Relief of the negative effects of heat stress on semen quality, reproductive efficiency and oxidative capacity of rabbit bucks using different natural antioxidants

Objective The potential of extra virgin olive oil (EVOO), betaine (BET), and ginger (GIN), as natural antioxidants, in reducing negative effects of heat stress on physiological responses, antioxidant capacity, semen quality and fertility of bucks under heat stress were investigated. Methods Forty adult Animal Production Research Institute line rabbit bucks were distributed randomly into four experimental treatments of ten rabbits each. The first treatment was fed the commercial pellet diet (CPD) without supplementation and served as a control. The other three treatments were fed CPD supplemented with EVOO (300 mg), BET (1,000 mg), and GIN (200 mg) per kg diet for 3 consecutive months during the summer season. Results Supplementation of EVOO, BET, or GIN improved (p<0.05) the sexual desire, progressive motility, vitality, intact acrosome and membrane integrity, sperm cell concentration, sperm outputs and fertility. Seminal plasma total proteins, globulin, total antioxidant capacity, glutathione and glutathione S-transferase, and initial fructose increased (p<0.05), while total lipids, aspartate and alanine aminotransferases and malondialdehyde decreased (p<0.05) compared with the control. In comparing the natural antioxidants treatments, GIN evoked the largest improvement. Conclusion The inclusion of GIN (200 mg/kg diet) appeared to improve the sexual desire, semen quality and oxidative stress of bucks. This may be a beneficial supplement for the management of rabbit bucks used in natural mating or artificial insemination.


INTRODUCTION
In comparing with other livestock, rabbit are characterized small body size, efficient feed utilization, rapid growth rate, high quality nutritious meat, early maturity, high reproduc tion rate and high genetic selection potential [1]. The environmental and nutritional factors are affecting the economic intensive rabbit production. Under environmental tempera ture more than 25°C, reduce their productivity by several behaviour and physiological changes [2]. The reproductive efficiency of bucks is economically important because semen with high characteristics is required for avoiding the loss in valuable genotypes and achiev ing high fertility [3]. Oxidative stress plays an important role in sperm motility, function, quality and fertility, since lipid peroxidation increases under heat stress (HS) [4]. Improper environmental conditions lead to reduction in quality and fertility of sperm cells, so HS negatively influences the testicular function [5].
Rabbit spermatozoa display high metabolic activity and they are rich in polyunsaturated fatty acids in plasma membrane, which may cause increasing lipid peroxidation [6], so they

MATERIALS AND METHODS
The experimental work of this study was conducted at a private commercial rabbit farm, Mansoura City, Dakahlia Governorate, Egypt. The laboratorial work was carried out at Physiology and Biotechnology Laboratory, Animal Production Department, Faculty of Agriculture, Mansoura University, Egypt. The experimental procedures were conducted accord ing to the Directive 2010/ 63/EU of the European Parliament and of the Council of 22 September 2010 on the Protection of Animals Used for Scientific Purposes.

Animals
Forty sexually mature Animal Production Research Institute (APRI) line rabbit bucks with 8 monthold and an average live body weight (LBW) of 3.05±0.22 kg were used in this study. Bucks were individually housed in stainless steel cages batteries (40×50×35 cm) accommodated with feeders for pelleted rations and automatic freshwater drinkers within a naturally ventilated and lighted rabbitry. The experimental bucks were fed ad libitum on a commercial pellet diet (CPD) covering the daily nutritional requirements of rabbit bucks according to the national research council guidelines [17]. The ingredients and chemical analysis of CPD is represented in Table 1.

Experimental design
Bucks were distributed randomly into four homogenous experimental treatments (10 in each), in a straightrun ex perimental design. Bucks in the first treatment were fed the CPD without supplementation and served as the control. Bucks in the other three experimental treatments were fed the CPD supplemented with 300 mg of EVOO (ILIADA PDO Kalamata EVOO; AGRO. VI. M.S.A., Kalamata, Greece), 1,000 mg of BET (natural betafine, Adisseo, France) and 200 mg of GIN per kg, respectively. The weekly CPD of each treatment was well mixed with their additives in homogenous form. Bucks were fed the experimental diets throughout experimental treatments of 3 consecutive months from 1st June to 30th September.

