INTRODUCTION:

Sterility is a major clinical issue, influencing larger part of the general population medicinally also, psychosocially ¹. As of late, oxidative anxiety has been involved in the movement of male barrenness. Prove has demonstrated that these harming occasions are brought about by free radicals².

Oxidative stress comes about because of either a lessening of regular cell cancer prevention agent limit on the other hand an expanded measure of responsive oxygen species (ROS) in creatures. Carbon tetrachloride (CCl₄), is a reasonable, dismal, unstable, substantial and non-combustible modern fluid, broadly used to actuate free radical poisonous quality in different tissues of trial creatures, for example, liver, kidneys, heart, lung, testis, cerebrum and blood³. CCl₄ is changed over through hepatic microsomal cytochrome P450 into tri without chloromethyl radical (∙CCl₃ or ∙CCl₃OO)⁴ which thus, start lipid peroxidation handle⁵. The most broadly acknowledged system of CCl₄ is the development of free radicals which is a rate restricting procedure in tissue peroxidative harm⁶.

Keeping up the harmony between responsive oxygen species and normal cell reinforcements could fill in as a noteworthy system in anticipating harm by oxidative anxiety prompted by dangerous specialists. Cancer prevention agent guard frameworks that shield the body from free radical harm incorporate the cell reinforcement supplements and catalysts⁷. Cancer prevention agent and radical scroungers have been utilized to concentrate the system of CCl₄ lethality and additionally to shield tissue cells from CCl₄ actuated harm by breaking the chain of lipid peroxidation⁸.

In view of the fact that CCl₄ has been shown to induce testicular toxicity, the effect being through oxidative stress and the antioxidant properties of chemical constituents of royal jellyinfluence oxidative stress in tissue of rats, we therefore aimed at investigating the protective efficacy of royal jellyon CCl₄ induced testicular derangement in rat.

MATERIALS AND METHODS:

Chemicals: Carbon tetrachloride (CCl₄) was obtained from the Dept. of Chemistry/University of Kufa. Royal jelly was obtained from the local market. The rats received equal dose of CCl₄ that was suspended in olive oil (1:1v/v) by oral administration method. Besides, Royal jelly was given at a dose of 150 mg/kg and 300 mg/kg by the same method.

Experimental design:

Thirty six healthy adult albino rats (Rattusnovegicus) weighting between 220-235 gm were used in this experiment. They were housed in separated plastic cages at Faculty of Science/University of Kufa/Iraq and kept in controlled environment of 22-25°C. Commercial food (pellets) and tap water were provided to animals. Rats were left to acclimatize for at least two weeks before the start of the experiment. Then the animals were divided into six groups of six rats in each. The first group was fed on the basal diet and saved as a normal control. The three groups were given carbon tetrachloride (CCl₄) for induction of toxicity in reproductive system; CCl₄ was diluted in an equal volume of olive oil as a vehicle and given by oral administration method. The first CCl₄ group was fed basal diet and kept it as toxicity group while the other toxicity groups were fed on basal diets and received royal jelly by oral administration at doses 150 mg/kg and 300 mg/kg respectively. The rest of two groups were fed on basal diets that received royal jelly only by oral administration at doses 150 mg/kg and 300 mg/kg respectively. At the end of experiment period (50 days)rats were anaesthetized by ketamine with xylazine⁹and blood samples were collected by heart puncture and put into jell tubes in the room temperature for several minutes and were centrifuged for 20 minutes at 3000 rpm. The sera, which were kept at-10°C till hormonal analysis were separated. testes and epididymis of the sacrificed rats were taken for measurement of sperm parameters.

Semen Analysis:

Sperm count in testes were measured by Seed ¹⁰ and epididymial contents of treated rats were obtained after cutting the tail of epididymis, squeezing it gently on clean slide and the sperm progressive motility and sperm count were determined¹¹. Microscopical examination of the seminal smears (stained with Eosin Nigrosin stain) were conducted to determine the percentage of sperm viability (ratio of alive/dead) and sperm cell abnormality¹². Hormones analysis were measured by ELISA method¹³.

Histological study:

For male fertility parameters, the second testis in the same animal was removed, cleaned from connective tissue and fixed immediately in 10% formalin solution for later histological preparation according to Bancroft and Stevens ¹⁴.

Statistical Analysis:

Data were expressed as mean±S.E., and Statistical Analysis was conducted by using computerized SPSS program version (17) with one way ANOVA ¹⁵.

