تأثیر غلظت‌های مختلف ژله ‌رویال بر ریخت شناسی و آپوپتوزیس اسپرماتوزوای زنبوران نر در زمان‌های مختلف پس از انجماد شیشه‌ای بدون سرما- محافظ به روش قطره‌ای

Introduction: Honeybees are among the most important pollinators, playing a vital role in the pollination of cultivated plants and wild species. It is estimated that nearly 70% of crop plant species worldwide rely on honeybee pollination. The Farm-to-Fork strategy emphasizes that food production should have a neutral or positive environmental impact, mitigate climate change effects, prevent biodiversity loss, and ensure access to mineral-rich and sustainable food for all citizens. Beekeeping is one agricultural sector that aligns with all these sustainability criteria, as it is essentially climate-neutral and waste-free. However, recent reports of significant colony losses worldwide have raised concerns about the future of honeybees. Additionally, many native honeybee species have been replaced by commercial honeybees, reducing genetic diversity within subspecies of Apis mellifera. Colony losses directly impact genetic diversity. Successful sperm cryopreservation is an effective strategy for preserving honeybee genetic diversity, aiding in the selection of pest- and disease-resistant lines, and preventing further colony losses. The decline or loss of native and non-native bee species can negatively affect agricultural production and ecosystem functioning. Increasing honeybee production globally through improved genetic lines and enhanced colony management is feasible, with genetic traits identified using semen from superior drones. Cryopreservation of bee sperm has been explored as a means of maintaining genetic diversity in honeybee populations. Cryopreserved drone sperm retains viability and fertilization capacity, supporting successful artificial insemination outcomes and contributing to disease-resistant breeding lines and genetic conservation. Royal jelly (RJ), produced by young worker bees, contains water, carbohydrates, proteins, amino acids, lipids, minerals, vitamins, and polyphenols. In 2002, a novel cryoprotectant-free vitrification method for human spermatozoa was introduced, using non-permeable additives like sugars to avoid the osmotic and toxic effects of high cryoprotectant concentrations, preserving sperm acrosome integrity and cytoskeletal structure. Our study aimed to enhance drone sperm preservation by incorporating royal jelly into the cryoprotectant-free vitrification extender.
Materials and Methods: Drone sperm from adult drones (36 days old) was collected using manual inversion. Various concentrations of royal jelly (0%, 0.5%, 1%, 2.5%, and 5%) were added to the extender. After preparation, a micropipette was used to drop 10 µL aliquots of the sperm suspension directly into the cooling agent from a distance of 10 cm. A sphere immediately formed and floated to the surface. After 6 seconds in liquid nitrogen, the sphere solidified and sank to the bottom of the strainer. Once solidified, the spheres were collected, packaged into 1.8 mL cryotubes, and stored in a liquid nitrogen tank for 90 to 300 days before thawing. Thawing was performed by quickly submerging five spheres, one by one (no more than five at a time), into 3 mL of pre-warmed (37°C) Modified Hopkins extender solution, followed by gentle vortexing for 10 seconds. The warmed sperm suspension was then maintained at 37°C in a 5% CO₂ environment for 10 minutes, followed by centrifugation at 1000 g for 10 minutes. The resulting cell pellet was resuspended in 20 µL of Modified Hopkins extender solution for quality evaluation. Data were analyzed using a completely randomized design with five replications. Sperm quality parameters, including morphology and apoptosis, were evaluated in vitro. To assess morphology, two eosin staining methods-crystal violet (acidic) and silver nitrate (alkaline)-were used. Eosin-crystal violet staining was performed according to Kondracki et al. (2005) as this acidic dye effectively stains sperm and is useful for detecting morphological characteristics. Silver nitrate staining was conducted based on the method of Andraszek and Smalec (2011), with morphology evaluation performed on 500 spermatozoa using phase-contrast microscopy at 400× magnification. Results from silver nitrate staining were compared with those from eosin-crystal violet complexes, and only the percentage of normal sperm was reported. Apoptosis was measured using the fluorescein isothiocyanate annexin V apoptosis detection kit with 7-aminoactinomycin D. A total of 500 spermatozoa per sample were analyzed, with only the percentage of apoptotic sperm reported for each treatment.
Results and Discussion: Sperm morphology and apoptosis after vitrification/thawing with different RJ concentrations were evaluated at 0, 90, and 300 days. Our results showed that supplementation with 1% RJ significantly increased the percentage of normal sperm morphology (75.40 ± 3.50) compared to other groups across all storage durations. Lower to moderate concentrations of royal jelly improved sperm morphology, suggesting a stabilizing effect on cell structure and protection against vitrification-induced damage. This effect is likely due to RJ's bioactive compounds, which maintain cell membrane integrity and prevent structural abnormalities. Our findings, in agreement with previous studies, support RJ's antioxidant benefits in reducing apoptosis. This effect was particularly evident with 1% RJ across all time points post-thawing, significantly lowering sperm apoptosis compared to controls. The use of non-penetrating cryoprotectants likely enhanced plasma membrane flexibility during vitrification, reducing apoptosis by preventing damage to sperm DNA and membrane integrity. Conclusion: This study demonstrates that adding royal jelly at a concentration of 1% to vitrification diluents significantly enhances the quality of drone bee sperm preserved via cryoprotectant-free vitrification using the drop method. The notable improvements in normal morphology and reduction in apoptosis highlight RJ’s potential as a beneficial additive to sperm vitrification protocols. Research into drone bee sperm quality presents promising applications, warranting further exploration of advanced methods such as computer-aided sperm analysis, novel fluorescent dyes, multiparametric tests, flow cytometry, and image analysis techniques. These efforts aim to refine vitrification techniques and investigate RJ’s long-term effects on sperm quality and queen fertility-critical factors for sustainable beekeeping and biodiversity conservation.
Keywords: Apoptosis, Cryoprotectant, Drone, Morphology, Royal jelly, Sperm, Vitrification.