Testicular morphology and seminal fluid parameters of adult Wistar rats following honey administration

Purpose: Honey has a long history of use in the traditional medical systems This objective of this study was to find out the effects of honey on quality and quantity of sperm and testicular microstructure when compared to fertility boosting drug and controls. Methods: A total number of thirty (30) matured male Wistar rats that were sexually active weighing 200 280g were used for this study. The animals were grouped into five as A E. Group A was the control; Group B (standard group) was the standard group that received 0.3ml of follicle stimulating hormone (FSH) drug for 6 days; groups C, D and E received 1ml, 2ml, and 2.5ml of honey daily for 21days respectively. After 21 days of administration, the testes were removed for analysis of the sperm parameters and the histology. Results: Honey significantly improved the sperm quality and spermatogenesis rate (denser seminiferous tubule lumen) of exposed animals compared to control animals, but most improvement was seen in the standard group that received 0.3ml FSH. Also no sign of degeneration or cellular loss was observable in the testicular histo-architecture of experimental animals. Conclusion: This research showed that honey possesses some fertility boosting properties in exposed animals compared to controls and honey is not associated with increased sperm abnormalities.


INTRODUCTION
Fertility in males is largely dependent on adequate production of healthy spermatozoa by the testes, these days many men have resolved to the use of alternative substances (like fruits, herbs, honey etc.) for fertility boost [1].It has been recorded that approximately 70 % of men worldwide use alternative medicinal substances that are cheap and readily available to increase their sperm content [1].The World health organization [2] endorsed honey and other sperm boosting supplement as being safe for usage [3].Studies have shown that honey increased spermatogenesis in rats i.e. sperm boosting properties [4].A report from Malawi suggested that long term administration of honey had no negative effect on the histopathology of Wistar rats' testis [5], and Igbokwe et al [6] found increase in sperm parameters in honey treated animals, while a contrary result was obtained by Dare et al [3] who reported reduced motility and increased percentage of dead and abnormal spermatozoa in honey treated animals.
Bees produce this sweet food (honey) by using nectars from flowers.Honey has a long history of use in the traditional medical systems [7].It has a density of about 1.36 kg/litre (i.e.36 % denser than water).The major active constituent which has been found to have strong fertility effect is the Chrysin [8] which is a bioflavonoid compound.This compound is in high quantity in honey and propolis.Chrysin is also known for its testosterone boosting activity Dhawan et al [9].It also inhibits the conversion of testosterone to estrogen [10].It appears to be poorly absorbed and also sufficiently metabolized, hence low levels are found in testes and blood.These levels are insufficient to exert the beneficial effects of testosterone boosting Kohut et al [11].
Chrysin, like most bioflavonoids, is poorly bioavailable in its isolated form.This makes its actual use to be decreased [12].Its bioavailability is thought to be due to its extensive metabolism and intestinal transportation.
Chrysin, similar to most bioflavonoids, is poorly bioavailable in its isolated form.This makes its actual use to be decreased [12].Its poor bioavailability is thought to be due to its extensive metabolism and intestinal transportation.
Organs containing the P450 system such as the liver, easily conjugates chrysin into chrysin glucuronidase and chrysin sulphate, which are its metabolites though may not be bioactive.Glucuronidation rates are increased in the intestinal cells via induction of UGT1A1 expression by chrysin, which further supports its low bioactive levels [12].Therefore, this work aims to determine the effect of honey on seminal fluid parameters and morphometry of the seminiferous tubules.

METHODS
Thirty (30) male Wistar rats weighing between 200g-280g were used for the study.They were kept in the animal house of the College of Health Sciences, University of Ilorin under normal atmospheric condition of 25 o C. The animals were divided into five (5) groups randomly.Each group consist of six (6) rats.Group A was the control; group B was administered 0.3ml of follicle stimulating hormone drug for 6 days [13] (standard group), group C, D and E received 1ml, 2ml, and 2.5ml of honey daily for 21days respectively (increasing quantity of administered honey) [10]

Sample collection
The Wistar rats were sacrificed after the experiment.They were administered inhalation anaesthetic (ether), then sacrificed by cervical dislocation.
Open castration method was used for orchidectomy via a midline scrotal incision.Then the testicles were gently milked out through the incision site.The testicles were exposed by incising the tunica vaginalis, followed by exposure, ligation and incision of the spermatic cord.The cauda epididymis was then identified, from which the semen samples were collected.Saba et al [14] and Oyeyemi and Ubiogoro [15] described a similar method of semen collection.The semen samples were then analysed immediately.

