Evaluating the effect of adding vitamins E & C to the extender for Barki ram semen by cooling

— The present work aimed at evaluating adequacy of adding different non-enzymatic additives; i.e. vitamins E and C, in to semen diluent on physical and morphological characteristics as well as oxidative status of ram semen stored at 4 o C. Ejaculates (n=80) were collected from Five sexually mature Barki rams during the period from January to April. After initial evaluation, each adequate raw ejaculate was diluted (1:10) with Tris-citrate egg yolk extender. Each diluted ejaculate was further split into seven aliquots to evaluate addition of three levels of either ascorbic acid or alpha tocopherol (0.1, 0.2 or 0.3 mM for each treatment) against control (untreated) specimens for their ability to maintain sperm viability criteria over 48 hr (T 0 and T 48 ) of chilled storage at 4 o C.The changes in seminal plasma oxidative status indices as well as enzymatic activates were also determined throughout the period of cooled storage.The results showed that addition of adequate concentrations of ascorbic acid (0. 1 mM) or α -tocopherol (0.3 mM) to semen diluent can reduce (P<0.05) the oxidative stress and, hence,maintained(P<0.05) physical characteristics of ram spermatozoa compared to controlthroughout the 48 h period of cold storage.


INTRODUCTION
Generally, liquid-chilled preservation of semen has been reported to reduce the fertilizing capacity of spermatozoa along with both sperm motility and morphology over time of storage (Perumal et al., 2013)as results of accumulated reactive oxygen species (ROS) which leads to the death of the sperm cells (Amidi et al., 2016). The development of assisted reproductive techniques, including artificial insemination (AI) and in vitro fertilization (IVF), require improving spermatozoa physical characteristics by supplementing the diluent with a variety of antioxidant substances such ascorbic acid (Vitamin C) and α-tocopherol (vitamin E), which were reported toplay important roles as non-enzymatic antioxidant for spermatozoa (Abd El-Hamid et al., 2018; Zeitoun and Al-Damegh, 2015),by protecting sperm cells from generated ROS that induce damage to sperm DNA over preservation time (Alvarez and Storey, 2005; Dandekar et al., 2002).Further, both ascorbic acid and α-tocopherol were used to reduce ROS related testicular impairments in animal tissues (Acharya et al., 2008), and improve the antioxidant enzymatic activity of seminal fluid (Foote et al., 2002), thereby enhancing sperm motility and viability during semen processing (Soren et al., 2016). Therefore, the present study aimed to investigate the effect of supplementing semen diluent with different levels of Ascorbic acid (Vitamin C) or α-tocopherol(Vitamin E) on maintaining ram sperm traits during 48 h of chilled preservation at 4 o C,as well asdetermination of some antioxidant enzyme activities throughout the period of chilled storage.

II. MATERIALS AND METHODS
This study was carried out at Artificial Insemination Lab, Mariout Research Station located 34 km west of Alexandria and belonging to the Desert Research Center (DRC), Egypt.

Animal and management.
Five sexually mature Barki rams, aged from 2 -3 years with averaged body weight of 45.0 ±2.0 kg were used in this study from January toApril. The animals were hosed in closed pens throughout the experimental period. The rams were fed concentrate according to their body weight

Semen collection.
A total number of 80 ejaculates were collected from the rams twice weekly using artificial vagina as previously described (El-Bahrawy et al., 2004). Mean values of raw ejaculates' physical traits included in the study were volume 0.96 ± 0.06ml, progressive motility ≥ 90%, pH 7.2 ± 0.1, mass motility 4.26 ± 0.16 and sperm concentration 2374.4 ±26.2 (×10 6 sperm/ml). Each raw ejaculate was diluted (1:10) with Tris-citrate egg yolk extender, and was further divided into two major groups. The first major group of diluted semen was then split into three aliquots, where each was supplementedwith one of three levels (0.1, 0.2 or 0.3 m M) alpha tocopherol (Vit E, Sigma-Aldrich, St. Louis MO, USA).Another similar set of aliquots was supplemented with with the same three levelsbut of ascorbic acid (vitamin, Sigma-Aldrich, St. Louis MO, USA). All six supplemented spicemens were evaluated, throughout the period of the study against control (non-supplemented) group.(table1). All diluted semen groupswere evaluated for physical characteristics and some biomarkers of enzyme activities during 48 hr (T0 and T48) at 4 o C of cooling period.

Semen evaluation.
Total sperm motility was estimated in all diluted samples byusing a phase-contrast microscope (Leica) at 40 X magnification, where an average of 5 random fields was obtained to the nearest 5%. Sperm vitality (live and dead sperm, %) were examined using the differential staining technique, where a mixture of 10 µl of semen and 5 µl of freshly-prepared eosin-nigrosin stain was smeared on a warm stage, and were examined under high power magnification (100×). Sperm abnormalities and acrosome integrity were evaluated using Romanowski's triple-stain technique (DIFF-QUICK III, Vertex, Egypt). Smears preparation and staining procedure were conducted following instructions provided by the manufacturer, and the stained smears were evaluated using a phase-contrast microscope at 100x magnification. Sperm plasma membrane integrity was determined by the hypo-osmotic swelling test (HOST) as described by Mosaferi et al. (2005), where at least 200 sperm were evaluated at 40 X magnification.

