Animal (2018), 12:3, pp 559–568 © The Animal Consortium 2017

doi:10.1017/S1751731117001811
animal

Age-related differences of semen quality, seminal plasma, and

spermatozoa antioxidative and oxidative stress variables in bulls
during cold and warm periods of the year
S. Vince1, I. Žura Žaja2, M. Samardžija1†, I. Majić Balić3, M. Vilić2, D. Ðuričić4, H. Valpotić5,
F. Marković6 and S. Milinković-Tur2
1
Clinic of Obstetrics and Reproduction, Veterinary Faculty University of Zagreb, Heinzelova 55, 10000 Zagreb, Croatia; 2Department of Physiology and Radiobiology,
Veterinary Faculty University of Zagreb, Heinzelova 55, 10000 Zagreb, Croatia; 3Center for Reproduction and Animal Breeding of Croatia, Bani 83, 10000 Zagreb,
Croatia; 4Veterinary Practice Ðurđevac, Malinov trg 7, 48350 Ðurđevac, Croatia; 5Department of Animal Nutrition and Dietetics, Veterinary Faculty University of
Zagreb, Heinzelova 55, 10000 Zagreb, Croatia; 6Belupo, Danica 5, 48000 Koprivnica, Croatia

(Received 1 June 2016; Accepted 21 June 2017; First published online 24 July 2017)

The aims of this study were to determine the presence and quantities of antioxidative status and oxidative stress (OS) variables in the
seminal plasma and spermatozoa of bulls of varying age during cold and warm periods of the year, and to establish the correlation of
these variables with semen quality parameters. The study was conducted on two groups each comprising nine Simmental bulls: one
group contained younger animals (aged 2 to 4 years) and the second older animals (aged 5 to 10 years). Semen samples were collected
using an artificial vagina for biochemical analysis. Seminal plasma and spermatozoa activities of total superoxide dismutase (TSOD),
manganese superoxide dismutase (MnSOD), copper–zinc superoxide dismutase (CuZnSOD), catalase (CAT), selenium-dependent
glutathione peroxidase, reduced glutathione and concentrations of total protein (TP), thiobarbituric acid reactive substances (TBARS)
and protein carbonyl content (PCC) were determined. Several antioxidants in seminal plasma were also determined: total glutathione
peroxidase (TGSH-Px), selenium-independent glutathione peroxidase (Non-SeGSH-Px), uric acid, albumins (ALB) and alkaline
phosphatase (ALP). Significantly higher spermatozoa motility was observed during the cold v. warm period, and a significantly higher
volume and total number of spermatozoa per ejaculate was observed in older than in younger bulls. Significantly higher values of ALP,
TP and ALB were found in seminal plasma of older bulls than in younger bulls during the warm period. The seminal plasma of younger
bulls showed significantly higher activities of TSOD, MnSOD, CuZnSOD, TGSH-Px and Non-SeGSH-Px. Younger bulls had significantly
higher PCC concentration and activity of CAT in seminal plasma than older bulls during the cold period. Significantly higher
concentrations of PCC and TBARS, and activities of TSOD, MnSOD and CuZnSOD were established in spermatozoa of the younger than
in older bulls during the warm period. It could be concluded that antioxidative and OS variables differ significantly depending on bull
age and time of year. Younger bulls were more sensitive to elevated ambient temperatures during the warm period, when the higher
enzymatic antioxidative protection in seminal plasma and spermatozoa were insufficient to counteract the intensive oxidative processes
in spermatozoa, which eventually resulted in decreased spermatozoa motility. The estimation of antioxidative and OS variables in
seminal plasma and spermatozoa may have practical value for the assessment of bull semen quality.

Keywords: semen, antioxidative/oxidative status, age, period of the year, breeding bulls

Implications service of bull semen. The estimation of the antioxidative and

OS variables in seminal plasma and spermatozoa may have
Influence of age of bulls and periods of the year on seminal
practical value for the assessment of semen quality, and thus
plasma and spermatozoa antioxidative status and oxidative
may serve to improve reproductive efficiency of breeding bulls.
stress (OS) variables were studied in order to establish potential
differences in these variables as well as their correlations with
semen quality parameters. Such analysed data would serve to Introduction
obtain insight into significance of these factors upon evaluating
Breeding animals are exposed to a large number of biological
and environmental factors, such as variations in feed and
†
E-mail: smarko@vef.hr husbandry practices, climatic variables, transportation,

