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An attempt at alleviating heat stress infertility in male rabbits with some antioxidants
El-Tohamy M.M., Kotp,M.S.Z., El-Nattat,W.S. Amira, H.M.&Soliman,S.I. Department of Animal Reproduction,National Research Centre,Cairo Faculty of Veterinary Medicine,Beni Suef University ABSTRACT The present experiment was conducted to study the effect of summer heat stress and the selective antioxidants on spermogram, serum and seminal plasma biochemical and endocrinal parameters, and oxidative/antioxidative status of NZW rabbits buck.
Forty-eight sexually mature NZW male rabbits were randomly divided into six equal groups. The study was performed in two experimental periods (winter and summer), each period lasting 12 weeks. In winter, one group was kept as a winter control group and receives basal diet only. Other five groups reared in summer and serve as heat-stressed groups. Heat-stressed five groups were fed on basal diet and the first group kept as control positive and given distal water. The second group was given ascorbic acid 40 mg/kg BW/day. The third group was given zinc methionine 10 mg/kg BW/day. The forth group was given co enzyme Q10 10 mg/kg bw/day. Finally, the fifth group was given l-carnitine 40 mg/kg/day.
The climatic data have continuously recorded among the experimental period
and the weekly averages of temperature–humidity index (THI) was calculated. Semen samples have collected weekly. Three ejaculates were collected for
each buck. One served as row semen sample for spermogram. The other two consecutive ejaculates were pooled for seminal plasma separation, which stored at -80 °C ultra freezer, until further analysis.
The season's averages of temperature–humidity indices (THI) were
18.52±0.22 in winter and 34.38±0.46 in summer, indicating absence of heat stress in winter and exposure to very severe heat stress in summer.
Summer heat stress reversely affected both qualitative and quantitative traits
of spermogram but bucks remain within fertile limit. Of antioxidants used in present study, zinc and l-carnitine were found to be the most beneficial antioxidants in the relief of spermogram heat stress-induced effects.
In contrast to serum testosterone and cortisol, which showed no changes in
summer, seminal plasma testosterone and cortisol showed significant increase in summer group compared to winter group. Only ascorbic acid restored seminal plasma cortisol to the winter level.
In the present study, serum and seminal plasma oxidative/antioxidant status seem to
take an identical (same, equivalent) pattern in response to heat stress. TBARS showed
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a significant increase while TAC and catalase showed significant decreases as
affected by summer heat stress. Concerning to antioxidants role, contrary to our
expectation, selected antioxidants did not restore these parameters to their winter
levels.
INTRODUCTION
I. Heat Stress
The definition of 'stress' established by Dobson et al. (2001), identifies those
animals that are exposed to changes in their environment that prevent them from
expressing full genetic potential, for example within the current context, maximizing
reproductive efficiency, consequently, stress is often blamed for suboptimal
reproductive efficiency.
Fuquay (1981) and Morrison (1983) documented that environments of high
temperatures and humidity were detrimental to the productivity and reproductivity of
commercial animal. Farm animals have known zones of thermal comfort (ZTC) that
are primarily dependent on the species, the physiological status of the animals, the
relative humidity, velocity of ambient air and the degree of solar radiation (NRC,
1981). Nearly every life form affected in some way by heat stress, and rabbits are no
exception, contrariwise, it is more sensitive. It is not high temperatures alone that
causes stress to the rabbits; but it is the combination of temperature and humidity,
among others. When some crucial limit has been reached, all bodily functions other
than those critical for survival, could shuts down.
Regardless of the precise mechanism (In conclusion), the most prominent
characteristic of summer HS infertility is its multifactorial nature. Although the
impact of the various direct and indirect effects of HS on reproductive processes has
never quantified, it believed that the direct effect of hyperthermia in impairing cellular
functions is the predominant one (Wolfenson, et al., 2000).
Oxidative stress (OS), as one of HS-induced response, has long believed to influence
male reproductive function. Although OS was suggested as an important factor in
disruption of sperm function over 50 years ago, the importance of OS has gained recently
a wider understanding. Reactive oxygen species (ROS) are normal physiological event
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in various organs including the testis. Paradoxically, the production of ROS is both
essential and detrimental to life; hence, numerous studies indicate that ROS play an
important role in normal sperm function and that an imbalance in ROS production (over-
production) and/or degradation (under-scavenging) by antioxidants may have serious
adverse effects on sperm (Akiyama, 1999). These findings explain the importance of
a balance between ROS scavenging and small, physiologic levels of ROS that are
necessary for normal sperm function.
In biological systems, a diversity of antioxidant defense systems operates to
control levels of ROS. Some antioxidants synthesized within the cells themselves
(endogenous) and others need to be provided in the diet (exogenous). These ROS
scavengers have an important protective action on the membrane integrity and lipid
stability in both seminal plasma and spermatozoa.
Therefore, to improve our comprehension of the relationship between antioxidant
status and the response to HS-induced OS, the present study aimed to:
1- Evaluate semen characteristics during summer.
2- Monitor the detrimental effect of heat stress on (male reproductive efficiency
the possible role for antioxidants supplementation in elimination or
minimization (alleviation) of the detrimental effects (consequences) of heat
stress on rabbit bucks fertility.