Seminal plasma biomarkers and enzymes activity assessment.
Seminal plasma samples were obtained during the cooling period (T0 and T48) and were subjected to biomarkers and enzymes activities using commercial kits. Glutamic oxaloacetic transaminase (GOT), Glutamic pyruvic transaminase (GPT) were analyzed using colorimetric kits (Spectrum, Egypt) according to Reitman and Frankel, (1975). Alkaline phosphatase (AKP), Lipid peroxide (LP) and hydrogen peroxidase (HP) were determined using colorimetric kits (Biodiagnostic, Egypt) according to Belfield and Goldberg, 1971  The statistical significance threshold was set at 0.05 and the differences between means were detected byDuncan's multiple range test..

Effect of different levels of Vitamin E on chilled semen traits
Means values ± SEM of physical characteristics sperm including sperm motility (%), normal sperm (%), sperm abnormalities (primary and secondary, %) acrosome integrity (%) and hypo-osmotic swelling test (HOST, %) are presented in table (2).
Overall mean of sperm motility (%) was significant higher (P<0.05) in level one (L1) and level two(L2) of vitamin E (87.66, 92.22 ± 0.93 %, respectively) compared with both level three (L3) (87.00±0.93%) or control (85.66±0.93%)at 48 of chilled preservation at 4 o C. There was a significant interaction (P<0.05) between time of preservation and levels were recorded. The higher (P<0.05) value of sperm motility was recorded in all levels of vit (E) at T0 of chilled preservation, while the low value was recorded in control (66.00±1.00 %) at T48 of chilled preservation at 4 o C (table 2).Mean value of normal sperm (%) was significantly higher (P<0.05) in (L1) and (L2) of vitamin E (93.77, 92.22 ± 0.57 %, respectively) followed byL3(90.33±0.57%) compared with control (79.88±0.57%)atT48of chilled preservation. Moreover, the higher (P<0.05) value of normal sperm was recorded in all levels of vit (E) at T0of chilled preservation, while the low value was recorded in control (66.00±1.00 %) in T48 of chilled preservation at 4 o C (table 2).
The results also showed that no significant was found among either levels of Vit. E and control groups in values of primary sperm abnormalities (%).However, the higher (P<0.05) values of primary sperm abnormalities (%) were recorded in control and(L1) (2.00, 2.00 ± 0.25 %, respectively) atT48, while the low value was recorded in control (1.00, 1.00 ± 0.25 %) at T0and T24 throughout cooling period (T0-T48)as illustrated in Table (

Effect of different levels of Vitamin E on oxidative status during preservation.
Changes in seminal plasma biomarkers and enzymes activities are presented in Figure (1).
Overall mean value of acrosome integrity percent (%) was significantly higher (P<0.05) in both (L1) and (L2) groups of vit C, while the lowest values (P<0.05)were recorded in control and L3 (87.44, 88.88 ±1.03 %) at 48 hr of chilled preservation (Table 3). A significant (P<0.05) interaction was noted between time and treatment. All levels of Vit C-supplemented or control specimens over storage period at T0 and gradualdecline thought preservation period at T48 of chilled preservation 4 o C Table (3). Mean value of hypo-osmotic swelling test (HOST, %) was significantly higher (P<0.05) in L1 and L2 (85.77, 85.77 ± 0.97 % respectively)compared with control (61.77±0.97 %). The interaction between time and levels were significant (P<0.05), the highest value was recorded in L1 (95.33±1.68 %) at T0, while the lowest (P<0.05) value was recorded in control (46.00±1.68%) atT48 of chilled preservation at 4 o C ( Table 3).

Effect of different levels of Vitamin C on oxidative status during preservation.
The changes in seminal plasma biomarkers and enzymes activities are presented in Figure (2).

IV. DISCUSSION
The results of the present work showed a significant enhancement in semen viability criteria in terms of sperm motility, normal sperm, sperm abnormalities (primary and secondary), acrosome integrity and HOST-reacted spermatozoa percentages in specimens supplemented with ascorbic acid or α-tocopherol. This finding is in agreement with previous studiesin different species ( Additionally, it improves sperm metabolic activity and viability, as well as mitochondrial membrane potential in sperm cells (Hu et al., 2010)in the present work, Vitamin C supplementation led to decrease in pH due to its strongly acidic properties (10% solution: pH 2). Similar finding has been previously reported to induce reversible or irreversible reductions in motility (Acott and Carr, 1984).
In the present study the enzyme activities ofGOT in specimens supplemented with both additives increased at 48 h of preservation at 4 o Calongside AKP activity. This agrees with previous observations of Zeiton& Mona(2015), and may directly indicate sperm membrane damage (Peschet al., 2006), and increased percentages of dead and abnormal spermatozoa (Gundoganet al., 2010).
Contrariwise, the results showed that values of LP, HP and GPT concentrations declined in specimens

V. CONCLUSIONS
The results indicated that addition of adequate concentrations of ascorbic acid or α-tocopherol to semen diluent can reduce the possible damage induced by oxidative stress, hence, maintain physical characteristics of ram spermatozoa during 48 h of cold storage. Nevertheless, inclusion of the optimum levels of both vitamins combined in the diluent may further improve the fertilization potential of spermatozoa prior to application of artificial insemination (AI) and in vitro fertilization (IVF) in sheep.