Climatic condition
Throughout the experimental treatments, ambient tempera ture (AT) and relative humidity (RH) were recorded daily at 1 p.m. using a hydrothermograph located inside the rabbitry. The daily values of AT and RH were estimated. Tempera turehumidity index (THI) was calculated according to the following equation [18]: Where T, dry bulb temperature (°C) and RH, relative humidity. THI va Where T, dry bulb temperature (°C) and RH, relative humidity. THI values of <27.8 (absence of HS); 27.8 to 28.9 (moderate HS); 29.0 to 30.0 (severe HS); and >30.0 (very severe HS). Table 2 show AT, RH, and THI during the experimental treatments. Results of the calculated THI value indicate that the experimental bucks were under HS, being severe during JuneAugust and moderate in September.

Body weight and feed intake
The LBW (kg/buck) and feed intake (g/buck) were recorded for the entire experimental treatments.

Physiological response parameters
The temperature of skin (ST), rectal (RT), and ear (ET) were individually measured, at the same time of measuring AT and RH, using a digital thermometer (Type "K" Thermocouple, ±0.01°C). The ST was measured at one location between the loin and neck on the body surface. The RT was measured by inserting the probe of thermometer at a depth of 2 cm into the rectum. The ET was measured by placing the probe of digital thermometer in direct contact with the internal cen tral area of the auricle. The measurement durations were minimized as possible to be all in similar times.

Semen collection and evaluation
Rabbit bucks were trained for semen collection by artificial vagina prior to the main collection period. From 1st August to 30th September, as semen collection period, semen was collected from all rabbit bucks (n = 10) in each group twice/ week for 9 successive weeks. Therefore, 180 ejaculates (10 bucks ×2 ejaculates/h/wk×9 weeks) were collected and evalu ated per group, and then the weekly data of each semen parameter were averaged for each rabbit during the whole collection period to avoid using them as experimental rep lications.
On day of collection, ejaculates were obtained in the morn ing (8 a.m.) by using an artificial vagina of rabbits (40°C to 41°C) and a teaser doe. The reaction time was recorded by measuring the time elapsed from a doe insertion into the cage of buck until complete ejaculation (as an indication of libido). After collection, the semen volume of each ejaculate was recorded using a graduated collection tube after removal of the gel mass and semen pH value was immediately de termined using a pH paper (Universalindikator pH 0 to 14 Merck, Merck KgaA, 64271 Darmstadt, Germany). Semen was maintained in a water bath (37°C) and then transferred to the laboratory for semen evaluation. Special attention was given to protect semen from cold or heat shocks and direct light. Throughout the course of semen collection, time and place of collection, and collector were kept constant.
Semen was diluted with saline (0.9% Nacl) at a rate of 1 semen: 10 saline, then percentage of sperm progressive mo tility was determined in five microscopic fields per semen sample using a phasecontrast microscope (Leica DM 500, Leica Mikrosysteme Vertrieb GmbH, Wetzlar, Germany) supplied with a hot stage at 37°C. Aliquots of raw semen (5 μL) were fixed using a vital stain of eosin (5%) and nigrosine (10%) to determine percentages of vitality, abnormality and normality in 200 spermatozoa in 5 microscopic fields by using phase contrast microscopy (Leica DM 500, Leica Mikrosys teme Vertrieb GmbH, Germany) at 400× magnification. Vital sperm cells (unstained sperm cells) and morphological  The hypoosmotic swelling test (HOSt) was used to evaluate the functional integrity of the sperm plasma cell membrane. The assay was performed by incubating a mix ture of 30 μL semen with 300 μL hypoosmotic solution (0.90 g fructose+0.49 g sodium citrate/100 mL of distilled water; osmolarity of 50 mOsm/L) in a water bath (37°C) for 30 minutes. Spermatozoa with swollen and curled tails (mem brane integrity), were considered responded to HOSt and were calculated in 200 sperm cells by using phase contrast microscopy (Leica DM 500, Leica Mikrosysteme Vertrieb GmbH, Germany) at 400× magnification. Sperm outputs as total (TSO), motile (MSO), normal (NSO), vital (VSO) and functional (FSO) calculated according to the following equations: TSO = net semen volume (mL) ×sperm cell con centration (×10 6 /mL); MSO = progressive motility %×TSO.

