Effects of high and low glucose DMEM extenders on sperm cryopreservation in honey bees

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Published: Apr 19, 2025
DOI: https://doi.org/10.12681/jhvms.37530
Keywords:
Cryoprotectant DMEM honey bee semen extender
AO Özkök
Amasya University Suluova Vocational School, Department of Veterinary, Amasya, Turkey
B Esin
Department of Reproduction and Artificial Insemination, Faculty of Veterinary Medicine, University of Ondokuz Mayis, Samsun/Turkey
A Eser
Department of Reproduction and Artificial Insemination, Faculty of Veterinary Medicine, University of Ondokuz Mayis, Samsun/Turkey
Abstract

Honey bee sperm can be stored and used at room temperature for up to a week. However, freezing storage methods used for long-term storage negatively affect the sensitive honey bee semen. In order to reduce this negative effect, the effects of many different diluents and various freezing protocols are being investigated. This study aims to evaluate the effect of DMEM (Dulbeco's Modified Eagle Medium) diluents with different glucose ratios on the spermatological parameters of frozen honeybee semen. The study used two different DMEM diluents containing high and low glucose. Honey bee sperm samples, diluted 13 -fold with high glucose DMEM (4.5 g/L glucose, L glutamine and pyruvate) and low glucose DMEM (1 g/L glucose, L-glutamine and sodium bicarbonate) at a ratio of 1:12, were frozen in liquid nitrogen. It was kept for three months. At the end of the period, the thawed samples were evaluated in terms of spermatological parameters (motility, viability, plasma membrane integrity, and spermatozoa morphology). Motility, viability, and plasma membrane integrity parameters were high in honeybee semen frozen with DMEM diluent with high glucose content (P<0.001). As a result, we think that DMEM diluent with high glucose content can be used effectively in the cryopreservation of honey bee semen.

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References
Alcay S, Cakmak S, Cakmak I, Mulkpinar E, Gokce E, Ustuner B, Sen H,
Nur Z (2019). Successful cryopreservation of honey bee drone spermatozoa
with royal jelly supplemented extenders. Cryobiology. 87:
-31. https://doi.org/10.1016/j.cryobiol.2019.03.005
Auth CA, Hopkins BK (2021). Nitrogen vapor immersion: An accessible
alternative for honey bee (Apis mellifera L.) semen cryopreservation.
Cryobiology. 100: 12-18. https://doi.org/10.1016/j.cryobiol.
04.006
Babich H, Liebling EJ, Burger RF, Zuckerbraun HL, Schuck AG, (2009).
Choice of DMEM, formulated with or without pyruvate, plays an
important role in assessing the in vitro cytotoxicity of oxidants and
prooxidant nutraceuticals. Vitr. Cell Dev. Biol. Anim. 45: 226-233.
https://doi.org/10.1007/s11626-008-9168-z
Birnie JH, Mitchell PH, Yeager Y, Hager A, Blum MS, Glowska Z, Taber
S (1957). Chemistry of the Drone Honey Bee Reproductive System.
II. Carbohydrates in the Reproductive Organs and Semen 1. In Newman)
larvae. Physiol. Zool 55: 135-139.
Bratu IC, Igna V, Simiz E, Dunea IB, Pătruică S (2022). The Influence of
Body Weight onSemen Parameters in Apis mellifera Drones. Insects.
: 1141. https://doi.org/10.3390/insects13121141
Cobey SW, Tarpy DR, Woyke J (2013). Standard methods for instrumental
insemination of Apis mellifera queens. J Apic Res. 52: 1-18.
https://doi.org/10.3896/IBRA.1.52.4.09
da Silva Morais, L, de Araujo Neto ER, da Silva AM, Bezerra LGP, da
Cunha AFS, de Sousa Chagas NO, dos Santos RP, Bergamo GC,
Façanha DAE, Gramacho KP, Silva AR (2023). “Africanized Honeybee
(Apis Mellifera) Semen Freezing Using Tris-Based and Collins
Extenders.” Tropical Animal Health and Production 55(5): 329.
