Sperm production native Thai stallions
Research internship Veterinary Medicine 2009
Manon Struik
0249106
1
Index
- Introduction
page 3
- Hypothesis
page 5
- Material and methods
page 6
- Results
page 9
- Discussion
page 21
- Conclusion
page 25
- Appendix
page 27
- Literature
page 28
2
Abstract
Thailand possesses a small population of native pony's. There's no studbook or any other kind
of registration for this breed. There seemed to be some problems involving fertility according
to local Thai people living in the provence of Khon Kaen.
The study involved five adult native Thai stallions who were examined in the months January
and February 2009. Total testicular volume and total testicular width were measured followed
by assessment of two ejaculates gathered with an hour in between. Volume and concentration
of the ejaculates were examined as well as the total sperm number (TSN) of the ejaculate,
motility, vitality, morphology, total normal built moving sperm (TNM) and the sperm plasma
membrane function by the HOS-test (hypo-osmotic swelling test). These parameters were
compared with those of sires of Dutch Studbooks; Dutch Warmblood horse, Friesian horse,
Welsh Pony and New Forest.
Despite a relative big total testicular width and total scrotal volume of the native Thai pony,
the average TSN (2425.50x10^6 SD:234.05x10^6) was significantly lower compared to the
other breeds, as well as the average TNM (1234.37x10^6 SD:372.41x10^6).
HOS-test outcome was 22.70% (SD:3.26%) HOS+ which is assumed to be very low,
indicating a low sperm plasma membrane quality.
This outcomes are indicative of a poor sperm production and quality of the native Thai pony.
Since only five pony's are involved in this study, more research should be done to confirm
this. Whether inbreeding and climate play a role in these results should be subject to near
investigation.
3
Introduction
Thailand has a small population of horses, probably only a few thousands.
There are two main horse groups in Thailand: one of them is the group of the sport, full-size
purebred horses including Arabians, Warmbloods and Thoroughbreds. These were all
originally imported. The native Thai horse is a pony horse and may originally have originated
from a breed of the Burmese ponies.12, 13 This native breed is generally used in religious
ceremonies, for recreational activities, and occasionally for transportation in highland areas.13
Natural breeding is still commonly performed to increase horse numbers. Chilled semen or
frozen semen are not yet available for the native Thai pony’s. Artificial inseminations with
chilled or frozen-thaw semen may be an important method for increasing population numbers
of this horse, especially looking at the small size of the population.
Nothing is known about the offspring of the pony`s or how the fertility is. At the moment of
writing the author is aware of only two studies involving the sperm production of crossbreds
of the native Thai pony.
The purpose of this study was to investigate the sperm production of the native Thai pony and
so to predict fertility.
Breeding Soundness Evaluation
The best way to assess fertility is simply to calculate the foaling rate of a stallion that he has
achieved and relate this to the number of mares he has served. Since it will take at least a year
to gather those data there are some other ways to evaluate fertility. The second best way is to
calculate the number of services needed per pregnancy or the number of cycles needed for
repeated insemination to lead to pregnancy. To gather these data one should have a good
registration of the services of the stallions. Since these data are rarely available, especially not
in a country like Thailand, there is another method to predict fertility. This method includes a
physical examination and semen evaluation. For a lot of breeds and studbooks, conditions
involving the semen, are created on which the stallions have to conform to. The Society for
Theriogenology developed and published in 1983 a recommended protocol for assessing the
breeding potential of stallions. This protocol is called the Breeding Soundness Evaluation
(BSE). This protocol includes the following components:
- Obtaining a general and reproductive history
- Performing a general physical examination
- Collecting a blood sample for testing for Equine Infectious Anemia
- Performing a physical examination of the penis
- Obtaining swab samples
- Evaluating breeding behaviour and libido
- Examining the scrotal contents, including measurements of the length, width and
height of each testicle and measurement of the scrotal width
- Examining the internal accessory glands and inguinal rings per rectum
- Collecting two semen ejaculates samples, 1 hour apart, each ejaculate is then analyzed
to determine volume, concentration, pH, motility (and its longevity when stored), total
sperm number and total number of normal built, good moving sperms (further called
TNM).
For classification as a Satisfactory Prospective Breeder, a stallion must be satisfactory in all of
the above points. A Satisfactory Prospective Breeder can be expected to successfully render
pregnant a minimum of 75% of a book of 40 mares if bred by natural cover as in this study, or
120 mares if bred by artificial insemination under good management conditions and given
mares of reasonable fertility.
4
To be classified as a Satisfactory Prospective Breeder, the stallions are also ought to have two
normal, scrotal testes and epididymides with a recommended minimum total scrotal width of
8 cm.9 Other classifications are Questionable Prospective Breeders or Unsatisfactory
Prospective Breeders (such as cryptorchids).
The purpose of a stallion breeding soundness evaluation is to identify those stallions that can
be expected to be incapable of achieving specific minimum levels of fertility, or at least alert
owners of potential problems. In addition, the examination assists in identifying potential
causes of reduced fertility so that measures can be taken to maximize stallion fertility. It is
crucial to understand that the main objective of semen evaluation is to identify infertile and
subfertile stallions, those of which fertility is expected to be <40% of the average for stallions
of the same type (breed, age).
The whole protocol of BSE is quite extensive, so the most studbooks have a selected part of
the BSE in their standard evaluation of the stallions. The stallions of the Dutch Studbooks are
evaluated for their testicular volume and consistency, and the TNM before they can be added
to the Studbook. The motility of the sperm is evaluated; 50% has to be motile. Also the
morphology is evaluated; 50% of the sperm has to be of normal morphology.
The motility of the spermatozoan is determined by the conformation of the tail. The tail is
divided into midpiece, principal piece and end piece. The axoneme extends through the entire
length of the tail and is formed by nine doublets of microtubules arranged around a central
pair of microtubules, which are the elements that contract to produce sperm tail movement.
The principal piece is the longest segment of the tail, with dense fibers and a fibrous sheath to
provide structural support and flexibility. The end piece is the short terminal segment of the
tail containing only the axoneme.
When multiplying the concentration with the total amount of sperm with a normal
morphology and normal motility one gets the TNM: total number of normal built, good
moving sperms. The minimum norm for the TNM of the Dutch Warmblood stallion is 1500 x
106 over the average of two ejaculates taken with an hour in between. In this Studbook they
make differentiations in sperm quality since 2006. There are four categories now: good,
sufficient, poor and insufficient. Only stallions with insufficient sperm (less then 1000 TNM,
less then 30% living cells and less then 30% moving with a normal morphology) are rejected
because of the insufficient sperm quality.
