos successfully.
These Wilsher Equine Embryo Transfer Forceps have won widespread acclaim and
commercial application in the equine veterinary and scientific communities. A
pharmacological method to extend donor-recipient synchrony was developed with
both commercial and scientific application. Further work also showed the unique
ability of the equine embryo to tolerate a very wide window of donor-recipient
asynchrony and it provided a valuable research tool with which to study the relevant
roles of the conceptus and uterine environment in regulating embryonic
differentiation and fetal growth in the mare.
CONTENTS
Preface ……………………………………………………………………………. I
Abstract …………………………………………………………………………... II
Acknowledgements ………………………………………………………………. V
CHAPTER 1
Epidemiological surveys of the efficiency of Thoroughbred racing and breeding
1.1 Introduction ……………………………………………………………… 2
1.2 Reproductive efficiency in the Thoroughbred ………………………… 6
1.3 Factors affecting racing wastage ……………………………………….. 14
1.4 Summary of work and findings by the candidate ……………………... 19
CHAPTER 2
The morphology and functions of the equine placenta
2.1 Introduction …………………………………………………………….. 22
2.1.1 Development of the equine placenta ………………………………… 22
2.1.2 The implications of deficiencies in placental morphology ………….. 24
2.1.3 Stereology …………………………………………………………… 24
2.2 Gross and stereological assessment of the equine placenta ………….. 25
2.2.1 The influence of maternal size and genotype on placental
development ………………………………………………………….. 26
2.2.2 The influence of maternal age and parity on placental development … 29
2.2.3 The influence of maternal nutritional on placental development …….. 33
2.3 Placental efficiency ……………………………………………………… 37
2.4 Vascularity of the placenta ……………………………………………... 39
2.5 Placental influences on the endocrinology of pregnancy ……………… 43
2.6 The influence of placental morphology on post-natal development
of the foal ………………………………………………………………… 45
2.7 Summary of work and findings by the candidate …………………….. 49
CHAPTER 3
Embryo transfer in the horse
3.1 Introduction ……………………………………………………………… 54
3.2 The historic development of equine embryo transfer techniques ……. 56
3.2.1 Development of a new transcervical embryo transfer method ……….. 60
3.3 Donor-recipient synchrony ……………………………………………… 64
3.3.1 The importance of donor-recipient synchrony ……………………….. 64
3.3.2 Historic attempts to extend donor-recipient asynchrony …………….. 66
3.3.3 The use of meclofenamic acid to extend donor-recipient asynchrony .. 67
3.3.4 Transfer of day 10 embryos to asynchronous recipient mares ……….. 70
3.4 Summary of work and findings by the candidate …………………….. 74
References ………………………………………………………………………. 77
Appendix 1: The candidate’s publications to be considered for the degree of PhD by
Publication that relate to; Chapter 1) epidemiological surveys of the efficiency of
Thoroughbred racing and breeding; Chapter 2) the morphology and function of the
equine placenta; and Chapter 3) embryo transfer in the horse.
Appendix 2: The contribution of the author to the submitted papers and citation of
these works.
Appendix 3: Full bibliography of the candidate
1
CHAPTER 1
Epidemiological surveys of the efficiency of Thoroughbred racing and breeding
1.1 Introduction …………………………………………………………….. 2
1.2 Reproductive efficiency in the Thoroughbred ……………………….. 6
1.3 Factors affecting wastage in the Thoroughbred racing industry …… 14
1.4 Summary of work and findings by the candidate ……………………. 19
2
1.1 Introduction
The Thoroughbred racing and breeding industries are of considerable
economic importance. For example, in 2004/05 British racing generated some £282
million in tax revenues for the Government and had an overall economic impact of
£2.86 billion (Table 1; Deloitte 2006). Although this same analysis reported that
insufficient data was available to accurately estimate the total revenue of the British
Thoroughbred breeding industry, as an indicator of scale it was estimated that there
were 2,200 studfarms in Britain in 2005, of which around 300 operated on a full-time
basis to produce some 5,500 live Thoroughbred foals annually, the 6th
highest in the
world based on 2003 comparatives. The value of these foals and other Thoroughbred
horses passing through public auctions in the UK in 2007 exceeded £240m (Figure
1). Furthermore, the racing and breeding industries were directly responsible for
18,000 full time jobs and supported a further 69,500 positions (Deloitte 2006).
Thoroughbred mares and stallions are selected for breeding on the bases of
racecourse performance and pedigree, not on fertility. Therefore, physiological and
managerial factors may both contribute to the failure of mares to conceive and/or
carry a pregnancy to term. The mare is a long day seasonal breeder (Osborne 1966)
and a disparity exists between the physiological breeding season (April to October in
the Northern Hemisphere) and the covering season that is arbitrarily imposed for the
purposes of age-related racing (mid-February to mid-July). Mares are mated naturally
‘in-hand’ and artificial insemination is banned worldwide in the Thoroughbred
breeding industry. Mare age (Hutton and Meacham 1968; Laing and Leech 1975;
Jeffcott et al 1982; Sanderson and Allen 1987; Woods et al 1987; Ricketts and
3
Alsonso 1991; Morris and Allen 2002; Allen et al 2007a) and status (i.e. maiden,
barren or lactating; Sanderson and Allen, 1987; Woods et al 1987; Ricketts and
Alonso 1991; Morris and Allen 2002; Allen et al 2007a) have both been shown to
influence the chances of a mare producing a live foal. Combined with these mare
factors, management of the stallion (Sullivan and Pickett 1975) also contributes to the
overall reproductive efficiency of the Thoroughbred breed.
Table 1: Economic impact of British Racing in 2005 (adapted from Deloitte 2006).
Core industry expenditure £ millions
Racecourses 1 298
Levy 2 103
Owners 3
185
Breeding 4 202
Other racing 82
Total core 870
Off-course racing expenditure 180
Secondary expenditure
Business to business 851
Consumer 955
Total secondary 1,806
Total economic impact of British Racing 2,856
Notes: 1 Expenditure of £298m from racegoers, corporate customers and sponsors.
2 Generated from gambling as a statutory 10% levy applied to profits of
bookmakers, the Tote and betting exchanges paid to the Horserace Betting
Levy Board.
3 Owners incurred direct gross expenditure of £274m whilst receiving income
of £89m through prize money and sponsorship. This resulted in a net
expenditure of £185m (excluding horse purchases).
4 The 2,200 British studfarms combined, incurred expenditure totaling
approximately £202m.
4
0
50
100
150
200
250
300
2000 2001 2002 2003 2004 2005 2006 2007 2008
Year
To
tal
sa
les
(G
ns
mil
lon
s)
30
35
40
45
50
Avera
ge s
ale
pri
ce
(Gn
s '000
s)
Total sales
Average sale price
Figure 1: Total sales and average sale price in Guineas* (Gns) of horses sold via
public auction at Tattersalls, Newmarket, UK between 2000 and 2008 inclusive. Data
from Tattersalls (2008). *The traditional unit of money that horses are sold (1 Gn ≈
£1.05).
During the last 20 years the Thoroughbred breeding industry has changed
dramatically. This is well illustrated by two epidemiology surveys, carried out 13
years apart, which examined the reproductive efficiency of intensively managed
mares in and around Newmarket, the principal Thoroughbred breeding area of
England. They showed increases in conception and foaling rates, and decreases in
matings per oestrus, over that time period (Sanderson and Allen 1987; Morris and
Allen 2002). The differences in reproductive efficiency recorded between the two
surveys occurred chiefly as a result of the application and routine use of ultrasound
scanning to monitor follicular growth and ovulation, the occurrence of uterine
pathology and early, accurate diagnosis and monitoring of singleton and twin
5
pregnancy (Palmer and Driancourt 1980; Simpson et al 1982), coupled with the use
of artificial light regimes (Palmer et al 1982) and hormone treatments to induce and
manipulate oestrous cyclicity (see Allen 1992 for review). In addition, both surveys
showed that increasing mare age is the major limiting factor to mare fertility and
fecundity. Other surveys carried out worldwide over the same period reported similar
trends within the Thoroughbred breeding industry (Bruck et al 1993; Morley and
Townsend 1997; Schulman et al 2003; Hemberg et al 2004).
Despite the considerable financial and physical resources expended on
Thoroughbred racing and the relatively poor return on expenditure for racehorse
owners, there have been relatively few studies on the wastage and financial losses
associated with racing. Two related surveys stood out as seminal for British racing.
Namely, those of Jeffcott et al (1982) and Rossdale et al (1985). Although other more
recent surveys have highlighted training problems in Thoroughbreds in other
countries an update on the British racing industry was well overdue.
Thus, two surveys were undertaken at the request of the Veterinary Advisory
Committee of the Horserace Betting Levy Board (HBLB) to provide up-to-date
statistics on the current ‘state-of-play’ in the British Thoroughbred racing and
breeding industries. The first of these focused on reproductive efficiency and reported
on differences between studs breeding Flatrace (Flat) horses versus those breeding for
National Hunt (NH) steeplechase and hurdling racing (Allen et al 2007a). The second
concentrated upon the costs and veterinary and other causes of wastage in the
Flatracing industry (Wilsher et al 2006a). In addition, it was hoped that both surveys
6
would allows comparisons to be drawn between different management regimes,
would identify causative factors where problems were found and would therefore
pinpoint areas for future research effort.
1.2 Reproductive efficiency in the Thoroughbred
The data analysed and discussed by the candidate from the survey of
reproductive efficiency in the Thoroughbred (Allen et al 2007a) was gleaned from
3519 mated oestrous cycles exhibited by 2321 Thoroughbred mares visiting Flatrace
stallions in and around Newmarket and on other studfarms in the south and west of
England, and 1486 mated oestrous cycles exhibited by 1052 Thoroughbred mares
visiting National Hunt (NH) stallions in Leicestershire, Nottinghamshire and
Shropshire, all during the 2002 breeding season. These mares were all mated to one
of 84 Flatrace stallions standing on 24 public studfarms or one of 43 NH stallions on
9 studfarms. The age distribution of the two populations of mares differed
significantly, thereby reflecting the differing ages at which the two types of horses
retire to stud. Accordingly, the Flatrace mares had a median age (IQR = Interquartile
range) of 8 (6 – 12) and the NH mares a median age of 11 (8 – 14) years.
Furthermore, mare status (i.e. maiden, barren or lactating) varied between the two
populations with the NH mares comprising more barren and fewer foaling mares than
the Flatrace cohort.
In the latest survey Allen et al (2007a) reported that NH mares were mated
significantly more times per oestrus than Flatrace mares (1.4 versus 1.1) and
7
consequently the number of matings per day 15 pregnancy was also significantly
higher in NH (2.3) compared to Flatrace (1.7) mares. The number of matings per
oestrus was not affected by mare age although the number of matings per day 15
pregnancy was (Table 2). Overall, Flatrace and National Hunt mares were mated one
or more times in 3519 and 1486 oestrous periods, respectively, to give per cycle early
pregnancy rates of 63.2 and 65.3%, respectively. The figure for Flatrace mares was
similar to the 59.9% reported by Morris and Allen (2002) but considerably higher
than the 53.3% reported by Sanderson and Allen (1987) thereby reflecting the
advances made in veterinary management of Thoroughbreds in the last 20 years. All
three surveys (Sanderson and Allen 1987; Morris and Allen 2002; Allen et al 2007a)
illustrated the deleterious effects of increasing mare age on the per cycle early
pregnancy rate (Table 2).
Table 2: Effects of mare age on pregnancy loss rates, the number of matings per
oestrus and per day 15 pregnancy in 3373 Thoroughbred mares (adapted from Allen
et al 2007a).
a, b Values in the same row with different superscripts differ significantly. χ
2 test,
Bonferroni p <0.001.
Mare age group 3 – 8yrs 9 – 13yrs 14 – 18yrs >18yrs
No. mares 1546 1108 573 146
No. oestrous cycles 2245 1633 892 235
No. matings per oestrus 1.2 1.2 1.2 1.2
No. matings per day 15 pregnancy 1.7a 1.9
b 2.1
b 2.2
b
Per cycle conception rate (%) 67.3a 63.4
a 58.3
b 54.9
b
% pregnancies lost days 15 – 42 5.9a 7.1
a 10.0
b 18.6
b
% pregnancies lost day 42 – Oct 1st 2.9
a 5.0
b 6.5
b 7.8
b
% pregnancies lost Oct 1st – foaling 1.8 3.1 3.7 2.3
Overall % pregnancies lost 10.6a 15.2
b 20.2
c 27.9
c
8
Regardless of subdivision on the bases of age or status, the more frequently
mated NH mares consistently had a slightly higher conception rate per cycle than the
Flatrace mares suggesting that the present day doctrine of ‘one mating per oestrus’,
which is driven by both the large number of mares booked to popular stallions and
the fear of mating-induced endometritis (Ashbury et al 1980; Katila 1995; Troedsson
et al 2000), may need re-examining, especially in view of the finding that pregnancy
rates in both groups of mares increased as the frequency of mating per oestrus
increased (Table 3; Allen et al 2007a).
Table 3: Effects of the number of matings per oestrus on per cycle conception rates,
multiple pregnancy rate and early pregnancy loss rate in 5005 oestrous cycles in
which mares were mated. From Allen et al 2007a.
Number of
matings/oestrus
Number of
oestrous
cycles
(% of total)
Pregnant at
day 15 (%)
Multiple
pregnancies
(%)
Pregnancy loss
rate days 15-42
(%)
1 4278 (85.5) 62.6a 11.1 7.4
2 581 (11.6) 70.6b 13.4 8.8
3 109 (2.2) 69.7ab
3.9 3.9
>4 31 (<1.0) 83.8b 12.9 0.0
a,b Values in the same column with different superscripts differ significantly. χ
2 test,
Bonferroni p <0.001.
The data from the study revealed that 14.3% of pregnancies were lost pre-
partum and, as previously reported, the highest proportion of these losses occurred
between days 15 and 42 after ovulation (Figure 2; Merkt and Gunzel 1979; Bruck et
al 1993; Morris and Allen 2002). Early pregnancy loss rates increased with
increasing mare age (Table 2). Morris and Allen (2002) similarly reported increases
in early pregnancy loss rate (15 – 35 days of gestation) with mare age, from 4.7% in 3
9
– 8 year old mares, 13.8% in 9 – 13 year old mares to 22.9% in mares > 18 years of
age. The same age-related trend was observed for fetal loss rates throughout the
whole of pregnancy, rising from 11.8% in the 3 – 8 year old mares to 31.4% in mares
>18 years of age. Similar figures showing a pronounced influence of mare age on
pregnancy loss rate have been reported worldwide by Bruck et al (1993), Morley and
Townsend (1997), Schulman et al (2003) and Hemberg et al (2004). They are, no
doubt, a reflection of age-related degenerative changes in the endometrium reducing
the latter’s nutritive capacity for the developing conceptus and its ability to establish
adequate placentation (Bracher et al 1996).
0
2
4
6
8
10
12
Days 15 - 42 Day 42 - October October - Term
% p
reg
na
nc
ies
lo
st
Figure 2: The percentage of diagnosed pregnancies lost during various periods of
gestation in 3373 Thoroughbred mares mated in the 2002 breeding season (adapted
from Allen et al 2007a).
The use of intrauterine therapy in the 2002 breeding season had risen
dramatically from that reported for the 1998 breeding season by Morris and Allen
(2002), even though these authors had demonstrated a lack of correlation between
10
uterine therapy and conception rate. As noted previously, the more frequent use of
intrauterine antibiotics in the NH mares may have contributed, in part, to the
increased conception rates per cycle in this group, despite their higher mean age
(Allen et al 2007a). However, since it was not possible to determine if the mares
treated with intrauterine antibiotics had shown clinical signs of bacterial endometritis
prior to treatment, it is difficult to judge the efficacy of such therapy. The initial
advantage exhibited by the NH mares in terms of their increased conception rate per
cycle was not sustained during gestation as pregnancy loss rates at all 3 stages of
gestation (days 15 – 42, day 43 – Oct, Oct – foaling) were higher in the NH mares
than the Flatrace mares, presumably as a consequence of their higher mean age
(Allen et al 2007a).
Other common veterinary treatments, for example, the use of prostaglandin
and other hormones to induce oestrus, also increased in the period between the two
surveys. Morris and Allen (2002) reported that 27.8% of mated oestrous cycles had
been induced by hormone therapy, whereas the equivalent figure reported by Allen et
al (2007a) was 39.6%. Such increases in veterinary treatments to give more intensive
management of the mares’ oestrous cycles no doubt reflected the increasing pressure
placed on stud veterinary clinicians to ensure that mares under their control are ready
to be mated within the tight time windows offered to them by stallion managers, in
turn, reflecting the increasing workload of popular stallions.
