Bull 1 Amer M. Hussin Bull. Iraq nat. Hist. Mus. (2015) 13 (4): 1-9 STUDY ON THE EFFECT OF ROYAL JELLY OF BEES (APIS MELLIFERA) ON THE MORPHOLOGY AND SPERM FUNCTION PARAMETERS IN MICE (SWISS ALBINO) Amer M. Hussin Assist. Prof. Dr., college of Veterinary Medicine, Baghdad University, Iraq. ABSTRACT The objective of this study was to investigate the effect of Royal jelly RJ on morphology and motility of mice sperms. Sperms were collected from the cauda region of the epididymis of each 10 mice from the treatment and control groups. Direct activation techniques and evaluation of sperm morphology were carried out. Dhino microscope was used for sperm measurement. The inspection was carried out in Salamatic laboratory for pathological analysis in 2015.The result revealed that all of the sperm function parameters registered significant activation in the treatment group. There was a significant increase in both the percentage of the sperm motility grade A and the progressive motility (A+B) of the treatment group. Spermatozoon with different lengths were noticed. The study explains that this difference may be due to the presence of different sperm developmental stages and not to the increase in the number and sizes of mitochondria in the mid-peace during activation or not to the sliding movement of microtubules in the axon of spermatozoon. These findings provide evidence that RJ can play an important role only in improving the male mice motility and which may open the way for further researches to demonstrate the possibility of using RJ in artificial insemination in field animals. Key words: Apis mellifera, Bees mice, Sperm morphology royal jelly. INTRODUCTION Royal jelly is a substance that is secreted from worker honey bees. It is the main food source for only the first 3 days of worker larva. One larva that is to be the Queen bee is fed only RJ its entire life, so that bee worker alives only for 6 months while Queen alives for 2-7 years. This exclusive feeding triggers the full development of her ovaries which is needed to lay the millions of eggs she will lay in her lifetime. This may be related to the extremely high nutritional contents of the RJ. (Mishima et al., 2005), (Narita et al., 2006), (Okuda et al., 1988), and (Taylor, 2004). Royal jelly is rich in amino acids, lipids, simple sugars (Monosaccharides), some enzymes, antibacterial and antibiotic components, vitamins such as pantothenic acid (Vitamin B5), vitamin B6 (pyridoxine), high levels of vitamins D and E. It contains ample levels of iron and calcium, fatty acids and most importantly, proteins. RJ also contains acetylcholine, which is needed to transmit nerve messages from cell to cell (Viuda-Martos et al., 2008). Also the objective of this study is to examine the in vitro effect of RJ on morphology and motility of sperms. Similar literature on this rasped were not available. The aim of this study is to investigate the effect of (RJ) in morphology and activity of sperms in mice. 2 Study on the Effect of Royal Jelly of Bees on sperm activity MATERIALS AND METHODS Experimental animals: Twenty mature apparently healthy male mice (Balb/C St Can BR Strain) 8-12 weeks old were purchased from the higher institute for the diagnosis of infertility & techniques of assisted production of Al-Nahrain university in 2015. Mice were divided into control and treatment groups. 1- Sperms Collection: Animals were sacrificed by cervical dislocation, The caudal region of the epididymis were removed aseptically and placed immediatly into 1ml of in vitro fertilization (IVF) medium in a 35-mm culture dish. The collected pieces of epididymis were minced by using forceps and scissors. The sperms were allowed to disperse by gently shaking the dish by hand for 3 to 5 minutes at room temperature. The sperms suspension was divided into two parts. 