Abstract
This study is aimed at evaluating the effect of different extenders on the cryopreservation of semen from Africanized honeybees (A. mellifera). Semen from honeybee drones from 10 different colonies was obtained by endophallus exposure technique and immediately evaluated for motility, viability using fluorescent probes, functional membrane integrity using the water test, and morphology. Samples from each colony were divided in three aliquots and subjected to a dilution ratio of 12:1 (diluent: semen) using Tris, Tris + egg yolk (Tris+EY), and Collins extender. Samples were cryopreserved and stored in liquid nitrogen for one week and then rewarmed and reevaluated. Immediate dilution provoked no significant effect on sperm motility and functional membrane integrity, regardless of the extender used; however, the greatest values (P < 0.05) for normal sperm morphology were found at the use of isolate Tris (69.3 ± 1.9%). After thawing, there were no significant differences among extenders with relation to the preservation of sperm motility, viability, and functional membrane integrity, but the Tris extender provided the highest post-thawing values (P < 0.05) for sperm normal morphology (49.2 ± 4.9%) while the Collins extender provoked the highest amounts (P < 0.05) of curled tail defects (67.5 ± 3.2%). Moreover, the Tris was the only extender at preserving the proportion of normal sperm after thawing similar to what was verified for fresh samples. In summary, we suggest the use of a Tris-based extender for the cryopreservation of Africanized honeybee semen.
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The data obtained during the current study are presented in the manuscript and available from the corresponding author.
References
Aboagla, E. M. E., & Terada, T., 2004. Effects of egg yolk during the freezing step of cryopreservation on the viability of goat spermatozoa. Theriogenology, 62 (6), 1160–1172. https://doi.org/10.1016/j.theriogenology.2004.01.013
Ahn, J. Y., Park, J. Y., & Lim, H. K., 2018. Effects of different diluents, cryoprotective agents, and freezing rates on sperm cryopreservation in Epinephelus akaara. Cryobiology, 83, 60–64. https://doi.org/10.1016/j.cryobiol.2018.06.003
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, 28–31. https://doi.org/10.1016/j.cryobiol.2019.03.005
Alcay, S., Ustuner, B., Cakmak, I., Cakmak, S., & Nur, Z., 2015. Effects of various cryoprotective agents on post-thaw drone semen quality. Kafkas Universitesi Veteriner Fakultesi Dergisi, 21 (1), 31–35. https://doi.org/10.9775/KVFD.2014.11515
Almeida, R., & Soares, E. E., A., 2002. Usage of green coconut water and different tissue culture media for in vitro honey bee semen storage (Apis mellifera; Hymenoptera: Apoidea). Interciencia, 27 (6), 317-321.
Bergeron, A., & Manjunath, P., 2006. New insights towards understanding the mechanisms of sperm protection by egg yolk and milk. Molecular Reproduction and Development, 73 (10), 1338–1344. https://doi.org/10.1002/mrd.20565
Castilhos, D., Dombroski, J. L. D., Bergamo, G. C., Gramacho, K. P., & Gonçalves, L. S., 2019. Neonicotinoids and fipronil concentrations in honeybees associated with pesticide use in Brazilian agricultural areas. Apidologie, 50 (5), 657–668. https://doi.org/10.1007/s13592-019-00676-x
Collins, A. M., & Donoghue, A. M., 1999. Viability assessment of honey bee, Apis mellifera, sperm using dual fluorescent staining. Theriogenology, 51 (8), 1513–1523. https://doi.org/10.1016/S0093-691X(99)00094-1
Collins, A. M., 2000. Survival of honey bee (Hymenoptera: Apidae) spermatozoa stored at above-freezing temperatures. Journal of Economic Entomology, 93 (3), 568–571. https://doi.org/10.1603/0022-0493-93.3.568
Dadkhah, F., Nehzati-Paghaleh, G., Zhandi, M., Emamverdi, M., & Hopkins, B. K., 2016. Preservation of honey bee spermatozoa using egg yolk and soybean lecithin-based semen extenders and a modified cryopreservation protocol. Journal of Apicultural Research, 55 (4), 279–283. https://doi.org/10.1080/00218839.2016.1243292
Evison, S. E. F., Roberts, K. E., Laurenson, L., Pietravalle, S., Hui, J., Biesmeijer, J. C., Smith, J. E., Budge, G., & Hughes, W. O. H., 2012. Pervasiveness of parasites in pollinators. PLOS ONE, 7 (1), e30641. https://doi.org/10.1371/journal.pone.0030641
Forouzanfar, M., Sharafi, M., Hosseini, S. M., Ostadhosseini, S., Hajian, M., Hosseini, L., Abedi, P., Nili, N., Rahmani, H. R., & Nasr-Esfahani, M. H., 2010. In vitro comparison of egg yolk-based and soybean lecithin-based extenders for cryopreservation of ram semen. Theriogenology, 73 (4), 480–487. https://doi.org/10.1016/j.theriogenology.2009.10.005
Gül, A., Şahinler, N., Onal, A. G., Hopkins, B. K., & Sheppard, W. S., 2017. Effects of diluents and plasma on honey bee (Apis mellifera L.) drone frozen-thawed semen fertility. Theriogenology, 101, 109–113. https://doi.org/10.1016/j.theriogenology.2017.06.020
Harbo, J. R., 1985. Instrumental insemination of queen bees – part 1. American Bee Journal, 125,197–202.
