Tierärztliche Hochschule Hannover Investigation of antioxidative capacity in bovine seminal plasma– Effects of Omega-3 fatty acids INAUGURAL – DISSERTATION zur Erlangung des Grades eines Doktors der Veterinärmedizin - Doctor medicinae veterinariae - ( Dr. med. vet. ) vorgelegt von Oguz Calisici Samsun / Türkei Hannover 2010
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Tierärztliche Hochschule Hannover
Investigation of antioxidative capacity in bovine seminal plasma–
Effects of Omega-3 fatty acids
INAUGURAL – DISSERTATION zur Erlangung des Grades eines
Doktors der Veterinärmedizin
- Doctor medicinae veterinariae -
( Dr. med. vet. )
vorgelegt von
Oguz Calisici Samsun / Türkei
Hannover 2010
Wissenschaftliche Betreuung: Prof. Dr. Heinrich Bollwein
Klinik für Rinder
1. Gutachter: Prof. Dr. H. Bollwein
2. Gutachter: Prof. Dr. H. Sieme
Tag der mündlichen Prüfung: 17.05.2010
To Duygu and my Parents
Contents 1 INTRODUCTION 1
2 REVIEW OF LITERATURE 2
2.1 Reactive Oxygen Species 2
2.1.1 Definition and existence of reactive oxygen species 2
2.1.2 Oxidative stress 4
2.1.3 Significance of oxidative stress in male reproduction 4
2.1.3.1 Sources of ROS in semen 4
2.1.3.2 Targets and pathological role of ROS in semen 6
Lipids of sperm plasma membrane and lipid peroxidation 7
Damage of DNA 10
Damage of proteins 10
Apoptosis 10
2.1.3.3 Physiological role of ROS in semen 11
2.2 Antioxidants 11
2.2.1 Enzymatic antioxidants 12
2.2.1.1 Superoxide Dismutase 12
2.2.1.2 Glutathione Peroxidase 13
2.2.1.3 Catalase 15
2.2.2 Non-enzymatic antioxidants 15
2.3 Seminal plasma and its antioxidative significance for male reproduction 17
2.3.1 Accessory glands and seminal plasma 17
2.3.2 Antioxidative properties of seminal plasma 18
2.4 Measurement of oxidative stress and antioxidants using chemiluminescence 19
3 OWN EXPERIMENTAL STUDIES 20
3.1 Establishment of a new assay for the determination of total antioxidative
capacity of bovine seminal plasma 20
3.1.1 Abstract 20
3.1.2 Introduction 21
3.1.3 Materials and Methods 22
3.1.3.1 Chemicals 22
3.1.3.2 Semen collection, dilution and freezing 22
3.1.3.3 Handling of seminal plasma 23
3.1.3.4 Antioxidant assays 23
Total Antioxidant Capacity 23
Instrumentation and automated measurement of TAC SOD and GPx 24
Superoxide Dismutase (SOD) Assay 24
Glutathione Peroxidase (GPx) Assay 25
Determination of protein concentrations 26
Measurements of intra- and inter-assay Variations 26
3.1.3.5 Flow cytometric analyses 26
Plasma Membrane Integrity and Acrosomal Integrity 27
Lipid Peroxidation (LPO) 27
Sperm Chromatin Structure Assay 28
3.1.3.6 Statistical analysis 28
3.1.4 Results 28
3.1.4.1 Reproducibility of TAC-, SOD- and GPx assays 28
3.1.4.2 TAC, SOD and GPx levels in seminal plasma 29
3.1.4.3 Volume and sperm concentration of ejaculates 30
3.1.4.4 Sperm quality 32
3.1.4.5 Relationships between antioxidant levels and between antioxidant levels
and sperm quality 34
3.1.5 Discussion 34
3.2 Effects of feeding omega-3-fatty acids on sperm quality of Holstein Friesian bulls
before and after cryopreservation: Effects on seminal plasma 37
3.2.1 Abstract 37
3.2.2 Introduction 38
3.2.3 Materials and Methods 39
3.2.3.1 Bulls 39
3.2.3.2 Dietary supplementation of bulls 39
3.2.3.3 Semen collection, dilution and freezing 40
3.2.3.4 Handling of seminal plasma and measurement of TAC, GPx and SOD 40
3.2.3.5 Flow cytometric analyses 41
Plasma Membrane Integrity and Acrosomal Integrity 41
Sperm Chromatin Structure Assay 41
3.2.3.6 Fatty acid extraction and analysis 41
3.2.3.7 Statistical analysis 42
3.2.4 Results 43
3.2.4.1 Fatty acid analysis 43
3.2.4.2 Dietary effects on sperm quality and fatty acid composition 43
3.2.4.3 Effect of feeding ALA on antioxidant levels of seminal plasma 45
3.2.4.4 Correlation between fatty acids and antioxidants 45
3.2.5 Discussion 46
4 SUMMARIZING DISCUSSION AND CONLUSIONS 49
5 SUMMARY 52
6 ZUSAMMENFASSUNG 54
7 REFERENCES 56
8 APPENDIX 75
List of abbreviations AD acrosome-damaged sperm ALA α-linolenic acid AO acridine orange BP C-11 BODIPY581/591 BSA bovine serum albumin CAT catalase CuZn-SOD copper-zinc SOD CV coefficient of variation DFI DNA fragmentation index DHA docosahexaenoic acid DMSO dimethyl sulfoxide DNA deoxyribonucleic acid EPA eicosapentaenoic acid EDTA ethylene diamin tetra acetic acid FITC-PNA fluorescein isothiocyanate labeled peanut from Arachis h hour HRP horse radish peroxidase H2O2 hydrogen peroxide GPx glutathione peroxidase GRx glutathione reductase GSH glutathione GSSG oxidized GSH L litre LPO lipid peroxidation mg milligram ng nanogram min. minute ml millilitre mM millimole Mn-SOD manganese SOD NADH nicotinamin dinucleotid NADPH nicotinamide adenine dinucleotide phosphate NADP+ oxidized form of NADPH NBT nitro blue tetrazolium NO. nitrite oxide radical OH. hydroxyl radical ONOO- peroxinitrite anion O2
SD standard deviation SD-DFI standard deviation of DFI SOD superoxide dismutase TAC total antioxidant capacity TBHP t-butyl hydroperoxide μL micro litre 1O2 singlet oxygen
1
1 INTRODUCTION
Bovine semen has been cryopreserved since more than a half century for artificial
insemination and nowadays it is being widely used all over the world. However, it is well
known that the cryopreservation procedure is detrimental to sperm particularly because of
chemical and physical stress factors which are occurring during this process
(HAMMERSTEDT et al. 1990; WATSON 1995; CHATTERJEE and GAGNON 2001). One
important factor is oxidative stress which, in turn, affects biological membranes and DNA of
sperm (AITKEN and KRAUSZ 2001; BALL 2008). Bovine sperm themselves have only few
amounts of endogenous antioxidants for the protection against reactive oxygen species (ROS)
and the main antioxidant source is the seminal plasma (DAWRA and SHARMA 1985;
BILODEAU et al. 2000). Therefore, the development of sensitive techniques for monitoring
the activity of antioxidants in seminal plasma is of clinical importance. Sensitive
chemiluminescence techniques have been employed to monitor total antioxidant capacity of
human seminal fluid (SHARMA et al. 1999).
Polyunsaturated fatty acids (PUFA) play an important role in regulating sperm membrane
fluidity and spermatogenesis (HAIDL and OPPER 1997; OLLERO et al. 2000). After
freezing and thawing, the portion of PUFA in sperm plasma membrane decreases
significantly due to lipid peroxidation (CEROLINI et al. 2001). Low portions of C20 and C22
PUFAs in sperm in old bulls were related to reductions in sperm quality and -fertilizing
ability (KELSO et al. 1997b).
In various feeding experiments polyunsaturated fatty acids (PUFA) have been supplied to
change the fatty acid composition of sperm membrane in order to improve sperm quality and
fertility. Indeed, the fatty acid profile of sperm membranes can be modified with diet and an
improvement of sperm quality was observed in a variety of livestock species including
chicken, turkey, boar and stallion (KELSO et al. 1997a; ROOKE et al. 2001; BLESBOIS et al.
2004; BRINSKO et al. 2005). However, it is possible that feeding of PUFAs reduces also the
antioxidative capacity of semen which, in turn, can (SURAI et al. 2000b; CASTELLINI et al.
2003) disturbs sperm quality in the case of excessive ROS production.
The aims of this study were: (i) to determine total antioxidant capacity of bovine seminal
plasma and its relationship with other antioxidants and sperm quality; (ii) to ascertain whether
feeding omega-3-fatty acid reduces the antioxidative status of seminal plasma.
2
2 REVIEW OF LITERATURE
2.1 Reactive Oxygen Species
2.1.1 Definition and existence of reactive oxygen species
A free radical is any species capable of independent existence that contains one or more
unpaired electrons (HALLIWELL 1989). A radical can be neutral or positively or negatively
charged. HALLIWELL and GUTTERIDGE (1989) defined reactive oxygen species (ROS) as
a collective term which includes oxygen radicals (e.g. Superoxide radical and Hydroxyl
radical) as well as some highly reactive derivates of O2 that do not contain unpaired electrons
(non-radicals) such as hydrogene peroxide (H2O2), singlet oxygen (1O2) and hypochlorous
acid (HOCl). ROS have different abilities to react with many structural and functional
important molecules in living organisms.
There are some nitrogen-derived free radicals called reactive nitrogen species (RNS, e.g.,
nitrite oxide (NO.) and peroxinitrite anion (ONOO-) which are considered as a subclass of
ROS (SIKKA 2001). Some of ROS and RNS are summarized in Table 1-1.