3- Assess some hormonal and biochemical changes in serum and seminal plasma
during heat stress and after the administration of antioxidants.
MATERIAL AND METHODS
Animals and Husbandry
Forty-eight sexually mature White New Zealand (WNZ) male rabbits, which
proven fertile, aged 26-30 (28±2) weeks, were used in this experiment. Bucks were
individually housed in metal wire mesh cages provided with separate facilities for
feeding and watering.
General Layout of Experiment
The experiment was carried out at the experimental rabbitry of lab animal
house of National Research Center, Dokki, Giza-Egypt. The first experimental period
started at 23rd December and terminated at 22nd March for summer group. The second
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experimental period started at 23rd June and terminated at 22nd September for winter
groups.
Feeding System
All rabbits under experiment have offered a commercial ration pellets.
The chemical analysis of the pellets according to A.O.A.C. (1980) showed
that it contained 17.5% crude protein, 14.0% crude fiber, 2.7% crude fat and 2200
kcal/kg diet. Bucks fed an amount of pellet ration (60gm/kg BW/day) that provide
normal growth and maintain adult body weight. Fresh tap water has supplied ad
libitum.
Climate Data
The climatic data have continuously recorded among the experimental period,
using a thermometer and hygrometer (hydro thermograph); the weekly averages of
ambient temperature and relative humidity values at midday inside the rabbit building
were estimated. The temperature–humidity index (THI) computed using the formula
established (cited) by Marai et al., (2001) for rabbits as following:
THI = db °C – {(0.31 – 0.31 RH) (db °C – 14.4)}
Where db °C = dry bulb temperature in degrees Celsius and RH = relative humidity expressed in percentage. Experimental Design
The experiment has conducted in two periods, the first period in winter and the
second in summer, each period lasting 12 weeks. The rabbits were randomly divided
into six equal groups (n=8) as showed in table (1).
Table (1): Experimental design
Periods Groups Supplementations
1st period
(winter)
I. Winter Control Basal diet
II. Summer Control (Heat
Stress) Basal diet
III. Ascorbic Acid Ascorbic acid 40 mg/kg bw/day
2nd period
(summer)
Antioxidan
t
Suppleme
IV. Zinc Zinc methionine 10 mg/kg
bw/day
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V. Coenzyme
Q10
Coenzyme Q10 10 mg/kg
bw/day
nt-ations
VI. L-carnitine L-carnitine 40 mg/kg bw/day
Oral administration has applied via oral gavages at the base of the tongue. The
previous dosages have given five consequent days per week, along the experimental
period. The control groups received the same volume of distilled water.
Semen Collection and Analyses
Adult males were trained to serve an artificial vagina (IMV, France) and a
teaser doe two weeks prior to experiment period, as preliminary period in order to
assure that the males were reproductively normal according to their libido and semen
characteristics, also to establish same regular semen collection schedule regime and to
avoid the collection of ancient spermatozoa owing to prolonged storage within the
epididymis. Semen was collected simultaneously weekly over a period of 12 weeks
from all males within the experiment.
Ejaculates containing urine and/or calcium carbonate deposits were discarded,
also ejaculates have technical error were subside from examination and statistics. Gel
plugs if found, were removed from ejaculate before evaluation.
Three ejaculates collected for each buck, with an interval of 30 min in
between. One served as row semen sample for spermogram. The other two
consecutive (successive) ejaculates were pooled and centrifuged at 12000 rpm for 10
min at 4 °C in cooled centrifuge to separate seminal plasma, which stored at -80 °C
ultra freezer, until further analysis.
Fertility Evaluation: 1. Sexual Activity
Sexual drive has graded as excellent, good, or fair according to the criteria of
Lohiya and Sharma (1984). Libido evaluation based upon willingness and eagerness
of the bucks to mount a teaser doe, ability to ejaculate and other subjective
observation of the mating behaviors.
In addition, reaction time has recorded from the moment of subjecting a doe to
the buck and completion of ejaculation, measured in seconds using a stopwatch.
(Spermogram) Semen Examination:-2-1. Quantitative Parameters
Ejaculate volume (ml) EV determined by a graduated tube that directly connected to
the artificial vagina, to the nearest 0.10 ml. Gel plugs, when present, have removed
before volume evaluation.
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Spermatozoa concentration (106/ml) Spermatozoa concentration assayed using a
weak aqueous eosin solution to kill sperm cells and stained the head purple color,
thus facilitate spermatozoa counting by the Thomas ruling double counting improved
Neubauer hemocytometer slide (GmbH + Co., Hamburg, Germany).
Total sperm output (106/ejaculate) TSO calculated by multiplying semen ejaculate
volume by semen concentration.
Packed sperm volume (%) PSV has recorded using capillary tubes and
microhematocrit centrifuge adjusted at 12000 rpm for 15 min and read by
microhematocrit reader. Semen sample must gently shaken before tube filling.-2.
Qualitative Parameters
Mass motility MM were estimated immediately after semen collection, by visual
examination under low-power magnification (100x) using a warm plate light
microscope with heated stage and take a grade ranged from 0 to 9 according to
Petitjean (1965) notation scale showed in table (2).
