Department of Production Animal Medicine
Faculty of Veterinary Medicine
University of Helsinki
Finland
Components of fractionated stallion seminal plasma and
the effects of seminal plasma on sperm longevity
Maria Kareskoski
ACADEMIC DISSERTATION
To be presented for public criticism,
with permission of the Faculty of Veterinary Medicine, University of Helsinki,
in Lecture Room 4, Latokartanonkaari 7 (B-building), Helsinki,
on April 8th, 2011, at 12 noon.
Supervisors
Professor Terttu Katila DVM, MS, PhD, Dipl. ECAR
Department of Production Animal Medicine
Faculty of Veterinary Medicine
University of Helsinki
Finland
Professor Magnus Andersson DVM, PhD, Dipl. ECAR
Department of Production Animal Medicine
Faculty of Veterinary Medicine
University of Helsinki
Finland
Professor Heriberto Rodriguez-Martinez DVM, PhD, Dipl. ECAR
Department of Clinical and Experimental Medicine
Faculty of Health Sciences
Linköping University
Sweden
Reviewers
Professor Anne-Marie Dalin DVM, PhD, Dipl. ECAR
Department of Clinical Sciences
Division of Comparative Reproduction
Swedish University of Agricultural Sciences
Sweden
Senior researcher Michèle Magistrini PhD
Joint Research Unit for Reproductive and Behavioural Physiology
Institut National de la recherche Agronomique (INRA)
France
Opponent
Professor Harald Sieme
Reproduktionsmedizinische Einheit der Kliniken
Tierärztliche Hochschule Hannover
Germany
Chairman
Professor Terttu Katila DVM, PhD, Dipl. ECAR
Department of Production Animal Medicine
Faculty of Veterinary Medicine
University of Helsinki
Finland
ISBN 978-952-92-8738-3 (Paperback)
ISBN 978-952-10-6899-7 (PDF)
http://ethesis.helsinki.fi
Unigrafia Oy
Helsinki 2011
2
I dedicate this work to a friend lost too early.
The grief of those who lost you
The pride of having known you
Makes the rain seem colder now
Author unknown
3
Contents
Abstract................................................................................................................................................... 7
List of original publications ..................................................................................................................... 9
Abbreviations ........................................................................................................................................ 10
1.
Introduction ................................................................................................................................... 12
2. Review of the literature...................................................................................................................... 13
2.1. Ejaculation of the stallion and characteristics of stallion semen ................................................... 13
2.2. Anatomy and function of the accessory glands ............................................................................ 14
2.3. Semen collection and semen processing for storage ..................................................................... 15
2.4. The composition of stallion seminal plasma ................................................................................ 16
2.4.1. Enzymes .............................................................................................................................. 16
2.4.2. Other protein compounds ..................................................................................................... 18
2.4.3. Prostaglandins ...................................................................................................................... 20
2.4.4. Electrolytes and trace elements ............................................................................................. 21
2.4.5. Other components studied in stallion seminal plasma ........................................................... 22
2.5. The effects of seminal plasma on sperm during storage ............................................................... 23
2.6. The effects of seminal plasma in the genital tract of the mare ...................................................... 25
2.7. Research objectives ..................................................................................................................... 26
3. Aims ................................................................................................................................................. 27
4. Material and methods ........................................................................................................................ 28
4.1. Animals ...................................................................................................................................... 30
4.1.1. Experiment A (I: Trial 1, II: Trial 1) and Experiment B (I: Trial 2) ....................................... 30
4.1.2. Experiment C (II: Trial 2, III: Trial 1)................................................................................... 30
4.1.3. Experiment D (II: Trial 3, III: Trial 2) .................................................................................. 31
4.1.4. Experiment E (IV) ................................................................................................................ 31
4.2. Semen collection......................................................................................................................... 31
4.2.1. Experiment A (I: Trial, II: Trial 1) ........................................................................................ 32
4.2.2. Experiment B (I: Trial 2) ...................................................................................................... 32
4.2.3. Experiment C (II: Trial 2, III: Trial 1)................................................................................... 32
4.2.4. Experiment D (II: Trial 3, III: Trial 2) .................................................................................. 34
4.2.5. Experiment E (IV) ................................................................................................................ 34
4.3. Semen handling and preparation of samples ................................................................................ 36
4
4.3.1. Experiment A (I: Trial 1, II: Trial 1) ..................................................................................... 36
4.3.2. Experiment B (I: Trial 2) ...................................................................................................... 37
4.3.3. Experiment C (II: Trial 2, III: Trial 1)................................................................................... 37
4.3.4. Experiment D (II: Trial 3, III: Trial 2) .................................................................................. 37
4.3.5. Experiment E (IV) ................................................................................................................ 38
4.4. Sperm quality assessment............................................................................................................ 38
4.4.1 Sperm motility (I, III, IV) ...................................................................................................... 38
4.4.2. Plasma membrane integrity (PMI) (I) ................................................................................... 39
4.4.3. Sperm morphology staining and evaluation (IV) ................................................................... 39
4.4.4. Sperm chromatin structure assay (IV) ................................................................................... 39
4.5. Determination of enzyme and electrolyte levels in seminal plasma (II, IV) .................................. 39
4.6. Protein analyses of seminal plasma (III) ...................................................................................... 40
4.7. Fertility data (IV) ........................................................................................................................ 41
4.8. Statistical analyses ...................................................................................................................... 41
4.8.1. Experiments A-D (I, II, III) .................................................................................................. 41
4.8.2. Experiment E (IV) ................................................................................................................ 42
5. Results .............................................................................................................................................. 43
5.1. Effects of seminal plasma fractions and seminal plasma removal on sperm survival during storage
(I, IV) ................................................................................................................................................ 43
5.1.1. Effects of seminal plasma removal on sperm longevity (I, IV) .............................................. 43
5.1.2. Differences between seminal plasma fractions in the effects on sperm survival during storage
(I, IV) ............................................................................................................................................ 46
5.1.3. Differences between stallions in sperm survival during storage (I) ........................................ 46
5.1.4. The effects of seminal plasma exchange between stallions on sperm motility (III) ................ 49
5.2. Levels of alkaline phosphatase, acid phosphatase, -glucuronidase and electrolytes in seminal
plasma fractions (II, IV) ..................................................................................................................... 50
5.2.1. Differences between seminal plasma fractions (II, IV) .......................................................... 50
5.2.2. Differences between stallions (II) ......................................................................................... 51
5.2.3. Correlations of the levels of enzymes and electrolytes in seminal plasma with semen quality,
sperm longevity, and fertility (II, IV) ............................................................................................. 52
5.3. Proteins in seminal plasma fractions and correlations with sperm concentration and motility (III) 54
5.3.1. Differences in protein levels between stallions and seminal plasma fractions (III) ................. 54
5.4. The association between seminal plasma components and semen quality, sperm longevity, and
fertility (III, IV) ................................................................................................................................. 56
5
6. Discussion ......................................................................................................................................... 58
6.1. Effects of the removal of seminal plasma on sperm longevity (I, IV) ........................................... 58
6.2. Sperm survival during storage in sperm-rich and sperm-poor fractions (I, IV) ............................. 60
6.3. Levels of seminal plasma components in sperm-rich and sperm-poor fractions (II, III, IV) .......... 61
6.4. Contents of seminal plasma in relation to sperm survival during storage and fertility (III, IV) ...... 62
7. Conclusions ....................................................................................................................................... 65
Acknowledgments ................................................................................................................................. 66
References............................................................................................................................................. 68
Original articles ..................................................................................................................................... 83
6
Abstract
Seminal plasma (SP) is the fluid portion of semen, secreted by the epididymides and the
accessory glands before and during ejaculation. The functions of SP include acting as a
vehicle transporting ejaculated spermatozoa through the urethra, and it also plays a role in
the transport, protection and nutrition of spermatozoa in the female genital tract. Seminal
factors participate in the regulation in sperm capacitation, the acrosome reaction and
gamete interaction.
The stallion’s ejaculate is a series of jets that differ in sperm concentration, semen volume
and biochemical composition. Before the actual ejaculation, a clear and watery pre-sperm
fluid is secreted. The first three jets form the sperm-rich fractions, and contain ¾ of the
total number of sperm. The semen volume and sperm concentration in each of the jets
decrease towards the end of the ejaculation, and the last jets are sperm-poor fractions with
a low sperm concentration.
The aims of these studies were to examine the effects of the different SP fractions, and the
presence of SP, on sperm survival during storage. Pre-sperm fluid, and semen fractions
with a high (sperm-rich) and low (sperm-poor) sperm concentration were collected in five
experiments. The levels of selected enzymes, electrolytes and proteins in different SP
fractions were determined. These studies also aimed at assessing the individual variation in
the levels of the selected SP components and in the effects of SP on spermatozoa. The
association between the components of SP and semen quality, sperm longevity, and
fertility was examined with a stepwise linear regression analysis.
Sperm motility and plasma membrane integrity (PMI) in fractionated ejaculates were
evaluated after 24 h of cooled storage (Experiment A) or freezing and thawing (Experiment
B), with or without the presence of SP. In Experiment C, four fractionated ejaculates were
collected from each of two reproductively normal stallions and two subfertile stallions.
Sperm motility was determined after 24h of cooled storage with or without SP, and SP was
also exchanged pairwise between normal and subfertile stallions. In experiment D, six
fractionated ejaculates were collected from each of seven stallions, and SP was separated
from spermatozoa through centrifugation and filtration. The levels of alkaline phosphatase,
acid phosphatase, calcium, magnesium, sodium, potassium and inorganic phosphate were
determined in SP samples collected in Experiments A, C, D and E. The level of glucuronidase was determined in Experiments A, C, and D, and prostaglandin E2 in
Experiment E. The samples in Experiment C were subjected to reverse-phase highperformance liquid chromatography (RP-HPLC), N-terminal amino acid sequence
analysis, and mass spectrometry in order to study the protein content in the SP fractions.
The protein profiles in the SP fractions collected in Experiment D were analyzed with
sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) and
densitometry.
In Experiment E, fractionated ejaculates were collected from 43 stallions (one
ejaculate/stallion) at nine stud farms. DNA integrity, measured with the sperm chromatin
structure assay, and normal sperm morphology were determined before storage. After 24h
7
of cold storage, sperm motility and DNA integrity were determined in samples stored with
or without SP. The first cycle pregnancy rate was calculated for all the reported matings
with the studied stallions in the year the samples were collected.
Compared to samples containing SP during storage, centrifugation and the subsequent
removal of SP reduced sperm motility parameters during 24 h of cooled storage in all SP
fractions, but sperm membrane integrity was not affected. Some of the measured post-thaw
motility parameters were also higher in samples containing SP compared to samples stored
without SP. In contrast, the proportion of DNA-damaged spermatozoa was greater in the
samples stored with SP than those without SP, and this effect was seen in both sperm-rich
and sperm-poor fractions. There were no differences in DNA integrity between fractions
stored with SP, but the sperm-rich fraction showed less DNA damage than the sperm-poor
fraction after SP removal. The differences between fractions in sperm motility after cooled
storage were non-significant.
The levels of alkaline phosphatase, acid phosphatase and -glucuronidase were higher in
the sperm-rich fractions compared to the sperm-poor fractions, while the concentrations of
calcium and magnesium were higher in sperm-poor fractions than in sperm-rich fractions.
The concentrations of sodium and chloride were highest in pre-sperm fluid.
Three horse seminal proteins (HSP-1, HSP-2, and HSP-4) were detected in the SP samples
of all stallions. HSP-1 was the major protein in all fractions, all stallions, and all ejaculates.
No significant differences emerged between fractions or stallions in the number of peaks in
liquid chromatography. The levels of HSP-1 and HSP-2 were correlated with the sperm
concentration. Gel electrophoresis revealed 6–12 protein bands in stallion SP. The amount
of proteins at 60–70 kD was greater in sperm-poor fractions than in sperm-rich fractions
and whole ejaculates.
In the sperm-poor fraction, the level of potassium was associated with the maintenance of
sperm motility during storage. The levels of alkaline and acid phosphatase were associated
with sperm concentration and the total number of spermatozoa in the ejaculates. None of
the measured SP components were correlated to the first cycle pregnancy rate.
In summary, the removal of SP improved DNA integrity after cooled storage compared
with samples containing SP. There were no differences in the maintenance of sperm
motility between the sperm-rich and sperm-poor fractions and whole ejaculates during
cooled storage, irrespective of the presence of SP. The lowest rate of DNA damage was
found in the sperm-rich fractions stored without SP. In practice, the results presented in
this thesis support the use of individual modifications of semen processing techniques for
cooled transported semen from subfertile stallions.
8
List of original publications
This thesis is based on the following original publications referred to in the text by their
Roman numerals:
I
Kareskoski AM, Reilas T, Andersson M, Katila T, 2006: Motility and plasma
membrane integrity of spermatozoa in fractionated stallion seminal plasma.
Reprod. Domest. Anim. 41 33-38.
II
Kareskoski AM, Reilas T, Sankari S, Andersson M, Güvenc K, Katila T, 2010:
Alkaline and acid phosphatase, -glucuronidase, and electrolyte levels in
fractionated stallion ejaculates. Reprod. Domest. Anim. 45 e369-e374.
III
Kareskoski AM, Rivera del Alamo MM, Güvenc K, Reilas T, Calvete JJ,
Rodriguez-Martinez H, Andersson M, Katila T, 2010: Protein composition
of seminal plasma in fractionated stallion ejaculates. Reprod. Domest.
Anim. 46 e79-e84.
IV
Kareskoski AM, Sankari S, Johannisson A, Kindahl H, Andersson M, Katila T:
The association of the presence of seminal plasma and its components with
stallion fertility and sperm longevity in fractionated ejaculates. Submitted for
publication.
9
Abbreviations
ACP
acid phosphatase
AP
alkaline phosphatase
AV
artificial vagina
AUC
area under the curve
BG
-glucuronidase
BSP
bovine seminal protein
CASA
computer assisted sperm analysis
CFDA
carboxyfluorescein diacetate
CRISP
cysteine-rich seminal protein
DFI
DNA fragmentation index
DIFF
difference between two samples
GPX
glutathione peroxidase
HIGH
ejaculate fraction with the highest sperm concentration
HSP
horse seminal protein
KE
Kenney’s extender (skim milk extender)
KMT
KE supplemented with a modified Tyrode’s medium
LIN
linearity
LOW
ejaculate fraction with the lowest sperm concentration
MMP
matrix-metalloproteinase
MW
molecular weight
NAG
-N-acetylglucosaminidase
PGE2
prostaglandin E2
PGF
prostaglandin F
PI
propidium iodide
PMI
plasma membrane integrity
PMN
polymorphonuclear cell
PMOT
progressive sperm motility
PRE
pre-sperm fluid
PSA
prostate-specific antigen
PSP
porcine seminal protein
RAP
percentage of rapid sperm cells
ROS
reactive oxygen species
RP-HPLC reverse-phase high performance liquid chromatography
SCSA
sperm chromatin structure assay
SDS-PAGE sodiumduodecyl-sulphate polyacrylamide gel electrophoresis
SOD
superoxide dismutase
SP
seminal plasma
SP1
sample stored with seminal plasma present
SP0
sample stored without SP
STR
straightness
10
TI
TMOT
VCL
VSL
WE
2D-PAGE
testicular index
total sperm motility
average sperm velocity
straight-line sperm velocity
whole ejaculate
two-dimensional polyacrylamide gel electrophoresis
11
1. Introduction
Seminal plasma (SP) is the fluid portion of semen, secreted by the epididymides and the
accessory glands shortly before and during ejaculation. It is a fluid composed of several
different types of compounds, such as proteins, ions, amino acids, monosaccharides, lipids,
polyamines, prostaglandins and steroid hormones. The main function of seminal fluid is to
act as a vehicle that flushes the urethra and simultaneously propels spermatozoa through
the stallion’s genital tract during ejaculation. Further functions include the transport,
protection and nutrition of spermatozoa in the female genital tract; seminal factors also
regulate sperm capacitation, the acrosome reaction and gamete interaction (reviewed by
Töpfer-Petersen et al. 1998).
The research presented in this thesis provides in part a new look into an old area of
investigations, as many of the studies on the SP of domestic animals date back to the 1960s
and 1970s. Although SP has been the subject of research for decades, the available
information still seems lacking, partly because of the immense species-specific variation
(Aumüller et al. 1990, Massanyi et al. 2003) in reproductive physiology, making
extrapolations between species a minefield. Recently, progress has been made especially in
the fields of proteomics and genomics, but the physiological role of SP is still not fully
understood. While artificial insemination using cooled transported semen has become an
integral part of modern horse breeding, enabling international semen transport and a wider
choice of stallions available for mare owners, an increased need for reliable and practical
techniques for both breeding soundness evaluation and semen processing has emerged.