https://doi.org/10.1007/s11250-023-03762-6
Dadkhah F, Nehzati-Paghaleh G, Zhandi M, Emamverdi M, Hopkins BK
(2016). Preservación de espermatozoides de la abeja de la miel usando
extensores de semen basados en la yema de huevo y la lecitina de
soja y un protocolo de criopreservación modificado. J. Apic. Res. 55:
-283. https://doi.org/10.1080/00218839.2016.1243292
Darr CR, Varner DD, Teague S, Cortopassi GA, Datta S, Meyers SA
(2016). Lactate and pyruvate are major sources of energy for stallion
sperm with dose effects on mitochondrial function, motility,
and ROS production. Biology of Reproduction, 95: 1-11 https://doi.
org/10.1095/biolreprod.116.140707
El-Sheshtawy RI, El-Badry DA, El-Sisy GA, El-Nattat WS, Abo Almaaty
AM (2016). Natural honey as a cryoprotectant to improve Arab stallion
post-thawing sperm parameters. Asian Pac J Reprod. 5: 331-334.
https://doi.org/10.1016/j.apjr.2016.06.004
Gibb Z, Lambourne, SR, Quadrelli J, Smith ND, Aitken RJ (2015). L-carnitine
and pyruvate are prosurvival factors during the storage of stallion
spermatozoa at room temperature. Biol. Reprod. 93: 1-9. https://
doi.org/10.1095/biolreprod.115.131326
Gulov AN, Berezin AS, Larkina EO, Saltykova ES, Milyausha K (2023).
Creation of a Biobank of the Sperm of the Honey Bee Drones of Different
Subspecies of Apis mellifera L. Using a Honey Extender. 13:
https://doi.org/10.20944/preprints202311.0022.v1
Gulov AN, Bragina YE (2022). Morphofunctional Changes in Frozen-
Thawed Sperm of Apis Mellifera Linnaeus Drones. Russ. Agric.
Sci. 48(6): 512-520. https://doi.org/10.3103/s1068367422060040
Hashem NM, Hassanein EM, Simal-Gandara J (2021). Improving reproductive
performance and health of mammals using honeybee products.
Antioxidants 10: 1-24. https://doi.org/10.3390/antiox10030336
Hernández-Avilés C, Love CC, Serafini R, Ramírez-Agámez L, Friedrich
M, Ghosh S, Teague SR, LaCaze KA, Brinsko SP, Varner DD (2020).
Effects of glucose concentration in semen extender and storage temperature
on stallion sperm quality following long-term cooled storage.
Theriogenology, 147: 1-9. https://doi.org/10.1016/j.theriogenology.
02.007
Hopkins BK, Herr C (2010). Factors affecting the successful cryopreservation
of honey bee (Apis mellifera) spermatozoa. Apidologie, 41:
-556. https://doi.org/10.1051/apido/20010006
Ioan Bratu C, Pătruică S (2021). Influence of the Use of Diluents in the
Technique of Homogenization of Beekeeping Seminal Material-Review.
Animal Sci. Biotechnol. 54: 156-160.
Judycka S, Dietrich MA, Szczepkowska B, Szczepkowski M, Liszewska
E, Ciereszko A (2024). Evaluation of the effects of glucose concentration
in extender and sperm concentration in straw for cryopreservation
of Atlantic sturgeon (Acipenser oxyrinchus) semen. Aquaculture
Reports. 35: 101995. https://doi.org/10.1016/j.aqrep.2024.101995
Judycka S, Nynca J, Liszewska E, Dobosz S, Grudniewska J, Ciereszko A
(2018). Optimal sperm concentration in straws and final glucose concentration
in extender are crucial for improving the cryopreservation
protocol of salmonid spermatozoa. Aquaculture. 486: 90-97. https://
doi.org/10.1016/j.aquaculture.2017.12.019
Kairo G, Provost B, Tchamitchian S, Ben Abdelkader F, Bonnet M, Cousin
M, Sénéchal J, Benet P, Kretzschmar A, Belzunces LP, Brunet JL
(2016). Drone exposure to the systemic insecticide Fipronil indirectly
impairs queen reproductive potential. Sci Rep. 6: 31904 https://doi.
org/10.1038/srep31904
Lamers ML, Almeida MES, Vicente-Manzanares M, Horwitz AF, Santos
MF (2011). High glucose-mediated oxidative stress impairs cell
migration. PLoS ONE, 6: e22865. https://doi.org/10.1371/journal.
