Dietary quercetin maintains the semen quality in rabbits under summer heat stress

Accepted Manuscript Dietary quercetin maintains the semen quality in rabbits under summer heat stress Zahid Naseer, Ejaz Ahmad, Hande Sultan Şahiner, Erkmen Tuğrul Epikmen, Muhammad Fiaz, Muhamad Rizwan Yousuf, Shahzad Akbar Khan, İlker Serin, Ahmet Ceylan, Melih Aksoy PII:

S0093-691X(18)30785-4

DOI:

10.1016/j.theriogenology.2018.09.009

Reference:

THE 14694

To appear in:

Theriogenology

Received Date: 1 February 2018 Revised Date:

11 September 2018

Accepted Date: 11 September 2018

Please cite this article as: Zahid Naseer, Ejaz Ahmad, Hande Sultan Şahiner, Erkmen Tuğrul Epikmen, Muhammad Fiaz, Muhamad Rizwan Yousuf, Shahzad Akbar Khan, İlker Serin, Ahmet Ceylan, Melih Aksoy, Dietary quercetin maintains the semen quality in rabbits under summer heat stress, Theriogenology (2018), doi: 10.1016/j.theriogenology.2018.09.009 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

ACCEPTED MANUSCRIPT

Revised non-highlighted

Dietary quercetin maintains the semen quality in rabbits under summer heat stress

RI PT

Zahid Naseera, d, *, Ejaz Ahmade, Hande Sultan Şahinerb, Erkmen Tuğrul Epikmenc, Muhammad Fiazd, Muhamad Rizwan Yousuff, Shahzad Akbar Khang, Đlker Serina, Ahmet Ceylana, Melih Aksoya, a

M AN U

SC

Department of Reproduction and Artificial Insemination, b Department of Pharmacology and Toxicology, c Department of Pathology, Faculty of Veterinary Medicine, Adnan Menderes University, Aydin, Turkey d Faculty of Veterinary and Animal Sciences, Pir Mehr Ali Shah Arid Agriculture University, Rawalpindi, Pakistan e Department of Clinical Sciences, Faculty of Veterinary Sciences, Bahauddin Zakariya University, Multan, Pakistan f Department of Theriogenology, University of Veterinary and Animal Sciences, Lahore, Pakistan g Faculty of Veterinary and Animal Sciences, The University of Poonch, Rawalakot, Azad Kashmir, Pakistan

*

TE D

Corresponding Author. E.mail: [email protected]

ABSTRACT

EP

Running title: Quercetin protects rabbit sperm against heat stress

AC C

This study focused to determine beneficial impact of feeding quercetin supplemented diet on semen quality in summer heat imposed rabbits. Twelve heat stressed (HS) adult rabbits bucks were either fed with basal diet (HS; n = 06) or quercetin supplemented diet (QU-HS; n = 06) for a period of 56 days. Semen samples were collected and evaluated for volume, osmolality, morphology, concentration, motility, motion kinetics, viability, acrosome integrity, mitochondrial potential, and seminal plasma MDA level. Semen volume, concentration, motility and sperm kinetics parameters were affected by diet supplementation. Diet affected the sperm mitochondrial potential and day of treatment affected the viable sperm percentage, whereas, there was an effect of diet, day of treatment and diet by day interaction (P < 0.05) on acrosome reaction rate. Sperm head abnormalities were influenced diet provision, sperm mid-piece abnormalities were affected by diet and day of treatment, whereas, a diet effect and

ACCEPTED MANUSCRIPT day by treatment interaction was observed for total sperm abnormalities. There was an effect of diet and diet by day interaction for seminal plasma MDA level. In conclusions, quercetin reduces the damaging effects of HS and maintains the semen quality by lowering the oxidative stress in rabbits. Keywords: Quercetin, Heat stress, Sperm quality, Rabbit

RI PT

1. Introduction The male reproductive soundness is influenced by several stress factors like disease, toxins and climate. The heat stress (HS) is the main factor that elicits notable alterations in testes which ultimately insult sperm structural and functional integrity [1,2]. The altered testicular

SC

texture/weight, low sperm output, reduced sperm quality or increased sperm morphologies, are prominent anomalies in HS mammals. Moreover, the spermatocytes and spermatids have great susceptibility to HS and readily undergo apoptosis and DNA fragmentation [2].