The superoxide radical (O2.-) is the main free radical and an example of free radicals with
intermediate reactivity (FRIDOVICH 1983; SIKKA 2001). One-electron reduction of O2
produces the superoxide radical, O2-. This is frequently written as O2
.- where the dot denotes a
radical species, that is an unpaired electron. It is negatively charged and produced in
biological systems during electron transport (during respiration) in mitochondria. It can not
rapidly cross the lipid membrane bilayer (KRUIDENIER and VERSPAGET 2002). However,
superoxide is a precursor of other, more powerful ROS. It can participate in the production of
more powerful radicals as an oxidizing agent or by donating an electron and thereby reducing
Fe3+ and Cu2+ to Fe2+ and Cu+, as follows:
O2.- + Fe3+/Cu2+ Fe2+/Cu+ + O2 (step 1, reduction of ferric ion to ferrous).
Further reactions between transition metals (Fe2+ or Cu+) and H2O2 are the source of the
hydroxyl radical (OH.) and called as Fenton reaction:
The NADPH is mainly provided in animal tissues by a complex metabolic pathway known as
the oxidative pentose phosphate pathway (or called as Hexose Monophosphate Shunt). In this
pathway, NADPH stems from the reaction (Figure 1-3) catalysed by glucose-6-phosphate
dehydrogenase (HALLIWELL and GUTTERIDGE 1989).
Figure 1-3: Schematic glutathione redox-cycle adapted from OCHSENDORF and FUCHS
(1997): H2O2 is reduced by GPx, glutathione (GSH) is oxidized and thereafter reduced by
glutathione reductase (GRx) utilizing NADPH. NADPH is available by the
Hexosemonophosphate-shunt system.
In somatic cells, 60-75 % of GPx activity is found in the cytoplasm and 25-40 % in the
mitochondria (ZAKOWSKI et al. 1978). At least four types of selenium-dependent GPx exist
in mammalian body, called GPx1, GPx2, GPx3, and GPx4. Selenium is essential for catalysis
in all four types of GPx (BRIGELIUS-FLOHE and TRABER 1999). The GPx4 is called
phospholipid hydroperoxide glutathione peroxidase (PHGPx) because of its ability to reduce
not only H2O2 but also fatty acid hydroperoxides to alcohols (FORESTA et al. 2002). On the
other hand, in the male genital tract, an selenium-independent isoenzyme of GPx (GPx5) has
GRx
GSSG
GSH
H2O2
NADPH
NADP+
Hexose Monophosphate Shunt-system
H2O
GPx
15
been identified at both protein and mRNA level (HALL et al. 1998; VERNET et al. 2004;
CHABORY et al. 2009).
In bovine seminal plasma a non-selenium-dependent GPx and a selenium-dependent GPx
have been detected (KANTOLA et al. 1988; BILODEAU et al. 2000). It has been reported
that the selenium-dependent GPx activity accounted for one third of total GPx activity and is
negatively correlated with the malondialdehyde level and acrosomal damage (SLAWETA and
LASKOWSKA-KLITA 1985; KANTOLA et al. 1988; SLAWETA et al. 1988).
2.2.1.3 Catalase
Catalase is present in all major body organs of animals, being especially localized in liver and
erythrocytes. It removes H2O2 within cells according to the following equation:
2H2O2 2H2O + O2
It consists of four protein subunits, each of which contains a heme (Fe (III)-protoporphyrin)
group bound to its active site (AEBI 1984). Each subunit also usually contains one molecule
of NADPH bound to it. This helps to stabilize the enzyme (KIRKMAN et al. 1987). Disso-
ciation of catalase into its subunits causes loss of its activity. Dissociations can occur easily
on storage, freeze-drying, or exposure of the enzyme to acid or alkali (HALLIWELL and
GUTTERIDGE 1989). The catalase activity of animal tissues is largely located in subcellular
organelles known as peroxisomes. Mitochondria and the endoplasmic reticulum contain little
catalase actvity. (MARKLUND et al. 1982; HALLIWELL and GUTTERIDGE 1989).
An activity of catalase has been found in seminal plasma of different species (BALL et al.
2000; BILODEAU et al. 2000; ZINI et al. 2002). However, its presence in sperm is a matter
of controversy (TRAMER et al. 1998). Catalase activity in human sperm and seminal plasma
was demonstrated using different methods (JEULIN et al. 1989; ZINI et al. 2002).
BILODEAU et al. (2000) noticed a low level of catalase activity in bovine seminal plasma but
they did not find any catalase activity in sperm.
2.2.2 Non-enzymatic antioxidants
In addition to enzymatic defence systems, there are extensive non-enzymatic antioxidant
compounds consisting of several different types of lipid and water-soluble small molecules
(SIKKA et al. 1995; AGARWAL et al. 2005). These molecules are able to scavenge free
16
radicals. Lipid-soluble scavengers are especially vitamin E and β-Carotene. Water-soluble
scavengers are vitamin C (ascorbic acid) and GSH. Furthermore, there are a lot of other
antioxidant compounds like selenium, zinc, uric acid, ceruloplasmin, taurine etc. (SIES 1993).
EVANS and BISHOP (1922) have discovered vitamin E as a “fertility vitamin (or fertility
factor)”. They showed that some plant oils reduced the occurrence of foetal mortality in diet-
restricted rats. This “fertility factor” was later isolated and characterized as tocopherol (BELL
1987). Vitamin E is a collective name of 8 fat-soluble vitamins (tocopherols and tocotrienols)
with antioxidant properties (HERRERA and BARBAS 2001). Of these, -Tocopherol has the
highest biological activity and is the most abundant form in nature (BRIGELIUS-FLOHE and
TRABER 1999). The ability of vitamin E to quench ROS and its hydrophobicity have led to
its common definition as the single most important essential lipid-soluble antioxidant
(BURTON 1994). When tocopherol quenches a lipid peroxyl radical, it is oxidated to the
tocopheroxy radical. Tocoperoxy radical accepts hydrogen to regenerate the tocopherol,
which makes this first oxidation step fully reversible. Vitamin E is present both in sperm
membranes and in seminal plasma (AGARWAL and PRABAKARAN 2005). Vitamin E and
Selenium act synergistically and protect the biomembranes from oxidative attack. Vitamin E
reduces alkyl peroxyl radicals of unsaturated lipids of cell membranes, thereby generating
hydroperoxides that can be removed by the Se-dependent peroxidases (MAIORINO et al.
1989; BRIGELIUS-FLOHE and TRABER 1999).
Carotene is the precursor of vitamin A, an important lipophilic antioxidant in animal tissues.
Carotenoid pigments such as β-Carotene are able to function as effective quenchers of ROS
(AGARWAL et al. 2005). β-Carotene was found to act synergistically with tocopherol and it
is capable of regenerating tocopherol from the tocopheroxyl radical (PALOZZA and
KRINSKY 1992). Large amounts of carotenoid pigments occur in male gonads and accessory
genital glands of mammals. The significance of vitamin A in developing and maintaining the
normal germinal epithelium in animals has been emphasized by several studies (PALLUDAN
1966). It is an essential vitamin for spermatogenesis in rats (HUANG and HEMBREE 1979).
Selenium, as a cofactor for glutathione peroxidase, is an important trace element and plays a
crucial role in enzymatic antioxidantive defence system. It is an essential trace element for
animals. Selenoenzymes (e.g. glutathione peroxidases, thioredoxin reductases) contain Se in
the form of selenocysteine, an amino acid that is identical to cysteine, except that selenium
17
replaces sulphur (PAPPAS et al. 2008). In spermatozoa, Selenium is abundantly localized in
midpiece region (CALVIN 1981). In rat sperm, Selenium deficiency causes reduction of
spermatogenesis and abnormal sperm morphology characterized by morphological midpiece
alterations (WU et al. 1979).
In spermatids, selenium-containing PHGPx occurs as an active peroxidase. In mammalian
tissues, the highest PHGPx acitivity was found in the testis (URSINI et al. 1995). It plays an
important role during spermiogenesis, maturation of spermatozoa and embryonic develop-
ment. On the other hand, this enzyme is transformed to an oxidatively inactivated protein in
mature sperm and is contributed as a main constituent of the mitochondrial capsule in the
midpiece (FORESTA et al. 2002). The activity of PHGPx is lower in sperm of infertile men
sperm compared to that of fertile men (FORESTA et al. 2002).
Although the GPx activity in bovine seminal plasma is higher than in spermatozoa (BROWN
et al. 1977; BILODEAU et al. 2000), selenium is found also in the spermatozoa in
considerable concentrations. In bulls, the Selenium concentration in seminal plasma is about
10 times higher than in serum (SAARANEN et al. 1986). Furthermore, it is known that the 75Se is faster incorporated into seminal plasma than into the spermatozoa of bulls (SMITH et
al. 1979). This may explain the fact that seminal plasma GPx levels increase rapidly after an
injection of Selenium to bulls (BARTLE et al. 1980).
2.3 Seminal plasma and its antioxidative role in male reproduction
2.3.1 Accessory glands and seminal plasma
Seminal plasma is the name for secretes originating from accessory glands (PETZOLT 2001).
Immature sperm complete their passage through the epididymis and get matured. Thereafter,
they will be transported via vas deferens and mixed with the seminal plasma before
ejaculation. There are developmental differences of accessory glands between species.
According to these differences, the amounts of secretions from the particular accessory glands
vary between species (DÖCKE 1963). Seminal plasma is produced by epididymidis, ampulla
ductus deferentis, prostate, vesicula seminalis, bulbo-urethral glands and urethral glands. The
size and secretory output of accessory glands are regulated by testosterone (PETZOLT 2001).
The amounts of seminal plasma vary considerably between species. The secretions of the
18
seminal vesicles and the prostate are qualitatively and quantitatively the most important ones
in domestic animals (DÖCKE 1963).