559
Vince, Žura Žaja, Samardžija, Majić Balić, Vilić, Ðuričić, Valpotić, Marković and Milinković-Tur
regrouping, therapeutic and prophylactic activities, various
stressors, etc. (Brito et al., 2002; Majić-Balić et al., 2012). The
ability of breeding bulls to cope with these factors is important
to maintain health and reproductive efficiency (Rahal et al.,
2014). Any disturbance in homoeostasis leads to an increased
production of reactive oxygen species (ROS), much higher than
the detoxifying capability of local tissues or the organism,
resulting in OS conditions (O’Flaherty, 2014). Strong and sus-
tained exposure to OS may result in a higher negative energy
balance, reduction in adaptation mechanisms, increased
susceptibility to infection, decline in reproductive efficiency
and ultimately to great economic loss. The overall physiolo-
gical impact of these factors and the adaptation ability of the
organism may depend on animal species, breed, age, gender,
etc. (Rahal et al., 2014; Žura Žaja et al., 2016a).
Historically, emphasis has been focussed on assessing bull
fertility by evaluating sperm characteristics, whereas little
attention has been paid to physiological function and/or
fertility markers, that is, antioxidants/proteins in the blood,
seminal plasma and spermatozoa of breeding animals
(Kumar et al., 2015). Spermatozoa concentration, motility
and morphology are the most common semen metrics used
at breeding stations worldwide. However, these semen
parameters have limited value for predicting bull fertility
(Samardžija et al., 2006; Dogan et al., 2013).
The ejaculated spermatozoon, as an aerobic cell, must be
resistant to toxic levels of ROS generated by its own meta-
bolism but also by exogenous products (O’Flaherty, 2014).
Reactive oxygen species are recognized for playing a
dual role and have beneficial or detrimental effects upon
spermatozoa functions, depending on their nature and
concentration (Kohen and Nyska, 2002). Mammalian
spermatozoa, in their cell membranes phospholipids, are
rich in polyunsaturated fatty acids and have a relatively
low antioxidant capacity, making them highly susceptible
to injuries caused by high ROS concentrations (Agarwal
et al., 2006; Žura Žaja et al., 2016d). The excessive ROS
concentration generates substantial damage to all compo-
nents of spermatozoa and thus, significant levels of lipid
peroxidation, protein and DNA oxidation may be observed
(Brouwers and Gadella, 2003; Bansal and Bilaspuri, 2011)
that are often associated with altered membrane function,
together with impaired metabolism, morphology, motility
and male infertility (O’Flaherty, 2014). The spermatozoa
oxidative status is maintained by intracellular and extra-
cellular mechanisms composed of non-enzymatic and enzy-
matic antioxidants to provide optimal amounts of ROS
that are necessary for the physiological functions of
spermatozoa (Bansal and Bilaspuri, 2011; Žura Žaja et al.,
2016b). Numerous antioxidants in seminal plasma and
spermatozoa are related to ROS detoxification, including
superoxide dismutase (SOD), glutathione peroxidase (GSH-Px),
catalase (CAT), reduced glutathione (GSH), albumins (ALB),
uric acid (UA), vitamins C and E, and other compounds
and minerals (Majić-Balić et al., 2012), which might
be useful in predicting spermatozoa fertilizing potential
(Kasimanickam et al., 2007).
560
Determination and quantification of OS by direct detection
of ROS and other free radicals is difficult as these molecules
are short-living and highly reactive in a non-specific manner.
Ongoing oxidative damage, that is, the intensity of OS, is
generally analysed by measurement of the content of sec-
ondary oxidation products, including derivatives of amino
acids/proteins (such as protein carbonyl content (PCC)),
nucleic acids and lipid peroxidation (such as thiobarbituric
acid reactive substances (TBARS)), or the evaluation of
enzymatic and non-enzymatic antioxidants (Kohen and
Nyska, 2002; Guéraud et al., 2010; Žura Žaja et al., 2016a).
Interaction and interrelation of enzymatic and non-
enzymatic antioxidants in seminal plasma and spermatozoa
and their relations with OS variables in bulls of varying age at
different times of year have not previously been investigated,
to the extent of our knowledge. Therefore, the aims of this
study were to determine the presence and quantities of
seminal plasma and spermatozoa antioxidative status and OS
variables, and to establish the correlation of these variables
with semen quality parameters of breeding bulls, in order to
obtain better insight into the significance of these factors and
variables on evaluating the service potential of bull semen.
Material and methods
The research protocol and animal management were in
accordance with Directive 2010/63/EU (European Union 2010)
on the protection of animals used for scientific purposes.
Feeding and handling of animals
This study was performed on 18 Simmental breeding bulls
aged from 2 to 10 years. Animals were divided into two
groups by age, each with nine bulls. The first group consisted
of younger bulls, with a mean age of 3.1 ± 0.58 years
(mean ± SD), and the second group consisted of older bulls
with a mean age of 7.4 ± 1.91 years (mean ± SD). Bulls were
kept in open concrete stables with solid floors (windows
were closed only in winter) in separate sections and tied with
leather collars. Sections were located in two facilities, one for
the group of older bulls with proven fertility results, and the
other for younger bulls in the testing process. Zoohygienic
measures were regularly conducted at the facilities, including
cleaning and washing of floors with the addition of dry straw
litter on the bay of each bull. Animals were fed with a
standard fodder mixture produced and quality controlled by
Department for Nutrition of Domestic Animals at the Faculty
of Agriculture of the University of Zagreb in Croatia. Feeding
was performed twice per day and water was provided ad
libitum (Table 1). Before each semen collection, bulls were
