Protective Role of Onion ( Allium cepa L.) on Caffeine Induced Spermatotoxicity in Albino Rats

Onion is widely consumed and is used for several medicinal and therapeutic purposes such as management of diabetes and also as antimutagenic, antimicrobial and antioxidant agents. Hence, this study investigated the protective role of onion on caffeine induced spermatotoxicity in albino rats as mammalian model. Thirty healthy male albino rats of 12 weeks old were divided into five groups with six rats in each group using a completely randomized design (CRD). The daily caffeine and onion juice treatments were administered orally via oral gavage for sixty five days. Result obtained indicated a significant (P<0.05) reduction in the weight of epididymes, sperm viability, sperm motility and sperm count while sperm head abnormality increased in caffeine-treated animals. No significant differences were observed in semen pH and weight of testes. Onion juice protected mammalian model from caffeine induced toxicities on the weight of testes, sperm viability, sperm motility, sperm count and sperm head abnormality. These results show that onion is effective in protecting albino rat models against caffeine induced spermatotoxicity in a dose dependent manner.

Onion (Alliun cepa L.) is grown primarily for use as food, adding flavor and taste. Young, green leaves with their white bases are also eaten raw in salads. It has several medicinal importances such as; in the management of asthma and diabetes; and their complications [1,3], also as antimicrobial, antiplatelet, antioxidant and antimutagenic agent [1,[3][4][5]. Onion contains plenty of enzymes, vegetal hormone (glycoquinine), trace elements, vitamins (A, Bcomplex, C, E) and flavonoids [1]. It is also used as aphrodisiac, vermifuge, blood thinner, alkalinizer, hypotensive and diuretic agent [3], as well as decongestant, heart and arterial protector [1].
Caffeine is one of the world's most widely consumed psychoactive substances and is present in several foods, drugs and beverage products such as energy drinks, coffee and tea [6][7][8].
Unlike most other psychoactive substances, it is legal and unregulated in most part of the world [9][10][11] with an estimated 80% of the world's population consuming a caffeinecontaining substance daily [6,10]. Caffeine dependency has a wide range of unpleasant physical and mental conditions such as nervousness, irritability, restlessness, insomnia, headache and heart palpitations [12].
Many medicinal plants with antioxidant properties have been shown to confer protective effects from toxicities induced by substances such as caffeine [14][15][16][18][19][20]. It is on this premise that this study set out to ascertain the protective role of onion (Allium cepa L.) on caffeine induced spermatotoxicity in albino rat as a mammalian model.

Collection and Preparation of Onion Juice
The onion bulbs were obtained from Watt market, Calabar, Cross River State of Nigeria and authenticated at the Department of Botany, University of Calabar, Calabar. The onion bulbs were washed thoroughly, weighed and cut into small pieces and then liquidized into onion juice using mammonlex juice extractor, model: JD 1004. The onion juice was stored in glass bottles and preserved in a refrigerator at 2-4°C until used for the experiment.

Chemicals
All chemicals used in the course of the study were of analytical grade.

Experimental Animals
Thirty healthy young adult male albino rats of 12 weeks old with average body weight of 182.5 g ranging from165-215 g were obtained from the animal house of the Department of Zoology and Environmental Biology, University of Calabar, Calabar. The rats were housed in well ventilated conventional wire mesh cages under standard laboratory conditions. They were allowed free access to water and pellet feed throughout the period of the experiment. Generally, the study was conducted in accordance with the recommendations from the declarations of Helsinki on guiding principles in care and use of animals and the local ethical committee.

Experimental Design and Procedure
The thirty rats were divided into five groups of six rats each using a completely randomized design. The daily treatments were given orally via oral gavage which lasted for 65 days and the protocol is shown in Table 1. The rats were sacrificed under chloroform anaesthesia 24 hours after the last treatment. The epididymes and testes were dissected out and weighed using Scout Pro SPU 601 electronic weighing balance. The epididymes were processed for epididymal sperm count, motility, viability and sperm head abnormality. Onion juice, 250 mg kg -1 BW in 2mL via oral gavage C+ OJ 250 Caffeine, 200 mg kg -1 BW and onion juice , 250 mg kg -1 BW in 2 mL via oral gavage C+OJ 500 Caffeine, 200 mg kg -1 BW and onion juice , 500 mg kg -1 BW in 2 mL via oral gavage

Semen pH
Immediately after dissection, a puncture was made in the epididymes with a sterile pin. The semen smeared on the pin was rubbed on a pH paper of the range 4.0-10.0. The colour change corresponding to the pH of the semen was read from the paper.

Sperm motility
The sperm motility was evaluated according to the method of Ekaluo et al. [14,15]. Two drops of sperm suspension were put on a microscope slide and cover slip was placed on it. The number of progressively motile cell was recorded and divided by the total number of spermatozoa counted under x40 objective lens and expressed in percentage.