The use of ovulation inducing hormones appeared to offer a small decrease in
the rate of early embryonic death in treated mares (Table 4). One such ovulatory
drug, the GnRH analogue, buserelin (Receptal, Intervet UK Ltd, Milton Keynes,
11
Bucks, UK) has been shown to increase pregnancy rates in dairy cattle when given as
a single intramuscular (i.m) injection on day 11 or 12 after insemination (Sheldon and
Dobson 1993; Drew and Peters 1994), and similarly, in mares, when given as a single
i.m injection between days 8 and 12 after ovulation (Newcombe et al 2001). It is
thought that the treatment may suppress follicular development and the consequential
rise in serum oestrogen concentration that can occur during early pregnancy, thereby
dampening the luteolytic drive mechanisms and promoting maternal recognition of
pregnancy (Drew and Peters 1994). In view of the findings that early embryonic loss
(i.e. days 15 – 42) represents by far the largest percentage of pre partum losses in the
mare, (Figure 2; Sanderson and Allen 1987; Morris and Allen 2002; Schulman et al
2003; Hemberg et al 2004; Allen et al 2007a), this apparently beneficial effect of
hormonal induction of ovulation during oestrus warrants further investigation
.
Table 4: Effects of hormonal induction of ovulation on reproductive parameters in
3373 Thoroughbred mares surveyed (adapted from Allen et al 2007a).
Hormone-induced
ovulation
Spontaneous
ovulation
No. of oestrous cycles (% of total) 2957 (59.1) 2048 (40.9)
No. of matings (% of total) 3290 (55.4) 2648 (44.6)
No. day 15 pregnancies (% of total) 1886 (59.0) 1310 (41.0)
No. matings per oestrus 1.1a 1.3
b
% pregnancies lost days 15-42 6.6a 8.7
b
a,b Values in the same row with different superscripts differ significantly, χ
2 test
Bonferroni p<0.005.
The results reported by Allen et al 2007a also highlighted the need for more
investigations into Thoroughbred stallion fertility, especially in view of the lack of
selection of stallions for their fertility potential when they go to stand at stud;
12
selection is based entirely on performance on the racecourse and pedigree. The
inherent biological variation in Thoroughbred stallion fertility is illustrated by the
tremendous variation in per cycle conception rate in mares covered by both Flatrace
(79 – 36%; Figure 3) and National Hunt (78 – 55%; Figure 4) stallions standing at
public studs (Allen et al 2007a).
0
10
20
30
40
50
60
70
80
90
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36
Flathorse stallion identification
Perc
enta
ge
Figure 3: Overall per cycle pregnancy rate (■), multiple pregnancy rate as a
percentage of conceptions (■) and early pregnancy loss rate per cycle (□) recorded in
mares mated by one of 36 Flatrace stallions that mated ≥30 mares (adapted from
Allen et al 2007a).
It has been noted in both cattle (López-Gatius et al 2002; Pegorer et al 2007),
and goats (Engeland et al 1997), that certain sires can influence embryonic loss rates.
Morris and Allen (2002) reported that 8 of 41 Thoroughbred stallions mated to 1144
mares were associated with increased rates of fetal loss above the norm in the mares
they covered. In these 8 horses, the mean number of mated oestrous cycles that were
13
followed by pregnancy loss before the October pregnancy test was 26%, while the
equivalent figure for the 33 remaining stallions was 6.8%. These authors postulated
that chromosomal incompatibilities between maternal and paternal genotypes
resulting from such factors as homozygous lethal genes and high inbreeding
coefficients might be responsible for the high rates of early fetal death associated with
these stallions. Certainly, in sheep, embryos obtained from relatively inbred lines of
ewes show a higher mortality after transfer than embryos from non-inbred lines
(Martal et al 1997).
0
10
20
30
40
50
60
70
80
90
100
1 2 3 4 5 6 7 8 9 10 11 12 13
National Hunt stallion identification
Pe
rce
nta
ge
Figure 4: Overall per cycle pregnancy rate (■), multiple pregnancy rate as a
percentage of conceptions (■) and early pregnancy loss rate per cycle (□) recorded in
mares mated by one of 13 National Hunt stallions that mated ≥30 mares (adapted
from Allen et al 2007a).
Similarly, in the Allen et al (2007a) study, early pregnancy loss rates in the
mares mated to the majority of the 49 Flatrace and NH stallions that covered 30 or
14
more mares ranged from 0 to 12%. However, 10 stallions were associated with higher
than normal early pregnancy loss rates of 13 – 23% although early embryonic loss
rate was not related to the per cycle conception rate or the multiple pregnancy rate
(Figure 3 & 4). Stallion age was also not related to pregnancy loss rate in the mares
covered. Clearly, further work is needed to determine if these marked differences in
early pregnancy loss rates are truly related to stallion genotype and are not merely a
reflection on differences in the age, status and reproductive health of the mares
covered by individual stallions. Collation of similar data from consecutive breeding
seasons would help greatly to determine more accurately the precise role of the
stallion in cases of high early pregnancy loss rate.
Whatever the causes, a relatively high incidence of early pregnancy failure
still represents a major loss to the Thoroughbred breeding industry so therefore
remains a high priority area for future research effort.
1.3 Factors affecting wastage in the Thoroughbred racing industry
The ‘sister’ racing survey (Wilsher et al 2006a) followed a cohort of 1022
foals, identified in the previous stud efficiency survey of Morris and Allen (2002),
from birth through to the end of their 3rd
year, with the aim of identifying time points
and causes of losses during their growth and training phases. In addition, comparisons
were drawn between this and the last authorative racing wastage survey conducted in
Britain some 20 years previously by Jeffcott et al (1982).
The 1022 Thoroughbred foals born in 1999 were surveyed at the beginning of
their 2-year-old year to determine their status (Table 5). This revealed a fall in the
15
proportion of youngstock that entered training in the UK, from the 62% reported by
Jeffcott et al (1982) to 52% in the present survey; the fall could be attributed to a
marked increase in the number of horses exported during the interval, rising from
11% in 1975 (Jeffcott et al 1982) to 37% in 1999. This, in turn, reflected the rapid
development of racing in other countries, such as the Middle East and Turkey, and
the continuing high demand for British-bred Thoroughbreds.
Horses that died or were destroyed by 4 years of age made up 8% of the total
cohort surveyed and the majority of these deaths (73%) occurred before 2 years of
age. This overall mortality rate was double the figure of 3.5% recorded by Jeffcott et
al (1982) for Thoroughbreds up to 4 years of age, but seemed relatively modest
compared to the 11% loss rate reported by Morley and Townsend (1997) for
Thoroughbred foals in Western Canada during the first year post partum.
Table 5: The status of 1022 Thoroughbred foals born in 1999 at the beginning of their
2-year-old year in 2001, as determined by a questionnaire sent to their owners (from
Wilsher et al 2006a).
No. %
Born live in 1999 1022 -
Entered training as a 2-year old 537 52
Known to have been exported 289 28
Died or destroyed (aged 0 – 2 years) 58 6
‘In store’ for NH racing 60 6
Waiting to race as a 3-year-old 25 2
Not intended for racing 17 2
Untraceable 36 4
Wilsher et al (2006a) reported that only 32% of the 1022 foals surveyed
started in a race at 2 years of age. This high level of non-performance at 2 years was
16
similar to the 30% of 2-year-old non-runners reported by Bourke (1995) in Australia
and it highlighted the low chance of an owner recuperating his or her training costs
during a horse’s 2-year-old career. For example, when setting a modest arbitrary
figure of £10,000 per annum for training costs, only 5% of 2-year-olds in training in
the Wilsher et al (2006a) racing survey were able to cover their basic costs of
training in prize money. This figure compares unfavourably with other racing
countries like Australia where More (1999) reported that 13% of 1567 two-year-olds
surveyed in Queensland earned their training costs in prize money. However, the 2-
year-olds in Queensland had a mean of 4.2 starts (More 1999) compared to the 3.2
starts for Thoroughbreds in the UK (Wilsher et al 2006a).
The percentage of 3-year-old horses in training that won (30%) or were placed
(48%) in a race in 2001 (Wilsher et al 2006a) was identical to that reported by
Jeffcott et al (1985) for 3- to 6-year old horses in 1980. In contrast, horses that raced
as 3-year-olds in Queensland in 1997 had a 54% chance of winning a race if they ran
(More 1999), appreciably more than the 40% chance for runners in Britain. As with
the 2-year-olds, horses in training at 3 years of age in the UK ran fewer times than
their counterparts in Queensland (5.3 versus 7.7 starts) and only 17% recouped their
training costs in prize money.
The Wilsher et al (2006a) survey also dealt with veterinary problems
experienced by horses in training as both 2- and 3-year-olds (Table 6). The most
common of these ailments in 2-year-olds was dorsometacarpal disease (‘sore shins’)
which affected no fewer than 29% of the cohort. The same condition was also rated
the most frequently occurring problem in 2-year-olds in Australia, with Sydney
17
trainers recording 60% of ‘lost training days’ resulting from the condition (Bailey et
al 1997). Differences in training strategies (Jeffcott et al 1982; Boston and
Nunamaker 2000; Verheyen et al 2005) and track conditions (Dyson 1987) have been
reported to influence the occurrence of sore shins in 2-year-olds and there is clearly a
case for innovative changes to be made in the training regimes of these youngsters.
Table 6: Incidence of specific ailments suffered by 514* two-year-olds during
training in 2001 and 424* three-year-olds during 2002 (adapted from Wilsher et al
2006a)
2-year-olds 3-year-olds
Ailment No.
affected
% of
514
No.
affected
% of
424
Dorsometacarpal disease (sore shins) 150 29 53 12
Inflammatory airway disease (IAD) 68 13 35 8
Joint problems 57 11 55 13
Fractures 52 10 40 9
Rhabdomyolysis (tying-up) 27 5 17 4
Knee chips 17 3 17 3
Tendon problems 12 2 16 4
Soft palate problem 8 2 13 3
Colic 8 2 3 <1
Laryngeal hemiplagia (roaring) 6 1 7 2
Other 68 13 49 12
*For 537 two-year olds, 514 (96%) completed forms were returned; 195 (38%) had
no veterinary problem, 319 (62%) had one or more veterinary problem. For 456
three-year-olds, 424 (93%) completed forms were returned; 213 (50%) had no
veterinary problems, 211 (50%) had one or more veterinary problems.
Although a high incidence of inflammatory airway disease (IAD) was present
in 2-year-olds, this had reduced by 3 years of age. The stresses experienced by a 2-
year-old entering the new environment of the training yard, coupled with exposure to
new pathogens from the other resident horses, no doubt, underlay the higher
incidence of IAD in the younger animals. Wood et al (2005) similarly noted a
18
reduction in the incidence in older horses in training, again suggesting infection is the
likely cause of the problem. However, the percentage of both 2- and 3-year-olds
suffering IAD may well have been higher than the figures reported by Wilsher et al
(2006a) as poor performance has been associated with asymptomatic IAD in NH
horses (Allen et al 2006).
A number of the commonly occurring veterinary problems identified in the
survey were influenced by gender. For example, colts and geldings were more injury
prone than fillies as 3-year-olds, especially with problems like sore shins, fractures,
joint inflammation and tendon strain. A similar conclusion was reached by
Kasashima et al (2004) who noted that entire colts were at greater risk of developing
tendonitis and suspensory ligament desmitis than geldings or fillies. Gender, and
hence by implication cyclic hormonal fluctuations, have been proposed as the cause
of the significant increase in the incidence of rhabdomyolysis (‘tying-up’) in fillies
versus colts (MacLeay et al 1999; McGowan et al 2002; Wilsher et al 2006a).
A genetic factor was suggested as being associated with laryngeal hemiplagia
(roaring) as 5 of the 6 two-year-olds affected by this disorder had been sired by the
same stallion. Although information of this type would be of considerable interest to
mare owners, the breeding industry as a whole must decide if such findings should be
divulged widely.
Half (148/295) of the fillies surveyed retired to stud at the end of their racing
career. Of these, 116 had been in training as both 2- and 3-year olds, while the
remaining 32 had retired to stud as 2-year-olds. In contrast to colts retiring to stud,
where very good racecourse performance is essential to achieve stallion status, 72%
19
of fillies that retired to stud had failed to win a race while in training and 38% of
them did not even appear on a racecourse. A comparison of fillies that retired to stud
versus those that did not revealed no significant difference in the number of times
they had run, the number of races won, the amount of prize money won or their
Timeform rating. However, the nomination fees of the sires of the fillies that retired
to stud were significantly higher than for those that did not (median £12,500 vs.
£7000). When considering the costs of breeding and racing a filly, it is perhaps
understandable that a disappointed owner should try and recoup some of that wasted
expenditure at stud. However, more discriminating selection of breeding fillies on the
bases of racecourse performance and soundness, rather than an expensive pedigree,
would undoubtedly produce progeny more likely to be able to survive the rigours of
training.
In the two decades that elapsed between the present survey (Wilsher et al
2006a) and the earlier one by Jeffcott et al (1982), the levels of wastage within
British flatracing, in terms of the proportions of horses in training that fail to reach
the racecourse, win a race or win any prize money at all, have remained completely
unchanged. Thus, it is clear that, if the racing industry is to reduce these high wastage
rates in the future, some novel and radical changes will have to be implemented.
1.4 Summary of work and findings by the candidate
In collaboration with others, the candidate undertook two extensive surveys;
one on reproductive efficiency in Thoroughbreds at stud (Allen et al 2007a) and
another on factors associated with the failure of Thoroughbred horses to train and
20
race (Wilsher et al 2006a). Increasing mare age was shown to be the biggest limiting
factor to an otherwise high rate of fertility on well-managed English Thoroughbred
studfarms, with the persistence of a high incidence of early pregnancy failure worthy
of further study. Comparing the results from this latest reproductive efficiency survey
(Allen et al 2007a) with those of the previous survey (Morris and Allen 2002)
showed few changes. In the Allen et al (2007a) study, minor differences between the
reproductive efficiency were highlighted between the Flat and NH mares but these
were largely accountable for by the higher mean age of the mares in the NH group.
The racing wastage survey confirmed previous reports of high non-run and
non-placed rates, a high incidence of injury and general cost-ineffectiveness of 2-
year-olds in flat racing. Both surveys pinpointed problem areas and thereby laid the
groundwork for further studies.
The results of both surveys have been presented by the candidate at national
and international scientific and veterinary conferences and at lay meetings of horse
breeders. The interest generated by the racing wastage survey within the horse-racing
industry led to it being highlighted in the racing press (Pagones, Racing Post, 2005),
and invitations for the candidate to present this work both in the UK (Thoroughbred
Racing and Breeding Seminar, Cheltenham, UK) and abroad (Ghent Veterinary
School, Belgium and 5th
International Conference on Equine Reproductive Medicine,
Leipzig, Germany). Wilsher et al (2006a) has, to date, been cited by 6 peer-
reviewed papers in veterinary journals, and Allen et al (2007a) by 5.Both papers are
likely to remain the seminal reference works on these topics for some years to come.
__________
21
CHAPTER 2
The morphology and functions of the equine placenta
2.1 Introduction ………………………………………………………….… 22
2.1.1 Development of the equine placenta ………………………………... 22
2.1.2 The implications of deficiencies in placental morphology ……......... 24
2.1.3 Stereology …………………………………………………………… 24
2.2 Gross and stereological assessment of the equine placenta …………. 25
2.2.1 The influence of maternal size and genotype on placental
development ………………………………………………………… 26
2.2.2 The influence of maternal age and parity on placental development .. 29
2.2.3 The influence of maternal nutritional on placental development …… 33
2.3 Placental efficiency …………………………………………………….. 37
2.4 Vascularity of the placenta ……………………………………………. 39
2.5 Placental influences on the endocrinology of pregnancy …………….. 43
2.6 The influence of placental morphology on post-natal development
of the foal ………………………………………………………………… 45
2.7 Summary of work and findings by the candidate …………………….. 49
22
2. 1 Introduction
Despite increased understanding of placental structure and its influence on
fetal growth and development in other species, there is a paucity of such data for the
horse. This is surprising when considering that pre- and post-natal foal deaths
represent a considerable loss to the equine breeding industry (Whitwell 1980;
Sanderson and Allen 1987; Morley and Townsend 1997; Morris and Allen 2002;
Allen et al 2007a). Furthermore, the potential implications of placental deficiency
can be even more disadvantageous in a species which is bred primarily for its athletic
performance (Platt 1978; Jeffcott et al 1982; Rossdale et al 1985; Morley and
Townsend 1997; Rossdale and Ousey 1998).