There are other conditions though for an other Dutch Studbook, which is The Friesch
Studbook. The minimum TNM sires of the Friesch Studbook have to conform to, is lower as
it is for the Studbook of the Dutch Warmblood because of the lover TNM produced by most
of the sires of the Friesian Studbook. The stallions of this Studbook should have a minimum
TNM of 600 x 106 to have access to the Studbook.
This study aims to investigate the sperm production of the native Thai pony and compare the
results of some semen characteristics with the characteristics of stallions of registered
Studbooks; the Welsh Pony, New Forest, Dutch Warmblood and the Friesian horse.
5
Hypothesis
I expect the average total scrotal width of the Thai pony to be smaller then the total scrotal
width of the Dutch Warmblood horses and Friesian horses, for the height of the Thai pony is
way smaller, and so in turn I expect their testicular volume to be smaller as well. I expect the
testicular width of the Thai Pony to be comparable with the testicular width of the Welsh
Pony and New Forest, for they are more or less in the same height-category. The scrotal width
is correlated with the total sperm number, so I expect the average total sperm number of the
Thai pony to be smaller compared to the Dutch Warmblood horses, but comparable with the
average total sperm number of the Welsh Pony and the New Forest.
Compared to the Friesian horse I expect the Thai pony to have a comparable average TSN,
despite an expected smaller total scrotal width. This because Friesians are known to have a
lower TSN as a result of a high level of inbreeding within this breed.
According to the TNM I expect the Thai Pony to have a significantly lower TNM compared
to the Dutch Warmblood stallions and the Welsh Pony’s and New Forests. This because of the
small pool of local stallions, what imputes a high level of inbreeding. Higher inbreeding
coefficients are correlated with a significantly lower sperm quality resulting in lower
percentages of progressively motile and morphologically normal sperm, and as a result a
significantly lower TNM value, following a study involving the Shetland Pony.26
So in this study I expect the Thai pony to have an average high Total Sperm Count, but an
average low percentage of progressively motile and morphologically normal sperm, and so a
low TNM.
6
Materials and methods
-
Stallions
During January and February 2009, 5 adult Thai stallions (T) were examined and semen was
collected. The stallions had no breeding record, but they had all experience in mounting
mares. The semen was collected three and if possible four times, every other day, with the aid
of an artificial vagina (Hannover model) while allowing them to mount a teasing mare. Every
time two ejaculates were collected with an hour in between. The ejaculates collected on day 3,
so the third and fourth ejaculates, were evaluated and used for the results, to make a good
comparison with the results of the stallions that jump in Holland for the Studbooks.
The age of the stallions varied between 3 and 15 years, with an average of 9.6 years.
Because there were no official papers belonging to the stallions, the horses were aged by their
teeth.
Physical examination showed that all stallions had normal reproductive tracts; testes with a
normal consistency and no cryptorchids were present.
All stallions showed a high libido.
Table 1 Data Thai stallions
Age (yrs)
Height (cm)
History
T1
T2
T3
T4
9
15
3
15
120
115
120
119
T5
6
123
-*
-*
-*
Transport +- 60
days before
samples
Transport day
before first
sample
Experienced
Y/N
Y
Y
Y
Y
Y
119.4 ± 2.88
(SD)
* = no relevant information effecting fertility
Average:
9.6 ± 5.37(SD)
The parameters evaluated in this study are:
- Total scrotal width and Total testicular volume
- Volume of ejaculate
- Motility
- Concentration of ejaculates
- Total Sperm Number (TSN)
- Vitality
- Morphology
- TNM
- Plasma membrane function
Total scrotal width and Total testicular volume
The testicular width, length and height were measured manually between finger and thump
next to a ruler to have a rough measure of the testicular size. This examination showed that
there were no cryptorchids.
7
Testicular size or volume is a direct measure of the amount of testicular parenchyma present,
which in turn determines potential sperm production. Small testicular size is associated with a
reduced sperm production.24
A formula for calculating the testicular volume is the formula for the volume of an ellipsoid.
So for each testicle, testicular volume equals 4/3π X length/2 X width/2 X height/2.
Total testicular volume is the sum of the volumes of the left and right testicles together.25
Since research showed there is a direct correlation between total testicular volume and Daily
Sperm Output (DSO), a formula is available to calculate the expected DSO for a stallion
based on that individuals testicular volume:
DSO (billions/109) = 0,2 (total testicular volume) – 1.26.25
One gets Daily Sperm Output after collecting semen once a day for a period of ten days.
Total scrotal width is calculated by the formula: TSW(cm) = 1,74 + 0,696 (LW+RW), with
LW left scrotal width, and RW right scrotal width.
Volume of the ejaculate
After the ejaculate was taken, the gel fraction was separated by a gauze. After, the gel-free
volume of the ejaculate was determined in millimetres.
Motility
The semen was diluted 1:3 immediately after the removal of the gel fraction with non-fat dry
milk semen extender. Total motility was assessed by examine a drop of this fresh diluted
semen on a prewarmed slide under the microscope with an objective of 400x. The motility
was estimated and written down in percentages.
Concentration
To determine the concentration of the ejaculate, the sperm was usually diluted 1:20 with
formal saline. Using a Bürker-Türk counting chamber, 10 μl of the diluted semen was added
at both sides of the Bürker-Türk. After waiting for about a minute, while the sperm was
spreading under the glass, five chambers of both sides of the Bürker-Türk were counted for
the amount of spermatozoa.
TSN
When volume and concentration of the ejaculate is known, one can calculate the TSN (total
sperm number) of the ejaculate by multiplying these two parameters.
Vitality
Immediately after, a drop of 5 μl of fresh sperm was put on a prewarmed slide (37°C) and a
drop of prewarmed eosine-aniline blue solution was put on this slide as well and gently mixed
with the sperm drop. A smear was made and dried for a about a minute on the warm stage
(37°C). For viability, 200 spermatozoa were determined, and either classified as dead
(stained, red) or alive (white, unstained).
Morphology
This blue-coloured eosine-aniline slides were taken back to the Netherlands, Utrecht, for
morphological examination. Here the slides were examined under an 1000x objective and
classified as normal or abnormal using the Brettschneider-table, not distinguishing dead or
alive spermatozoa.