The main organic and inorganic constituents of bovine seminal plasma are sodium, potassium,
calcium and chloride, bicarbonate, phosphate, citrate, fructose, zinc, iron, copper
(ROTHSCHILD and BARNES 1954; KIRTON et al. 1964). These constituents maintain the
osmotic pressure in the semen and provide the required energy.
Seminal plasma plays crucial regulative roles in the processes prior to oocyte penetration of
the spermatozoa. The functions of seminal plasma are based on direct or indirect interactions
with spermatozoa (WABERSKI 1995):
direct functions: nutrition of spermatozoa, protection against oxidative and osmotic
stresses, regulation of motility, capacitation, gamete recognition and binding
indirect functions: enhancement of uterine contractions, relaxation of the tubal isthmus,
immune modulation of the uterus.
2.3.2 Antioxidant properties of seminal plasma
The major antioxidative defence mechanisms in cells are the enzymes SOD, GPx and CAT.
However, in spermatozoa, the cytoplasmic space available for these enzymes is very limited.
For example mouse fibroblasts have an intracellular water space of 150 µl/108 cells
(WOHLHUETER et al. 1978) whereas the total water space of human spermatozoa ranges
between 1.7 and 2.6 µl/108 cells (GLANDER and DETTMER 1978; FORD and HARRISON
1983). As sperm have only limited antioxidative defence mechanisms, seminal plasma is the
main antioxidant source in semen (BILODEAU et al. 2000). Deficiencies in this protective
milieu have been associated with oxidative stress and male infertility. For example, the
surgical ablation of the male accessory glands is associated with oxidative stress followed by
high levels of DNA damage in sperm and embryonic loss (O et al. 2006).
A variety of factors like diet can deplete the antioxidative status of seminal plasma. Feeding
experiments with more PUFAs have shown that the antioxidant status and sperm quality can
be impaired. On the other hand, it has been demonstrated that the negative effects of PUFA
supplementation can be reversed with vitamin E addition (CASTELLINI et al. 2003).
19
2.4 Measurement of oxidative stress and antioxidants using chemiluminescence
The susceptibility of sperm to oxidative stress is an important factor of defective sperm
function and impaired fertility (AITKEN and CLARKSON 1987). Therefore, the
development of sensitive techniques for monitoring the activity of antioxidants and the levels
of ROS generation is of clinical importance. In general, the determination of the rate of ROS
production using different tests can be used to quantify oxidative stress (SHARMA et al.
1999; PASQUALOTTO et al. 2008). Nevertheless, due to a very short half-life of ROS,
clinical examination of ROS production is limited and needs attention under specific
controlled conditions (SIKKA et al. 1995).
Sensitive chemiluminescence techniques have been developed to monitor total antioxidant
capacity of seminal fluid and ROS generation in semen (AITKEN et al. 1992; AITKEN et al.
2004). Luminol is most commonly used in these chemiluminescence techniques. AITKEN et
al. (1992) suggested that the basic luminol signal depends upon the intracellular oxidation of
this probe by ROS of the whole ejaculate. The hydrogen peroxide oxidizes luminol through
mediation of an intracellular peroxidase. Furthermore, it was noticed that the addition of horse
radish peroxidase to the incubation medium resulted in an elevated signal since it allows
extracellular hydrogen peroxide to contribute to the chemiluminescence. This let to the
conclusion that the combination of luminol and horse radish peroxidase constitutes an
excellent assay for routine chemiluminescence analysis of ROS at intra- and extracellular
levels (SHARMA et al. 1999).
20
3 OWN EXPERIMENTAL STUDIES
3.1 Establishment of a new assay for the determination of total antioxidative capacity of
bovine seminal plasma
3.1.1 Abstract
The aim of the present study was to establish an assay for the measurement of total
antioxidant capacity (TAC) of bovine seminal plasma. In addition, relationships between
TAC and other antioxidants in seminal plasma and parameters of sperm quality, respectively,
should be investigated. Eight consecutive ejaculates were collected from 9 Holstein-Friesian
bulls. TAC-, glutahione peroxidase- (GPx) and superoxide dismutase assays (SOD) were
modified for use in an automated 96-well microplate reader. Plasma membrane integrity
(PMI) and acrosomal damage (AD) were evaluated flow cytometrically before and
immediately after cryopreservation. The DNA fragmentation index (DFI) was determined
after cryopreservation by using sperm chromatin structure assay (SCSA). The level of
membrane lipid peroxidation (LPO; oxidation of lipiphilic probe C11-BODIPY581/591) was
assessed without and with induction of LPO using t-butyl hydroperoxide (TBHP) before
cryopreservation, immediately (0h) and 3 hours (3h) after thawing. The intra-assay
coefficients of variation (CV) for TAC-, SOD- and GPx were 5.0 %, 3.5 % and 6.2 %,
respectively. The inter-assay CVs for the TAC-, SOD- and GPx were 8.3 %, 3.6 % and
8.3 %, respectively. Levels of TAC, SOD and GPx differed (P < 0.0001) between bulls but
not (P > 0.05) between ejaculates within bulls. A negative correlation (r=-0.76; P ≤ 0.05) was
observed between SOD and GPx, but no other relationships (P > 0.05) were detected between
antioxidants. TAC was negatively related with TBHP-LPO 0h (r=-0.85; P ≤ 0.01). Positive
correlations were found between SOD and LPO at 0h (r=0.71; P ≤ 0.05) and 3h (r=0.80;
P ≤ 0.05). There were no (P > 0.05) associations between DFI and antioxidants levels of
seminal plasma. The results of this study show that the total antioxidant capacity differs
between bulls and gives valuable information about the sensitivity of sperm against lipid
peroxidation of cryopreserved sperm.
21
3.1.2 Introduction
The cryopreservation procedure is detrimental to sperm particularly because of chemical and
physical stress factors which are occurring during this process (HAMMERSTEDT et al. 1990;
ASHWORTH et al. 1995). The susceptibility of cryopreserved sperm to damage induced by
reactive oxygen species (ROS) is well known (AITKEN and FISHER 1994; BILODEAU et al.
2002; AITKEN and BAKER 2004). Toxic levels of ROS can affect sperm quality and
function. In particular, two reasons for this susceptibility have been emphasized in the past.
These are the increased generation of ROS and the lower levels of antioxidants after
cryopreservation (AITKEN and FISHER 1994; BILODEAU et al. 2000). This imbalance
between production of ROS and cellular antioxidants is defined as oxidative stress (SIKKA et
al. 1995).
Mammalian sperm are endowed with a high content of polyunsaturated fatty acids within the
plasma membranes which are particularly susceptible to free radical attack and consequently
lipid peroxidation (POULOS et al. 1973; JONES and MANN 1977; JONES et al. 1979;
AITKEN and FISHER 1994; DE LAMIRANDE and GAGNON 1995; SIKKA et al. 1995).
Membrane lipid peroxidation (LPO) can lead to alterations of membrane fluidity and these
alterations have deleterious effects on sperm function, e.g. ability to fuse with oocytes
(STOREY 1997; BALL 2008).
It has been shown that bovine sperm themselves have only few amounts of endogenous
antioxidants for the protection against ROS and the main antioxidant source is the seminal
plasma (DAWRA et al. 1984; DAWRA and SHARMA 1985; BILODEAU et al. 2000).
Seminal plasma contains both enzymatic ROS scavengers, including superoxide dismutase
Values during the feeding period with asterisk (*) differ (P < 0.05) compared to the values before the feeding period.
45
3.2.4.3 Effect of feeding ALA on antioxidant levels of seminal plasma In both groups the levels of TAC, SOD and GPx did not change (P > 0.05) during the feeding
of PO and ALA (P > 0.05). The values of TAC, SOD and GPx did not differ (P > 0.05)
between PO - and ALA bulls during the study periods (Table 3-2).
Table 3-2: Effects of diets containing palmitic acid (PO) or alpha-linolenic acid (ALA) on
levels of total antioxidant capacity (TAC = µmol/L), glutathione peroxidise (GPx = nmol
NADPH oxidized/min/mg of protein) and superoxide dismutase (SOD = U/ml) in bovine
seminal plasma. Values are means±SD from 8 consecutive ejaculates (twice weekly for 4
weeks) of PO (n=8) and ALA (n=9) bulls before feeding and after 9 weeks (mean of weeks 9-
12) of feeding.
PO ALA Before feeding feeding Before feeding Feeding
were evaluated by flow cytometry in sperm before cryopreservation and in frozen sperm
immediately after thawing. The DNA-fragmentation was determined on frozen-thawed sperm
using the sperm chromatin structure assay (SCSA). Membrane lipid peroxidation (LPO;
oxidation of lipiphilic probe C11-BODIPY581/591) was quantified with and without induction of
LPO using t-butyl hydroperoxide (TBHP) before cryopreservation, 0 and 3 h after thawing.
For TAC, SOD and GPx, intra-assay coefficients of variation (CV) were 5.0, 3.5 and 6.2 %,
respectively, and inter-assay CV were 8.3, 3.6 and 8.3 %. Levels of TAC, SOD and GPx
differed among bulls (P < 0.0001), but not among ejaculates within bulls (P > 0.05). There
was a negative correlation (r=-0.76; P ≤ 0.05) between SOD and GPx, but there were no
(P > 0.05) other relationships between antioxidants. The TAC was negatively correlated with
TBHP-LPO at 0 h (r=-0.85; P ≤ 0.01) and there were positive correlations between SOD and
LPO at 0 h (r=0.71; P ≤ 0.05) and 3 h (r=0.80; P ≤ 0.05). There were no (P > 0.05)
relationships between DFI and antioxidant levels.
In the second part of the study, semen was collected from 17 Holstein Friesian bulls for four
weeks twice a week. Thereafter, the basal ration of nine bulls (ALA bulls; age: 3.2±1 yrs) was
53
supplemented with 800 g/day coated alpha-linolenic acid and the basal ration of eight bulls
(PA bulls; age: 3.7±0.8 yrs) was supplemented with 400 g/day palmitic acid (Bergafat®).