2.1.1 Development of the equine placenta
The equine placenta is classified as diffuse, non-invasive and epitheliochorial
in structure (Grosser 1909; Amoroso 1952; Mossman 1987). Many features of its
development are unique to the genus Equus and are of considerable academic interest
and practical significance. Until day 40 after ovulation in the mare, the non-invasive
trophoblast of the allantochorion lies in simple apposition to the lumenal epithelium
of the endometrium (Ewart 1897; van Niekerk and Allen 1975) and the conceptus
survives and grows by imbibing the exocrine secretions of the endometrial glands,
called histotroph or ‘uterine milk’ (Amoroso 1952). Beyond day 40, the outermost
trophoblast layer of the allantochorion establishes a microvillous attachment to the
lumenal epithelium of the endometrium and true allantochorionic placentation
23
commences (Samuel et al 1974; 1975). Blunt primary villi of allantochorion
interdigitate with corresponding upgrowths of endometrium (sulci) and, as gestation
advances, several adjacent primary villi may coalesce to form one branched and
complex microcotyledon with a common stem (Samuel et al 1974), or a single
primary villus may become multibranched to achieve the same result (Amoroso 1952:
Leiser et al 1998). Continued secondary and tertiary branching of the chorionic villi
and their accommodating endometrial sulci leads to the increasingly complex
structure of the mature microcotyledon, the architecture of which maximises the
surface area contact between maternal and fetal epithelia (Figure 1). Development is
essentially complete by mid gestation when each microcotyledon exhibits a diameter
of 1 – 2 mm (Samuel et al 1974; 1975). Nevertheless, further remodelling of the
microcotyledons continues right up until term, with a progressive lengthening and
sub-branching of the villi in the last two months of gestation (Macdonald et al 2000).
Figure 1: On the left, the branched structure of the developing fetal microcoytledons
which interdigitate with corresponding crypts in the maternal endometrium, shown on
the right. (Image on left courtesy of Verena Bracher; image on right author’s
unpublished scanning electron micrograph).
24
2.1.2 The implications of deficiencies in placental structure and function
Any deficiencies in placental structure and function may be reflected in
corresponding deficits of fetal growth and maturity leading, in very severe
disturbances, to fetal death and abortion (Whitwell 1980). Furthermore, long-term
adverse changes in the physiology and metabolism of the foal may result from an
inappropriate intrauterine existence, which can lead to sub-optimal performance in
later life. In domestic livestock species it has been demonstrated that low birthweight
has negative effects on postnatal growth and productivity (Bell 1992), while in
humans, analysis of epidemiological studies has shown a strong association between
low birthweight and the development of a number of metabolic diseases in later life
(Barker 1995; Harding 2001; Barker et al 2002; Barker 2006). Studies in the sheep
(Alexander 1964; Emmanoulides et al 1968; Alexander and Williams 1971; Robinson
et al 1979; Mellor 1983; Bell et al 1987; Wallace et al 1996) have provided the basis
for our current understanding of the origins and consequences of intrauterine growth
restriction (IUGR).
2.1.3 Stereology
The word stereology is taken from the Greek for solid, stereos, and the term
came into the scientific vernacular in the 1960’s when it was adopted by biologists,
geologists and material scientists to describe a discipline that focused on the
quantification of three-dimensional (3-D) objects from their appearance in two-
dimensional (2-D) sections (Mouton 2002). Hence, in biology, unbiased stereology
can be used to describe the 3-D structural composition and spatial arrangement of
25
biological specimens from 2-D thin sections. By undertaking such measurements on
microscopic sections, stereology facilitates the interpretation of normal and perturbed
structure from whole organs.
Researchers working on aspects of human placental function have used a
stereological approach to describe and interpret the function of the placenta, from the
whole organ to the molecular level. Examples include diffusive transport, villous
growth, fetoplacental angiogenesis, trophoblast turnover, arterial vascular
remodelling and high-resolution immuno-localization experiments (see Mayhew
2006 for review). Such studies have shed light on how the human placenta grows and
develops its form (Mayhew 1997) and they have given an improved understanding of
placental function in both normal pregnancies and those complicated by external
factors such as high altitude, maternal diabetes mellitus, pre-eclampsia and maternal
smoking (Mayhew and Burton 1997; Dockery et al 2000).
2.2 Gross and stereological assessment of the equine placenta
In the horse, gross placental morphology at term has been described in both
normal pregnancies (Whitwell and Jeffcott 1975; Cotrill et al 1991; Rossdale and
Ricketts 2002) and compromised pregnancies (Whitwell 1980; Whitwell 1987;
Cotrill et al 1991; Ball et al 1993). However, until relatively recently, no reports
existed that quantified the 3-D morphology of the placenta. Preliminary studies using
stereology were performed by Gerstenberg (1998) on samples of allantochorion
recovered from 70-day pregnancies in 1- and 2-year-old maiden fillies, and in
extraspecific donkey-in-horse pregnancies created by embryo transfer. However, this
26
initial experiment did not include term placentae. To address this shortfall the
candidate used stereological techniques to measure the surface density (surface area
per unit volume; Sv) of the microcotyledons (Figure 2) on the allantochorion and, by
multiplication of this value by the volume of the chorion, calculated the total
microscopic area of feto-maternal contact (Ta) at the placental interface (Allen et al
2002a; Wilsher and Allen 2002; 2003). Thus, reference stereological values were
established for the equine placenta at term which could be used in further studies to
identify specific factors that might modify placental development and function.
Figure 2: Diagrammatic representation of surface density (Sv) of the microcotyledons
on the equine placenta. Sv is the surface area of the microcotyledons within a unit
reference volume (represented by the white cube). Hence, Sv = units2/units
3 = units
-1.
From Wilsher and Allen (2003).
2.2.1 The influence of maternal size and genotype on placental development
The gross area of the allantochorion, and the macroscopic and microscopic
structure of this placental layer, have been positively correlated with foal birthweight
27
(Rossdale 1966; Cotrill et al 1991; Bracher et al 1996). Maternal size, and hence
uterine size, also profoundly affects the birthweight of the foal, as demonstrated
originally by Walton and Hammond (1938) in their classical between-breed crossing
of small Shetland ponies with large Shire horses. Tischner and Klimszak (1989)
observed a similar effect when they transferred Pony embryos into the uteri of larger
draft-type recipient mares and compared birth size and subsequent development of
the resulting foals with sex-matched full siblings born from the genetic Pony
mothers. Although both the previous experiments clearly demonstrated that uterine
size profoundly influenced pre-and post-natal growth of the foal, neither study
investigated placental parameters in any detail, or any adaptive changes that the
placental microcotyledons may have undergone.
Figure 3: The reciprocal embryo transfer experiment using large Thoroughbreds and
smaller Ponies to create models for intrauterine growth restriction (IUGR; restricted)
and intrauterine growth enhancement (IUGE; luxurious). From Allen et al (2002a).
28
To address these shortfalls, a reciprocal embryo transfer model (Allen et al
2002a; 2002b; 2004) was created by transferring small Pony embryos to the uteri of
large Thoroughbred mares and visa versa (Figure 3). Accordingly, Thoroughbred-in-
Pony (Tb-in-P) pregnancies, in which the genetically larger Thoroughbred fetus
endured cramping and nutritional deprivation in utero (an intrauterine growth
restriction model; IUGR), and reciprocal Pony-in-Thoroughbred (P-in-Tb)
pregnancies in which the smaller Pony fetus was exposed to nutritional and spatial
excesses in utero (an intrauterine growth enhancement model; IUGE), were
established. Control Thoroughbred-in-Thoroughbred (Tb-in-Tb) and Pony-in-Pony
(P-in-P) pregnancies were created using artificial insemination. The mass, gross area
and volume of the allantochorion were all significantly greater in the Tb-in-Tb than
P-in-P pregnancies, whereas intermediate values for these parameters were obtained
for the experimental P-in-Tb and Tb-in-P groups. Analysis of the data showed that
both maternal and fetal genotypes played significant roles in determining these
parameters. For example, stereological assessment of the surface area per unit
volume (Sv) of the microcotyledons showed that Thoroughbred mares exhibited a
significantly higher mean Sv than Pony mares, regardless of the genotype of the fetus
they were carrying (Figure 4). Hence, it could be concluded that the plexiform
structure of the microcotyledons is under maternal control. However, in the
‘deprived’ Tb-in-P pregnancies, the microcotyledonary villi were longer than those in
the control P-in-P pregnancies although the Sv remained unchanged; this had
presumably occurred as a compensatory measure to increase the area of fetomaternal
contact. But despite this attempt to redress the shortfall, a reduction in the total area
29
of fetomaternal contact across the placental interface was still apparent; namely, 42.0
(± 4.4) m2 in the Tb-in-Tb control pregnancies falling to 29.4 (± 2.6) m
2 in the Tb-in-
P pregnancies. This was reflected in the significantly lower birth weight of the Tb-in-
P foals compared to their Tb-in-Tb controls.
This study demonstrated that, in equids, maternal size and genotype of both
the dam and the fetus can act to restrict or enhance growth of the allantochorion and,
hence, the available area for fetomaternal exchange.
0.005
0.015
0.025
0.035
0.045
Tb-in-Tb P-in-Tb Tb-in-P P-in-P
Me
an
mic
roc
oty
led
on
Sv (m
m-1
)
a
b b
a
Figure 4: The mean microcotyledonary Sv value calculated for the 4 types of
pregnancy, illustrating the influence of maternal genotype on microcotyledon
formation. Different letters indicate significant differences between groups (p< 0.001;
adapted from Allen et al 2002a).
2.2.2 The influence of maternal age and parity on placental development
Age-related degenerative changes in the mare’s endometrium may also limit
placentation by reducing the effective area for fetomaternal exchange, and thereby
30
inducing IUGR in the foal (Kenny 1993). The literature gives few examples of the
relationship between age and placental parameters although Bracher et al (1996)
demonstrated delayed and abnormal development of the microcotyledons, resulting
in reduced weight gain in the fetus, occasioned by fewer and shorter chorionic villi in
those areas of the allantochorion that were apposed to degenerate areas of
endometrium in the aged mares. Since Thoroughbred mares, and also Sporthorse
mares, are often retained in the breeding herd until late in life, knowledge of the
changes in placental structure and function that are influenced by maternal age and
parity would be useful to these sections of the horse breeding industry. Hence, a large
cohort of term placentae were analysed by the candidate using both gross and
stereological examination techniques (Wilsher and Allen 2002; 2003). In addition,
light and scanning electron microscopy, and vascular casting (Figure 5) were
employed to examine development of the microcotyledons throughout gestation in
mares of varying age and parity status (Abd-Elnaeim et al 2006).
Figure 5: On left, a photograph showing a corrosion cast of the chorionic surface of a
mare’s placenta at 309 days of gestation with just the placental vascaulture left in
situ. On right, a scanning electron micrograph of the same placenta showing the
vascaulture within an individual microcotyledon. (Author’s unpublished images.)
31
The candidate’s stereological assessment of the placental microcotyledons of
84 Thoroughbred mares that were separated into 4 groups on the bases of age and
parity, namely, primiparous mares aged ≤ 6 years (n = 24) and multiparous mares
aged 5 – 9 (n = 41), 10 – 15 (n = 10) and ≥ 16 (n = 9) years, showed significantly
lower Sv values in the oldest animals (Figure 6), presumably due to age-related
degenerative changes in the apposing endometrium. However, the maiden
primiparous mares also showed significantly lower Sv values than their
secundiparous and younger multiparous counterparts, despite the healthy, virginal
endometrium in the maiden animals. Similarly, the term placentae of 11 young
Thoroughbred mares followed in their first and second successive pregnancies
showed significantly lower Sv values at the end of their first pregnancy than the
second (Wilsher and Allen 2003). In a similar manner, pregnant fillies aged only 1
or 2 years of age from which the conceptuses were removed surgically on day 70 of
gestation also showed a significant increase in Sv values between the first and second
consecutive pregnancies, despite the confounding factors of immaturity of both the
dams and the placentae (Gerstenberg 1998). It would appear, therefore, that the
equine uterus needs to be ‘primed’ in some way by a first pregnancy for
microcotyledon development to reach its full potential.
During pregnancy in the mare, large quantities of mitogenic growth factors,
such as epidermal growth factor (EGF), are secreted by the endometrial glands
(Stewart et al 1994) and differences in the quantity of such factors produced may
play a significant role in bringing about the observed reduction in microcotyledon Sv
in the older animals. However, although Gerstenberg et al (1999) reported a
32
reduction in EGF mRNA expression in damaged and degenerate glands in older
mares, an ultrastructural study of the secretory endometrium of oestrous mares by
Tunon (1995) failed to show any differences between nulli-, primi-, and
secundiparous animals. It is interesting to speculate that the dramatic remodelling of
the endometrium which occurs during a first pregnancy may not completely resolve
after parturition so that pregnancy results in permanent anatomical changes. Khong et
al (2003) proposed this theory as the basis for the increases in birthweight measured
in human infants born from second and subsequent gestations.
0.005
0.015
0.025
0.035
0.045
Primiparous
aged < 6 yrs
Multiparous
aged 5 - 9 yrs
Multiparous
aged 10 - 15 yrs
Multiparous
aged >16yrs
Me
an
mic
roc
oty
led
on
Sv (m
m-1
)
a d
ac b
Figure 6: The mean microcotyledonary Sv value for 4 groups of Thoroughbred mares
divided on the basis of age and parity. Different letters indicate significant
differences between groups (p< 0.001; adapted from Wilsher and Allen 2003).
Although undertaken on a limited number of animals, vascular casting of the
placental blood vessels in placentae from mares of differing age and parity likewise
33
demonstrated the disadvantageous influence of age-related degenerative endometrial
changes on microplacentome development, and on both the extent and intimacy of
physical and haemotological contact at the feto-maternal interface (Abd-Elnaeim et
al 2006).
These experiments undertaken by the candidate (Wilsher and Allen 2002;
2003; Abd-Elnaeim et al 2006) demonstrated clearly that placental development
reflects the age and parity of the mare. Such changes in placental structure, and the
total area of feto-maternal contact at the placental interface, are further reflected in
the birthweight of the foal, with smaller foals being born from maiden and older
mares.
2.2.3 The influence of maternal nutrition on placental development
From work in other animal species it had been established that placental
development may be enhanced or compromised by a range of external influences. For
example, a critical period of sensitivity occurs between 40 and 80 days of gestation in
the ewe which coincides with the time of rapid proliferative growth of the placenta
(Edhardt and Bell 1995). During this period, placental growth may be modulated
nutritionally by the size (Russell et al 1981; McCrabb et al 1992), body condition
(Clarke et al 1998) and degree of maturity of the ewe (Wallace et al 1996, 1999).
These last authors showed that maternal overfeeding of adolescent sheep during their
first pregnancy results in nutrient partitioning in favour of the ewe at the expense of
the fetus; this surprising finding is modulated by major reductions in placental mass
in the overfed animals.
34
There is, however, a paucity of experimental data relating maternal nutrition
with placental development and function in the horse. Hence, a study (Wilsher and
Allen 2006) was undertaken to investigate the common industry practice of rapidly
increasing the body condition score of lean Thoroughbred fillies fresh out of athletic
training at 3 or 4 years of age to that of well-rounded broodmares seen on studfarms.
Specifically, whether the combination of maternal immaturity and over feeding
during their first pregnancy might underlie the reductions in microcotyledon surface
density (Sv), total microscopic surface area of the allantochorion (Ta) and foal
birthweight observed between primigravid versus young multiparous mares by
Wilsher and Allen (2002; 2003). Hence, two groups of maiden primigravid
Thoroughbred fillies were subjected to either moderate or excessive maternal
nutrition throughout gestation. During the course of the experiment all 20 fillies
became infected by Streptococcus equi (‘Strangles’) when they were between 90 and
150 days of gestation and, hence, their placentae were still in the phase of
proliferative growth. The illness caused pyrexia and inappetance during a 7 – 10 day
period and therefore the influence of a disease-mediated maternal nutritional insult in
mid gestation could be examined in the same study (Wilsher and Allen 2006).
The experiment failed to demonstrate any major differences in placental or
fetal structure and function between fillies maintained on moderate versus excessive
planes of nutrition throughout gestation. Although the effects of the nutritional insult
induced by the S.equi infection, and other aspects of the infection per se, could not be
ruled out as possibly negating nutritional differences between the diets, it did
nevertheless appear that Thoroughbred fillies aged 3 – 4 years did not exhibit the
35
same degree of biological immaturity as the adolescent sheep studied by Wallace et
al (1996; 1999).