M AN U

However, the sperm morphology is not affected abruptly by HS because it is linked with duration of HS exposure and spermatogenesis cycle. The HS male has capacity to produce viable sperm but these sperm could produce lower number of embryo or high proportion of abnormal offspring due to high incidence of mutations and DNA damage [3]. HS induced changes are totally reversible [4,2]; therefore, by adopting certain management practices, the

TE D

improvement in sperm parameters during HS months can be ensured. Quercetin is well-known antioxidant amongst the flavonoids and largely present in numerous fruits. It scavenges the free reactive oxygen species (ROS) by suppressing lipid peroxy radical synthesis, metal ion chelating through enzyme inhibition and adopting the

EP

repair mechanisms. It also contributes well to maintain endogenous antioxidant defence system of the cell. Its antioxidant capacity relies on the particular chemical structure with the

AC C

phenolic hydroxyl groups located at the B-ring and the three positions providing its free radical-scavenging activity [5]. The antioxidant capacity of quercetin has been tested for providing protection to sperm during chilling and freezing process [6,7]. A protective role of quercetin against different stressors [8,9] and as an antitoxic property on sperm quality and testicular alterations [10-14] have been documented. In view of above mentioned protective role of quercetin against the different stressors, it has been hypothesized that quercetin provision could be an ameliorative strategy for the sperm against the heat-induced damages. Therefore, the current study was carried out to determine the beneficial impact of feeding quercetin supplemented diet on sperm quality in summer heat imposed rabbit bucks.

ACCEPTED MANUSCRIPT

2. Materials and methods 2.1. Location The study was carried out at the Department of Reproduction and Artificial Insemination, Faculty of Veterinary Medicine, Adnan Menderes University, Aydin, Turkey (37°43’42N 27°56’14’E), during July to September, 2015. An approval was sanctioned by the Animal

RI PT

Ethics Committee of Adnan Menderes University (ADÜ-HADYEK No. 64583101/2014/153) to use rabbits for this study. 2.2. Temperature humidity index calculation

The daily ambient temperature and relative humidity were recorded and temperature-

SC

humidity index (THI) was calculated by the equation described by Marai et al. [15]. 2.3. Animals

Twelve male White New Zealand rabbits, age 10-12 month, body weight (2.9±0.1) and

M AN U

regular semen donors, were used in the present study. The bucks were caged individually in wire enclosures with a provision of normal daylight (16-17 h). All the rabbits were spaced in the experimental area for 1 week to acclimatize the heat conditions. The experimental rabbits were fed either a basal diet (HS; n = 06) or the basal diet supplemented with quercetin (QUHS; n = 06; Quercetin hydrate, 337951, Sigma-Aldrich, St. Louis, USA; 30mg/kg/BW) for a

TE D

period of 08 weeks. The feed and fresh clean water were provided ad libitum. The basal diet was formulated according to the recommended values [16; Table 1]. 2.4. Semen collection

The semen samples from each rabbit buck were collected on Day 0, 15th, 30th, 48th and

EP

56th during the feeding trial. The semen was collected through artificial vagina by exposing a matured cyclic doe to the bucks as a teaser at collection time.

AC C

2.5. Semen evaluation

Reaction time was recorded as a spell between the introductions of teaser doe in the pen to mount. After collection, the semen volume for each ejaculate was recorded in microliters (µL) and, then, a semen sample of 50 µL was submitted to determine the osmolality using freezing-point depression osmometer (Osmomat-3000, Gonotec, Germany). The sperm concentration was evaluated using standard Thoma chamber and denoted in million per milliliter. The semen samples were diluted with TCG [Tris 276.5mM (hyrdoxymethyl)-aminomethane, Glucose 76.8mM and Citric acid 90.9mM] solution. The motility assessment was performed by using CASA system (SCA®-Sperm Class Analyser, Microptic S.L. Viladomat, Barcelona,

ACCEPTED MANUSCRIPT Spain) connected with a phase contrast microscope (Olympus, CX41, Japan). The particle size was set 10 to 80 micron to focus the sperm head. Multiple fields (up to 100 sperms/field at ×10) in less than 2 seconds/field were analyzed. Different fields were selected over the slide and at least 500 sperm were recorded for motility characteristics in each sample. The measured variables included i.e. Total motility (TM, %); Progressive motility (PM, %);

RI PT

Average Path Velocity (VAP, µm/s); Straight Line Velocity (VSL, µm/s); Curvilinear Velocity (VCL, µm/s); Amplitude of Lateral Head displacement (ALH, µm); Beat Cross Frequency (BCF, Hz); Straightness (STR, %) and Linearity (LIN, %).

Sperm viability was determined by using propidium iodide (PI) staining procedure. A

SC

sample of 50 µL diluted semen was incubated with 2.5 µL (5 µL; 200µg/mL stock solution) PI (Propodium iodide, P4170 Sigma-Aldrich, St. Louis, USA) for 5 min at 37 °C. A fixative (2.5 µL; 4% Paraformaldehyde dissolved in PBS) was added to halt the sperm motion. A

M AN U

minimum of 200 sperm were tallied under fluorescence microscope. The cells exhibit the red fluorescence, were considered nonviable with damaged plasma membrane, whereas, viable sperm with normal plasma membrane do not emit any florescence. To determine acrosome integrity, diluted semen samples (50 µL) were incubated with Arachis hypogea (peanut) agglutinin Fluorescein isothiocyanate conjugate [(5 µL; 200µg/mL

TE D

stock solution); FITC-PNA, L7381 Sigma-Aldrich, St. Louis, USA) for 5 min at 37 °C. After incubation, the samples were fixed using 4% paraformaldehyde solution. This stain intensely labels the acrosomal region of acrosome-reacted sperm and emits a uniform apple-green fluorescence while acrosome-intact spermatozoa do not show any fluorescence at the

EP

acrosome region.