taken outside the barn and tied to a carousel to encourage
them to move for about 30 min.
Semen sampling and preparation for analysis
The study was conducted at the Centre for Reproduction in
Livestock Breeding, Križevci, Croatia, where bulls were
exploited for semen collection two times per week for

Table 1 The composition and quantity of the daily ration for bulls
Ingredients Amount CP (%)
Concentrate mix 8 kg 18.0
Haylage 6 kg 10.2
Hay 10 kg 5.8
Staw Ad libitum 2.3
Misol* Ad libitum
Water Ad libitum
*1% Mg, 39% Na, 3000 mg/kg Zn, 1200 mg/kg Mn, 24 mg/kg Co, 20 mg/kg Se, 20 to 30 mg/kg J (Žura Žaja et al., 2016a).
artificial insemination. For experimental purposes semen was
collected from each breeding bull at routine exploitation
during the cold (late November to late January) and warm
(mid-May to mid-July) period of the year two times within the
same interval in each period of the year. Average air tem-
peratures (mean ± SD) in the cold and warm periods were
−0.09 ± 2.38°C and 17.89 ± 3.94°C, respectively. Average
air humidity (mean ± SD) in the cold and warm periods were
84.14 ± 2.78% and 76.21 ± 9.04%, respectively (source:
Weather Service, Croatia). The weather station was located
at 350 m from the barn. Bull ejaculates were collected using
an artificial vagina between 0700 and 0900 h. Immediately
after semen collection, semen vials were placed in a water
bath at 37°C for the duration of the evaluation process.
Macroscopic evaluation included ejaculate volume mea-
surement by direct reading on a graduated semen vial, and
microscopic assessment was performed for estimation of
spermatozoa motility percentage. Concentration of sperma-
tozoa in the ejaculate (4.0 ml 0.9% NaCl and 0.4 ml native
semen) was determined using a Photometar SDM 5
(Minitube, Tiefenbach, Germany). Spermatozoa motility in
small drop of native semen was estimated at 200 × magni-
fication on two separate visual fields, using a binocular
phase-contrast microscope (Olympus BX, Japan) with incor-
porated spermotherm. All of the semen samples meeting the
criteria of the macroscopic and microscopic evaluations
(concentration 1 to 2 × 109 spermatozoa/ml and motility
>85%) were further processed. The total number of sper-
matozoa per ejaculate per bull was calculated by multiplying
spermatozoa concentration and ejaculate volume. Each
native ejaculate sample was centrifuged at 1500 × g for
20 min at 20°C. After centrifugation, separated seminal
plasma (1.5 ml) and spermatozoa were obtained, and the
seminal plasma was stored at −80°C until analysis. The
separated spermatozoa were washed three times in saline,
centrifuged at 500 × g for 5 min at room temperature (about
20°C), and stored at −80°C until analysis. Prior analysis the
equal amount of redistilled water was applied on precipitate
containing sperm cells to induce destruction of sperm cell
membranes in hypotonic conditions. Following centrifuga-
tion supernatants were used for biochemical analysis.
Analysis of seminal plasma and spermatozoa samples
The antioxidative status in the seminal plasma and sperma-
tozoa samples of breeding bulls was determined by measuring
Semen antioxidative/oxidative variables in bulls
Chemical composition
Crude fat (%) Crude fibre (%) Ash (%) Dry matter (%)
– 15.0 10.0 13.5
1.8 32.4 6.15 31.5
2.1 28.5 6.02 14.4
1.1 39.8 5.3 15.6
the activity of total superoxide dismutase (TSOD), manganese
superoxide dismutase (MnSOD), copper–zinc superoxide dis-
mutase (CuZnSOD), CAT, selenium-dependent glutathione
peroxidase (SeGSH-Px), GSH and total protein (TP). Several
antioxidants were determined in the seminal plasma of
breeding bulls as follows: total glutathione peroxidase
(TGSH-Px), selenium-independent glutathione peroxidase
(Non-SeGSH-Px), UA, ALB and alkaline phosphatase (ALP).
The intensity of OS in the seminal plasma and spermatozoa
samples of the bulls was established by lipid peroxidation, that
is, by determining the concentration of TBARS. The intensity of
oxidative damages of proteins was established by determining
the concentration of PCC. The resulting values of antioxidative
variables in spermatozoa were expressed in grams of proteins.
The obtained values for the variables ALP, TP, ALB, UA, GSH
were expressed per litre of seminal plasma, whereas for the
variables TSOD, MnSOD, CuZnSOD, CAT, TGSH-Px, SeGSH-Px,
Non-SeGSH-Px, PCC, TBARS as grams of proteins of seminal
plasma.
Determination of antioxidative status variables
The activities of TGSH-Px, SeGSH-Px, TSOD and MnSOD
in semen samples of bulls were determined spectro-
photometrically with the SABA 18 analyser (AMS, Rome, Italy)
using Randox commercial kits (Crumlin, UK). The activity
of GSH-Px was determined using Ransel commercial kits
(Randox, Crumlin, UK; catalogue no. RS505) at 340 nm,
whereas TSOD activity was determined by using Ransod kits
(Randox, Crumlin, UK; catalogue no. SD125) at 505 nm.
Hydrogen peroxide was used for the detection of TGSH-Px
activity, and cumene hydroperoxide was used for the detection
of SeGSH-Px activity. Activity of Non-SeGSH-Px was derived by
calculating the difference between TGSH-Px and SeGSH-Px
activities. Further, the activity of MnSOD was determined
similarly as for TSOD with exception that samples were pre-
viously incubated for 30 min. with 1 M KCN to suppress
CuZnSOD activity. Copper–zinc superoxide dismutase activity
was calculated as the difference of activities between TSOD
and MnSOD (Spitz and Oberley, 1989; Ibrahim et al., 2000).
Uric acid was also determined spectrophotometrically at
520 nm with the SABA 18 analyser using Randox commercial
reagents (catalogue no. UA 1613). In spermatozoa and semi-
nal plasma, the concentration of GSH was determined
according to the method by Beutler et al. (1963), protein
concentration by the method of Lowry et al. (1951) using
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Vince, Žura Žaja, Samardžija, Majić Balić, Vilić, Ðuričić, Valpotić, Marković and Milinković-Tur