As sperm longevity and stallion fertility vary markedly between individuals, it is of interest
for the equine industry to investigate the factors producing these effects. Stallion SP
influences the function and survival of ejaculated sperm during in vitro storage and in the
female genital tract, and some of the stallion variation could perhaps be explained by
inherent differences in the composition of SP. Knowledge of the composition of SP is
needed in order to develop semen handling procedures and to perhaps even predict the
storage tolerance of spermatozoa. The differences in the composition of SP between
consecutive parts of the ejaculated fluid may also provide information on the sequence of
fluid release from the accessory sex glands during ejaculation.
The following literature review is partly based on previous reviews by Kareskoski and
Katila (2008) and Katila and Kareskoski (2006), and presents research on selected aspects
of stallion reproductive physiology, mainly on the sequence of ejaculation and on the
composition of SP and the effects of SP on sperm longevity and pre-fertilization events.
Compared to humans and other animal species, fairly limited data are available on stallion
SP, and research on other mammalian species is therefore also discussed.
12
2. Review of the literature
2.1. Ejaculation of the stallion and characteristics of stallion semen
A reproductively sound stallion expresses normal sexual behavior and produces an
adequate number of progressively motile, morphologically normal spermatozoa, at least 1
x 109 spermatozoa at any time of the year (Kenney et al. 1983). A stallion with normal
libido shows an immediate interest in the mare and mounts her within just a few minutes.
The mean reaction time (the time from visual contact with the mare to mounting) has been
reported to be 221 s, and the mean number of mounts was 1.8 mounts/ejaculate (Pickett et
al. 1976). Excessive sexual preparation, teasing, or false mounts increase the amount of SP
and the total volume of semen, without increasing the sperm output (Ionata et al. 1991).
Emission of fluids from the cauda epididymis, ductus deferens and the accessory sex
glands occurs before ejaculation, through smooth muscle contractions in these organs. The
emission reflex is under -adrenergic control, involving sympathetic fibres from the
lumbosacral segment of the spinal cord. Ejaculation is a series of 6-9 jets, produced by
rhythmic contractions of the ischiocavernosus, bulbospongiosus, and urethralis muscles,
and this process is controlled by the pudendal nerve and the sacral segment of the spinal
cord (reviewed by Tibary 2007).
The ejaculatory jets differ in sperm concentration, semen volume and biochemical
composition. Before the actual ejaculation, a clear and watery pre-sperm fluid is secreted.
As this often occurs before intromission, this pre-sperm fluid is probably not present in
large amounts in collected semen or in the uterus after ejaculation. The first three jets, the
sperm-rich fractions, contain ¾ of the total number of sperm, even though these fractions
represent less than half of the total ejaculate volume. The volume of semen and the sperm
concentration in each of the jets decrease towards the end of the ejaculation (Tischner et al.
1974, Kosiniak 1975, Varner et al. 1987).
Horses are long-day seasonal breeders, and seasonal differences are seen in the amount and
composition of SP. The seasonal effects on seminal plasma seem more pronounced than
the effects on sperm characteristics (Gebauer et al. 1976, Abou-Ahmed et al. 1993).
Seasonal variation has been detected in the semen volume, total sperm count, seminal pH,
and in sexual behavior depicted by the reaction time and mounts per ejaculate (Pickett et
al. 1976). The volume of semen, sperm count and sperm motility of fresh semen have been
shown to be higher in summer than in winter (Janett et al. 2003). These effects could be
associated with changes in sex steroid levels, which vary according to the season in both
blood and SP (Braun et al. 1996, Hoffmann and Landeck 1999). However, the correlations
between the chemical characteristics of SP and plasma concentrations of testosterone have
been reported to be weak (Gebauer et al. 1976).
13
In a study on over 550 ejaculates, the mean volume of gel-free semen in stallion ejaculates
was 58.4 ml, ranging from 44.8 ml in March, to 80.8 ml in July. The mean volume of gel
was 7.1 ml for first ejaculates and 2.0 ml for second ejaculates collected one hour after the
first ejaculation. First ejaculates contained 281 x 106 sperm/ml compared with 170 x 106
sperm/ml in second ejaculates. The total amount of spermatozoa was 14.7 x 109 in first
ejaculates and 7.9 x 109 in second ejaculates (Pickett et al. 1976). The endocrinological
profile of stallions is also affected by disease, hard physical exercise (Janett et al. 2005)
and medications, such as dexamethasone, which increases the total protein and iron
concentrations, and decreases the alkaline phosphatase (AP) levels of SP (Danek 2001).
2.2. Anatomy and function of the accessory glands
The stallion has a full set of accessory glands (i.e. paired bulbourethral glands, ampullar
glands, seminal vesicles and a prostate gland), as opposed to some of the other domestic
animal species. All of the accessory glands are also present in the bull and in man, but
there are marked differences between species in the amount of secretions produced by
these glands, reflected in ejaculate volumes ranging from 2–8 ml in the bull to 50–150 ml
in the stallion. Dogs do not have seminal vesicles or bulbourethral glands, and boars have
prominent seminal vesicles and bulbourethral glands, but lack ampullae (Nickel et al.
1979). Because of these fundamental species differences, comparison between species is
rarely fruitful because the seminal fluid varies in composition, as the gland contributions
vary. Even if there are macroscopic similarities in some of the accessory glands between
different species, the composition of the produced secretions is often not comparable
(Beyler and Zaneveld 1982).
The ampullae are enlargements of the distal part of the vas deferens, and they have been
presumed to function as a storage site of spermatozoa, but also in the transport of
spermatozoa through the urethra during ejaculation (Nickel et al. 1979). The ampullae are
quite large in the stallion compared with other species, and they contain a variable amount
of yellowish, creamy fluid. Ergothioneine in SP has been found to originate in the
ampullae (Mann et al. 1956). Some absorption, such as transport of proteins through
pinocytosis, can take place in the ampullae, but the secretory functions are better
documented. The stallion’s ampullae secrete several different types of compounds, e.g.
proteins, Na+ and K+ ions, water, ergothioneine, glycerylphosphorylcholine and
monosaccharides, and a variety of glycosidases, such as -N-acetylglucosaminidase and mannosidase (Beyler and Zaneveld 1982).
The seminal vesicles of the stallion are pear-shaped sacs with muscular walls lined with a
thick mucous membrane. Each seminal vesicle is 10–15 cm long and has a diameter of 3–6
cm (Nickel et al. 1979). Seminal vesicle secretions are transparent or slightly opaque and
viscous fluid (Mann et al. 1956). In many other species, the seminal vesicles secrete large
amounts of fructose and ergothioneine, whereas citric acid and lactic acid are more
14
prominent in the stallion (Mann et al. 1956, Beyler and Zaneveld 1982). The prostate gland
contains only a small amount of fluid (Mann et al. 1956). The prostate gland has two
nodular lobes connected over the midline by an isthmus. The prostate and the seminal
vesicles are united by connective tissue, and they are thus difficult to palpate transrectally
(Nickel et al. 1979). The two bulbourethral glands are club-shaped and situated at the
pelvic outlet, dorsolateral to the urethra (Nickel et al. 1979).
The composition of SP in the consecutive ejaculate fractions varies, as the accessory
glands release their contents in a specific order during the emission of fluid into the pelvic
urethra and the subsequent ejaculation. This sequence has been studied by identifying
certain markers for particular accessory gland secretions in different ejaculate fractions. An
early study by Mann et al. (1956) showed that the first sperm-rich ejaculate jets contained
mainly spermatozoa and ampullar secretions high in ergothioneine, while the latter spermpoor fractions contained vesicular secretions high in citric acid. Carnitine,
glycerylphosphorylcholine and choline have later been used as markers for the
epididymides, the N-acetyl function of glycoproteins and spermine for ampullae, acetic
acid for the bulbourethral glands, citric acid for seminal vesicles, and lactic acid for
prostate secretions (Magistrini et al. 1995). Magistrini et al. (2000) used an automated
semen collection device (Lindeberg et al. 1999) to collect fractionated ejaculates and 1H
magnetic resonance spectroscopy to quantify these markers of accessory gland secretions.
Bulbourethral fluid was secreted first (pre-sperm fluid), and then epididymal and ampullar
secretions formed the first sperm-rich fractions of the ejaculate, while seminal vesicle fluid
appeared by the end of the ejaculation. The concentration of acetic acid did not differ
between the ejaculate fractions, suggesting that it does not serve as a specific marker for
the bulbourethral glands. Similar results on the sequence of ejaculation have been shown
earlier using transrectal ultrasonography. Ampullar and prostatic activity began prior to the
start of ejaculation, with fluid release from the prostate continuing throughout the first
urethral contractions. The seminal vesicles did not release their contents until the end of
prostatic secretion (Weber and Woods 1993). There is always a certain amount of overlap
between the fractions, and the secretions from the different glands do not appear by
themselves at any point during the ejaculation (Mann et al. 1956).
2.3. Semen collection and semen processing for storage
Several different models of artificial vaginas (AVs) are available for semen collection in
stallions. These AVs are either open-ended, with semen collection into a separate
receptacle, or closed, with semen collection into a bottle attached to the AV (Love 1992).
Fractionated ejaculates can be collected using an open-ended AV, although modifications
of closed AVs such as the Missouri model can also facilitate the collection of ejaculatory
fractions. Collection of only the sperm-rich fractions with an open-ended AV can be
beneficial, especially in stallions producing semen with a low sperm concentration
(Tischner et al. 1974), as it will render centrifugation unnecessary. Low sperm
15
concentrations are problematic when semen is prepared for cooled storage, because
adequate dilution of semen ( 1:4 = semen:extender) recommended to achieve adequate
sperm longevity (see section 2.5.) would result in too low sperm concentrations (Varner et
al. 1987).
A more elaborate form of fractioning is achieved using a computer-controlled phantom
such as the Equidame® (Haico Oy, Loimaa, Finland), where the ejaculate is collected in
several cups according to a pre-set target weight of each cup corresponding to a particular
semen volume. The Equidame® phantom is equipped with a detachable open-ended AV, a
metallic funnel, and a semen collection tray with 5 plastic cups. The computer-controlled
tray moves horizontally as the cups reach their target weight during the ejaculation
(Lindeberg et al. 1999).
After the collection of semen, any gel is promptly removed, the semen volume is recorded
and a sample is taken to determine the sperm concentration. For cooled storage, prewarmed extender (+37 ºC) is then added to lower the volume proportion of SP. The final
volume is calculated based on the sperm concentration. To lower the volume proportion of
SP in stored semen, more extender is added once the sperm concentration is known and the
needed volume can be calculated. A total number of 500–1000 x 106 progressively motile
spermatozoa, with a concentration of 25–100 x 106 spermatozoa/ml, is often considered an
adequate insemination dose for semen transport that lasts less than 24 h (Katila 1997,
Blanchard et al. 2003). The volume as such has no influence on maintenance of motility of
fertility, but for practical reasons, such as the limited size of the commonly used Styrofoam
transport boxes, the total volume of the insemination dose usually varies between 10–40
ml. The role of SP, extenders and sperm concentration during cooled storage (+5 °C) of
semen is discussed in section 2.5.
Because of the limited volume available in the plastic straws (0.25 to 2.5 ml) used for
storing frozen semen, the superfluous and possibly harmful SP has to be removed before
semen freezing in order to reduce the semen volume. To this end, semen is diluted with
extender, centrifuged, and the supernatant is removed. Because semen is not washed by
repeated centrifugations, a small amount of SP (< 5%) will be left, even though the semen
has been centrifuged. The main proportion of SP, however, is removed before freezing
stallion sperm in liquid nitrogen (Blanchard et al. 2003).
2.4. The composition of stallion seminal plasma
2.4.1. Enzymes
Several enzymes have been identified in stallion accessory glands and in SP, and efforts
have been made to study the correlations of enzyme activities with semen quality and
stallion fertility. The origin of a few of the SP enzymes in the various reproductive organs
16
and accessory glands has been identified, but in many cases the physiological function of
these compounds has remained elusive. Alkaline phosphatase (AP) is a widely distributed
phosphomonoesterase (reviewed by Coleman 1992). There are high levels of AP activity in
the SP of reproductively normal stallions, and spermatozoa are not a significant source of
the enzyme. As AP activity is mainly derived from the testes and epididymides,
measurement of AP can be used to differentiate ejaculatory failure and excurrent duct
blockages from testicular azoospermia (Turner and McDonnell 2003). In dogs, AP activity
is similarly found in the epididymides and the seminiferous tubules (Frenette et al. 1986,
Kutzler et al. 2003), and can be used as a marker for ejaculation and tubular patency
(Kutzler et al. 2003, Stornelli et al. 2003). Activity of AP has been found in seminal vesicle
fluid in bulls and boars, and in prostatic secretions in many mammals (Beyler and
Zaneveld 1982). High levels of acid phosphatase (ACP) are found in the prostatic fluid of
man, monkeys and dogs, and lower levels are found in bull prostate fluid. The two
phosphomonoesters, AP and ACP, differ in their pH optima and in substrate specificity,
but both hydrolyze different types of phosphate monoesters (Beyler and Zaneveld 1982,
Kramer and Hoffman 1997). The levels of acid phosphatase (ACP) activity reflect prostate
function in dogs (James et al. 1979), and increased levels of ACP are associated with
prostate cancer in humans (Mahan and Doctor 1979, Tanaka et al. 2004, Tappel 2005). In
the stallion, studies on ACP have been limited to epididymal tissue (Lopez et al. 1989).
The levels of both AP and ACP in SP are positively correlated with the sperm count and
negatively with semen volume (Pesch et al. 2006). Both AP and ACP are under androgenic
control (Beyler and Zaneveld 1982). In the boar, AP levels in seminal plasma vary
according to the season (Kozdrowski and Dubiel 2004).
Several different types of glycosidases have been identified in mammalian SP. Stallion
seminal vesicle secretion contains -glucuronidase (BG), -glucosidase, -glucosidase, galactosidase, -galactosidase and -N-acetylglucosaminidase (NAG), and the ampullae
are a significant source of NAG and -mannosidase (Beyler and Zaneveld 1982). In
stallion SP and in the epididymides, -1,4-glucosidase activity has been detected, and the
activity levels have been observed to increase from the proximal caput epididymis to the
cauda epididymis. The acid form of the enzyme was predominant in the cauda epididymis,
while the neutral form dominated in the caput epididymis and in SP (Dias et al. 2004).
Stallion epididymal fluid also contains NAG (Fouchécourt et al. 2000).
In humans, -galactosidase, BG and NAG bind to the surface of the sperm head in the
epididymal duct and may be involved in modifying the sperm surface before fertilization
(Sosa et al. 1991, Barbieri et al. 1994, Barbieri et al. 1996), with NAG also participating in
the acrosome reaction (Brandelli et al. 1994). Together with other enzymes, acrosomal BG
acts on glycosaminoglycans and participates in causing cumulus dispersion (Rethinaswamy
et al. 1994). Despite their essential function in the fertilization process, the over-expression
of certain glycosidases is associated with subfertility and oligo-astheno-teratozoospermia
in men (Corrales et al. 2000, Corrales et al. 2002). In the bull, low sperm concentrations
and morphologically abnormal sperm are seen in conjunction with decreased levels of
17
NAG, -fucosidase,
Perttula 1986).
-galactosidase, BG and
-mannosidase (Jauhiainen and Vanha-
Lipocalin-type prostaglandin D2 synthase and the angiotensin-I-converting enzyme have
been shown to be strongly correlated with stallion fertility, whereas the total protein
concentration in SP was not (Barrier-Battut et al. 2005). Lipocalin-type prostaglandin D2
synthase activity has been found in stallion epididymal fluid (Fouchécourt et al. 2000).
Lipase activity has been demonstrated in stallion seminal plasma, and it is negatively
correlated with sperm motility (Carver and Ball 2002). Other enzymes demonstrated in
equine seminal plasma include three carbonic anhydrase isoenzymes detected in the
seminal vesicles, prostate and bulbourethral glands. These enzymes appear to regulate the
bicarbonate concentration and pH of seminal plasma (Asari et al. 1996). Angiotensinconverting enzyme has been found in the plasma membranes of ejaculated and epididymal
spermatozoa and in postpubertal testes. The influence of angiotensin II on sperm motility
has also been evaluated and it affected some motility parameters, such as the average path
velocity (VAP) and curvilinear velocity (VCL), but not total sperm motility (TMOT) (Ball
et al. 2003). Pesch (2005) measured the activities of lactate dehydrogenase, aspartate
aminotransferase, glutamate dehydrogenase, gamma-glutamyltransferase and creatine
kinase in stallion SP. Of the mentioned enzymes, only lactate dehydrogenase was
correlated with motility, the sperm count and volume. Platelet-activating factor (PAF)
acetylhydrolase activity has also been detected in the semen of several mammalian species,
including the stallion. Sperm-derived PAF stimulates sperm motility and is involved in
capacitation (Hough and Parks 1994).