The mitochondrial activity in the mid-piece of sperm was determined using the

AC C

mitochondrial-specific dye rhodamine 123 (Rh123; R8004 Sigma-Aldrich, St. Louis, USA). Briefly, 10 µL of Rh123 (0.01mg/mL diluted in PBS) was added to 500 µL diluted semen sample aliquots and samples were left for incubation for 30 min at 37°C in water bath. A fixative solution (4% formaldehyde) was added to the incubated sample to check the sperm motility. A wet mount was prepared and observed about 200 sperm per sample immediately under fluorescence microscopy. Sperm with bright fluorescence at the mid-piece were considered to have high mitochondrial activity, whereas, the sperm with no or little fluorescence were supposed to have low mitochondrial activity at the mid-piece region. Briefly, the percentage of morphologically abnormal sperm was determined by fixing a 25 µL portion of each fresh sperm sample in 250 µL of Hancock’s solution. A drop of 5 µL was placed over the slide and observed at ×100 magnification under phase contrast microscope.

ACCEPTED MANUSCRIPT Counted 200 sperm from each sample were differentiated for head, mid-piece and tail abnormalities. These abnormalities were numbered and classified as acrosomal changes (missing or knobbed acrosome), head abnormalities (pyriform, micro, macro or elongated head), mid-piece (double or retroaxial), proximal or distal cytoplasmic droplet, and tail defects (curved, coiled, dag defect or double tail).

RI PT

2.6. Seminal plasma MDA estimation The semen ejaculates were centrifuged (1000×g, 10min) and supernatant (seminal plasma) was collected. The seminal plasma samples were kept at -20°C until assayed. MDA was determined by Thiobarbituric Acid (TBA) assay. The MDA-TBA reaction has been used to

SC

determine the lipid peroxidation extent in biological material. The reaction gives a red MDATBA complex and this colored is evaluated through spectrophotometrically (Shimadzu UV1601, Kyoto, Japan) at 532 nm. The values of MDA are presented in nmol/mL.

M AN U

2.7. Statistical analyses

The data were analyzed using Statistical Analysis Software (SAS version 9.1, Cary, NC, USA). Data were tested for normality (UNIVARIATE procedure) and physical and microscopic sperm variables at different time periods between control and quercetin supplemented groups were analyzed using repeated measures ANOVA. Diet, day of

TE D

sampling, sampling day and diet interactions, were included in the model. Animal (rabbits) was termed as the experimental unit and sampling time (day of semen collection) was considered as a repeated term in the statistical model. A level P < 0.05 was considered as the significance. The data were expressed as mean ± standard error (S.E).

EP

3. Results

No significant (P > 0.05) change was observed in ambient temperature and humidity

AC C

during the trial. The average temperature, relative humidity, and THI were 33.5 ± 1.7 °C, 49.5 ± 5.7 % and 29.9 ± 1.2, respectively. The recorded THI value (29.9 ± 1.2) indicated the severe heat stress conditions for rabbits. In addition, there was no (P > 0.05) difference between the body weights of rabbits at the start (2.9 ± 1.0) or end of the trial (3.1 ± 1.0). The average reaction time (QU-HS; 5.4 ± 2.3 vs. HS; 4.2 ± 1.2) and semen osmolality (QU-HS; 368.5 ± 47.2 vs. HS; 400.1 ± 29.6) did not change (P < 0.05) between the QU-HS and HS rabbits throughout the study duration. The effects of dietary quercetin supplementation on semen quality from heat stressed rabbits are presented in Table 2. There was diet effect (P < 0.05) on semen volume, sperm concentration (day 48 and 56) and progressive motility (day 48) in QU-HS groups. The diet

ACCEPTED MANUSCRIPT and day of treatment affected (P < 0.05) the total motility; however, no significant change in total sperm motility pattern in QU-HS groups was recorded. Neither diet by day interaction nor effect of day of treatment on sperm kinetics was observed. The sperm kinetic parameters (VCL, VSL, and VAP) were comparatively higher (P < 0.05) in QU-HS group at day 48 due to the diet effect. There was no change in sperm kinetics (VCL, VSL and VAP) within HS

RI PT

groups, however, these parameters were improved (P < 0.05) within QU-HS groups at day 48. The values for LIN, STR, WOB, ALH and BCF were similar between the groups and among the point of observations (Table 2).