Figure 7: The pregnant filly in the moderate food intake group that lost the highest
percentage of bodyweight (19.5%) as a consequence of Streptococcus equi infection,
pictured, left before the outbreak and, right at the peak of the weight loss. (From
Wilsher and Allen 2006)
The disease resulted in a dramatic weight loss in all the infected fillies (Figure
7) and taken as a percentage of pre-infection body weight, it was possible to quantify
the degree of nutritional insult the fillies had suffered. This correlated negatively to
the weight and volume of the placenta at term but, surprisingly, positively with
placental efficiency (kilograms of foal birthweight per square metre of feto-maternal
contact via the microcoytledons; Wilsher and Allen 2006). Evidence has emerged
that pigs selected for post natal survival exhibit an increase in placental efficiency
(Wilson et al 2003), thereby suggesting that the above changes in placental efficiency
measured in the Strangles infected, nutritionally stressed pregnant mares may have
resulted from an adaptive strategy to increase the provision of nutrients to the fetus at
critical times in gestation. For example, to meet the challenge of increasing fetal
36
demands in late gestation or to make up for reduced feto-maternal contact across the
placental interface.
The plexiform nature of the microcotyledons in the maiden fillies that
suffered Strangles, as indicated by their mean Sv values, was not modified by the
nutritional insult or by maternal nutrition, and was equivalent to the mean values
obtained in healthy primiparous Thoroughbred fillies (Wilsher and Allen, 2003). In
women, the Sv of the placental villi can be modified by many factors, including
IUGR (Mayhew et al 2003), pre-eclampsia (Boyd and Scott 1985; Teasdale 1985;
Burton et al 1996), hypoxia at high altitude (Mayhew 2003) and exercise (Jackson et
al 1995). By contrast, the only factors that appear able to influence the Sv of the villi
of the microcotyledons in the mare are maternal age, parity and genotype (Allen et al
2002a; Wilsher and Allen 2003; 2006), the three factors that also influence the
architecture and health of the maternal endometrium (Kenney 1993; LeFranc and
Allen 2007). Thus, the maternal endometrium seems to exert the over-riding
influence on microcotyledon development in equids. It is likely that the
epitheliochorial architecture of the equine placenta, with the intimate contact that
exists between the placental villi and the maternal endometrial crypts or sulci, in
contrast to the haemochorial architecture of the human placenta with the associated
breakdown of maternal tissues (Steven 1975), probably influences the ability of the
chorionic villi to modify themselves under different circumstances.
37
2.3 Placental efficiency
Studies in the pig have shown major differences in placental structure and
function between different breeds (Rivera et al 1996; Ford 1997; Wilson and Ford
1997; Biensen et al 1998). These are reflected in placental efficiency, a measure of
the ability of the placenta to support fetal growth, which is heritable and is negatively
associated with placental, but not fetal, weight (Wilson et al 1999; Wilson and Ford
2001). In addition, evidence is emerging that pigs selected for post-natal survival
exhibit increased placental efficiency (Wilson et al 2003). This parameter of placental
efficiency had not been described previously in the horse before the experimental
studies described herein by the candidate.
Assessment of placental efficiency in terms of kilograms of foal birthweight
per square metre of microscopic fetomaternal contact in the previously described
equine IUGR and IUGE models showed no significant effect of perturbed in utero
environment on this parameter; an overall mean value of 1.23 kg/m2 was measured in
both groups of experimental controls and the controls (Allen et al 2002a). However,
calculation of placental efficiency showed that the allantochorion of both primiparous
and multiparous mares aged ≥ 16 years appeared to be more ‘productive’, in terms of
kilograms of foal birthweight per square metre of placental contact, than those of the
other groups of multiparous mares (Wilsher and Allen 2003). Indeed, regardless of
the age and parity of the mare, any increase in total microscopic surface area of
fetomaternal contact was correlated with decreases in placental efficiency. The same
correlation was also evident when plotting the Sv of the microcotyledons against
placental efficiency (Wilsher and Allen 2003). Such findings indicate that smaller
38
placentae, or structurally less well developed microcotyledons, become markedly
more efficient in extracting nutrients for the growing fetus. In this context, it is
important to remember that placental efficiency does not relate to foal birthweight
and large and small foals can develop from both efficient and inefficient placentae
(Figure 8; Wilsher and Allen 2003).
30
40
50
60
70
0.5 0.7 0.9 1.1 1.3 1.5 1.7 1.9
Placental efficiency (kg foal birthweight/m2 of
fetomaternal contact via the microcotyledons)
Fo
al
bir
thw
eig
ht
(kg
)
Figure 8: No correlation is exhibited between foal birthweight and placental
efficiency (y = 0.6676x + 51.207; r = 0.025, p = 0.923, n = 84; adapted from Wilsher
and Allen 2003).
These same correlations matching increases in placental weight with
decreases in placental efficiency have been reported in the pig (Wilson et al 1999;
Wilson and Ford 2001), in which placental efficiency correlated strongly with both
placental and endometrial vascularity (Vonnahme et al 2001). Additionally,
differences in placental efficiency and vascularity have been noted between different
39
breeds of pig. For example, the prolific Meishan breed achieves an increase in litter
size over other breeds with the same ovulation rate by virtue of smaller, but more
vascular, placentae (Ford 1997). The same example also highlights the concept of
placental efficiency whereby the Meishan placenta, although smaller, has the
capacity to produce, weight-for-weight, more piglet per placenta as a result of
enhanced placental and endometrial vascularity.
It remains to be determined if the differences observed in placental efficiency
between horses may be related to placental vascularity. Alterations in endometrial
vascularity and maternal uterine blood flow have been observed in mares of varying
age and parity (Stolla and Bollwein 1997; Bollwein et al 1999) and such differences
may influence the ability of the placenta to become more or less efficient.
Furthermore, experimental reduction of placental size in the ewe has been shown to
enhance placental transport of antipyrine and facilitate diffusion of glucose (Owens
et al 1987).
2.4 Vascularity of the placenta
In normal human pregnancy development of the placental capillary networks
begins with vasculogenesis whereby vascularisation of the first rudimentary villi
results from local de novo formation of capillaries from blood islands, rather than the
extension of embryonic vessels into the placenta. Fetoplacental angiogenesis then
shapes development of the villi so that an initial phase of branching angiogenesis,
resulting in the formation of tightly looped capillaries followed by a second phase of
increased non-branching angiogenesis leading to the formation of longer capillaries,
40
is the norm (Kaufmann et al 2004; Charnock-Jones et al 2004). Work undertaken by
the candidate (Abd-Elnaeim et al 2006) showed that this essentially biphasic
development of placental vascularity also occurs in the equine placenta, commencing
with complex interbranched capillary networks within the developing chorionic villi
in early gestation that change increasingly in the second half of pregnancy to
elongated non-branching capillaries which run parallel to one another within the villi.
Furthermore, the same authors demonstrated by histological analysis that the fetal
interface of the placenta consists of no more than a single thin layer of low columnar-
to-cuboidal trophoblast cells which overlies and provides the essential structural
framework for an incredibly densely packed mass of fetal capillaries supported in
minimal amounts of allantoic mesoderm. In women, the relationship between the
placental villi and their capillary networks suggests that the villous trophoblast is a
plastic layer which adapts in parallel, or in response to, the changing structure of the
underlying vasculature. This idea implies that the trophoblast does not sculpt the
vasculature and angiogenesis is responsible for villous development and
differentiation (Kaufmann et al 2004). To what extent the scheme might hold true in
the horse, where the villi are held in corresponding crypts in the maternal
endometrium rather than the blood pool of the human placenta, remains to be
determined (Figure 9).
The major driving force behind the development of capillary networks is
vascular endothelial growth factor (VEGF), a homodimeric glycoprotein that exists in
5 alternatively spliced forms containing 121 – 206 amino acids in each of the
monomers (Houck et al 1991; Charnock-Jones et al 1993). It is a potent mitogen,
41
morphogen and chemo-attractant for endothelial cells and it is widely recognised as
the most potent stimulator of vasculogenesis and angiogenesis (Charnock-Jones et al
2004). VEGF, and its two main receptor molecules, VEGF-I (Flt) and VEGF-II
(KDR), have been shown to be expressed in the endometrium, decidual tissue and
trophoblast of humans (Sharkey et al 1993; Charnock-Jones et al 1994; Jackson et al
1994; Ahmed et al 1995; Clarke et al 1996; Sharaishi et al 1996; Torry et al 1996;
Vuckovic et al 1996; Shore et al 1997) and non-human primates (Wulff et al 2002)
and it is also in the syndesmochorial placenta of the sheep (Cheung et al 1995) and
the epitheliochorial placenta of the pig (Charnock-Jones et al 2001).
Figure 9: On left, a scanning electron micrograph of the villi within the equine
microtyledons and, on right, villi from the same placenta with the external one-cell
layer of trophoblast cells stripped away. It is easy to imagine how growth of the
villous tree could dictate the shape of the villi. (Author’s unpublished images.)
42
Localization of VEGF and its two receptors, Flt-I and KDR, in the
endometrium and placenta of the mare during the oestrous cycle and pregnancy has
also been demonstrated immunohistochemically by the candidate (Allen et al 2007b).
As in the pig with its similar non-invasive epitheliochorial placenta (Winther et al
1999; Dantzer and Winther 2001; Charnock-Jones et al 2001), the two tissues in the
pregnant mare uterus that labelled strongly and consistently throughout gestation
were the maternal and fetal epithelial layers, the lumenal and glandular epithelia in
the endometrium on the maternal side and the outermost trophoblast layer of the
allantochorion on the fetal side.
In addition to their shared epitheliochorial placentation, porcine and equine
embryos both secrete appreciable quantities of oestrogens from as early as day 10
after ovulation (Bazer and Thatcher 1977; Heap et al 1982). Charnock-Jones et al
(2001) postulated that the increase in angiogenesis observed in the sow at the
mesometrial side of the uterus where the embryonic disk first establishes contact with
the endometrium (Keys and King 1988; Dantzer and Leiser 1994) is likely to be
stimulated by the paracrine action of blastocyst oestrogens stimulating VEGF release
in the endometrium. Since oestrogens have been shown to stimulate VEGF
production in the rat, human and sheep (Cullinan-Bove and Koos 1993; Ahmed et al
1995 and Reynolds et al 1998) it is likely that blastocyst oestrogens have a similar
angiogenic action in the pregnant horse uterus. Indeed, strong staining for KDR
became apparent in the sub-epithelial stroma of the endometrium directly beneath the
conceptus from as early as day 18 after ovulation (Allen et al 2007b). Furthermore,
Doppler ultrasonographic examinations of the mare’s uterus have shown a
43
pronounced increase in endometrial blood flow in the gravid versus the non-gravid
uterine horn around the same stage (Silva et al 2005).
2.5 Placental influences on endocrinology during gestation
Since the fetus, placenta and maternal endometrium together orchestrate the
equine fetoplacental unit to produce all the gonadotrophic and steroid hormones
necessary to maintain the pregnancy state and promote placental and fetal growth
(Pashen and Allen 1979; Allen 1984, 2001, 2004), placental structure influences the
production of placental hormones and visa versa. Hence, the influences of maternal
size and fetal genotype on the endocrinology of pregnancy were also examined by the
candidate (Allen et al 2002b) using the previously described reciprocal embryo
transfer model (Tb-in-P; P-in-Tb; P-in-P and Tb-in-Tb) and employing amplified
enzyme-linked immunoassays (AELIA) and radioimmunoassays to measure the
candidate used both radio- and immuno-assay to measure both gonadotrophic and
steroid hormones.
Measurement of equine Chorionic Gonadotrophin (eCG) concentrations in the
blood of pregnant mares demonstrated that the amount of this equine-unique placental
gonadotrophin produced during the first half of gestation is dependent more upon the
genotype of the mare than the genotype of the fetus being carried. Other factors that
have been suggested to affect eCG production include mare age and parity (Day and
Rowlands 1947), fetal genotype (Bielanski et al 1955; Allen 1969) and uterine
environment (Allen et al 1993). Further, as yet unpublished, work by the candidate
had indicated that maternal diet and exercise may also influence eCG production.
44
However, the work described in Allen et al (2002b) and in Chapter 3 of this thesis
detailing the transfer of day-10 horse embryos to asynchronous recipients mares
(Wilsher and Allen 2009), support strongly the concept that the uterine environment
exerts the over-riding influence on the development of the progenitor chorionic girdle
(Allen et al 1993) and, hence, upon the size and productivity of the eCG secreting
endometrial cups in the mare’s uterus.
Figure 10: The fetal ovaries (top), which undergo tremendous enlargement during the
second half of gestation followed by regression in the last weeks before term, are the
source of large quantities of C-19 precursors which are aromatized by the placenta to
produce oestrogens. Note the tiny under-developed uterus below the fetal gonads and
the inactive maternal ovaries (bottom), all from a mare at 263 days of gestation
(Photograph courtesy of WR Allen).
In contrast, the mean amount of total conjugated oestrogens secreted during
the second half of pregnancy, as measured by area under the curve, were significantly
greater when a Thoroughbred fetus was being gestated regardless of the genotype of
45
the mother. This suggested either or both the Thoroughbred fetus has larger gonads
(Figure 10) than its smaller Pony counterparts and thereby secretes greater quantities
of C-19 precursor steroids for aromatization to oestrogens by the placenta, or it has a
larger placenta that is able to carry out more aromatisation. The functions of these
large quantities of feto-placental oestrogens produced during mid- to late-gestation in
the mare are not altogether clear, although the fetal gonadectomy studies of Pashen
and Allen (1979) indicated that, as in sheep (Resnick et al 1974; Pupkin et al 1975),
oestrogen production may be important to stimulate the development of both the
maternal and fetal placental vasculatures.
Further findings from this study showed that, in the final weeks of pregnancy,
mean serum progestagen concentrations rose much earlier, and to significantly higher
levels, in the Tb-in-P (IUGR) pregnancies than in the P-in-Tb (IUGE) pregnancies,
thereby perhaps reflecting increased fetal stress in the latter Tb-in-P pregnancies
(Allen et al 2002b) caused by premature maturation of the fetal adrenal gland,
resulting in increased secretion of pregnenolone by the adrenal cortex for conversion
to 5-reduced progestagens by the placenta (Holtan et al 1991).
2.6 The influence of placental morphology on post-natal development of the
foal
Ultimately, the structure of the placenta must be related to its function and,
hence, the long-term consequences on post-natal growth following perturbations in
placental development must be considered. Although the horse is born a precocious
46
neonate, able to stand and run alongside its mother within an hour of birth (Rossdale
1966), the equine skeletal system does not mature completely until 5 years of age,
although most limb development is completed by 36 months (Frape 1998; Ott 2004).
Postnatal growth of the musculoskeletal system can be influenced by a host of
conditions and circumstances (Ellis and Lawrence 1978; Hintz et al 1979; Platt 1984;
Pagan et al 1996; Bhuvanakumar and Satchidanandam 1989) many of which, for
example age and parity, have also been shown to influence placental development
(Bracher et al 1996; Wilsher and Allen 2002, 2003) and performance ability on the
racecourse (O’Sullivan 1980; Finnocchio 1986; Barron 1995). Furthermore,
conditions in utero have been implicated in both postnatal growth per se, and as an
underlying aetiology for equine developmental diseases (Rossdale and Ousey 1998,
2003; Rossdale 2005).
Considering that injuries to, and functional shortcomings of, the
muscloskeletal system are one of the major causes of wastage in the Thoroughbred
racing industry (Jeffcott et al 1982; Rossdale et al 1985; Wilsher et al 2006), it
would be beneficial to identify relationships between perturbations in placental
development and subsequent post-natal growth and development.