In humans, the levels of matrix metalloproteinases (MMPs) are correlated with semen
quality characteristics such as sperm concentration, morphology and motility (Baumgart et
al. 2002, Buchman-Shaked et al. 2002). The two gelatinases, MMP-2 and MMP-9, and
their pro-forms are present in human seminal plasma (Shimokawa et al. 2002). Proteinases
and proteinase inhibitors have been characterized in different regions of the epididymis in
rams, boars and stallions, and one of the main metalloproteinases from the caput
epididymis of the ram and stallion was a pro-form of MMP-2. In all three species,
antibodies against MMP-2, MMP-3, and MMP-9 and proteinase inhibitors were detected in
epididymal fluids (Métayer et al. 2002).
2.4.2. Other protein compounds
In addition to enzymes, the SP of most domestic animal species contains protein
compounds similar to those present in blood plasma, such as prealbumin, albumin, -, and -globulins, transferrin and immunoglobulins (Polakoski and Kopta 1982). The
suggested functions of SP proteins include their involvement in several essential steps
preceding fertilization, such as regulating capacitation, establishment of the oviductal
sperm reservoir, modulation of the uterine immune response, and sperm transport in the
18
female genital tract, and also in gamete interaction and fusion (reviewed by TöpferPetersen et al. 2005).
Calvete et al. (1994) described eight types of proteins among stallion SP proteins, and
these were named and numbered horse seminal protein (HSP)-1 to HSP-8 according to the
order of appearance of peaks on the reverse-phase liquid chromatography charts. Most of
the proteins in SP were of low molecular weight (14-30 kDa) and formed multi-protein
aggregates. All of the 8 protein groups, except HSP-4, were found to attach to the sperm
surface. The two major proteins, HSP-1 and HSP-2, accounted for 70-80% of the total
protein. They were shown to be heparin-binding proteins, and hypothesized to modulate
capacitation. In fractionated ejaculates, the amount of total protein is lowest in pre-sperm
fluid, and highest in the sperm-rich fractions (Koskinen et al. 2002).
Structurally, the main proteins in stallion SP belong to three groups: proteins carrying
fibronectin type II (Fn-2) modules, cysteine-rich secretory proteins (CRISPs), and
spermadhesins. The most abundant SP proteins in many species, including the stallion, are
short Fn-2 type proteins, such as HSP-1 and HSP-2 (also called SP-1 and SP-2). These
proteins are similar to the major bovine heparin-binding proteins (BSP) associated with
capacitation (reviewed by Töpfer-Petersen et al. 2005). At the time of ejaculation, the Fn-2
type proteins typically bind to sperm membrane phospholipids with a phosphorylcholine
head group, preferentially phosphatidylcholine or sphingomyelin, causing changes in the
membrane structure (Greube et al. 2004, Ekhlasi-Hundrieser et al. 2005). The ampullae are
the major source of HSP-1 and HSP-2, while a larger Fn-2 type protein (EQ-12) is mainly
secreted in the epididymis (Saalmann et al. 2001), but expression of Fn-2 type proteins
occurs along the entire genital tract of stallions (Ekhlasi-Hundrieser et al. 2005). A member
of the CRISP family in stallion SP is HSP-3 (equine CRISP-3), primarily produced in the
ampullar glands and in the seminal vesicles (Magdaleno et al. 1997, Schambony et al.
1998). An association of polymorphism of the CRISP3 gene with stallion fertility has been
suggested (Hamann et al. 2007). Recent findings suggest that CRISP-3 is the protein
responsible for the selective protection of live spermatozoa from polymorphonuclear
(PMN) cell binding and allows the sperm to survive and travel to the oviduct, despite an
ongoing breeding-induced endometritis (Troedsson et al. 2010).
In stallions, the protein profiles of whole ejaculates have been characterized in a few
studies. In sodium dodecyl-sulphate polyacrylamide gel electrophoresis (SDS-PAGE),
Frazer and Bucci (1996) found 14 protein bands and described a protein profile typical for
the stallion. Seven of these bands were present in all of the samples, but the relative
amounts of each protein varied between individual stallions. The findings of a twodimensional (2D)-PAGE study by Brandon et al. (1999) were similar, with 14 protein
groups common in all stallions. Four of these proteins (named SP-1, SP-2, SP-3 and SP-4)
were correlated to the breeding scores of the stallions. The breeding score was calculated
by dividing the number of conceptions per cycle by the number of breedings for each
stallion for four successive breeding seasons. Based on molecular mass, it was
19
hypothesized that SP-3 and SP-4 are similar to the two major proteins found in the 2DPAGE study by Frazer and Bucci (1996) and to HSP-1 and HSP-2 identified by Calvete et
al. (1994). Three proteins (SP-2, SP-3 and SP-4) were found in higher concentrations in
stallions with low fertility scores, while SP-1 was positively correlated with fertility and
was suggested to be homologous to a bovine fertility-associated protein described by
Killian et al. (1993). Proteins associated with high fertility in bovine SP are osteopontin,
derived from the ampulla and the seminal vesicle (Cancel et al. 1997), and prostaglandin
D2 synthase originating in the testis and epididymis (Gerena et al. 1998). Protein profiles in
2D-PAGE were compared between SP from stallions exhibiting high and low fertility by
Jobim et al. (2005). One protein band (protein number 19) was detectable only in
ejaculates from stallions in the high fertility group, whereas the amount of another protein
(number 17) was higher in ejaculates of stallions with low fertility. The authors suggested
that protein spot 17 could correspond to CRISP-3.
Of the other stallion SP proteins described by Calvete et al. (1994), HSP-4 was presumed
to be related to a calcitonin gene-like product. Calcitonin levels are correlated with sperm
motility in the semen of infertile men (Mungan et al. 2001). In the horse, HSP-7 has been
identified as the only member of the spermadhesin family, and like its porcine homologue
AWN-1, it shows zona-pellucida-binding activity (Reinert et al. 1996). Hoshiba and
Sinowatz (1998) detected synthesis of the AWN protein homologue in the testes, the
ductus epididymis and the seminal vesicles of stallions. In the boar, the non-heparinbinding spermadhesins PSP-I/PSP-II are of special interest because of their effects on
sperm function. These spermadhesins have a dose- and time-dependent beneficial effect on
the viability of highly extended boar spermatozoa, whereas the heparin-binding proteins in
porcine SP decrease sperm viability at high extension ratios (Centurión et al. 2003). The
supplementation of PSP-I/PSP-II in the freezing extender does not affect post-thaw sperm
survival (Cremades et al. 2004), and it has a deleterious effect on the ability of boar
spermatozoa to penetrate oocytes in vitro (Caballero et al. 2004). In the study by Calvete et
al. (1994), HSP-6 and HSP-8 appeared to be different isoforms of a kallikrein-like protein
homologous with human prostate-specific antigen.
2.4.3. Prostaglandins
Early studies have shown that prostaglandin E 1 and E2 (PGE2) are present in human SP,
and in larger quantities than other prostaglandins (Jonsson et al. 1975). Claus et al. (1992)
verified the presence of prostaglandin F (PGF ) in stallion semen. No differences in the
levels of the immunosuppressive PGE2 were detected between groups of fertile and
infertile men in a study by Camejo (2003), but an earlier paper reported lower PGE2 levels
in oligoterato-asthenozoospermic patients with genital infections compared with fertile
controls (Huleihel et al. 1999).
The transport of spermatozoa may be influenced by PGE2, as it controls oviductal smooth
muscle function (Weber et al. 1991), but it is not known whether SP-derived PGE 2 could
20
reach the uterotubal junction at levels high enough to have any effect on oviductal muscle.
The infusion of PGE2 into the proximal end of the uterine horn prior to insemination
improved the pregnancy rate in mares bred by a stallion with good quality semen, but did
not affect pregnancy rates in mares bred by a stallion with poor quality semen (Woods et
al. 1999).
Other effects of PGE2 include the modulation of the immune response in the female genital
tract, and it may also influence sperm function more directly. Both SP and PGE2 stimulate
the release of the proinflammatory neutrophil chemotactic factor interleukin-8 (IL-8) and
the anti-inflammatory cytokine interleukin-10 (IL-10) from peripheral blood and
monocytes, suggesting both anti- and proinflammatory effects of SP (Denison et al. 1999).
In rams, PGE2 levels are inversely related to the capacity of sperm to undergo the
acrosome reaction (Archbald et al. 1990). Porcine SP also contains components that
regulate the synthesis of prostaglandins in the oviduct (Kaczmarek et al. 2010).
2.4.4. Electrolytes and trace elements
Calcium is one of the most extensively studied components of mammalian semen, as it is
involved in many physiological events surrounding sperm function and fertilization.
Extracellular Ca2+ regulates sperm capacitation through decreasing the availability of
intracellular ATP (Baker et al. 2004). Hyperactivation of sperm (Marquez and Suarez
2004) and induction of the acrosome reaction are also mediated, at least in part, by Ca2+.
Abnormal levels of ionized Ca2+ can induce a premature acrosome exocytosis and lead to a
decrease in fertility (Yanagimachi and Usui 1974). Spontaneous acrosome exocytoses are
also associated with increased concentrations of ionized Ca2+ in stallion SP (Pesch 2005).
As stated earlier, notable species differences exist in the composition of SP. About 60-75%
of the total amount of Ca is ionized (as Ca2+) in stallion SP (Pesch et al. 2006), whereas in
human semen, only 2-4% is in the ionized form (reviewed by Owen and Katz 2005). These
marked species differences make the extrapolation of results from other species of
questionable use, but some research on human reproduction will be presented here, since
knowledge of the composition of SP, and the consequences of this variation, in domestic
animal species is incomplete. It has been shown in humans that post-thaw motility values
are lower with increased levels of Ca2+ (Meseguer et al. 2004), but the levels of total Ca in
SP did not vary between fertile and subfertile men in a study by Wong et al. (2001). In
stallions, Barrier-Battut et al. (2002) reported individual variation in the concentrations of
Ca, Mg and Cu, but not of Zn, in sperm-rich fractions, but these differences did not explain
the variation in post-thaw motility parameters. In bulls, post-thaw motility values increased
when MgCl2 was added to the freezing extender (Lapointe et al. 1996). In ram semen, Ca
and Mg concentrations were higher in sperm than in SP, and both components were
positively correlated with the sperm concentration (Abdel-Rahman et al. 2000), as opposed
to the negative correlation that is seen in stallions. In the stallion, the concentrations of
total Ca and ionized Ca2+ were positively correlated with ejaculatory volume, indicating
21
the accessory sex glands as the main contributors to Ca in SP (Pesch et al. 2006). In
humans, the levels of Ca and Mg have also been suggested to reflect the secretory function
of the accessory glands, because the concentrations of these components were lower in
men with accessory gland infections (Adamopoulos and Deliyiannis 1983, Edorh et al.
2003).
The addition of K to semen extenders seems to improve motility of stallion and human
sperm, but the effect depends on the proportion of SP retained in the sample during storage
(Padilla and Foote 1991, Karow et al. 1992). The ionic composition and osmolarity of SP
are not correlated (Rossato et al. 2002). In sheep, increasing potassium levels are
negatively correlated with progressive motility, while the reverse is true for sodium and
chloride (Abdel-Rahman et al. 2000). Significant stallion variation has been reported in Na,
K, Ca and Cl levels (Amann et al. 1987).
There is some evidence that the concentrations of Cu and Fe in stallion SP vary between
normal samples and samples with decreased semen quality as assessed with routine semen
evaluation methods (Pesch 2005). The concentrations of Fe, Zn, and Cu are negatively
correlated with semen volume, with Fe and Zn concentrations also correlating with the
sperm concentration (Pesch et al., 2006). Massanyi et al. (2003) assessed the
concentrations of various trace elements in the SP of several animal species. Zinc and Fe
levels were positively correlated in stallion and bull semen, and boar semen contained
more Zn than semen from stallions or bulls. In addition, Cu and Fe concentrations were
higher in ram semen compared with stallions and boars. Sperm-chromatin stability in
human ejaculates is Zn-dependent, and Zn levels can also affect pregnancy rates (as
reviewed by Björndahl and Kvist 2003). In human semen samples, high Zn concentrations
were associated with a decrease in progressive motility, but Zn concentrations did not
affect the time to pregnancy (Sorensen et al. 1999).
2.4.5. Other components studied in stallion seminal plasma
The secretions of the accessory glands contain many low molecular weight organic
constituents such as citric acid, glycerylphosphorylcholine, glucose, glycerol,
glycerylphosphorylinositol, inositol, lactic acid, sorbitol and small amounts of fructose
(Polakoski and Kopta 1982). The carbohydrate content in stallion SP differs from that of
bulls, with a considerably lower amount of free carbohydrates in stallions (Gebauer et al.
1976). Some inositol and glucose are found in stallion SP, but only negligible amounts of
fructose and no galactose. Galactose is the main type of bound carbohydrate in stallion
seminal plasma, but glucose, mannose and fucose have also been detected (Baronos et al.
1971).
Stallion SP also contains hormones, ergothioneine, small amounts of ammonia and urea,
free amino acids, and several different forms of lipids (Polakoski and Kopta 1982). The
hormonal content of SP is beyond the scope of this text. Variation between species in
22
sperm membrane composition may be related to the cold shock sensitivity of sperm.
Cholesterol is the main sterol in the membranes of boar, bull, stallion, and rooster
spermatozoa, and choline and ethanolamine phosphoglycerides and sphingomyelin are the
major types of phospholipids (Parks and Lynch 1992). Stallion semen also contains
prostasome-like particles with a characteristic lipid profile and high amounts of cholesterol
and sphingomyelin (Arienti et al. 1998). These membrane vesicles are mainly found in the
sperm-rich part of the ejaculate, but their origin and function are not well known (El-Hajj
Ghaoui et al. 2004), although effects on sperm motility, the acrosome reaction and the
immune system have been proposed (reviewed by Sostaric et al. 2008).
Oxidative damage to sperm is of significance for sperm survival during storage.
Spermatozoa are protected against oxidative damage caused by reactive oxygen species
(ROS), such as hydrogen peroxide, mainly by catalase, glutathione peroxidase (GPX) and
superoxide dismutase (SOD). Cryodamaged or morphologically abnormal sperm, and also
neutrophils, are significant sources of ROS (Ball et al. 2001, Baumber et al. 2002). Limited
GPX- and SOD-activity exists in sperm, and thus the main protective activity is due to the
protective enzymes in SP (Baumber and Ball 2005). The prostate gland is likely to be the
main source of catalase in the reproductive tract of the stallion (Ball et al. 2000). In
fractionated stallion ejaculates, catalase is found in high amounts in all fractions of the
ejaculate, except for in the pre-sperm fluid (Koskinen et al. 2002), which could indicate
that the prostate contributes to several ejaculatory fractions or that catalase also originates
in other glands. The testes and epididymides are the main sources of GPX (Fouchécourt et
al. 2000).
Sperm motility is reduced by ROS, (Baumber et al. 2000), and they also cause DNA
fragmentation and membrane damage in spermatozoa (Baumber et al. 2003) due to lipid
peroxidation (Ball et al. 2000). It is likely that increased ROS production is responsible for
these effects, rather than a decrease in the amount or function of the ROS protective
enzyme systems (Zini et al. 1993). Despite an apparent physiological function of these
enzymes, adding antioxidant to the freezing extender does not seem to improve sperm
survival during storage (Baumber et al. 2005).
The production of ROS may have a physiological role in promoting capacitation and
tyrosine phosphorylation (Baumber et al. 2003), involved in the signalling pathway
controlling capacitation and the acrosome reaction (Leyton et al. 1992). In a review by
Griveau and Le Lannou (1997), it was concluded that small amounts of ROS are needed
for normal sperm function, but excessive ROS production is highly toxic to sperm.