There was an effect of diet, day of treatment and diet by day interaction (P < 0.05) on

SC

acrosome reaction rate at day 48 and 56 in QU-HS. The day of treatment affected the proportion of viable spermatozoa (P < 0.05) at day 30, 48 and 56. Similarly, there was effect (P < 0.05) of diet on sperm mitochondrial potential at day 30, 48 and 56 of observation in

M AN U

QU-HS. However, the effect of day of semen collection and diet by day interaction was nonsignificant.

There was an effect of diet (P < 0.05) on head and total abnormalities at day 48 and 56, whereas, both diet and day of treatment have effect (P < 0.05) on mid-piece abnormalities at day 30 and 48.

TE D

There was an effect of diet and diet by day interaction for seminal plasma MDA level. In QU-HS rabbits, the seminal plasma MDA concentration was higher (P > 0.05) than HS rabbits at the start of trail and lower (P > 0.05) on day 48 and 56 of trial (Fig. 1).

4. Discussion

EP

The present study examined the effect of dietary quercetin on semen characteristics in rabbits during summer months. The semen volume, sperm concentration, sperm motility,

AC C

viability, acrosome integrity and sperm morphology, tended to improve with supplementation of quercetin during extended period. To the best of our knowledge, this is the first available report in the literature, evaluating dietary quercetin provision on sperm quality during hot summer months.

In the present study, the semen volume and sperm concentration improved after the quercetin supplementation in HS rabbits. Similar pattern was observed in the earlier studies where HS animals were fed different dietary supplements [17-20]. Association of high environmental temperature and low semen volume is well-known phenomenon in HS rabbits which is exacerbated by low activity of accessory glands under lower testosterone level [15] or increased ROS production in accessory glands [21]. Improved seminal volume upon

ACCEPTED MANUSCRIPT quercetin supplementation, ascertain the ROS scavenge role of quercetin in the accessory glands. Moreover, the improvement of sperm concentration under heat stress also signify the antioxidant role of quercetin and similar observations have been reported in earlier studies by using various supplements [17-20]. It is well reported that reduced motile sperm subpopulation in fresh ejaculates under HS

RI PT

[1,22-25] is linked to lower mitochondrial activity induced by excessive ROS production [26]. Additionally, excessive ROS production decreases motility by lowering axonemal protein phosphorylation and sperm immobilization [27] that further hinders the activity of glucose-6- phosphate-dehydrogenase in sperm which play a role to maintain the glucose level

SC

for motility [28]. In the present study, the motility pattern was significantly maintained by the quercetin supplementation in heat imposed rabbits. In earlier studies, positive [17,20] or no [18,19] impact of supplements have been observed under HS. The improved motility rate

M AN U

might be linked to the activity of endothelial nitric oxide synthase, elicited by dietary antioxidants supplementation [29] or ROS scavenging effect of quercetin [10]. In addition, the enhancement of the sperm motility could be ascribed to the excessive release of fructose in semen during sperm transfer from testis to ejaculation site [17,30]. A negative impact on sperm viability has been observed in heat exposed rabbits. The

TE D

similar fashion has been documented in rabbits during short or long-term HS exposure [1,2325]. In the present study, viable sperm percentage increased after four weeks quercetin supplementation. It has been reported that use of dietary antioxidants agent like pomegranate peel [17], ascorbic acid and betaine [31], royal jelly [20], selenium and folic acid [19],

EP

vitamin E and propolis [18], mitigate the sperm against the damaging effects of excessive ROS produced during HS. Higher proportion of polyunsaturated fatty acids and intrinsic

AC C

antioxidant capacity of sperm, make it more prone to the ROS damage; therefore, plasma membrane next to the acrosome or tail region is protected by antioxidants present in seminal plasma [32]. The maintenance of sperm membrane and DNA integrity against excessive ROS [33] could be brought about by quercetin intake under stress conditions. In the present study, a significant improvement in acrosome integrity by quercetin supplementation during HS, was evident its antioxidant property. Maya-Soriano et al. [1] have documented that chronic heat stress had no influence on acrosome integrity due to the adaptation of animals to the heat stress. On the other hand, most of the authors reported that HS induce the acrosome reaction in a great extent [23-25]. The elevated ambient temperatures influence the epididymis milieu which in turn hasten the lipid peroxidation rate

ACCEPTED MANUSCRIPT and alter the content of polyunsaturated fatty acids and lipoproteins of sperm plasma membranes that enforce the sperm to undergo the premature acrosome reaction [34]. High ambient temperatures significantly influence the functional potential of mitochondria owing to the excessive production of ROS. Earlier report also denotes the detrimental effect of ROS on the mitochondria structure under high temperature [35]. Generally, the

RI PT

mitochondria do not contain the GSH [36] that’s why the susceptibility of mitochondrial structure to ROS increased twice during the stress. It has been reported recently that quercetin localize in mid-piece of sperm that help to maintain the integrity of sperm as well as mitochondrial machinery [37] In the present study, an improvement in mitochondrial activity