Hopkins, B. K., & Herr, C., 2010. Factors affecting the successful cryopreservation of honey bee (Apis mellifera) spermatozoa. Apidologie, 41 (5), 548–556. https://doi.org/10.1051/apido/20010006
Jeyendran, R. S., Van der Ven, H. H., Perez-Pelaez, M., Crabo, B. G., & Zaneveld, L. J., 1984. Development of an assay to assess the functional integrity of the human sperm membrane and its relationship to other semen characteristics. Journal of Reproduction and Fertility, 70(1), 219–228. https://doi.org/10.1530/jrf.0.0700219
Kaftanoglu, O., & Peng, Y. S., 1984. Preservation of honeybee spermatozoa in liquid nitrogen. Journal of Apicultural Research, 23 (3), 157–163. https://doi.org/10.1080/00218839.1984.11100625
Kline, O., & Joshi, N. K., 2020. Mitigating the effects of habitat loss on solitary bees in agricultural ecosystems. Agriculture, 10 (4), 115. https://doi.org/10.3390/agriculture10040115
Lim, H.-K., Lee, C.-H., Min, B.-H., Lee, J.-U., Lee, C.-S., Seong, K.-B., & Lee, S.-M., 2008. Effects of diluents and cryoprotectants on sperm cryopreservation of mason salmon, Oncorhynchus masou masou. Korean Journal of Fisheries and Aquatic Sciences, 41(4), 267–271. https://doi.org/10.5657/kfas.2008.41.4.267
Maxwell, W. M., & Salamon, S., 1993. Liquid storage of ram semen: a review. Reproduction, Fertility, and Development, 5(6), 613–638. https://doi.org/10.1071/rd9930613
Morais, L. S., Araujo Neto, E. R., Silva, A. M., Marinho, D. E. L., Bezerra, L. G. P., Velarde, J. M. D. S., Silva, A. R., Gramacho, K. P., & Message, D., 2022. Sperm characteristics of Africanized honey bee (Apis mellifera L.) drones during dry and wet seasons in the Caatinga biome. Journal of Apicultural Research, 0 , 1–8. https://doi.org/10.1080/00218839.2022.2113328
Moreira, S. S. J., Santos, C. S., Castelo, T. S., Bezerra, L. G. P., Praxedes, É. C. G., Matos, T. M., Souza-Junior, J. B. F., Feijó, F. M. C., Comizzoli, P., & Silva, A. R., 2022. Investigating the need for antibiotic supplementation to the extender used for semen cryopreservation in collared peccaries. Frontiers in Veterinary Science, 9, 1354. https://www.frontiersin.org/articles/10.3389/fvets.2022.954921
Murray, J. F., van der Horst, G., Allsopp, M., & Kotzé, R. C. M., 2022. A new fluorescent method to determine honey bee sperm motility parameters with computer-aided sperm analysis. Journal of Apicultural Research, 0, 1–9. https://doi.org/10.1080/00218839.2022.2090729
Nishijima, K., Kitajima, S., Koshimoto, C., Morimoto, M., Watanabe, T., Fan, J., & Matsuda, Y., 2015. Motility and fertility of rabbit sperm cryopreserved using soybean lecithin as an alternative to egg yolk. Theriogenology, 84 (7), 1172–1175. https://doi.org/10.1016/j.theriogenology.2015.06.018
Nur, Z., Dogan, I., Yerlikaya, H., & Soylul, M., 2004. Relationship between the canine sperm membrane integrity and other semen characteristics. The Indian veterinary journal, 81, 1119–1121.