Both fat supplementations were fed for 12 weeks. Volume, sperm concentration, and total
sperm number of each ejaculate were determined. PMI and AD were evaluated by flow
cytometry before cryopreservation and after thawing. Levels of TAC, GPx and SOD were
determined in seminal plasma before and after fat supplementation. Fatty acid content in
shock frozen sperm samples was determined using gas chromatography before and after fat
supplementation. Feeding ALA increased (P < 0.05) the content of docosahexaenoic acid in
ALA bulls whereas the DHA content in PA bulls did not (P > 0.05) change. The increase of
DHA in ALA bulls had no (P > 0.05) effect on the seminal plasma levels of TAC, GPx and
SOD. Before cryopreservation, values of PMI- and AD were not (P > 0.05) affected by the
additional feeding of fatty acids, neither by PA nor by ALA. PMI increased and AD
decreased after cryopreservation in ALA bulls as well as in PA bulls during the study period
(P < 0.005).
In summary, a reliable assay for assessment of TAC in bovine seminal plasma was
established. The measurement of TAC gave valuable information about the sensivity of sperm
against lipid peroxidation of cryopreserved sperm. The feeding of neither saturated nor
polyunsaturated fatty acids affected the antioxidant levels in bovine seminal plasma. Both
types of fatty acids increased the quality of cryopreserved sperm, although the content of
docosahexaenoic acid in sperm membranes increased only in ALA bulls.
54
6. ZUSAMMENFASSUNG
Oguz Calisici
Untersuchung der antioxidativen Kapazität des bovinen Seminalplasmas– Effekte der
Fütterung von Omega-3 Fettsäure
Das erste Ziel der vorliegenden Arbeit war es, einen Test zu etablieren, um die totale
antioxidative Kapazität (TAC) im Seminalplasma von Bullen zu bestimmen und zu prüfen, ob
ein Zusammenhang zwischen TAC und anderen Antioxidantien im Seminalplasma und der
Spermaqualität besteht. Das zweite Ziel der Studie lag darin, zu überprüfen, ob die Fütterung
der mehrfach ungesättigten Fettsäuren die antioxidative Kapazität im Seminalplasma
beeinflusst.
Für den ersten Teil der Studie wurden von neun Bullen der Rasse Holstein Frisian über 4
Wochen je zweimal wöchentlich Ejakulate gewonnen. Die Untersuchungsmethoden von TAC,
Glutathion-Peroxidase (GPx) und Superoxid-Dismutase (SOD) wurden für den Gebrauch in
einem automatischen 96-Well-Mikroplatten-Reader modifiziert. Die
Plasmamembranintegrität (PMI) und die akrosomale Schädigung (AD) von frisch verdünntem
und kryokonserviertem Sperma wurden mittels Durchflusszytometrie bestimmt. Die
Bestimmung der DNA-Fragmentierung erfolgte an kryokonserviertem Sperma mit Hilfe des
Sperm-Chromatin-Struktur Assays (SCSA™). Die Membranlipidperoxidation (LPO) wurde
mit und ohne Induktion der LPO durch tertiäres Butylhydroperoxid (TBHP) vor der
Kryokonservierung, unmittelbar (0h) und 3 Stunden (3h) nach dem Auftauen quantifiziert.
Für die Bestimmungen von TAC, SOD und GPx lagen die Intra-Assay-Variations-
koeffizienten (CV) bei 5,0, 3,5 und 6,2 % und die Inter-Assay-Variationskoeffizienten (CV)
bei 8,3, 3,6 und 8,3 %. Der Gehalt von TAC, SOD und GPx unterschied sich zwischen den
Bullen (P < 0,0001) aber es bestanden keine (P > 0,05) Unterschiede zwischen den einzelnen
Ejakulaten. SOD und GPx korrelierten negativ (r=-0,76; P ≤ 0,05) miteinander. Es waren
keine (P > 0,05) Zusammenhänge zwischen den anderen Antioxidantien festzustellen. Eine
negative Korrelation gab es zwischen TAC und TBHP-LPO unmittelbar nach dem Auftauen
(r=-0,85; P ≤ 0,01). Die SOD-Werte korrelierten positiv mit den unmittelbar (r=0,71;
55
P ≤ 0,05) und 3 Stunden nach dem Auftauen erhobenen LPO-Werten (r=0,80; P ≤ 0,05).
Keine (P > 0,05) Zusammenhänge waren zwischen DFI und den Antioxidantien festzustellen.
Für den zweiten Teil der Studie wurden zunächst über einen Zeitraum von 4 Wochen 8
Ejakulate von 17 Holstein Frisian Bullen gewonnen. Anschließend wurde der Grundration
von 9 Bullen 800 g gecoateter Alpha-Linolensäure (ALA) pro Tag zugesetzt. Die Grundration
der 8 Bullen der Kontrollgruppe wurde mit 400 g Palmitinsäure (PO) pro Tag angereichert.
Die Fettsupplementation wurden über 12 Wochen durchgeführt. Das Volumen, die
Spermienkonzentration und die Gesamtspermienzahl jedes Ejakulates, sowie PMI und AD
vor und nach der Kryokonservierung wurden bestimmt. Die Seminalplasmagehalte an TAC,
SOD und GPx (vier Wochen lang vor Versuchsbeginn und vier Wochen lang am Ende des
Versuches) sowie die Fettsäureanteile in der Spermienmembranen wurden am Anfang (1.
Versuchswoche) und nach der Fettsupplementation (12 Wochen nach Versuchsbeginn)
ermittelt.
Durch die ALA-Fütterung erhöhte sich der Anteil von DHA (P < 0,05) in der Spermien-
membran, während der DHA-Anteil in den Spermien der PA-Bullen gleich blieb (P > 0,05).
Der DHA-Anstieg in der ALA-Gruppe hatte keine (P > 0,05) Auswirkungen auf die
Antioxidantien (TAC, SOD und GPx). Die vor der Kryokonservierung ermittelten PMI- und
AD-Werte änderte sich auch nicht (P > 0,05) durch die Fütterung der beiden Fettsäuren, ALA
und PO. Im Gegensatz dazu war nach dem Auftauen bei den Spermien beider
Fütterungsgruppen ein Anstieg von PMI und ein Abfall von AD nachweisbar (P < 0,005).
Zusammenfassend wurde im Rahmen dieser Arbeit eine verlässliche Methode zur Messung
von TAC in bovinem Seminalplasma etabliert. Der TAC-Wert lieferte wichtige Informationen
über die Sensibilität von Spermien gegenüber LPO bei der Kryokonservierung. Die Fütterung
von gesättigten als auch von mehrfach ungesättigten Fettsäuren hatte zwar keine Auswirkung
auf die antioxidative Kapazität in bovinem Seminalplasma, jedoch hatten beide
Fettsäuretypen einen positiven Einfluss auf die Spermaqualität.
56
7. REFERENCES
ABU-ERREISH, G., L. MAGNES and T. K. LI (1978): Isolation and Properties of Superoxide Dismutase from Ram Spermatozoa and Erythrocytes. Biol Reprod 18, 554-560 ADEEL, M., A. IJAZ, M. ALEEM, H. REHMAN, M. S. YOUSAF and M. A. JABBAR (2009): Improvement of Liquid and Frozen-Thawed Semen Quality of Nili-Ravi Buffalo Bulls (Bubalus Bubalis) through Supplementation of Fat. Theriogenology 71, 1220-1225 AEBI, H. (1984): Catalase in Vitro. Methods Enzymol 105, 121-126 AGARWAL, A. and S. A. PRABAKARAN (2005): Mechanism, Measurement, and Prevention of Oxidative Stress in Male Reproductive Physiology. Indian J Exp Biol 43, 963-974 AGARWAL, A., S. A. PRABAKARAN and T. M. SAID (2005): Prevention of Oxidative Stress Injury to Sperm. J Androl 26, 654-660 AGARWAL, A. and R. A. SALEH (2002): Role of Oxidants in Male Infertility: Rationale, Significance, and Treatment. Urol Clin North Am 29, 817-827 AITKEN, R. J. and M. A. BAKER (2004): Oxidative Stress and Male Reproductive Biology. Reprod Fertil Dev 16, 581-588 AITKEN, R. J., M. A. BAKER and M. O'BRYAN (2004): Shedding Light on Chemiluminescence: The Application of Chemiluminescence in Diagnostic Andrology. J Androl 25, 455-465 AITKEN, R. J., D. W. BUCKINGHAM and K. M. WEST (1992): Reactive Oxygen Species and Human Spermatozoa: Analysis of the Cellular Mechanisms Involved in Luminol- and Lucigenin-Dependent Chemiluminescence. J Cell Physiol 151, 466-477 AITKEN, R. J. and J. S. CLARKSON (1987): Cellular Basis of Defective Sperm Function and Its Association with the Genesis of Reactive Oxygen Species by Human Spermatozoa.
