Progress in the field of drone semen cryopreservation

Compared with colleagues working with other animal species, honey bee breeders must overcome additional major difficulties, in particular:

• the reproductive mode of Apis mellifera. This involves a single mating period, limited to a few days, which nevertheless must suffice to fertilise the eggs laid throughout the queen’s entire life. For this purpose, the number of spermatozoa in the spermatheca of inseminated queens is about 3–4 million, sometimes more;
• the short lifespan of queens: evaluating the value of a queen requires at least one year of production. Generally, only one further season then remains in which she can be used as a breeder.

Would it therefore not be interesting to preserve the alleles of the most productive or most important queens after their death? It is thus not surprising that the cryopreservation of drone semen has been the subject of research since the 1970s. The risk of losing natural biodiversity is another motivation for this research. There are almost thirty subspecies of Apis mellifera, most of which are threatened by introgression of imported genetic material. These “local” bees, however, carry genes that may provide solutions to future apicultural problems. Finally, semen cryopreservation could become a valuable tool for the selection of rare traits, such as resistance to Varroa. In such cases, cryopreservation could increase the number of possible matings and thus limit the level of inbreeding.

Unlike most animal species, the spermatozoa of the honey bee are naturally adapted for long-term storage – even at room temperature they remain alive and fertile for several weeks, a feature that is extremely useful for inseminators. In collaboration with the Institute for Wildlife Research in Berlin and the University of Leipzig, the laboratory in Hohen Neuendorf (Germany) showed that this naturally long lifespan is probably partly due to the composition of the cell membranes, which are extremely poor in polyunsaturated fatty acids (Wegener et al., 2013). However, the quality requirements for preserved spermatozoa are also higher than in other animal species – a queen inseminated with preserved semen must lay thousands of fertilised eggs, and the spermatozoa received must remain viable in the spermatheca for a long time.

Consequently, Soviet and later American researchers in the 1970s and 1980s rapidly developed methods to freeze drone semen at the temperature of liquid nitrogen (−196 °C) and then thaw it alive and sometimes even highly active (Melnichenko and Vavilov, 1975; Harbo, 1983). After insemination, however, the number of cells found in the spermatheca was low (generally < 300,000), the proportion of worker brood (an indicator of egg fertilisation rate) was low, and queen lifespan was often short. More recently, improvements made by researchers at the University of Washington increased the proportion of worker brood in queens inseminated with thawed semen to an average of 50 %, but queen lifespan remained very short (Hopkins et al., 2012). In France, INRA in Avignon also obtained encouraging results in the 2000s with a few queens inseminated with cryopreserved semen that entered egg laying. Unfortunately, the method used did not work consistently.

The Bee Research Institute in Hohen Neuendorf became interested in the cryopreservation of drone spermatozoa in 2009, with the support of a local SME (AMP-Lab GmbH) and the German Ministry of Agriculture*. After many unsuccessful attempts, the researchers questioned whether the very active state of spermatozoa after thawing might be problematic. In other species, it is known that sperm hyperactivity is induced only briefly and just before contact with the oocyte. This hyperactive state is generally irreversible, and activated spermatozoa do not survive for long. Fertilisation in bees of the genus Apis, however, is notably different: at the moment of ejaculation, spermatozoa form an extremely dense mass (about 7 million spermatozoa per microlitre!), in which the only possible movements are parallel undulations of the flagella of sperm bundles (Figure 2). All “traditional” cryopreservation methods involve adding a diluent to the semen containing substances necessary to prevent intracellular ice formation (cryoprotectants). Dilution generally leads to sperm activation and destroys most of the bundles. Therefore, an alternative method was developed to add cryoprotectants not by dilution but by dialysis (Figures 3–5). In this case, the semen is transferred into a tube, which is submerged in a concentrated cryoprotectant solution. The material of the tube allows the cryoprotectant to pass through, but not larger molecules or spermatozoa. Thus, the protectant penetrates the semen without destroying the bundles. In addition, dialysis reduces cellular water content, thereby decreasing the risk of ice crystal formation during freezing and thawing.

Using this method, recently published in the journal Cryobiology (Wegener et al., 2014), worker brood rates generally above 50 % were achieved and maintained for several months. Some overwintered queens were still producing worker brood the following spring. These results do not yet allow the use of thawed semen for inseminating “production” queens, but they are sufficient to inseminate queens with frozen semen and obtain fertilised eggs from them. This makes it possible to reintroduce frozen alleles. Cryopreservation of drone semen can therefore now be used for biodiversity protection or in specific breeding programmes.

In 2014, the Hohen Neuendorf institute greatly simplified the protocol and developed a commercial cryopreservation kit**. However, the process requires handling liquid nitrogen and having a cryogenic container for long-term storage. Such equipment involves significant cost, and its use also requires training to avoid risks. For this reason, interested breeders are advised to contact either a public institute or a commercial cryobank.

A first drone semen cryobank has recently been established in the United States, still working with a “conventional” cryopreservation protocol, i.e. without dialysis. In Europe, discussions are underway to set up another one. Such a tool could provide important support for the preservation of apicultural genetic resources as well as for breeders’ work, for example with the aim of creating a honey bee strain truly resistant to Varroa destructor.

 

* via the Federal Office for Agriculture and Food, within its innovation support programme (FKZ 2813500408).
** available at www.amplab.de

 

References

Harbo, J. (1983) Survival of honey bee (Hymenoptera: Apidae) spermatozoa after two years in liquid nitrogen (−196°C). Ann Entomol Soc Am 76, 890–891.

Hopkins, B.K., C. Herr, W.S. Sheppard (2012) Sequential generations of honey bee (Apis mellifera) queens produced using cryopreserved semen. Reproduction, Fertility and Development 24, 1079–1083.

Melnichenko, A.N., I.L. Vavilov Long term storage of drone sperm by freezing in liquid nitrogen. In: Apimondia, Prague, 1975. p. 311–314.

Wegener, J., K. Zschörnig, K. Onischke, B. Fuchs, J. Schiller, K. Müller (2013) Conservation of honey bee (Apis mellifera) sperm phospholipids during storage in the bee queen – a TLC/MALDI-TOF MS study. Exp Gerontol 48, 213–222.

Wegener, J., T. May, G. Kamp, K. Bienefeld (2014) A successful new approach to honeybee semen cryopreservation. Cryobiology 69, 236–242.

 

The lead author of this article is Dr Jakob Wegener, an agricultural engineer graduated from the Institut Supérieur Agricole de Beauvais. He has been a beekeeper for 25 years and has worked for 12 years at the Bee Research Institute in Hohen Neuendorf (Germany). You can contact him by e-mail: wegenerj@hu-berlin.de.

Source: http://itsap.asso.fr/

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See also:

• Single-Drone Insemination: A Review of the Practice
• All About the Drone
• Drone Rearing
• Rearing Male Bees
• Principles and Methods of Queen Rearing
• Practical Guide: 4.7 Colony Assessment and Selection