Impact of Methanolic Bee Pollen Extract on Ram Sperm Quality in Short- and Long-Term Storage

Document Type : Research Paper

Authors

1 1. Department of Animal Sciences, Faculty of Animal Science and Fisheries, Sari Agricultural Sciences and Natural Resources University, Sari, Iran

2 Department of Animal Sciences, Faculty of Agriculture, University of Guilan, Rasht, Iran

3 3. Department of Animal Sciences, Faculty of Animal Science and Fisheries, Sari Agricultural Sciences and Natural Resources University, Sari, Iran

4 4. Department of Animal Sciences, Faculty of Animal Science and Fisheries, Sari Agricultural Sciences and Natural Resources University, Sari, Iran

Abstract

This study evaluated the effect of supplementing a Tris–egg yolk-based extender with methanolic bee pollen extract on the sperm quality of Zel rams during short-term storage and after cryopreservation. Semen was collected twice a week from four mature Zel rams using an artificial vagina. After initial evaluation, qualified ejaculates were pooled to eliminate individual variation. Samples were diluted with extenders containing 0 (control), 5, 10, and 15 mg/mL of methanolic bee pollen extract. Additionally, 0.1 mM vitamin E was used as a positive control, and 5 µL of dimethyl sulfoxide (DMSO) was added as the solvent for the extract, whose effect was also evaluated. Short-term storage was carried out at 4°C for 5, 24, 48, and 72 hours. Cryopreserved samples were assessed 14 days after storage in liquid nitrogen. Sperm parameters were evaluated, including motility, viability, plasma membrane integrity, mitochondrial activity, morphological abnormalities, and lipid peroxidation (MDA levels).  Results showed that supplementation with bee pollen extract significantly (P<0.05) improved all evaluated parameters compared to the control. The 5 mg/mL treatment yielded the best outcomes during short-term storage, while 15 mg/mL was most effective after thawing. Moreover, the 5 mg/mL concentration was superior in reducing oxidative stress by lowering MDA levels. These findings suggest that adding 5 mg/mL methanolic bee pollen extract to the extender improves ram sperm quality and may be an effective strategy for semen preservation.

Keywords

Main Subjects

Extended Abstract

Introduction

     Honey bees play an important role in meeting the needs of human societies. Their primary role is pollination, and their products, such as honey, pollen, royal jelly and wax, are also important. However, the evolution of human societies, the destruction of natural habitats, and some management problems have caused the population of this insect to decrease. One of these management issues is an increase in homozygosity of sexual alleles, which has led to a reduction in the number of sexual alleles and an increase in homozygosity, as well as its associated effects and consequences, including reduced adaptation power, decreased fertility and reduced production. The purpose of this study is therefore to investigate the level of homozygosity in bee colonies in areas with a high density of beekeepers in Sistan and Baluchistan province, and its impact on honey production. Materials and methods     The study was conducted at the level of apiaries in the cities with the highest concentration of honey bee breeding in the Sistan and Baluchistan provinces (Delgan, Khash, Taftan and Iran-shahr) during the spring and summer of 2024. Two hundred hives from four cities (Delgan, Khash, Taftan and Iran-shahr), five apiaries from each region, ten hives from each apiary and three hives from each hive were randomly selected and subjected to field studies. For this purpose, brown-colored healthy pomace was given to each of the studied colonies. Three days later, the state of queen spawning was evaluated, and the desired queens were coded once it was confirmed that the queen had spawned. Twelve days after the queen hatched, the combs in question were removed from the hive. Evaluation of the empty cells was performed using a template in six areas of each comb (three from each side), and the number of empty cells was counted and recorded. 300 cells from each side were examined, making a total of 600 cells from each comb and 1,800 cells from each hive. The homozygosity of sex alleles was calculated using Ruttner's (1988) and Page and Laidlow's (1985) instructions. To count the empty cells in each hive, three frames containing closed-headed babies (pupae) were removed and the number of empty cells among the full cells was counted by placing a template in the pupal area. The average number of empty cells indicates the average homozygosity of each sex allele. The ratio 100/s-100 was used to estimate the number of homozygous sex alleles, where s is the average percentage of vitality of babies due to the action of the homozygosity of sex alleles. During the next stage of the honey harvesting season, the weight of the harvested honey was calculated and recorded based on the difference in weight of each hive before and after honey extraction. Finally, after taking the measurements, the data were analyzed using a nested design in SAS software version 9.4. To check the effect of the homozygosity coefficient on honey yield, regression analysis was used. In this model, honey production was considered the dependent variable and the homozygosity percentage the auxiliary variable. Results    In apiaries across the province, the average homozygosity and number of homozygous alleles were 3.46% and 32.81, respectively, while honey production was 10.75 kg. While the percentage of homozygosity in different cities within the province did not differ significantly (P>0.05), significant differences were observed within each city's departments (P<0.05). The range of changes in the homozygosity coefficient in different cities was between 3.31% and 3.57%, indicating a low homozygosity percentage. The average honey production per hive in the cities of the province was between 9.56 and 12.07 kg, showing a statistically significant difference (P < 0.05). Due to the low homozygosity percentage, it is expected that the number of sexual alleles will be high. The average number of alleles in different cities was between 31.32 and 35.8, with the highest number belonging to Taftan city, which showed the lowest homozygosity coefficient. The variation in the coefficient of hemozygosity in different parts of each city was greater, ranging from 2.06% to 5.08%. Average honey production ranged from 6.13 to 18.79 kg, indicating a wide variation in production across the province. The variation in the number of alleles was also significant, ranging from 20.39 to 50.2. The highest number of alleles was found in different parts of Taftan city, which had the lowest coefficient of homozygosity and the highest average honey production. Correlation analysis showed no significant correlation between honey production performance and percentage of homozygosity (P<0.05).

 

Conclusions

In the present study, the correlation between the percentage of homozygosity and the honey production trait was insignificant. Therefore, the adverse effects of homozygosity on honey bee performance and reduction in colony population size were not observed, thus indicating the favorable state of beekeeping and high diversity in honey bee colonies in the Sistan and Baluchistan province, and enabling breeding programs by selecting for the production of queens with high performance and other desirable traits.

 Key words: Complementary Sex Determiner gene, Honey production, Honey bee, Homozygosity of sexual alleles, Sistan and Baluchistan

Author Contributions

Methodology, Z. M., G. R. D. and K. S.; software, G. R. D. and Z. M.; formal analysis, G. R. D. and Z. M.; writing—original draft preparation, Z. M., G. R. D., K. S. and M. R.; writing—review and editing, Z. M., G. R. D., K. S. and M. R.; supervision, G. R. D., K. S. All authors have read and agreed to the published version of the manuscript.

Data Availability Statement

Data available on request from the authors.

Acknowledgements

The authors would like to thank all participants of the present study.

Ethical consideration

The study was approved by the Ethics Committee of the University of Zabol (Ethical cod: IR-UOZ-4398). The authors avoided data fabrication, falsification, plagiarism, and misconduct.

Conflict of interest

The author declares no conflict of interest.

References

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Iranian Journal of animal Science
Volume 57, Issue 1
April 2026
Pages 59-77

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History

  • Receive Date: 10 May 2025
  • Revise Date: 26 August 2025
  • Accept Date: 10 September 2025

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