A non-activating diluent to prolong in vitro viability of Apis mellifera spermatozoa: Effects on cryopreservation and on egg fertilization

Authors: Rajamohan, Arun; Danka, Robert; HOPKINS, BRANDON - Washington State University; Rinehart, Joseph - Joe

Submitted to: Journal of Cryobiology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 11/29/2019
Publication Date: 2/1/2020
Citation: 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. Journal of Cryobiology. 92:124-129. https://doi.org/10.1016/j.cryobiol.2019.11.045.
DOI: https://doi.org/10.1016/j.cryobiol.2019.11.045

Interpretive Summary: An active sperm cell has a short lifetime. In the case of honey bees, the sperm cell that is naturally produced in an inactive state, and can therefore survive months to years, even after transfer to a queen bee. When honey bee semen is manipulated for cryopreservation or artificial insemination, it is often diluted with a saline that is thought to cause the sperm cells to become highly active, thereby reducing their longevity. In this study USDA-ARS researchers in Fargo developed a saline medium (identified as Fargo Extender Medium) that was specifically designed to not activate the spermatozoa to improve the effectiveness of sperm cryopreservation and queen artificial insemination. Under laboratory conditions, this medium kept sperm cells viable for more than 500 days at 14°C. Next, Fargo Extender Medium was tested with artificial insemination, and the egg laying patterns and the number of workers produced was used as indicators of queen and sperm health. With the new extender medium, 53% of the samples resembled that of untreated control queens. Queens that were artificially inseminated with the conventional medium, currently used by the industry, resulted in only 30% of the samples with egg laying patterns similar to the untreated queens. This represents a significant step forward in the development of reliable honey bee cryopreservation protocols, and for the improvement of honey bee artificial insemination.

Technical Abstract: A non-activating diluent is a semen extender that does not activate the spermatozoa. In other words, it does not cause motility, acrosomal reaction or capacitate the sperm cell. The effects of such a diluent on the viability of the spermatozoa stored in ambient environs, both prior to and post-cryopreservation were assessed. Subsequently the treated semen was used to inseminate virgin queens to study the brood characteristics versus when inseminated with untreated semen. Seven variations of the TRIS-based non-activating diluents were designed (FEM1 – FEM7) for the current study. In addition, a conventional activating diluent [13] was also assessed. The seminal viability was assessed after short- and long-term storage at 14±0.2°C. The diluent (FEM7) that yielded the best results was used as the non-activating medium to cryopreserve bee semen and inseminate queens. The hive quality was assessed 30 days after insemination. The non-activating medium only held a statistically insignificant advantage over the conventional extender in terms of percentage worker eggs laid and percentage drone emergence. On the other hand, the non-activating medium had very significant effect on the prolonged in vitro longevity of the spermatozoa with or without cryopreservation.