Table (2): Notation scale used to measure mass motility Grade Note Description
0 No spermatozoa or motionless spermatozoa (spz) 1 Few stirring spz without notable movement 2 Important number of stirring spz without any movement 3 Few spz moving slowly 4 Few motionless spz, few stirring spz without any movement, few
mobile spz 5 Same as 4, but higher proportion of mobile spz. Rather high, but not
homogeneous motility in the observed field 6 Nearly all spz moving. High and homogeneous motility 7 Same as 6, wavelike movements begin 8 Same as 7, with slow wave movements 9 Strong waves; whirlwind appearance
Individual progressive motility (%) IM assessed within 5 min after collection using a
37 ºC warm plate light microscope at 200x and 400x magnification. Semen sample
was diluted at a rate of 1:4 with a commercial rabbit semen extender GALAB (IMV,
France) and the result was expressed as the percentage of spermatozoa (nearest to 5
%) exhibiting progressive rectilinear movement.
Sperm motility index SMI calculated by multiplying motility grade by individual
motility.
Total motile sperm (106/ejaculate) TMS calculated by multiplying percentage of
motile sperm and total sperm output.
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Abnormalities and Live sperm (%) (physical membrane integrity) Stained smear was
performed as soon after ejaculation, using an eosin-nigrosine staining mixture at 1:4
dilution rate (Blom, 1977). Two hundred spermatozoa per sample have examined for
morphology and viability in stained smear at 1000x magnification (oil immersion).
The principle of these techniques is dye exclusion; red eosin stained dead sperm head
while nigrosine provided a blue-black background.
Total functional sperm fraction (106/ejaculate) TFSF is a parameter described by
Correa and Zavos (1994). It was calculated as the product of multiply total sperm
output (106) by individual motility (%) by normal morphology (%).
3. Special Semen Tests
1. Functional membrane integrity: hypo-osmotic swelling (HOS) test is a relatively
simple test to evaluate the functional integrity of the spermatozoa membrane. During
the HOS test, the biochemically active spermatozoa, due to the influx of water, will
undergo swelling and increase in volume to establish equilibrium between the fluid
compartment within the spermatozoa and the extracellular environment. This volume
increase is associated with the spherical expansion of the cell membrane covering the
tail, thus forcing the flagellum to coil inside the membrane. Coiling of the tail begins
at the distal end of the tail and proceeds towards the mid-piece and head as the
osmotic pressure of the suspending media is decreased (Jeyendran et al.1984). The
plasma membrane surrounding the tail fibers appears to be more loosely attached than
the membrane surrounding the head.
The optimal hypo-osmotic medium should exert an osmotic stress large
enough to cause an observable increase in volume, but small enough to prevent the
lysis of the sperm membrane. Results of HOS test founded to give good correlation
with progressive motility Neild et al., 1999, 200).
Michele et al. (2002) developed this assay for rabbit spermatozoa. The HOS
test for rabbit was performed immediately after ejaculate collection by mixing 100 ul
of each semen ejaculate with 900 ul 60 mOsmol fructose solution, differing from the
original test used by Jeyedran et al. (1984) because preliminary test showed a form
of sodium citrate toxicity on rabbit spermatozoa. Then, diluted semen examined
immediately (zero time) and after 30 minutes after sample smearing. One hundred
spermatozoa examined for swallowed coiled tail at 1000× magnification (oil
immersion) (Michele, et al., 2002).
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Acrosome integrity: In present study, Giemsa stain was used to stain the acrosome
dark purple. Staining technique briefed as following:
1) Fresh ejaculate diluted at 1:5 in commercial rabbit semen extender GALAB
(IMV, France).
2) Diluted semen smeared and air-drayed.
3) Smear was fixed in 10% neutral formal saline for 15 minutes.
4) Fixed smear washed in running water for 20 minutes.
5) Fixed smear immersed in Giemsa working solution overnight.
6) Stained smear rinsed in two changes of distilled water and air-drayed.
One hundred spermatozoa per sample have examined at 1000x (oil immersion)
for acrosome integrity in each stained smear.
Semen quality test: The test based on the ability of metabolically active spermatozoa
to reduce the blue resazurine dye (with maximum absorption at wavelength 580 nm)
to pink resorufin (with maximum absorption at wavelength 615 nm). This reduction
ability has evaluated to judge the sperm viability, therefore the test called Sperm
Viability Test (SVT) as well as Resazurine Reduction Test (RRT). In addition, RRT
use spectrophotometer that provides a tool of seminal diagnosis more accurately than
the routine semen analysis (Reddy and Bordekar, 1999).
The results represented as RRT ratio that equal absorption at 580 nm/
absorption at 615 nm. Higher RRT ratio observed to correlate with sperm motility,
count, morphology and viability. The test kit obtained from Bio Diagnostic Research
office (Dokki, Giza, Egypt).
Semen Biochemical Analysis:
. Hydrogen ion concentration PH was determined immediately after semen collection
using PH paper (Spezial-Indikatorpapier pH 5.5-9.0 MACHEREY-NAGEL W.
German) with 0.5 degree intervals.
. Initial fructose concentration (mg/dl) IF determined immediately after ejaculation
or later in stored seminal plasma according to Foreman et al. (1973). Seminal
Plasma Biochemical Analysis:
Oxidative and antioxidant status were methodology measured (evaluated)
similar to that of serum.