2.5. The effects of seminal plasma on sperm during storage
Reducing the amount of SP in cooled semen has been considered beneficial for sperm
survival during storage, and this can be accomplished through centrifugation and
23
subsequent removal of the supernatant, extension of semen, or through collecting only the
sperm-rich fraction of the ejaculate. Several studies have shown that when the volume
proportion of SP is lowered to less than 5 or 10%, sperm motility is better maintained
compared to samples containing a higher proportion of SP (10–30%), during both cooled
storage and freezing and thawing (Jasko et al. 1991, Braun et al. 1994, Todd et al. 2001,
Alghamdi et al. 2002). A high SP content (40–50%) in cooled semen during storage for
24–48 h was shown to be detrimental to sperm motility (Jasko et al. 1991), whereas a
proportion of 20% SP did not have any negative effects on semen quality during storage
for 24 to 48 h (Jasko et al. 1992). Lower SP proportions (1.25%, 2.5% and <10%) seem to
maintain sperm motility even better (Pruitt et al. 1993, Todd et al. 2001). The removal of
SP before semen storage also had beneficial effects on DNA integrity compared to samples
containing 20% SP during cooled storage in a study by Love et al. (2005). The removal of
SP has also been shown to increase the resistance to hypo-osmotic shock, and to reduce the
responsiveness to induced acrosome exocytosis (Barrier-Battut et al. 2010). Short term (15
min) exposure of sperm to SP did not reduce post-thaw semen quality, but longer periods
of exposure (2, 4 or 6 hours) of sperm to 5% or 20% SP in the freezing extender had
negative effects on sperm (Moore et al. 2005). The proportion of SP included in
insemination doses did not affect pregnancy rates when semen was stored at +5 °C for less
than 12 h (Katila et al. 2010a). In a recent study, the sperm concentration did not affect
sperm survival when the effect of the proportion of SP was eliminated. Sperm were stored
at +5 °C for 24 h at sperm concentrations of 25 or 250 x 106 sperm/ml and a SP content of
20%, and no differences between the samples were detected in total sperm motility,
progressive sperm motility, membrane integrity or DFI (Voge et al. 2010).
The choice of extender affects the results of storage trials, depending on whether the
samples contain SP or not. There is some evidence that the commonly used skim milk
extender (Kenney et al. 1975) may not be the best alternative for cooled storage of semen
after complete SP removal. Padilla and Foote (1991) compared sperm motility values of
centrifuged and non-centrifuged semen samples stored 4 ºC for 24–72 h in either skim milk
extender (KE) containing glucose, non-fat dry skim milk, Na penicillin, streptomycin and
water, or KE supplemented with a modified high-potassium Tyrode’s medium (KMT).
This extender contained 65% KE extender and 35% modified Tyrode’s medium, which
consisted of NaCl, KCl, NaHCO3, NaH2PO4, Na lactate syrup, CaCl2. MgCl2, HEPES, Na
pyruvate, bovine serum albumin and deionized water. The osmotic pressure and pH for KE
and KMT were 330 and 315 mOsm/kg and 6.8 and 7.2, respectively. The Na+ and K+
content in SP (120 and 26 mM) was higher than in KMT (43 and 10 mM) and KE (1 and 1
mM). When SP was removed after centrifugation, the motility values were higher in the
samples that were stored in KMT compared with the samples in KE, but when SP was
present (in samples extended to a sperm concentration of 50 x 106), KE seemed superior to
KMT. In another study, KMT gave higher motility values after storage than KE,
irrespective of the presence of SP (Rigby et al. 2001). The KMT extender together with
removal of SP also improved the longevity of sperm motility compared with KE in a more
recent study (Akcay et al. 2006).
24
The effect of SP on sperm longevity in the different ejaculate fractions has been examined
in a few studies. Sperm-rich fractions extended to a sperm concentration of 25 x 106
sperm/ml showed superior motility values compared with whole ejaculates and lower
extension rates (100 x 106 and 50 x 106; Varner et al. 1987). Sieme et al. (2004) found that
centrifuged sperm-rich fractions had higher post-thaw motility and viability compared with
both non-centrifuged sperm-rich fractions (i.e. samples containing SP) and centrifuged
whole ejaculates. Thus, it seems that the fluid in the last ejaculate fractions, mainly
produced by the seminal vesicles, has a more pronounced negative effect on sperm
survival. Vesiculectomy has been shown to improve sperm motility and viability values
after 4 h of cooled storage compared to semen collected before surgery from the intact
stallions (Webb et al. 1990). On the other hand, Akcay et al. (2006) reported that SP from
the sperm-rich fraction decreased progressive sperm motility and plasma membrane
integrity (PMI) compared with SP from the sperm-poor fractions.
Variation between stallions in the effects of SP on spermatozoa is evident. It has been
reported that spermatozoa from individual stallions react differently to the presence or
removal of SP, and to the choice of extender (Padilla and Foote 1991, Jasko et al. 1992,
Akcay et al. 2006). Exchange studies, where spermatozoa from one stallion were stored in
another stallion’s SP, have also been carried out; the effects of SP varied between stallions,
and SP from some of the stallions seemed to have less negative effects on sperm survival
during storage (Aurich et al. 1996). On the other hand, adding autologous or heterologous
SP to cooled semen did not affect sperm motility at 24 or 48 h in a study by Todd et al.
(2001). It has been recommended that stallions producing semen with poor tolerance to
cooling and storage should be evaluated for the effects of their SP and different extenders
on semen quality, thus enabling alterations of semen processing techniques for individual
stallions (Brinsko et al. 2000, Love et al. 2005). Brinsko et al. (2000) studied the effect of
SP on sperm motility during cooled storage in stallions considered “good coolers” and
“bad coolers”, grouping the stallions according to the reduction in progressive sperm
motility after 24 h of cooled storage. Centrifugation and partial removal of SP (90%)
increased the percentage of progressively motile spermatozoa of “bad coolers” after 48 h of
cooling. At 24 h the effect was not as obvious. The authors concluded that components of
SP adversely affect sperm motion characteristics after cooling and storage. These effects
are independent of their concentration, and some stallions will benefit from centrifugation
and SP removal, especially if the semen is stored > 24 h.
2.6. The effects of seminal plasma in the genital tract of the mare
A transient inflammatory reaction, caused mainly by the presence of sperm, occurs after
natural breeding and artificial insemination (Kotilainen et al. 1994), and SP contains
factors that modulate this process. In in vitro experiments, SP has been shown to suppress
chemotaxis of (PMNs), opsonization and phagocytosis of spermatozoa and complement
25
activity (Troedsson et al. 1999, Troedsson et al. 2000, Troedsson et al. 2005). The
suppression of PMN chemotaxis was caused to a large degree by a factor with a molecular
weight in the range of 50–100 kDa, but other SP components may also affect PMN
chemotaxis (Troedsson et al. 1999). The presence of SP in insemination doses seems to
protect spermatozoa within the uterus, perhaps through a reduction in the binding of
spermatozoa to PMNs (Troedsson et al. 2002). A protein fraction <35 kDa has been shown
to suppress PMN binding to both viable and dead sperm, and a greater molecular weight
fraction promotes binding between PMN and dead sperm (Troedsson et al. 2005).
Subsequently, the SP protein CRISP-3 has been shown to be responsible for the protection
of live spermatozoa from PMN binding (Troedsson et al. 2010).
In contrast, in vivo experiments have shown a stimulatory effect of SP on the inflammatory
response and an increase in the number of PMNs in the uterus after insemination compared
with extender or saline (Kotilainen et al. 1994, Parlevliet et al. 1997). Pregnancy rates
have, however, been shown to be equal in normal mares inseminated with or without SP
(Portus et al. 2005).
Effects of SP on sperm transport have been described in swine. The number of
spermatozoa found in the oviducts 4 h after insemination was higher when the spermatozoa
had been thawed in SP instead of a buffer (Einarsson and Viring 1973). The presence of SP
also affects the blood flow to the uterus and oviducts in mares (Bollwein et al. 2001). The
functions of SP in the sperm-uterus interaction and fertilization do not directly correspond
to fertility rates. When mares were inseminated with semen stored without SP or with
semen containing 20% SP, there were no differences between the pregnancy rates of the
two mare groups (Rigby et al. 2001).
2.7. Research objectives
Based on earlier research on the effects of SP on spermatozoa during storage, it was
hypothesized that the removal of SP would clearly improve sperm longevity. As reviewed
here, research on the effects of SP on sperm survival during storage has mainly been
carried out on whole ejaculates, or the sperm-rich fractions have been compared to whole
ejaculates. The studies presented in this thesis compared the effects of the presence of SP
from different ejaculate fractions on sperm survival during storage. The goal was to
improve semen handling procedures in order to achieve better sperm longevity and
fertility. The information on the composition of SP has also been incomplete, and further
studies were needed to examine the differences in composition between different SP
fractions, and how the differences in the composition of SP between stallions are linked to
sperm survival during storage.
26
3. Aims
The studies forming this thesis focused on the composition of fractionated stallion SP and
on the effects of ejaculatory fractions on sperm survival during storage in an attempt to
examine the physiology of stallion SP and how it affects sperm longevity and fertility. The
ultimate goal was to provide a sound basis for improved methods of storing stallion semen.
The specific aims of these studies were to:
1. determine which SP fraction could best maintain sperm survival during storage (I,
IV);
2. study whether the removal of most of the SP before storage would improve sperm
survival during storage (I, II);
3. analyze and compare the levels of selected enzymes, electrolytes and proteins in
different SP fractions (II, III, IV);
4. assess stallion variability in the effects of SP and in the composition of SP (I, II,
III); and
5. examine the association between the components of SP and semen quality, sperm
longevity and fertility (IV).
27
4. Material and methods
Presented here is an overview of the material and methods used in each study, with an
overview of the experiments summarized in Table 1. More detailed descriptions are
available in the original publications (I–IV). Five experiments (Experiments A–E) were
conducted to collect data for the four original publications, each including one, two or
three trials that were parts of different experiments (A-E).
In Experiment A, the purpose was to collect semen and SP samples to evaluate the effects
of the presence of SP and different ejaculate fractions on sperm survival during cooled
storage.
In Experiment B, the effects of SP and the different ejaculate fractions on sperm survival
were studied after freezing and thawing.
Experiment C was designed to compare the effects of SP from two fertile and two
subfertile stallions on sperm survival during cooled storage in different ejaculate fractions.
Seminal plasma was also exchanged between stallions.
In Experiment D, SP samples were collected for the determination of the levels of selected
biochemical components in the different fractions. Similar SP samples were also collected
in Experiments A, C, and E.
In Experiment E, samples were collected for analysis of the association between
components of SP and semen quality, sperm longevity, and fertility.
28
29
II: Trial 2
C
(Feb 2003)
III: Trial 2
II: Trial 3
E
IV
(Aug 2006)
D
(Feb 2005)
1, 5, 6
I: Trial 2
B
(Oct 2001)
III: Trial 1
1, 5, 6, 7
A
I: Trial 1
(Aug 2001) II: Trial 1
43 stallions Sperm motility and DFI after cooled storage in
semen fractions stored and with or without SP;
Na, Cl, K, Mg, Ca, Pi, AP, ACP, PGE2 in SP
fractions
SDS-PAGE of SP fractions
Katila et al. 2001,
Koskinen et al.
2002
Published
abstracts
1-4 fractions/ejaculate:
HIGH: fraction with highest sperm
concentration;
LOW: fraction with lowest sperm concentration
Kareskoski et al.
2008a,
Kareskoski et al.
2008b
1-4 fractions/ejaculate:
Kareskoski et al.
HIGH: fraction with highest sperm
2009
concentration;
LOW: fraction with lowest sperm concentration;
WE: whole ejaculate
1-4 fractions/ejaculate:
Kareskoski et al.
PRE: pre-sperm fluid;
2005b
HIGH: fraction with highest sperm
concentration;
LOW: fraction with lowest sperm concentration;
WE: whole ejaculate
Kareskoski et al.
2005a,
Katila et al. 2006
1-3 fractions/ejaculate:
Katila et al. 2002
HIGH: fraction with highest sperm
Katila et al. 2003
concentration;
LOW: fraction with lowest sperm concentration
Cups 1 and 2: sperm-rich fractions;
Cups 3 and 4: sperm-poor fractions;
WE: whole ejaculate
Cup 1: pre-sperm fluid;
Cups 2 and 3: sperm-rich fractions;
Cups 4 and 5: sperm-poor fractions;
WE: whole ejaculate
Fractioning
Proteins (RP-HPLC, mass spectrometry) in SP 1-3 fractions/ejaculate:
fractions
HIGH-1 and HIGH-2: sperm-rich fractions;
LOW: sperm-poor fractions; WE: whole
ejaculate
Na, Cl, K, Mg, Ca, Pi, AP, ACP, BG in SP
fractions
Sperm motility and PMI after freezing and
thawing in semen fractions stored with or
without SP
Sperm motility and PMI after
cooled storage in semen fractions stored with
or without SP;
Na, Cl, K, Mg, Ca, Pi, AP, ACP, BG in SP
fractions
Analyses
1, 2, 4, 5, 8, Na, Cl, K, Mg, Ca, Pi, AP, ACP, BG in SP
9, 10
fractions
1, 2, 3, 4
Stallion
identity
Experiment Original
(time)
article
Table 1. Overview of experiments, material, methods and published abstracts.
4.1. Animals
The stallions in Experiments A-D were housed at the Equine Research Station at MTT
Agrifood Research Finland in Ypäjä. The stallions in Experiment E were privately owned
breeding stallions, and semen was collected at the stud farm where the stallion was placed
for the breeding season.
4.1.1. Experiment A (I: Trial 1, II: Trial 1) and Experiment B (I: Trial 2)
Four reproductively normal stallions were used for semen collection. Three of the stallions
(Finnhorses, Stallions 1 and 5, aged 14 and 17 years, and one Standardbred, Stallion 7,
aged 15 years) had previously been used for breeding, while the third Finnhorse stallion
(Stallion 6, aged 7 years) had no breeding history. After one week of daily semen
collections, the total sperm counts on the first day of Experiment A were 5.8 x 109
spermatozoa (Stallion 1), 10.0 x 109 spermatozoa (Stallion 5), 7.4 x 109 spermatozoa
(Stallion 6), and 5.2 x 109 spermatozoa (Stallion 7).
4.1.2. Experiment C (II: Trial 2, III: Trial 1)
Semen was collected from two stallions that were considered to be reproductively normal
and had good breeding records, with seasonal pregnancy rates of 80% and 82% in the
previous season (Stallion 1: Finnhorse aged 16 years; Stallion 3: Standardbred aged 12
years), and two stallions producing semen with a low sperm concentration and low
progressive motility and considered to be subfertile (Stallion 2: Finnhorse aged 19 years;
Stallion 4: Finnhorse aged 24 years). The book of stallions 2 and 4 had to be limited
because of subfertility: Stallion 2 and Stallion 4 served only three and eight mares in 2002,
and thus, fertility data are not meaningful. Stallion 2 was the son of Stallion 4. A breeding
soundness examination was performed on all of the stallions before collecting semen
samples for analyses. The testicular index (TI, Kenney et al. 1983) was calculated based on
measurements of the testes, and the sperm motility of an extended fresh semen sample was
assessed subjectively using a phase contrast microscope (Olympus BH2, Olympus Optical
Co., Hamburg, Germany). At the time of the examinations, Stallion 1 had a TI of 5.9 and
progressive sperm motility of 50%, and the corresponding figures for Stallion 3 were 4.9
and 60%. Stallion 2 had a TI of 5.7 and motility of 20%, and Stallion 4 correspondingly 1.9
and 30%. After one week of daily semen collections, the total daily sperm counts were 5.5
x 109 spermatozoa (Stallion 1), 2.0 x 109 spermatozoa (Stallion 2), 4.5 x 109 spermatozoa
(Stallion 3), and 1.3 x 109 spermatozoa (Stallion 4).
30
4.1.3. Experiment D (II: Trial 3, III: Trial 2)
Semen samples were collected from seven Finnhorse stallions (ages 3–26) varying in
breeding history.
4.1.4. Experiment E (IV)
Semen was collected from 43 stallions (ages 4–23 years) at nine stud farms in Finland. A
total of 30 stud farms, housing at least one stallion that had served over 20 mares in the
previous season, were invited to offer their residing stallions to participate, and all of the
available stallions were included in the study. All of the samples were collected during the
breeding season in 2006. Twenty stallions were Standardbred trotters (ages 4–23 years), 17
were Finnhorses (ages 8–23 years), four were warmblood riding horses (ages 9–19 years)
and two were ponies (ages 8 and 9 years). The breeding history and current use of the
stallions was variable, with the stallions serving 2 to 150 (median 14) mares in 2006.
4.2. Semen collection
The semen collections in Experiments A, B, C and D (I–III) took place at the equine
research station in Ypäjä (MTT Agrifood Research Finland), using a computer-controlled
fractioning phantom (Equidame®, Haico Oy, Loimaa, Finland) that collects portions of the
ejaculate separately into five cups (Fig. 1). In this collection device, the fractioning is
based on a pre-set target weight of each cup, and the settings can be adjusted individually
to optimize the separation of fractions. The stallion ejaculates into the metallic funnel that
is in front of the AV inside the phantom (Fig. 2). The plastic cups are attached to a moving
tray and are located below the metallic funnel, so that semen fractions can be collected into
the plastic cups that move horizontally below the funnel (Lindeberg et al. 1999). Before the
actual experiments, the stallions were collected daily to empty the extra-gonadal sperm
reserves.
Fig. 1. A fractionated ejaculate
collected with the Equidame®phantom. Pre-sperm fluid is seen in
the first cup on the right, followed
by sperm-rich and sperm-poor
fractions. The last small cup and the
large cup contained mostly gel in
this ejaculate.
