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

Parsamehr, Hedyeh; Deldar, Hamid; Ansari Pirsaraie, Zarbakht; Dirandeh, Essa

doi:10.22059/ijas.2025.394211.654072

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

Document Type : Research Paper

Authors

Hedyeh Parsamehr ¹

Hamid Deldar ²

Zarbakht Ansari Pirsaraie ³

Essa Dirandeh ⁴

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

10.22059/ijas.2025.394211.654072

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

Bee pollen

Cryopreservation

Flavonoids

Oxidative stress

Ram sperm

Subjects

Animal Physiology

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

Agarwal, A., Durairajanayagam, D., & Du Plessis, S. S. (2014). Utility of antioxidants during assisted reproductive techniques: an evidence based review. Reproductive Biology and Endocrinology, 12, 1-19. https://doi.org/10.1186/1477-7827-12-112

Aitken, R. J., & Sawyer, D. (2003). The human spermatozoon—not waving but drowning. Advances in male mediated developmental toxicity, 85-98. https://doi.org/10.1007/978-1-4419-9190-4_8

Aksoy, Y., Aksoy, H., Altinkaynak, K., Aydin, H. R. & Ozkan, A. (2006). Sperm fatty acid composition in subfertile men. Prostaglandins, Leukotrienes, and Essential Fatty Acids. Sciencedirect, 75(2): 75-79. https://doi.org/10.1016/j.plefa.2006.05.002

Allai, L., Benmoula, A., da Silva, M. M., Nasser, B., & El Amiri, B. (2018). Supplementation of ram semen extender to improve seminal quality and fertility rate. Animal Reproduction Science, 192, 6-17. https://doi.org/10.1016/j.anireprosci.2017.12.014

Amidi, F., Pazhohan, A., Shabani Nashtaei, M., Khodarahmian, M., & Nekoonam, S. (2016). The role of antioxidants in sperm freezing: a review. Cell and tissue banking, 17, 745-756. https://doi.org/10.1007/s10561-016-9566-5

Al-Amery, K. S., & Banana, H. J. (2020). Effect of adding aqueous and alcoholic extract of bee pollen on semen characteristics of Awassi ram semen stored at 5° C. Biochemical & Cellular Archives, 20(1). . https://doi.org/10.35124/bca.2020.1.723

Blokhina, O., Virolainen, E., & Fagerstedt, K. V. (2003). Antioxidants, oxidative damage and oxygen deprivation stress: a review. Annals of botany, 91(2), 179-194. https://doi.org/10.1093/aob/mcf118

Chang, C.-C., Yang, M.-H., Wen, H.-M., & Chern, J.-C. (2002). Estimation of total flavonoid content in propolis by two complementary colorimetric methods. Journal of food and drug analysis, 10(3). https://doi.org/10.38212/2224-6614.2748

Chen, S., Fang, L., Xi, H., Guan, L., Fang, J., Liu, Y., Wu, B., & Li, S. (2012). Simultaneous qualitative assessment and quantitative analysis of flavonoids in various tissues of lotus (Nelumbo nucifera) using high performance liquid chromatography coupled with triple quad mass spectrometry. Analytica Chimica Acta, 724, 127-135. https://doi.org/10.1016/j.aca.2012.02.047

Chen, Z., Xi, G., Fu, Y., Wang, Q., Cai, L., Zhao, Z., Liu, Q., Bai, B., & Ma, Y. (2021). Synthesis of 2, 3-dihydro-3, 5-dihydroxy-6-methyl-4H-pyran-4-one from maltol and its taste identification. Food Chemistry, 361, 130052. https://doi.org/10.1016/j.foodchem.2021.130052

Das, A. B., Goud, V. V., & Das, C. (2019). Phenolic compounds as functional ingredients in beverages. In Value-added ingredients and enrichments of beverages (pp. 285-323): Elsevier. https://doi.org/10.1016/B978-0-12-816687-1.00009-6

Dayrit, F. M. (2015). The properties of lauric acid and their significance in coconut oil. Journal of the American Oil Chemists' Society, 92, 1-15. https://doi.org/10.1007/s11746-014-2562-7

