Towards Sustainable Storage Solutions for Drone Sperm of the European Honey Bee (Apis mellifera)

The month of May has been incredibly busy, not only because we have fully launched the queen bee breeding season and the first queens have already been sent to their new homes, but also because I had the wonderful opportunity to participate in the international scientific conference CONECT 2023, which took place from May 10, 2023, to May 12, 2023.

CONECT is an international scientific conference organized by the Institute of Environmental Protection and Heat Systems of Riga Technical University (RTU VASSI), dedicated to environmental and climate technologies. This was the 16th year of the conference, and this year 125 researchers from 24 countries gathered in Riga to present their achievements in environmental engineering.

I was also honored to present my scientific publications, which I developed as part of my doctoral studies. The topic of my doctoral dissertation focuses on enhancing the efficiency of selective breeding in the European honey bee by developing various engineering and biotechnological solutions. One of the main objectives of my work is to explore methods for the long-term storage of honey bee drone sperm, which would allow the preservation and restoration of unique genetic material as needed. Active research in this field is ongoing worldwide, and I hope that we will contribute to making simple, affordable, accessible, and reliable drone sperm storage a reality in the near future. Such a breakthrough would be a major advancement for the beekeeping industry globally!

The first publication on this topic has already been published under the title: "Review of sustainable cryopreservation and above-freezing storage solutions of European honey bee (Apis mellifera) drone semen.". Authors: Agnese Smilga-Spalviņa, Krišs Spalviņš, and Ivars Veidenbergs. This publication is already available in open access in the international scientific journal Environmental and Climate Technologies. The full original article in English can be found on the publisher's website, SCIENDO, at: sciendo.com/article/10.2478/rtuect-2023-0014 or by searching for the DOI number in any online search engine: doi.org/10.2478/rtuect-2023-0014. 

In this issue of "Biškopis," I have prepared a translated summary of the publication to introduce a broader audience of Latvian beekeepers to the latest scientific achievements in beekeeping.

Sincerely, Agnese Smilga-Spalviņa

Photo: Agnese Smilga-Spalviņa and Krišs Spalviņš at the CONECT 2023 conference. (11.05.2023)

Introduction

One of the priorities of the European Green Deal is the sustainable development of agriculture to halt the decline of biodiversity, establish a healthy and sustainable food production system, and mitigate risks related to food availability. These concerns became especially relevant during the COVID-19 crisis, which exposed vulnerabilities in logistics chains, and were further exacerbated by Russia's invasion of Ukraine. As part of the "Farm to Fork" strategy, which aims to ensure a sustainable food production system, it has been defined that food production should: have a neutral or positive impact on the environment; help reduce climate change effects; prevent the loss of biodiversity; provide nutrient-rich and sustainable food accessible to all citizens.

One of the agricultural sectors that fully meets these sustainability criteria is beekeeping, which is inherently a climate-neutral and zero-waste industry. Climate neutrality is indirectly achieved by sequestering carbon in beekeeping products. Plants absorb CO₂ from the atmosphere to produce carbon-rich compounds such as nectar, pollen, and plant sap, which honey bees utilize to produce honey, bee bread, propolis, beeswax, and royal jelly. Additionally, beekeeping commonly uses wooden hives, which can serve beekeepers for several decades, further enhancing sustainability. As of 2020, according to FAOSTAT data, there were approximately 19.6 million bee colonies registered in Europe, producing around 389,000 tons of honey.

In addition to honey production, beekeeping plays a crucial role in pollination, supporting other agricultural sectors reliant on plant-based production. According to FAOSTAT (2020), 10.6% of Europe’s total plant-based agricultural production depends directly on pollination (e.g., fruits, berries, buckwheat, rapeseed, and other crops). It is estimated that honey bees, along with other pollinators, contribute approximately €22 billion annually to European agriculture. However, due to diseases, pesticide use, and climate change, bee colonies are weakened, making them harder to maintain. Beekeepers worldwide continue to experience unexplained colony losses, often referred to as Colony Collapse Disorder (CCD), which threatens pollination-dependent crops and biodiversity. In the European Union (EU), 50% of agricultural land dependent on pollinators is already suffering from a shortage of pollinators.

The only sustainable solution for preserving bee colonies, restoring genetic material, and ensuring genetic diversity is consistent selective breeding, focusing on disease resistance and other economically significant traits. However, selective breeding comes with its own challenges. A queen bee typically lives 3 to 4 years, in some cases even longer. The evaluation of a queen’s colony performance can usually be completed by the third year of her life. In the first year, the queen is raised or purchased and overwinters with her worker bees. In the second year, the colony’s performance is monitored throughout the season. If the colony demonstrates exceptional performance, a decision must be made in the third year about preserving its genetic material by raising the next generation of queens, whose average lifespan will also be around 3 years. The first-generation offspring (F1) queens can transmit their exceptional genetic material by producing drones (male bees), as drones develop from unfertilized eggs and carry only the genetic information of their grandmother’s colony. Controlled queen insemination can then be carried out through isolated mating stations or instrumental insemination. When performing instrumental insemination, the age of reproductive material plays a crucial role in insemination quality. Queen bees should be 5–14 days old for successful insemination. Drones should be 2–3 weeks old for optimal sperm quality. Using older individuals may reduce sperm quality, result in lower sperm storage in the queen’s spermatheca, and increase the risk of queen mortality due to the accumulation of sperm residues in the oviducts. Given the seasonality of beekeeping, the weather-dependent mating process, and the complex reproductive mechanisms of honey bees, selective breeding, reproductive material evaluation, and controlled breeding must be carried out within a short timeframe. Drone rearing alone requires 40 days, and there is always a risk of losing valuable breeding queens prematurely or producing unexpectedly poor offspring. Additionally, a lack of new genetic material may lead to inbreeding. 