III. Hormones Assay
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Testosterone (ng/ml) Total serum and seminal plasma testosterone levels were
measured using imported commercial solid phase enzyme amplified sensitivity
immunoassay kit (TESTO-EASIA KAP1701, BioSource Europe S.A., Belgium).
The test is a based on competition between fixed amounts of horseradish
peroxidase (HRP) labeled testosterone and unlabelled testosterone present in the
calibrators and samples for a limited number of binding sites on a specific antibody
(Maruyama et al., 1987). The amount of substrate turnover is determined
colormetrically by measuring the absorbance at wavelength 450 nm, which inversely
proportional to testosterone concentration. A calibration curve is plotted and
testosterone concentrations in samples are determined by interpolation from the
calibration curve. Assay has sensitivity (detection limit) of 0.05 ng/ml.
III-b. Cortisol (ug/dl) Microplate Enzyme Immunoassay kit (Monobind Inc. Lake
Forest, CA 92630, USA) used for estimation of total serum and seminal plasma
Cortisol concentration.
The test is a based on competition between serum native antigen and enzyme-
antigen conjugate for a limited number of antibody binding sites. The inter action is
illustrated by the followed equation:
EnzAg + SerAg + AbBin ↔ SerAgAbBin + EnzAgAbBin
A simultaneous reaction between the antibody attached biotin and microwell
immobilized streptavidin occurs. SerAgAbBin + EnzAgAbBin + streptavidin → immobilized complex
The enzyme activity in the antibody-bound fraction is inversely proportional
to the serum native antigen concentration. By utilizing several different serum
references of known antigen concentration, a dose response curve can generated, from
which the antigen (Cortisol) concentration of unknown can be ascertained Burtis and
Ashweed, 1994 ).
IV. Statistical analysis
All data were subjected to statistical analysis including the calculation of the mean (M), standard error of mean (SE) and F-test (one way ANOVA) at a confidence limit of 95% (P<0.05) according to the method of Armitage (1971) using practicing statistical analysis program (SPSS, Edition 11). Duncan’s multiple range test was used for testing pairs for Comparisons among means at probability 5%.
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Result. Climate Data
Monthly and season's averages of ambient temperature, relative humidity and
temperature-humidity indices during summer and winter months were illustrated in
table 3 The season's averages of ambient temperature and relative humidity were
19.06 ºC ±0.26 and 63.24 % ±0.88 during the winter and 36.17 ºC ±0.44 and 73.40 %
±1.00 during the summer.
The season's averages of temperature–humidity indices, were 18.52±0.22 in
winter and 34.38±0.46 in summer, indicating absence of heat stress in winter (less
than 27.80) and exposure to very severe heat stress in summer (more than 30.00)
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Table (3)) Monthly and seasonal means (±SE) of climate data during the two experimental periods.
SE =
Standard error
Sexual activity
Summer control group showed significant increase in reaction time compared to winter control
one. All antioxidant supplemented groups significantly decreased reaction time than summer control
values, but only l-carnitine supplemented bucks restored this parameter to winter group values.
Spermogram
Hydrogen ion concentration:
Summer control group revealed significant decrease in pH values in comparison with winter
group. Different supplementations did not affect pH values compared to summer control group, except
for zinc supplemented bucks, which restored pH to its winter group values
Ejaculate volume:
There was a significant decrease in ejaculate volume in summer heat stressed bucks compared to
winter group. Ascorbic acid and zinc supplemented groups got back ejaculate volume to winter group
values.
Mass motility:
Summer control group showed non significant decrease in mass motility compared to winter
group. Ascorbic and co enzyme Q10 supplemented bucks showed significant decrease in mass motility,
while zinc and l-carnitine showed non significant increase in MM, in comparison with summer control
group.
eriods 1st Period (Winter) 2nd Period (Summer)
Months
parameters
23rd December – 22nd
January
23rd January – 22nd February
23rd February – 22nd March
Average
23rd June – 22nd July
23rd July – 22nd
August
23rd August – 22nd September
Average
Ambient temperature
(°C) 18.23 ±0.27
19.03 ±0.29
19.93 ±0.30
19.06 ±0.26
35.48 ±0.46
37.73 ±0.19
35.30
±0.84
36.17
±0.44
Relative humidity (%)
66.10 ±1.12
63.63 ±0.58
60.00 ±0.84
63.24 ±0.88
73.63 ±1.37
76.03 ±1.03
70.55
±1.75
73.40
±1.00
Temperature- humidity
index 17.82 ±0.25
18.50 ±0.25
19.24 ±0.25
18.52 ±0.22
33.76 ±0.51
35.99 ±0.10
33.41
±0.87
34.38
±0.46
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Individual motility:
There was non significant decrease in individual motility percentage noticed in summer control
bucks compared to winter group. Ascorbic and co enzyme Q10 supplementations significantly decreased
IM, while L-carnitine supplemented group showed non significant increase in individual motility
percentage, in comparison with summer control group.
Motility index:
Motility index values were recorded to be decreased significantly in summer heat stressed bucks
compared to winter ones. Among different antioxidants supplementations, zinc and l-carnitine
supplemented groups restored motility index to winter group values.
Alive spermatozoa:
Summer control bucks noticed to significantly decreased life sperm percentage than winter
group. Ascorbic supplemented bucks showed non significant increase in life sperm percentage, while
zinc and l-carnitine supplemented groups restored this parameter to winter group values.