Much of the work in this area had previously looked at the effect of maternal
size on post-natal growth of the foal. For example, Walton and Hammond (1938), by
crossing Shire horses with Shetland ponies using artificial insemination,
demonstrated a profound influence of maternal size on foal birthweights and post-
natal growth and Tischner and his colleagues similarly highlighted the influence of a
‘spacious’ uterine environment on fetal growth (Tischner 1985; 1987; Tischner and
47
Klimczak 1989). The experiment described in Allen et al (2004) extended the work
of these former authors and examined particularly how conditions in utero can
influence post-natal growth. Hence, the candidate measured, from birth to 3 years of
age, the growth parameters of either Thoroughbred (Tb) foals that had experienced
intrauterine growth restriction (IUGR) following transfer as embryos to the uteri of
smaller Pony (P) mares or P foals that had experienced intrauterine growth
enhancement (IUGE) after transfer to larger Tb mares. The growth parameters of
these animals were compared to those of control Tb and P foals, which were
conceived by within-breed artificial insemination and gestated in the uteri of their
genetic mother. At birth, an approximate 15% reduction or increase in the parameters
measured was observed in the Tb-in-P and P-in-Tb foals compared to their within-
breed controls, which declined to approximately 5% by 3 years of age. Growth in the
first 6 months post partum was enhanced in the previously restricted Tb-in-P foals
and curbed in the previously enhanced P-in-Tb foals, compared to their respective
controls. Allowing for differences in uterine effects it was evident that the genotype
of the foal influenced postnatal size, insofar as the Tb offspring were larger than P
offspring, both at birth and at 3 years of age. However, changes in the measured
parameters that occurred during this 3-year growth period, judged as percentage
increases, were influenced by the recipient and, hence, uterine environment (Allen et
al 2004).
Furthermore, Wilsher and Allen (2006) demonstrated that a disease-mediated
nutritional insult to mares during mid-gestation could influence placental, and hence
fetal, development. Examination of the post-natal growth of the foals in that
48
experiment correlated to altered placental morphology that has been occasioned by
maternal weight loss in mid-gestation. Although the birthweights of these foals was
not correlated to the percentage of maternal weight loss, both their crown-rump (CR)
length and ponderal index (PI = bwt/CR3) were positively correlated at birth, and 6
months later at weaning. This change in CR length probably resulted from the timing
of the insult. In human pregnancy, reduced nutrition in the second trimester exerts its
effects mostly on body length, while food shortage in the third trimester negatively
affects fetal bodyweight (Villar and Belizan 1982).
20
60
100
140
0 5 10 15 20
Maternal weight loss during S.equi infection (%)
Fo
al
pla
sm
a I
GF
-1 c
on
c.
at
bir
th (
ng
/ml)
Figure 11: Relationship between percentage weight losses exhibited by fillies infected
with S.equi and foal plasma IGF-1 concentrations at birth (y = -3.137x + 98.80; r =
0.67; n = 20; p = 0.001; from Wilsher and Allen 2006).
In addition, plasma insulin-like growth factor 1 (IGF-1) concentrations in the
foals at birth correlated with maternal weight loss during gestation (Figure 11), but
49
not with foal birthweight. In other animals, plasma IGF-1 levels increase rapidly after
birth, primarily as a result of growth hormone-stimulated IGF-1 production by the
liver. Furthermore, there is a shift from IGF-2 before birth to IGF-1 after birth, and
this change has led to the concept that IGF-2 is the member of the IGF family
responsible for fetal growth (Fowden 2003). Hence, any lack of a relationship
between plasma IGF-1 concentrations in the foals and their birthweight was not
surprising. The possibility exists that differences in liver function between the fetal
foals may have modulated their IGF-1 concentrations, since maternal nutrient
restriction in ewes in early-to-mid gestation has been shown to reduce the abundance
of IGF-1 mRNA in the fetal liver (Brameld et al 2000).
2.7 Summary of work and findings by the candidate
In conclusion, the principal aim of this series of experiments was to
characterise the structure of the placenta and to relate the findings to fetal
development and post-natal growth. For the first time, the candidate applied
stereology to term equine placentae harvested from a reciprocal embryo transfer
model in which both intra-uterine growth restriction (IUGR) and intra-uterine growth
enhancement (IUGE) had been created experimentally (Allen et al 2002a). From this
initial study reference values were established in Thoroughbreds and Ponies for both
the stereologically-derived surface density of their microcotyledons (surface area per
unit volume: Sv) and for their total area of fetomaternal contact via the
microcotyledons (Ta). Also, an insight was gained into factors that influence
microcotyledon surface area by demonstrating that the Sv of the microcotyledons is
50
controlled solely by maternal genotype, whereas other placental parameters are
governed by both maternal and fetal genotypes. The surface density of the
microcotyledons was shown not to change in either the IUGR or IUGE pregnancies,
but morphological adaptation of the length of the villi did occur in the face of intra-
uterine growth restriction. Foal birthweight was found to be determined primarily by
the total area of fetomaternal contact which, in turn, was dependent on both maternal
and fetal genotypes. Analysis of the hormone changes in these pregnancies provided
useful data on the effects of IUGR versus IUGE on feto-placental hormone secretion
rates (Allen et al 2002b). Further study of the resulting foals demonstrated that both
pre- and post-natal growth in the horse is influenced markedly by placental
development (Allen et al 2004).
Additional experiments established stereologically-derived values (Sv and Ta)
for normal placentae recovered from Thoroughbred mares of varying age and parity
(Wilsher and Allen 2003). In addition, they provided reference values for later inter-
study comparisons. Increasing mare age and parity were shown to cause significant
reductions in the complexity of the microcotyledons. It was also demonstrated that
maximum microcotyledon formation requires some form of ‘priming’ by a first
pregnancy. Foal birthweight in these different groups of mares reflected the balance
between fetomaternal contact and placental efficiency. For the first time, evidence
was gleaned from this study which showed that increases in the area of fetomaternal
contact lead to decreases in placental efficiency. Furthermore, placental efficiency
bears no relation to birthweight.
51
Another study undertaken by the candidate examined how maternal nutrition
and disease could modify both placental and fetal development (Wilsher and Allen
2006). The results showed that the microcotyledon surface density remained
unaffected by these factors, but the development of the placenta was modified in
other ways to influence its efficiency. Furthermore, this study illustrated the long-
term consequences of nutritional deprivation during pregnancy on the overall growth
and development of the foal. This was the first report in the literature to describe the
potential consequences of maternal weight loss on pre- and post-natal development in
the horse.
Furthermore, the candidate examined the vasculature of the equine placenta
using vascular casting to demonstrate and quantify the fine structure of the capillary
network in the placenta at selected stages throughout gestation (Abd-Elnaeim et al
2006). She also collaborated in an immunohistochemical study to examine the
expression of vascular endothelial growth factor (VEGF) and its two receptors
molecules, Flt-1 and KDR, throughout pregnancy in the mare (Allen et al 2007b) to
present the first published report of this angiogenic stimulator in the equine placenta
and endometrium.
This work has already been presented, both by invitation and submission (see
Appendix 3), to scientific and lay audiences around the world to educate and update
them on factors in pregnancy that are important to optimise the growth and athletic
potential of foals. It has been widely received with interest and appreciation. The
innovative work undertaken by the candidate on stereological assessment of the
equine placenta (Allen et al 2002a; Wilsher and Allen 2003) has been particularly
52
well cited (see Appendix 2), not only in the equine-based literature but also in relation
to other species (Dwyer et al 2005; Bowen et al 2006; Gootwine et al 2007; Swali
and Wathes 2007; Coan et al 2008; Ribeiro et al 2008; Sandoval-Castillo and
Villavicenco-Garayzar 2008). In addition, the candidate’s work in this area has been
incorporated into major reviews and veterinary texts on the subject (Morresey 2005;
Bucca 2006; Wooding and Fowden 2006; Wu et al 2006), and has had an impact on
the considerations for the size, age and parity of recipient mares used in embryo
transfer programmes (Stout et al 2006).
____________
53
CHAPTER 3
Embryo transfer in the horse
3.1 Introduction ……………………………………………………………... 54
3.2 The historic development of equine embryo transfer techniques …… 56
3.2.1 Development of a new transcervical embryo transfer method ………. 60
3.3 Donor-recipient synchrony …………………………………………….. 64
3.3.1 The importance of donor-recipient synchrony ……………………….. 64
3.3.2 Historic attempts to extend donor-recipient asynchrony ……………... 66
3.3.3 The use of meclofenamic acid to extend donor-recipient asynchrony .. 67
3.3.4 Transfer of day 10 embryos to asynchronous recipient mares ……….. 70
3.4 Summary of work and findings by the candidate …………………….. 74
54
3.1 Introduction
In its simplest terms, embryo transfer in the horse is the process whereby a 7-
or 8-day old embryo (Figure 1) is flushed from a donor mare and transferred to the
uterus of a well-synchronised recipient mare. Embryo transfer enables the breeding
of foals from all types of genetically superior Sportshorse mares (polo, eventing,
showjumping, dressage, endurance and showing), while allowing them to continue
their competitive careers. This not only shortens the generation interval so that
offspring from such donor mares will be ready to compete before the donor herself
finishes her competitive career, but it also allows production from a younger fertile
animal prior to her retirement from competition, when her fertility could well be
compromised. Post-pubertal yearling and 2-year-old fillies can also be used as donors
before they are physically mature enough to carry a foal to term (Steiner and Jordan
1988; Fleury et al 1989; Camillo et al 2000; Panzani et al 2007) and, at the other end
of the spectrum, older genetically valuable mares that are either incapable of
maintaining a pregnancy themselves or produce inferior foals due to age-related
degenerative changes in their uterus (Bracher et al 1996), may also be suitable
candidates for embryo transfer.
However, unlike the situation in cattle where embryo transfer was embraced
keenly and exploited widely from its original development, the technique was much
slower to be utilised in the horse breeding industry. Argentina was alone in
foreseeing the benefits offered by embryo transfer to develop the Polo Argentina
breed and, hence, improve the quality of polo ponies. The strong demand for elite
polo ponies, both within Argentina and for export to the rest of the world, led to the
55
rapid growth of commercial equine embryo transfer centres. Hence, in the 2007-2008
breeding season, over 4000 polo pony embryos were recovered and transferred in 11
embryo transfer centres (Lascombes and Pashen 2008).
Figure 1: An 8-day old equine embryo which at this developmental stage, is
composed of an outermost trophoblast layer overlain by the equine-unique capsule,
the inner cell mass (at 10 o’clock) and a blastocoelic cavity. (Author’s unpublished
photomicrograph).
Acceptance of embryo transfer offspring by the majority of horse registration
authorities, with the notable exception of the Thoroughbred, has led to an increase in
the use of the technique in recent years, particularly in South America and the USA.
By contrast, Europe has lagged behind to the extent that, in 2005, the International
Embryo Transfer Society recorded the transfer of only 500 equine embryos
throughout Europe (IETS 2006).
56
3.2 The historic development of embryo transfer techniques in the mare
Surgical and non-surgical embryo transfer had been successfully undertaken
in sheep (Lopyrin et al 1950; Hunter et al 1955; Averill and Rowson 1958; Moore et
al 1960), pigs (Kvasnitski 1951; Polge and Day 1968) and cattle (Willett et al 1951;
Mutter et al 1964; Rowson and Moor 1966) by the late 1960’s but it was not until the
early 1970’s that the initial reports by Allen and Rowson (1972) and Oguri and
Tsutumi (1972) of the recovery and transfer of day 7 embryos between donor and
recipient mares appeared in the literature. Original embryo recovery and transfer
techniques in the horse were adapted from those used in the other large animal
species (Hunter et al 1955; Harper et al 1961; Moore and Shelton 1964; Rowson and
Moor 1966) and, as in the cow, surgical transfer initially gave much better pregnancy
rates than non-surgical, transcervical transfer (Douglas 1982; Squires et al 1982;
Iuliano et al 1985; Lagneaux and Palmer 1989).
The development of non-surgical embryo transfer in the horse followed a
similar developmental path to that of the cow and, like the cow, various methods of
depositing the embryo in the uterine lumen have been attempted (Figure 2). The first
two reports of successful embryo transfer in the horse used different transfer
techniques; Allen and Rowson (1972) employed a midline surgical route whereas
Oguri and Tsutsumi (1972) used the non-surgical transvaginal approach previously
described by Sugie (1965) and developed for use in cattle. Adaptations of the
transvaginal embryo transfer method were reported by Muller and Cunat (1993), who
achieved 3 pregnancies from 8 transfers (38%) and, more recently, Gastal et al (2002)
who obtained 18 pregnancies from 23 attempts (78%) when transferring embryos
57
through the vaginal wall under transrectal ultrasound guidance. Both these authors
hoped that, by using the transvaginal rather than the transcervical route, they would
avoid cervical manipulation and the possible consequential release of PGF2 and
oxytocin, uterine infection and reflux of the embryo backwards through the cervix, all
widely suspected of contributing to the lower pregnancy rates achieved following
transcervical non-surgical transfer.
Figure 2: Different approaches to embryo transfer; a) surgical either by midline
laparotomy, flank incision or laparoscopy, b) transcervical or, c) transvaginal
bypassing the cervix (from Muller and Cunat 1993).
Circumventing the cervix to accomplish deposition of the embryo in the
uterus was also tried using a laparoscopic approach through the flank wall of the
recipient mare but low pregnancy rates combined with the disadvantages of cost and
the need for laparoscopic expertise curtailed any widespread application of the
method (Muller and Cunat 1993; Neal et al 2000).
In the 1980’s the use of embryo transfer in horses increased for both research
and commercial purposes and transfer was routinely performed surgically, either by
58
placement of the embryo in the lumen of an exteriorized uterine horn via midline
laparotomy under general anaesthesia (Allen 1982), or via flank incision under local
anaesthesia (Squires et al 1985). Several papers published during this decade
compared pregnancy rates following surgical versus transcervical non-surgical
transfer. In one such study only 4 of 15 (27%) recipient mares that received an
embryo non-surgically became pregnant compared to 8 out of 15 (53%) following
surgical transfer (Imel 1981). Iuliano et al (1985) reported a 72% pregnancy rate for
43 embryos transferred surgically versus 45% for 18 embryos transferred
transcervically and McKinnon et al (1988b) obtained pregnancy rates of 66% versus
39% for 175 embryos transferred surgically and 97 transferred non-surgically. In a
detailed study, Squires et al (1985) were unable to show significant differences
between different types of transfer gun but they did demonstrate convincingly that
protecting (guarding) the transfer pipette in a plastic sleeve (chemise) as it was passed
through the vagina significantly increased the pregnancy rate, thereby implicating
contamination of the transfer pipette with commensal vaginal bacteria as a cause of
the lower pregnancy rates achieved with the unguarded transfer pipette.
Many suggestions were put forward to explain the lower pregnancy rates
associated with transcervical transfer in the horse including; i) the release of
prostaglandinF2 (PGF2, Kask et al 1997; Aurich et al 2008; Koblischka et al 2008)
and/or oxytocin (Handler et al 2002) by manipulation of the cervix during the transfer
procedure, leading to premature luteolysis (Hurtgen and Ganjam 1979; Lagneaux and
Palmer 1989); ii) localised inflammatory responses in the endometrium and/or reflux
59
of the embryo back through the cervix (Squires et al 1999); iii) infective endometritis
established at the time of transfer (Allen 1982; Lagneaux and Palmer 1989; Aurich et
al 2008; Kobischka et al 2008). Despite these suggestions, other studies demonstrated
that pregnancy loss did not necessarily follow cervical dilation in the mare (Handler
et al 2002), and that PGF2 released during sham embryo transfers did not induce
luteolysis and a return to oestrus (Betteridge et al 1985; Kask et al 1997).
These perceived problems, and the continued reports in the literature of
mediocre and variable pregnancy rates following non-surgical transfer, led to the
continued use of surgical transfer via flank incision in the 1990’s. The situation was
summarised by Squires (1993) when he noted that pregnancy rates following the
transfer of horse embryos via flank incision had varied, and had averaged 65% over
the 9-year period between 1981 and 1990, whereas, in the same laboratory and over
the same time period, non-surgical transfer via the cervix had given very variable
pregnancy rates that were, on average, 15 – 20 % lower. However, non-surgical
transcervical transfer was used exclusively in the developing commercial embryo
transfer centres in Argentina as surgical transfer had never been an option under their
management systems. Anecdotal and published data coming from Argentina soon
made it apparent that transcervical transfer could indeed deliver pregnancy rates
equivalent to those achieved surgically, but only by skilled operators with large
numbers of recipient mares at their disposal (Pashen et al 1993). Over the next decade
substantial improvements in transcervical transfer success rates were reported by
Losinno et al (2001; 81%), Jasko (2002; 83%), Lisa and Lisa personal
60
communication (80%) and McCue and Troedsson (2003; 70%) and the benefits of the
non-surgical method in terms of cost, practicality and welfare of the recipient mares,
resulted in complete abandonment of surgical transfer techniques. Indeed, in some
European countries, surgical embryo transfer in horses has now been banned by
statute (Stout 2006).