De Luca, M. N., Colone, M., Gambioli, R., Stringaro, A., & Unfer, V. (2021). Oxidative stress and male fertility: role of antioxidants and inositols. Antioxidants, 10(8), 1283. https://doi.org/10.3390/antiox10081283

Díaz, R., Torres, M., Bravo, S., Sanchez, R., & Sepúlveda, N. (2016). Determination of fatty acid profile in ram spermatozoa and seminal plasma. Andrologia, 48(6), 723-726. https://doi.org/10.1111/and.12506

Esterbauer, H., & Cheeseman, K. H. (1990). Determination of aldehydic lipid peroxidation products: malonaldehyde and 4-hydroxynonenal. In Methods in enzymology (Vol. 186, pp. 407-421). Elsevier. https://doi.org/10.1016/0076-6879(90)86134-H

Evans, G., & Maxwell, W. C. (1987). Salamons' artificial insemination of sheep and goats.

Fry, C. J., & Peterson, C. L. (2001). Chromatin remodeling enzymes: who's on first? Current Biology, 11(5), R185-R197. https://doi.org/10.1016/S0960-9822(01)00090-2

Gholami, H., Chamani, M., Towhidi, A., & Fazeli, M. H. (2011). Improvement of semen quality in holstein bulls during heat stress by dietary supplementation of omega-3 fatty acids. International journal of fertility & sterility, 4(4), 160.

Gurung, A. B., Ali, M. A., Al-Hemaid, F., El-Zaidy, M., & Lee, J. (2022). In silico analyses of major active constituents of fingerroot (Boesenbergia rotunda) unveils inhibitory activities against SARS-CoV-2 main protease enzyme. Saudi journal of biological sciences, 29(1), 65-74. https://doi.org/10.1016/j.sjbs.2021.11.053

Hancock, J. (1952). The morphology of bull spermatozoa. Journal of Experimental Biology, 29(3), 445-453. https://doi.org/10.1242/jeb.29.3.445

Haydak, M. H. (1970). Honey bee nutrition. Annual review of entomology, 15(1), 143-156. https://doi.org/10.1146/annurev.en.15.010170.001043

Huang, A., Isobe, N., Obitsu, T., & Yoshimura, Y. (2016). Expression of lipases and lipid receptors in sperm storage tubules and possible role of fatty acids in sperm survival in the hen oviduct. Theriogenology, 85(7), 1334-1342. https://doi.org/10.1016/j.theriogenology.2015.12.020

Ijaz, M. U., Shahzadi, S., Hamza, A., Azmat, R., Anwar, H., Afsar, T., Shafique, H., Bhat, M. A., Naglah, A. M., & Al-Omar, M. A. (2023). Alleviative effects of pinostrobin against cadmium-induced renal toxicity in rats by reducing oxidative stress, apoptosis, inflammation, and mitochondrial dysfunction. Frontiers in Nutrition, 10, 1175008. https://doi.org/10.3389/fnut.2023.1175008

Johnson, L. V., Walsh, M. L., & Chen, L. B. (1980). Localization of mitochondria in living cells with rhodamine 123. Proceedings of the National Academy of Sciences, 77(2), 990-994. https://doi.org/10.1073/pnas.77.2.990

Kanchanapiboon, J., Kongsa, U., Pattamadilok, D., Kamponchaidet, S., Wachisunthon, D., Poonsatha, S., & Tuntoaw, S. (2020). Boesenbergia rotunda extract inhibits Candida albicans biofilm formation by pinostrobin and pinocembrin. Journal of Ethnopharmacology, 261, 113193. https://doi.org/10.1016/j.jep.2020.113193

Kiernan, M., Fahey, A., & Fair, S. (2013). The effect of the in vitro supplementation of exogenous long-chain fatty acids on bovine sperm cell function. Reproduction, Fertility and Development, 25(6), 947-954. https://doi.org/10.1071/RD12204

Leja, M., Mareczek, A., Wyżgolik, G., Klepacz-Baniak, J., & Czekońska, K. (2007). Antioxidative properties of bee pollen in selected plant species. Food chemistry, 100(1), 237-240. https://doi.org/10.1016/j.foodchem.2005.09.047

Lenzi, A., Gandini, L., Maresca, V., Rago, R., Sgro, P., Dondero, F., & Picardo, M. (2000). Fatty acid composition of spermatozoa and immature germ cells. Molecular human reproduction, 6(3), 226-231. https://doi.org/10.1093/molehr/6.3.226

Lima, E., & Abdalla, D. S. P. (2001). Peroxidação lipídica: mecanismos e avaliação em amostras biológicas. Braz J Pharm Sci, 37(3), 293-303.