Spermatozoa abnormalities:
There was non significant increase in sperm abnormalities percentage in summer control group
compared to winter group. All antioxidant supplemented groups showed non significant decrease in
sperm abnormalities except co enzyme Q10 supplemented bucks, which showed non significant increase
in sperm abnormalities in comparison with summer control group percentage. Spermatozoa concentration:
There was no difference in sperm concentration between summer and winter control groups.
While only l-carnitine supplemented bucks showed significant increase in sperm concentration
compared to summer control group.
Packed sperm volume:
No significant difference in packed semen volume was noticed in all experimental groups.
Total sperm output:
There was marked significant decrease in TSO in summer heat stressed bucks compared to bucks
reared in winter. Ascorbic and zinc supplemented bucks showed significant increase in TSO in
comparison with summer control group. Total motile sperm:
Notable significant decrease in TMS was recorded in summer control group compared to winter group. Zinc supplemented bucks significantly increased TMS, while ascorbic and l-carnitine supplemented Bucks showed non significant increase in TMS, in comparison with summer control group. Total functional sperm fraction:
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Summer control group showed significant decrease in TFSF compared to winter group.
However, zinc, ascorbic and l-carnitine supplemented groups showed increase in TFSF in comparison
with summer control group, which was significant in zinc supplemented group. Table (4): Overall means (±SE) of reproductive efficiency (sexual activity and spermogram) of rabbit bucks as affected by different
seasons and antioxidant supplementations (N = 84).
Seasons Winter Summer Groups
Parameters Control Control Ascorbic Zinc Co
enzyme Q10
L-carniti
ne
P ≤
Reaction time (s)
11.49±1.12a
23.39±0.47d
15.14±0.81b
15.62±0.63b
19.07±0.45c
12.11±0.66a 0.000
pH 7.50±0.04b 7.38±0.03a 7.36±0.04a 7.41±0.04ab 7.39±0.0
3a 7.34±0.0
5a 0.038 Ejaculate Volume (ml)
1.02±0.03b
0.74±0.03a
0.96±0.09b
0.91±0.04b
0.73±0.02a
0.76±0.03a 0.000
Mass Motility
7.46±0.15b
7.21±0.08ab
6.90±0.14a
7.24±0.09b
6.90±0.09a
7.46±0.09b 0.000
Individual Motility
(%) 67.14±1.95
b 63.82±1.18
ab 61.43±1.79
a 64.10±1.28ab
59.67±1.42a
66.98±1.33b 0.002
Motility Index
5.20±0.22d
4.67±0.14abc
4.41±0.19ab
4.73±0.15bc
d 4.21±0.
15a 5.08±0.15cd 0.000
Alive sperm (%)
73.52±1.09c
69.92±0.65a
70.62±1.01ab
70.94±0.79a
bc 68.35±0.84a
73.10±0.88bc 0.000
Abnormalities (%)
15.40±0.60a
16.79±0.62ab
15.40±0.57a
15.72±0.61a
18.14±0.62b
15.54±0.63a 0.007
Concentration
(106/ml) 282.92±3
.12a 276.59±1
.96a 275.65±2
.78a 282.97±2.34a
277.53±1.88a
290.31±2.34b 0.000
Packed sperm vol.
(%) 16.14±0.
16a 15.97±0.
10a 15.80±0.
14a 16.13±0.11a
15.88±0.086a
16.49±0.12a NS
TSO (106/ejacu
late) 289.03±9
.04d 208.92±7
.90a 240.70±6.75bc
255.79±9.48c
202.18±6.36a
220.64±8.62ab 0.000
TMS (106/ejacu
late) 199.98±10.44d
136.91±6.73ab
147.92±6.30bc
162.05±6.46c
123.32±5.94a
150.23±7.71bc 0.000
TFSF (106/ejacu
late) 172.86±9
.93d 116.16±6.30ab
127.02±5.95bc
137.62±5.91c
103.16±5.65a
128.85±7.22bc 0.000
Means within the same row followed by the different superscripts are significantly different at p ≤ 0.05. NS = non significant TSO = Total sperm output TMS = Total motile sperm TFSF = Total functional sperm fraction SE = Standard error
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Special Semen Tests
Overall mean values of intact acrosome percentage, hypoosmotic swelling test results, semen
quality test and initial fructose concentration in winter and summer (control and antioxidants
supplemented) groups were shown in table (5).
Acrosome integrity:
There was no difference in intact acrosome percentage in summer heat stressed bucks compared
to winter ones. Regarding to antioxidant supplemented groups, ascorbic and co enzyme Q10
supplemented bucks showed non significant decrease in intact acrosome percentage, while zinc
supplemented group showed non significant increase in this regard, in comparison with summer control
group.
Hyposomotic swelling test:
No difference in swollen coiled sperm percentage among all experimental groups was observed.
Semen quality test:
Summer control group showed no significant difference in semen quality test results compared to
winter values. Only zinc supplemented group showed significant increase in quality test results in
comparison with summer control group.
Table (5): Overall means (±SE) of intact acrosome, hypoosmotic swelling test, semen quality test and seminal plasma initial fructose level
of rabbit bucks as affected by different seasons and antioxidant supplementations (N = 84).