Fertility study
Total of 120 sexually mature APRI rabbit does were distrib uted into four groups. Does in each group (n = 30) were naturally mated with five bucks from each treatment (6 does/ buck) at 3 days mating interval. Reproductive criteria includ ing pregnancy and parturition rates and litter size at birth (total born and total born alive) and at weaning (on day 28 of age) were recorded, then viability rate of kits at birth and weaning was calculated. Pregnancy diagnosis was performed manually by abdominal palpation to calculate pregnancy rate (PR) using the following equation: PR = (number of pregnant does/number of mated does) ×100. After birth, parturition rate (PR) was calculated as the following equa tion: PR = (number of delivered does/number of pregnant does) ×100.

Analytical procedures
At the last week of semen collection, seminal plasma were separated by centrifugation at 1,500 rpm for 20 min and stored at -20°C, pending biochemical analysis. After col lection, concentration of initial fructose, total proteins (TP), albumin (AL), and total lipids were determined. However, globulin (GL) was calculated by subtracting the AL values from the corresponding TP values. Activity of aspartate aminotransferase (AST), alanine aminotransferase (ALT), alkaline phosphatase (ALP), and acid phosphatase (ACP) in seminal plasma were determined. In addition, total antioxi dant capacity (TAC), glutathione content (GSH), glutathione peroxidase (GPx), glutathione Stransferase (GST), super oxide dismutase (SOD), and malondialdehyde (MDA) were assayed. Concentration of biochemicals, enzyme activity and oxidative capacity were determined by using commer cial available kits (Biodiagnostic Co., Recycling Crusher SBM, www.Biodiagnostic.com) and spectrophotometer (Spectro UVVIS Auto, UV2602, Labomed, Los Angeles, CA, USA).
Blood samples were collected from five bucks in each group from ear vein into heparinized tubes and were placed immedi ately on ice box. Plasma was obtained by blood centrifugation at 3,000 rpm for 20 min and stored at -20°C until assaying the testosterone concentration by enzymeimmunoassay using commercial kits (BiosourceEurope S.A. 8, rue de L'Lndustrie. B1400 Nivelles, Belgium). The intra and interassay coeffi cients of variation were 7.8% and 8.4%, respectively. The minimum detectable limit was 0.1 ng/mL and the maxi mum limit was 18.0 ng/mL.

Statistical model and analysis procedure
Data were subjected to analysis of variance using general linear model procedure (GLM) of statistical analysis system SAS [19] (Cary, NC, USA).
The following statistical model was applied for analysis of all measurements Where, Y ij = Observations, μ = Overall mean, TRT = ef fect of ith antioxidant material (i, 1 to 4), e ij = random error. The differences between the control and antioxidants were investigated according to orthogonal comparisons, while odds ratio and 95% confidence intervals for pregnancy and parturition cases were determined according to binary logistic regression using the same program mentioned before. The dif ferences between treatment means were separated by Tukey's studentized range (HSD) test. The statistical significance was accepted at p<0.05. ShapiroWilk test was conducted in order to check for normality [20]. The association between natural antioxidants supplementation and each of pregnancy rate and parturition rate was detected by ChiSquare test (χ 2 ). Person correlation coefficients were done according to CORR Procedure within SAS program [19].

Performance and physiological response
Based on the results presented in Table 3, the effect of treat ment with different types of natural antioxidants (EVOO, BET, and GIN) was not significant (p>0.05) on the mean final LBW and feed intake of rabbit bucks during the experimental period. In comparing with other treatments and the control, only BET supplementation significantly (p<0.05) reduced rectal, skin and ear temperature degrees.