RESULTS AND DISCUSSION:

Sperm concentration:

Regenerative danger from substantial and non-combustible modern fluid in guys is one of the zones of worry in toxicology today. Carbon tetrachloride lethality is extremely very much announced in writing and one of the principle instruments behind this danger has been depicted to oxidative anxiety^16,17.

The outcomes acquired from the present review demonstrate a critical reduction (P<0.05) in sperm centralization of testicles in CCl₄ treated gathering in contrast with typical control aggregate. Then again, treatment with RJ at focuses 150 mg/kg and 300 mg/kg in mix with CCl₄ demonstrated a noteworthy increment (P<0.05) in sperm check in contrasting and CCl₄ assemble just, while no critical distinction were seen in the rats that got 150 and 300 mg/kg of RJ just (Figure 1).

Figure (1): Effect of carbon tetrachloride (CCl₄) and royal jelly (RJ) on sperm concentration in testes after 60 days.

Number of rats in each group = 5

Data are expressed as mean ± standard error.

One-way ANOVA with LSD test at P<0.05.

Figure (2): Effect of carbon tetrachloride (CCl₄) and royal jelly (RJ) on sperm concentration in epididymis after 60 days.

Number of rats in each group = 5

Data are expressed as mean ± standard error.

One-way ANOVA with LSD test at P<0.05.

Sperm number is a standout amongst the most touchy tests for spermatogenesis and ripeness assessment. In rats, CCl₄ effectively affects the testis and epididymis, bringing about capture of spermatogenesis. Jaffatet al.¹⁸have announced that the diminishing in the sperm tally in male rats after organization of CCl4 might be related with decreased gonadotropins (LH and FSH) and testosterone hormones, since these hormones are basic for spermatogenesis. LH empowers Leydig cells to mystery testosterone while FSH is important for incitement of spermatogenesis ¹⁹.

Changes in the spermatogenic cycle and degeneration in seminiferous tubules has been accounted for with CCl4 in rats ²⁰. In the CCl4 treated gathering, seminiferous tubules and germ cells were worsened, interstitium mostly vanished and was supplanted by fibroblast and provocative cells in a portion of the ranges of testicles ²¹. Additionally, the present review is in concurrence with past reviews^22,23. The perceptions from this review uncovered that RJ enhanced the epididymal sperm number and this might be because of the essential part for the bioactive segments which display in this bumble bee item.Hidaka et al.²⁴ proposed that nearness of steroidal hormones inside the RJ may be viewed as one purpose behind upgrade in the sperm number on the grounds that the real part of testosterone in the spermatogenesis. Moreover, the zinc is another essential element of RJ, which prompts to arrangement of colossal advantages on the body and regenerative framework can incorporate an expansion in the number and versatility of sperm cells and direction of prostate capacity ²⁵.

Sperm viability and motility:

Figure (3) and figure (4) shows the different effects of CCL₄ and RJ on sperm viability and motility in epididymis. The result of the present study exhibited a marked decrease (p<0.05) in percentage of sperm viability and motility in the male rats of CCl₄ treated group. While the administration of RJ at concentrations 150 and 300 mg/kg resulted in significant increases (p<0.05) in the percentage of sperm viability and motility in comparison to CCl₄ treated group.

Figure (3): Effect of carbon tetrachloride (CCl₄) and royal jelly (RJ) on sperm viability after 60 days.

Number of rats in each group = 5

Data are expressed as mean ± standard error.

One-way ANOVA with LSD test at P<0.05.

Figure (4): Effect of carbon tetrachloride (CCl₄) and royal jelly (RJ) on sperm motility after 60 days.

Number of rats in each group = 5

Data are expressed as mean ± standard error.

One-way ANOVA with LSD test at P<0.05.

Besides, Ojoet al.²⁶ exhibited that movement of some essential mitochondrial proteins (e.g. aconitase) which is viewed as vital amid Krebs cycle and can be impacted within the sight of CCL4. Consequently, mitochondrial brokenness may prompt to unsettling influence in sperm reasonability and motility²⁷.

The present review showed that oral organization of RJ in blend with CCl₄ limited its risks as to sperm reasonability. This outcome concurs with various reviews which have shown that RJ with its cell reinforcement properties has beneficial outcomes for increment the movement of epididymis and afterward upgrade sperm feasibility and general regenerative status²⁸.

Royal jelly intragastric organization can advance the improvement of male regenerative organs, for example, hypothalamus and testicular, enhancing the motility and thickness of sperm in rabbits²⁹.