57
J Reprod Fertil 81, 459-469 AITKEN, R. J., J. S. CLARKSON and S. FISHEL (1989): Generation of Reactive Oxygen Species, Lipid Peroxidation, and Human Sperm Function. Biol Reprod 41, 183-197 AITKEN, R. J. and H. FISHER (1994): Reactive Oxygen Species Generation and Human Spermatozoa: The Balance of Benefit and Risk. Bioessays 16, 259-267 AITKEN, R. J., D. S. IRVINE and F. C. WU (1991): Prospective Analysis of Sperm-Oocyte Fusion and Reactive Oxygen Species Generation as Criteria for the Diagnosis of Infertility. Am J Obstet Gynecol 164, 542-551 AITKEN, R. J. and C. KRAUSZ (2001): Oxidative Stress, DNA Damage and the Y Chromosome. Reproduction 122, 497-506 AITKEN, R. J., J. K. WINGATE, G. N. DE IULIIS and E. A. MCLAUGHLIN (2007): Analysis of Lipid Peroxidation in Human Spermatozoa Using Bodipy C11. Mol Hum Reprod 13, 203-211 ALVAREZ, J. G. and B. T. STOREY (1984): Lipid Peroxidation and the Reactions of Superoxide and Hydrogen Peroxide in Mouse Spermatozoa. Biol Reprod 30, 833-841 ASHWORTH, P. J., R. A. HARRISON, N. G. MILLER, J. M. PLUMMER and P. F. WATSON (1995): Flow Cytometric Detection of Bicarbonate-Induced Changes in Lectin Binding in Boar and Ram Sperm Populations. Mol Reprod Dev 40, 164-176 AUSTIN, C. R. and M. W. BISHOP (1958): Capacitation of Mammalian Spermatozoa. Nature 181, 851 BALL, B. A. (2008): Oxidative Stress, Osmotic Stress and Apoptosis: Impacts on Sperm Function and Preservation in the Horse. Anim Reprod Sci 107, 257-267 BALL, B. A., C. G. GRAVANCE, V. MEDINA, J. BAUMBER and I. K. LIU (2000): Catalase Activity in Equine Semen. Am J Vet Res 61, 1026-1030
58
BALLACHEY, B. E., W. D. HOHENBOKEN and D. P. EVENSON (1987): Heterogeneity of Sperm Nuclear Chromatin Structure and Its Relationship to Bull Fertility. Biol Reprod 36, 915-925 BANSAL, A. K. and G. S. BILASPURI (2008): Oxidative Stress Alters Membrane Sulfhydryl Status, Lipid and Phospholipid Contents of Crossbred Cattle Bull Spermatozoa. Anim Reprod Sci 104, 398-404 BARTLE, J. L., P. L. SENGER and J. K. HILLERS (1980): Influence of Injected Selenium in Dairy Bulls on Blood and Semen Selenium, Glutathione Peroxidase and Seminal Quality. Biol Reprod 23, 1007-1013 BEISKER, W. (1994): A New Combined Integral-Light and Slit-Scan Data Analysis System (Das) for Flow Cytometry. Comput Methods Programs Biomed 42, 15-26 BELL, E. F. (1987): History of Vitamin E in Infant Nutrition. Am J Clin Nutr 46, 183-186 BILLIG, H., I. FURUTA, C. RIVIER, J. TAPANAINEN, M. PARVINEN and A. J. HSUEH (1995): Apoptosis in Testis Germ Cells: Developmental Changes in Gonadotropin Dependence and Localization to Selective Tubule Stages. Endocrinology 136, 5-12 BILODEAU, J. F., S. BLANCHETTE, N. CORMIER and M. A. SIRARD (2002): Reactive Oxygen Species-Mediated Loss of Bovine Sperm Motility in Egg Yolk Tris Extender: Protection by Pyruvate, Metal Chelators and Bovine Liver or Oviductal Fluid Catalase. Theriogenology 57, 1105-1122 BILODEAU, J. F., S. BLANCHETTE, C. GAGNON and M. A. SIRARD (2001): Thiols Prevent H2o2-Mediated Loss of Sperm Motility in Cryopreserved Bull Semen. Theriogenology 56, 275-286 BILODEAU, J. F., S. CHATTERJEE, M. A. SIRARD and C. GAGNON (2000): Levels of Antioxidant Defenses Are Decreased in Bovine Spermatozoa after a Cycle of Freezing and Thawing. Mol Reprod Dev 55, 282-288 BLESBOIS, E., V. DOUARD, M. GERMAIN, P. BONIFACE and F. PELLET (2004): Effects of N-3 Polyunsaturated Dietary Supplementation on the Reproductive Capacity of Male Turkeys.
59
Theriogenology 61, 537-549 BLIGH, E. G. and W. J. DYER (1959): A Rapid Method of Total Lipid Extraction and Purification. Can J Biochem Physiol 37, 911-917 BOLLWEIN, H., I. FUCHS and C. KOESS (2008): Interrelationship between Plasma Membrane Integrity, Mitochondrial Membrane Potential and DNA Fragmentation in Cryopreserved Bovine Spermatozoa. Reprod Domest Anim 43, 189-195 BONGALHARDO, D. C., S. LEESON and M. M. BUHR (2009): Dietary Lipids Differentially Affect Membranes from Different Areas of Rooster Sperm. Poult Sci 88, 1060-1069 BRADFORD, M. M. (1976): A Rapid and Sensitive Method for the Quantitation of Microgram Quantities of Protein Utilizing the Principle of Protein-Dye Binding. Anal Biochem 72, 248-254 BRIGELIUS-FLOHE, R. and M. G. TRABER (1999): Vitamin E: Function and Metabolism. Faseb J 13, 1145-1155 BRINSKO, S. P., D. D. VARNER, C. C. LOVE, T. L. BLANCHARD, B. C. DAY and M. E. WILSON (2005): Effect of Feeding a Dha-Enriched Nutriceutical on the Quality of Fresh, Cooled and Frozen Stallion Semen. Theriogenology 63, 1519-1527 BROUWERS, J. F. and B. M. GADELLA (2003): In Situ Detection and Localization of Lipid Peroxidation in Individual Bovine Sperm Cells. Free Radic Biol Med 35, 1382-1391 BROWN, D. V., P. L. SENGER, S. L. STONE, J. A. FROSETH and W. C. BECKER (1977): Glutatione Peroxidase in Bovine Semen. J Reprod Fertil 50, 117-118 BURTON, G. W. (1994): Vitamin E: Molecular and Biological Function. Proc Nutr Soc 53, 251-262 CALVIN, H. I. (1981): Comparative Labelling of Rat Epididymal Spermatozoa by Intratesticularly Administered 65zncl2 and [35s]Cysteine. J Reprod Fertil 61, 65-73
60
CASTELLINI, C., P. LATTAIOLI, A. DAL BOSCO, A. MINELLI and C. MUGNAI (2003): Oxidative Status and Semen Characteristics of Rabbit Buck as Affected by Dietary Vitamin E, C and N-3 Fatty Acids. Reprod Nutr Dev 43, 91-103 CEROLINI, S., A. MALDJIAN, F. PIZZI and T. M. GLIOZZI (2001): Changes in Sperm Quality and Lipid Composition During Cryopreservation of Boar Semen. Reproduction 121, 395-401 CEROLINI, S., P. F. SURAI, B. K. SPEAKE and N. H. SPARKS (2005): Dietary Fish and Evening Primrose Oil with Vitamin E Effects on Semen Variables in Cockerels. Br Poult Sci 46, 214-222 CHABORY, E., C. DAMON, A. LENOIR, G. KAUSELMANN, H. KERN, B. ZEVNIK, C. GARREL, F. SAEZ, R. CADET, J. HENRY-BERGER, M. SCHOOR, U. GOTTWALD, U. HABENICHT, J. R. DREVET and P. VERNET (2009): Epididymis Seleno-Independent Glutathione Peroxidase 5 Maintains Sperm DNA Integrity in Mice. J Clin Invest 119, 2074-2085 CHATTERJEE, S. and C. GAGNON (2001): Production of Reactive Oxygen Species by Spermatozoa Undergoing Cooling, Freezing, and Thawing. Mol Reprod Dev 59, 451-458 CHEN, S. S., L. S. CHANG and Y. H. WEI (2001): Oxidative Damage to Proteins and Decrease of Antioxidant Capacity in Patients with Varicocele. Free Radic Biol Med 30, 1328-1334 CONQUER, J. A., J. B. MARTIN, I. TUMMON, L. WATSON and F. TEKPETEY (2000): Effect of Dha Supplementation on Dha Status and Sperm Motility in Asthenozoospermic Males. Lipids 35, 149-154 DARLEY-USMAR, V., H. WISEMAN and B. HALLIWELL (1995): Nitric Oxide and Oxygen Radicals: A Question of Balance. FEBS Lett 369, 131-135 DAWRA, R. K. and O. P. SHARMA (1985): Effect of Seminal Plasma Antioxidant on Lipid Peroxidation in Spermatozoa, Mitochondria and Microsomes. Biochem Int 11, 333-339 DAWRA, R. K., O. P. SHARMA and H. P. MAKKAR (1984): Evidence for a Novel Antioxidant in Bovine Seminal Plasma.