Libido of rabbit bucks
Results of Figures 1 and 2 show that the effect of natural antioxidants (EVOO, BET, and GIN) supplementation sig nificantly (p<0.05) decreased reaction time ( Figure 1) and significantly (p<0.05) increased blood plasma testosterone ( Figure 2) compared with the control. Correlation coefficient between testosterone and reaction time was significantly negative (r = -0.89, p<0.0001). This reflects positive impact of EVOO, BET, and GIN supplementation on improving the sexual desire of rabbit bucks, particularly GIN supple mentation.

Semen production
According to the results of Table 4, EVOO, BET, or GIN supplementation significantly (p<0.05) improved semen production, quantitatively and qualitatively. Net semen volume, percentages of progressive motility, vitality, intact acrosome and membrane integrity, sperm cell concentration, TSO, MSO, NSO, VSO, and FSO increased (p<0.05), while percentage of tail abnormality decreased (p<0.05) in treatments compared to the control. On the other hand, semen pH value increased Table 3. Effect of extra virgin olive oil, betaine, and ginger supplementations on body weight, feed intake and body temperature degrees of APRI rabbit bucks during the experimental treatment (n = 10 in each group)   (p<0.05) by GIN and BET, while, head abnormality percentage decreased (p<0.05) only by GIN supplementation as com pared to the control. In comparing the natural antioxidants treatments, GIN evoked the largest (p<0.05) improvement in semen production parameters, followed by BET and EVOO, respectively.

Biochemicals, enzyme activity and oxidative capacity in seminal plasma
The effect of dietary EVOO, BET, or GIN supplementa tion on biochemical constitutes, enzyme activity, and oxidative capacity in seminal plasma of rabbit bucks is presented in Table  5. The natural antioxidants (EVOO, BET, or GIN) supplemen tation increased (p<0.05) concentration of TP, GL, and initial * p-value of treatment was 0.012 according to one-way ANOVA analysis. fructose compared to the control. However, total lipids showed an opposite trend. Activity of AST and ALT was significantly (p<0.05) decreased, while ALP activity was significantly (p< 0.05) increased by EVOO, BET, or GIN supplementation in comparing with the control. Concentration of AL was sig nificantly (p<0.05) increased only by GIN compared with the control and other treatments. However, AL/GL ratio and ACP activity were not affected significantly (p>0.05) by EVOO, BET, or GIN treatment. In comparing all the natural antioxi dants studied, GIN evoked additional reduction (p<0.05) in total lipids, AST and ALT compared with EVOO and BET.
Respecting to the effect of the natural antioxidants addition on oxidative capacity (Table 5), it can be observed that the inclusion of EVOO, BET, or GIN significantly (p<0.05) in creased levels of GSH and GST, and significantly (p<0.05) decreased MDA level as compared to the control. While, TAC level significantly (p<0.05) increased by BET and GIN supplementation compared with EVOO and the control. However, GPX and SOD were not affected significantly (p> 0.05) by the natural antioxidants additives.

Fertility
Results illustrated in Figure 3 clearly indicated that pregnan cy rate of does mated by bucks treated with BET and GIN significantly (p<0.05) increased compared with EVOO and the control. Meanwhile, parturition rate showed nonsignifi cant differences, although does mated by bucks treated with GIN showed higher parturition rate than the control, followed by BET and EVOO due to the differences in the numbers of pregnant rabbit does between different treatments. Cal culated values for odds ratios indicated that pregnancy cases of does mated by bucks treated with EVOO, BET, and GIN were higher 1.3, 4.0, and 5.0 times than the control, respec tively. The corresponding values of parturition cases were higher 1.63, 3.5, and 6.3 times than the control (Table 6). Total and live litter size at birth as well as litter size at wean ing significantly (p<0.05) improved in rabbit does mated by bucks received dietary natural antioxidants compared to the control, but nonsignificant differences were observed be tween GIN and BET treatments. Generally, does mated by bucks treated with GIN showed the highest litter size at birth and weaning. Viability rate at birth was significantly (p<0.05) improved only for kits produced by does mated by bucks in GIN treatment, while viability rate at weaning was not affect ed significantly (p>0.05) by treatment (Table 7).