Linford, E., Glover, F.A., Bishop, C. & Stewart, D.L. (1976). The relationship between semen evaluation methods and fertility in the bulls. Reproduction and Fertility, 47: 283-291. https://doi.org/10.1530/jrf.0.0470283

MAIA, M. d. S., & Bicudo, S. (2009). Radicais livres, antioxidantes e função espermática em mamíferos: uma revisão.

Maxwell, W., & Salamon, S. (1993). Liquid storage of ram semen: a review. Reproduction, Fertility and Development, 5(6), 613-638. https://doi.org/10.1071/RD9930613

Mohammad, S. M., Mahmud-Ab-Rashid, N.-K., & Zawawi, N. (2020). Botanical origin and nutritional values of bee bread of stingless bee (Heterotrigona itama) from Malaysia. Journal of Food Quality, 2020(1), 2845757. https://doi.org/10.1155/2020/2845757

Nemati, Z., Sattari, M., Karimi, A., Besharati, M., & Kamrani, N. (2022). Improving of frozen-thawed sperm quality in commercial broiler breeder roosters challenged with dexamethasone using diet vitamin E supplementation. Iranian Journal of Animal Science Research, 14(1), 109-120. https://doi.org/10.22067/IJASR.2021.68725.1007

Partyka, A., Łukaszewicz, E., & Niżański, W. (2012). Effect of cryopreservation on sperm parameters, lipid peroxidation and antioxidant enzymes activity in fowl semen. Theriogenology, 77(8), 1497-1504. https://doi.org/10.1016/j.theriogenology.2011.11.006

Raeeszadeh, M., Karimfar, B., Amiri, A. A., & Akbari, A. (2021). Protective Effect of Nano‐Vitamin C on Infertility due to Oxidative Stress Induced by Lead and Arsenic in Male Rats. Journal of Chemistry, 2021(1), 9589345. https://doi.org/10.1155/2021/9589345 (in persian)

Revell, S., & Mrode, R. (1994). An osmotic resistance test for bovine semen. Animal Reproduction Science, 36(1-2), 77-86. https://doi.org/10.1016/0378-4320(94)90055-8

Salamon, S., & Maxwell, W. (1995). Frozen storage of ram semen I. Processing, freezing, thawing and fertility after cervical insemination. Animal Reproduction Science, 37(3-4),185-249. https://doi.org/10.1016/0378-4320(94)01327-i

Rafeie, F., Abdoli, R., Hossein-Zadeh, N.G. et al. (2023). Interaction networks and pathway analysis of genetic resistance to gastrointestinal nematodes in sheep. Tropical Animal Health and Production, 55, 34. https://doi.org/10.1007/s11250-022-03448-5

Rodríguez-Pólit, C., Gonzalez-Pastor, R., Heredia-Moya, J., Carrera-Pacheco, S. E., Castillo-Solis, F., Vallejo-Imbaquingo, R., Barba-Ostria, C., & Guamán, L. P. (2023). Chemical properties and biological activity of bee pollen. Molecules, 28(23), 7768. https://doi.org/10.3390/molecules28237768

Šarić, A., Balog, T., Sobočanec, S., Kušić, B., Šverko, V., Rusak, G., Likić, S., Bubalo, D., Pinto, B., & Reali, D. (2009). Antioxidant effects of flavonoid from Croatian Cystus incanus L. rich bee pollen. Food and Chemical Toxicology, 47(3), 547-557. https://doi.org/10.1016/j.fct.2008.12.007