The TNM (in millions) was calculated by multiplying the volume, percentage of motility,
concentration and percentage of normal morphological spermatozoa.
8
The mean value of the TNM for the Thai native crossbred is compared with the values for
some others breeds; Welsh Pony, New Forest, Dutch Warmblood and the Friesian horse.
Therefore a Dutch database is used in which parameters are registered for young stallions who
come to the Utrecht University Reproduction for a breeding soundness evaluation.
The TNM is the final result in this evaluation, which is used for comparison with the results
of this study.
The stallions from the database are usually young stallions being 2.5 – 3 years old. Some of
them jumped already before, some of them never did.
The number of Welsh Pony’s used is 49, 48 New Forests, 127 Dutch Warmbloods and 101
Friesian horses.
HOS-test
Since the integrity and the functionality of the sperm plasma membrane are very important in
the fertilisation process, these parameters are evaluated as well by accomplishing the Hypo
Osmotic-Swelling (HOS) test. This test is based on the capacity of sperm plasma membranes
to establish osmotic equilibrium when exposed to a hypo osmotic solution, producing a
typical swelling of the tail.
For this HOS-test 50 μl of fresh semen was added to 450 μl of a citrate buffer hypoosmotic
solution (100 mOsm/l) and placed on the warm stage at 37 °C for 30-45 minutes. After, 500
μl of the HOS-solution (100 mOsm lactose/l) with 5% formal saline was added to be able to
keep the samples for long-term storage. At least 200 spermatozoa were observed at
magnification 400x and classified by the presence or absence of a swollen tail (curled/coiled
principal or end piece classified as swollen, but bented tails classified as unswollen).
For the final results only the third and fourth ejaculates are being calculated. This is because
the data these results are compared with, are all from stallions who gave their ejaculates to be
determined to get access to the Dutch Studbooks. There is a protocol in which one ejaculate is
collected when the stallions arrive at the clinic where the semen will be assessed, and a
second ejaculate after an hour. The average of the two ejaculates is the final result used for the
Studbook. Because many of these stallions mounted a few times before, the stallions in this
study also mounted two times before, two days before the third and fourth ejaculates.
So in this study we also used the averages of the third and fourth ejaculates, with an hour in
between.
9
Results
Standard deviation in the following results are characterized by the symbol ±.
1. Total testicular volume (TTV) and total scrotal width (TSW)
The average total scrotal width of the testicles of the Thai pony in this study is 9.26 cm with a
standard deviation of 0.81 cm. The testicular volume is for the left testicle 66.50 cm3 ±18.40
cm3 and for the right testicle 93.96 cm3 ±20.69 cm3.
The average total testicular volume is 160.46 cm3.
Table 2 Testicular sizes Thai stallions ± SD
Stallion
T1
L
testis
length
(cm)
7
L
testis
width
(cm)
5
L
testis
height
(cm)
6
R
testis
length
(cm)
7
R
testis
width
(cm)
5
R
testis
height
(cm)
9
T2
7
5
5
9.5
5
6
T3
7.5
5.5
5.5
8
5.5
6.5
T4
8.5
6
6.5
9
7
7
T5
7.5
4.5
5.5
7.5
6.5
5.5
Average:
7.5 ±
0.61
5.2 ±
0.57
5.7 ±
0.57
8.2 ±
0.93
5.8 ±
0.81
6.8 ±
1.21
5.2
5.3
8.3
5.3
5.1
Thai
8.9
Crossbred
(n = 5)19
Testicular Total
volume
Testicular
3
(cm )
volume
(cm³)
L:61.85
154.63
R:92.78
L:51.54
8.7
135.48
R:83.94
9.396 L:66.82
151.05
R:84.23
10.788 L:97.63
227.52
R:129.89
L:54.67
8.7
133.64
R:78.97
9.26 ± L:66.50 ± 160.46 ±
18.40
0.81
38.61
R:93.96 ±
20.69
L:131.3 ± 251.1
9.05
14.5
R:119.8 ±
14.5
Total
scrotal
width
(cm)
8.7
Scrotal Volume = 4/3π X length/2 X width/2 X height/2
10
Table 3 shows the average testicular sizes of other breeds, from other studies.
Table 3 Testicular sizes ±SD
Breed
L testis
length (cm)
L testis
width (cm)
R testis
length (cm)
R testis
width (cm)
Welsh Pony and
New Forest
(n=56)
Warm blood
(n=398)
Friesian (n=84)
8.6 ± 1.0
4.9 ± 0.7
8.5 ± 1.0
4.9 ± 0.7
Total scrotal
width (TSW)
in cm
8.56
9.8 ± 0.9
5.6 ± 0.8
9.9 ± 0.9
5.7 ± 0.8
9.60
8.9 ± 1.2
5.0 ± 0.8
8.9 ± 1.1
5.2 ± 0.8
8.84
Thai pony (n=5)
7.5 ± 0.6
5.2 ± 0.57
8.2 ± 0.93
5.8 ± 0.81
9.26 ± 0.81
8.3
5.3
9.05
6.63 ± 0.07
4.14 ± 0.05
7.56
Thai Crossbred
8.9
5.2
19
(n=5)
Shetland Pony
6.74 ± 0.07
4.2 ± 0.05
(n=216)17
TSW(cm) = 1,74 + 0,696 (LW + RW)
There was a difference in total testicular volume for the stallions of this study compared to the
5 stallions of the study of Phetudomsinsuk et al. (2008). Some difference can be explained by
the fact that the testicular size of each horse in that study was measured with callipers, while
in this study the testicular size is measured manually, between thumb and finger, next to a
ruler. The pony’s used in the study of Phetudomsinsuk et al. are crossbreds, so possibly
crossed with full-size horses, what might explain a bigger scrotal volume.
The total scrotal width of the Thai Pony is bigger than those of the other breeds, except for the
Dutch Warmblood horse. The size of the testicles is expected to be in accordance with the
height of the horse. According to the height of the Thai Pony, which was 1.19 meter average
for these five stallions, a smaller testicular size was to be expected.
Since total scrotal width and total testicular volume are parameters for the sperm production,
the comparative big testicles of these Thai Ponies should produce quite a lot of sperm
(predicted DSO in Table 7). The total amount of spermatozoa of the stallions in this
experiment though, is even smaller than the average total amount found for the Shetland
Pony. So, apparently they do not produce that much sperm as one should expect according to
their testicular volume.