2- In vitro Sperm Activation Technique: Direct Activation Technique was used where the sperms were allowed to swim-up through the medium for at least 60 minutes at 37 by 5% CO2 incubation (Cross and Overstreet 1987). This technique for sperm activation is characterized by direct effect of the culture medium on sperm parameters (Fig.1). Then the sperms were counted. 3- Royall Jelly preparation: Royal jelly was used as 10% concentration, was prepared by adding 0.1ml of RJ suspension to 0.9 ml of mouse IVF media (Gain medium., FertiPro NV, Industriepark Noord 32,8730 Beernem, Belgium v.C1). The 10% solution was filtered with pore size 0.45μm and 0.22 μm, and then pH was adjusted to reach 7.2-7.4 with HCl one molar (CYBOW 10, DFI Co., Ltd. Korea). Then activate the sperms using Direct Activation Technique that described previously. 4-Sperm concentration, motility, grade of activity and normal morphology: The motility of spermatozoa were graded according to (Mishima et al. 2005): A-Rapid linear progressive motility, B-Rapid nonlinear or linear non rapid progressive motility, C-Non progressive motility (localized) and D- Immotile. 5-Evaluation of sperm morphology: Sperms recovered from the treatment and control groups were fixed in zinker's fluid. The Nigrosin-Eosin stain was used for examining the sperm morphology. Live sperms appear does not take the Eosin stain and looks colorless under light microscope; whereas dead sperms take up eosin and appear pinkish in color (Graham, 2004). The lengths of sperms from each of the treatment and control were examined and measured by Digital microscope Dinocapture 2.0 (Made in Taiwan) (Check et al., 1992). RESULTS In vitro sperm activation technique: (Table 1) revealed that the mean sperm concentration (x10 6 sperm/ml) following direct activation with 10% R.J-IVF medium was significantly (P<0.05) higher than before activation. Active sperm motility (grade A and grade B) was significantly (P<0.05) increased. The percentage of morphologically normal sperms following the addition of 10% R.J-IVF medium showed no significant (P< 0.05) difference when compared with the R.J-free IVF medium. The morphological results: With the digital Dino microscope, the sperm appears to consist of only two portions: The head and tail. The measurement of the different parts of 3 Amer M. Hussin spermatozoa was difficult as the head of mice sperm was irregular and characterized by severe polymorphism. Although, the morphological study couldn't detect any apparent increase in the head of treated spermatozoa, we could observe a non significant total elongation in the length of treated mice sperm (71±0.98) compared to control ones (67±1.07) (Table 2). . Fig. 1: Sperms before activation. Head of sperm (short arrows), Tail of sperm (long arrow). x400. Eosin-Nigrosin stain. Fig. 2: Sperms after activation. Head of sperm (short arrows). Tail of sperm (Long arrow). x400. Eosin-Nigrosin stain. 4 Study on the Effect of Royal Jelly of Bees on sperm activity Fig. 3: Sperms after activation. Head of the sperm (short arrows). Tail of the sperm (long arrow). x400. Eosin stain. Table (1): Comparison between control and treatment groups with 10% R.J-IVF medium on certain sperm function parameters following in vitro direct activation. (Mean SE) In vitro sperm activation Grouping with and without R.J. After 1 hour incubation Mean± SE Significance Sperm concentration (10 6 /ml) Without R.J 25.22±1.405 S With R.J 36.32±4.323 Sperm motility grade A (%) Without R.J 12.40±2.315 S With R.J 18.73±0.918 Sperm motility grade B (%) Without R.J 21.33±2.622 S With R.J 30.73±2.472 Sperm motility Grade C (%) (Localized) Without RJ 26.14±0.05 S With RJ 36.21±0.123 Sperm motility Grade D (%) (Immotile) Without RJ 40.13±0.04 S With RJ 14.33±0.984 Progressive motility (A+B)% Without R.J 33.73±4.362 S With R.J 49.07±3.065 Morphologically normal sperms(%) Without R.J 30.43±4.410 NS With R.J 31.93±3.084 