SC

has been observed in HS rabbit sperm when the quercetin was supplemented in diet and the increment in mitochondrial activity by quercetin is indicative of its antioxidant characteristic. Alike to the other rabbit sperm parameters, sperm morphological abnormalities are also

M AN U

greatly influenced by high ambient temperature in the present study. This observation has a harmony with previous studies [24,25,38]; however, few reports [1,38] inferred that longer HS exposure has no influence on sperm morphology as observed during the short exposure. It has been suggested that chronic heat exposure might be related to a defending mechanism, which can be termed as an adaptation to higher temperatures [38]. A modulatory effect of

TE D

quercetin on sperm abnormalities has been inferred. A similar action of royal jelly [20], selenium and folic acid [19] have been observed on HS rabbit sperm morphology. The sperm morphological abnormalities occur during the course of maturation process ‘between caput epididymis to the cauda epididymis’. The caput epididymis is rich in blood supply and

EP

incidence of ROS is also increased under stress condition; therefore, a well-protected mechanism to combat the excessive ROS is required. In this context, the incorporation of quercetin could be ameliorative strategy against the oxidative stress by increasing

AC C

intracellular GSH, directly reducing the ROS level, and preventing the influx of Ca+2 under high ambient temperature.

The higher volume in quercetin supplemented group indicate the protective effect of quercetin on seminal gland that in turn contributed the higher seminal volume level after three week supplementation. Increase sperm concentration with normal sperm after six weeks of quercetin supplementation provide a clue influence at spermatogenesis level. Sustained motility percentages along higher rate of viable with intact mitochondrial sperm and acrosomal integrity after six to seven week of quercetin provision clearly indicate its beneficial impact of spermatogenesis and protection in epididymis against harmful effects of oxidation elicited by HS.

ACCEPTED MANUSCRIPT The results of discussed study suggest that dietary quercetin is beneficial for male rabbit reproductive efficiency during hot summer months. Most probably, the improved sperm quality in term of motility, viability, acrosome integrity, mitochondrial activity, sperm morphology, seminal volume, sperm concentration in supplemented animals, coupled with the heat stress; ensure the antioxidant activity of quercetin. The current findings provide a

RI PT

relatively simple and economical strategy to extending the reproductive efficiency of animals under heat stress. It is also possible that the introduction of some unknown factors, other than the antioxidant activity, which play significant role in cells protection, mediated by dietary quercetin. Therefore, the further investigations regarding the molecular mechanisms by which

SC

quercetin modulates germ cell growth, intracellular signaling and cell differentiation under stress, are required.

Conclusions

M AN U

In conclusion, HS deteriorate sperm quality in exposed male rabbits and, intake of quercetin, which has antioxidant characteristics, maintains the semen quality against HS induced damages by lowering the oxidative stress.

Conflict of interest Acknowledgement

TE D

The authors declare that there are no conflicts of interest.

This work was supported by ADU-BAP, Project number VTF-15034, Adnan Menderes University, Aydin, Turkey. Thanks extended to Scientific and Technological Research

Ph.D. program.

AC C

References

EP

Council of Turkey (TUBITAK-BIDEB 2215) for providing a fellowship to Zahid Naseer for

1. Maya-Soriano, M. J., Taberner, E., Sabés-Alsina, M., Ramon, J., Rafel, O., Tusell, L., Piles, M., López-Béjar, M., 2015. Daily exposure to summer temperatures affects the motile subpopulation structure of epididymal sperm cells but not male fertility in an in vivo rabbit model. Theriogenology 84, 384–389. 2. Pei, Y., Wu, Y., Cao, J., Qin, Y., 2012. Effects of chronic heat stress on the reproductive capacity of male Rex rabbits. Livestock Sci. 146, 13-21. 3. Yaeram, J., Setchell, B. P., Maddocks, S., 2006. Effect of heat stress on the fertility of male mice in vivo and in vitro. Reprod. Fertil. Dev. 18, 647–653. 4. Sabés-Alsina, M., Planell, N., Torres-Mejia, E., Taberner, E., Maya-Soriano, M.J., Tusell, L., Ramon, J., Dalmau, A., Piles, M., Lopez-Bejar, M., 2015. Daily exposure

