Key findings:

Royal jelly at a dose of 5.25 mg/day effectively increases the number of tertiary and de Graaf follicles in noise-stressed mice. Lower doses (1.75 mg/day and 3.50 mg/day) also show significant improvements compared to the control, indicating a dose-dependent effect on fertility under stress conditions.

What is known and what is new?

Royal jelly has been recognized for its potential health benefits, including its impact on fertility. Noise stress negatively affects the folliculogenesis process in mice. This study demonstrates that royal jelly, particularly at a dose of 5.25 mg/day, significantly enhances the number of tertiary and de Graaf follicles in noise-stressed mice, suggesting its effectiveness in mitigating stress-induced fertility issues.

What is the implication, and what should change now?

Implication of the study Royal jelly can mitigate the adverse effects of noise stress on fertility by enhancing folliculogenesis, specifically increasing the number of tertiary and de Graaf follicles. Further research should explore the mechanisms behind royal jelly's effects on folliculogenesis and evaluate its potential applications in addressing fertility issues in stressed populations. Additionally, optimal dosing and long-term effects should be investigated to validate its therapeutic use.

Stressed is a condition where the individual's body fails to control the body's balance because sudden environmental changes [1]. Continuous exposure to noise not only results in the organs of hearing, but also has side effects on neurophysiological and psychological [2]. Exposure to noise that occurs over a long period of time can induce the assetsof the hypothalamic-pituitary-adrenal (HPA) axis continuously, causing reproductive disorders in folliculogenesis, because there is a disturbance in the secretion of FSH and LH will cause disturbances in folliculogenesis [3].

The number of follicles that develop in the ovaries is influenced by the response of follicular granulosa cells to the hormone FSH. Follicles affected by FSH stimuli will grow to the stage of maturation of oocytes [4]. Research conducted by Taixeriaet al., (2017) [5] has proven that royal jelly can reduce corticosterone levels in mice induced by cold stress because royal jelly contains 10-HDA fatty acids that can inhibit the production of corticosterone stimulated by ACTH.  In addition, royal jelly contains a protein that is predominantly known as Major Royal Jelly Proteins (MJRPs). 

Royal jelly is currently widely used as a dietary supplement, as it is believed to increase the body's resistance.  Therefore, this study aims to see if royal jelly can also prevent the negative impact of exposure to stress due to noise stressors on the number of mice follicles.

This research was conducted for two months starting from May to July 2022. This research was conducted in the experimental animal cage of the Faculty of Veterinary Medicine, Universitas Airlangga Surabaya for the maintenance and treatment of experimental animals. The manufacture of histopathological preparations was carried out in the Pathology Division of the Faculty of Animal Medicine, Universitas Airlangga. Examination of histopathological preparations was carried out in the Division of Veterinary Anatomy, Faculty of Veterinary Medicine, Universitas Airlangga.

Animal 

This study used 25 female mice (Mus musculus) strain Balb/C aged 2-3 month old with an average body weight of 20g were purchased from Pusvetma Surabaya. Mice were acclimatized in the laboratory for 7 days before being placed in a single cage and fed a conventional diet with plenty of water for a total of 12 days of treatment.

Material

The ingredients used in this study were royal jelly, sterile aquades, husk, pellets Hi-Pro Vite Medicated 593, PDAM water ad libitum, NaCl 0.9%, Buffered Neutral Formalin (BNF) 10%, constituent alcohol graded constellation 70%, 80%, 90% and 96%, xylol, paraffin; dye Haematoxylin-Eosin (HE), entelan (adhesive), glass object; and cover glass. The equipment used in this study was a soundproofed wooden box measuring 1.5m x 1.5m x 1m which was flexed by the speaker, Sound Level Meter (SLM), 4 pieces of insulated glass boxes measuring 100cm x 15cm x 15cm, sonde needles and syringes measuring 1cc, cages measuring 60cm x 40cm x 15cm, wire mesh covers, squeaking drinking places with a capacity of 80mL,  porcelain cups, mixing rods, measuring cups,  glass beakers, Pasteur pipettes, cotton buds, masks, gloves, jars, surgical tools (Scapel, blade, anatomical tweezers and surgical scissors), glass objects, glass covers, and Nikon Eclipse-E100 light microscopes, computers.

Methods

Experimental animals that were adapted and divided into 5 treatment groups performed vaginal swabs. Experimental animals that experience estrus can be carried out research methods: K-: given aquades as much as 0.2mL perorally, K+: given aquades as much as 0.2mL perorally and continued to provide exposure to 94db noise for 1 hour which was carried out, P1: a solution of royal jelly was given at a dose of 1.75mg perorally and an interval of 1.5 hours followed by exposure to noise 94db for 1 hour which was carried out, P2: a solution of royal jelly was given at a dose of 3.50mg perorally and an interval of 1.5 hours followed by exposure to noise 94db for 1 hour which was carried out, P3: given royal jelly solution at a dose of 5.25mg perorally and an interval of 1.5 hours followed by exposure to noise 94db for 1 hour which was carried out, all control and treatment groups were given for 28 days. On the 29th day the mice were euthanized by means of cervical dislocation and then dissected and taken one of the ovarian organs and continued the process of making preparations.

Statistical analysis

The data obtained in the form of a figure on the number of ovarian follicles of the sample was then carried out data analysis. To determine the change in the histopic picture of pathology, statistical analysis was carried out using Analysis of Variance (ANOVA). Data is processed using the SPSS (Statistical Programs for Social Scientific) program. If there are significant differences between treatment groups, it is continued with the Duncan test.

Ethical approval

Ethics approval of this research project was obtained from the Committee of Ethics in Health Research Faculty of Veterinary Medicine, Universitas Airlangga Surabaya with certificate number: 2.KE.116.12.2022. All research work has been completed in the same institute.