5 Amer M. Hussin Table (2). Morphometric measurement (μm) of the spermatozoon with and without adding RJ. Head Tail & middle piece Total Without RJ 4±0.17 63±1.56 67±1.07 NS With RJ 4±0.42 67±1.92 71±0.98 NS DISCUSSION In the current study, the results referred to the improvement of sperm motility in the treatment group. This is confirmed by (Rodriguez, 2007) who reported that (RJ) increase libido and supports egg and sperm health. This may be related to the components of the RJ present in the activation fluid (Svoboda et al. 1986). The presence of different lengths of the sperms taken from the treatment may be attributed to the different developmental stages of spermiogenesis and not due to the proliferation of mitochondria and proteinaceous materials present in the mid-piece and principal part of the tail respectively (Ganong, 2005) or the slide movement of microtubules (Samuelson, 2007). This is confirmed by the finding of (Noguchi and Koizumi, 2011). This could also be in agreement with (Lercker et al., 1982) who stated that mitochondria aggregate around the proximal part of each flagellum, forming a thickened region known as the middle piece, the region where the ATP for flagellar movements of spermatozoa is generated. However the present findings were within the limits of the spermatozoon length of domestic species (Samuelson, 2007). Further studies under electron microscope might shade more light on the effect of RJ on the mitochondria or other structures that are involved in the sperm motility. The current study shows that increased sperm length is unlikely to be driven by selection for increased swimming speed, and that the relative lengths of a sperm's constituent parts, rather than their absolute lengths are likely to be the target of selection. All else being equal, we suggest that a simple measurement of the ratio of head to tail length should be used to assess the possible link between morphology and speed. However, this is mostly likely to be the case for external fertilizers in which females have relatively limited opportunity to influence a sperm's motility. In this study, there was an enhancement in certain sperm function parameters, and that was attributed firstly to the direct activation technique with IVF medium. The medium provided the same culture components which found in the female genital tract and that will trigger the sperm hyperactivity motility (Mishima et al., 2005). The technique sustains the epididymal sperms to get rid of the decapacitating factors in the seminal plasma and makes the sperm ready for successful fertilization in vitro (Tournaye et al., 2003). Secondly, adding of RJ to the culture media enhances different sperm function parameters following 60 minutes of activation, mainly sperm concentration, total sperm motility percentage and grade activity of forward progressive movement. The results of the present study found that, in vitro activation of caudal epididymal sperms by direct activation technique with 10% RJ-IVF medium resulted in a significant increase in the concentration of the recovered spermatozoa by swim-out after 60 minutes of incubation. The differences in sperm concentration between RJ-free IVF and 10% RJ-IVF medium may be explained by the booster effect of R.J one epididymal sperms to move out and to release from the epididymal tissue. Moreover, culturing of the semen sample with 10% RJ-IVF medium result a significant increase in the percentages of sperm motility and grade activity of forward movement to reach in the last one to 50% (Grade A+Grade B) of the semen sample. 