The early reports of high pregnancy rates achieved from transcervical transfer
came from the commercial embryo transfer centres in which high numbers of
transfers were undertaken annually and recipient mares were not in short supply. The
free choice of an ideal recipient for each donor in terms of both the synchrony of their
oestrous cycles and the normality of the recipient’s reproductive tract no doubt
influenced pregnancy rates positively. In addition, the experience and manipulative
expertise of the operators also played an important part and Squires et al (1999)
opined that the large variations reported in the success of transcervical embryo
transfer stemmed mostly from differences in the technical skill of the operator
performing the procedure
3.2.1 Development of a new transcervical embryo transfer technique
In view of the above mentioned reliance of the success of embryo transfer in
horses upon operator experience, the decision was made to attempt to alter and
simplify the transfer method so as to dispense with this need for the traditional
‘learning curve’. As mentioned, much of the methodology used in equine embryo
transfer had been adopted from the earlier work in cattle (Hunter et al 1955; Harper et
61
al 1961; Rowson and Moor 1966; Rowson et al 1969) and it seemed likely that these
techniques were not optimum for the horse.
Figure 3: The traditional method of non-surgical transcervical embryo transfer in the
mare. The operator must shield the transfer pipette to pass it through the vagina and
then guide it through the cervix to enable deposition of the embryo in the uterine
lumen. (Diagram courtesy of E.L. Squires)
Most of the manipulative skill involved in successful transcervical embryo
transfer lay in the operator’s ability to manipulate the transfer gun or pipette through
the recipient mare’s cervix without causing trauma or bacterial contamination (Figure
3). Therefore, the first line of change focused on altering the method of passing the
transfer gun through the tight, dioestrous cervix. Some 50 years previously, German
equine veterinary surgeons had often grasped and straightened the relaxed cervix of
oestrous mares with a large pair of Velsellum forceps when performing artificial
62
insemination and in human gynaecological procedures, cervical manipulation had
also been accompanied by the use of Velsellum forceps. Accordingly, using a pair of
German equine Velsellum forceps as the starting point, and working in conjunction
with a British surgical instrument maker, Holburn Surgical of Margate, Kent, a pair
of forceps were designed whereby the pointed, unhinged upper jaw with a single
sharp tooth could be inserted into the external os of the mare’s cervix while hinged
lower jaws containing two sharp teeth could be closed so as to grasp the ventral
quadrant of the cervical os; pulling backwards on the forceps should then straighten
the cervical canal (Figure 4). In addition, to minimise potential bacterial
contamination from the wall of the vagina, an old-fashioned duck-billed Polansky’s
vaginal speculum was inserted into the vagina and opened to assess and visualise the
external cervical os (Figure 5).
Figure 4: A close-up view of the jaws of the modified Wilsher Embryo Transfer
Forceps showing the pointed upper ‘nose’ to aid insertion into the external os of the
cervix, and the hinged lower jaw.
Other minor changes were instigated. Namely, the embryo was transferred in
a much larger volume of transfer medium (2.5ml) than previously (0.1 – 0.3ml) and a
63
simple, disposable insemination pipette, rather than the traditional 0.5ml straw was
used to transfer the embryo. The larger volume of medium would help both to
prevent the embryo ‘sticking’ to the tip of the transfer pipette following its expulsion
(Jasko 2002) and to buffer the embryo against temperature fluctuations prior to
transfer.
Figure 5: Distension of the vagina by the arms of the Polansky speculum to enable
sight-directed insertion of the Wilsher Forceps to grasp and straighten the cervix to
enable easy passage of the pipette through the cervical canal. (From Wilsher and
Allen 2004)
The new method was tested initially in 20 experimental recipient mares, of
which 17 (85%) became pregnant (Wilsher and Allen 2004). Accordingly, during
2004 – 2007 the method was used routinely at the Equine Fertility Unit for both
commercial and research purposes and pregnancy rates during the period averaged
80% (180/224), a marked improvement on the previous non-surgical transfer rates of
~50%. The transfer forceps, now named the Wilsher Embryo Transfer Forceps, were
64
produced commercially, originally by Holburn Surgical, and latterly by Surgical
Holdings at Southend-on-Sea, Essex. (www.surgicalholdings.com). They have been
sold worldwide and the modified transfer technique has been adopted by several
commercial embryo transfer centres in Argentina; Brazil and elsewhere (L Losinno; J
Oriol personal communications). Furthermore, the method has been cited in reviews
of embryo transfer methodology (Stout 2006), in text books on reproductive
technologies (Gordon 2004) and in the ‘Material and Methods’ sections of research
papers (Révora et al 2008).
3.3 Donor-recipient synchrony in the mare
A level of synchrony between the embryo and its uterine environment is
essential for the establishment and maintenance of pregnancy and maximum
pregnancy rates are achievable when transferring horse embryos to recipient mares
that ovulated +1 to –3 days with respect to the donor mare (Allen 1982; Squires et al
1982; McCue and Troedsson 2003; Stout 2003 and 2006). Even though this window
of acceptable donor-recipient asynchrony is greater in equids than in cattle, sheep and
pigs, the considerable variation in oestrous length exhibited by individual mares can
make synchronization between donors and recipients difficult, particularly if only
small numbers of recipient mares are available.
3.3.1 The importance of donor-recipient asynchrony
It was first demonstrated in the rat that altering the degree of synchrony
between donor and recipient influenced the success of embryo transfer (Nickolas
65
1933). Further work in laboratory species demonstrated that rabbit blastocysts
degenerated when transferred to a uterine environment that was >2 days
asynchronous with their stage of development (Chang 1950) and rat blastocysts older
than the recipient uterus to which they were transferred showed delayed development
but nevertheless implanted when the uterus became receptive, while blastocysts
younger than the recipient uterus degenerated rapidly when transferred (Dickmann
and Noyes 1960; Noyes and Dickmann 1960). Similarly, in the mouse,
asynchronously transferred embryos that were older than the recipient uterus were
able to implant while those that were younger degenerated (Doyle et al 1963).
Subsequent work in livestock species demonstrated that their embryos also
lacked the flexibility to respond to a large degree of uterine asynchrony (see Pope
1988 for review). In cattle, optimal pregnancy rates were achieved when embryos
were transferred to recipients that had ovulated only + 1 to –1 days relative to the
donor (Lawson et al 1975) while in sheep the window of acceptable donor-recipient
asynchrony could be widened to + 2 to –2 days (Moore and Shelton 1964). The horse,
possibly due to its long delay in implantation and placentation compared to
ruminants, has an even wider window of acceptable donor-recipient asynchrony of +
1 to – 3 days (Allen 1982; Squires et al 1982; McCue and Troedsson 2003; Stout
2003 and 2006).
Under conditions of embryo-uterine asynchrony a variety of complications
can occur, including failure of the embryo to implant, early embryonic mortality or
retarded or accelerated embryonic growth (see Pope 1988; Barnes 2000 for reviews).
For example, sheep embryos transferred to a more advanced uterus increase their rate
66
of cell division (Wilmut et al 1985) and slow the rate when transferred to a less
advanced uterus (Wilmut and Sales 1981; Lawson et al 1983; Wilmut et al 1986). In
the horse, Carnevale et al (2000) reported that pregnancy failure following embryo
transfer was more likely to occur if the embryo was transferred to the uterus of a
recipient mare that was >7 days after ovulation, possibly due to an inability of the
embryo to prevent luteolysis occurring in the recipient, or not being able to stimulate
adequate production of histotroph by the endometrial glands so late in the luteal
phase. Indeed, the traditional view is that maternal recognition of pregnancy in the
mare must occur prior to day 10 after ovulation in order to prevent the upregulation of
ocytocin receptors and the initiation of the luteolytic cascade (Stout et al 1999).
3.3.2 Historic attempts to extend donor-recipient asynchrony in the horse
Commercial equine embryo transfer has increased in recent years, particularly
in America and Europe (McCue and Troedsson 2003; Stout 2003), with the result that
the availability of mares suitable for use as recipients has declined proportionally. To
try and meet this shortfall a variety of hormone treatment regimes have been
employed to make either ovariectomised or seasonally anoestrous mares available as
alternative embryo recipients (Hinrichs et al 1985; Hinrichs and Kenny 1987;
McKinnon et al 1988a; Lagneaux and Palmer 1993; Rocha Filho et al 2004).
In parallel, a number of authors have reported upon pharmacological attempts
to extend donor-recipient asynchrony in the horse, both for commercial and scientific
reasons. For example, the synthetic progestagen, altrenogest, has been administered
to recipient mares to obviate the need for the transferred embryo to establish maternal
67
recognition of pregnancy and luteal maintenance. However, although altrenogest
administration did indeed prevent a return to oestrus in recipient mares, Pool et al
(1987) showed that the transfer of day-7 embryos to recipient mares treated daily with
altrenogest from the time of ovulation was successful when the transfer was
performed in mares that had ovulated 2 – 6 days previously but unsuccessful when
the mares had ovulated 7 – 12 days prior to transfer. Clark et al (1987) likewise failed
to achieve and maintain pregnancy in altrenogest-treated recipient mares that
ovulated 2 or 3 days after the donor and Hinrichs and Kenny (1987) demonstrated
that ovariectomised recipient mares which received exogenous progesterone in
approximate synchrony with the donor mare’s ovulation had significantly higher
pregnancy rates than those that were treated for 4 to 11 days prior to ovulation in the
donor. In attempting to explain their findings, Hinrichs and Kenny (1989) went on to
examine the effect of the duration of progesterone treatment on the secretion of
uterine proteins in ovariectomised mares. Their findings concurred with those of
Zavy et al (1979; 1982) in cycling mares that the composition of uterine secretory
proteins changed over time and the concentration of the proteins increased with the
duration of progesterone treatment.
3.3.3 The use of meclofenamic acid to extend donor-recipient asynchrony
Using the new embryo transfer technique described above (Wilsher and
Allen 2004), a study was undertaken to attempt to manipulate the donor-recipient
window of asynchrony (Wilsher et al 2006b). Such an extension, if still able achieve
68
acceptable pregnancy rates, would allow more efficient use of recipient mares and
would provide valuable information on embryo-maternal interactions.
0
20
40
60
80
100
+2 days +3 days +4 days +5 days
Asynchrony of recipient relative to donor
% o
f re
cip
ient
mare
s pre
gnant
Figure 6: Pregnancy rates 7 – 8 days after embryo transfer in meclofenamic acid-
treated (■) and untreated (□) control mares at different stages of asynchrony with
respect to the donor mare. For all groups at 2 or 3 days asynchrony, n = 10; for all
groups at 4 or 5 days asynchrony, n = 8. Significantly more meclofenamic acid-
treated mares versus untreated controls became pregnant with an asynchrony of +3
days (p = 0.025; from Wilsher et al 2006b)
Embryos were transferred to recipient mares that had ovulated 2, 3, 4 or 5
days before the donor, half of which were administered the prostaglandin synthetase
inhibitor, meclofenamic acid (Arquel V Granules, Pfizer, Sandwich, Kent, UK),
beginning from 9 days after ovulation and continuing for 7 days after embryo
transfer. The results showed that meclofenamic acid treatment improved the rate of
establishment of pregnancy in recipient mares that ovulated before the donor (Figure
69
6). However, whereas the pregnancies were maintained in the mares that ovulated 2
or 3 days before the donor, there was a greatly increased incidence of early
embryonic death in the mares that ovulated 4 or 5 days before the donor. Since
meclofenamic acid-treated mares that failed to become pregnant after embryo transfer
returned to oestrus at the expected time it could be concluded that the support
afforded by the meclofenamic acid in the asynchronous transfers was not by the
prostaglandin synthetase inhibitory action of the drug suppressing luteolysis.
However, in addition to its ability to suppress cyclo-oxygenase and 5-lipoxygenase
(Brogden 1986; Civelli et al 1991), meclofenamic acid has also been shown to block
the binding of PGE2 to its receptor (Rees et al 1988) and to modulate a variety of ion
channels and block gap junction intracellular communication (Harks et al 2001).
Although a definitive conclusion could not be reached on how the pharmacological
properties of meclofenamic acid might be aiding the embryo to overcome the
problems associated with an asynchronous uterine environment, they may provide
clues to understanding maternal-embryo interactions, especially during the period of
maternal recognition of pregnancy.
It has been proposed subsequently by Koblischke et al (2008) that
meclofenamic acid and other non steroidal anti-inflammatory drugs (NSAID’s) may
suppress subclinical endometritis in the mare and, hence, prevent premature luteolysis
and subsequent embryo loss following transcervical embryo transfer. However,
although NSAID treatment of recipient mares with the normal acceptable +1 to -3
day asynchrony inhibited the release of prostaglandin F2 from the endometrium
70
compared to untreated controls, treatment with NSAID’s did not increase pregnancy
rates in the recipients. Hence, although the authors had demonstrated a potential
therapeutic use for meclofenamic acid in preventing PGF2 release, and hence
potential luteolysis when undertaking conventional embryo transfer, their findings did
not explain the candidates finding of the action of meclofenamic acid in extending
donor-recipient asynchrony (Wilsher et al 2006b).
3.3.4 Transfer of day-10 embryos to asynchronous recipient mares
The extent to which donor-recipient asynchrony could be stretched in equine
embryo transfer had not been fully investigated, nor had the effects of such
asynchrony on embryonic development. Hence, large (2 – 5mm diameter), visible-to-
the-naked-eye day-10 embryos were flushed from the uteri of donor mares and
transferred to recipient mares that had ovulated 9, 7, 5, 3, or 1 day after the donor, on
the same day as the donor, or 2 or 4 days before the donor, thereby creating a 13-day
window over which the transfers were performed (Wilsher and Allen 2007; Wilsher
et al 2008; Wilsher and Allen 2009).
Putting aside the asynchrony aspect of the experiment for the moment, the
transfer of such large day-10 embryos was, in itself, a radical change from
conventional embryo transfer practice since, anecdotally, day-10 equine embryos had
previously been considered too large and too fragile to be handled safely for routine
embryo transfer procedures (Figure 7). The flushing and transfer of such late stage
embryos could potentially provide some practical advantages for commercial embryo
71
transfer centres; namely, most of the embryos would be visible ultrasonographically
in the uterus of the donor mare prior to flushing (S Wilsher and WR Allen,
unpublished data), they would be readily visible to the naked eye in the embryo filter
during recovery of the flushing medium from the donor mare and a positive or
negative pregnancy diagnosis could be made within a very few days of transfer to the
recipient mare.
Figure 7: A day-10 equine embryo, 4mm in diameter. Note the embryonic disk
visible in the equatorial region of the embryo (Photograph courtesy of Tim Flach).
Previous reports in the literature had reported higher pregnancy rates
following the transfer of day-7 or -8 embryos compared to the transfer of day-9 or -10
embryos. For example, Squires et al (1982), when comparing the results of both
surgical and non-surgical embryo transfer methods, concluded that more mares
became pregnant following the transfer of day-8 embryos (32%) than day-9 embryos
(9%). Similarly, Vogelsang et al (1985) reported no pregnancies following the
72
transfer of day-9 or -10 embryos compared to a 61% pregnancy rate for day-6
embryos, 55% for day-7 embryos and 25% for day-8 embryos. On the other hand
Fleury et al (1989) did achieve a 69% success rate when transferring 16 day-9 horse
embryos. One further study (Sirios et al 1987) involved transferring 4 day-10
embryos straight back into the donor mares from which they had been flushed. A
single ongoing pregnancy was achieved whereas no pregnancies resulted from the
transfer of a further 4 day-10 embryos to recipients that were +1 to –1 days
asynchronous with the donor. In attempting to explain these poor results, Squires et al
(1985) suggested that the increased fluid volume-to-surface ratio of day-9 and -10
embryos made them more prone to damage during the collection and transfer
procedures. However, it should be borne in mind that these earlier studies had been
undertaken before the use of non-surgical embryo transfer had become widespread
and the operators had acquired much practical experience.