DISCUSSION
In tropical and subtropical regions, alleviation of the deleteri ous effects of HS is the most challenges of animal production. The HS can evoke multiple biological and physiological re sponses [2]. Under HS conditions, reactive oxygen species were increased, while antioxidant capacity decreased leading to oxidative stress [21]. Oxidative stress negatively affects semen quality and sperm function, as a result of lipid per oxidation in the plasma membrane [4]. The HS negatively influences the testicular function, which may suppress tes tosterone production [5] lead to reduction in quality and  Table 7 for doe numbers of total, mated, and delivered does). * p-value was 0.009 for pregnancy rate and 0.167 for parturition rate according to χ 2 -test.  Table 7 for doe numbers of total, mated, and delivered does).
* p-value was 0.009 for pregnancy rate and 0.167 for parturition rate according to χ2 -test.  fertility of sperm cells. Damage of sperm DNA and decreased motility, membrane integrity, antioxidant defense system and fertility are the main causes of oxidative stress [10]. In the rabbit production, productivity of animals was affected by HS, which is considered as an important stressor [8].
According to the climatic conditions in the environment of rabbit bucks in our study, the calculated THI value indi cates that the experimental bucks were under HS, being severe during JuneAugust and moderate in September. The supplementation of natural antioxidants is essential to relief the adverse effects of HS [15]. In our study, the hypothesis is that HS negatively affects physiological responses, antioxidant capacity, immunity, semen quality and fertility of rabbit bucks, and supplemen tation of natural antioxidants such as EVOO, BET, or GIN may show promising relieving effects in improving semen quality and sperm fertility of rabbit bucks. Antioxidant sup plementation improved the sexual desire of rabbit bucks, which was depressed by HS in the control bucks, through suppress testosterone production, causing destroy Leydig cell function [22]. In this concern, antioxidant supplemen tation increased testosterone profile and reduced reaction time by improving the testosterone synthesis [23]. In addition, antioxidant administration (EVOO, BET, or GIN) improved semen production, quantitatively and qualitatively, including net semen volume, motility, vitality and normal morphol ogy of spermatozoa, acrosomal and membrane integrities, sperm cell concentration, as well as TSO, MSO, NSO, VSO, and FSO, which adversely affected by HS in rooster [4].
This improvement was mainly related to their properties as natural antioxidants. In this respect, all EVOO, BET, and GIN decreased MDA level as lipid peroxidation marker, while increased TAC in term of GSH and GST contents in the seminal plasma. These antioxidants act as ROS scaven gers within the testicular tissues by decreasing the oxidative damage in tissues through the protective effects of antioxi dant enzymes, which play an important modulatory role against endogenous oxidative damage [10]. It is worthy not ing that natural antioxidants supplementation (EVOO, BET, or GIN) improved the chemical composition of the seminal plasma from TP, AL, GL and initial fructose, while decreased total lipids, AST and ALT, which may has a vital role in func tion and metabolism of sperm cells. Similar results were reported by ElSpeiy et al [10], who found that dietary treat ment with natural antioxidant (0.5% or 1% GIN) resulted in higher TP, AL, and GL levels, and lower AST and ALT activity in rabbit seminal plasma. The observed reduction in AST and ALT activities in seminal plasma of treated bucks is in association with improving membrane integrity of sperma tozoa [24].
The obtained results indicated the highest antioxidant properties for GIN administration in comparing with EVOO and BET. In this context, GIN extract contain different active phytochemical components such as volatile oils, gingerol, gingerone, piperine, shogaols,acid, terpenoids, and zinge rone [9]. It contains polyphenols (flavonoids and flavones glycosides) and antioxidants like βcarotene, ascorbic acid, terpenoids, and alkaloids [10]. According to these properties, increased TAC level and decreased MDA level [11] in rabbit seminal plasma were observed due to the effect of GIN as natural antioxidant. Generally, GIN increased the testicular antioxidant enzymes activities (SOD, catalase, and GPx) and has protective effects against the oxidative stress and testicular damage [25]. The present results are in accordance with El Speiy et al [10], who reported significant decreased in reaction time from 15.9 to 6.9 s in rabbit bucks, fed diet supplemented with GIN powder. Orally administration of rabbit bucks with GIN aqueous extract increased testosterone level from 3.45 to 6.29 ng/mL and increased sperm production in terms of increased concentration, motility and normality of sperma tozoa [11].