The results of this study showed that royal jelly could not maintain the number of primary follicles and secondary follicles in mice that were stressed due to exposure to noise (p>0.05). However, there was a significant difference in tertiary follicles and de Graff follicles between the normal control group without exposure and the group without royal jelly with exposure, the royal dose group jelly 1.75 mg/day, the royal jelly dose group was 3.50 mg/day, but not significantly different from the royal jelly dose group 5.25 mg/day (p<0.05).

The test results and the comparative values of the average number of primary follicles, secondary follicles, tertiary follicles and de Graff follicles in mice can be seen successively in Table 1 and figure 1.

Table 1. Average number of primary follicles, secondary follicles, tertiary follicles and de Graff follicles of royal jelly administration that exposed to noise.

Description: Different superscripts in the same column showed marked differences between treatments (p<0.05). K-: given aquades, P1: a solution of royal jelly was given at a dose of 1.75mg, P2: 3.50mg, P3: 5.25mg perorally and followed by exposure to noise 94db.

Based on the results of the Shapiro-Wilk test on the number of tertiary follicles, it is stated that the data is normally distributed (p>0.05) so that parametric data testing of the ANOVA test can be carried out and the average number of tertiary follicles is obtained, showing a noticeable difference between the treatment variables shown and the results (p< 0.05).  Then continued with the Duncan test which showed that the dose of royal jelly on P3 could maintain the number of de Graff follicles in mice that were stressed due to exposure to noise. The test results and the comparative values of the average number of de Graff follicles in mice can be seen in Table 1 and Figure 1.

Figure1. Histology of primary follicles, secondary follicles, tertiary follicles and de Graff follicles in the ovaries of mice that are stressed due to exposure to noise. Description: The red arrow (→) indicates the de Graff follicle, the yellow arrow (→) indicates the tertiary follicle, the green arrow (→) indicates the secondary follicle, and the blue arrow (→) indicates the primary follicle. (H.E; Magnification 100x; Nikon Eclipse-E100 light microscope).

Based on the results of the study, it can be seen that perorally administration of royal jelly solution can maintain the number of primary follicles, secondary follicles, tertiary follicles and de Graff follicles. This is based on the morphological features of each follicle. The number of primary follicles and secondary follicles in the ovaries of stress mice due to exposure to noise given by royal jelly cannot be maintained because the amount of granulosa cells in the primary follicles is still small, the reception of FSH due to the administration of royal jelly to the primary follicles is also still not enough for the development process to the next stage. This can be understood because in the development of follicles, FSH is needed to be able to initiate the development of follicles [6].

An increase in the mice group exposed to noise in the primary and secondary follicles appeared to be a slight increase in the number of follicles and did not differ markedly from the between of the treatment group could be possible due to the presence of growth factors produced locally through the mechanisms of autocrine and paracrine, namely kit ligands (KL) which played a role in oogenesis and folliculogenesis [7].  In Bogdanov (2017) [8] it is said that royal jelly contains progesterone, this can be possible as a cause of inhibition of the early development of follicles.

The number of tertiary follicles and de Graff follicles in the ovaries of mice stress due to exposure to noise given by royal jelly can be maintained, because in royal jelly there are contents that can improve the hormonal function of the reproductive system through the metabolism of lipids, proteins and carbohydrates. Lipids in royal jelly that play a role in the reproductive system include 10HDA, and 10H2DA, while proteins that play a role in the reproductive system are MRJPs 1 [9].

Taking Gonadotropin Releasing Hormone (GnRH) can cause increased secretion of the hormones Follicle Stimulating Hormone(FSH) and Luteinizing Hormone (LH) which play a role in the development of tertiary follicles and de Graff follicles in the ovaries (Stamatiades and Kaiser, 2017) [3]. In the mice group given royal jelly 3.50mg / day and 5.25mg / day had a noticeable effect inmaintaining the number of de Graff follicles, this was due to the presence of 10H2DA fatty acids. 10H2DA fatty acids in royal jelly have a role in modulating estrogen activity by increasing the interaction activity of estrogen receptors (ERα) [10]. Estrogen synthesized by tertiary follicles also plays a role in increasing the work of FSH hormone follicular cell receptors to increase the LH hormone response for the function of maturation of follicles into de Graff follicles that are ready for ovulation [4].

The development of ovarian function in terms of defense of the number of follicles or folliculogenesis in the mice group there is a noticeable difference can be due to the content of Major Royal Jelly Protein 1 (MRJP1) or royal actin in royal jelly. MJRP1 or Royal actin is the most dominant protein feed in royal jelly, containing monomeric glycoproteins that can activate p70 S6 kinase. p70 S6 kinase is a protein as an integrator of nutrient signals in growth factors in the ovaries. This kinase is responsible for the development of ovarian function, by optimizing the work of the hormone estrogen in the ovaries [11]. 

Apart from the three important components above, royal jelly also contains various hormones, such as testosterone, progesterone, prolactine, and estradiol, so that it can optimize the function of the gonadal organs including the ovaries (Bogdanov, 2017) [8]. This is in line with research from Rahayu (2007) [12] that royal jelly has an effect on increasing the number of follicles in mice. 

Based on the results of the research that has been carried out, it can be concluded that: the administration of royal jelly cannot maintain the number of primary and secondary follicles, but can maintain the number of tertiary follicles and de Graff follicles in the ovaries of mice that are stressed due to exposure to noise.

Conflict of Interest:

The authors declare that they have no conflict of interest.

Funding: No funding sources 

Ethical approval: The study was approved by the Institutional Ethics Committee of University of Airlangga

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