Nur, Z., Seven-Cakmak, S., Ustuner, B., Cakmak, I., Erturk, M., Abramson, C. I., Sağirkaya, H., & Soylu, M. K., 2012. The use of the hypo-osmotic swelling test, water test, and supravital staining in the evaluation of drone sperm. Apidologie, 43 (1), 31–38. https://doi.org/10.1007/s13592-011-0073-1
Paillard, M., Rousseau, A., Giovenazzo, P., & Bailey, J. L., 2017. Preservation of domesticated honey bee (Hymenoptera: Apidae) drone semen. Journal of Economic Entomology, 110 (4), 1412–1418. https://doi.org/10.1093/jee/tox149
Painel Brasileiro de Mudanças Climáticas e (PBMC)., 2013. Contribuição do Grupo de Trabalho 1 ao Primeiro Relatório de Avaliação Nacional do Painel Brasileiro de Mudanças Climaticas [Contribution of Working Group 1 to the First National Assessment Report of the Brazilian Panel on Climate Change]. Rio de Janeiro, Brasil: Sumário Executivo GT1. PBMC. http://www.pbmc.coppe.ufrj.br/pt/noticias/82-destaque.
Pearodwong, P., Suwimonteerabutr, J., Rungruangsak, J., & Tummaruk, P., 2019. Comparison of egg yolk-based and soybean lecithin-based extenders for cryopreservation of boar semen. Veterinarska Stanica, 50 (6), 531-540.
Pettis, J. S., Rice, N., Joselow, K., vanEngelsdorp, D., & Chaimanee, V., 2016. Colony failure linked to low sperm viability in honey bee (Apis mellifera) queens and an exploration of potential causative factors. PLOS ONE, 11 (2), e0147220. https://doi.org/10.1371/journal.pone.0147220
Potts, S. G., Biesmeijer, J. C., Kremen, C., Neumann, P., Schweiger, O., & Kunin, W. E., 2010. Global pollinator declines: trends, impacts and drivers. Trends in Ecology & Evolution, 25 (6), 345–353. https://doi.org/10.1016/j.tree.2010.01.007
Rajamohan, A., Danka, R. G., Hopkins, B. K., & Rinehart, J. P., 2020. A non-activating diluent to prolong in vitro viability of Apis mellifera spermatozoa: effects on cryopreservation and on egg fertilization. Cryobiology, 92, 124–129. https://doi.org/10.1016/j.cryobiol.2019.11.045
Requier, F., Antúnez, K., Morales, C. L., Aldea Sánchez, P., Castilhos, D., Garrido, P. M., Giacobino, A., Reynaldi, F. J., Rosso Londoño, J. M., Santos, E., & Garibaldi, L. A., 2018. Trends in beekeeping and honey bee colony losses in Latin America. Journal of Apicultural Research, 57 (5), 657–662. https://doi.org/10.1080/00218839.2018.1494919
Tarliyah, L., Boediono, A., Walujo., & D. Pergerakan., 1999. Spermatozoa leb ah madu Apis mellifera L. (hymenoptera: Apidae) pada berbagai suhu penyimpanan dalam media pengencer dengan kadar glukosa yang berbeda. Media Veteriner, 6,15–20.
Taylor, M. A., Guzmán-Novoa, E., Morfin, N., & Buhr, M. M., 2009. Improving viability of cryopreserved honey bee (Apis mellifera L.) sperm with selected diluents, cryoprotectants, and semen dilution ratios. Theriogenology, 72 (2), 149–159. https://doi.org/10.1016/j.theriogenology.2009.02.012
Wegener, J., May, T., Kamp, G., & Bienefeld, K., 2014. A successful new approach to honeybee semen cryopreservation. Cryobiology, 69(2), 236–242. https://doi.org/10.1016/j.cryobiol.2014.07.011
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 (4), 472–481. https://doi.org/10.1007/s13592-019-00659-y
Funding
This study was financed in part by the Coordenacão de Aperfeiçoamento de Pessoal de Nível Superior-Brazil (CAPES, Financial Code 001). Dr. Alexandre R. Silva is a recipient of grants from the National Council for the Scientific Development (CNPq).
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LdSM, AMdS, LGPB, AFSdC, and NOdSC carried out the conceptualization, data collection, and initial investigations. GCB and RPdS conducted the data analysis. DAEF, KPG, and ARS carried out the supervision of the work. Furthermore, all authors equally contributed to the concept of the manuscript at all stages and approved the submitted version.
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da Silva Morais, L., de Araujo Neto, E.R., da Silva, A.M. et al. Africanized honeybee (Apis mellifera) semen freezing using Tris-based and Collins extenders. Trop Anim Health Prod 55, 329 (2023). https://doi.org/10.1007/s11250-023-03762-6
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DOI: https://doi.org/10.1007/s11250-023-03762-6