The sizes of the testicles of the ponies were not exactly corresponding to their TNM, but there
is a positive correlation of 0.022 between the TTV and TNM of the individual pony’s. Using
the Spearman’s rank correlation coefficient (rs) with n=5 and a correlation coefficient of
0.022, the P > 0.1. This means the correlation between total testicular volume and total sperm
number in this study is not significant though.
11
Table 4 Total testicular volume compared to total sperm number
Stallions
Total Testicular
Total sperm number
volume (cm³)
(x106)
T1
154.63
1613.5
T2
135.48
2139.5
T3
151.05
1387.5
T4
227.52
2260
T5
133.64
2591
DSO predicted
(x106)
2966.66
2583.60
2895
4424
2546.8
A positive correlation coefficient of 0.058 is shown for the TSN and TSW. Using the table
for the Spearman’s rank correlation coefficient, with n=5 and an coefficient of 0.058 gives
P>0.1. This means there’s no significant correlation for the TSW and the TSN seen in the
averages of these breeds.
Table 5 TSW compared to TSN in different breeds ±SD
Total scrotal width Total sperm
(TSW) in cm
number (x 106)
Welsh Pony and New 8.56
Forest (n=56)
Warm blood (n=398) 9.60
14717
Friesian (n=84)
8.84
8964
Thai pony (n=5)
9.26 ± 0.81
2425.50 ± 234.05
Thai Crossbred
(n=5)19
Shetland Pony
(n=216)17
9.05
13596
7.56
4940 (SE:90)
13390
12
2. Total Sperm Number
a. Volume
The average volume of the ejaculates of the Thai stallions was 56.2 ml with a standard
deviation of 30.29 ml. This value is within the normal range for a stallion which is 50-200 ml.
Stallion number one had an average volume under 50 ml, the others above (table 6).
Table 6 Volume (ml) ejaculates of the native Thai Pony ±SD
T1
T2
T3
T4
Stallion
T5
3rd ejaculate
4th ejaculate
44
17
48
55
20
55
120
95
70
38
Average
30.5
51.5
37.5
107.5
54
56.20±30.29
Volume and concentration both are subject to many circumstances, for example the amount of
ejaculates the stallion produces per week or per day at the time of sampling, and the fact
whether the stallion had ever mounted anyways. Other factors that influence the volume are
degree of sexual stimulation before collection, breeding conditions and foreign material in the
ejaculate. The semen volume can be increased for example by excessive precopulatory teasing
because of increasing of the accessory sex gland secretions, which increase volume but
decrease spermatozoa concentration in the ejaculate. So volume and concentration should
never be evaluated separately.
In this project all the stallions had mounted at least once, but it was unknown when they had
mounted for the last time. The history according to the frequency of mounting however, of the
stallions which are examined for the Dutch Studbooks, aren’t known either.
Table 7 Parameters compared with different breeds ± SD
Breed
Volume
Motility (%) Vitality (%) Concentration Normal
(ml)
(x 106)/mL
morphology
(%)
Welsh/
New
Forest5
(n=56)
Dutch
Warm
blood5
(n=398)
Friesian5
(n=84)
Thai Pony
(n=5)
Thai
Crossbred
(n=5)19
Shetland17
(n=285)
46.5
67.5
-
316.5
-
Total
sperm
number
(x 106)
14717*2
65
69.5
-
206
-
13390*2
50.5
64
-
177.5
-
8964*2
56.2
±30.29
44
(SEM*1:2.1)
76.67±
7.64
77.8
(SEM*1:1.3)
82.23±
6.01
75.5
(SEM*1:1.3)
42.95±
14.37
309
(SEM*1:30.7)
77.75±
2425.50
5.02
±234.05
49.7
13596*2
1
(SEM* :1.3)
23.5
66.05
71.6
230.05
57.45
5406*2
13
*1 SEM: Standard error of the mean
*2 B. Colenbrander et al. (1992)
b. Concentration
The average concentration was 42.95 x 106 spermatozoa per ml with a standard deviation of
14.37 x 106 /mL. The sperm concentration in stallions is typically 100-400 x 106 /mL.1
All of the stallions had a ejaculate concentration under 60x106/ml, which is considered to be
low, but concentration cannot be criticized without taking volume into account. The result of
the concentration multiplied by the cell-free volume gives the total sperm number of the
ejaculate; TSN, which is a more important parameter.
Table 8 Sperm concentration (x10/ml6) of the native Thai Pony ±SD
T1
T2
T3
T4
Stallion
3rd
57.5
60.5
108.5
25
th
4
41
25
11
16
Average
49.25
42.75
59.75
20.5
T5
61
24
42.5
42.95±14.37
Total Sperm Number
The total sperm number of the Thai pony in this project is 2425.50 x 106 spermatozoa per ml
(±234.05 x 106) average, which is significantly lower compared to the other breeds (table 10
& graphics 1), and even lower than the average of the Shetland Pony which has the lowest
average TSN (5406 x 106).
This despite their quite big testicular size. As visible in table 9 especially the second ejaculate
has a much lower TSN for all of the stallions.
Table 9 TSN (x106) of the native Thai Pony ±SD
T1
T2
T3
Stallion
rd
3
2530
2904
2170
4th
697
1375
605
T4
3000
1520
T5
4270
912
Average
2260
2591
1613.5
2139.5
1387.5
3555.5±
1364.01
Table 10 Total sperm number different breeds ±SD
Breed
TSN (x 106)
Min/Max
Welsh Pony (n=49)
9967.18 ± 6684.13
New Forest (n=48)
7736.01 ± 4861.97
KWPN (n=127)
10257.15 ± 6873.74
Fries (n=101)
7606.57 ± 5430.50
Native Thai Pony
(n=5)
2425.50 ± 234.05
Min: 810
Max: 39168
Min: 1330
Max: 22952
Min: 1380
Max: 36135
Min: 546
Max: 26506
Min: 605
Max: 4270
14
Graphics 1 Total sperm number different breeds
TSN
(x106)
Ras
3. Quality
a. Motility
The average motility was 76.67% ±7.64 %. Since motility above 50% is considered to be
good, the average of 76.67% is a good value. The stallions were consistent in having a high
percentage good moving sperms.
This average motility is consistent with the findings of Phetudomsinsuk et al. (2008) for five
Thai native crossbred stallions. This study shows an average total motility of 77.8%
(SE=standard error: 1.3%).
These findings are higher than the average percentage of motility found for the other breeds
listed in table 7.