bovine albumin as a standard, and CAT activity was measured
at by the method of Johansson and Borg (1988). Probe
absorbencies were measured spectrophotometrically by
Thermospectronic Helios delta (Unicam, Cambridge, UK)
apparatus (for GSH at 412 nm, for TP at 750 nm and for CAT at
240 nm). Alkaline phosphatase activity and ALB concentration
were determined spectrophometrically with the SABA 18
analyser using commercial reagents (catalogue no. TR-1104)
at 405 nm and (catalogue no. T-1000) at 630 nm, respectively,
produced by Herbos Dijagnostika (Sisak, Croatia).
Determination of oxidative stress variables
The concentration of TBARS in semen samples was deter-
mined according to the methods of Trotta et al. (1982) and
Placer et al. (1966), and PCC concentration was determined
by the method of Levine et al. (1990). Probe absorbance
were measured spectrophotometrically by Thermospectronic
Helios delta apparatus (for TBARS at 532 nm, for PCC at
370 nm). In the current study we have performed single
biochemical analysis of the samples using automatic bio-
chemistry analyser SABA 18 which is reliable and regularly
calibrated. However, in our research all data that extremely
differ from the most frequently obtained values for any par-
ticular analysis for individual bull/sample (only two samples
in this study, but not for all biochemical parameters tested)
were subjected to repetition of the analysis. Manually per-
formed methods (CAT, TBARS, PCC, GSH and TP) were made
in triplicates.
Statistical analysis
Statistical analysis were performed using SAS 9.4 software
(2002 to 2012 SAS Institute Inc., Cary, NC, USA). Each vari-
able was tested for normality of distribution and homo-
scedasticity of variance estimating λ with the BOXCOX model
using the TRANSREG procedure of SAS. The general linear
mixed model (PROC GLIMMIX) was used to analyse the
antioxidative status and OS variables in seminal plasma and
spermatozoa. The statistical model included the fixed effects
of age groups and time periods and their interactions. The bull
effects on repeated measures over time were also included in
the model by the RANDOM statement. The Tukey–Kramer
multiple comparison test was performed using the SLICE
option to compare each age group level within each period
and vice versa. The correlation coefficient was calculated
between variables using the CORR procedure of SAS. The
results in tables were presented as the least square mean and
pooled standard error. The level of statistical significance was
set at P < 0.05.
Results
Conventional semen analysis
The results on ejaculate volume, total number of spermato-
zoa per ejaculate, concentration and motility of spermatozoa
of younger and older bulls at different times of year are
shown in Table 2. Ejaculate volume and total number of
spermatozoa per ejaculate differed between younger and
older bulls during different periods. Namely, older bulls had a
higher ejaculate volume and total number of spermatozoa
per ejaculate than younger bulls during both cold and warm
periods (P < 0.05). Younger bulls had a greater concentration
of spermatozoa in ejaculate than older bulls, though these
differences were statistically significant only during the warm
period (P < 0.05). Moreover, statistical analysis indicated
that there were significant differences in spermatozoa moti-
lity between periods, and thus greater spermatozoa motility
was found during the cold v. warm period in older and
younger bulls (P < 0.05).
Seminal plasma non-enzymatic antioxidant variables and
alkaline phosphatase activity
The mean values of seminal plasma non-enzymatic anti-
oxidant concentrations and ALP activity in bulls of different

Table 2 The ejaculate volume, total number of spermatozoa per ejaculate (TNSE), concentration and motility of spermatozoa of younger and older
bulls in relation to different periods
Age group Period of the year Group × period

Variables YB OB SE Cold Warm SE Cold Warm SE

Number of ejaculates 36 36 36 36 VB 18 18
OB 18 18
Volume (ml) 4.23a 7.34b 0.60 5.41 5.52 0.69 YB 4.22A 4.23A 0.86
OB 7.17B 7.52B 0.85
Concentration (109/ml) 1.30 1.14 0.07 1.17 1.28 0.07 YB 1.20 1.40A 0.11
OB 1.13 1.16B 0.10
TNSE (109 spermatozoa) 5.41a 8.48b 0.92 6.36 7.38 0.92 YB 4.84A 6.01A 1.29
OB 8.09B 8.88B 1.26
Motility (%) 80.1 77.9 1.9 81.8a 75.9b 1.8 YB 82.5a 77.4b 2.5
OB 81.0a 74.2b 2.9
The results are expressed as a least squared mean and pooled standard error.
YB = younger bulls (aged 2 to 4 years); OB = older bulls (aged 5 to 10 years).
a,b
Values with different superscript differ significantly within rows at P < 0.05.
A,B
Values with different superscript differ significantly between rows at P < 0.05.