Schuster, R., Holzer, W., Doerfler, H., Weckwerth, W., Viernstein, H., Okonogi, S., & Mueller, M. (2016). Cajanus cajan–a source of PPARγ activators leading to anti-inflammatory and cytotoxic effects. Food & function, 7(9), 3798-3806. https://doi.org/10.1039/c6fo00689b

Shahin, M. A., Khalil, W. A., Saadeldin, I. M., Swelum, A. A.-A., & El-Harairy, M. A. (2020). Comparison between the effects of adding vitamins, trace elements, and nanoparticles to shotor extender on the cryopreservation of dromedary camel epididymal spermatozoa. Animals, 10(1), 78. https://doi.org/10.3390/ani10010078

Silva, P., & Gadella, B. (2006). Detection of damage in mammalian sperm cells. Theriogenology, 65(5), 958-978. https://doi.org/10.1016/j.theriogenology.2005.09.010

Sinclair, S. (2000). Male infertility: nutritional and environmental considerations. Alternative medicine review: a journal of clinical therapeutic, 5(1), 28-38.

Sinha, M., Sinha, A., Singh, B., & Prasad, R. (1996). The effect of glutathione on the motility, enzyme leakage and fertility of frozen goat semen. Animal Reproduction Science, 41(3-4), 237-243. https://doi.org/10.1016/0378-4320(95)01450-0

Sopanaporn, J., Suksawatamnuay, S., Sardikin, A., Lengwittaya, R., Chavasiri, W., Miyakawa, T., & Yompakdee, C. (2020). Pinostrobin suppresses the Ca2+-signal-dependent growth arrest in yeast by inhibiting the Swe1-mediated G2 cell-cycle regulation. FEMS yeast research, 20(4), foaa026. https://doi.org/10.1093/femsyr/foaa026

Wang, H., Gao, X. D., Zhou, G. C., Cai, L., & Yao, W. B. (2008). In vitro and in vivo antioxidant activity of aqueous extract from Choerospondias axillaris fruit. Food chemistry, 106(3), 888-895. https://doi.org/10.1016/j.foodchem.2007.05.068

Watson, P. F. (2000). The causes of reduced fertility with cryopreserved semen. Animal Reproduction Science, 60, 481-492. https://doi.org/10.1016/s0378-4320(00)00099-3

Wojdyło, A., Oszmiański, J., & Czemerys, R. (2007). Antioxidant activity and phenolic compounds in 32 selected herbs. Food chemistry, 105(3), 940-949. https://doi.org/10.1016/j.foodchem.2007.04.038

Xu, X., Sun, L., Dong, J., & Zhang, H. (2009). Breaking the cells of rape bee pollen and consecutive extraction of functional oil with supercritical carbon dioxide. Innovative food science & emerging technologies, 10(1), 42-46. https://doi.org/10.1016/j.ifset.2008.08.004

Yanishlieva, N. V., & Marinova, E. M. (1996). Antioxidative effectiveness of some natural antioxidants in sunflower oil. Zeitschrift für Lebensmittel-Untersuchung und Forschung, 203, 220-223. https://doi.org/10.1007/bf01192867

Yıldız, O., Can, Z., Saral, Ö., Yuluğ, E., Öztürk, F., Aliyazıcıoğlu, R., Canpolat, S., & Kolaylı, S. (2013). Hepatoprotective potential of chestnut bee pollen on carbon tetrachloride‐induced hepatic damages in rats. Evidence‐Based Complementary and Alternative Medicine, 2013(1), 461478. https://doi.org/10.1155/2013/461478

Yonis, M., & Hassan, A. (2023). The Effect of adding bee pollen on the sexual efficiency of quail males. Journal of Applied Veterinary Sciences, 8(1), 32-37. https://doi.org/10.21608/javs.2022.164168.1180

Yousif, A. I., Abd-Elaziz, M. A. & Shehabeldin, A. M. (2024). Nano-oleic Acid in Tris-Extender: Effects on Bull Sperm Cryopreservation, Fertility, and Post-thaw Antioxidant Status. Journal of Sustainable Agricultural and Environmental Sciences,3(4): 9-18. https://doi.org/10.21608/jsaes.2024.299701.1091