31
Fig. 2. The Equidame® phantom consists of the following parts: a detachable open-ended
artificial vagina (1A) and a rubber ring (1B) inside the vagina, a detachable metallic funnel
(2), 5 detachable metallic holders (3A) and 5 plastic cups (3B), small styrofoam shields (3C)
that envelope the plastic cups to isolate semen, and a computer control panel for adjusting the
settings for semen fractioning (Lindeberg et al. 1999).
4.2.1. Experiment A (I: Trial, II: Trial 1)
Each stallion was collected twice a week for two weeks in two trials. In Trial 1, pre-sperm
fluid without any spermatozoa was collected in cup 1, the sperm-rich fractions into cups 2
and 3, and the last fractions with a low sperm concentration into the last two cups
(combined in cup 4). Small amounts of pre-sperm fluid may also have been included in cup
2, which contained the first jets of the ejaculate.
4.2.2. Experiment B (I: Trial 2)
Cup 1 contained sperm-rich fractions, and the following cups (2–5 cups per ejaculate)
contained fractions with successively decreasing sperm concentration. Pre-sperm fluid was
not collected separately in this trial.
4.2.3. Experiment C (II: Trial 2, III: Trial 1)
Semen was collected twice a week for two weeks. The fractioning phantom collected the
ejaculates in 4–5 cups (Table 2).
32
Table 2. Semen volume (ml; mean, min-max) and sperm concentration (106
spermatozoa/ml; mean, min-max) in fractionated ejaculates collected in 3 cups
(Experiment C).
Volume
Stallion
Cup 1
Sperm
concentration
Cup 2
Cup 3
Volume
Sperm
concentration
Volume
Sperm
concentration
1
15.5
194.5
13.8
238.8
7.8
15.3
2
(5-24)
9.5
(5-16)
(160-253)
90.8
(54-154)
(9-21)
15.5
(10-25)
(135-340)
154.3
(103-213)
(4-12)
11.0
(7-16)
(11-20)
44.8
(4-95)
3
9.0
(8-10)
294.0
(273-315)
9.5
(8-11)
147.0
(158-441)
4.7
(4-5)
234.3
(86-273)
4
10.8
(9-14)
13.3
(5-26)
34.3
(20-41)
24.3
(8-45)
16.3
(10-21)
6.5
(2-13)
In Trial 2 (II), the cups with the highest (HIGH) and lowest (LOW) sperm concentration
were selected from each ejaculate for the SP analyses. In Trial 1 (III), cup 1 contained presperm fluid and the first sperm-rich jets (portion 1 of the ejaculate, HIGH-1), while Cup 2
contained the rest of the sperm-rich part of the ejaculate (portion 2 of the ejaculate, HIGH2). The following cups (3–5) were fractions with a low sperm concentration, and they were
combined to form portion 3 of the collected ejaculate (LOW). The HIGH-fractions always
preceded the LOW fractions by definition.
33
4.2.4. Experiment D (II: Trial 3, III: Trial 2)
Semen was collected twice a week for three weeks. The fractioning phantom collected the
ejaculates in 4–5 cups, as in Experiment C (Table 3). The cups with the highest (HIGH)
and lowest (LOW) sperm concentration were selected from each ejaculate for the SP
analyses, and pre-sperm fluid (PRE) samples were collected separately.
Table 3. Semen volume (ml; mean, min-max) and sperm concentration (106
spermatozoa/ml; mean, min-max) in all fractionated ejaculates collected in Experiment D,
and the total sperm count (109 spermatozoa) for each stallion after one week of daily
ejaculations.
Stallion
Total sperm
count
Fraction
Sperm
concentration
Volume
1
8.3
HIGH
LOW
317.7 (294-380)
110.5 (12-223)
14 (8-20)
7.3 (5-10)
2
6.2
PRE
HIGH
LOW
142.5 (108-172)
48.7 (17-115)
11.5 (5-20)
19.7 (8-26)
12.0 (5-27)
4
0.8
PRE
HIGH
LOW
13.0 (8-20)
2.8 (2-5)
12.2 (8-20)
25.8 (23-28)
18.3 (6-34)
5
8.0
PRE
HIGH
LOW
319.0 (281-387)
174.7 (76-279)
2.5 (2-3)
8.6 (6-13)
5.3 (4-6)
8
8.5
PRE
HIGH
LOW
238.2 (175-323)
67.8 (15-159)
7.0 (4-14)
17.3 (10-28)
9.8 (2-15)
9
7.4
PRE
HIGH
LOW
98.0 (12-194)
359.0 (212-422)
9
8.7 (2-18)
13.7 (10-25)
10
2.4
HIGH
LOW
204.5 (177-232)
171.0 (153-189)
8.5 (7-10)
6.5 (4-9)
PRE: pre-sperm fluid; HIGH: fraction with the highest sperm concentration; LOW:
fraction with the lowest sperm concentration.
4.2.5. Experiment E (IV)
One ejaculate was collected from each stallion. Ejaculatory jets were collected as 1–4
fractions depending on individual differences in the ejaculates. The ejaculates were
collected using either an open-ended artificial vagina (AV), a modified closed AV or the
Equidame® phantom, depending on stallion preference and stud farm. The Equidame®
34
phantom was available only at one stud farm (6 stallions). The open-ended and closed AVs
were from variable manufacturers, because the AV available at the stud farm was
preferably used. The modified closed AV (8 stallions) was a Missouri type AV with an
ultrasound probe cover (Kruuse A/S, Langeskov, Denmark) attached at the end of the AV
instead of the semen collection bottle, and the same probe covers were also attached to the
funnel used in semen collection with the open-ended AV. Using this technique, the
ejaculate fractions could be separated with hemostatic forceps or by hand, as the fractions
could be seen entering the probe cover (Fig. 3). The sperm concentrations (measured with
a Bürker counting chamber) in the HIGH and LOW fractions collected with the different
methods in Experiment E (IV) are shown in Table 4.
Fig. 3. The fractioning method
used in Experiment E (IV). The
ejaculate fractions were
separated with forceps or by
hand as the fractions could be
seen entering the probe cover.
Table 4. The sperm concentrations (106 spermatozoa/ml; median, min – max) in the HIGH
and LOW fractions collected with an open-ended AV (29 stallions), a Missouri-type closed
AV (8 stallions) and the Equidame®-phantom (6 stallions).
Semen collection method
Open-ended AV
Closed AV
Equidame®-phantom
All methods
Fraction: HIGH
300.5 (24-933)
259.0 (30-429)
288.0 (38-463)
300.0 (24-983)
35
Fraction: LOW
54.0 (6-522)
85.0 (21-280)
12.0 (7-88)
54.0 (6-522)
As a description of the collected ejaculates, the initial sperm motility and sperm
morphology is shown in Table 5. The methods of motility and morphology assessment are
described in section 4.4.
Table 5. Sperm morphology (%; median, min-max) and sperm
motility (%; median, min-max) before storage in Experiment E.
Sperm morphology
Normal sperm
Abnormal heads
Abnormal acrosomes
Tailless heads
Proximal droplets
Distal droplets
Abnormal midpieces
Bent tails
Coiled tails
Median
Min-max
52.5
5.0
4.0
2.5
5.5
12.5
2.0
3.0
3.0
2.5-82.0
0.5-41.5
0.0-11.0
0.0-83.0
1.0-24.5
0.0-42.0
0.0-27.5
0.5-10.0
0.0-43.5
80
50
5-90
0-80
Sperm motility
Total motility
Progressive motility
4.3. Semen handling and preparation of samples
After collection, a percentage (5–10% depending on the study) of the volume of each cup
was combined to form a sample representing the whole ejaculate (WE) (I, II, III).
4.3.1. Experiment A (I: Trial 1, II: Trial 1)
Trial 1 in original article I compared the effects of different SP fractions on the
maintenance of sperm motility and membrane integrity during cooled storage. The semen
was extended in a ratio of 1:1 with skim milk extender (Kenney et al. 1975). Half of each
sample was centrifuged (500 g, 15 min) and the supernatant removed. Extender was added
to the centrifuged samples to a sperm concentration of 50 x 106 sperm/ml after the removal
of supernatant. The other half of the sample was not centrifuged, but immediately diluted
to a final sperm concentration of 25–50 x 106 sperm/ml, with a semen to extender ratio of
1:1–3. All of the samples were stored for 24 h and transported to the laboratory in 1.5-ml
vials in an Equitainer transport container (Hamilton-Thorne Research, South Hamilton,
MA, USA), which maintains a temperature of 5–10 ºC for 48 h (Katila et al. 1997). After
storage, the samples were incubated for 5 min at 37 ºC prior to the sperm motility and PMI
analyses.
36
To separate SP, a part of all samples were centrifuged at 3000 g for 20 min, and filtered
through a 0.45 µm filter (Minisart, Sartorius, Göttingen, Germany). The filtered SP was
frozen in 1-ml aliquots at -75 ºC.
4.3.2. Experiment B (I: Trial 2)
Skim milk extender (Kenney et al. 1975) was added to all cups at a ratio of 1:1, and the
samples were placed in a refrigerator at 5 ºC for 2 h to increase the effects of SP. After
centrifugation at 500 g for 15 min, the supernatant was removed and skim milk extender
with or without SP was added. Egg yolk and glycerol (2% and 2.5% of the final volume,
respectively) were also added. The contents of each cup were extended further to a final
concentration of 200 x 106 sperm/ml by adding either more of the mentioned freezing
extender, or freezing extender and a part of the previousl y removed supernatant (50% SP
and 50% extender). The samples were frozen in 0.5-ml straws, at a rate of -50 ºC/min from
+13 ºC to -140 ºC using a programmable freezer (Planer Kryo 10 Series II, Sunbury of
Thames, UK) and plunged into liquid nitrogen. The straws were thawed for 30 s in a 37 ºC
water bath, and the samples were then diluted by adding skim milk extender at a ratio of
1:1, and the incubation at 37 ºC was continued for an additional 15 min.
4.3.3. Experiment C (II: Trial 2, III: Trial 1)
After semen collection, HIGH-2 was divided into two parts. Half of the semen in HIGH-2
was extended 1:1 using skim milk extender (Kenney et al. 1975) and centrifuged at 500 g
for 15 min. The supernatant was removed and the sperm pellet was re-suspended in a small
volume of skim milk extender. The sperm concentration was then determined using a
Bürker counting chamber, and the suspension was extended with skim milk extender to a
final concentration of 75 x 106 spermatozoa/ml. Semen was subsequently mixed in a ratio
of two parts extended semen to one part SP (2:1, v/v). The final sperm concentration was
40–50 x 106 spermatozoa/ml. A part of each sample was also suspended in extender and SP
from another stallion, as SP was exchanged between Stallions 1 and 2, and Stallions 3 and
4. The samples were packed in an Equitainer (Hamilton Research, Inc., South Hamilton,
MA, USA) in 1.5-ml vials and stored for 24 h while shipped to the laboratory. The
fractions HIGH-1, LOW, WE, and the remaining half of HIGH-2 were processed further to
be used as SP. The SP samples were centrifuged at 3000 g for 20 min, and filtered through
a 0.45 µm filter (Minisart, Sartorius, Göttingen, Germany). The filtered SP was frozen in
1-ml aliquots at -75 ºC.
4.3.4. Experiment D (II: Trial 3, III: Trial 2)
All of the samples were centrifuged at 3000 g for 20 min, and filtered through a 0.45 µm
filter (Minisart, Sartorius, Göttingen, Germany). The filtered SP was frozen in 1-ml
aliquots at -75 ºC.
37
4.3.5. Experiment E (IV)
After semen collection, the gel was removed and the volume of each fraction was
measured. The sperm concentration in each fraction was determined using a Bürker
counting chamber. One drop of semen was placed on two glass slides and smears for
morphological evaluation were prepared and air-dried. For the sperm chromatin structure
assay (SCSA) to be performed at a later date, a sample of 2–10 x 106 spermatozoa from
each fraction of raw semen was pipetted into cryovials and TNE-buffer (9.48 g Tris-HCl,
52.6 NaCl, 2.23 g disodium-EDTA, aqua ster. ad 600 ml, pH 7.4) was added (ad 1.5 ml in
each vial). The SCSA samples were placed in liquid nitrogen vapor (3 cm above the liquid
surface) for 10 min, and then plunged into liquid nitrogen.
Each fraction was divided into two parts: one half was centrifuged for preparation of SP
(SP samples), and the other half was processed for cooled storage (semen samples). The
semen samples were extended in a semen to extender ratio of 1:1 using skim milk
extender, and total and progressive sperm motility were assessed subjectively using phase
contrast microscopy. Each fraction of the semen samples was divided into two centrifuge
tubes, and centrifuged at 500 g for 10 min. After removal of the major part (about 5% was
left) of the supernatant (i.e. extender and SP), one part of each fraction was extended to a
concentration of 50 x 106 sperm/ml using only skim milk extender (SP0), and the other part
was extended to the same sperm concentration using a combination of supernatant and
skim milk extender at a SP to extender ratio of 1:2 (SP1). The semen samples were stored
in 1-ml vials at 5 °C for 24 h. After 24-h cooled storage, 5 x 106 spermatozoa from each
semen sample were pipetted into cryovials for SCSA analyses, TNE buffer was added (ad
1.5 ml in each vial), and the samples were placed in liquid nitrogen vapor (3 cm above the
liquid surface) for 10 min and plunged into liquid nitrogen.
The SP samples in Experiment E were centrifuged at 4000 g (15 min), and the supernatant
was filtered using 0.45 µm filters (Millex-HV, Millipore, Billerica, MA, USA). Protease
inhibitor (Trasylol 10000 Kallikrein Inactivator Units (KIU)/ml, Bayer Schering Pharma,
Berlin, Germany) was added to each sample (500 KIU/ml of SP) to reduce the proteolytic
activity on enzymes and other proteins. The SP samples were stored frozen in 1-ml aliquots
at -75 °C until analyzed.
4.4. Sperm quality assessment
Sperm motility, plasma membrane integrity and morphology were examined in the
laboratory of the Department of Production Animal Medicine at the Faculty of Veterinary
Medicine (University of Helsinki).
4.4.1 Sperm motility (I, III, IV)
The samples containing spermatozoa were warmed in a water bath (37 ºC, 5 min) after
storage for 24 h. In Experiment A, the sperm motility parameters total motility (TMOT),
38
progressive motility (PMOT), average path velocity (VAP) and the percentage of rapid
cells (RAP) were determined with a Hamilton-Thorne Motility Analyzer (HTM-S, version
7.2., Hamilton-Thorne Inc, MA, USA).
In Experiments C and E, the motility parameters TMOT, PMOT, VAP, curvilinear velocity
(VCL), straightness (STR) and linearity (LIN) were evaluated using the SpermVision
CASA system (SpermVision Minitüb, Tiefenbach bei Landshut, Germany).
4.4.2. Plasma membrane integrity (PMI) (I)
The membrane integrity of the stored spermatozoa was assessed with the method described
by Harrison and Vickers (1990), using staining with carboxyfluorescein diacetate and
propidium iodide (CFDA/PI) (Molecular Probes Inc., OR, USA) and counting 200 cells in
fluorescence microscopy.
4.4.3. Sperm morphology staining and evaluation (IV)
The morphology smears were fixed and stained using the Giemsa method according to
Watson (1975), and 200 sperm were assessed in each sample. The morphological
characteristics were classified in the following way: morphologically normal spermatozoa,
abnormal heads, abnormal acrosomes, abnormal midpieces, tailless heads, proximal
cytoplasmic droplets, distal cytoplasmic droplets, bent tails and coiled tails.
4.4.4. Sperm chromatin structure assay (IV)
Sperm chromatin stability of sperm from both the raw and stored semen samples was
measured as the susceptibility of sperm DNA to denaturation with the sperm chromatin
structure assay (SCSA) at the Department of Anatomy, Physiology and Biochemistry,
Swedish University of Agricultural Sciences (SLU, Uppsala, Sweden) as described by
Evenson et al. (1999). Acridine orange was added to each sample after exposure to an acid
detergent solution that induces partial DNA denaturation in situ, and flow cytometry was
then used to measure either red-colored (abnormal spermatozoa, denatured DNA) or greencolored (normal sperm) fluorescence. The sperm DNA fragmentation index (DFI),
formerly referred to as Cells Outside the Main Population (COMP t), was assessed by
calculating the amount of red fluorescence divided by the total (red plus green)
fluorescence, indicating the amount of denatured sperm DNA in relation to the total DNA
in each sperm cell.
4.5. Determination of enzyme and electrolyte levels in seminal plasma (II,
IV)
The enzyme and electrolyte levels were measured at the Central Laboratory of the Faculty
of Veterinary Medicine at the University of Helsinki (Clinicum, Viikki). Levels of AP
activity were measured according to the recommendations of the Scandinavian Society for
39
Clinical Chemistry and Clinical Physiology (SCE 1974), and total ACP using the method
described by Hillman (1971). The enzyme activities were measured using the reagents and
adaptations for serum applied by the manufacturer of the automatic chemistry analyser
(KONE Pro Selective Chemistry Analyser, Thermo Fisher Scientific, Vantaa, Finland).
