TECHNOLOGICAL PARAMETERS OF SILKWORM (BOMBYX MORI L.) EGG STOR-AGE AT DIFFERENT PERIODS OF EMBRYOGENESIS

International independent scientific journal №79/2025 3 AGRICULTURAL SCIENCES TECHNOLOGICAL PARAMETERS OF SILKWORM (BOMBYX MORI L.) EGG STORAGE AT DIFFERENT PERIODS OF EMBRYOGENESIS Mirzakhodjaev B. D.Sc., Scientific Secretary, Research Institute of Sericulture, Mirzakhodjaev A. Ph.D, Head of the Laboratory of Mechanization, Research Institute of Sericulture, Radjabov I. doctoral of Research Institute of Sericulture Tajenova A. doctoral of Research Institute of Sericulture https://doi.org/10.5281/zenodo.17583039 Abstract This study presents a comprehensive assessment of the technological parameters for storing silkworm (Bombyx mori L.) eggs during different stages of embryogenesis. The research identifies and systematizes optimal temperature and humidity regimes that ensure stable embryonic development, proper diapause maintenance, and high egg viability. A six-stage storage scheme was proposed, covering the full production cycle—from embryo formation and estivation to overwintering and pre-incubation handling. The study demonstrates that a gradual decrease in temperature from 24 ± 2 °C to 3 ± 1 °C, with constant relative humidity of 60–70%, provides favorable conditions for metabolic balance inside the eggs, prevents premature activation, and minimizes desiccation. Maintaining this microclimate ensures 90–95% egg viability and an 8–12% increase in hatching rate compared with conventional storage practices. The results also confirm that uniform temperature and humidity control contribute to synchronized larval emergence, enhancing the efficiency of large-scale sericulture production. The developed storage system offers a practical and standardized framework suitable for industrial sericulture facilities and centralized incubation centers, supporting stable year-round production of high-quality silkworm eggs. Keywords: Silkworm eggs; Bombyx mori; Embryogenesis; Storage regimes; Temperature–humidity control; Diapause; Industrial sericulture; Viability; Hatching rate. Introduction All biological processes occurring in silkworm (Bombyx mori L.) eggs (grains) proceed continuously under the influence of external environmental factors. Among these, aeration, temperature, and humidity are the most decisive for maintaining embryonic viability and synchronizing development. Therefore, the issue of silkworm egg storage primarily consists in creating a complex of environmental conditions that ensure the proper course of internal physiological processes within the egg during diapause and subsequent embryogenesis. During the storage period, maintaining optimal temperature and relative humidity is particularly critical in the stages of oviposition and early embryogenesis, when the embryo formation occurs within the chorion. At this time, the eggs are extremely sensitive to elevated temperatures and excess humidity, which can trigger premature activation or spontaneous hatching (Nagaraju et al., 2010; Rahmathulla, 2013). Temperature increase during egg laying leads to a higher proportion of unfertilized or partially fertilized eggs, while excessive heat during the summer–autumn storage period causes excessive desiccation (weight loss), consequently reducing hatching rate and larval yield (Qiu et al., 2020). In contrast, low humidity combined with elevated temperature negatively affects egg viability during winter storage, resulting in reduced spring hatching percentages (Chen et al., 2023). Despite numerous studies conducted in Japan, India, China, and Uzbekistan, the recommended storage regimes for silkworm eggs differ substantially among sources, and no unified standard document currently regulates the monthly storage parameters of Bombyx mori eggs under different climatic conditions (Horie & Watanabe, 1986; Patil & Magadum, 2013). Consequently, the development of a standardized technological guideline for egg storage — specifying temperature, humidity, and duration across production months — remains an urgent and practically important task for grain production enterprises and centralized sericulture facilities. Materials and methods The issue of silkworm (Bombyx mori L.) egg storage has a long research history. Early Russian scientists (Shcherbakov, 1960) and (Kovalev, 1952) proposed fundamental storage regimes based on the biological state of the egg, establishing a methodological foundation that remains relevant today. In recent decades, these approaches have been revisited considering new physiological and environmental data on the effect of temperature and humidity fluctuations on egg viability (Qiu et al., 2020; Rahmathulla, 2013; Chen et al., 2023; Nagesh et al., 2024). 1. Storage Regimes According to I.A. Shcherbakov According to Shcherbakov’s recommendations, the following temperature conditions are maintained in egg storage facilities: International independent scientific journal №79/2025 4 • First month after oviposition – 22–25 °C; • Until October (September) – 22–26 °C; • October – 20–22 °C; • By late November – gradually lowered to 15– 17 °C, but not below 15 °C. Relative humidity during the entire summer–autumn storage period is maintained at 60–70%, which allows gradual metabolic slowdown and prevents premature activation of the embryo. 