pone.0022865
Akandi A, Machebe NS, Ugwu S (2015). Survivability of boar sperm
stored under room temperature in extenders containing some natural
products. Open Access Anim Physiol. 7: 57-64. https://doi.
org/10.2147/oaap.s71360
Morais LS, Araujo Neto ER, Silva AM, Marinho DEL, Bezerra LGP,
Velarde JMDS, Silva AR, Gramacho KP, Message D (2022). Sperm
characteristics of Africanized honey bee (Apis mellifera L.) drones
during dry and wet seasons in the Caatinga biome. J. Apic. Res. 2022:
-8. https://doi.org/10.1080/00218839.2022.2113328
Moreira, DR, de Souza THS, Galhardo D, Puentes SMD, Figueira CL,
Silva BG, Chagas F das, Gigliolli AAS, Toledo V de, Ruvolo-Takasusuki
AA de MCC (2022). Imidacloprid Induces Histopathological
Damage in the Midgut, Ovary, and Spermathecal Stored Spermatozoa
of Queens After Chronic Colony Exposure. Environ Toxicol Chem.
: 1637-1648. https://doi.org/10.1002/etc.5332
IBM, Corp (2011) IBM SPSS Statistics for Windows, Version 21.0. IBM
Corporation, Armonk, NY.
Paillard M, Rousseau A, Giovenazzo P, Bailey JL (2017). Preservation of
Domesticated Honey Bee (Hymenoptera: Apidae) Drone Semen. J.
Econ. Entomol. 110: 1412-1418. https://doi.org/10.1093/jee/tox149
Sharaf AE, Khalil WA, Khalifa EI, Nassan MA, Swelum AA, El-Harairy
MA (2022). The Supplementation of Bee Bread Methanolic Extract to
Egg Yolk or Soybean Lecithin Extenders Can Improve the Quality of
Cryopreserved Ram Semen. Cells. 11: 3403. https://doi.org/10.3390/
cells11213403
Sieme, H, Oldenhof, H, Wolkers WF. (2016). Mode of action of cryoprotectants
for sperm preservation. Animal reproduction science, 169:
-5. https://doi.org/10.1016/j.anireprosci.2016.02.004
Slater GP, Smith NM, Harpur BA (2021). Prospects in connecting genetic
variation to variation in fertility in male bees. Genes. 12: 1251 https://
doi.org/10.3390/genes1208125
Smilga-Spalvina A, Spalvins K, Veidenbergs I (2023). Review of Sustainable
Cryopreservation and Above-Freezing Storage Solutions of
European Honey Bee Apis mellifera Drone Semen. Environ. Clim.
Technol. 27: 177-194. https://doi.org/10.2478/rtuect-2023-0014
Taylor MA, Guzmán-Novoa E, Morfin N, Buhr MM (2009). Improving
viability of cryopreserved honey bee (Apis mellifera L.) sperm with
selected diluents, cryoprotectants, and semen dilution ratios. Theriogenology,
: 149-159. https://doi.org/10.1016/j.theriogenology.
02.012
Wegener J, Bienefeld K (2012). Toxicity of cryoprotectants to honey
bee semen and queens. Theriogenology, 77: 600-607. https://doi.
org/10.1016/j.theriogenology.2011.08.036
Wegener J, May T, Knollmann U, Kamp G, Müller K, Bienefeld K (2012). In vivo validation of in vitro quality tests for cryopreserved honey
bee semen. Cryobiology, 65: 126-131. https://doi.org/10.1016/j.cryobiol.
04.010
Wegener J, May T, Kamp G, Bienefeld G (2014). A successful new approach
to honeybee semen cryopreservation. Cryobiology, 69: 236-
https://doi.org/10.1016/j.cryobiol.2014.07.011
Verma LR (1973). Osmotic analysis of honeybee (Apis mellifera L.) semen
and haemolymph. American Bee Journal. 11: 412.
Yániz JL, Silvestre MA, Santolaria P (2020). Sperm quality assessment
in honey bee drones. Biology 9: 1-16. https://doi.org/10.3390/biology9070174
Yániz J, Palacín I, Santolaria P (2019). Effect of chamber characteristics,
incubation, and diluent on motility of honey bee (Apis mellifera)
drone sperm. Apidologie, 50: 472-481. https://doi.org/10.1007/
s13592-019-00659-y
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