Analyses of BG were performed on microtitration trays. Activity levels of BG were
analyzed by the method described by Linko-Löppönen and Mäkinen (1985) using 4methyl-umbelliferyl- -glucuronide (Sigma M-9130, Sigma Chemical Co., St Louis, MO,
USA) as substrate and measuring the resulting fluorescence (Fluoroskan Ascent,
Labsystems, Helsinki, Finland).
Colorimetric methods were used for the determination of Ca (Bauer 1981), Mg (Gindler
and Heth 1971) and Pi (Daly and Ertigshausen 1972) and concentrations of Na+, K+ and Clwere measured directly by ion selective electrodes. The analyses were performed on the
same automatic chemistry analyzer that was used for the enzyme analyses.
The concentration of prostaglandin E2 (PGE2) was determined with a commercial enzyme
immunoassay kit (Correlate-EIA, Assay Designs Inc., Ann Arbour, MI, USA) according to
the manufacturer’s specifications. The PGE2 determinations were performed by Prof. Hans
Kindahl at SLU in Sweden.
4.6. Protein analyses of seminal plasma (III)
In Experiment C (III: Trial 1), peptides were isolated as described by Calvete et al. (1994)
by reverse-phase high-performance liquid chromatography (RP-HPLC) on an RP-100
Lichrospher (Merck, Germany) C18 column (25 x 4 mm, 5- m particle size) eluted at 1
ml/min with a gradient of 0.1% trifluoroacetic acid in water and acetonitrile. N-terminal
amino acid sequence analyses were performed using an Applied Biosystems 472
automated protein sequencer (Applied Biosystems, Langen, Germany) to identify HSP-1,
HSP-2, and HSP-4 in relation to the RP-HPLC curves. The area-under-the-curve (AUC) on
the chromatograms was used for comparing the relative amounts of proteins within each
fraction. Samples of SP were also analyzed using matrix-assisted laser desorption
ionization – time of flight (MALDI-TOF) mass spectrometry. The RP-HPLC and mass
spectrometry were performed at the Institute of Biomedicine (CSIC) in Valencia, Spain.
In Experiment D (III: Trial 2), the SP samples were analyzed by SDS_PAGE
electrophoresis in 12% polyacrylamide gels. The molecular weight (MW) was estimated
using a protein standard (Precision Plus Protein All Blue Standard 161-0373, Bio-Rad
Laboratories, Hercules, CA, USA). Densitometry gave a quantitative estimate of each band
(AlphaImager HP, Alpha Innotech, San Leandro, CA, USA). The SDS-PAGE was done at
the Universidad Autonoma de Barcelona, Spain.
40
4.7. Fertility data (IV)
Fertility data were provided by the stud farms and the central Finnish trotting and breeding
association (Suomen Hippos ry). Pregnancy rates were calculated from all reported
matings in the year 2006 for the stallions included in the study. All mares that foaled or
aborted were considered pregnant. First cycle pregnancy rates were the main fertility
parameter used for statistical comparisons, and were calculated separately for mares
inseminated with fresh semen or transported semen using data obtained from the stud
farms and Suomen Hippos ry. Inseminations using frozen semen were excluded from the
study.
4.8. Statistical analyses
4.8.1. Experiments A-D (I, II, III)
Repeated measures analysis of variance was used to analyze the differences between SP
fractions and the whole ejaculate in sperm motility parameters, membrane integrity and
sperm concentration, and the effects of centrifugation and SP removal on motility and
membrane integrity in publication I.
Nonparametric tests were used to compare the means in publications II and III. The
Friedman test, followed by the Wilcoxon signed-rank sum test was used to analyze the
differences between SP fractions in the levels of the measured components, and the
Kruskal-Wallis test for several independent samples was used to evaluate the differences
between stallions in each semen fraction.
The Mann-Whitney U-test was used in Trial 2 of publication II to compare the means of
HIGH to LOW ratios of the measured SP components of the two stallion pairs (fertile vs.
subfertile).
Spearman’s rank correlation coefficient was used to evaluate the relationships between the
measured components of SP and sperm concentration in Trial 2 of publication II, and the
association between sperm concentration, total motility (TMOT), progressive motility
(PMOT), and average path velocity (VAP) to the AUC of the peaks containing HSP-1,
HSP-2, and HSP-4 in Trial 1 of publication III. Spearman’s rank correlation coefficient
was also used in Trial 2 of publication III to study the correlation between sperm
concentration and relative band density percentages.
41
4.8.2. Experiment E (IV)
In Experiment E (IV), semen fractions with the highest (HIGH) and lowest (LOW) sperm
concentration within each ejaculate were included in the statistical analyses. The
differences between fractions in the levels of ACP, Na, K, and Cl, and in the mean
difference (DIFF) in sperm motility parameters between SP1 and SP0 samples were
evaluated using a two-sample t-test. The t-test was also used to compare mean values in
sperm motility and DFI between fractions within the storage groups (SP1 and SP0), and
between the storage groups within fractions. Because of the skewed distribution of the
levels of PGE2, Ca, Pi and AP, and the mean DIFF in DFI, the nonparametric MannWhitney U-test was used for fraction comparisons.
The sperm longevity parameters included in the correlation analyses were the DIFF in
motility between SP1 and SP0 samples, and the DIFF in DFI between samples before and
after storage (separately for SP0 and SP1 samples). The semen quality parameters were
sperm concentration, total number of sperm, total and progressive motility before storage,
and the number of morphologically normal sperm. The first cycle pregnancy rate depicted
fertility. The correlations of sperm longevity, semen quality, and fertility parameters with
the levels of the measured SP components were evaluated using the Pearson correlation
coefficient for normally distributed parameters (all sperm motility parameters, the DIFF in
motility between SP1 and SP0 samples in all fractions, the DIFF in DFI before and after
storage with or without SP in WE, total number of sperm and number of morphologically
normal sperm in all fractions, ACP in HIGH, Ca in WE, Cl, K, Mg, Na and first cycle
pregnancy rate in all fractions, and AP in SR) and Spearman’s rank correlation coefficient
for the other parameters.
The dependent variables (i.e. the sperm longevity, semen quality, and fertility parameters)
that correlated significantly with a minimum of two SP components were included in a
stepwise linear regression analysis for the assessment of the association of the levels of
these SP components (explaining variables) with each dependent variable.
Significance was set at p < 0.05 in all studies. The statistical analyses in Studies I, II, and
III were performed using SPSS 13.0 software (SPSS Inc., Chicago, IL, USA). In
Experiment E (IV), SAS software (SAS Institute Inc., Cary, NC, USA) was used.
42
5. Results
5.1. Effects of seminal plasma fractions and seminal plasma removal on
sperm survival during storage (I, IV)
5.1.1. Effects of seminal plasma removal on sperm longevity (I, IV)
In Experiment A (I), centrifugation and the subsequent removal of SP decreased all sperm
motility parameters measured after 24 h of storage in all SP fractions, except for TMOT
and PMOT in cup 4, containing the last fractions with a low sperm concentration,
compared to samples containing SP. Sperm membrane integrity was not affected by
centrifugation and SP removal.
In Experiment E (IV), VAP, VCL, and VSL were significantly higher in SP1 than in SP0
in all fractions (Fig. 3). The DFI was higher in SP1 than in SP0 in HIGH and LOW, but not
in WE. The DFI was higher in SP1 compared with samples taken before storage in all
fractions, and the DFI was higher in SP0 compared with the samples taken before storage
in LOW, but no differences were detected in HIGH or WE (Fig. 4).
43
SP1
200
SP0
Sperm motility
*
150
*
HIGH
100
*
*
* *
50
0
TMOT
PMOT
VAP
VCL
VSL
SP1
Sperm motility
200
*
150
*
100
SP0
*
LOW
*
* *
50
0
TMOT
PMOT
VAP
VCL
SP1
200
*
Sperm motility
VSL
*
150
SP0
WE
100
* *
* *
50
0
TMOT
PMOT
VAP
VCL
VSL
Fig. 3. Mean (± SE) values of total motility (TMOT, %), progressive motility (PMOT, %),
average path velocity (VAP, µm/s), curvilinear velocity (VCL, µm/s), and straight-line velocity
(VSL, µm/s) in two cold-storage groups (with seminal plasma, SP1, and without seminal plasma,
SP0) and in ejaculate fractions with high (HIGH) and low (LOW) sperm concentration, and in
whole ejaculates (WE) in Experiment E (IV).
*Asterisks indicate significant differences between SP1 and SP0 groups.
44
b
35
b
c*
30
DFI
25
a
a*
b
Before storage
a
a
20
SP1 after storage
15
10
SP0 after storage
5
0
HIGH
LOW
WE
Fig. 4. Mean (± SE) values of the DNA fragmentation index (DFI) in ejaculate fractions
with high (HIGH) and low (LOW) sperm concentration, and in whole ejaculates (WE), in
samples taken before storage, and in two cold-storage groups (with seminal plasma, SP1,
and without seminal plasma, SP0) in Experiment E (IV).
abc
Different letters indicate significant difference between storage groups (before storage,
SP1 and SP0) within an ejaculate fraction.
*Asterisks indicate significant differences between fractions within a storage group (SP1
and SP0).
In HIGH, the DIFF between SP1 and SP0 (DIFF = SP1 – SP0) in sperm motility was
positive (SP1 > SP0) in the following proportion of stallions (stallions with SP1 > SP0 / all
stallions): TMOT 53%, PMOT 88%, VAP 65%, VCL 65%, STR 40% and LIN 53%. In
LOW, the corresponding figures were: TMOT 57%, PMOT 46%, VAP 83%, VCL 86%,
STR 26%, LIN 31% (Fig. 5).
The DIFF between DFI after storage and before storage (DIFF = after storage – before
storage) was positive (after storage > before storage) in 87% of stallions in HIGH, and in
91% of stallion in LOW (Fig. 6).
The mean DIFF between SP1 and SP0 (DIFF = SP1 – SP0) samples in TMOT, PMOT,
VAP, VCL, VSL and DFI was positive in both HIGH and LOW fractions. There was a
positive mean DIFF in DFI between samples taken after storage and before storage (DIFF
= after storage – before storage), in both SP1 and SP0 groups. A negative mean DIFF
between SP1 and SP0 in STR and LIN was found in both HIGH and LOW fractions.
In Experiment B (I), samples containing SP showed significantly higher post-thaw TMOT,
PMOT and RAP in cup 1 (sperm concentration 234 ± 142 x 106) and cup 2 (sperm
concentration 170 ± 96 x 106), and in WE, compared with samples stored in extender
without SP. The presence of SP did not significantly affect VAP or membrane integrity.
45
In summary, the removal of SP decreased motility and decreased the amount of DNA
damage during cooled storage.
5.1.2. Differences between seminal plasma fractions in the effects on sperm survival
during storage (I, IV)
In Experiment A (I), the differences between ejaculate fractions in the measured sperm
motility parameters after cooled storage were non-significant.
In Experiment E (IV), there were no significant differences between HIGH and LOW in
any of the measured sperm motility parameters within the two storage groups (Fig. 3). In
samples taken before storage, and in SP1, the DFI did not differ between HIGH and LOW.
In SP0, the DFI was higher in LOW than in HIGH (Fig. 4).
There were no significant differences between the two fractions in the mean DIFF of any
of the motility parameters. The DIFF in DFI in the SP0 samples was lower in HIGH than
in LOW, whereas no differences between fractions could be seen in the SP1 samples.
After freezing and thawing in Experiment B (I), the mean values of TMOT, PMOT and
RAP were significantly higher in cup 1 (the sperm-rich fractions) compared with WE. The
differences between fractions in VAP and membrane integrity were non-significant.
In summary, the least amount of DNA damage was found in the sperm-rich fraction stored
without SP. Sperm motility after cooled storage did not differ between fractions.
5.1.3. Differences between stallions in sperm survival during storage (I)
The differences between individual stallions were significant in several parameters in
Experiments A and B (all motility values in Experiment A, and TMOT, PMOT, RAP and
membrane integrity in Experiment B). The DIFF in total and progressive motility, and in
straightness and linearity, between samples stored with or without SP (DIFF = SP1 – SP0)
for each individual stallion in Experiment E (IV) is illustrated in Fig. 5 and 6, respectively.
The DIFF in DFI between samples stored with or without SP (DIFF = SP1 – SP0) for each
stallion in shown in Fig. 7.
46
30
Difference in motility (%)
25
HIGH
20
15
10
5
0
-5
1 2 3 4 5 6 7 8 9 10111213141516171819202122232425262728293031323334353637383940
-10
-15
Stallion
-20
-25
Difference in total motility
Difference in motility (%)
30
Difference in progressive motility
LOW
20
10
0
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
-10
Stallion
-20
-30
Difference in total motility
Difference in progressive motility
Fig. 5. The difference in total and progressive motility between samples stored with or without
seminal plasma for each individual stallion in sperm-rich (HIGH) and sperm-poor (LOW) semen
fractions in Experiment E (IV).
47
Difference in velocity (µm/s)
80
HIGH
60
40
20
0
1 2 3 4 5 6 7 8 9 10111213141516171819202122232425262728293031323334353637383940
-20
-40
Stallion
Difference in average path velocity
Difference in velocity (µm/s)
120
100
Difference in curvilinear velocity
LOW
80
60
40
20
0
-20
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
-40
-60
-80
Stallion
Difference in average path velocity
Difference in curvilinear velocity
Fig. 6. The difference in average path velocity and curvilinear velocity between samples stored
with or without seminal plasma for each individual stallion in sperm-rich (HIGH) and spermpoor (LOW) semen fractions in Experiment E (IV).
48
HIGH
Difference in DFI (%)
40
30
20
10
0
-10
-20
1 2 3 4 5 6 7 8 9 101112131415161718192021222324252627282930313233343536373839
Stallion
Difference in DFI
LOW
Difference in DFI (%)
40
30
20
10
0
-10
-20
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
Stallion
Difference in DFI
Fig. 7. The difference in the DNA fragmentation index (DFI) between samples stored with or
without seminal plasma for each individual stallion in sperm-rich (HIGH) and sperm-poor
(LOW) semen fractions in Experiment E (IV).
5.1.4. The effects of seminal plasma exchange between stallions on sperm motility (III)
In Experiment C, significant differences were seen between stallions in TMOT and PMOT
after storage in the stallion’s own SP in HIGH-2. The SP of Stallion 3 seemed to reduce
both the TMOT and PMOT of spermatozoa from Stallion 4, with a decrease of 14–28%,
depending on the ejaculate fraction, compared with the samples stored in the stallion’s own
SP. These differences were not tested statistically, because of the low number of samples
(three or four ejaculates per stallion).
49
5.2. Levels of alkaline phosphatase, acid phosphatase, -glucuronidase and
electrolytes in seminal plasma fractions (II, IV)
5.2.1. Differences between seminal plasma fractions (II, IV)
The levels of AP and ACP were higher in HIGH than in LOW in all experiments. In
Experiment D (II), the lowest levels of AP and ACP were found in PRE. The levels of BG
were higher in HIGH than in LOW.
The concentrations of Ca and Mg were higher in LOW than in HIGH, with the lowest
concentrations found in PRE.
The concentration of Pi was highest in HIGH and lowest in PRE in Experiment D (II), but
in Experiment E (IV), the difference between fractions was non-significant.
The K concentration did not differ between fractions in Experiment D (II), whereas in
Experiment E (IV), the concentration of K was higher in LOW than in HIGH.
The highest concentration of Na and Cl was found in PRE. There were no significant
differences between HIGH, LOW, and WE in the concentration of Na and Cl in
Experiment D (II), but in Experiment E (IV), the concentration of Na was higher in HIGH
compared with LOW.
The levels of PGE2 did not differ between fractions.
The levels of the studied components in Experiments D and E are presented in Table 6.
50
Table 6. The levels (mean ± SE) of biochemical components of stallion seminal plasma in
pre-sperm fluid (PRE), ejaculate fractions with the highest (HIGH) and lowest (LOW) sperm
concentrations, and in whole ejaculates (WE). The studied components were prostaglandin E2
(PGE2), alkaline phosphatase (AP), acid phosphatase (ACP), Ca, Cl, K, Mg, Na and inorganic
phosphate (Pi). N = number of samples.