Undeterred by the negative findings in the earlier studies, the present
experiment was undertaken with some optimism due to the simplicity and high
degree of success of the new embryo transfer method (Wilsher and Allen 2004). In
the event, the horse proved remarkable compared to the other large domestic species
in that day-10 embryos were shown to be able to survive in a uterine environment
that was up to 7 days behind, and up to 2 days ahead of embryonic age (Table 1). As
had been shown previously in the mouse and sheep, when an embryo was placed into
a uterus that was ‘temporally behind’ the embryo’s stage of development, embryonic
growth and differentiation slowed down (Bowman and McLaren 1970; Wilmut and
Sales 1981; Lawson et al 1983; Wilmut et al 1985, 1986), thereby causing the late
73
Dame Anne McLaren FRS to remark that ‘the embryo will wait for the uterus, but the
uterus will not wait for an embryo’. Although a high degree of pregnancy failure
occurred in the recipient mares that were either 1 or 3 days post ovulation (OV) when
a day-10 embryo was transferred to them, the 3 embryos that did survive in the OV +
3 recipients showed delayed growth and development. So much so that the stage-
specific invasion of the equine-unique chorionic girdle portion of the fetal membranes
into the maternal endometrium, and the consequential secretion of equine Chorionic
Gonadotrophin (eCG) into the maternal bloodstream occurred when the uterus, not
the embryo, reached the appropriate stage of 36 days after ovulation. Further novel
insights into the events leading to maternal recognition of pregnancy in the mare were
revealed by this experiment. For example, the survival of all 6 day-10 embryos
transferred to OV + 12 recipient mares completely negated the former widely held
belief that that the luteolytic cascade in the mare is initiated on or before day-10 post
ovulation (Goff et al 1987; Stout et al 1999, 2000). The present experiment showed
that either the luteolytic cascade is stoppable once initiated or, more likely, it does not
begin until after day-12 after ovulation.
The asynchronous embryo transfer model provides exciting possibilities to
study more precisely the role played by uterine environment in many of the stage-
specific events that occur in early embryogenesis and fetal development in the mare.
For example, little is presently known about the mechanisms involved in the
desialylation of the equine-unique blastocyst capsule (Betteridge 1989; Oriol et al
1991) around day-21 of gestation and whether this loss is initiated by embryo or the
uterine environment.
74
Table 1: The influence of recipient asynchrony on pregnancy and early embryonic
loss rates after transfer of day-10 embryos. (From Wilsher et al 2008).
Stage of recipient
(days after ovulation)
No. of recipient mares pregnant/No. of transfers (%)
2 days post ET 10 days post ET Embryo with a heartbeat
OV + 1 4/6 (67%) 2/6 (33%) 0/6 (0%)
OV + 3 8/8 (100%) 6/8 (75%) 3/8 (38%)
OV + 5 6/8 (75%) 6/8 (75%) 6/8 (75%)
OV + 7 6/8 (75%) 6/8 (75%) 6/8 (75%)
OV + 9 6/8 (75%) 5/8 (63%) 5/8 (63%)
OV + 10 6/6 (100%) 6/6 (100%) 6/6 (100%)
OV + 12 6/6 (100%) 6/6 (100%) 6/6 (100%)
OV + 14 3/6 (50%) 0/6 (0%) 0/6 (0%)
OV = ovulation (day of ovulation = 0); ET = embryo transfer
3.4 Summary of work and findings by the candidate
The candidate developed a novel and very practical method for undertaking
transcervical embryo transfer in the horse, primarily by designing a pair of cervical
forceps (Wilsher Embryo Transfer Forceps) that enable inexperienced operators to
transfer embryos in a relatively large volume of transfer medium in a simple,
disposable insemination pipette without the need to acquire high levels of experience
and manipulative skill (Wilsher and Allen 2004). The success of the method has
resulted in the commercial production of the Wilsher Forceps and their widescale use
within the equine veterinary and scientific communities.
75
The candidate also investigated the application of a pharmacological method
to extend donor-recipient synchrony in embryo transfer programmes (Wilsher et al
2006b), which has proved successful in a commercial setting (Michaela Kölling,
unpublished observations). The results of the study are also of considerable scientific
interest in the field of embryo-maternal interactions, particularly in relation to the
maternal recognition of pregnancy in the mare.
More recent work by the candidate demonstrated the apparently unique ability
of the equine embryo to tolerate a very wide window of donor-recipient asynchrony
(Wilsher and Allen 2009). This has provided not only a useful practical advance in
commercial equine embryo transfer but also a valuable model to study the relevant
roles of the conceptus and uterine environment in regulating early embryonic and
fetal development in the horse.
In addition to their successful application to commercial equine embryo
transfer, the Wilsher Forceps have been used to assist in the delivery of drugs,
hormones and other experimental compounds into the mare’s uterus without causing
endometritis or luteolysis. The results of such studies are currently being written up
and part of the work has been described in a refereed abstract (Wilsher and Allen
2008).
There has been worldwide interest in the candidate’s work on embryo transfer
in the horse and she has been invited to communicate her findings in this area in
Argentina, Chile, Czech Republic, Germany, Portugal and New Zealand, (see
Appendix 3). The candidate’s papers in this area have been cited in peer-reviewed
76
journals (see Appendix 2), including a review on the developing potential of equine
embryo transfer (Stout 2006).
________________
77
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Appendix 1: The candidate’s publications to be considered for the degree of PhD by
Publication. Where they are cited in the text, they appear in bold.
Chapter 1: Epidemiological surveys of the efficiency of Thoroughbred racing and
breeding.
1. Wilsher S, Allen WR and Wood JLN (2006) Factors associated with failure of
Thoroughbred horses to train and race. Equine Veterinary Journal 38 113-118
2. Allen WR, Brown L, Wright M and Wilsher S (2007) Reproductive efficiency of
Flatrace and National Hunt mares and stallions in England. Equine Veterinary
Journal 39 438-445
Chapter 2: The morphology and function of the equine placenta.
3. Allen WR, Wilsher S, Turnbull C, Stewart F, Ousey J, Rossdale PD and Fowden
A (2002) Influence of maternal size on placental, fetal and postnatal growth in the
horse: I Development in utero. Reproduction 123 445-453
4. Allen WR, Wilsher S, Stewart F, Ousey J and Fowden A (2002) The influence of
maternal size on placental, fetal and postnatal growth in the horse. II
Endocrinology of pregnancy. Journal of Endocrinology 172 237-246
5. Wilsher S and Allen WR (2003) The effects of maternal age and parity on
placental and fetal development in the mare. Equine Veterinary Journal 35 476-
483
6. Allen WR, Wilsher S, Tiplady C and Butterfield RM (2004) The influence of
maternal size on pre- and postnatal growth in the horse. III Postnatal growth.
Reproduction 127 67-77
7. Abd-Elnaeim MMM, Leiser R, Wilsher S and Allen WR (2006) Structural and
haemovascular aspects of placental growth throughout gestation in young and
aged mares. Placenta 27 1103-1113
8. Wilsher S and Allen WR (2006) Effects of a Streptococcus equi infection
mediated nutritional insult during mid gestation in Thoroughbred mares: I.
Placental and fetal development. Equine Veterinary Journal 38 549-557
9. Allen WR, Gower S and Wilsher S (2007) Immunohistochemical localization of
vascular endothelial growth factor (VEGF) and its two receptors (Flt-1 and KDR)
in the endometrium and placenta of the mare during the oestrous cycle and
pregnancy. Reproduction in Domestic Animals 42 516-526
Chapter 3: Embryo transfer in the horse.
10. Wilsher S and Allen WR (2004) An improved method for non-surgical embryo
transfer in the mare. Equine Veterinary Education 16 39-44
11. Wilsher S, Kölling M and Allen WR (2006) Meclofenamic acid extends donor-
recipient asynchrony in equine embryo transfer. Equine Veterinary Journal 38
428-432
12. Wilsher S and Allen WR (2009) Uterine influences on embryogenesis and early
placentation in the horse revealed by transfer of day 10 embryos to day 3 recipient
mares. Reproduction 137 583-593
PAPER 1
Wilsher S, Allen WR and Wood JLN (2006) Factors associated with failure of
Thoroughbred horses to train and race. Equine Veterinary Journal 38 113-118
PAPER 2
Allen WR, Brown L, Wright M and Wilsher S (2007) Reproductive efficiency of
Flatrace and National Hunt mares and stallions in England. Equine Veterinary
Journal 39 438-445
PAPER 3
Allen WR, Wilsher S, Turnbull C, Stewart F, Ousey J, Rossdale PD and Fowden A
(2002) Influence of maternal size on placental, fetal and postnatal growth in the
horse: I Development in utero. Reproduction 123 445-453
PAPER 4
Allen WR, Wilsher S, Stewart F, Ousey J and Fowden A (2002) The influence of
maternal size on placental, fetal and postnatal growth in the horse. II Endocrinology
of pregnancy. Journal of Endocrinology 172 237-246
PAPER 5
Wilsher S and Allen WR (2003) The effects of maternal age and parity on placental
and fetal development in the mare. Equine Veterinary Journal 35 476-483
PAPER 6
Allen WR, Wilsher S, Tiplady C and Butterfield RM (2004) The influence of
maternal size on pre- and postnatal growth in the horse. III Postnatal growth.
Reproduction 127 67-77
PAPER 7
Abd-Elnaeim MMM, Leiser R, Wilsher S and Allen WR (2006) Structural and
haemovascular aspects of placental growth throughout gestation in young and aged
mares. Placenta 27 1103-1113
PAPER 8
Wilsher S and Allen WR (2006) Effects of a Streptococcus equi infection mediated
nutritional insult during mid gestation in Thoroughbred mares: I. Placental and fetal
development. Equine Veterinary Journal 38 549-557
PAPER 9
Allen WR, Gower S and Wilsher S (2007) Immunohistochemical localization of
vascular endothelial growth factor (VEGF) and its two receptors (Flt-1 and KDR) in
the endometrium and placenta of the mare during the oestrous cycle and pregnancy.
Reproduction in Domestic Animals 42 516-526
PAPER 10
Wilsher S and Allen WR (2004) An improved method for non-surgical embryo
transfer in the mare. Equine Veterinary Education 16 39-44
PAPER 11
Wilsher S, Kölling M and Allen WR (2006) Meclofenamic acid extends donor-
recipient asynchrony in equine embryo transfer. Equine Veterinary Journal 38 428-
432
PAPER 12
Wilsher S and Allen WR (2009) Uterine influences on embryogenesis and early
placentation in the horse revealed by transfer of day 10 embryos to day 3 recipient
mares. Reproduction 137 583-593
Appendix 2: The contribution of the author to the submitted papers and citation of these
works.
The contribution the candidate has made to the papers submitted for the degree of PhD by
Publication and the number of citations for each paper.
Chapter 1 1 2 2 2 2 2 2 2 3 3 3
Paper identification* 1 2 3 4 5 6 7 8 9 10 11 12
Category Percentage contribution by the candidate
Experimental design
and planning 80 50 20 20 75 50 50 50 75 80 100 100
Practical scientific
work 75 80 25 60 90 50 40 30 75 75 60 75
Supervision of
scientific / technical
staff 60 100 10 20 20 20 0 50 50 40 50 100
Writing of draft
manuscript 50 40 25 40 100 50 30 40 100 80 100 75
Statistical analyses 80 100 100 100 100 100 n/a n/a 100 n/a 100 100
Final writing of
manuscript 70 50 20 40 75 50 50 60 80 70 90 75
Number of citations† 6 5 37 12 22 7 4 1 1 4 3 0
† Source Scopus and ISC Web of Science (accessed April 2009)
* Papers are listed in the order they appear in the Chapters:
1 = Wilsher, Allen and Woods (2006)
2 = Allen, Brown, Wright and Wilsher (2007)
3 = Allen, Wilsher, Turnbull, Stewart, Ousey, Rossdale and Fowden (2002)
4 = Allen, Wilsher, Stewart, Ousey and Fowden (2002)
5 = Wilsher and Allen (2003)
6 = Allen, Wilsher, Tiplady and Butterfield (2004)
7 = Abd-Elnaeim, Leiser, Wilsher and Allen (2006)
8 = Allen, Gower and Wilsher (2007)
9 = Wilsher and Allen (2006)
10 = Wilsher and Allen (2004)
11= Wilsher, Kölling and Allen (2006)
12 = Wilsher and Allen (2009)
Appendix 3: Full bibliography of the candidate.
Bibliography
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placental area and foal birthweights in the mare. Pferdeheilkunde 15 599-
602.
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function in the mare. Proceedings of the 50th
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Rossdale PD (2000) Effects of maternal size on pre- and postnatal
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parity and age on morphometric placental development and function in the
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and Fertility Abstract Series 25 14 (Abstr)
6. Morris L H-A, Tiplady C, Cook E, Wilsher S, Xihe L and Allen WR
(2000) Hysteroscopic insemination of mares with low numbers of frozen-
thawed ejaculated and epididymal spermatozoa. Journal of Reproduction
and Fertility Abstract Series 25 29 (Abstr)
7. Neal H, Morris LHA, Wilsher S and Allen WR (2000) Laparoscopic
embryo transfer. Proceedings of 39th
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8. Allen WR, Wilsher S, Turnbull C, Stewart F, Ousey J, Rossdale PD and
Fowden A (2002) Influence of maternal size on placental, fetal and
postnatal growth in the horse: I. Development in utero. Reproduction 123
445-453.
9. Allen WR, Wilsher S, Stewart F, Ousey J and Fowden A (2002) The
influence of maternal size on placental, fetal and postnatal growth in the
horse: II. Endocrinology of pregnancy. Journal of Endocrinology 172
237-246.
10. Wilsher S and Allen WR (2002) The influences of maternal size, age and
parity on placental and fetal development in the horse. Theriogenology 58
833-835.
11. Allen WR and Wilsher S (2002) Influence of maternal uterine size on pre-
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37 223 (Abstr)
12. Li X, Morris LH-A, Wilsher S and Allen WR (2003) Fetal development
after intracytoplasmic sperm injection (ICSI) in the horse. In: From
Epididymis to Embryo. Eds LH-A Morris, L Foster and JF Wade.
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13. Allen WR and Wilsher S (2003) Influence of maternal size on pre- and
post natal growth of the foal. In: Comparative Neonatology and
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14. Wilsher S and Allen WR (2003) Influences of maternal and fetal
genotypes on placental and fetal development in the horse. In: Comparative
Neonatology/Perinatology. Eds. P Sibbons, L Foster and JF Wade.
Havemeyer Foundation Monograph Series 8 51-53 (Abstr.)
15. Wilsher S and Allen WR (2003) The effects of maternal age and parity on
placental and fetal development in the mare. Equine Veterinary Journal 35
476-483
16. Wilsher S and Allen W R (2003) The effects of maternal nutrition on
placental and fetal development in maiden Thoroughbred mares. In:
Embryonic and Fetal Nutrition Eds. S Wilsher and J Wade. Havemeyer
Foundation Monograph Series 10 70-71 (Abstr)
17. Wilsher S and Allen WR (2003) A novel method for non-surgical embryo
transfer in the mare. Reproduction in Domestic Animals 38 320 (Abstr).
18. Allen WR, Wilsher S, Tiplady C and Butterfield RM (2004) The influence
of maternal size on pre- and postnatal growth in the horse: III Postnatal
growth. Reproduction 127 67-77.
19. Wilsher S and Allen WR (2004) An improved method for non-surgical
embryo transfer in the mare. Equine Veterinary Education 16 39-44.
20. Wilsher S and Allen WR (2004) Factors controlling microcotyledon
development and placental efficiency in Thoroughbreds: Influences of
mare age, parity and nutritional insults. In: Proceedings of a Workshop on
Equine Placenta. Kentucky Agricultural Experiment Station, Lexington,
Kentucky, SR-2004-1, pp58-64.
21. Xihe Li, A-C Lefranc, S Wilsher and W R Allen (2004) What is different
about the horse embryo? In: European Equine Gamete Group (EEGG).
Eds J Müller, Z Müller and JF Wade. Havemeyer Foundation Monograph
Series 13 62 (Abstr.).
22. Wilsher S and Allen WR (2004) A new method for non-surgical embryo
transfer in the horse. In: European Equine Gamete Group (EEGG). Eds J
Müller, Z Müller and JF Wade. Havemeyer Foundation Monograph Series
13 42-43 (Abstr.).
23. Wilsher S and Allen WR (2004) Factors influencing placental and fetal
development in the mare: important considerations for the selection of
embryo recipients. In: Emerging Equine Science. Eds: J Alliston, S Chadd,
A Ede, A Hemmings, J Hyslop, A Longland, H Moreton and Moore-
Colyer M. Nottingham University Press, Nottingham, UK. pp87-89
24. Xihe L, Lefranc AC, Wilsher S and Allen WR (2004) The characteristics
of horse trophectoderm cells and their ability to support embryonic
development following nuclear transfer. Proceedings of 15th
International
Conference on Animal Reproduction. Abstracts Vol 2 561 (Abstr.)
25. Wilsher S and Allen WR (2004) The effects of maternal nutrition on
placental development in maiden mares. Proceedings of 15th
International
Conference on Animal Reproduction. Abstracts Vol 1 94 (Abstr.)