562
 Semen antioxidative/oxidative variables in bulls

ages during different period of the year are presented in
Table 3. The mean values of ALP and ALB were significant
higher in seminal plasma of older bulls v. younger bulls during
the warm period (P < 0.05). Moreover, the concentration of TP
in seminal plasma of bulls differed depending on age, that is,
there was a significantly higher concentration of TP in seminal
plasma of older than in younger bulls during the warm period
(P < 0.05). Concentrations of UA and GSH of seminal plasma
in bulls did not differ significantly depending on age or time of
year (P > 0.05).
Seminal plasma enzymatic antioxidant and oxidative
stress variables
The activities of enzymatic antioxidant and concentrations of
OS variables in the seminal plasma of bulls by age and time
of year are shown in Table 4. The mean values of TSOD and

Table 3 Concentration of non-enzymatic antioxidants and alkaline phosphatase (ALP) activity in the seminal plasma of younger and older bulls in
relation to different periods
Age group Period of the year Group × period

Variables YB OB SE Cold Warm SE Cold Warm SE

ALP (U/l) 47.2a 72.5b 6.4 61.5 55.6 6.6 YB 55.5 40.2A 8.7
OB 68.2 77.0B 9.0
TP (g/l) 55.9 63.1 3.2 60.8 58.0 3.2 YB 59.0 53.0A 4.6
OB 62.6 63.6B 4.6
ALB (g/l) 14.5a 17.0b 1.0 16.2 15.3 1.0 YB 15.5 13.6A 1.4
OB 17.0 17.0B 1.4
UA (μmol/l) 3.40 2.69 0.49 3.44 2.65 0.49 YB 3.92 2.91 0.70
OB 2.99 2.40 0.70
GSH (μmol/l) 0.41 0.38 0.02 0.40 0.39 0.02 YB 0.40 0.42 0.04
OB 0.40 0.36 0.03
The results are expressed as a least squared mean and pooled standard error.
YB = younger bulls (aged 2 to 4 years); OB = older bulls (aged 5 to 10 years); TP = total protein; ALB = albumins; UA = uric acid; GSH = glutathione.
a,b
Values with different superscript differ significantly within rows at P < 0.05.
A,B
Values with different superscript differ significantly between rows at P < 0.05.

Table 4 Activities of enzymatic antioxidants and concentrations of oxidative stress variables in the seminal plasma of younger and older bulls in
relation to different periods
Age group Period of the year Group × period

Variables YB OB SE Cold Warm SE Cold Warm SE

TSOD (U/g protein) 686.6a 570.6b 52.7 578.2 677.6 53.7 YB 582.1a 809.9Ab 73.0
OB 574.3 566.9B 75.7
MnSOD (U/g protein) 46.2 40.4 4.2 44.4 42.1 4.3 YB 43.1 49.3A 5.9
OB 45.8 35.0B 6.1
CuZnSOD (U/g protein) 640.5a 529.9b 50.3 533.7a 635.9b 51.35 YB 539.0a 761.1Ab 69.7
OB 528.5 531.3B 72.3
CAT (mU/g protein) 329.5 253.6 51.8 310.2 271.1 52.0 YB 411.4Aa 256.6b 77.3
OB 223.2B 286.0 68.6
TGSH-Px (U/g protein) 1501.5a 1342.7b 70.2 1381.1 1460.0 63.5 YB 1412.4a 1596.9Ab 89.4
OB 1350.5 1334.8B 93.1
SeGSH-Px (U/g protein) 416.5 340.2 57.9 398.6 353.2 56.0 YB 418.5 414.5 94.7
OB 380.4 307.7 68.7
Non-SeGSH-Px (U/g protein) 1070.2 972.0 63.3 953.1a 1091.4b 64.9 YB 978.8a 1170.2b 91.5
OB 928.2 1017.9 91.5
PCC (μmol/g protein) 393.9a 317.7b 25.7 482.2a 246.9b 24.3 YB 516.6Aa 287.8Ab 34.4
OB 448.9Ba 209.1Bb 33.9
TBARS (μmol/g protein) 0.52 0.55 0.04 0.55 0.52 0.04 YB 0.50 0.54 0.05
OB 0.61 0.50 0.05
The results are expressed as a least squared mean and pooled standard error.
YB = younger bulls (aged 2 to 4 years); OB = older bulls (aged 5 to 10 years); TSOD = total superoxide dismutase; MnSOD = manganese superoxide dismutase;
CuZnSOD = copper–zinc superoxide dismutase; CAT = catalase; TGSH-Px = total glutathione peroxidase; SeGSH-Px = selenium-dependent glutathione peroxidise;
Non-SeGSH-Px = selenium-independent glutathione peroxidise; PCC = protein carbonyl content; TBARS = thiobarbituric acid reactive substances.
a,b
Values with different superscript differ significantly within rows at P < 0.05.
A,B
Values with different superscript differ significantly between rows at P < 0.05.