Experiment D (II)
7 stallions
AP
ACP
Ca
Cl
K
Mg
Na
Pi
PRE
HIGH
LOW
WE
N = 16
N = 36
N = 33
N = 36
487 ± 114a
1.0 ± 0.2a
0.13 ± 0.01a
129.2 ± 0.6a
17.8 ± 0.6a
0.46 ± 0.03a
140.1 ± 0.6a
0.12 ± 0.01a
40374 ± 2906b
14.1 ± 1.4b
0.98 ± 0.07b
116.2 ± 2.3b
19.4 ± 0.6b
1.78 ± 0.17b
123.1 ± 2.8b
0.69 ± 0.04b
13738 ± 2501c
7.5 ± 1.1c
2.24 ± 0.26c
119.0 ± 1.3b
20.2 ± 0.8b
3.95 ± 0.55c
121.7 ± 2.2b
0.44 ± 0.04c
30396 ± 2719d
11.0 ± 1.1d
1.30 ± 0.10d
117.5 ± 1.3b
19.5 ± 0.6b
2.12 ± 0.11d
123.4 ± 1.9b
PRE
PGE2
AP
ACP
Ca
Cl
K
Mg
Na
Pi
Experiment E (IV)
43 stallions
HIGH
LOW
N = 33
N = 32
303.6 ± 118.7
95052 ± 8331a
22.2 ± 1.8a
1.23 ± 0.16a
111.4 ± 1.3
19.5 ± 0.7a
1.83 ± 0.16a
125.0 ± 1.6a
0.59 ± 0.02
272.2 ± 98.1
30819 ± 5268b
11.1 ± 1.3b
3.90 ± 0.45b
114.7 ± 1.4
24.3 ± 1.6b
5.57 ± 0.67b
118.9 ± 2.6b
0.54 ± 0.04
0.60 ± 0.04d
WE
N = 29
383.3 ± 134.2
57743 ± 6868c
13.6 ± 1.8b
2.24 ± 0.19c
111.5 ± 4.1
20.0 ± 1.2a
3.10 ± 0.34c
121.6 ± 4.6ab
0.58 ± 0.03
Different superscripts within a row indicate a significant difference (p < 0.05).
5.2.2. Differences between stallions (II)
The HIGH:LOW ratios of Pi and Cl varied significantly between the fertile and subfertile
stallion pairs. In the HIGH fraction, there were significant differences between the two
stallion pairs in the levels of ACP, BG, Pi and Cl, and in the sperm concentration. The
stallion pair differences were non-significant in the LOW fraction in Experiment C.
The levels of AP, ACP, BG, Na and K differed significantly between stallions in HIGH,
LOW and WE, but not in PRE. The stallion differences in Ca, Pi and Cl concentrations
51
were significant in HIGH and WE, but not in PRE or LOW. The concentration of Mg
varied between stallions only in HIGH in Experiment D.
5.2.3. Correlations of the levels of enzymes and electrolytes in seminal plasma with
semen quality, sperm longevity, and fertility (II, IV)
In Experiments C and D (II), the correlations between the measured components of SP and
the sperm concentration in WE were significant. The significant correlations of the levels
of SP components with semen quality parameters and sperm longevity detected in
Experiment E (IV) are listed in Table 7. None of the measured SP components were
correlated to the first cycle pregnancy rate in Experiment E.
52
Table 7. The association between levels of SP components and the listed variables (Experiment E (IV).
Only variables with a significant correlations are listed in sperm-rich ejaculate fractions (HIGH), spermpoor fractions (LOW) and whole ejaculates (WE), and the correlation coefficient are shown for each
significantly correlated variable. Non-significant correlations are not shown. The analyzed SP
components were: alkaline phosphatase (AP), acid phosphatase (ACP), prostaglandin E2 (PGE2),
inorganic phosphate (Pi), Ca, Cl, Mg and Na. The measured variables were: the mean difference (DIFF)
in sperm motility in samples stored with SP (SP1) and without SP (SP0), the DIFF in the DNA
fragmentation index (DFI) before and after storage in SP0 and SP1 samples, sperm concentration, the
total number of sperm, total and progressive motility before storage, the number of morphologically
normal sperm, and the first cycle pregnancy rate.
*Marked variables were significantly associated with the dependent variable in the stepwise linear
regression analysis.
HIGH ( )
DIFF in
motility
Total motility
Other
variables
WE ( )
PGE2 (-0.46)
PGE2 (-0.41)
PGE2 (-0.59)
PGE2 (-0.54)
-
K (-0.51)*
ACP (-0.45)
ACP (-0.37)
ACP (0.53)
ACP (0.45)
ACP (0.56)
-
PGE2 (-0.42)
Mg (-0.46)
Ca (-0.41)
-
-
Ca (-0.49)
-
Sperm concentration
AP (0.81)*
ACP (0.67)
Cl (-0.62)
K (0.36)
Na (-0.55)
AP (0.84)*
ACP (0.63)*
Cl (-0.53)
K (0.37)
Na (-0.44)
AP (0.86)*
ACP (0.56)*
Cl (-0.49)
Na (-0.45)
Total nr of sperm
AP (0.63)*
ACP (0.38)
Ca (-0.35)
Cl (-0.50)
Na (-0.44)
AP (0.61)*
ACP (0.52)
Pi (0.36)*
AP (0.36)
PGE2 (-0.42)
Cl (-0.43)
Na (-0.38)*
K (-0.36)
Pi (0.39)
-
-
-
-
-
Pi (0.40)
-
-
Progressive motility
Average path velocity
Velocity, curved line
Velocity, straight line
Straightness
Linearity
DIFF in DFI
LOW ( )
SP1
SP2
Total motility before
storage
Progressive motility
before storage
Morphologically normal
sperm
-
53
-
5.3. Proteins in seminal plasma fractions and correlations with sperm
concentration and motility (III)
5.3.1. Differences in protein levels between stallions and seminal plasma fractions (III)
In Experiment C (III: Trial 1), the proteins HSP-1, HSP-2, and HSP-4 (at <30 kD) were
detected in the RP-HPLC and N-terminal sequencing of SP samples of all of the stallions.
A <10-kD protein (probably acrosine inhibitor) and prostate specific antigen (PSA), or
kallikrein, at 26 kD were also found in all stallions. HSP-1 was the major protein in all
fractions, all stallions, and all ejaculates. The relative amounts of proteins are expressed as
the area-under-the-curve (AUC) of the chromatograms. The chromatogram of ejaculate 1
of stallion 3 is presented in Fig. 8. No significant differences emerged between fractions or
stallions in the number of peaks in RP-HPLC. The number of peaks varied from 4 to 8 in
each sample; 8 peaks were found most frequently, in 38% of all samples.
The peak containing HSP-4 followed HSP-1 and HSP-2, and it was one of the three highest
peaks in all of the samples. This peak is likely to contain both HSP-3 and HSP-4, as HSP-3
is N-terminally blocked and not detectable with the N-terminal sequencing used in this
experiment, and thus, this peak will be termed HSP-3/4. In mass spectrometry, HSP-1,
HSP-2, HSP-3, HSP-4, PSA and the <10-kD protein were detected in all ejaculate
fractions. No significant differences were present between fractions in the AUC values of
HSP-1, HSP-2 or HSP-3/4. The AUC values of HSP-2 and HSP-3/4 were significantly
different between stallions only in LOW.
In Experiment D (III: Trial 2), the SP samples contained 6–12 protein bands. The amount
of proteins at 60-70 kD was greater in LOW than in HIGH and WE, but no other
significant differences existed between ejaculate fractions in the amount of other proteins.
54
Fig. 8. A RP-HPLC chromatogram, showing the fraction-specific protein peaks (cup 1 =
HIGH, cup 3 = LOW). Peaks containing HSP-1, HSP-2 and HSP-4 are marked with
respective N-terminal amino acid sequences.
55
5.4. The association between seminal plasma components and semen
quality, sperm longevity, and fertility (III, IV)
Descriptive data on fertility and the number of stallions and mares in Experiment E (IV)
are provided in Table 8. There were no significant differences in pregnancy or foaling rates
between breeding methods (insemination with fresh or transported semen).
Table 8. Mean (± SE) values of pregnancy rate/season, foaling rate, number of cycles/pregnancy
and first cycle pregnancy rate for all matings: inseminations with fresh semen, transported
semen, and including natural breeding in Experiment E (IV).
Stallions serving 20 mares
All
Fresh
Transported
inseminations
semen
semen
N (stallions)
N (mares)
N (mares/stallion, min-max)
N (inseminated cycles)
1st cycle pregnancy rate (%)
Pregnancy rate/season (%)
Foaling rate (%)
Number of cycles/pregnancy
16
937
20-150
1538
51.6 ± 3.4a
80.0 ± 2.4a
70.2 ± 2.4a
2.15 ± 0.1a
7
281
20-69
663
60.5 ± 7.6a
83.0 ± 4.1a
73.4 ± 4.3a
1.98 ± 0.3a
9
483
20-113
853
50.1 ± 5.3a
74.2 ± 6.5a
65.2 ± 6.3a
2.15 ± 0.2a
a,b
Different letters indicate a significant difference within a row.
In addition to the correlations listed in Table 7, the correlation between normal
morphology and DFI before storage was found to be negative (-0.63, p = 0.000) in
Experiment E (IV).
In Experiment C (III: Trial 1), the AUC values of HSP-1, HSP-2 and HSP-3/4 were
significantly and positively correlated with sperm concentration, but not with TMOT,
PMOT or VAP. In Experiment D (III: Trial 2), proteins at the 70- to 90-kD peak were
negatively correlated with the sperm concentration; otherwise, no significant correlations
between protein levels and the sperm concentration could be detected.
In the LOW fraction in Experiment E (IV), a significant association of K (coefficient of
determination (R2) = 0.26) with the DIFF in TMOT emerged in the linear regression
analysis, whereas none of the SP components affected the mean DIFF in any of the other
motility parameters or in DFI in any of the fractions. The concentration of K was not
significantly correlated with any of the sperm motility parameters in any of the ejaculate
fractions. The sperm concentration was associated with the levels of AP in HIGH (R2 =
0.47), and both AP and ACP in LOW (AP: R2 = 0.50, ACP: R2 = 0.61) and WE (AP: R2 =
0.68, ACP: R2 = 0.74). The total number of sperm was associated with AP in HIGH (R2 =
0.28) and LOW (R2 = 0.63), Pi in LOW (R2 = 0.55) and Na in WE (R2 = 0.19) (Table 7).
56
None of the studied components of SP were significantly correlated with the first cycle
pregnancy rate.
In summary, the levels of AP, ACP and the AUC values of HSP-1, HSP-2 and HSP-3/4 are
associated with sperm concentration. In the LOW fraction, the levels of K are associated
with the DIFF in TMOT, while none of the components showed any correlation with the
first cycle pregnancy rate.
57
6. Discussion
6.1. Effects of the removal of seminal plasma on sperm longevity (I, IV)
According to our results, the removal of SP causes a decrease in several motility
parameters, but also a concurrent and perhaps paradoxical decrease in DFI and a decrease
in STR and LIN. Previous reports have suggested an association between DNA integrity
and fertility (Love and Kenney 1998, Morrell et al. 2008). Experiments on SP removal
have yielded contradicting results, as summarized in the literature review of this thesis.
Several studies have shown a beneficial effect of SP removal on sperm motility (Jasko et
al. 1991, Pruitt et al. 1993, Braun et al. 1994, Todd et al. 2001, Alghamdi et al. 2002), but
as stated earlier, the effects of SP removal are dependent on the choice of extender, which
will inevitably vary in osmolarity, pH and composition (Padilla and Foote 1991, Bedford et
al. 1995, Akcay et al. 2006). Several factors, such as choice of extender, variation in the
composition of SP between stallions and ejaculations, variation in semen quality between
stallions, and also the interactions between all these, are likely to have an effect on the
results of storage studies. Sperm concentration does not seem to have an effect on sperm
survival when the effect of the proportion of SP is eliminated (Voge et al. 2010). Recently,
Barrier-Battut et al. (2010) demonstrated that sperm motility and acrosome integrity
without ionophore challenge were unaffected by SP removal after 48 h of cooled storage,
but PMI was greater, and the responsiveness to induction of acrosome exocytosis was
reduced, in samples stored without SP. The proportion of SP varied between 4.5% and
33% in the mentioned study, and the effects of SP did not appear to be dose-dependent.
The intention in both Experiment A and E was not to remove SP completely, but to leave
approximately 5% of SP in the samples, because sperm motility after complete removal of
SP has been reported to be affected by the type of extender (Padilla and Foote 1991, Webb
and Arns 1995, Akcay et al. 2006). A plausible explanation for the decrease in motility in
the samples stored without SP in Experiments A and E is that the proportion of SP was too
low (<5%), as the final volume of SP could not be accurately measured at the time of the
final extension.
The higher rate of DNA integrity in samples stored without SP seen in Experiment E (IV),
despite the maintenance of sperm motility, is in accordance with earlier work by Love et al.
(2005). The conflicting effects on sperm motility and DNA integrity, and especially the
increase in the proportion of sperm with high VCL and low STR and LIN, raise the
suspicion that hyperactivation and premature capacitation of sperm could have affected the
mean motility values in these studies. In humans, heat induction of hypermotility has been
proposed as a means of improving total sperm motility, and also pregnancy rates (Kücük et
al. 2008). In stallion semen incubated in a capacitating bicarbonate-containing media for 5
h at 37 °C, 5.3 ± 2.4% of motile spermatozoa were hyperactive, when sperm with a VCL of
180 µm/s and an amplitude of lateral head displacement of
12 µm were considered
hyperactive. This was a smaller proportion than the proportion of capacitated sperm (Rathi
58
et al. 2001), indicating that hyperactivity may not depict the capacitation status very
clearly. In Experiment E (IV), the proportion of hyperactive sperm (VCL 180 µm/s) in
SP1 was 7.5% in HIGH, 26.5% in LOW and 22.0% in WE. In SP0 the corresponding
values were 0%, 8.3% and 9.8%. The higher proportion of SP may thus have inhibited the
hyperactivation of sperm, but the capacitation status was not evaluated in this study. As SP
is known to contain decapacitation factors (Suzuki et al. 2002), it could prevent premature
capacitation, and it has been reported that the removal of SP increases the capacitationassociated Ca++ influx into sperm cells (Magistrini et al. 1997). The hyperactivation of
stallion spermatozoa requires further investigation, but at this point it is useful to recognize
the fact that sperm motility does not always reliably portray the fertilizing capacity of
spermatozoa. Spermatozoa need numerous functional attributes to fertilize an ovum, and it
would be useful to include more semen quality parameters in studies evaluating the effects
of SP removal on sperm survival. Further studies in this area using modern applications are
certainly warranted, as many studies have mostly focused on sperm motility, which is not a
reliable predictor of fertility (Kuisma et al. 2006, Varner et al. 2008).
In Experiment A (I), the samples stored with SP were not centrifuged, and sperm longevity
in the samples stored without SP could have been harmed by centrifugation. However, a
similar effect of the removal of SP was again seen in Experiment E (IV), where the
samples in both storage groups (SP1 and SP0) were centrifuged, and the number of
stallions was greater. Other studies have shown that centrifugation at a similar force (500
g) does not have an adverse effect on sperm survival during storage (Jasko et al. 1991,
Barrier-Battut et al. 2010).
The low number of stallions in Experiments A–D is one of the main challenges in
interpreting the results of these experiments. Some of the stallions in Experiments A-C
were older stallions with suboptimal semen quality, and thus the true effect of SP on sperm
longevity may not have been detected in those experiments. Considering the evident
stallion variation, the characteristics of an individual stallion could easily have affected the
results. As shown in Figures 4 and 5, the effects of SP on motility could be exactly the
opposite in two randomly selected stallions. Furthermore, samples were collected outside
of the breeding season. Season does affect semen quality parameters, such as semen
volume, sperm concentration, total sperm count, motility of fresh semen (Janett et al.
2003), and freezability (Janett et al. 2003, Wrench et al. 2010), but on the other hand, the
current experiments compared different treatments on the same samples. The results of the
larger experiment (Experiment E) were well in line with the other experiments (A–D).
Because of the inherent variability between stallions, it may be useful to modify the semen
processing procedures for stallions producing semen with poor tolerance of storage or with
SP “toxicity”, as has previously been recommended (Brinsko and Varner 1992, Brinsko et
al. 2000, Love et al. 2005).
59
6.2. Sperm survival during storage in sperm-rich and sperm-poor fractions
(I, IV)
Based on earlier reports (Webb et al. 1990, Varner et al. 1987, Sieme et al. 2004), it was
hypothesized that the sperm-rich fractions would maintain sperm longevity better than
whole ejaculates. The results of Experiments A and E (I, IV) indicate that sperm motility
after cooled storage is similar in sperm-rich and sperm-poor fractions, and that the lowest
rate of DNA damage was found in the sperm-rich fractions after the removal of SP. When
SP was present during storage, the amount of DNA damage was more equal in the
ejaculate fractions, but higher than in the sperm-rich fraction stored without SP. Sieme et
al. (2004) reported that centrifuged sperm-rich fractions had higher sperm motility and
PMI than non-centrifuged sperm-rich fractions and centrifuged whole ejaculates. Lower
plasma membrane integrity and PMOT has been reported in sperm stored in SP from the
sperm-rich fractions compared with that stored in SP from the sperm-poor fractions, and it
seems that the sperm-rich fraction may benefit more from SP removal than the sperm-poor
fraction, and that SP from the sperm-rich fractions may be more harmful for sperm survival
during storage (Akcay et al. 2006). The presence of pre-sperm fluid in the sperm-rich
fractions was mentioned by Akcay et al. (2006) as an explanation for the decrease in sperm
survival in the sperm-rich fraction, and this could also have been the case in Experiment E.