Season Winter Summer Groups
Parameters Control Control Ascorbic Zinc Co enzyme Q10
L-carnitine
P ≤
Intact acrosome (%)
44.14ab ±0.98
44.45ab ±1.44
41.95a ±1.01
48.05b ±1.25
42.90a ±1.18
45.65ab ±2.05
0.037
Hyposomotic swelling test
(%) 63.11a ±2.21
65.75a ±1.22
65.35a ±0.87
65.15a ±0.94
63.45a ±0.84
67.05a ±0.72 NS
Semen quality test
1.81a ±0.09
1.66a ±0.11
1.76a ±0.11
2.27b ±0.15
1.76a ±1.40
1.83a ±0.12
0.012
Initial fructose
concentration(mg/dl)
223.49a ±3.99
223.53a ±3.22
218.43a ±2.96
224.24a ±3.34
224.08a ±3.43
230.18a ±3.43 NS
Means within the same row followed by the different superscripts are significantly different at p ≤ 0.05. NS = non significant SE = Standard error
Initial fructose:
There was no difference in initial fructose levels in all experimental groups.
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Oxidative and Antioxidant Status
Overall means of serum and seminal plasma malondialdehyde levels, total antioxidant capacity
and catalase activities in winter and summer (control and antioxidants supplemented) groups were
shown in table (6)
Serum Oxidative and Antioxidant Status
Lipid peroxides:
There was a significant increase in serum TBARS levels in summer control group compared to
winter group. All antioxidants supplemented bucks showed no change in serum TBARS levels in
comparison with summer heat stressed bucks.
Total antioxidant capacity:
Summer heat stressed bucks significantly decreased serum TAC level than winter ones. As in
serum MDA, all antioxidants supplemented groups showed no change in serum TAC levels in
comparison with summer control group.
Catalase:
There was a significant decrease in serum catalase activity in summer heat stressed bucks
compared to winter group. Ascorbic, co enzyme Q10 and l-carnitine supplemented bucks showed
significant increase in serum catalase activities, while zinc supplemented bucks showed non significant
increase in serum catalase activity, in comparison with summer control bucks. Table (6) Overall means (±SE) of serum and seminal plasma oxidative and antioxidant status of rabbit bucks as affected by different
seasons and antioxidant supplementations (N = 36). Seasons Winter Summer
Groups Parameters Control Control Ascorbic Zinc
Co enzyme Q10
L-carnitine
P ≤
TBARS (nmol/ml)
2.14a ±0.09
3.68b ±0.10
3.48b ±0.18
3.54b ±0.12
3.57b ±0.13
3.47b ±0.13 0.000
TAC (mmol/L)
1.23b ±0.10
0.68a ±0.02
0.76a ±0.06
0.73a ±0.03
0.82a ±0.05
0.80a ±0.05 0.000 Serum
Catalase (U/ml)
19.36d ±0.75
13.76a ±0.23
15.14bc ±0.39
14.33ab ±0.36
15.90c ±0.33
15.61bc ±0.46 0.000
TBARS (nmol/ml)
1.21a ±0.09
1.93d ±0.04
1.62bc ±0.09
1.82cd ±0.08
1.64bc ±0.07
1.58b ±0.05 0.000
TAC (mmol/L)
1.71c ±0.05
1.12ab ±0.03
1.07a ±0.04
1.07a ±0.07
1.26b ±0.09
1.24b ±0.04 0.000 Seminal
plasma
Catalase (U/ml)
24.84c ±0.66
13.55a ±0.27
15.11b ±0.62
15.03b ±0.39
16.26b ±0.59
16.09b ±0.49 0.000
Means within the same row followed by the different superscripts are significantly different at p ≤ 0.05.
TBARS = Thiobarbituric acid-reactive substances TAC = Total antioxidant capacity
SE = Standard error
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2. Seminal plasma Hormones
Testosterone:
There was significant increase in seminal plasma testosterone levels in summer control group
compared to winter group. Asc orbic and zinc supplemented bucks showed significant increase in
testosterone levels in comparison with summer control bucks.
Cortisol:
Summer heat stressed bucks showed significant increase in seminal plasma cortisol level
compared to winter bucks. Ascorbic and zinc supplemented bucks significantly decreased seminal
plasma cortisol levels. On contrary, l-carnitine supplemented group showed non significant decrease in
cortisol levels, in comparison with summer control group. Table (7) Overall means (±SE) of serum and seminal plasma testosterone and cortisol of rabbit bucks as affected by
different seasons and antioxidant supplementations (N = 36).
Season Winter Summer
Groups Parameters Control contro
l Ascorbi
c Zinc Co
enzyme Q10
L-carnitin
e
P ≤
Testosterone
(ng/ml) 3.03a ±0.16
3.01a ±0.16
2.86a ±0.18
2.93a ±0.18
2.49a ±0.14
2.73a ±0.18 NS
Serum
Cortisol (µg/dl)
3.84a ±0.24
3.21a ±0.25
3.86a ±0.11
3.80a ±0.21
4.65b ±0.38
3.79a ±0.12
0.004
Testosterone
(ng/ml) 1.87a ±0.21
3.18b ±0.33
4.98c ±0.18
4.34c ±0.37
2.87b ±0.42
2.99b ±0.23
0.000
Seminal
Plasma Cortisol (µg/dl)
1.08a ±0.06
2.45d ±0.20
1.46ab ±0.16
1.70bc
±0.07
2.23d ±0.16
2.06cd ±0.11
0.000
Means within the same row followed by the different superscripts are significantly different at p ≤ 0.05. NS = non significant
SE = Standard error
In present study, the results obtained in the chemistry of seminal plasma components were in
harmony with those of spermogram characteristics. The changes in seminal traits due to variations in
climate may be due to differences in food intake and other factors such as fertility, number of ejaculates
and sexual desire.