Table 11 Motility (%) of the sperm of the native Thai Pony
T1
T2
T3
Stallion
rd
3
70
85
85
4th
70
80
85
±SD
T4
70
80
T5
80
75
Average
75
77.5
70
82.5
85
76.67±7.64
15
b. Vitality
The average vitality was 82.23% with a standard deviation of 6.01%.
Motility should reflect vitality, since vital sperms usually move. So the average motility
should be very close to the average vitality. In this study there is a slight difference, with an
average percentage motility of 76.67% ±7.64 %, but it shows similarity with the average
vitality in being close to the 80%. All the individuals have vitality percentages close to 80%
as well (table 11).
Table 12 Vitality(%) of the sperm of the native Thai Pony ±SD
T1
T2
T3
T4
Stallion
T5
3rd
4th
Average
75.71
73.6
74.66
76.4
84.4
80.40
83.5
90
86.75
90
89.5
89.75
67.2
92
79.60
82.23
±6.01
There are not many studies in which vitality is taken into account, mostly because of the
similarity with the motility, which is of greater importance. So more emphasis will be put on
motility.
The study of Phetudomsinsuk et al. (2008) shows an average vitality for the native Thai
crossbred of 75.5% (SEM=standard error of the mean: 1.3%). In this study also full-size
purebred stallions were involved (n=4), representing an average vitality of 73.9% (SEM:
1.3%).
Another study involving Shetland Pony’s shows an average vitality of 71.6% (n=285).26
c. Normal Morphology
The average of spermatozoa with a normal morphology in this study was 77.75% ±5.02%. A
stallion should have an average normal morphology above 50%. So all the stallions in this
study meet this requirement easily.
The average percentage of normal morphologic spermatozoa is quite different though from
the 49.7% found by Phetudomsinsuk et al. (2008), involving Thai crossbreds.
In that study four full-size purebred stallions are examined as well, having a mean value of
48.1% morphologically normal sperm. This corresponds to earlier findings by Long et al.
(1993) 47.5% and Pickett (1993) 51.3% and Kavak et al. (1994) Tori; 57.5%, but are lower
than the findings of Parlevliet et al. (1994) Dutch Warmblood; 66%, Dowset and Knott (1996)
Standardbreds; 67.8% and Kavak et al. (2004) Estonian; 74.4%.
For the Shetland pony a percentage of 57.45 was found for normal morphological
spermatozoa.
Colenbrander et al. (1992) also showed that Warmblood, Welsh and New Forest stallions
evaluated at 3 years of age for breed registry seemed to have a greater percentage of normal
sperm (approximately 70%) than Friesian and Shetland stallions indeed (approximately 55%).
So there seems to be a quite big difference between breeds.
Within this study there’s a range between averages of 72.5% normal morphology and 85.5%
(also see appendix 1).
16
Table 13 Morphology (%) of the sperm of the Thai Pony ±SD
T1
T2
T3
T4
Stallion
T5
3rd
4th
Average
81
90
85.5
75
70
74.5
86.5
72
79.25
72.5
72.5
72.5
75
79
77
77.75
±5.02
d. TNM
As described before, the Total Number of Morphologically normal motile spermatozoa, short:
TNM, is calculated by multiplying the volume with the concentration, percentage normal
morphologic sperm and percentage sperm with a normal motility.
The native Thai stallions in this study had an average TNM of 1234.37x106 ±372.41x106
which is significantly lower than the averages of the other Dutch breeds.
Table 4 shows the results of the Thai stallions when all ejaculates are taken in account. This
gives an average TNM of 1352.78 with a standard deviation of 116.91.
Table 14 TNM Thai Pony all ejaculates (n=5)
Stallion TNM (x 106)
T1
T2
T3
T4
T5
1I
1596.4
2131.4
1432.3
1I
1161.6
2028.3
1II
440.2
1271.6
901.5
1II
1126.8
3204.4
2I
1348.7
2152.8
1343.4
2I
1575
2767
2II
361.0
792.0
372.8
2II
1015.4
615.6
3I
2144.8
2002.8
1284.6
3I
1589.8
897.4
3II
711.7
1792.8
683.1
3II
1239.3
685.4
4I
2220.80
1358.6
2282
4II
492.80
949.2
747.4
1234.37
±372.41
Average:
1392.53
1525.85
1249.28
1354.88
1241.35
1352.78
± 116.91
Fat printed values are the 3th and 4th ejaculates, and used in this study for the final results.
Looking at the minima and maxima, we only find in the Friesian Studbook one stallion with
an ejaculate who has even a lower TNM (235x106) than the lowest TNM of the Thai stallions
(361x106) in this project, though this group is much bigger (n=101).
According to the BSE (The Standard Breeding Soundness Evaluation) as proposed by the
Society for Theriogenology, stallions that fail to produce 1 billion morphologically normal
progressively motile sperm in each of the 2 ejaculates at any time during the year, should be
classified as questionable or unsatisfactory prospective breeders, depending on the severity of
the problem.25
That means for the stallions in this study, all of them would be classified as a satisfactory
prospective breeder, looking at this point.
17
Table 15 TNM different breeds
Breed
TNM (x 106) ±
SD
Min/Max
Welsh pony
(n = 49)
New Forest
(n = 48)
KWPN
(n = 127)
Friesian
(n = 101)
Thai pony
(n = 5)
Min: 910
Max: 12238
Min: 901
Max: 7552
Min: 518
Max: 13916
Min: 235
Max: 9252
Min: 361
Max: 3204
4684 ± 2444.88
SE: 345.57
3779 ± 2030.95
SE: 349.15
4699 ± 2815.89
SE: 214.65
3005 ± 2052.61
SE: 240.70
1234.37 ±
372.41
SE: 1081.81
Thai Crossbred
(n = 5)12
Shetland pony
(n = 285)6
9031
2051
With the aid of SPSS 16.0 the standard error of the mean, the Standard Deviation and the 95%
Confidence interval are calculated. The TNM of the native Thai pony is consistently under the
95% confidence interval of the other breeds which is clearly shown in the boxplot (graphics
2).
18
Graphics 2 Boxplot TNM different breeds
TNM
(x109)
When analyzing the data to define whether there are significant differences with the aid of
SPSS 16.0 using the Mann-Whitney test, it becomes clear there is a significant difference
between all of the breeds and the native Thai pony, listed in table 16.