61
Biochem Int 8, 655-659 DE LAMIRANDE, E. and C. GAGNON (1995): Impact of Reactive Oxygen Species on Spermatozoa: A Balancing Act between Beneficial and Detrimental Effects. Hum Reprod 10 Suppl 1, 15-21 DE LAMIRANDE, E., H. JIANG, A. ZINI, H. KODAMA and C. GAGNON (1997): Reactive Oxygen Species and Sperm Physiology. Rev Reprod 2, 48-54 DEHNING, F. (2008) Einfluss Der Fütterung Von Omega-3-Fettsäure Auf Die Spermaqualität Von Bullen. Hannover, Tierärztliche Hochschule Hannover, Vet. Med. Dissertation. DÖCKE, F. (1963): Seminalplasma. In: Die Künstliche Besamung Bei Den Haustieren Gustav Fischer Verlag, Jena, 227-235 DUTEAUX, S. B., T. BERGER, R. A. HESS, B. L. SARTINI and M. G. MILLER (2004): Male Reproductive Toxicity of Trichloroethylene: Sperm Protein Oxidation and Decreased Fertilizing Ability. Biol Reprod 70, 1518-1526 ERENPREISS, J., M. SPANO, J. ERENPREISA, M. BUNGUM and A. GIWERCMAN (2006): Sperm Chromatin Structure and Male Fertility: Biological and Clinical Aspects. Asian J Androl 8, 11-29 EVANS, H. M. and K. S. BISHOP (1922): On the Existence of a Hitherto Unrecognized Dietary Factor Essential for Reproduction. Science 56, 650-651 EVENSON, D. and L. JOST (2000): Sperm Chromatin Structure Assay Is Useful for Fertility Assessment. Methods Cell Sci 22, 169-189 EWING, J. F. and D. R. JANERO (1995): Microplate Superoxide Dismutase Assay Employing a Nonenzymatic Superoxide Generator. Anal Biochem 232, 243-248 FAGAN, J. M., B. G. SLECZKA and I. SOHAR (1999): Quantitation of Oxidative Damage to Tissue Proteins. Int J Biochem Cell Biol 31, 751-757 FERNANDEZ-SANTOS, M. R., F. MARTINEZ-PASTOR, V. GARCIA-MACIAS, M. C. ESTESO, A. J. SOLER, P. PAZ, L. ANEL and J. J. GARDE (2007):
62
Sperm Characteristics and DNA Integrity of Iberian Red Deer (Cervus Elaphus Hispanicus) Epididymal Spermatozoa Frozen in the Presence of Enzymatic and Nonenzymatic Antioxidants. J Androl 28, 294-305 FISCHER, A., E. HASENPUSCH, A. WEYAND and H. BOLLWEIN (2010): [Effects of Different Dilution Levels on Bull- and Ejaculate Related Variability of Plasmamembran Integrity, Acrosomal Damage and DNA-Integrity of Cryopreserved Bull Spermatozoa]. Berl Munch Tierarztl Wochenschr 123, 83-88 FOOTE, R. H. and E. HARE (2000): High Catalase Content of Rabbit Semen Appears to Be Inherited. J Androl 21, 664-668 FORD, W. C. and A. HARRISON (1983): D-[1-14c]Mannitol and [U-14c]Sucrose as Extracellular Space Markers for Human Spermatozoa and the Uptake of 2-Deoxyglucose. J Reprod Fertil 69, 479-487 FORESTA, C., L. FLOHE, A. GAROLLA, A. ROVERI, F. URSINI and M. MAIORINO (2002): Male Fertility Is Linked to the Selenoprotein Phospholipid Hydroperoxide Glutathione Peroxidase. Biol Reprod 67, 967-971 FORSTROM, J. W., J. J. ZAKOWSKI and A. L. TAPPEL (1978): Identification of the Catalytic Site of Rat Liver Glutathione Peroxidase as Selenocysteine. Biochemistry 17, 2639-2644 FRAGA, C. G., P. A. MOTCHNIK, A. J. WYROBEK, D. M. REMPEL and B. N. AMES (1996): Smoking and Low Antioxidant Levels Increase Oxidative Damage to Sperm DNA. Mutat Res 351, 199-203 FRIDOVICH, I. (1983): Superoxide Radical: An Endogenous Toxicant. Annu Rev Pharmacol Toxicol 23, 239-257 FRIDOVICH, I. (1995): Superoxide Radical and Superoxide Dismutases. Annu Rev Biochem 64, 97-112 FRIEDMAN, A. and S. MOE (2006): Review of the Effects of Omega-3 Supplementation in Dialysis Patients. Clin J Am Soc Nephrol 1, 182-192
63
GARNER, D. L., C. A. THOMAS and C. H. ALLEN (1997): Effect of Semen Dilution on Bovine Sperm Viability as Determined by Dual-DNA Staining and Flow Cytometry. J Androl 18, 324-331 GARNER, D. L., C. A. THOMAS, C. G. GRAVANCE, C. E. MARSHALL, J. M. DEJARNETTE and C. H. ALLEN (2001): Seminal Plasma Addition Attenuates the Dilution Effect in Bovine Sperm. Theriogenology 56, 31-40 GAVELLA, M. and V. LIPOVAC (1992): Nadh-Dependent Oxidoreductase (Diaphorase) Activity and Isozyme Pattern of Sperm in Infertile Men. Arch Androl 28, 135-141 GLANDER, H. J. and D. DETTMER (1978): Monosaccharide Transport across Membranes of Human Spermatozoa. I. Development of a Radiochemical Method of Measuring Monosaccharide Uptake by Spermatozoa. Andrologia 10, 69-73 GÜNZLER, A. and L. FLOHÉ (1985): Glutathione Peroxidase. In: Crc Handbook of Methods for Oxygen Radical Research CRC Press, Boca Raton, Fla. , 285-290 HAIDL, G. and C. OPPER (1997): Changes in Lipids and Membrane Anisotropy in Human Spermatozoa During Epididymal Maturation. Hum Reprod 12, 2720-2723 HALL, L., K. WILLIAMS, A. C. PERRY, J. FRAYNE and J. A. JURY (1998): The Majority of Human Glutathione Peroxidase Type 5 (Gpx5) Transcripts Are Incorrectly Spliced: Implications for the Role of Gpx5 in the Male Reproductive Tract. Biochem J 333 ( Pt 1), 5-9 HALLIWELL, B. (1989): Free Radicals, Reactive Oxygen Species and Human Disease: A Critical Evaluation with Special Reference to Atherosclerosis. Br J Exp Pathol 70, 737-757 HALLIWELL, B. (1990): How to Characterize a Biological Antioxidant. Free Radic Res Commun 9, 1-32 HALLIWELL, B. (2006): Reactive Species and Antioxidants. Redox Biology Is a Fundamental Theme of Aerobic Life. Plant Physiol 141, 312-322
64
HALLIWELL, B. and C. E. CROSS (1994): Oxygen-Derived Species: Their Relation to Human Disease and Environmental Stress. Environ Health Perspect 102 Suppl 10, 5-12 HALLIWELL, B. and J. M. GUTTERIDGE (1989): Free Radicals in Biology and Medicine Clarendon Press Oxford, UK HALLIWELL, B. and J. M. GUTTERIDGE (1990): Role of Free Radicals and Catalytic Metal Ions in Human Disease: An Overview. Methods Enzymol 186, 1-85 HAMMERSTEDT, R. H. (1993): Maintenance of Bioenergetic Balance in Sperm and Prevention of Lipid Peroxidation: A Review of the Effect on Design of Storage Preservation Systems. Reprod Fertil Dev 5, 675-690 HAMMERSTEDT, R. H., J. K. GRAHAM and J. P. NOLAN (1990): Cryopreservation of Mammalian Sperm: What We Ask Them to Survive. J Androl 11, 73-88 HARTREE, E. F. and T. MANN (1961): Phospholipids in Ram Semen: Metabolism of Plasmalogen and Fatty Acids. Biochem J 80, 464-476 HENGARTNER, M. O. (1997): Apoptosis and the Shape of Death. Dev Genet 21, 245-248 HERBETTE, S., P. ROECKEL-DREVET and J. R. DREVET (2007): Seleno-Independent Glutathione Peroxidases. More Than Simple Antioxidant Scavengers. Febs J 274, 2163-2180 HERRERA, E. and C. BARBAS (2001): Vitamin E: Action, Metabolism and Perspectives. J Physiol Biochem 57, 43-56 HUANG, H. F. and W. C. HEMBREE (1979): Spermatogenic Response to Vitamin a in Vitamin a Deficient Rats. Biol Reprod 21, 891-904 HUNTER, R. H. and H. RODRIGUEZ-MARTINEZ (2004): Capacitation of Mammalian Spermatozoa in Vivo, with a Specific Focus on Events in the Fallopian Tubes. Mol Reprod Dev 67, 243-250 IWASAKI, A. and C. GAGNON (1992):
65
Formation of Reactive Oxygen Species in Spermatozoa of Infertile Patients. Fertil Steril 57, 409-416 JANUSKAUSKA, A., A. JOHANNISSON and H. RODRIGUEZ-MARTINEZ (2001): Assessment of Sperm Quality through Fluorometry and Sperm Chromatin Structure Assay in Relation to Field Fertility of Frozen-Thawed Semen from Swedish Ai Bulls. Theriogenology 55, 947-961 JEULIN, C., J. C. SOUFIR, P. WEBER, D. LAVAL-MARTIN and R. CALVAYRAC (1989): Catalase Activity in Human Spermatozoa and Seminal Plasma. Gamete Res 24, 185-196 JONES, R. and T. MANN (1977): Damage to Ram Spermatozoa by Peroxidation of Endogenous Phospholipids. J Reprod Fertil 50, 261-268 JONES, R., T. MANN and R. SHERINS (1979): Peroxidative Breakdown of Phospholipids in Human Spermatozoa, Spermicidal Properties of Fatty Acid Peroxides, and Protective Action of Seminal Plasma. Fertil Steril 31, 531-537 KANTOLA, M., M. SAARANEN and T. VANHA-PERTTULA (1988): Selenium and Glutathione Peroxidase in Seminal Plasma of Men and Bulls. J Reprod Fertil 83, 785-794 KASIMANICKAM, R., V. KASIMANICKAM, C. D. THATCHER, R. L. NEBEL and B. G. CASSELL (2007): Relationships among Lipid Peroxidation, Glutathione Peroxidase, Superoxide Dismutase, Sperm Parameters, and Competitive Index in Dairy Bulls. Theriogenology 67, 1004-1012 KASIMANICKAM, R., K. D. PELZER, V. KASIMANICKAM, W. S. SWECKER and C. D. THATCHER (2006): Association of Classical Semen Parameters, Sperm DNA Fragmentation Index, Lipid Peroxidation and Antioxidant Enzymatic Activity of Semen in Ram-Lambs. Theriogenology 65, 1407-1421 KELSO, K. A., S. CEROLINI, R. C. NOBLE, N. H. SPARKS and B. K. SPEAKE (1996): Lipid and Antioxidant Changes in Semen of Broiler Fowl from 25 to 60 Weeks of Age. J Reprod Fertil 106, 201-206 KELSO, K. A., S. CEROLINI, B. K. SPEAKE, L. G. CAVALCHINI and R. C. NOBLE (1997a): Effects of Dietary Supplementation with Alpha-Linolenic Acid on the Phospholipid Fatty Acid Composition and Quality of Spermatozoa in Cockerel from 24 to 72 Weeks of Age. J Reprod Fertil 110, 53-59