Sperm abnormality:

The effects of CCl₄ and RJ on sperm abnormality in rats are shown in Figure (5). The results indicate a significant increase (p<0.05) in percentage of sperm abnormality of CCl₄ treated group in comparing with control group, whereas treatment male rats with RJ showed significant decreases (p<0.05) in sperm abnormality in compare with CCl₄ treated group.

Figure (5): Effect of carbon tetrachloride (CCl₄) and royal jelly (RJ) on sperm abnormality after 60 days.

Number of rats in each group = 5

Data are expressed as mean ± standard error.

One-way ANOVA with LSD test at P<0.05.

The expansion in the sperm variation from the norm might be because of the rise of the oxidative anxiety which initiated by CCl₄. Besides, the relationship between the oxidative anxiety prompted by xenobiotics and sperm variation from the norm has been accounted for ³⁰. As indicated by Abdul-Rasoulet al.³¹ the height in oxidative anxiety or inadequacy of androgen hormones is viewed as an imperative variable for the expansion of the sperm variation from the norm of male rats prompted by CCl₄, thus, these elements prompt to diminishment in sperm development and secretary elements of epididymal cells.

For the most part the sperm cells deliver controlled centralizations of ROS required for treatment. In any case, when the free radicals were created in high fixations can straightforwardly harm the sperm cells. Indeed, sperms have high substance of phospholipids and Polyunsaturated unsaturated fats consider the primary constituents of sperm cell layer and these mixes helpless against oxidative harm actuated by free radicals ³².

It is notable that the free radicals can incite DNA harm in meiotic chromosomes ³³ and furthermore meddle with the separation procedure amid the spermatogenesis ³⁴. In parallel, the expansion in sperm deformation of male rats in the wake of being treated with CCl₄ might be ascribed to the expansion in thiobarbituric corrosive receptive substances (TBARS), in light of the fact that any expansion in TBARS level can prompt to destructive impact on midpiece of sperm ³⁵.

This review uncovered a checked diminishing in the unusual sperm rate when the orally RJ was presented in blend with CCl₄ for male pale skinned person rats, and this might be because of the cancer prevention agent impact of RJ against the unfavorable impact of CCl₄ on spermatogenic cells. In this manner, our outcome is in concurrence with past reviews Hassan ³⁶ and Amirshahiet al.³⁷found that RJ has a useful part for lessened the irregular sperm in male grown-up rats which treated with hydrogen peroxide.Inoue et al.³⁸revealed that the nearness of various amino acids and vitamins in segment of RJ lessen or keep some variation from the norm to happen fit as a fiddle,Moreover, RJ lessened tissue DNA oxidative harm and expanded life expectancy.

Cavusogluet al.³⁹ found that genotoxicity and oxidative harms prompted by cadmium danger can be forestalled in pale skinned person mice by treatment with RJ and this recuperation can be credited to the part of RJ against the chromosomal abnormalities.Past review El-Hanounet al.⁴⁰ revealed when RJ administrated in rabbits at any dosages prompted to a critical and progressively expanded (P<0.05) live sperm and ordinary sperm number contrasted with the control gather. The inverse pattern was appeared in the anomalous sperm and dead sperm, which demonstrated a noteworthy diminishing, and this impact was dosage subordinate.

Hormonal study:

Testosterone hormone:

The effect of CCl₄ and RJ on testosterone levels are shown in Figure (6). The results revealed a significant decrease (p<0.05) in CCl₄-treated group in comparison with control group. Treated rats with RJ at concentrations150 and 300 mg/kg of body weight showed significant increases (p<0.05) in comparison with CCl₄ -treated group.

Figure (6): Effect of carbon tetrachloride (CCl₄) and royal jelly (RJ) on testosterone hormone level after 60 days.

Number of rats in each group = 5

Data are expressed as mean ± standard error.

One-way ANOVA with LSD test at P<0.05.

As it is known, testosterone hormone is discharged from the interstitial (cells of Leydig) by incitement of LH. Along these lines, the decrease in LH level in this review initiated by CCl₄ introduction bringing about decay of serum testosterone focus. It has been accounted for that CCl₄ applied a noteworthy unfriendly impact on the steroidogenesis and in addition expanded creation of nitric oxide, instigated by intemperate CCl₄, and might hinder testosterone levels ⁴¹. The consequence of our review corresponds with the discoveries of Sandaraet al.⁴² recorded a checked lessening in testosterone in serum of male rats.