Table 16 Significance TNM
Breed
Significance
Welsh Pony
0.001
New Forest
0.001
KWPN
0.001
Friesian
0.019
e. HOS-test
The results of the HOS-test for the individual stallion are shown in table 5. The average gives
a percentage of 22.70% positively responding spermatozoa to the test, with a standard
deviation of 3.26%. There is no official range yet, so this result can only be compared with
some results from other studies.
19
Table 17 HOS (+)% of the sperm of the Thai Pony ±SD
T1
T2
T3
T4
Stallion
T5
3rd ejaculate
4th ejaculate
Average
11.65
19.82
15.74
0
19.4
9.7
51.2
43.6
47.4
12.02
15.1
13.56
27.1
27.1
27.1
22.70
±3.26
The correlation between HOS(+)% and fertility is confirmed by Neild et al. (2000) who found
a correlation of r = 0.08 (P=0,75) with pregnancy rate (%) for 25 Thoroughbred stallions.
Stallions were divided in two groups: stallions of acceptable fertility (n=18) and stallions of
doubtful fertility (n=7) based on threshold levels of fertility proposed by Dowsett and Pattie
(1982).
The first group had a mean swollen spermatozoa with the HOS test of 56.1% ±14.2% and the
second group with doubtful fertility 28.0% ± 11.9%, which was significantly different
(P<0.05).
The HOS test also proved to have a correlation of r = 0.82 with progressive motility (%), r =
0.53 with morphologically normal spermatozoa (%), r = -0.13 with tail abnormalities and r = 0.40 with number of services per pregnancy.
The results obtained here show stallions with low swelling scores (HOS <40%) to be of
doubtful fertility. None of the stallions in this study has HOS scores of 40% or more and
according to Neild et al. they should be considered as sires with doubtful fertility.
An earlier study with 30 horses from different breeds, of Neild et al. (1999) shows an average
of 45.6% swollen tails ±6.7% (at 100 mosm lactose). So the percentage of functional
spermatozoa which undergo swelling to establish osmotic equilibrium in the hypo osmotic
swelling test is more than twice as high as it is for the Thai pony.
This study of Neild et al. also shows a positive relationship between HOS and progressive
motility of 0.75 and also a positive relationship between HOS and morphologically normal
spermatozoa; r = 0.51 which is consensus the study in 2000.
So according to the low outcomes of the HOS-tests in this study, one should expect the
progressive motility and the morphology of the samples of these five stallions to be low as
well. But even the samples with the very low percentages of swollen tails don’t have that low
percentages of progressive motility and morphology.
In another study, involving the Thai crossbred, the HOS positive sperm percentage was 58.7%
± 1.9% at 50 mOsm/l lactose solution.19
The study of Nie and Wenzel (2001) showed a HOS(+) % of 64.1% ± 3.0% for a 100 mOsm/l
sucrose-solution in an experiment with 10 mature stallions of various horse breeds.
Human ejaculates are considered normal when 60% or more HOS(+) spermatozoa are
observed. When fewer than 50% are HOS(+) the sample is classified as abnormal. Results
between 50 and 59% are considered suspect, but no definitive statement can be made about
the sample quality. Abnormal HOS test results, as we have here, are felt to provide reasonable
certainty that the ejaculate is infertile or sub fertile.
But normal values for the percentage of HOS(+) cells of stallions, have been reported for only
a limited number of research animals. Thus, normal ranges for the purpose of clinical assays
for stallions have not been validated.
Still, in this study the HOS(+)% is much lower as it is for stallions in other studies. This
would suggest a poor plasma membrane quality, what would suggest infertile or sub fertile
ejaculates.
20
This is in contrast though with the study of Phetudomsinsuk et al. (2005), where the HOS(+)
percentages of the Thai crossbreed are more then twice as high as in this study. Since this
breed is involves native Thai pony’s crossed with other breeds this might explain the
difference.
Graphics 5 HOS(+)% different studies
Light-horse Thai CrossbredFull-size horses
breeds
Thai native
pony
70
60
50
40
30
20
10
0
HOS(+)%
Light-horse breeds: G.J. Nie (2001) HOS(+)%: 64,1 ± 3,0 (100 mOsm/l sucrose), n=10
Thai Crossbred: K. Phetudomsinsuk (2009) HOS(+)%: 58,7 ± 1,9 (50 mOsm/l lactose), n = 5
Full-size horses: D. Neild (1997) HOS(+)%: 45,6% ± 6,7 (100 mOsm/l lactose) 45,7 ± 4 (100
mOsm/l sucrose) n= 30
21
Discussion
The correlation between total testicular volume and total sperm number in this study was
found to be not significant.
This can be explained by the small number of stallions used in this study.
The Total scrotal width was quite big compared to other breeds, even though the Thai pony is
a small pony. This can be coincidence because of the small number of stallions as well.
Dowsett and Knott found out in 1996 in a study involving 168 stallions representing 9 breeds
and ranging in age from 2 to 26 years that semen quality (gel-free volume, sperm
concentration, total sperm numbers and sperm abnormalities) was poorest in stallions under 3
yr of age and over 11 yr. In this study two stallions (T2, T4) are about 15 years old and so the
semen quality of these two stallions can be poor due to age.
Total scrotal width is smaller in 2- to 3-year-old stallions than in their older counterparts.
Stallions that are 2 or 3 years old are expected to have smaller daily sperm outputs and
spermatozoa reserves.
In this study one out the five stallions (T3) is 3 years old, and might have a smaller total
scrotal width and a smaller daily sperm output due to his age. This pony doesn’t seem to have
a smaller scrotal width compared to the other stallions in this study though (see table 2). Since
Daily Sperm Output is not subject of research in this study, we can only look at the TSN to
see whether the age might have an effect on sperm production. The TSN for this stallion is
indeed the lowest compared to the TSN of the other stallions.
The difference in volume between the right and left testicles in this study is consistent in all
five stallions. An explanation for this difference might be found in the fact that the stallions
were approached from the right side, where the right testicle was measured first and secondly
the left testicle. This may cause the cremaster muscle to contract in response, raising the left
testicle making measuring the size of the left testicle hard.
Since the data about the amount of horses in Thailand show us a very small population, the
expectation is there are not that much native Thai pony’s, most probably only a few hundreds.
This could implicate a quite high inbreeding coefficient in this breed. A recent study
(Lindgren et al., 2004) indicated that only one Y chromosome lineage has been found in a
large number of horse breeds, including the Thai native breed. Microsatellite markers used in
another study also showed a highly-polymorphic, genetic characteristic (Tawatsin et al.,
2005). To confirm the presumption that there is quite a high inbreeding coefficient within the
Thai native crossbred, more genetic investigation is needed, since offspring is unknown.