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Table 5 Values of antioxidant and oxidative stress variables in the spermatozoa of younger and older bulls in relation to different periods
Age group Period of the year Group × period

Parameters YB OB SE Cold Warm SE Cold Warm SE

TSOD (U/g protein) 3094.1 3244.9 1136.0 2617.5 3835.9 951.8 YB 1834.3a 5219.1b 1486.0
OB 3734.9 2819.3 1142.9
MnSOD (U/g protein) 1055.9 960.1 356.9 855.9 1178.9 288.5 YB 724.6a 1498.4b 404.5
OB 1005.5 916.3 377.0
CuZnSOD (U/g protein) 2101.8 2290.3 914.6 1769.7 2696.9 897.0 YB 1100.9a 3725.8b 949.1
OB 2730.0 1909.0 888.3
SeGSH-Px (U/g protein) 868.2a 623.7b 73.5 1042.4a 519.4b 66.9 YB 1124.1a 670.5Ab 92.9
OB 966.7a 402.4Bb 90.8
GSH (μmol/g protein) 0.64a 0.97b 0.13 0.73 0.86 0.14 YB 0.53A 0.77 0.19
OB 0.99B 0.95 0.18
PCC (μmol/g protein) 1885.1a 356.2b 146.1 333.1a 1908.3b 146.2 YB 283.1a 3487.1Ab 209.5
OB 383.0 329.5B 203.7
TBARS (μmol/g protein) 0.68 0.60 0.25 0.43a 1.03b 0.21 YB 0.35a 1.85Ab 0.25
OB 0.55 0.66B 0.24
The results are expressed as a least squared mean and pooled standard error.
YB = younger bulls (aged 2 to 4 years); OB = older bulls (aged 5 to 10 years); TSOD = total superoxide dismutase; MnSOD = manganese superoxide dismutase;
CuZnSOD = copper–zinc superoxide dismutase; CAT = catalase; TGSH-Px = total glutathione peroxidase; SeGSH-Px = selenium-dependent glutathione peroxidise;
GSH = glutathione; Non-SeGSH-Px = selenium-independent glutathione peroxidise; PCC = protein carbonyl content; TBARS = thiobarbituric acid reactive substances.
a,b
Values with different superscript differ significantly within rows at P < 0.05.
A,B
Values with different superscript differ significantly between rows at P < 0.05.

TGSH-Px activities in seminal plasma of bulls differed sig-
nificantly depending on age. Namely, younger bulls had
higher activity of TSOD and TGSH-Px v. older bulls during the
warm period (P < 0.05). Total superoxide dismutase and
TGSH-Px activities in seminal plasma of younger bulls were
significantly higher during the warm v. cold period
(P < 0.05). The activities of MnSOD and CuZnSOD in seminal
plasma of younger bulls were significantly higher in com-
parison to that in older bulls during the warm period
(P < 0.05). The seminal plasma of younger bulls also
recorded higher CuZnSOD activity during the warm period
(P < 0.05). However, CAT activity was significantly higher in
younger than in older bulls during the cold period. Signi-
ficantly higher CAT activity in seminal plasma of younger
bulls was established during the cold period (P < 0.05).
Similar results were obtained for PCC concentration, that is,
significantly higher concentration was recorded in seminal
plasma of younger v. older bulls in both periods (P < 0.05).
Furthermore, younger and older bulls had significantly higher
PCC concentration during cold v. warm period (P < 0.05).
The mean value of on-SeGSH-Px activity was significantly
higher during the warm period, but only in seminal plasma of
younger bulls (P < 0.05). Seminal plasma SeGSH-Px and
TBARS values did not differ significantly by bull age or time
of year.
Spermatozoa antioxidant and oxidative stress variables
The results obtained for values of antioxidant and OS vari-
ables in the spermatozoa of bulls of different ages at differ-
ent times of year are shown in Table 5. Total superoxide
dismutase, MnSOD and CuZnSOD activities in spermatozoa
of younger bulls were significantly higher during the warm v.
cold period (P < 0.05). During the warm period, younger
bulls had significantly higher SeGSH-Px activity in sperma-
tozoa than older bulls. Significantly higher SeGSH-Px activity
in spermatozoa of younger and older bulls was determined
in the cold v. warm period (P < 0.05). Older bulls had a
significantly higher GSH concentration in spermatozoa
than younger bulls during the cold period (P < 0.05). The
PCC concentration in spermatozoa of younger bulls was
significantly higher during the warm v. cold period
(P < 0.05). A significantly higher PCC concentration was
determined in spermatozoa of younger v. older bulls during
the warm period (P < 0.05). During that period, younger
bulls had a significantly higher concentration of TBARS in
comparison to older bulls (P < 0.05), whereas CAT activity
was not measurable in spermatozoa.
Correlations among tested semen variables
Correlations were found among the tested semen variables,
and the correlations of the tested seminal plasma enzymatic
and non-enzymatic antioxidant variables of bulls are shown
in Table 6. The correlations of investigated bull spermatozoa
antioxidant and OS variables in relation to conventional
semen analysis are shown in Table 7, and the correlations of
tested bull spermatozoa antioxidant variables in relation to
seminal plasma antioxidant variables are shown in Table 8.
Discussion
The influence of genetics, physiological status, age, man-
agement and climatic conditions on seminal plasma and
spermatozoa antioxidative status and OS variables, and
whether potential changes in these variables are correlated
with the semen quality parameters of breeding bulls have not
yet been fully elucidated.

564
 Semen antioxidative/oxidative variables in bulls

Table 6 Coefficient of correlations among the investigated seminal plasma enzymatic and non-enzymatic antioxidant variables of bulls
TP ALB UA GSH TSOD MnSOD CuZnSOD CAT Non-SeGSH-Px

TP 0.97 Ns −0.80 −0.63 −0.46 −0.63 Ns −0.67

<0.0001 <0.0001 <0.0001 <0.001 <0.0001 <0.0001
ALB 0.97 Ns −0.79 −0.58 −0.40 −0.58 Ns −0.69
<0.0001 <0.0001 <0.0001 <0.01 <0.0001 <0.0001
UA Ns Ns Ns Ns Ns Ns 0.58 Ns
<0.0001
GSH −0.80 −0.79 Ns 0.66 Ns 0.66 Ns 0.66
<0.0001 <0.0001 <0.0001 <0.0001 <0.0001
TSOD −0.63 −0.58 Ns 0.66 0.71 0.99 Ns 0.46
<0.0001 <0.0001 <0.0001 <0.0001 <0.0001 <0.0001
MnSOD −0.46 −0.40 Ns Ns 0.71 0.67 Ns Ns
<0.001 <0.01 <0.0001 <0.0001
CuZnSOD −0.63 −0.58 Ns 0.66 0.99 0.67 Ns 0.47
<0.0001 <0.0001 <0.0001 <0.0001 <0.0001 <0.0001
CAT Ns Ns 0.58 Ns Ns Ns Ns Ns
<0.0001
Non-SeGSH-Px −0.67 −0.69 Ns 0.66 0.46 Ns 0.47 Ns
<0.0001 <0.0001 <0.0001 <0.0001 <0.0001
TP = total protein; ALB = albumins; UA = uric acid; GSH = glutathione; TSOD = total superoxide dismutase; MnSOD = manganese superoxide dismutase; CuZnSOD =
copper–zinc superoxide dismutase; CAT = catalase; Non-SeGSH-Px = selenium-independent glutathione peroxidise.
In the table only data with correlation coefficient higher than ±0.400 regardless statistical significance are shown.