6 Study on the Effect of Royal Jelly of Bees on sperm activity Results of this study also showed that, there was no significant difference in the morphologically normal sperms between the treated and control groups, and the mean was near 30% as an average. These results were compatible with the improvements in sperm concentration and grade activity of forward movement; because it was very difficult to make any enhancement on either sperm parameters when the semen sample considered morphologically abnormal (Coetzee et al., 1998). It was concluded from the present study that the addition of RJ to the culture media of sperm can enhance the sperm quality in mice. This result can be utilized for other mammalian IVF programs. LTERATURE CITED Al-Dujaily, S. S.; Al-Janabi, A. S. and Nori, M. 2006. Effect of Glycyrrhiza extract on in vitro sperm activation of asthenospermic patients. Journal of Babylon University, 11(3): 477-483. Al-Jarah, I. A. N. 2002. Study of some exogenous hormones on sperm in vitro activation of asthenozoospermia patients. MSc Thesis. College Science. University of Babylon, Iraq. Check, J. H; Adelson, H. G.; Schubert, B.R. and Bollendrof, S. 1992. Evaluation of sperm morphology. 28.1 P: 15-17. Claire, L. Borg. 2009. Phenotyping male infertility in the mouse: how to get the most out of a' non-performer'. Hum. Reprod. Update, 16 (2): 205-24. Coetzee, K.; Kruger, T. F. 1998. Predictive value of normal sperm morphology: a structural literature review. Human Reproduction Update.73-82. Cross, N. L. and Overstreet, J. W. 1987. Glycoconjugates of the human sperm surface: distribution and alterations that accompany capacitation in vitro. Gamete Res., 16 (1):23–35. Dellman, H. D. and Brown E. M. 1976. Veterinary histology. Land Febiger. Philadelphia, 300pp. Ganong, W. F. 2005. Medical physiology, 22th, Lange; 425-426. Graham, J. K. 2004. Effect of adding cholesterol to bull sperm membranes on sperm capacitation, the acrosome reaction and fertility. Biol. Reprod. 1(2): 522-7. Hassan, A. A. 2009. Effect of royal jelly on sexual efficiency in adult rats. Iraqi Journal of veterinary sciences, vol. 23, supplement π .pp:155-160. proceeding of the 5th scientific conference, college of Vet. Medicine, University of Mosul. Hidaka, S.; Okamoto, Y.; Uchiyama, S.; Nakatsuma, A.; Hashimoto, K. and Ohnishi, S.T. 2006. Royal jelly prevents osteoporosis in rats: beneficial effects in ovariectomy model and in bone tissue culture model. Evid Based Complement Alternat Med., 3:339. 7 Amer M. Hussin Katja, M. Wolski; Gerard M. Gibbs; Moira K. O'Bryan; Anna, G. Sabatini; Gian, L. Marcazzan; Maria, F. Caboni; Stefan, B. L.; Bicudo and de Almeida-Muradian. 2009. Quality and standardization of Royal Jelly, Journal of Apiproduct and Apimedical science, 1(1): 1-6. Koya-Miyata, S.; Okamoto, I.; Ushio, S.; Iwaki, K.; Ikeda, M. and Kurimoto, M. 2004. Identification of a collagen production-promoting factor from an extract of royal jelly and its possible mechanism. Biosci Biotechnol Biochem, 68: 767-73. [PubMed] Lercker, G.; Capella, P.; Conte, L. S.; Ruini, F. and Giordani, G. 1982. Components of royal jelly II. The lipid fraction, hydrocarbons and sterols. J. Apicult Res, 21:178-84. Mateescu, C. and Barbulescu, D. 1999. Enhanced nutritive, functional and therapeutic action of combined bee products in complex food supplements. Roum Biotechnol Lett, 4: 163-72. Mesher, A. L. 2010. Junqueira's Basic Histology. 12ed. Mac Graw Hill Medical, 373-378. Mishima, S.; Miyata, T.; Suzuki, K .M.; Araki, Y.; Akao, Y. and Isohama, Y. 2005. Estrogenic effects of royal jelly. J. Tradit Med. 22: 171-5. Mishima, S.; Suzuki, K. M.; Isohama, Y.; Kuratsu, N.; Araki, Y. and Inoue, M. 2005. Royal jelly has estrogenic effects in vitro and in vivo. J. Ethnopharmacol, 101: 215- 20. [PubMed] Nagy, A.; Gertsenstein, M.; Vintersten, K. and Behringer, R. 2003. Manipulating the mouse embryo, A laboratory manual. 