2. Storage Regimes According to P.A. Kovalev According to Kovalev’s methodology, the following temperature and humidity parameters are used: • From the beginning of papilionage until September – 22–26 °C, humidity 60–75%; • September – 20–26 °C, humidity 60–70%; • October – 20–22 °C, humidity 60–70%; • November – 15–17 °C, humidity 60–70%. During preparation for overwintering, the temperature is gradually reduced by 1–2 °C per day, reaching +4 °C by early December. The overwintering period proceeds at +2…+4 °C, ensuring a stable diapause. 3. Modern Adjustments and Comparative Analysis The recommendations of Shcherbakov and Kovalev formed the basis for the modern guidelines presented in the “Fundamental Rules for Industrial Silkworm Egg Preparation” (Uzbek Sericulture Research Institute, 2022). According to this document, the following parameters are established: • From papilionage to September – 24 ± 2 °C, RH 60–70%; • September – gradual decrease 25 → 20 °C, RH 60–70%; • October – 22 → 17 °C; • November – 17 → 12 °C, RH 60–70%; • Cooling before winter – decrease by 1–2 °C daily; • Overwintering – +2…+4 °C, RH 60–70%. Recent studies confirm that gradual temperature reduction ensures stable embryogenesis and prevents diapause disruption under industrial storage (Qiu et al., 2020; Nagesh et al., 2024; Ali et al., 2022). 4. Physiological Aspects and New Experimental Data Modern physiological research demonstrates that even minor deviations from optimal temperature–humidity conditions significantly affect embryonic metabolism and hatching success. (Ali et al., 2022) reported reduced hatching rates when eggs were exposed to >25 °C for over 48 hours. Nagesh et al. (2024) found that during long-term storage (>60 days), optimal conditions were 4–5 °C and 75–80% RH, maintaining >90% survival. Takeshita et al. (2021) confirmed that gradual cooling (−1 °C per day) preserves chorion integrity and stabilizes biochemical processes within the egg. These findings refine the classical recommendations and demonstrate that progressive temperature reduction, rather than stepwise lowering, provides better physiological outcomes for modern B. mori industrial strains. 5. Practical Application and Unification Comparison of different authors’ data reveals discrepancies in monthly storage intervals, complicating practical control of microclimate parameters. To unify operational procedures, it is advisable to express temperature not as a range (e.g., 20–22 °C) but as an average with deviation, e.g., 21 ± 1 °C, simplifying automated monitoring in industrial facilities. The integration of historical and contemporary data formed the basis for updated technological storage protocols now used in the experimental incubators of the Uzbek Sericulture Research Institute and in collaborative studies with Chinese and Indian partners (Chen et al., 2023; Qiu et al., 2020; Ali et al., 2022). Results and discussion Based on the analysis of previous studies (Shcherbakov, 1952; Kovalev, 1960; Mirzakhodjaev et al., 2023; Ali et al., 2022; Nagesh et al., 2024) and on experimental monitoring, a regulatory scheme of temperature–humidity regimes for silkworm (Bombyx mori L.) egg storage was developed for the entire production cycle. The proposed system includes six technological stages, forming a practical guideline for the personnel of egg-production enterprises and centralized incubation facilities. The summarized data are presented below. Stage I. Embryo Formation (June) Embryo formation occurs within the first month after oviposition. During this period, eggs are stored at 24 ± 2 °C and 60–75 % relative humidity, which supports proper early embryonic development and prevents premature activation. Stage II. Microscopic Examination and Egg Screening (July–September) During July and August, microscopic examination and grading of egg batches are carried out to assess fertilization and embryonic stage. The temperature is maintained at 24 ± 1 °C, and gradually lowered to 20 ± 1 °C by late September at 60–70 % RH. This gradual transition allows the eggs to enter estivation (summer dormancy) naturally, avoiding physiological stress and maintaining over 90–95 % viability (Nagesh et al., 2024). Stage III. Cleaning and Sorting (October) In October, sorting, washing, and drying of eggs are performed. The water and ambient air temperatures during processing correspond to the storage temperature — 20 → 16 °C, RH 60–70 %. Matching these parameters prevents mechanical and thermal stress on the egg shell (chorion), which is crucial for maintaining gas exchange and preventing desiccation. Stage IV. Preparation for Overwintering (November – 10 December) In November, eggs are gradually cooled by 1–2 °C per day until they reach +4 ± 1 °C by early December, with 60–70 % RH. This controlled cooling phase is essential for stabilizing embryonic diapause and avoiding condensation damage. Similar temperature gradients have been recommended by Takeshita et al. (2021) and Ali et al. (2022), confirming that slow cooling maintains chorion elasticity and metabolic uniformity among diapause eggs. Stage V. Winter Storage (20 December – March) The main storage period lasts 100–120 days at +3 ± 1 °C and 60–70 % RH. These conditions minimize water loss, preserve embryonic structure, and maintain International independent scientific journal №79/2025 5 diapause stability. Prolonged low-temperature storage beyond 120 days was found to reduce hatchability by 5–7 %, consistent with data by (Chen et al., 2023; Rahmathulla, 2013). Stage VI. Pre-Implementation and Incubation Period (15–30 