ACCEPTED MANUSCRIPT

AC C

EP

TE D

M AN U

SC

RI PT

to summer circadian cycles affects spermatogenesis, but not fertility in an in vivo rabbit model. Theriogenology 83, 246–252. 5. Nimse, S. B., Pal, D., 2015. Free radicals, natural antioxidants, and their reaction mechanisms. RSC Adv. 5, 27986-28006. 6. Seifi-Jamadi, A., Ahmad, E., Ansari, M., Kohram, H., 2017. Antioxidant effect of quercetin in an extender containing DMA or glycerol on freezing capacity of goat semen. Cryobiology 75, 15-20. 7. Johinke, D., de Graaf, S. P., Bathgate, R., 2014. Quercetin reduces the in vitro production of H2O2 during chilled storage of rabbit spermatozoa. Anim. Reprod. Sci. 151, 208–219. 8. Moretti, E., Mazzi, L., Terzuoli, G., Bonechi, C., Iacoponi, F., Martini, S., Rossi, C., Collodel, G., 2012. Effect of quercetin, rutin, naringenin and epicat-echin on lipid peroxidation induced in human sperm. Reprod. Toxicol. 34, 651–657. 9. Ben Abdallah, F., Zribi, N., Ammar-Keskes, L., 2011. Antioxidative potential of Quercetin against hydrogen peroxide induced oxidative stress in spermatozoa in vitro. Andrologia 43, 261-265. 10. Adedara, I,A., Subair, T.I., Ego, V.C., Oyediran,O., Farombi, E.O., 2017. Chemoprotective role of quercetin in manganese-induced toxicity along the brainpituitary-testicular axis in rats. Chemico-Biological Inter. 263, 88-98. 11. Nna V.U., Ujah, G.A., Mohamed, M., Etim, K.B., Igba B.I., Augustine, E.R., Osim, E.E., 2017. Cadmium chloride–induced testicular toxicity in male wistar rats; prophylactic effect of quercetin, and assessment of testicular recovery following cadmium chloride withdrawal. Biomed. Pharmacother. 94, 109–123. 12. Altintas, R., Ciftci. O., Aydin, M., Akpolat, N., Oguz, F., Beytur, A., 2015. Quercetin prevents docetaxel-induced testicular damage in rats. Andrologia 47, 248–256. 13. Bharti, S., Misro, M. M., Rai, U., 2014. Quercetin supplementation restores testicular function and augments germ cell survival in the estrogenized rats. Mol. Cell. Endocrinol. 383, 10–20. 14. Aldemir, M., Okulu, E., Kosemehmetoglu, K., Ener, K., Topal, F., Evirgen, O., Gurleyik, E., Avcı, A., 2014. Evaluation of the protective effect of quercetin against cisplatin-induced renal and testis tissue damage and sperm parameters in rats. Andrologia 46, 1089–1097. 15. Marai, I. F. M., Habeeb, A. A. M., Gad, A. E., 2002. Rabbits’ productive, reproductive and physiological performance traits as affected by heat stress: A review. Livestock Prod. Sci. 78, 71-90. 16. NRC. National Research Council. Nutrient Requiremnts of Rabbits. Nutrient Requirement of Dometsic Animals. Second Edition. National Academy of Sciences, Washington D.C., 1977. 17. Zeweil, H. S., Elnagar, S., Zahran, S. M., Ahmed, M. H., El-Gindy, Y., 2013. Pomegranate peel as a natural antioxidant boosts bucks’ fertility under Egyptian summer conditions. World Rabbit Sci. 21, 33-39. 18. Hashem, N. M., Abd El-Hady, A., Hassan, O., 2013. Effect of vitamin E or propolis supplementation on semen quality, oxidative status and hemato-biochemical changes of rabbit bucks during hot season. Livestock Sci. 157, 520–526.

ACCEPTED MANUSCRIPT

M AN U

SC

RI PT

19. El‐Tohamy, M. M., Kotp, M. S., El‐Nattat, W. S., Mohamed, A. H., 2012. Semen characteristics and oxidative/antioxidative status in semen and serum of male rabbits supplemented with antioxidants during heat stress. Iranian J Appl. Anim. Sci. 2, 175183. 20. Elnagar, S. A., 2010. Royal jelly counteracts bucks’ “summer infertility”. Anim. Reprod. Sci. 121, 174–180. 21. Alshahrani, S., McGill, J., Agarwal, A., 2013. Prostatitis and male infertility. J. Reprod. Immunol. 100, 30-36. 22. Sabés-Alsina, M., Tallo-Parra, O., Mogas, M.T., Morrell, J. M., Lopez-Bejar, M., 2016. Heat stress has an effect on motility and metabolic activity of rabbit spermatozoa. Anim. Reprod. Sci. 173, 18-23. 23. Safaa, H. M., Emarah, M. E., Saleh, N. F. A., 2008. Seasonal effects on semen quality in black Baladi and white New Zealand rabbit bucks. World Rabbit Sci. 16, 13–20. 24. Okab, A. B., 2007. Semen characteristics and plasma testosterone of New-Zealand male rabbits as affected by environmental temperatures. Slovak J. Anim. Sci. 40, 161–167. 25. Roca, J., Martınez, S., Orengo, J., Parrilla, I., Vazquez, J. M., Martınez, E. A., 2005. Influence of constant long days on ejaculate parameters of rabbits reared under natural environment conditions of Mediterranean area. Livestock Prod. Sci. 94, 169–177. 26. Agarwal, A., Makker, K., Sharma, R., 2008. Clinical relevance of oxidative stress in male factor infertility: an update. Amr. J. Reprod. Immunol. 59, 2-11.