Inbreeding can have a lot of consequences for the sperm production.
The volume of the ejaculates is with 56.2 ml average over 2 ejaculates of each stallion within
the normal range of 50-200 ml, but higher than those of the other studbooks, except for the
Dutch Warmblood with a higher average volume of 65 ml.
The study of van Eldik et al. (2006) with Shetland Ponies showed that an increase in
inbreeding coefficient was accompanied by a rise in ejaculate volume with a heritability of
0.57 (SE: 0.24).
So part of the rise in ejaculate volume of the Thai Pony in this study can be due to a high
inbreeding coefficient.
The big volume of the ejaculates in this study can be caused by precopulatory teasing. The
accessory sex gland secretions increase, when stallions are teased prior to mounting.
In this study the stallions were housed around the paddock where the ejaculates were
gathered. So the stallions could see the teasing mare, and the other stallions mounting
precopulatory. All of the stallions had a high libido.
22
The concentration of the ejaculates is with 42.95 x 106 (±14.37 x 106) spermatozoa per ml
average, very low compared to those of other breeds and to the common concentration of 40100 million per ml.
Part of this finding could possibly be associated with the quite large volume of the ejaculate,
but even when we correct the volume for the concentration, still the concentration is really
low (as shows the TSN).
The study of van Eldik et al. (2006) shows a slight decrease in concentration as inbreeding
coefficient increases, which is not significant though. As discussed for volume, it’s possible
the Thai Pony has a high inbreeding coefficient and as a result a decrease in ejaculate
concentration.
Since volume and concentration are subject to many factors, the total sperm number (TSN),
calculated as the product of spermatozoal concentration and semen volume, is a more
important measurement.
The total sperm number of the Thai pony in this project is 2425.50 x 106 (±234.05 x 106)
average, which is very low compared to the other breeds, and even lower than the average of
the Shetland Pony which has the lowest average TSN (5406 x 106) of all the other breeds
listed in table 10, due to the low concentration of the ejaculates. The low average TSN for the
Shetland Pony is thought to be the result of the high inbreeding coefficient in this breed, like
in the Friesian horse. A study in 2004 found an inbreeding coefficient of the total Friesian
horse population from 1879 to 2000 (involving 52,392 of which 21,991 stallions; 2.5% used
as sires) of 1.9% per generation.23 A negative correlation between the high (increasing)
inbreeding coefficient of the Friesian horse and the low average TSN compared to other
breeds, is not yet been proved, but is believed to play a role. So, the low TSN for the native
Thai pony can be an effect of a high inbreeding coefficient.
The average motility in this study was 76.67% ±7.64%. Though consistent with another study
involving five Thai crossbred stallions (Phetudomsinsuk et al., 2008), this result is higher than
the average motility for other breeds.
Higher inbreeding coefficients are correlated with lower sperm quality in the form of lower
percentages of progressively motile sperm, as resulted from a study of van Eldik et al. (2006)
involving 285 Shetland pony stallions.
The percentage of motile sperm in this study though is higher than seen in other breeds.
Motility has a lot to do with morphology, since abnormalities in morphology can cause
abnormal motility. This is especially the case for the tail, which is the most important part for
the movement of the spermatozoa. In this study the average percentage of spermatozoa with a
normal morphology was also quite high which corresponds with the high average motility.
Vitality of the Thai pony in this study is 82.23% ±6.01%, which is like most other normal
stallions, where over 70% of the sperm in a fresh ejaculate are viable.
The percentage vitality should reflect the percentage motility since living sperms are able to
move.
Van Eldik et al. (2006) also showed that coefficients of inbreeding above 2% were associated
with lower percentages of motile and morphologically normal sperm. The percentages of live
morphologically normal sperm decreased from 47.6% (n=132) in stallions with inbreeding
coefficient <1% to 32.6% in stallions with inbreeding coefficient >12% (n=19).
Among the live sperm, the fall in the number of morphologically normal sperm was primarily
attributable to an increase in the incidence of head abnormalities.
The average of spermatozoa with a normal morphology in this study was quite high though,
being 77.75% ±5.02%.
Differences in sperm production of the native Thai pony compared to Dutch stallions can be
found in the difference in season and climate.
23
A study in Europe, Switzerland, in 2002 showed a clear seasonal pattern for all semen quality
parameters. The volume, total sperm count and motility in fresh semen were significantly
higher in summer than in winter, while sperm concentration was significantly lower in
summer compared to the other seasons.8 Switzerland though, is just like The Netherlands
geographically part of the Northern Hemisphere, where big differences in day length
throughout the year influence the fertility of mammals.
A study performed in Thailand by Phetudomsinsuk et al. (2008)19 shows a total sperm number
of 13.596 x 106 average for five Thai crossbred stallions. This study was performed in the
months January to June 2007, where only one ejaculate was collected once a month. January
to June includes actually all three seasons; the Cool season (November – February), the Hot
season (March – May) and the Rainy season (June – October). Even though this study was
performed in the Cool season, it’s believed that the breeding season of the Thai Pony is to be
throughout the whole year since the day length has only a very low variability throughout the
year. So the influence of the time of the year at the sperm production should not be
significant.
Apart from the season, Thailand has a tropical climate, which implies that all twelve months
have mean temperatures above 18 °C. It is well known that a right temperature is essential for
a healthy sperm production. Both spermatogenesis and normal testicular function are
temperature dependent, and in most mammals the testes are kept between 2 and 8 °C below
core body temperature by virtue of being situated in the scrotum outside the main body cavity.
Experimental studies have suggested that a mild increase in testicular temperature could be a
potential contraceptive method for men and that nocturnal scrotal cooling could improve
sperm count in patients with elevated testicular temperatures who have been diagnosed with
oligoasthenoteratozoospermia. Several rodent models have been used to study the impact of
heat stress on the testis by housing males at elevated temperatures (e.g. 35–36 °C) for several
hours. In these studies, the common features of disturbances in testicular function that have
been recorded include decreased testicular weights, germ cell loss and increased rates of
apoptosis. The environment temperature in Thailand during this study was sometimes around
or even above 35-36°C. Localised scrotal heating of mice is also associated with detection of
DNA damage in sperm and reduced sperm counts. This might explain the low TSN in this
study. To see whether environment temperature has indeed a negative effect on the sperm
production, one should investigate the sperm production of Dutch stallions when housed in
Thailand. The mean value found in this study, being 1234.37 x 106 TNM, is significantly
lower compared to the mean value found for the Welsh Pony, New Forest, Dutch Warmblood
and Friesian. The total sperm count is responsible for this low value, since the percentages for
motility and morphology are even high.