3rd Ed. Cold Springer Harber Press. New York, 31-61pp. Narita, Y.; Nomura J.; Ohta S.; Inoh Y.; Suzuki K.M. and Araki Y. 2006. Royal jelly stimulates bone formation: physiologic and nutrigenomic studies with mice and cell lines. Biosci Biotechnol Biochem, 70:2508-14. Nassar, A.; Mahony, M.; Blackmore, P.; Morshedi, M. and Ozgur, K. 1998. Increase of intracellular calcium is not cause of Penteoxifylline-induced hyperactivation or acrosome reaction in human sperm. Fertility Sterility, 69: 745-749. Noguchi, T. and Koizumi, M. 2011. Sustained elongation of sperm tail promoted by local remodeling of giant mitochondria in Drosophila. Current biology, 21 (10): 805- 814. Okuda, H.; Kameda, K.; Morimoto, C.; Matsuura, Y.; Chikaki, M. and Jiang, M. 1998. Studies on insulin-like substances and inhibitory substances toward Angiotensin- converting enzyme in royal jelly. Honeybee Sci. 19: 9-14. (in Japanese). Rodriguez, H. 2007. Fertility super food-Royal Jelly, natural fertility info. com/royal- jelly.html. Sabatini, A. G. 2009. Quality and standardization of Royal Jelly. Journal of Api product & Api Medical Science, 1(1): 16-21. 8 Study on the Effect of Royal Jelly of Bees on sperm activity Samuelson, D. A. 2007. Textbook of veterinary histology. Saunders Elsevier, China, 427- 428. Suzuki, K.; Isohama, Y.; Maruyama, H.; Yamada, Y.; Narita, Y.; Ohta, S.; Araki, Y.; Miyata, T. and Mishima, S. 2008. Estrogenic activities of fatty acids and a sterol isolated from royal jelly. Evidence 30-based complementary and alternative medicine, 5(3): 295-302. Svoboda, J. A.; Herbert, E. W. Jr.; Thompson References M. J. 1986. Sterols of organs involved in brood food production and of royal jelly in honey bees. Insect Biochem. 16: 479-82. Tamir, S.; Eizenberg, M.; Songen, D. M.; Shelach, R.; Kaye, A. and Vaya J. 2000. Estrogenic and Anti-proliferative Properties of Glabridin from Licorice in Human Breast Cancer cells. Cancer Research, 15(60): 5704. Taylor, L. 2004. Database File for Licorice. Rain tree Nutrition Inc. (Home page on the internet) (Cited: 14 August, 2007). Tournaye, H.; Verheyen, G. and Albano, C. 2003. Intracytoplasmic sperm injection versus in vitro fertilization: a randomized controlled trial and a meta-analysis of the literature. Fertility Sterility, 78: 1030-1037. Viuda-Martos, M. Y.; Ruiz-Navajas, J.; Fernández-López and Pérez-Álvarez, J. A. 2008. Functional Properties of Honey, Propolis, and Royal Jelly. Journal of Food Science, 73: 117-124. 9 Amer M. Hussin Bull. Iraq nat. Hist. Mus. (2015) 13 (4): 1-9 على شكلياء ومعايير Apis mellifera عسللنمل االغذاء الملكي ل دراسة تأثير Swiss albinوظيفة نطف ذكور الفأر السويسري عامر متعب حسين العراق بغداد, ,جامعة بغداد, كلية الطب البيطري, فرع التشريح واألنسجة الخالصة صممت هذه الدراسة للمقارنة بين نتائج متغيرات النطف قبل وبعد التنشيط بالغذاء عشرة لكل من )الفئران البالغة إستخدم في هذه التجربة عشرون من ذكور. الملكي للنحل والتي جمعت من بيت الحيوان في المعهد العالي لتشخيص (مجموعتي التجربة والسيطرة أجريت التجربة والتحليالت في . العقم والتقنيات المساعدة على االنجاب بجامعة النهرين . أخذت النطف من ذيل البربخ. 5102الرصافة - مختبر سالمتك للتحليالت المرضية ببغداد , والنسبة المئوية لنشاط النطف, ف المتحركةوالنسبة المئوية للنط, تم تقييم تركيز النطف تم تسجيل األبعاد الشكليائية للنطفة في الحالتين . والنسبة المئوية لشكلياء النطف الطبيعي أظهرت نتائج الدراسة الحالية . تم تحليل النتائج إحصائيا. باستخدام الميكروسكوب نوع داينو خارج )مجموعة المعاملة بعد التنشيط في متغيرات النطفة في ( p<0.05)تحسن معنوي بمادة الغذاء الملكي مقارنة بمجموعة السيطرة حيث حصلت زيادة معنوية ( الجسم (P<0.05 ) بعد تنشيط النطف لكل من الحركة التقدميةA للنطف والحركة التقدمية الكلية (A+B )و أظهرت الدراسة وجود أطوال مختلفة للنطف في مجموعتي السيطرة . للنطف تعزي الدراسة هذه الزيادة , التجربة وزيادة غير معنوية في أطوال النطف لمجموعة التجربة بسبب إزدياد أعداد وأحجام المتقدرات وليسإلى المراحل التطورية المختلفة لنشأة النطف الموجودة في المنطقة الوسطية للنطفة بعد التنشيط أو بسبب حركة ألنبيبات الدقيقة أإلنزالقية ترى الدراسة أن إضافة الغذاء الملكي الى الوسط الزرعي . لموجودة في محور النطفةا وإمكانية إستخدامه لزيادة نشاط النطف , حركتها وقد يؤدي إلى زيادة نشاطها للنطف يحفز .في التلقيح االصطناعي في الحيوانات الحقلية