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Discussion The present study was performed to investigate the influence of heat stress on male rabbit
reproductive performance and an attempt to alleviate this heat stress effect using selected antioxidants.
Climate Data
The season's averages of temperature–humidity indices (THI) as heat stress index, were
18.52±0.22 in winter and 34.38±0.46 in summer, indicating absence of heat stress in winter (less than
27.80) and exposure to very severe heat stress in summer (more than 30.00). These values were similar
(very close) to that estimated by Marai et al. (2003). In addition, Marai et al. (2004, 2005, 2006)
estimated similar THI in winter and slightly lower values in summer.
Semen Analyses
Sexual activity (desir)
Summer control group showed significant increase in reaction time compared to winter control
one. This finding agrees with the observations of Seleem (2005), Nagwa et al., (2006). This is partly
confirmed by the literature. Alvarino, 2000), which reports that high temperatures (above 27C) depress
libido. Contrariwise, Marai et al. (2002b) recorded that effects of heat stress on reaction time in NZW
rabbits were not significant.
The decrease in bucks' libido with increasing ambient temperature may be due to delay in sexual
urge and/or due to low physical performance of bucks under heat stress Nagwa et al., (2006). In the
light of present result, the delayed reaction time in summer with constant serum testosterone level seems
to be due to low bodily activity in attempt to minimize metabolic activity, consequently minimize heat
production.
All antioxidant supplemented groups significantly decreased reaction time than summer control
values, but only l-carnitine supplemented bucks restored this parameter to winter group values.
Spermogram
Hydrogen ion concentration:
Seminal plasma is usually an isotonic neutral medium and it is a detrimental factor to sperm cell
survival. Contrary to most of previous literatures (El-Bashary et al., 2005; Nagwa et al., (2006) results
of the present study show that semen pH values significantly decreased in heat stressed bucks in
comparison with winter group (7.50 vs. 7.38). Other authors Chen et al., 2003; Nizza et al., 2003)
reported insignificant effect of heat stress on hydrogen ion concentration.
It seems that after rabbit's exposure to heat stress and developed mild hyperthermia in Bedouin
rabbits, the first process to develop is metabolic acidosis prior to the metabolic alkalosis that occurs in
advanced stage (Marder et al., 1990). The progressive metabolic acidosis strongly indicates a shift to
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anaerobic metabolism and suggests the existence of severe metabolic complications Kazemi and
Johnson, 1986) followed by an increased production of lactic acid in different body tissues. Taking
these findings and the current result into consideration, the significant decrease in semen hydrogen ion
concentration may be explained.
Different antioxidant supplementations did not affect pH values compared to summer control
group, except for zinc supplemented bucks, which restored pH to its winter group values.
Ejaculate volume: A significant effect of season on ejaculate volume of NZW rabbits is observed in the current
study. The lower bucks ejaculate volume in summer is similar to the results of El-Bashary et al. (2005),
and Nagwa et al., (2006) and contrast the results of Janet et al. (2003, b) and Nizza et al. (2003).
Meanwhile, in rabbit season of the year had no significant effects on semen ejaculate volume of rabbit
bucks.
The changes in ejaculate volume may be due to a low sperm concentration and a decrease in the
volume of seminal plasma as result of hypoactivity of the accessory glands and the testes due to the
adverse effect of high ambient temperature Zeidan et al., 1997).Ascorbic acid and zinc supplemented
groups got back ejaculate volume to winter group values. Using antioxidant supplemented groups
showed non significant decrease in sperm abnormalities except co enzyme Q10 supplemented bucks,
which showed non significant increase in sperm abnormalities in comparison with summer control
group percentage.
This apparently stability in sperm concentration may be due to the low ejaculate volume,
therefore the total sperm output is more really parameter as quantitative index. L-carnitine
supplementation significantly increases sperm concentration compared to summer control group.
Packed sperm volume:
No significant difference in packed semen volume was noticed in all experimental groups.
Because of the PSV is a relative parameter; i.e. depend on volume and concentration, only its elevation
or decline has true indication.
Zinc supplementation significantly increased TMS, while ascorbic acid and l-carnitine
supplementations insignificantly increased TMS, in comparison with summer control group.
Total functional sperm fraction:
Summer control group showed significant decrease in TFSF compared to winter group.
However, zinc, ascorbic and l-carnitine supplemented groups showed increase in TFSF in
comparison with summer control group, which was significant in zinc supplemented group.
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Special Semen Tests Hyposomotic swelling test:
Contrary to our expectation, results of the present study show no difference in HOS-positive
spermatozoa (swollen curled/coiled principal or end piece) percentage among all experimental groups.
No available literature studies the HOS in relation to different season. Janet et al., 2003) reported a
significant increase in summer in stallion (note the study in Switzerland where the summer is a
temperate season and winter represents cold stress).