AC C

EP

TE D

27. de Lamirande, E., Gagnon, C., 1995. Impact of reactive oxygen species on spermatozoa, a balancing act between beneficial and detrimental effects. Human Reprod. 10, 15-21. 28. Liu, J., Wang, Y., Gong L., Sun C., 2015. Oxidation of glyceraldehyde-3-phospahte dehydrogenase decreases sperm motility in diabetes mellitus. Biochem. Biophysic. Res. Commun. 465, 245-248. 29. Donnelly, E. T., Lewis, S. E. M., Thompson, W., Chakravarthy, U., 1997. Sperm nitric oxide and motility: the effects of nitric oxide synthase stimulation and inhibition. Mol. Human Reprod. 3, 755–762. 30. Yousef, M. I., Abdallah, G. A., Kamel, K. I., 2003. Effect of ascorbic acid and Vitamin E supplementation on semen quality and biochemical parameters of male rabbits. Anim. Reprod. Sci. 76, 99–111. 31. Hassan, R. A., Morsy, W. A., Abd El-Lateif, A. I., 2012. Effect of dietary ascorbic acid and betaine supplementation on semen characteristics of rabbit bucks under high ambient temperature. World Rabbit Science Association Proceedings 10th World Rabbit Congress – September 3 - 6, Sharm El- Sheikh –Egypt, 273 - 277 273. 32. Talwar, P., Hayatnagarkar, S., 2015. Sperm Function Test. J. Human Reprod. Sci. 8, 61–69. 33. Aitken, R. J., 1999. The Amoroso Lecture. The human spermatozoon-a cell in crisis? J. Reprod. Fertil. 115, 1-7.

ACCEPTED MANUSCRIPT

AC C

EP

TE D

M AN U

SC

RI PT

34. Li, Y., Huang, Y., Piao, Y., Nagaoka, K., Watanabe, G., Taya, K., Li, C., 2013. Protective effects of nuclear factor erythroid 2-related factor 2 on whole body heat stress-induced oxidative damage in the mouse testis. Reprod. Biol. Endocrinol. 11, 23. 35. Qian, L., Song, X., Ren, H., Gong, J., Cheng, S., 2004. Mitochondrial mechanism of heat stress-induced injury in rat cardiomyocyte. Cell Stress Chaperones 9, 281-293. 36. Fernandez, J. C., Kaplowitz, N., Colel, A., Garca, R. C., 1997. Oxidative stress and alcoholic liver disease. Alcohol Health Res. World 21, 321-324. 37. Yoshimoto, H., Takeo, T., Nakagata, N., 2017. Dimethyl sulfoxide and quercetin prolong the survival, motility, and fertility of cold-stored mouse sperm for 10 days. Biol. Reprod. 97, 883-891. 38. Finzi, A., Morera, P., Kuzminsky, G., 1995. Sperm abnormalities as possible indicators of rabbit chronic heat stress. World Rabbit Sci. 3, 157–161.

ACCEPTED MANUSCRIPT •

Heat stress affects the sperm quality in exposed male rabbits

•

Dietary intake of quercetin maintains the semen quality in rabbit

•

Quercetin provision the lower the HS induced damages by reducing the oxidative

AC C

EP

TE D

M AN U

SC

RI PT

stress

ACCEPTED MANUSCRIPT

EP

TE D

M AN U

SC

RI PT

(%) 87.8 17.4 4.8 10.73 25.65 11.4 4

AC C

Table 1. Ingredients and proximate analysis of nutrients Feed Ingredients (%) Chemical Composition Corn 31 Dry matter Wheat bran 25 Crude protein Dried distillers grains soluble 15 Crude Fat Sunflower meal (36% protein) 9.5 ME MJ/kg Soybean 7 NDF Safflower 5 ADF Sunflower meal (28% protein) 3.5 Ash Limestone 2.5 Salt 1 Vitamin-minerals mixture 0.1

ACCEPTED MANUSCRIPT

Table 2. Effect of dietary quercetin supplementation on semen quality (mean ± S.E) in heat stress (temperature ~ 33.5 °C and humidity ~ 49.5 %) exposed New Zealand White rabbits (age ~ 10-12 months with ~ 3 kg body weight).

1

RI PT

SC

13.1±1.4 8.9±1.6

13.6±1.2 2.2±0.2

11.6±1.0 8.3±1.0

7.3±0.5 9.1±1.2

6.7±1.0 8.3±1.1

4.9±0.2 26.9±2.5 3.05±0.18

3.7±0.4 19.5±1.6 2.54±0.3

4.0±0.4 23.9±1.0 2.82±0.19

4.9±0.4 20.3±1.7 NA

4.4±0.4 19.4±1.3 2.49±0.24

M AN U

Progressive motility (%) Total motility (%) VCL (µm/Sec) VSL (µm/Sec) VAP (µm/Sec) LIN (%) STR (%) WOB (%) ALH(µm) BCF Semen volume (µL) Sperm conc. (×106/mL) Acrosome reaction (%) Sperm viability (%) Sperm mitochondrial potential (%) Head abnormalities (%) Mid-piece abnormalities (%) Tail abnormalities (%) Total abnormalities (%) MDA nmol/mL 1