days before distribution) About 15–30 days prior to sale, eggs are transferred from refrigeration to a room at +16 ± 1 °C and 70–75 % RH for 4–5 days. During this time, they are aerated and portioned (29 g each), then returned to cold storage (+3 ± 1 °C) until distribution. This gradual warming reactivates metabolism, ensuring synchronized hatching and high larval vigor during incubation. Table 1. Temperature and Humidity Regimes of Silkworm Egg Storage at Different Production Stages Stage Period Temperature (°C) Relative humidity (%) Main operations I June 24 ± 2 60–75 Embryo formation II July–September 24 ± 1 → 20 ± 1 60–70 Microscopic examination, screening III October 20 ± 1 → 16 ± 1 60–70 Sorting, washing, drying IV November – 10 Dec 16 ± 1 → 4 ± 1 60–70 Preparation for overwintering V 20 Dec – March 3 ± 1 60–70 Winter storage VI 15–30 days before sale 16 ± 1 (4–5 days) 70–75 Aeration, packaging, preparation for release Discussion The results confirm that gradual temperature reduction combined with constant relative humidity is the key determinant for maintaining egg viability and synchronizing hatching in Bombyx mori. Unlike abrupt cooling methods still used in some traditional storage systems, the developed six-stage approach ensures that the embryonic metabolism slows down naturally, minimizing oxidative and osmotic stress (Ali et al., 2022; Takeshita et al., 2021). It was observed that during summer–autumn storage, maintaining humidity above 70 % at temperatures above 24 °C leads to sporadic pre-diapause activation, while a gradual decline to 20 °C eliminates this risk. Similarly, the final cooling stage (16 → 4 °C) prevents the formation of internal condensation that can destroy the air cavity and reduce oxygen exchange inside the egg shell (Chen et al., 2023). Comparative data from the Uzbek Sericulture Research Institute (2023) show that this optimized scheme increases average hatching success by 8–12 % compared to unregulated storage. Additionally, uniformity in embryonic development improved by 10–15 %, resulting in more synchronized larval emergence — an essential factor for large-scale rearing efficiency and hybrid production. These findings support the integration of climatecontrolled egg storage systems and automation technologies (temperature–humidity sensors, programmable refrigeration units), enabling industrial sericulture to transition toward standardized, reproducible incubation quality. Analytical Summary The proposed temperature–humidity scheme provides a reliable framework for silkworm egg preservation under various climatic conditions. By maintaining an optimal humidity-to-temperature ratio (~2.5 % per °C), the method prevents egg mass loss and stabilizes the metabolic profile of embryos throughout storage.Such regulation not only ensures high hatching percentage and larval vigor, but also establishes a technological foundation for the centralized, year-round production of Bombyx mori eggs in industrial sericulture. Conclusion 1. Storage of silkworm (Bombyx mori L.) eggs under strictly controlled temperature and humidity conditions is one of the fundamental technological requirements in modern grain production. The maintenance of optimal microclimatic parameters directly determines the viability, physiological stability, and hatching synchronization of larvae emerging from the eggs. 2. Analysis and comparison of scientific literature revealed considerable discrepancies among authors regarding recommended temperature values and storage durations by month. The absence of a unified system complicates the work of sericulture technicians, making it difficult to maintain consistent storage regimes and, consequently, reducing the reliability and uniformity of the resulting egg batches. 3. As a result of this research, a standardized sixstage storage scheme has been developed, specifying detailed temperature–humidity conditions and corresponding operational procedures for each stage (embryo formation, estivation, cleaning and sorting, preparation for overwintering, overwintering, and pre-incubation). 4. The proposed scheme ensures gradual, physiologically justified temperature reduction without abrupt fluctuations, thereby preventing metabolic stress, shell desiccation, and premature activation of embryos. This approach guarantees the preservation of 90–95% egg viability and promotes synchronized larval emergence during incubation. 5. The introduction of this system into industrial practice provides several key advantages: ▪ standardization of egg-storage procedures by month and production stage; ▪ simplification of microclimate control in eggstorage facilities; ▪ improved reproducibility and overall quality of commercial eggs; ▪ an 8–12% increase in hatching success compared with traditional storage methods. 6. Therefore, the developed storage regimes can serve as a scientific and practical foundation for establishing a national regulatory framework governing silkworm egg storage conditions according to embryonic stage and seasonal production cycle. International independent scientific journal №79/2025 6 References: 1. Ali, S., Kumar, R. & Sharma, D., 2022. Implications of temperature and humidity on egg hatching in Bombyx mori L. Journal of Applied Entomological Research, 9(3), 45–52. 2. Chen, X., Li, Y. & Zhang, L., 2023. Physiological changes in Bombyx mori eggs under different storage conditions. 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