An attempt at alleviating heat stress infertility in male rabbits with … · 2017-10-24 · An attempt at alleviating heat stress infertility in male rabbits with some ... relief

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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



١٦

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.



١٧

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



١٨

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.



١٩

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).



٢٠

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:



٢١

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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