Table 7 Coefficient of correlations among the investigated bull spermatozoa antioxidant variables, oxidative stress variables in relation to con-
ventional semen analyses
Concentration Motility GSH PCC TSOD MnSOD CuZnSOD SeGSH-Px TBARS

Concentration Ns Ns Ns 0.43 Ns 0.42 Ns Ns

<0.001 <0.01
Motility Ns −0.55 −0.50 −0.49 −0.48 −0.46 Ns −0.55
<0.0001 <0.0001 <0.0001 <0.0001 <0.001 <0.0001
GSH Ns −0.55 0.53 0.82 0.85 0.74 Ns 0.41
<0.0001 <0.0001 <0.0001 <0.0001 <0.0001 <0.01
PCC Ns −0.50 0.53 0.52 0.46 0.51 Ns 0.83
<0.0001 <0.0001 <0.0001 <0.001 <0.0001 <0.0001
TSOD 0.43 −0.49 0.82 0.52 0.85 0.97 0.41 Ns
<0.001 <0.0001 <0.0001 <0.0001 <0.0001 <0.0001 <0.01
MnSOD Ns −0.48 0.85 0.46 0.85 0.73 Ns Ns
<0.0001 <0.0001 <0.001 <0.0001 <0.0001
CuZnSOD 0.42 −0.46 0.74 0.51 0.97 0.73 0.41 Ns
<0.01 <0.001 <0.0001 <0.0001 <0.0001 <0.0001 <0.01
SeGSH-Px Ns Ns Ns Ns 0.41 Ns 0.41 Ns
<0.01 <0.01
TBARS Ns −0.55 0.41 0.83 Ns Ns Ns Ns
<0.0001 <0.01 <0.0001
GSH = glutathione; PCC = protein carbonyl content; TSOD = total superoxide dismutase; MnSOD = manganese superoxide dismutase; CuZnSOD = copper–zinc super-
oxide dismutase; SeGSH-Px = selenium-dependent glutathione peroxidise; TBARS = thiobarbituric acid reactive substances.
In the table only data with correlation coefficient higher than ±0.400 regardless statistical significance are shown.

The current study indicated that volume and total number
of spermatozoa per ejaculate were significantly affected by
age, that is, these semen characteristics tended to increase
with bull age, which has been previously reported (Mathevon
et al., 1998; Brito et al., 2002; Fuerst-Waltl et al., 2006;
Majić-Balić et al., 2012). This observation could be explained
by well-known fact that the main factor determining the
volume of ejaculate and total number of spermatozoa
produced is the size of the testes, which increases for at least
5 years after puberty (Mathevon et al., 1998; Majić-Balić
et al., 2012). The results obtained for spermatozoa con-
centration in the present study were partially consistent with
those of Brito et al. (2002) and Majić-Balić et al. (2012), who
reported that bull age had no effect on spermatozoa con-
centrations. In contrast to the present findings, other authors
(Mathevon et al., 1998; Fuerst-Waltl et al., 2006) observed

565
Vince, Žura Žaja, Samardžija, Majić Balić, Vilić, Ðuričić, Valpotić, Marković and Milinković-Tur
Table 8 Coefficient of correlations among the investigated bull sper-
matozoa antioxidants variables in relation to seminal plasma anti-
oxidant variables.
Spermatozoa
Seminal plasma TSOD MnSOD
TSOD 0.53 0.44
<0.0001 <0.001
MnSOD 0.43 0.41
<0.001 <0.01
CuZnSOD 0.53 0.44
<0.0001 <0.001
TSOD = total superoxide dismutase; MnSOD = manganese superoxide dis-
mutase; CuZnSOD = copper–zinc superoxide dismutase.
In the table only data with correlation coefficient higher than ±0.400 regardless
statistical significance are shown.
an increase in spermatozoa concentration with age. A sig-
nificantly higher spermatozoa concentration and significantly
smaller ejaculate volume were observed in the current study
in younger v. older bulls during the warm period, suggesting
that the smaller ejaculate volume could be compensated for
by a greater spermatozoa concentration in younger bulls
during the warm period. A testicular temperature below body
temperature is essential for the production of fertile sper-
matozoa. Higher ambient temperature may result in an
increased testicular temperature, thus decreasing semen
quality (Fuerst-Waltl et al., 2006). In the present study, of the
selected semen characteristics tested, spermatozoa motility
was most affected by ambient temperature and humidity.
Significantly higher spermatozoa motility was observed
during the cold v. warm period, which corroborates the
findings by Fuerst-Waltl et al. (2006) and similar results to
those presented here for spermatozoa motility were reported
by Majić-Balić et al. (2012) for Simmental bulls. However,
contrary to the present findings were those of Brito et al.
(2002) who reported that ambient temperature and humidity
did not significantly affect the semen production. Such
disagreement of our results and those of Brito et al. (2002)
who kept bulls in tropical conditions could be ascribed to
great differences in climatic conditions. Although above-
mentioned authors did not establish the influence of ambient
temperature and humidity on semen production in two
bovine species (Bos indicus and Bos taurus), more recent
data reported that season affected volume of semen and
total sperm output in four different breeds of bulls (Holstein,
Brown Swiss, Limousin and Charolais) of B. taurus in same
climatic zone as in our study (Snoj et al., 2013).
The seminal plasma, derived from testes, epididymis and
accessory sex glands, contains proteins that contribute to
spermatozoa motility, spermatozoa membrane protection,
protection from OS, etc. (Kumar et al., 2015; Žura Žaja et al.,
2016d). Non-enzymatic antioxidants, present predominately
in extracellular fluid, are known to eliminate free radicals
generated by OS, and some protect the membrane integrity
of spermatozoa, such as from heat shock, by acting inter-
actively and synergistically (Bansal and Bilaspuri, 2011).
566
In the current study we have tested OS parameters in bulls
not younger than 2 years which is essential due to the fact
that OS status of bulls younger than 2 years could be sig-
nificantly different, especially around puberty. In the present
study, significantly higher values of ALP, TP and ALB were
CuZnSOD found in seminal plasma of the older bulls than in younger
bulls during the warm period. Simultaneously, in the seminal
0.51 plasma of the younger bulls, significantly higher activities of
<0.0001 TSOD, MnSOD, CuZnSOD, TGSH-Px and Non-SeGSH-Px were
0.42
determined v. those in older bulls during the same period.
<0.01
The formation of the enzymatic antioxidant system, whose
0.51
<0.0001 components vary among species, provides the protection of
spermatozoa against OS with their interplay. Superoxide
dismutase and its two isozymes (CuZnSOD, MnSOD) spon-
taneously dismutase both extracellular and intracellular
superoxide anions to O− 2 and H2O2. In order to act against
H2O2, it essentially conjugated either with CAT or GSH-Px
family enzymes (SeGSH-Px, Non-SeGSH-Px) using GSH
(O’Flaherty, 2014). The results of this study could indicated
that non-enzymatic antioxidants in seminal plasma of older
as well as enzymatic antioxidants in younger bulls have
important and significant functions in protecting against OS
during the warm period, and that the mechanisms of this
protection differed for different age groups of bulls. Positive
correlations within non-enzymatic and enzymatic anti-
oxidative variables were established. However, the negative
correlations between non-enzymatic and enzymatic anti-
oxidative variables in seminal plasma were recorded and
confirmed by previously presented results (Table 6). Signi-
ficantly higher activities of CuZnSOD and Non-SeGSH-Px
observed in seminal plasma of bulls during the warm v. cold
period were not accompanied by increased values of OS
variables, which could indicate an adaptive antioxidative
protective response in seminal plasma of bulls in the warm
period. Significantly higher PCC concentrations found in
seminal plasma of bulls during cold v. warm period, sug-
gested that the intensity of oxidative processes in seminal
plasma were significantly higher during the cold period, as
recently reported by Žura Žaja et al. (2016a) in the serum of
breeding bulls. This finding is consistent with earlier reported
data by Kumar et al. (2015), who established that the
majority of seminal plasma constituents originated from
blood, whereas some were seminal plasma-specific con-
stituents. In the current study, younger bulls had significantly
higher PCC concentration and CAT activity in seminal plasma
than older bulls during the cold period, indicating that the
seminal plasma of younger bulls showed greater sensitivity
to OS during this period. CAT activity is increased in
situations when H2O2 concentration was greatly increased,
i.e. in the OS status (O’Flaherty, 2014; Žura Žaja et al.,
2016a) when CAT converts H2O2 into oxygen and water,
which was also established in this study. Immunoblotting
studies demonstrated that bull spermatozoa do not contain
CAT which was also observed in the current study. It is
assumed that CAT is not a significant player in the anti-
oxidant protection of bull spermatozoa against high levels of
H2O2 (O’Flaherty, 2014).