Sperm viability
A portion of the sperm suspension was mixed with 1% eosin Y solution (10:1) for 30 minutes and smeared on glass slides. The air-dried smears were washed with physiological saline and counter stained with 1% Malachite green stain. The slides were examined for percentage viability. Normal live sperm cells appeared colourless, while dead sperm cells took up stain and appeared pinkish in greenish background. The percentage viability was calculated based on the number of live sperm cells out of the total number of cells observed.

Sperm count
Epididymal sperm count was obtained by cytometry using the improved Neubauer Cytometer and will be expressed in million/ml of the sperm suspension [21].

Sperm head abnormality
A portion of the sperm suspension was mixed with 1% eosin Y solution

Statistical Analysis
Data obtained from weight of testes and epididymes, semen pH, sperm motility, viability, count, and sperm head abnormalities were subjected to analysis of variance (ANOVA) test. Statistical significance were considered if p<0.05 while least significant difference (LSD) test was used to separate the means.

Weight of Testes and Epididymes
There was no significant difference (P>0.05) in the weight of the testes of the animals. However, caffeine significantly (P<0.05) reduced the weight of epiddymes. The caffeine treated group had the lowest weight (0.49 g), closely followed by the C+ OJ 250 group (0.50 g). The weight of the epididymes significantly increased in the C+OJ 500 group indicating a dose dependent protective role of onion juice. The highest weight of epididymes was obtained in the OJ 250 group (0.74 g) while the control group recorded 0.64 g ( Table 2).

Semen pH
The treatments did not significantly affect the pH of the semen of the animals. It was within the range of 6.70 to 7.02 as shown in Table 2.

Sperm Motility
As shown in Table 2, sperm motility was significantly reduced in the caffeine group (65.06%) compared with the control (72.38%). A significant (P<0.05) increase was observed in C+ OJ 250 and C+OJ 500 groups (69.78 and 71.53% respectively) indicative of the protective role of onion juice. The highest value was obtained in the OJ 250 group (76.53%).

Sperm Viability
There was a significant (P<0.05) reduction in the percentage of viable sperm cells in caffeine treated animals when compared to the control (89.12%). The onion juice significantly protected and increased sperm viability in the groups treated with onion juice in a dose-dependent manner from caffeine induced toxicity as shown in Fig. 1 and Table 2. The sperm viability was increased from 74.18% in caffeine group to 93.49% in OJ 250 group with the following trend: C < C+ OJ 250 < C+OJ 500 < control < OJ 250 .

Sperm Count
The sperm count was significantly (P<0.05) reduced by the caffeine treatment when compared with the control. Fig. 1 and Table 2 show that the caffeine group has the lowest value of 5.80 x10 6 /ml and the onion juice protected the rats from the effect of caffeine in C+ OJ 250 and C+OJ 500 groups (6.99 and 7.330x10 6 /ml ) respectively in a dose-dependent manner with the following trend: C < C+A 250 < C+A 500 < control < A 250 .

Sperm Head Abnormality
The sperm head abnormality was also significantly (P<0.05) increased by the caffeine treatments as shown in Table 2. The percentage of sperm head abnormalities were significantly reduced from 9.24 to 3.66%; indicative of the protective role of onion juice in a dosedependent manner with the following trend: C < C+A 250 < C+A 500 < control < A 250 ( Fig. 1 and Table 2). Sperm count (x10 6 mL -1 ) and Sperm head abnormality (%)

Treatments
Sperm head abnormality Sperm count Sperm viability  [14][15][16]24], and suggest a distortion in the biosynthetic processes underlying spermatogenesis, hence distorts in the fertility indices of the male animals [25]. The significant (P<0.05) decrease in the weight of the epididymes is also corroborated by the reduction in the sperm count of the caffeine-treated animals. The caffeine treatment also significantly increased the percentage of sperm head abnormalities which suggests induced mutations on the sperm cells during spermatogenesis. This also agrees with the reports of Harris [26] and Ekaluo et al. [11].
Onion juice protected the rats from the caffeine induced toxicities on the weight of epidiymes, sperm viability, sperm count, sperm motility and sperm head abnormality. The protective role of onion juice could be attributed to its rich vitamin C [27], which agrees with the protective role of vitamin C reported by Nashwa and Venes [28], Karawya and El-Nahas [29]; Ekaluo et al. [14,15]. The protective role of onion might also be due to the antioxidant properties of the onion juice against oxidative stress, which has been implicated as one factor that affects fertility [30].
Epidemiological studies have revealed that consuming fruits and vegetables as well as their extracts reduced free radical oxidative damage [20,31] and promote fertility [18,19,32]. Increased reactive oxygen species (ROS) level has been correlated with decreased sperm count and motility [33]. Therefore, the protecting effect of onion juice on caffeine induced spermatotoxicity can be attributed to the protective roles of its constituents against oxidative stress and induced mutations.

CONCLUSION
The present study shows that onion juice is effective in protecting the albino rat models from caffeine induced spermatotoxicity in a dose dependent manner.