Sperm motility and count assay
A drop of semen was placed on a glass slide to assess sperm motility.2.9% sodium citrate was added.This was covered with a glass slip, then the microscopic fields were viewed under the light microscope to determine the motile and non-motile spermatozoa.This method was as described by Zemjanis [16].
Sperm count was with the use of a hemocytometer.The cauda epididymis was minced in normal saline, then filtered and an aliquot of 10ul was placed on the hemocytometer.The improved Neubauer chamber (Deep 1/10m, LABART, Munich, Germany) was used for the count, under a light microscope [17].

Sperm morphology and viability assay
Four hundred (400) spermatozoa count were used to determine this.The semen was placed on the slide, then Wells and Awa stains (which consist of 0.2g eosin and 0.6g fast green dissolved in distilled water and ethanol in ratio 2:1) was added, as documented by Wells and Awa [18]; Isaac and Ebenezer [19].This was then viewed under the microscope In determining the viability, a drop of semen was placed on the slide, then addition of a drop of eosin-nigrosin stain (which is 1% eosin with 5% nigrosin in 3% sodium citrate solution).This was mixed well then air dried.It was then observed under the microscope for the live and dead spermatozoa cells, which were counted as described by Wells and Awa [18] and Isaac and Ebenezer [19].

Statistical analysis
Data collected were analyzed using one-way analysis of variance (ANOVA) followed by Tukey's multiple comparison test with the aid of SPSS (V20; USA).The data were presented as means ± SEM (standard error of mean).P ≤ 0.05 was considered statistically significant.

Histology of the testes
The proliferation of spermatogonic series (SG) into matured spermatozoa in the seminiferous tubular lumen (L) was observed in the control rats (Figure 1).There was an improvement in the proliferation rate of the spermatogenic series (SG) in the FSH treated rats (Figure 2), shown by the presence of abundant spermatozoa in the seminiferous tubular lumen (L), i.e., a dense lumen.The basement membrane was also intact and there were Leydig cells present in the interstitium (I).Additional increases in the doses of honey in the honey treated groups (III, IV and V) showed progressive proliferation of spermatogonic series (SG) into matured spermatozoa in the seminiferous tubular lumen (L) with intact basement membrane and interstitial space (I).This is shown in Figure 1 (C-E).

DISCUSSION
The histological evaluation of the actions of honey on the microstructure of the testes revealed less dense packing of spermatogenic cells in control compared to the FSH and honey treated groups.Also, the lumen of control was less densely filled with seminiferous tubular lumen with spermatozoa compared to FSH and honey treated groups.The increase in the abundance of spermatozoa in the lumen is dose dependent among the groups that received varying doses of honey as most spermatozoa i.e. denser lumen, was seen in animals that received 2ml and 2.5ml Honey, which is similar to findings by Nworah et al [5].Previous studies revealed that honey of appropriate dose might enhance the third stage of spermatogenesis in rats [20] .Yousef and Salama [21] observed a similar finding of honey acting as a physiologic modulator for spermatogenic cell proliferation, which influence the cell cycle of the seminiferous epithelium thus, increasing spermatogenesis.
Likewise, follicle stimulating hormone has been tested and confirmed to cause an increase in spermatogenesis [22] The characteristics measured by semen analysis are some of the factors in semen quality that determine the viability of sperm [23].There was a significant dose-dependent increase in the percentage sperm motility and sperm count following treatment with honey.This corresponds to findings by Igbokwe et al [6], who reported the potency of honey as a fertility boosting agent and Salman et al [24], whose findings were also suggestive of the beneficial effects of honey on sperm count.Honey contains several metals, amongst which is zinc [25].These findings are suggestive of zinc accumulating in the testis during early spermatogenesis.Therefore, it may be important in DNA synthesis and regulation of spematogonial proliferation [26].

CONCLUSION
Though there is not enough established data concerning the medicinal use of honey in treating human male infertility currently, this study revealed that honey is potentially useful as a fertility boosting agent by improving on the qualitative and quantitative parameters of spermatogenesis in a dose dependent manner.