The study of Phetudomsinsuk et al. (2005) shows HOS(+) percentages of the ejaculates of
five Thai native crossbreed stallions being more then twice as high as in this study. In that
study a solution of 50 mOsm/l lactose is used in stead of the 100 mOsm/l lactose in this study.
But since Neild et al. (1999) found maximum numbers of swollenspermatozoa in solutions of
fructose, sucrose and lactose each at 100, 50 and 25 mOsm and since no differences were
found between these, the use of a solution of 50 mOsm/l lactose should not make any
difference.
Furthermore, Nie & Wenzel (2001) describe a basic protocol for the HOS test established in
their laboratory, where the semen after collection is added immediately to the 100 mOsm/l
sucrose solution. In this study it took more time because of the investigator working alone.3
This might influence the HOS(+) percentages.
24
Due to this long distance transport of the slides from Thailand back to the Netherlands, among
others by airplane, the eosine-aniline blue-coloured slides were a bit damaged. Because of this
it was quite hard to evaluate these slides. So there may be some defects missed because of the
bad quality of the slides, especially according to the morphology of the acrosome, which was
harder to evaluate in these slides.
Another point is a lack of experience for semen evaluation of the researcher in this study.
After examination of the slides by the researcher, one of the lab workers from the sperm
laboratory in Utrecht, who is very experienced, and is involved since years in the sperm
evaluation of the Dutch stallions, determined some of the glasses. He came to an average of
spermatozoa with a normal morphology of 72.7%, where for the same slides the researcher
came to an average of 86.9% normal morphologic, which is almost 15% higher.
25
Conclusion
The average total scrotal width of the Thai pony appeared to be pretty big, compared to the
other breeds, with an exception for the Dutch Warmblood horse. This is in contrast of what I
expected considering the height of the ponies.
Knowing the total scrotal width to be quite big, I expected the total sperm number (TSN) per
ejaculate to be quite high as well, compared to the other breeds.
The average TSN though, appeared to be the significantly lower compared to the other breeds.
The average vitality of the ejaculates of the Thai pony was pretty high, higher than other
known vitality percentages in other studies.
Motility is considered quite high as well, with an average percentage above the averages of
the other breeds we compared them to.
The average percentage normal morphology can only be compared with an another small
study of Thai pony’s and a bigger study involving Shetland pony’s. The stallions in this study
had a much higher percentage normal morphologic spermatozoa.
The final parameter, which is used by a lot of Studbooks to determine whether a stallion
should be admitted for breeding, is the TNM.
This is where volume, concentration, morphology and motility come together.
At this point it becomes clear that the Thai pony obviously has a significantly lower average
TNM, compared to the Dutch Warmblood, Friesian, Welsh Pony and New Forest. The
average TNM is sufficient according to the parameters recommended by the Society of
Theriogenology. The average TNM of stallion number 1 and 3 though, is not sufficient.
The minimum norm for the Dutch Warmblood stallion; 1500 x 106 TNB doesn’t seem to be a
achievable norm for the native Thai pony.
When looking at the integrity and functionality of the sperm plasma membrane, one should
have a look at the results for the HOS-test. Even though their are not yet normal ranges for the
outcomes of this test, the percentage of 22.7% gives the impression of semen with an
abnormal quality. One could say that this test implies infertility or subfertility for the
ejaculates.
Joining the results, we can conclude that we can have valid doubts about the fertility of this
group of Thai pony stallions.
Since the stallions on this farm don’t serve that many mares a year, most of them probably are
able to cover a few mares a year successfully.
And when considering the conditions for stallions formulated by the Society for
Theriogenology, all these stallions would pass the test. For their motility, morphology, vitality
and TNM is good enough.
There is some doubt though, whether these stallions will be able to cover a whole lot of
mares. This because the average TNM of each stallion just passes the billion, which is the
limiting value, but also considering the bad results in the HOS-test.
Since this study involves only five Thai pony stallions, it’s not possible to draw conclusions
for the whole breed. To map out the semen quality for the whole breed, more research is
recommended involving more stallions.
Repeating the HOS-test in more stallions of this breed would be interesting to see whether the
whole breed has a poor sperm plasma membrane quality.
26
To rule out the influence of season and climate one should examine sires from the Studbooks
of the KWPN, Friesch, Welsh and New Forest in Thailand in the same months; January and
February.
According to evaluate inbreeding in the native Thai pony breed, DNA studies would be
interesting to determine the inbreeding coefficient since it’s impossible to find out more about
the ancestors of the breed.
27
Appendix
Appendix 1 All parameters of the 3th and 4th ejaculates Thai Pony (n=5)
Stallion Volume Concentration Motility Normal
Vitality TNM
(ml)
(x106)
(%)
morphology (%)
(x106)
(%)
T1
T2
T3
T4
T5
Average
30.5
51.5
37.5
107.5
54
56.2±
30.29
49.25
42.75
59.75
20.5
42.5
70
82.5
85
75
77.5
42.95±
14.37
76.67±
7.64
74.5
79.25
72.5
77
85.5
77.75±
5.02
80.4
86.75
89.75
79.6
74.66
855
1472.4
858.1
1295.2
1691.3
Total
sperm
number
(x106)
1613.5
2139.5
1387.5
2260
2591
82.23± 1234.37± 2425.50±
6.01
372.41
234.05
28
Literature
1. Blanchard, T. L., Varner, D.D. & Lawrence, F.B., Manual of Equine Reproduction, St.
Louis: Mosby, Inc. 2003. Chapter 13: Examination of the Stallion for Breeding
Soundness and Chapter 14: Semen Preservation, p 143- 177.
2. Card, C. ‘Cellular associations and the differential spermiogram: Making sense of
stallion spermatozoal morphology’, Theriogenology 2005, 64, 558–567.
3. Catriona, P., Murray, A.A., Spears, N. & Saunders, P.T.K., A single, mild, transient
scrotal heat stress causes DNA damage, subfertility and impairs formation of
blastocysts in mice, Reproduction, 2008, 136, 73-84.
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