Aims: As optimization of farm animals reproductive performance is a main objective, the present study was, undertaken to investigate the possible protective effect of vitamin C, vitamin E and olive pomace against dizocilpine (MK-801) induced oxidative stress and its resultant alterations on antioxidant status, spermiogram, hormonal, enzyme markers and histomorphology of testes of rabbit bucks during first and second month of the experiment.
Study Design: Laboratory experimental design was performed.
Place and Duration of Study: The study was conducted in the experimental rabbitry of Physiology Department, Faculty of Veterinary Medicine Cairo University during the period from January to May 2014 in accordance with the Chinese guidelines for animal welfare and approved by the animal welfare committee of Animal Science College, Zhejiang University.
Methodology: Thirty- six male New Zealand White rabbits of about 5-months age and average body weight of 2500 g were randomly selected, used for the experimental work one month later. During this month, the rabbits were gradually adapted to natural mating. The experiment lasted for 2 months (length of spermatogenic cycle in rabbit), bucks were housed individually in commercial cages (55×60×34 cm), equipped with automatic drinkers and j-feeders. Clean and fresh water was available all time. The whole rabbitry was well ventilated through both natural windows and electric fans and illuminated to 14:10 light dark cycle through natural and fluorescent lighting. The rabbitry average ambient temperature and relative humidity ranged from 20 to 30ºC and 70 -80%, respectively during summer resting period. Basal and experimental diets were formulated to cover the nutrient requirements of rabbits as recommended in . Diets were subjected to chemical analysis according to . Offered for all animals ad libitum Table 1. The bucks were equally and randomly divided into six groups (6 in each) namely control which injected with 1.0 ml sterile saline intraperitoneal (i.p.), second group injected by dizocilpine (MK-801 purchased from Sigma-Aldrich, Egypt), at dose 0.1 mg/kg. The dose of MK-801 was dissolved in 1.0 ml sterile saline and was injected daily intraperitoneal (i.p.) for 5 consecutive days, third group was injected by dizocilpine and supplemented with “vitamin C” (1 g/L of drinking water), while the forth group was injected by dizocilpine and supplemented with “vitamin E” in drinking water (50 ml/100 L)( the international units of vitamin E contained in 50 ml be mentioned) and those of the fifth group was supplemented with “vitamin C” plus “vitamin E”. All five groups were fed the basal diets, while the sixth group was injected by dizocilpine and supplemented with the experimental diet that contained olive pomace (10%).
1) Samples collection: Semen was collected using artificial vagina . Semen collection was done by using a teaser female and artificial vagina (containing water at 50ºC) that was locally fabricated as described by Herbert and Adejumo . The ejaculate volumes were recorded (using a graduated collection tube) after removal of the gel mass; Semen pH was determined using pH paper 1-14; Mass motility (MM) was determined by placing a drop of semen on a clean, dry, warm slide and examining microscopically using thermostatically controlled hot stage adjusted at 38-40ºC. Mass activity of spermatozoa was scored (0-5) according to the intensity of the moving whirls  as follows: 0 = no current, 1 = few slow current, 2 = many moderate waves, 3 = many sweeping waves, 4 = numerous vigorous waves, 5 = numerous rapid and vigorous waves. Individual motility was assessed in semen sample diluted with 2.9% sodium citrate dehydrate solution, spread almost evenly under a glass cover slide and examined microscopically using adjusted hot stage at 38-40ºC. Individual sperm motility percent was determined on a subjective scale of 0-100% to the nearest 5% after viewing several microscopic fields. Sperm-cell concentration per ml was measured by counting the number of spermatozoa present on both sides of an improved Neubauer hemocytometer slide (GmbH & Co., Brands twiete 4, 2000 Hamburg 11, Germany).
Total sperm output was calculated by multiplying semen ejaculate volume and semen concentration. Assessment of live, dead, and abnormal spermatozoa was performed using an eosin–nigrosin blue staining mixture .
2) Samples analysis: Two bucks from each group one and two months later from beginning of experiment were slaughtered for obtaining the testes. The testes of each buck were dissected and weighed; one testis was kept in liquid nitrogen for testicular enzyme markers, antioxidant parameters and hormonal assay, while the other testis was fixed in 10% formol saline for histomorphology.
The antioxidant parameters and activities of selected testicular enzyme markers (alkaline phosphatase and lactate dehydrogenase) besides Sertoli cell index (gamma glutamyl transferase) were estimated in testes using testicular homogenate which was prepared according to the method adopted by Hodgen and Sherins ; Wherein testicular tissue was homogenized in 0.015 M disodium hydrogen phosphate (Na2HPO4), 0.15 M sodium chloride (Nacl), pH 7.8 at 4ºC. All assays were performed within 48 hours after homogenization.
Superoxide dismutase activity  glutathione peroxidase activity  glutathione-S-transferase , total antioxidant capacity  and lipid peroxidation expressed in Malondialdehyde  were performed using kits purchased from Biodiagnostic Company, Dokki, Egypt.
3) Hormonal assay: Radioimmunoassay was used for quantitative determination of testosterone and17beta-estradiol hormones in testes homogenate.
Determination of testicular testosterone was performed according to the method of  using kits purchased from “TESTO-RIA-CT” Belgium.Determination of testicular 17beta- estradiol was performed according to the method of  using kits purchased from “IMMUNOTECH”.
2.3.3 The Testicular Enzymes. Alkaline phosphatase (ALP)  gamma glutamyl transferase (GGT)  and lactate dehydrogenase (LDH)  using kit purchased from Spectrum Diagnostics.
4) Histomorphological studies: For qualitative analysis of testicular histology, the testes samples were fixed for 2 days at 10% formal-saline and dehydrated by passing successfully in different mixtures of ethyl alcohol-water, cleaned with xylene and embedded in paraffin. Sections of tissue (5-6 μm thickness) were prepared by using microtome and stained with haematoxylin and eosin and in neutral deparafinated xylene (DPX) medium for microscopic observations.
5) Statistical analysis: Data are presented as means ±S.E. and analyzed by one-way ANOVA using Costate computer program Costat version 6.400 (copyright© 1998-2008 CoHort software) according to the method of . Groups were compared using the calculated least significant difference test (LSD) at the at P value ≤ 0.05.
Results: The first month revealed (1) significant decrease in spermiogram, antioxidative parameters, testicular estradiol, testosterone, enzyme markers and clear pathological changes in testes of dizocilpine group; (2) Significant improvement in the measured parameters of groups subjected to dizocilpine and supplemented with “vitamin E”, “vitamin C” and olive pomace; (3) significant increase of all measured parameters in the “vitamin C” plus “vitamin E” supplemented group. On the other hand, results at second month showed no difference between all groups in these parameters.
Conclusion: It was obvious that the supplementation with vitamin C or/and vitamin E and olive pomace to male rabbits exposed to oxidative stress was associated with improved spermiogram, anti-oxidative parameters, hormonal and testicular enzymatic activities.
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