Semen quality test:
Summer heat stress showed no significant difference in semen quality test results in comparison with
winter values. Only zinc supplemented group showed significant increase in quality test results in
comparison with summer control group. This finding is in harmony with the current (present) significant
high total motile sperm in zinc supplemented bucks compared to other experimental groups, because the
test based on the reduction ability of metabolically active spermatozoa.
Initial fructose: There was no difference in initial fructose levels in all experimental groups. Similar result
obtained by Nagwa et al., (2006) in NZW bucks. Other investigations are conflicting among significant
increase in initial fructose concentration (El-Bashary et al., 2005) in rabbits and significant decrease. Special Semen Tests Acrosome integrity:
There was no difference in intact acrosome percentage in summer heat stressed bucks compared
to winter ones. This result is in agreement with that of Nizza et al. (2003) in rabbit, while Janet et al.
(2003) recorded significant increase in normal acrosome percentage in summer in study on stallion. The
effect of heat stress on acrosome integrity may be attributed to the high lipid peroxidation in epididymes
as a result of elevated oxidative stress, which altered the stability of plasma membrane that surrounds
the acrosome through the effect on its content of polyunsaturated fatty acids and lipoproteins (Zini et al,
1998).
Regarding to antioxidant supplemented groups, ascorbic and co enzyme Q10 supplemented bucks
showed non significant decrease in intact acrosome percentage, while zinc supplemented group showed
non significant increase in this regard, in comparison with summer control group.
Seminal Plasma Oxidative and Antioxidant Status
Literature regarding oxidative stress characteristics in semen in response to heat stress is very
limited; the objective of most experiments was to evaluate the effects of prooxidants or antioxidants on
the concentrations of TBARS, or on semen quality (Nichi et al., 2006).
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Lipid peroxides:
Summer control group showed significant increase in seminal plasma TBARS levels in summer
control bucks compared to winter ones. In bull, Nichi et al. (2006) reported similar result.
The high semen concentrations of TBARS in summer than winter suggested that there was more
lipid peroxidation in semen during the summer months, presumably due to increased production in the
testis (Gil-Guzman et al., 2001). Furthermore, the increased TBARS may be to the present increased
incidence of dead sperm and that may proposes an association between lipid peroxidation and sperm
quality. In addition, this finding suggests that the higher concentrations of TBARS found in seminal
plasma during the summer were apparently related to higher levels of ROS, and not due to a lower
antioxidant capacity in present results.
Fructose concentrations in seminal plasma could falsely affected concentrations of TBARS in
semen. This carbohydrate, an important source of energy for sperm cells, can react with thiobarbituric
acid, falsely increasing TBARS concentrations ( Sobenin et al., 1998). However, initial fructose
concentrations in present study results are not affected by heat stress and show no significant difference
between two seasons. Therefore, the increased TBARS concentrations during the summer were
apparently not due to higher fructose concentrations.
All antioxidants supplemented groups showed decrease in seminal plasma TBARS levels than
summer control group, which was significant in ascorbic, co enzyme Q10 and l-carnitine supplemented
groups. Total antioxidant capacity:
Seminal plasma TAC level show significant decrease in summer control bucks compared to
those reared in winter. Nichi et al. (2006) in bull, reported no change in seminal plasma TAC.
None of antioxidants supplemented groups showed significant effect in seminal plasma TAC
levels in comparison with summer control group.
Catalase:
Catalase activity in semen remains a matter of debate, presumably owing to varying digress of
sample purity. Most species have little protective catalase in their semen, but rabbit semen contains
unusually substantial amounts of catalase (Foote and Hare, 2000). In present results, seminal plasma
catalase activity showed markedly decline in summer control group compared to winter ones.
All antioxidant supplemented bucks showed significant increase in seminal plasma catalase
activities in comparison with summer control bucks.
Seminal plasma testosterone:
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A significant increase in seminal plasma testosterone in summer control group compared to
winter group is reported in the present study and harmonizes the findings of Kafi et al. (2004) in ram.
Summer heat stressed bucks showed significant increase in seminal plasma cortisol level
compared to winter bucks. This finding is in contrast to the result reported by Marai et al. (2002).
Ascorbic and zinc supplementations significantly decreased seminal plasma cortisol levels, while l-
carnitine supplemented group showed non significant decrease in cortisol level in comparison with
summer control group.
Conclusion In regard to summer heat stress consequence (effect), the present study shows that NZW rabbits
buck have continuous and acceptable spermatogenic activity and apparently normal body physiology
during summer as well as during winter. Contrary to our expectation, the examined semen traits of NZW
rabbits buck are not much inferior in summer compared to that obtained in winter. However, apparently
(relatively) seasonal variations in semen characteristics are detected.
To sum up, it should be said that (separate) protective administrations of zinc and l-carnitine
cause significant improvement in rabbit sperm characteristics, meanwhile, ascorbic acid and co enzyme
Q10 administrations have no considerable enhancement in such concern.
Taking the current results into consideration, it can be concluded that an adequate reproductive
performance in NZW rabbits buck can be achieved in summer and from male side of breeding, it is not
obligatory to stop breeding in summer months, but such some extraordinary (protecting) managements
should be attained.
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