Najafi et al.⁴³ recorded that RJ created a critical increment in level of testosterone in serum of male mice prompted by oxymetholone. In other hand, aftereffects of this review demonstrated that the testosterone hormone was drastically expanded with the expansion of the RJ dosage which administrated to the male rats. Our information exhibited that RJ was in all focuses brought about prominent increment in testosterone level when contrasted with CCl₄ assemble.The improvement in the level of testosterone might be ascribed to the part of RJ in expanding the grouping of LH, which is viewed as the most critical variable for incitement discharge of testosterone from interstitial cells ⁴⁴.

Luteinizing hormone (LH) and follicle stimulating hormone (FSH):

The results showed in Figures (7 and 8) indicate a significant decrease (P<0.05) in both LH and FSH levels in CCl₄-treated group in comparing with control group. The data of this study showed that interference of RJ with CCl₄ showed significant increases (P<0.05) in LH and FSH levels compared to CCl₄- treated group, while no significant different was recorded in rats that received 150 mg/kg and 300 mg/kg of RJ.

Figure (7): Effect of carbon tetrachloride (CCl₄) and royal jelly (RJ) on luteinizing hormone level after 60 days.

Number of rats in each group = 5

Data are expressed as mean ± standard error.

One-way ANOVA with LSD test at P<0.05

Figure (8): Effect of carbon tetrachloride (CCl₄) and royal jelly (RJ) on follicle stimulating hormone level after 60 days.

Number of rats in each group = 5

Data are expressed as mean ± standard error.

One-way ANOVA with LSD test at P<0.05

The results showed in Figures (7 and 8) indicate a significant decrease (P<0.05) in both LH and FSH levels in CCl₄-treated group in comparing with control group. The data of this study showed that interference of CCl₄ with RJ at concentrations 150 and 300 mg/kg of body weight showed significant increases (P<0.05) in LH and FSH levels compared to CCl₄- treated group, while no significant different was recorded in rats that received 50 mg/kg of RJ.

The decay of LH and FSH levels in blood serum might be credited to collection of CCl₄ in the pituitary organ and testis which might be prompt to capacity unsettling influence in the pituitary-testicular pivot in rats ⁴⁵.

In addition, the lessening in LH and FSH levels mirror the decrease in the level of gonadotropin discharging hormone (GnRH) which emitted from hypothalamus and assume a vital part in directing LH and FSH emission from pituitary organ ⁴⁶.

Comparable perceptions were noted in male rats by Shahrakiet al.⁴⁷recorded a lessening in the levels of LH and FSH that as it may, conflicting outcome has been accounted in male mice. Then again, RJ contains around 1 mg/g dry weight of acetylcholine inside its substance also It is intriguing that this nerotransmitters animates the emission of GnRH from the hypothalamus. Therefore, the utilizing of RJ causes expanding in emission of GnRH from hypothalamus, thus, this prompts to increment in the discharging of LH and FSH to blood serum ⁴⁸.

Histopathological changes in testes

Figure (9) Testes section of normal control rat showing cross section of seminiferous tubules (ST), with stratified epithelium consisting of spermatogenic cells (SG) and Leydig cells (LC) (400 X).

Figure (10) Testes section of rats treated with showing complete swallowing of seminiferous tubules with degenerated germ cells (D) and hypoplasia of Leydig cells (H) (400X).

Figure (11) Testes section of rats treated with CCl₄ and 150 mg/kg of royal jellyshowing moderate degeneration of seminiferous tubules and mild hypoplasia of leydig cells (400X).

Figure (12) Testes section of rats treated with CCl4 and 300 mg/kg of royal jellyshowing mild degeneration in spermatogenic cells (D) and moderat degeneration of leydig cells (H) (400X).

Figure (13) Testes section of rats treated with royal jelly 150 mg/kg showing normal seminiferous tubules with complete spermatogenic cells (400X).

Figure (14) Testes section of rats treated with royal jelly 300 mg/kgshowing normal seminiferous tubules with complete spermatogenic cells (400X).

The present review has built up the enhancing impact of RJ in the testicular structure and spermatogenesis prepare. The restorative adequacy of RJ might be credited to its cancer prevention agent movement against lipid peroxidation and free radical era. The helpful organic properties of RJ segments display anti-cancer prevention agent limit and enhanced endogenous cell reinforcement safeguard framework, along these lines, histopathologic picture of testicles were turned around by treatment with RJ to ordinary engineering. These outcomes are in concurrence with many reports^49,50.

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Received on 25.03.2017 Modified on 05.05.2017

Research J. Pharm. and Tech. 2017; 10(8): 2479-2486.

DOI:10.5958/0974-360X.2017.00438.3