66
KELSO, K. A., A. REDPATH, R. C. NOBLE and B. K. SPEAKE (1997b): Lipid and Antioxidant Changes in Spermatozoa and Seminal Plasma Throughout the Reproductive Period of Bulls. J Reprod Fertil 109, 1-6 KERR, J. F., A. H. WYLLIE and A. R. CURRIE (1972): Apoptosis: A Basic Biological Phenomenon with Wide-Ranging Implications in Tissue Kinetics. Br J Cancer 26, 239-257 KIRKMAN, H. N., S. GALIANO and G. F. GAETANI (1987): The Function of Catalase-Bound Nadph. J Biol Chem 262, 660-666 KIRTON, K. T., H. D. HAFS and A. G. HUNTER (1964): Levels of Some Normal Constituents of Bull Semen During Repetitive Ejaculation. J Reprod Fertil 8, 157-164 KOBAYASHI, T., T. MIYAZAKI, M. NATORI and S. NOZAWA (1991): Protective Role of Superoxide Dismutase in Human Sperm Motility: Superoxide Dismutase Activity and Lipid Peroxide in Human Seminal Plasma and Spermatozoa. Hum Reprod 6, 987-991 KOLETTIS, P. N., R. K. SHARMA, F. F. PASQUALOTTO, D. NELSON, A. J. THOMAS, JR. and A. AGARWAL (1999): Effect of Seminal Oxidative Stress on Fertility after Vasectomy Reversal. Fertil Steril 71, 249-255 KOPPERS, A. J., G. N. DE IULIIS, J. M. FINNIE, E. A. MCLAUGHLIN and R. J. AITKEN (2008): Significance of Mitochondrial Reactive Oxygen Species in the Generation of Oxidative Stress in Spermatozoa. J Clin Endocrinol Metab 93, 3199-3207 KRUIDENIER, L. and H. W. VERSPAGET (2002): Review Article: Oxidative Stress as a Pathogenic Factor in Inflammatory Bowel Disease--Radicals or Ridiculous? Aliment Pharmacol Ther 16, 1997-2015 LENZI, A., M. PICARDO, L. GANDINI and F. DONDERO (1996): Lipids of the Sperm Plasma Membrane: From Polyunsaturated Fatty Acids Considered as Markers of Sperm Function to Possible Scavenger Therapy. Hum Reprod Update 2, 246-256 LEPAGE, G. and C. C. ROY (1986): Direct Transesterification of All Classes of Lipids in a One-Step Reaction. J Lipid Res 27, 114-120
67
MACLEOD, J. (1943): The Role of Oxygen in the Metabolism and Motility of Human Spermatozoa. Am J Physiol 138, 512-518 MAIORINO, M., V. BOSELLO, F. URSINI, C. FORESTA, A. GAROLLA, M. SCAPIN, H. SZTAJER and L. FLOHE (2003): Genetic Variations of Gpx-4 and Male Infertility in Humans. Biol Reprod 68, 1134-1141 MAIORINO, M., M. COASSIN, A. ROVERI and F. URSINI (1989): Microsomal Lipid Peroxidation: Effect of Vitamin E and Its Functional Interaction with Phospholipid Hydroperoxide Glutathione Peroxidase. Lipids 24, 721-726 MARKLUND, S. L., N. G. WESTMAN, E. LUNDGREN and G. ROOS (1982): Copper- and Zinc-Containing Superoxide Dismutase, Manganese-Containing Superoxide Dismutase, Catalase, and Glutathione Peroxidase in Normal and Neoplastic Human Cell Lines and Normal Human Tissues. Cancer Res 42, 1955-1961 MARTI, E., J. I. MARTI, T. MUINO-BLANCO and J. A. CEBRIAN-PEREZ (2008): Effect of the Cryopreservation Process on the Activity and Immunolocalization of Antioxidant Enzymes in Ram Spermatozoa. J Androl 29, 459-467 MARTIN, G., O. SABIDO, P. DURAND and R. LEVY (2004): Cryopreservation Induces an Apoptosis-Like Mechanism in Bull Sperm. Biol Reprod 71, 28-37 MCCORD, J. M. and I. FRIDOVICH (1969): Superoxide Dismutase. An Enzymic Function for Erythrocuprein (Hemocuprein). J Biol Chem 244, 6049-6055 MEISTER, A. (1988): Glutathione Metabolism and Its Selective Modification. J Biol Chem 263, 17205-17208 MENNELLA, M. R. and R. JONES (1980): Properties of Spermatozoal Superoxide Dismutase and Lack of Involvement of Superoxides in Metal-Ion-Catalysed Lipid-Peroxidation and Reactions in Semen. Biochem J 191, 289-297 NEILD, D. M., J. F. BROUWERS, B. COLENBRANDER, A. AGUERO and B. M. GADELLA (2005): Lipid Peroxide Formation in Relation to Membrane Stability of Fresh and Frozen Thawed Stallion Spermatozoa. Mol Reprod Dev 72, 230-238
68
NEILL, A. R. and C. J. MASTERS (1972): Metabolism of Fatty Acids by Bovine Spermatozoa. Biochem J 127, 375-385 O, W. S., H. CHEN and P. H. CHOW (2006): Male Genital Tract Antioxidant Enzymes--Their Ability to Preserve Sperm DNA Integrity. Mol Cell Endocrinol 250, 80-83 OCHSENDORF, F. R. and J. FUCHS (1997): Antioxidants in Germinal Epithelium, Spermatozoa and Seminal Plasma. In: Oxidative Stress in Male Infertility Gardez! Verlag, St. Augustin, 85-129 OLIVER, C. N., B. W. AHN, E. J. MOERMAN, S. GOLDSTEIN and E. R. STADTMAN (1987): Age-Related Changes in Oxidized Proteins. J Biol Chem 262, 5488-5491 OLLERO, M., E. GIL-GUZMAN, M. C. LOPEZ, R. K. SHARMA, A. AGARWAL, K. LARSON, D. EVENSON, A. J. THOMAS, JR. and J. G. ALVAREZ (2001): Characterization of Subsets of Human Spermatozoa at Different Stages of Maturation: Implications in the Diagnosis and Treatment of Male Infertility. Hum Reprod 16, 1912-1921 OLLERO, M., R. D. POWERS and J. G. ALVAREZ (2000): Variation of Docosahexaenoic Acid Content in Subsets of Human Spermatozoa at Different Stages of Maturation: Implications for Sperm Lipoperoxidative Damage. Mol Reprod Dev 55, 326-334 ORRENIUS, S. (2007): Reactive Oxygen Species in Mitochondria-Mediated Cell Death. Drug Metab Rev 39, 443-455 PALLUDAN, B. (1966): Direct Effect of Vitamin a on Boar Testis. Nature 211, 639-640 PALOZZA, P. and N. I. KRINSKY (1992): Antioxidant Effects of Carotenoids in Vivo and in Vitro: An Overview. Methods Enzymol 213, 403-420 PAPPAS, A. C., E. ZOIDIS, P. F. SURAI and G. ZERVAS (2008): Selenoproteins and Maternal Nutrition. Comp Biochem Physiol B Biochem Mol Biol 151, 361-372 PARKS, J. E. and D. V. LYNCH (1992):
69
Lipid Composition and Thermotropic Phase Behavior of Boar, Bull, Stallion, and Rooster Sperm Membranes. Cryobiology 29, 255-266 PASQUALOTTO, F. F., A. SUNDARAM, R. K. SHARMA, E. BORGES, JR., E. B. PASQUALOTTO and A. AGARWAL (2008): Semen Quality and Oxidative Stress Scores in Fertile and Infertile Patients with Varicocele. Fertil Steril 89, 602-607 PAULENZ, H., O. TAUGBØL, E. KOMMISRUD and I. S. GREVLE (1999): Effect of Dietary Supplementation with Cod Liver Oil on Cold Shock and Freezability of Boar Semen. Reproduction in Domestic Animals 34, 431-435 PETZOLT, R. (2001): Akzessorisches Sekret In: Veterinärmedizinische Andrologie Schattauer Verlag, Stuttgart, New York, 55-66 POULOS, A., A. DARIN-BENNETT and I. G. WHITE (1973): The Phospholipid-Bound Fatty Acids and Aldehydes of Mammalian Spermatozoa. Comp Biochem Physiol B 46, 541-549 RODRIGUEZ, I., C. ODY, K. ARAKI, I. GARCIA and P. VASSALLI (1997): An Early and Massive Wave of Germinal Cell Apoptosis Is Required for the Development of Functional Spermatogenesis. Embo J 16, 2262-2270 ROOKE, J. A., C. C. SHAO and B. K. SPEAKE (2001): Effects of Feeding Tuna Oil on the Lipid Composition of Pig Spermatozoa and in Vitro Characteristics of Semen. Reproduction 121, 315-322 ROTHSCHILD, L. and H. BARNES (1954): Constituents of Bull Seminal Plasma. J Exp Biol 31, 561-572 SAARANEN, M., U. SUISTOMAA, M. KANTOLA, E. REMES and T. VANHA-PERTTULA (1986): Selenium in Reproductive Organs, Seminal Fluid and Serum of Men and Bulls. Hum Reprod 1, 61-64 SAARANEN, M., U. SUISTOMAA and T. VANHA-PERTTULA (1989): Semen Selenium Content and Sperm Mitochondrial Volume in Human and Some Animal Species. Hum Reprod 4, 304-308