During the warm period, elevated ambient temperatures
can increase testicular temperature, which results in
increased metabolism and oxygen consumption. Testicular
blood flow is limited and this increased O2 consumption
cannot be supplied, resulting in hypoxia, overproduction of
ROS, lipid peroxidation, OS and decrease in spermatozoa
motility (Brito et al., 2004; Majić-Balić et al., 2012).
According to these results, significantly higher concentra-
tions of PCC and TBARS were established in the spermatozoa
of younger v. older bulls during the warm period, indicating
more intensive OS in spermatozoa of younger bulls and their
higher sensitivity to OS. Majić-Balić et al. (2012) obtained
similar results, during the summer season. In the current
study, a positive correlation was obtained between sperma-
tozoa concentrations of PCC and TBARS (Table 7). Simulta-
neously, the higher TSOD, MnSOD and CuZnSOD activities in
the spermatozoa of younger bulls during the warm period
were insufficient to counteract the intensive oxidative pro-
cesses in spermatozoa, which eventually resulted in
decreased spermatozoa motility during the warm period
(Table 2). These results were additionally supported by the
negative correlations obtained between spermatozoa moti-
lity and activities of TSOD, MnSOD and CuZnSOD and con-
centrations of PCC and TBARS in spermatozoa (Table 7).
Similar results were obtained by Nichi et al. (2006), who
found positive correlations among bull spermatozoa primary
defects with TBARS concentration and SOD activity during
the summer. In this study, a significantly higher activity of
SeGSH-Px in spermatozoa (Table 5) and spermatozoa moti-
lity (Table 2) were obtained during the cold v. warm period.
This is in agreement with the statement of Stradaioli et al.
(2009), who suggested that SeGSH-Px plays an important
role in protecting mammalian spermatozoa from the loss of
motility caused by lipid peroxidation processes.
Based on these results, it could be concluded that anti-
oxidative and OS variables differ significantly depending on
bull age and time of year. Namely, younger bulls were more
sensitive to elevated ambient temperatures during the warm
period, when the higher enzymatic antioxidative protection in
seminal plasma and spermatozoa were insufficient to coun-
teract the intensive oxidative processes in spermatozoa. Dis-
ruption of the balance between prooxidants and antioxidants,
that is, development of OS in spermatozoa of younger bulls,
may eventually result in decreased spermatozoa motility, with
consequential semen quality deterioration during the warm
period. Furthermore, in the seminal plasma of younger bulls,
significantly intensive oxidative processes of proteins have also
been detected as compared with older bulls, which may indi-
cate that younger bulls show greater sensitivity to changing
climate conditions during the cold period, though these oxi-
dative processes were not reflected on spermatozoa motility
and semen concentration. In addition, this may indicate that
both oxidative damages caused on lipids and proteins may
have serious consequences on spermatozoa function, particu-
larly their motility. Estimation of the antioxidative and OS
variables in seminal plasma and spermatozoa may have prac-
tical value for the assessment of bull semen quality. Our results
Semen antioxidative/oxidative variables in bulls
show that it may be beneficial to avoid collecting bull semen at
the warmest period of the year. However, studies on largest
bull populations and on different breeds will be necessary to
strengthen this conclusion.
Acknowledgements
This research was supported by a grant from the Ministry of
Science, Education and Sports, Croatia (Number 053-0531854-
1866). The current study was performed according to animal
welfare law (NN 135/06) and was approved by the Ministry
of Agriculture, Fisheries and Rural Development, Croatia
(UP/I 322-01/07-01/59, 525-6-07-2 Lj.Z.).
References
Agarwal A, Gupta S and Sikka S 2006. The role of free radicals and antioxidants
in reproduction. Current Opinion in Obstetrics and Gynecology 18, 325–332.
Bansal AK and Bilaspuri GS 2011. Impacts of oxidative stress and antioxidants
on semen functions. Veterinary Medicine International 2011, 686137. 7 pp.
Beutler E, Duron O and Mikus KB 1963. Improved method for the determination
of blood glutathione. Journal of Laboratory and Clinical Medicine 61, 882–886.
Brito LFC, Silva A, Rodrigues EDF, Vieira LH, Vderagon F and Kastelic LAG 2002.
Effect of environmental factors, age and genotype on sperm production and
semen quality in Bos indicus and Bos taurus AI bulls Brazil. Animal Reproduction
Science 70, 181–190.
Brito LFC, Silva AEDF, Barbosa RT and Kastelic JP 2004. Testicular thermo-
regulation in Bos indicus, crossbred and Bos taurus bulls: relationship with
scrotal, testicular vascular cone and testicular morphology, and effects on semen
quality and sperm production. Theriogenology 61, 511–528.
Brouwers JF and Gadella BM 2003. In situ detection and localization of lipid
peroxidation in individual bovine sperm cells. Free Radical Biology and Medicine
35, 1382–1391.
Dogan S, Mason MC, Govindaraju A, Belser L, Kaya A, Stokes J, Rowe D and
Memili E 2013. Interrelationships between apoptosis and fertility in bull sperm.
Journal of Reproduction and Development 59, 18–26.
Fuerst-Waltl B, Schwarzenbacher H, Perner C and Sölkner J 2006. Effect of age
and environmental factors on semen production and semen quality of Austrian
Simmental bulls. Animal Reproduction Science 95, 27–37.
Guéraud F, Atalay M, Bresgen N, Cipak A, Eckl PM, Huc L, Jouanin I, Siems W
and Uchida K 2010. Chemistry and biochemistry of lipid peroxidation products.
Free Radical Research 44, 1098–1124.
Ibrahim W, Lee US, Yen HC, St Clair DK and Chow CK 2000. Antioxidant and
oxidative status in tissues of manganese superoxide dismutase transgenic mice.
Free Radical Biology and Medicine 28, 397–402.
Johansson LH and Borg LAH 1988. A spectrophotometric method for determi-
nation of catalase activity in small tissue samples. Analytical Biochemistry 74,
331–336.
Kasimanickam R, Kasimanickam V, Thatcher CD, Nebel RL and Cassell BG 2007.
Relationships among lipid peroxidation, glutathione peroxidase, superoxide
dismutase, sperm parameters, and competitive index in dairy bulls. Therio-
genology 67, 1004–1012.
Kohen R and Nyska A 2002. Oxidation of biological systems: oxidative stress
phenomena, antioxidants, redox reactions, and methods for their quantification.
Toxicologic Pathology 30, 620–650.
Kumar A, Kroetsch T, Blondin P and Anzar M 2015. Fertility-associated meta-
bolites in bull seminal plasma and blood serum: 1H nuclear magnetic resonance
analysis. Molecular Reproduction and Development 82, 123–131.
Levine RL, Garland D, Oliver CN, Amici A, Climent I, Lenz AG, Ahn BW, Shaltiel S
and Stadtman ER 1990. Determination of carbonyl content in oxidatively modi-
fied proteins. Methods in Enzymology 186, 464–478.
Lowry OH, Rosebrough NJ, Farr AL and Rendal RJ 1951. Protein measurement
with the Folin phenol reagent. Journal of Biological Chemistry 193, 265–267.
Majić-Balić I, Milinković-Tur S, Samardžija M and Vince S 2012. Effect of age and
environmental factors on semen quality, glutathione peroxidase activity and
oxidative parameters in Simmental bulls. Theriogenology 78, 423–431.
567
Vince, Žura Žaja, Samardžija, Majić Balić, Vilić, Ðuričić, Valpotić, Marković and Milinković-Tur
Mathevon M, Buhr MM and Dekkers JCM 1998. Environmental, management
and genetic factors affecting semen production in Holstein bulls. Journal of Dairy
Science 81, 3321–3330.
Nichi M, Bols PE, Züge RM, Barnabe VH, Goovaerts IG, Barnabe RC and
Cortada CN 2006. Seasonal variation in semen quality in Bos indicus and Bos
taurus bulls raised under tropical conditions. Theriogenology 66, 822–828.
O’Flaherty C 2014. The enzymatic antioxidant system of human spermatozoa.
Advances in Andrology 2014, 626374, 15 pp.
Placer ZA, Cushman LL and Connor Johnson B 1966. Estimation of product of
lipid peroxidation (malonyl dialdehyde) in biochemical systems. Analytical
Biochemistry 16, 359–364.
Rahal A, Kumar A, Singh V, Yadav B, Tiwari R, Chakraborty S and Dhama K 2014.
Oxidative stress, prooxidants, and antioxidants: the interplay. BioMed Research
International, pp. 19. http://dx.doi.org/10.1155/2014/761264
Samardzija M, Karadjole M, Getz I, Makek Z, Cergolj M and Dobranic T 2006.
Effects of bovine spermatozoa preparation on embryonic development in vitro.
Reproductive Biology and Endocrinology 4, 58.
Snoj T, Kobal S and Majdic G 2013. Effects of season, age and breed on semen
characteristics in different Bos taurus breeds in a 31-year retrospective study.
Theriogenology 79, 847–852.
Spitz DR and Oberley LW 1989. An assay for superoxide dismutase activity in
mammalian tissue homogenates. Analytical Biochemistry 179, 8–18.
568
Stradaioli G, Sylla L, Monaci M and Maiorino M 2009. Phospholipid
hydroperoxide glutathione peroxidase in bull spermatozoa provides a
unique marker in the quest for semen quality analysis. Theriogenology 72,
91–98.
Trotta RJ, Sullivan SG and Stern A 1982. Lipid peroxidation and hemoglobin
degeneration in red blood cells exposed to T-butyl hydroperoxide. Biochemical
Journal 204, 405–415.
Žura Žaja I, Samardžija M, Vince S, Majić-Balić I, Ðuričić D and Milinković-Tur S
2016a. The influence of different periods of the year and age on the parameters of
antioxidative status and oxidative stress in the blood serum of breeding bulls.
Reproductive Biology 16, 157–164.
Žura Žaja I, Samardžija M, Vince S, Majić-Balić I, Ðuričić D and Milinković-Tur S
2016b. Antioxidant system parameters in boar spermatozoa of different
morphology and motility. Veterinarski arhiv 86, 655–666.
Žura Žaja I, Samardžija M, Vince S, Majić-Balić I, Vilić M, Ðuričić D and Mili-
nković-Tur S 2016c. Influence of boar breeds or hybrid genetic composition on
semen quality and seminal plasma biochemical variables. Animal Reproduction
Science 164, 169–176.
Žura Žaja I, Samardžija M, Vince S, Vilić M, Majić-Balić I, Ðuričić D and Mili-
nković-Tur S 2016d. Differences in seminal plasma and spermatozoa
antioxidative systems and seminal plasma lipid and protein levels among
boar breeds and hybrid genetic traits. Animal Reproduction Science 170,
75–82.