26. Wilsher S and Allen WR (2005) A novel method for non-surgical embryo
transfer in the mare. In: Proceedings of the 6th
International Symposium on
Equine Embryo Transfer. Eds. M Alvarenga and JF Wade. Havemeyer
Foundation Monograph Series 14 110-112 (Abstr).
27. Allen WR, Wilsher S, Kölling M and Lefranc A-C (2005) Luteal and
endometrial interdependence for early embryonic development in the mare.
In: Maternal Recognition of Pregnancy in the Mare III. Eds TAE Stout and
JF Wade. Havemeyer Foundation Monograph Series 16 3-4 (Abstr).
28. Wilsher S, Kölling M and Allen WR (2005) The use of meclofenamic acid
to extend donor-recipient asynchrony in equine embryo transfer. In:
Maternal Recognition of Pregnancy in the Mare III. Eds TAE Stout and JF
Wade. Havemeyer Foundation Monograph Series 16 8-9 (Abstr).
29. Wilsher S and Allen WR (2005) Factors influencing placental and fetal
growth. Proceedings of 44th
Congress British Equine Veterinary
Association. 69 (Abstr.)
30. Wilsher S, Kölling M and Allen WR (2005) The use of meclofenamic acid
to extend donor-recipient asynchrony in equine embryo transfer.
Reproduction in Domestic Animals 40 344 (Abstr)
31. Wilsher S, Ousey J and Allen WR (2005) Gross and histological
observations on placentae from abnormal pregnancies. In: Proceedings of a
Workshop on Comparative Placentology. Eds. P Sibbons and JF Wade.
Havemeyer Foundation Monograph Series 17 57-58 (Abstr).
32. Wilsher S, Allen WR and Wood JLN (2005) Success and wastage rates in
Thoroughbred flat racing in Britain and Ireland. In: Thoroughbred Racing
and Breeding Seminar; Cheltenham 2005. 75 (Abstr.)
33. Wilsher S, Allen
WR and Wood
JLN (2006) Factors associated with failure
of Thoroughbred horses to train and race. Equine Veterinary Journal 38
113-118.
34. Xihe Li, Qin Y, Wilsher S and Allen WR (2006) Centrosome changes
during meiosis in horse oocytes and first embryonic cell cycle organization
following parthenogenesis, fertilization and nuclear transfer. Reproduction
131 661-667.
35. Wilsher S, Kölling M and Allen WR (2006) Meclofenamic acid extends
donor-recipient asynchrony in equine embryo transfer. In: International
Equine Gamete Group. Eds. H Alm, H Torner and JF Wade. Havemeyer
Foundation Monograph Series 18 56-57 (Abstr).
36. Abd-Elnaeim MMM, Leiser R, Wilsher S and Allen WR (2006) Structural
and haemovascular aspects of placental growth throughout gestation in
young and aged mares. Placenta 27 1103-1113
37. Wilsher S, Kölling M and Allen WR (2006) Meclofenamic acid extends
donor-recipient asynchrony in equine embryo transfer. Equine Veterinary
Journal 38 428-432.
38. Wilsher S and Allen WR (2006) Effects of a Streptococcus equi infection
mediated nutritional insult during mid gestation in Thoroughbred mares. I.
Placental and fetal development. Equine Veterinary Journal 38 549-557.
39. Wilsher S, Kölling M, Allen WR (2006) The effects of estrogen during
early pregnancy in the mare on serum progestagen and IFG-1 levels and
embryonic growth. Animal Reproduction Science 94 381-382 (Abstr.)
40. Allen WR, Wilsher S, Morris L, Crowhurst JS, Hillyer MH, Neal HN
(2006) Re-establishment of ovdicual patency and fertility in infertile mares.
Animal Reproduction Science 94 242-243 (Abstr.)
41. Villani M, Wilsher S, Carluccio A, Contri A and Veronesi MC (2006)
Stereology in the term placenta of the donkey, Thoroughbred and pony: A
comparative study. Animal Reproduction Science 94 411-412 (Abstr)
42. Allen WR, Wilsher S, Morris L, Crowhurst JS, Hillyer MH, Neal HN
(2006) Laparoscopic application of PGE2 to re-establish oviducal patency
and fertility in infertile mares: a preliminary study Equine Veterinary
Journal 38 454-459
43. Ellenberger C, Kölling, Wilsher S, Allen WR, Bazer FW, Klug J, Schoon
D and Schoon H-A. (2006) Immunolocalisation of uterine secretory
proteins in the mare during the oestrous cycle and pregnancy. Reproduction
in Domestic Animals 41 308 (Abstr)
44. Wilsher S, Kölling M and Allen WR (2006) The effects of exogenous
oestradiol on luteal function and embryonic growth during early pregnancy
in the mare. Reproduction in Domestic Animals 41 360 (Abstr)
45. Allen WR, Wilsher S, Morris L, Crowhurst JS, Hillyer MH and Neal HN
(2006) Re-establishment of oviducal patency and fertility in infertile mares.
Reproduction in Domestic Animals 41 360 (Abstr)
46. Allen WR, Gower S and Wilsher S (2007) Immunohistochemical
localisation of vascular endothelial growth factor (VEGF) and its two
receptors (Flt-I and KDR) in the endometrium and placenta of the mare
during the oestrous cycle and pregnancy. Reproduction in Domestic
Animals 42 516-526.
47. Allen WR, Brown L, Wright M and Wilsher S (2007) Reproductive
efficiency of Flatrace and National Hunt Thoroughbred mares and stallions
in England. Equine Veterinary Journal 39 438-445
48. Allen WR, Kölling M and Wilsher S (2007) An interesting case of early
pregnancy loss in a mare with persistent endometrial cups. Equine
Veterinary Education 19 539-544.
49. Wilsher S and Allen WR (2007) Intrauterine administration of plant oils
suppresses luteolysis in the mare. Proceedings of the 5th
Joint Meeting of
the UK Fertility Societies. 58. (Abstr)
50. Allen WR, Brown L, Wright M and Wilsher S (2007) Reproductive
efficiency of the Thoroughbred. Proceedings of the 5th
Joint Meeting of the
UK Fertility Societies. 74 (Abstr)
51. Wilsher S and Allen WR (2007) Successful transfer of Day 10 horse
embryos to asynchronous recipient mares. Reproduction in Domestic
Animals 42 74 (Abstr)
52. Allen WR and Wilsher S (2008) The equine endometrial gland: a bountiful
milch-cow of pregnancy in the mare. In: Second Workshop on Embryonic
and Fetal Nutrition. Eds. S Wilsher, WR Allen and JF Wade. Havemeyer
Foundation Monograph Series 21 47-50 (Abstr.)
53. Wilsher S and Allen WR (2008) Effects of a Streptococcus equi infection-
mediated nutritional insult during mid gestation on placental and fetal
development in primiparous Thoroughbred fillies. In: Second Workshop on
Embryonic and Fetal Nutrition. Eds. S Wilsher, WR Allen and JF Wade.
Havemeyer Foundation Monograph Series 21 47-50 (Abstr.)
54. Ousey J, Fowden AL, Wilsher S and Allen WR (2008) Effects of maternal
nutrition state during pregnancy on insulin secretion and sensitivity in
neonatal foals. In: Second Workshop on Embryonic and Fetal Nutrition.
Eds. S Wilsher, WR Allen and JF Wade. Havemeyer Foundation
Monograph Series 21 76-79 (Abstr.)
55. Allen WR, Wilsher S, Morris L H-A, Crowhurst JS, Hillyer MH and Neal
HN (2008) Re-establishment of fertility in ageing mares by laparoscopic
application of PGE2 to their oviducts. Pferdeheilkunde 24 117 (Abstr.)
56. Wilsher S and Allen WR (2008) Maternal recognition of pregnancy in the
mare: Inhibition of luteolysis by intrauterine administration of plant oils.
Pferdeheilkunde 24 118 (Abstr.)
57. Allen WR, Brown L, Wright M and Wilsher S (2008) Reproductive
efficiency of well managed Thoroughbred mares and stallions.
Pferdeheilkunde 24 125 (Abstr.)
58. Wilsher S, Allen WR and Woods JLN (2008) Success and wastage rates in
Thoroughbred flatracing in Britain. Pferdeheilkunde 24 125-126 (Abstr.)
59. Ellenberger C, Wilsher S, Allen WR, Hoffman C, Kölling M, Bazer FW,
Klug J, Schoon D and Schoon HA (2008) Immunolocalisation of the
uterine secretory proteins uterocalin, uteroferrin and uteroglobulin in the
mare’s uterus and placenta throughout gestation. Theriogenology 70 746-
757
60. Wilsher S, Clutton-Brock A and Allen WR (2008) Transfer of day 10
embryos to asynchronous recipient mares. Proceedings of the Havemeyer
7th
International Symposium on Equine Embryo Transfer. Ed: WR Allen.
R&W Publications, Newmarket, UK 50-51 (Abstr)
61. Wilsher S, Clutton-Brock A, Allen WR and Morris L.H-A. (2008)
Transfer of embryos to oestrous recipient mares treated with long-acting
progesterone or altrenogest. Proceedings of the Havemeyer 7th
International Symposium on Equine Embryo Transfer. Ed: WR Allen.
R&W Publications, Newmarket, UK. 77-78 (Abstr)
62. Ousey JC, Fowden AL, Wilsher S and Allen WR (2008) The effects of
maternal health and body condition on the endocrine responses of neonatal
foals. Equine Veterinary Journal 40 673-679
63. Abd-Elnaeim MM, Derar IR, Wilsher S, Allen R, Leiser R and Schuler G
(2009) Immunohistochemical localization of oestrogen receptors alpha and
beta, progesterone receptor and aromatase in the equine placenta.
Reproduction in Domestic Animals 44 312-319
64. Wilsher S and Allen WR (2009) Uterine influences on embryogenesis and
early placentation in the horse revealed by transfer of day-10 embryos to
day-3 recipient mares. Reproduction 137 583-593
65. Wilsher S and Allen WR (2009) Survey of fertility statistics of horse bred
for Flat and National Hunt racing with particular reference to equine
pregnancy failure. In: Rossdale and Partners Equine Pregnancy Failure
Course. Ed S. Ricketts, Lifelearn Ltd, Newmarket UK. 9-15
66. Wilsher S and Allen WR (2009) Does lymphocyte immunisation have a
role in the prevention of equine early pregnancy failure? In: Rossdale and
Partners Equine Pregnancy Failure Course. Ed S. Ricketts, Lifelearn Ltd,
Newmarket UK. pp 53-61
67. Ousey JC, Wilsher S and Allen WR (2009) A survey of placentae
associated with abnormal foals. In: Rossdale and Partners Equine
Pregnancy Failure Course. Ed S. Ricketts, Lifelearn Ltd, Newmarket UK.
pp 101-102
68. Ellenberger C, Muller K, Schoon HA, Wilsher S and Allen WR (2009)
Histological and immunohistochemical characterization of equine
anovulatory haemorrhagic follicles (AHFs). Reproduction in Domestic
Animals (in press)
69. Wilsher S, Lefranc A-C and Allen WR (2009) Postnatal oestrogen
administration stimulates precocious endometrial gland development in the
horse. Equine Veterinary Journal (in press)
70. Wilsher S, Ousey JC and Allen WR (2009) Abnormal umbilical cord
attachment sites in the mare: a review illustrated by three case reports.
Equine Veterinary Journal (in press)
71. Wilsher S and Allen WR (2009) Development and morphology of the
placenta. In: Equine Reproduction 2nd
Edn. Eds. AO McKinnon, E Squires,
D Vaala , D Varner and JL Voss. Wiley Blackwell (in press)
72. Allen WR, Gower S and Wilsher S (2009) Fetal membrane differentiation,
implantation and early placentation in the mare. In: Equine Reproduction
2nd
Edn. Eds. AO McKinnon, E Squires, D Vaala , D Varner and JL Voss.
Wiley Blackwell (in press)
Invited oral communications
1. 1st International Conference on Equine Reproductive Medicine, Leipzig,
Germany. September, 1999.
2. Havemeyer Foundation Workshop on Fetomaternal Control of Pregnancy,
Barbados, West Indies. November, 1999.
3. Havemeyer Foundation Workshop on Comparative Neonatology and
Perinatology, California, USA. March, 2002.
4. Argentinean Symposium on Equine Reproduction, Tandil, Argentina.
November, 2002.
5. New Zealand Link Foundation Breeders Symposium, Agricultural and
Technical College, Hamilton, New Zealand. February, 2003.
6. Havemeyer Foundation Workshop on Embryonic and Fetal Nutrition,
Ravello, Italy. May, 2003.
7. Irish Thoroughbred Breeders’ Association, Co. Kildare, Ireland. October,
2003.
8. Havemeyer Foundation Workshop, 3rd
Meeting of the European Gamete
Group (EEGG), Pardubice, Czech Republic. October, 2003.
9. Inner Mongolia University, Hohhot City, China. November, 2003.
10. Emerging Equine Science Conference, Royal Agricultural College,
Cirencester, UK. September, 2004.
11. Havemeyer Foundation Workshop on Maternal Recognition of Pregnancy
in the Mare III, Barbados, West Indies. November, 2004.
12. A Workshop on the Equine Placenta, Kentucky, USA. December, 2004.
13. Havemeyer Foundation Workshop on Comparative Placentology, Victoria,
Canada. April 2005.
14. 44th
British Equine Veterinary Association Congress, Harrogate,
Yorkshire, UK. September, 2005.
15. Havemeyer Foundation Workshop, 1st International Equine Gametes
Group Workshop, Rostock Germany. September, 2005.
16. Thoroughbred Racing and Breeding Seminar, Cheltenham, UK. November,
2005.
17. New Zealand Link Foundation and Equibreed Breeders Symposium,
Cambridge, New Zealand. April, 2006.
18. Havemeyer Foundation 2nd
Workshop on Embryonic and Fetal Nutrition,
Ravello, Italy. June, 2006.
19. Ghent Veterinary School, Ghent, Belgium. March, 2007.
20. International Scientist Forum on Mammalian Reproductive Biotechnology,
Inner Mongolia University, Hohhot City, Inner Mongolia. August, 2007.
21. 5th
International Conference on Equine Reproductive Medicine, Leipzig,
Germany. September, 2007.
22. Waikato Thoroughbred Breeders’ Association, Cambridge, New Zealand.
March, 2007.
23. Havemeyer Foundation Workshop, Peter Rossdale 80th
Birthday
Symposium, Cambridge, UK. September, 2007.
24. Equine Symposium, Alter Real Stud, Portugal. June, 2008.
25. Seminar of Biotechnology and Reproductive Biology, School of Veterinary
Medicine, Temuco, Chile. September, 2008.
26. 3rd
Annual Equine Science Conference, Open College of Equine Studies,
Boxted Hall, Suffolk, UK. November, 2008.
27. Rossdale and Partners Equine Pregnancy Failure Veterinary Course,
Newmarket, UK. January, 2009.
Submitted abstracts
Oral presentations
1. 50th
Annual Meeting of the European Association Animal Production, Basel,
Switzerland. December, 1999.
2. Society for Study of Reproduction and Fertility, Edinburgh, Scotland. July,
2000.
3. 5th
International Symposium on Equine Embryo Transfer, Saari, Finland.
July, 2000.
4. 9th
Conference of the European Placenta Group, Sorrento, Italy.
September, 2001.
5. 8th
International Symposium on Equine Reproduction, Colorado, USA.
August, 2002.
6. 7th
Annual Conference of the European Society for Domestic Animal
Reproduction, Dublin, Ireland. August, 2003.
7. 6th
International Symposium on Equine Embryo Transfer, Rio de Janeiro,
Brazil. August, 2004.
8. 9th
Annual Conference of the European Society for Domestic Animal
Reproduction, Murcia, Spain. September, 2005.
9. 9th
International Conference on Equine Reproduction, Kerkade, The
Netherlands. August, 2006.
10. 11th
Annual Conference of the European Society for Domestic Animal
Reproduction, Celle, Germany. September, 2007.
11. 67h
International Symposium on Equine Embryo Transfer, Cambridge, UK.
July, 2008.
Poster presentations
1. 5th
Annual Conference of the European Society for Domestic Animal
Reproduction, Vienna, Austria. August, 2001.
2. 8th
Meeting of the International Federation of Placenta Associations,
Melbourne, Australia. October, 2002.
3. 15th
International Conference on Animal Reproduction, Porto Segura,
Brazil. August, 2004.
4. 10th
Annual Conference of the European Society for Domestic Animal
Reproduction, Ljubljana, Slovenia. September, 2006.
5. Fertility 2007: Biennial Meeting of the UK Fertility Societies, York, UK.
July, 2007.