70
SALEH, R. A., H. KOBAYASHI, P. RANGANATHAN, R. K. SHARMA, D. R. NELSON and A. AGARWAL (2001): Assessment of Laboratory Variability in the Measurement of Total Non-Enzymatic Antioxidant Capacity of Semen Using an Enhanced Chemiluminescence Assay. Fertility and sterility 76, S246 SALEM, N. J., H.-Y. KIM and J. A. YERGEY (1986): Docosahexaenoic Acid: Membrane Function and Metabolism. In: Health Effects of Polyunsaturated Fatty Acids in Seafoods Academic Press, New York 319–351 SCOTT, T. W. and R. M. DAWSON (1968): Metabolism of Phospholipids by Spermatozoa and Seminal Plasma. Biochem J 108, 457-463 SENGOKU, K., K. TAMATE, T. YOSHIDA, Y. TAKAOKA, T. MIYAMOTO and M. ISHIKAWA (1998): Effects of Low Concentrations of Nitric Oxide on the Zona Pellucida Binding Ability of Human Spermatozoa. Fertil Steril 69, 522-527 SHANNON, P. and B. CURSON (1982): Site of Aromatic L-Amino Acid Oxidase in Dead Bovine Spermatozoa and Determination of between-Bull Differences in the Percentage of Dead Spermatozoa by Oxidase Activity. J Reprod Fertil 64, 469-473 SHARMA, R. K., F. F. PASQUALOTTO, D. R. NELSON, A. J. THOMAS, JR. and A. AGARWAL (1999): The Reactive Oxygen Species-Total Antioxidant Capacity Score Is a New Measure of Oxidative Stress to Predict Male Infertility. Hum Reprod 14, 2801-2807 SIES, H. (1991): Role of Reactive Oxygen Species in Biological Processes. Klin Wochenschr 69, 965-968 SIES, H. (1993): Strategies of Antioxidant Defense. Eur J Biochem 215, 213-219 SIKKA, S. C. (2001): Relative Impact of Oxidative Stress on Male Reproductive Function. Curr Med Chem 8, 851-862 SIKKA, S. C. (2004): Role of Oxidative Stress and Antioxidants in Andrology and Assisted Reproductive Technology. J Androl 25, 5-18
71
SIKKA, S. C., M. RAJASEKARAN and W. J. HELLSTROM (1995): Role of Oxidative Stress and Antioxidants in Male Infertility. J Androl 16, 464-468 SLAWETA, R. and T. LASKOWSKA-KLITA (1985): Glutathione Content in the Semen of Bulls of the Lowland Black-White Breed. Acta Physiol Pol 36, 107-111 SLAWETA, R., T. LASKOWSKA and E. SZYMANSKA (1988): Lipid Peroxides, Spermatozoa Quality and Activity of Glutathione Peroxidase in Bull Semen. Acta Physiol Pol 39, 207-214 SMITH, D. G., P. L. SENGER, J. F. MCCUTCHAN and C. A. LANDA (1979): Selenium and Glutathione Peroxidase Distribution in Bovine Semen and Selenium-75 Retention by the Tissues of the Reproductive Tract in the Bull. Biol Reprod 20, 377-383 STOREY, B. T. (1997): Biochemistry of the Induction and Prevention of Lipoperoxidative Damage in Human Spermatozoa. Mol Hum Reprod 3, 203-213 STRADAIOLI, G., T. NORO, L. SYLLA and M. MONACI (2007): Decrease in Glutathione (Gsh) Content in Bovine Sperm after Cryopreservation: Comparison between Two Extenders. Theriogenology 67, 1249-1255 SURAI, P. F. (1999): Vitamin E in Avian Reproduction. Poult. Avian Biol. Rev. 10, 1-60 SURAI, P. F., J. P. BRILLARD, B. K. SPEAKE, E. BLESBOIS, F. SEIGNEURIN and N. H. SPARKS (2000a): Phospholipid Fatty Acid Composition, Vitamin E Content and Susceptibility to Lipid Peroxidation of Duck Spermatozoa. Theriogenology 53, 1025-1039 SURAI, P. F., E. KUTZ, G. J. WISHART, R. C. NOBLE and B. K. SPEAKE (1997): The Relationship between the Dietary Provision of Alpha-Tocopherol and the Concentration of This Vitamin in the Semen of Chicken: Effects on Lipid Composition and Susceptibility to Peroxidation. J Reprod Fertil 110, 47-51 SURAI, P. F., R. C. NOBLE, N. H. SPARKS and B. K. SPEAKE (2000b): Effect of Long-Term Supplementation with Arachidonic or Docosahexaenoic Acids on Sperm Production in the Broiler Chicken. J Reprod Fertil 120, 257-264
72
TAVILANI, H., M. DOOSTI, K. ABDI, A. VAISIRAYGANI and H. R. JOSHAGHANI (2006): Decreased Polyunsaturated and Increased Saturated Fatty Acid Concentration in Spermatozoa from Asthenozoospermic Males as Compared with Normozoospermic Males. Andrologia 38, 173-178 TAVILANI, H., M. T. GOODARZI, M. DOOSTI, A. VAISI-RAYGANI, T. HASSANZADEH, S. SALIMI and H. R. JOSHAGHANI (2008): Relationship between Seminal Antioxidant Enzymes and the Phospholipid and Fatty Acid Composition of Spermatozoa. Reprod Biomed Online 16, 649-656 TOSIC, J. and A. WALTON (1950): Metabolism of Spermatozoa. The Formation and Elimination of Hydrogen Peroxide by Spermatozoa and Effects on Motility and Survival. Biochem J 47, 199-212 TRAMER, F., F. ROCCO, F. MICALI, G. SANDRI and E. PANFILI (1998): Antioxidant Systems in Rat Epididymal Spermatozoa. Biol Reprod 59, 753-758 TREMELLEN, K. (2008): Oxidative Stress and Male Infertility--a Clinical Perspective. Hum Reprod Update 14, 243-258 TURRENS, J. F. (2003): Mitochondrial Formation of Reactive Oxygen Species. J Physiol 552, 335-344 TWIGG, J., N. FULTON, E. GOMEZ, D. S. IRVINE and R. J. AITKEN (1998a): Analysis of the Impact of Intracellular Reactive Oxygen Species Generation on the Structural and Functional Integrity of Human Spermatozoa: Lipid Peroxidation, DNA Fragmentation and Effectiveness of Antioxidants. Hum Reprod 13, 1429-1436 TWIGG, J., D. S. IRVINE, P. HOUSTON, N. FULTON, L. MICHAEL and R. J. AITKEN (1998b): Iatrogenic DNA Damage Induced in Human Spermatozoa During Sperm Preparation: Protective Significance of Seminal Plasma. Mol Hum Reprod 4, 439-445 URSINI, F., M. MAIORINO, R. BRIGELIUS-FLOHE, K. D. AUMANN, A. ROVERI, D. SCHOMBURG and L. FLOHE (1995): Diversity of Glutathione Peroxidases. Methods Enzymol 252, 38-53 VERNET, P., R. J. AITKEN and J. R. DREVET (2004):
73
Antioxidant Strategies in the Epididymis. Mol Cell Endocrinol 216, 31-39 WABERSKI, D. (1995): Boar Seminal Plasma and Fertility. Reproduction in Domestic Animals 31, 87-90 WATERHOUSE, K. E., P. O. HOFMO, A. TVERDAL and R. R. MILLER, JR. (2006): Within and between Breed Differences in Freezing Tolerance and Plasma Membrane Fatty Acid Composition of Boar Sperm. Reproduction 131, 887-894 WATHES, D. C., D. R. ABAYASEKARA and R. J. AITKEN (2007): Polyunsaturated Fatty Acids in Male and Female Reproduction. Biol Reprod 77, 190-201 WATSON, P. F. (1995): Recent Developments and Concepts in the Cryopreservation of Spermatozoa and the Assessment of Their Post-Thawing Function. Reprod Fertil Dev 7, 871-891 WATSON, P. F. (2000): The Causes of Reduced Fertility with Cryopreserved Semen. Anim Reprod Sci 60-61, 481-492 WOHLHUETER, R. M., R. MARZ, J. C. GRAFF and P. G. PLAGEMANN (1978): A Rapid-Mixing Technique to Measure Transport in Suspended Animal Cells: Applications to Nucleoside Transport in Novikoff Rat Hepatoma Cells. Methods Cell Biol 20, 211-236 WU, A. S., J. E. OLDFIELD, L. R. SHULL and P. R. CHEEKE (1979): Specific Effect of Selenium Deficiency on Rat Sperm. Biol Reprod 20, 793-798 ZAKOWSKI, J. J., J. W. FORSTROM, R. A. CONDELL and A. L. TAPPEL (1978): Attachment of Selenocysteine in the Catalytic Site of Glutathione Peroxidase. Biochem Biophys Res Commun 84, 248-253 ZANIBONI, L., R. RIZZI and S. CEROLINI (2006): Combined Effect of Dha and Alpha-Tocopherol Enrichment on Sperm Quality and Fertility in the Turkey. Theriogenology 65, 1813-1827 ZANINI, S. F., C. A. TORRES, N. BRAGAGNOLO, J. M. TURATTI, M. G. SILVA and M. S. ZANINI (2003): Evaluation of the Ratio of Omega(6: Omega3 Fatty Acids and Vitamin E Levels in the Diet on the Reproductive Performance of Cockerels.
74
Arch Tierernahr 57, 429-442 ZINI, A., E. DE LAMIRANDE and C. GAGNON (1995): Low Levels of Nitric Oxide Promote Human Sperm Capacitation in Vitro. J Androl 16, 424-431 ZINI, A., M. A. FISCHER, V. MAK, D. PHANG and K. JARVI (2002): Catalase-Like and Superoxide Dismutase-Like Activities in Human Seminal Plasma. Urol Res 30, 321-323