Signification Day of treatment

Diet × Day of treatment

0.0123 0.041 0.0361 0.0777 0.0612 NS 0.094 0.043 0.001 0.0498 0.0022 0.06 NS 0.024

0.0348 0.08 NS NS NS NS 0.069 NS NS NS NS 0.022 0.033 NS

NS NS NS NS NS NS NS NS NS NS NS 0.06 NS NS

Diet

11.2±0.5 6.7±0.3

12.9±0.6 4.6±0.3

14.8±1.1 13.3±1.9

12.4±0.8 12.4±1.0

11.5±1.3 11.1±0.8

0.022 0.003

NS 0.021

NS NS

4.8±0.8 22.7±0.5 2.72±0.1

3.9±0.6 21.4±0.3 3.03±0.2

3.9±0.5 32.0±2.8 3.17±0.13

2.2±0.3 27.4±1.4 NA

3.4±0.2 26.0±1.0 3.24±0.24

NS 0.006 0.005

NS NS NS

NS 0.021 0.090

TE D

Variables

Treatments QU-HS ( n = 06; 30mg/kg quercetin supplemented heat stressed HS ( n = 06; non-supplemented heat stressed rabbits) rabbits) Day 0 Day 15 Day 30 Day 48 Day 56 Day 0 Day 15 Day 30 Day 48 Day 56 43.1±7.1 35.7±6.6 31.6±7.2 58.9±11.8 44.4±10.7 38.7±10.4 30.9±9.1 26.2±6.7 25.9±5.4 35.1±6.6 78.3±11.9 85.6±16.4 71.4±15.4 73.8±15.1 80.6±14.9 80.1±13.1 62.1±19.2 61.2±9.8 58.0±9.2 69.9±9.3 58.7±8.2 59.5±9.7 46.7±11.5 82.9±15.2 63.2±12.8 54.2±12.5 52.2±17.4 49.6±5.8 54.9±8.8 60.1±10.6 13.1±4.1 14.7±2.8 11.1±2.8 23.5±6.9 17.1±5.8 11.1±4.2 14.1±4.7 12.1±3.5 12.7±2.7 16.7±3.9 25.7±6.4 28.2±2.4 22.4±7.4 42.8±10.2 31.7±7.5 23.7±5.3 25.3±8.2 23.2±5.0 24.5±3.9 30.4±7.2 22.0±3.5 27.8±3.3 22.7±3.1 25.4±3.3 27.3±1.9 18.8±3.7 24.5±4.4 24.6±5.2 21.2±5.1 27.9±2.3 49.4±3.6 54.4±3.9 50.1±3.0 52.6±2.4 54.2±1.2 45.7±2.4 52.0±5.9 52.0±5.2 48.8±6.6 55.3±1.0 44.8±3.7 51.0±2.8 42.2±3.5 48.3±4.5 50.8±1.9 42.3±4.4 46.2±6.7 47.7±4.7 42.9±4.5 50.8±4.5 2.1±0.5 2.5±0.7 1.6±0.7 2.1±0.4 2.1±0.4 2.0±0.4 1.9±0.5 1.9±0.5 1.9±0.5 2.1±0.6 6.8±0.8 9.7±2.8 4.9±3.0 7.6±1.6 7.1±2.7 4.3±2.2 7.0±2.2 6.9±3.3 6.3±2.0 8.2±38 550.0±148.3 692.0±80.0 780.0±49.2 840.0±29.1 862.0±33.2 507.0±63.8 615.0±57.8 506.0±77.6 462.0±59.1 510.0±93.6 454.0±24.0 441.8±28.4 406.4±34.1 546.4±50.8 681.0±18.7 472.0±86.7 375.8±34.1 305.2±46.2 255.6±37.9 410.0±26.3 25.0±4.8 10.0±3.3 11.0±1.1 40.8±10.5 27.4±9.1 34.7±5.5 32.6±8.0 23.9±3.6 29.8±8.5 24.4±1.1 55.0±8.3 59.4±6.3 59.3±3.6 76.5±2.5 67.3±9.0 32.9±6.1 40.3±2.2 44.1±2.3 69.5±7.0 65.7±3.5 70.1±5.3 64.4±3.4 69.9±3.3 55.9±8.3 58.0±4.9 33.5±3.4 44.2±3.6 43.2±5.8 64.7±4.2 65.0±8.5

AC C

EP

MDA was not evaluated on day 48 of experiment NS = Non-significant; NA = Not available VCL = Curvilinear velocity; VSL = Straight linear velocity; VAP = Average path velocity; LIN = Linearity; STR = sperm track straightness; WOB = Wobble; ALH = Amplitude of lateral head displacement; BCF = Beat cross-frequency