It would be interesting to examine the effect of pre-sperm fluid on DNA integrity and
pregnancy rates. If there is a need to try to improve sperm longevity in an individual
stallion, the collection of the sperm-rich fraction separately followed by the removal of
most of the SP could be recommended based on these results. Avoiding the inclusion of
pre-sperm fluid in transported insemination doses may also be useful, but definitive
evidence of any harmful effects of pre-sperm fluid are lacking.
It is worth noting that the fractioning of the ejaculates differed between the experiments
presented in this thesis, and also between the works presented by other research groups,
making the comparison of the sometimes conflicting results difficult. Ejaculates of
stallions producing ejaculates with low volume and high sperm concentrations were often
difficult to fractionate, as the semen jets were nonexistent or virtually impossible to
separate. In the studies presented in this thesis, this type of ejaculate usually had to be
examined as a WE sample only. Ejaculates containing large volumes of SP were simple to
collect as fractions, as the semen jets were easily distinguishable. The difficulties in
fractioning decreased the number and volume of samples for analyses. The results of the
various published storage studies are affected by the method of fractioning, but also by
other factors, such as the length of storage, the type of extender, the proportion of SP in the
stored samples, and which semen quality parameters have been selected to depict sperm
longevity.
60
6.3. Levels of seminal plasma components in sperm-rich and sperm-poor
fractions (II, III, IV)
In Experiment D, the highest levels of Na were found in pre-sperm fluid, and thus the
presence of some pre-sperm fluid in the sperm-rich fraction in Experiments A and E may
have raised the concentration of Na in this fraction. The high levels of Na and Cl in presperm fluid were demonstrated in the early studies by Mann et al. (1956, 1963). The
electrolyte composition in SP and extenders has been suggested to be of importance to
sperm survival during storage (Padilla and Foote 1991). The effects of Na and pre-sperm
fluid are further discussed in Section 6.4.
The association between sperm concentration and AP has been documented in earlier
reports (Turner et al. 2003, Pesch et al. 2006), and was confirmed in the experiments
presented here. The levels of AP differed somewhat in magnitude between Experiments A,
D and E (II, IV), which is probably due to the small number of stallions in Experiment A
(II), allowing individual variation to affect the mean values. The samples in Experiment D
were collected in February, as opposed to the samples in Experiment E that were collected
in the end of the breeding season. Variation due to season has been documented for semen
quality parameters, such as semen volume, sperm concentration, and total sperm count
(Heckenbichler et al. 2010), and for the composition of SP (Abou-Ahmed et al. 1993,
Gebauer et al. 1976). The method of AP analysis was the same in all studies, and the same
person performed all of the analyses.
Characteristic of the sperm-rich fractions were the high levels of AP, ACP and BG,
whereas higher levels of Ca and Mg were seen in the sperm-poor fractions. As AP is
mainly derived from the testes and epididymis (Turner and McDonnell 2003), the high
levels of AP in the sperm-rich fractions suggests that most of the testicular and epididymal
SP components are released in the first ejaculatory jets. The seminal vesicles are a likely
source of Ca and Mg, as the last ejaculatory jets mainly contain vesicular secretions (Mann
et al. 1956).
The major SP proteins were detected in all ejaculate fractions, and there were no
differences between fractions in the number of peaks found in the chromatograms, or in the
relative amount of the studied proteins in the RP-HPLC. The total protein level is lower in
pre-sperm fluid than in the other fractions (Koskinen et al. 2002). Some fraction variation
was detected in the SDS-PAGE, but based on the methods used, it is not possible
determine which particular protein is involved. The difference in the origin of SP in the
sequentially formed fractions may have caused the variation in protein composition seen in
the SDS-PAGE, where the 60- to 70-kD proteins were more abundant in the sperm-poor
fraction. The proteins present in this electrophoretic band are likely to mainly originate
from the seminal vesicles, as the last fractions of the ejaculate are predominantly seminal
vesicle fluid (Weber and Woods 1993, Magistrini et al. 2000). It is difficult to draw any
61
conclusions about the origin of specific proteins or protein groups in the first ejaculate
fractions, as they are composed of fluids from several glands. However, HSP-1 and HSP-2
have been shown to mainly be produced in the ampulla, and a smaller portion in the cauda
epididymis (Ekhlasi-Hundrieser et al. 2005). Because the subsequent SP fractions come in
contact with each other at least indirectly during ejaculation, all of the proteins found in the
RP-HPLC were detected in all fractions.
6.4. Contents of seminal plasma in relation to sperm survival during storage
and fertility (III, IV)
An association between K and the difference in TMOT between samples stored with SP
and samples stored without SP emerged in Experiment E (IV). With decreasing
concentrations of K in SP, the presence of SP leads to an increase in TMOT, and the
removal of SP with a lower K content will reduce TMOT values. In the presence of higher
concentrations of K in the LOW fraction, TMOT in SP0 and SP1 were more equal. In other
words, it is plausible that the presence of SP with a low K content could be less harmful for
sperm motility than SP with a higher K content. The levels of K have not been shown to be
correlated with semen volume, sperm concentration, motility, viability or morphological
abnormalities in stallion semen (Pesch et al. 2006), or with sperm motility in men (Rossato
et al. 2002), and this is well in agreement with the present results.
The addition of Na and K to storage media has been shown to induce lipid peroxidation of
sperm membranes (Alvarez and Storey 1982). Padilla and Foote (1991) discussed K levels
as a potential factor affecting the maintenance of motility during cooled storage. They
reported that the levels of K were 26 mM in SP, 1 mM in skim milk extender, and 10 mM
in skim milk extender supplemented with a modified Tyrode’s solution (KMT). Thus, the
K content in skim milk extender is lower than in SP or KMT. The pH in a Kenney-type
skim milk extender has also been shown to be significantly lower, and the osmolarity
higher, than in KMT and in SP from the sperm-rich and sperm-poor fractions (Padilla and
Foote 1991, Akcay et al. 2006). A traditional Kenney’s skim milk extender is not the ideal
storage medium after complete (or nearly complete) removal of SP (Padilla and Foote
1991, Akcay et al. 2006). When non-centrifuged samples containing 20% SP were studied,
the samples stored in skim milk extender maintained motility better than samples stored in
the modified extender with higher potassium content. In centrifuged samples, the samples
stored in the high-potassium extender maintained higher motility than samples stored in
skim milk extender (Padilla and Foote 1991). Similar results have been reported by Webb
and Arns (1995). According to Rigby et al. (2001), KMT was a superior storage medium
compared to Kenney’s skim milk extender, irrespective of the presence of SP. All in all,
adjusting the ion content of cooled semen by adding extenders with lower K content than
in SP is likely to have a beneficial effect on sperm motility. Further investigations on the
ion content of SP and its effects on sperm survival are needed, however, before definitive
suggestions can be made on the ideal ionic composition of semen extenders.
62
Pre-sperm fluid contains higher levels of both Na and Cl than the other parts of the
ejaculate, but pre-sperm fluid did not seem to have any radical negative or positive effects
on motility or membrane integrity. In Experiment A, spermatozoa from the sperm-rich
fraction were stored cooled for 24 h in pre-sperm fluid and skim milk extender. Because of
difficulties collecting adequate amounts of pre-sperm fluid separately, the number of
samples was low (n = 6) and the pre-sperm fluid samples were not included in the
statistical analysis. Based on the descriptive statistics, the motility parameters TMOT,
PMOT and RAP were numerically only slightly lower in pre-sperm fluid than in the
sperm-rich fractions, but higher than in the sperm-poor fractions. The PMI was slightly
higher in pre-sperm fluid compared to both sperm-rich and sperm-poor fractions. In
Experiment E, the sperm-rich fraction benefited more from SP removal than the spermpoor fraction, as DNA integrity was maintained best in the sperm-rich fraction without SP,
and while this could be associated with the presence of pre-sperm fluid in the first jets of
the ejaculate, the direct effects of pre-sperm fluid on DNA integrity are not known. Further
research on the effects of pre-sperm fluid on sperm survival is needed.
The enzymes AP and ACP were positively correlated to sperm concentration in the present
study, and have earlier been shown to be negatively correlated with the semen volume
(Pesch et al. 2006). The positive correlation between AP and both sperm concentration and
the total number of sperm is well in accordance with previous work (Turner and
McDonnell 2003).
None of the measured components of SP served as reliable predictors of stallion fertility.
However, the fertility assessment should be interpreted with caution, as the number of both
stallions and mares was low. Experiment E aimed at scanning a larger population of
stallions for a SP composition profile compatible with good fertility, to try to reveal which
SP components, if any, would have a clear association with fertility. The draw back is the
use of single ejaculates. The analyzed ejaculates were not used for inseminations; instead,
the pregnancy rates were calculated based on data from the whole season. Because semen
quality can change over time, and many stallion-independent factors influence pregnancy
rates, it is challenging to interpret the significance of the effects of SP components on
fertility.
When only those stallions who served at least 20 mares were included in the statistical
analysis, the differences in pregnancy and foaling rates between inseminations with fresh
or transported semen were non-significant. As these stallions were in higher demand and
have higher breeding fees, it is likely that the more intensive veterinary care and breeding
management gave better results also for inseminations with transported semen. When all
stallions were included, the first cycle pregnancy rate was higher when fresh semen was
used, compared to inseminations with cooled semen. Recently, Brinkerhoff et al. (2010)
reported that a mare bred with fresh semen had a 2.5 times higher probability of becoming
pregnant than a mare bred with cooled, transported semen at a site other than the stud farm.
63
When high quality semen was used, sperm quality and the pre-breeding status of the mare
(i.e. barren, foaling or maiden) did not influence fertility, but mare age and the breeding
method were critical factors affecting fertility. Successful management at the receiving
station is therefore essential for achieving high pregnancy rates. It has also been reported
that inseminations with transported semen at artificial insemination centers result in higher
pregnancy rates than inseminations at the breeder’s own facilities (Rota et al. 2004), and if
the semen quality of transported semen used for insemination at the breeder’s facilities is
poor, the pregnancy rate could be expected to decrease even further. As the use of cooled
transported semen in Finland has increased, the foaling rates have decreased (Katila et al.
2010b). Stud farms need to ensure that the sperm longevity in their transported
insemination doses is optimized. This can be achieved by correct semen handling
procedures, such as avoiding cold shock and prolonged exposure to light (reviewed by
Katila 1997), applying individual modification of semen processing and storage conditions,
such as removing SP from semen before cooling and transport (Brinsko and Varner 1992,
Brinsko et al 2000, Love et al. 2005), and by selecting breeding methods suitable for
individual stallions, so that stallions producing semen with a poor tolerance of storage are
only recommended for insemination with fresh semen.
64
7. Conclusions
1. There were no differences in the maintenance of sperm motility between the sperm-rich and
sperm-poor fractions and whole ejaculates during cooled storage, irrespective of the presence of
seminal plasma (I, IV).
2. The removal of seminal plasma reduced sperm motility (I, IV) but improved DNA integrity
during cooled storage compared with samples containing seminal plasma (IV), suggesting that
the presence of seminal plasma may increase the amount of DNA damage in stored spermatozoa.
However, marked between-stallion variation exists in the effects of seminal plasma on sperm
longevity, and some stallions may need individual modifications of the semen processing
procedures.
3. The lowest rate of DNA damage was found in the sperm-rich fractions stored without seminal
plasma (IV), indicating that spermatozoa in the sperm-rich fraction may benefit more from the
removal of seminal plasma than those in the sperm-poor fraction.
4. Post-thaw sperm motility was highest in the sperm-rich fraction (I), suggesting that the collection
of fractionated ejaculates could be beneficial when freezing semen.
5. The sperm-rich and sperm-poor fractions differ in composition. The sperm-rich fractions
contained the highest levels of the enzymes alkaline phosphatase, acid phosphatase, and glucuronidase. The sperm-poor fractions contained the highest concentrations of calcium and
magnesium. The highest levels of sodium and chloride were found in the pre-sperm fluid (II, IV).
6.
The major proteins of stallion seminal plasma, HSP-1 and HSP-2, were found in all fractions
and were positively correlated with sperm concentration (III).
7. The level of potassium was higher in the sperm-poor fraction than in the sperm-rich fraction.
Lower concentrations of potassium in the sperm-poor fraction led to higher total motility after
storage when seminal plasma was present during storage, while the removal of seminal plasma
with lower potassium content reduced motility. The level of potassium may be of importance
when choosing a suitable extender and semen processing regimen for subfertile stallions (IV).
65
Acknowledgments
The research presented in this thesis was carried out at the Department of Production
Animal Medicine at the Faculty of Veterinary Medicine (University of Helsinki). Most of
the work was funded by the Mercedes Zachariassen foundation and the Foundation for
Veterinary Medicine in Finland (The Finnish Veterinary Association).
I would like to thank my mentor and supervisor Professor Terttu Katila for all her hard
work in guiding me through this project, for her wisdom and integrity, and for teaching me
about science and equine reproduction. I am truly grateful to Professor Katila and to the
Department of Production Animal Medicine for giving me the opportunity to participate in
this research.
I would also like to thank my other supervisors for their input in this project; Professor
Magnus Andersson at the Department of Production Animal Medicine, for always being so
funny and entertaining, but especially for his insights in the field of andrology, and for his
never-ending encouragement. I am also grateful and honored to have had Professor
Heriberto Rodriguez-Martinez (Linköping University) as one of my supervisors. His
scientific and critical approach in teaching has instigated much improvement in my
research.
I would like to thank my two pre-examiners Michèle Magistrini and Anne-Marie Dalin for
the time and effort they put into evaluating my work and providing constructive criticism
and helpful comments. Professor Harald Sieme is thanked for agreeing to act as my
opponent.
I am grateful to everyone who contributed to this work. Satu Sankari at the Central
Laboratory of the Faculty of Veterinary Medicine at the University of Helsinki spent a lot
of time on the biochemical analyses and discussing the methods and results with me. Maria
Rivera del Alamo at the University of Barcelona made a huge effort in getting our samples
from Valencia to Barcelona and doing the gel electrophoresis. Juan Calvete and his
colleagues at the Institute of Biomedicine in Valencia did all the advanced proteomics
work presented in this thesis, explained it to me (again and again), and helped in editing
the protein paper (publication III). Anders Johannisson and Hans Kindahl at SLU in
Uppsala kindly contributed to the analyses in Experiment E by performing the SCSA and
PGE2 assays. Tero Wahlberg at the University of Turku provided expert assistance with the
statistical analyses in Experiment E.
I am greatly indebted to Tiina “Skipper” Reilas at MTT Agrifood Research Finland, for
teaching me mare gynecology, for always helping me when I am lost, for diligently and
thoroughly going through version after version of maturing manuscripts, and for being a
good friend.
66
This research could not have been done without the help of the wonderful staff at MTT
Agrifood Research Finland; all of the barn managers and Marja Rauvola at the MTT lab
assisted in this study and have taught me a lot. I am also grateful to all of the cooperative
and helpful stud farms, stud farm veterinarians and horse owners who participated in
Experiment E: MTT Agrifood Research Finland, Metsäpirtti, Kallelan oriasema, Aria
Stable, Skyhorses, Sillanpään oriasema, Kylämäen Hevostila, Keskisen oriasema, and RokTalli. Merja-Liisa Toivonen and Metsäpirtti are especially acknowledged for all the
friendly help and cooperation during these years.
I would also like to acknowledge my dear friends, the horsey girls from the days of yore,
for supporting me during this time. A special thank you goes out to all of my dear friends
and colleagues at the Production Animal Hospital and the Department of Production
Animal Medicine; working with you is an inspiration. The friendly and fun atmosphere in
Saari is one of a kind. All of my colleagues who have given me strength and belief, and
shared these years with me; thank you, you know who you are.
Last, but definitely not least, I would like to thank my family; my husband Kim for putting
up with this sometimes grumpy “testicle doctor”, as someone so eloquently once called me,
and for everything we’ve shared together. It’s always good to come home. My parents, for
not allowing me to quit school and be a groom, and for always encouraging me and
believing in all the strange things I think of doing. My brother, for not bullying me
anymore, and for being my idol. And many thanks to my beloved daughter Kira, who I
never dared to think we’d be lucky enough to have.
Maria Kareskoski
67
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