The mechanisms through which GIN enhances testos terone production as an androgenic agent, are mainly by improving the activities of 3βhydroxysteroid dehydrogenase, 17αhydroxylase and 17, 20 lyase, and 17βhydroxysteroid dehydrogenase in the testis [26]. Also, GIN increase LH release via increasing the level of cholesterol and reducing lipid peroxidation in the testis. Furthermore, GIN normalizes the blood glucose, enhances nitric oxide production, and increases the blood flow into Leydig cells, besides increasing the testicular weight, and recycling testosterone receptors [25]. This action leads to an increasing testosterone profile, which has important role in maintenance of libido and im provement of semen production in rabbit bucks.
It is worthy noting that improvement in sexual desire and semen quality of rabbit bucks treated with BET was associ ated with increasing the physiological response by reducing body temperature degrees (RT, ST, and ET) which suggest the action of BET in heat regulation. In this respect, BET has been found to reduce body temperatures estimates (RT, ST, and ET) in growing rabbits [27] under a severe heat load. BET may maintain the thermoneutral state of animals by revers ing the heatinduced inhibition of the osmotic equilibrium and maintaining the tertiary structures of macromolecules in the kidney [28].
In parallel with the effect of BET and EVOO on semen production in our study, BET is reported to have antioxidant activity, which may protect different stages of spermatocytes from apoptosis, leading to an increase in sperm quality in rooster [4]. It is indicated that BET plus Vitamin C and E improved oxidative capacity (TAC and MDA) in rooster chickens reared under HS conditions [4]. In addition, olive oil contains omega3 polyunsaturated fatty acids [12] and has beneficial bioactive components (flavonoids and poly phenols) as bioactive organic molecules. These compounds have antimicrobial, antioxidant and antiinflammatory prop erties [13]. The antioxidant status in the testes of rat were improved in term of increased TAC and decreased MDA level by olive leaves extract [12]. These properties of BET and EVOO may explain the improvement occurred in semen quality of rabbit bucks in our study under HS conditions. High motility, normality and concentration of spermatozoa have been associated with the improving fertility rate [29]. Also, total litter size was influenced significantly by num ber of motile sperm output. Most of sperm parameters are essential for sperm fertilizability in mammals. Therefore, the foregoing results of rabbit does mated by bucks treated with EVOO, BET, and GIN, concerning the improved se men quality and antioxidant status, reflect improving litter size (total and live) as compared to control bucks. These results are proved by ElSpeiy et al [10], when bucks were supplemented with GIN. The positive effect of GIN as an enhancer of reproductive capacity of rabbit bucks may be due to its ability to protect mammal cells from oxidation as showed by Ulkowski et al [30].

CONCLUSION
In spite of dietary supplementation of EVOO, BET, or GIN can improve oxidative stress, sexual desire and semen quality of rabbit bucks, it is found that the GIN supplementation provides the best impact. Based on the obtained results, GIN may assist rabbit bucks to counter negative impacts of oxida tive stress, given the increase of motility, vitality, normality, acrosomal and membrane integrities, functional sperm out put and fertility as well as improving biochemical constitutes and antioxidant status. Therefore, dietary supplementation with GIN at a level of 200 mg/kg is recommended as a valu able strategy for enhancing the reproductive efficiency of breeding rabbit bucks used in natural mating or artificial in semination.