To preserve valuable genetic material, enable instrumental insemination throughout the beekeeping season, and enhance insemination service accessibility, the industry requires long-term honey bee drone sperm storage methods. Research on drone sperm preservation began in the 1970s and has significantly progressed over the past two decades, focusing on: improving cryopreservation techniques, reducing chemical toxicity, enhancing sperm quality and queen fertility. In 2021, the idea of establishing Europe’s first honey bee genetic bank was widely discussed to aid in genetic conservation.

The significance of sperm quality and queen bee fertility indicators.

The queen bee is the only individual in the bee colony that lays fertilized eggs and ensures the replacement of generations in the bee colony. Accordingly, from fertilized eggs develop the female individuals, that is, the worker bees, and from unfertilized eggs develop the male individuals, that is, the drones. Worker bees perform the functions of feeding, cleaning the cells, building the cells, collecting food, and protecting the hive, and if necessary, raise a new queen bee from a fertilized egg to replace the existing one. The lifespan of worker bees varies from 21 days during the active season to 6 months during the winter period. In turn, the sole task of drones is to successfully mate with new queen bees and pass on their genes to the next generations in another bee colony. The bee colony rears and maintains drones for only a few months, while natural mating of new queen bees is seasonally possible. Within the season, the proportion of drones in the bee colony is usually 5%, which can change depending on the age of the queen bee or the amount and quality of sperm present in the queen bee's body. In maintaining the functionality of the bee colony, the queen bee's fertility indicators play an essential role, which include the proportion of fertilized eggs, that is, worker bees, out of the total number of eggs, the quality of oviposition, and the duration of oviposition, which are directly affected by the quality of drone sperm.

A. The number of sperm cells in the queen bee’s spermatheca. The queen bee, at 5–14 days of age, goes on "wedding" flights to mate with several drones and accumulate sperm for the rest of her life for several years. The queen bee’s lifespan is directly dependent on the number of sperm cells in the spermatheca; if the queen begins to lay more unfertilized eggs, from which drones develop, the existing worker bees replace the queen to preserve the viability of the bee colony. Naturally mated queen bees accumulate an average of 4 to 5 million sperm cells in the spermatheca (which can vary from 2 to 7 million). On the other hand, a queen bee that has been instrumentally inseminated can be considered high quality if at least 3 million sperm cells have accumulated in the spermatheca, which would be a sufficient amount for the queen bee’s working life. If the number of sperm cells in the spermatheca is less than 0.5 million, there is a risk that the queen bee will start to lay only unfertilized eggs. In order to determine the number of sperm cells in the spermatheca, queen bees are prepared no earlier than 48 hours after instrumental insemination. This is mainly due to the fact that during the first 40 hours after mating, drone sperm migrates within the queen bee’s body from the median oviducts to a specialized organ designated for sperm storage – the spermatheca. The spermatheca is usually dissected and the sperm is diluted with a solvent, e.g., a potassium ion buffer solution (Kiev solution). Afterwards, the sample is examined under a microscope at 400x magnification, and cell counting is performed to calculate the number of cells in the spermatheca. The number of sperm cells in the queen bee’s spermatheca is an important indicator of drone sperm quality (as well as insemination quality), upon which the queen bee’s working lifespan and performance depend; therefore, it is used in research as one of the parameters characterizing sperm quality.

B. Sperm motility. In order for sperm to reach the queen bee’s spermatheca and later successfully fertilize the eggs, an important indicator of sperm quality is sperm motility. Motility is one of the most frequently tested indicators of sperm quality in studies. It is considered that conclusions about sperm performance can be drawn more accurately based on motility than solely on sperm viability indicators. Drone sperm of honey bees is characterized by both circular movements and sudden vibrations. Motility determination is carried out in the same way as sperm cell counting. The sample is diluted with a solvent and examined under a microscope at 400x magnification, and the percentage of the total sperm cell count in the sample that exhibits circular movements and vibrations is expressed. Motility is usually determined both in sperm samples before their use in instrumental insemination of queen bees and after queen bee insemination, by determining the motility in the spermatheca. It is extremely valuable to determine motility precisely before queen bee insemination in order to avoid inefficient use of labor and time resources devoted to instrumental insemination of the queen bee as well as subsequent monitoring and evaluation.

C. Sperm viability (plasma membrane integrity). The most frequently determined indicator of sperm quality is sperm viability. In studies using sperm samples for queen bee insemination, in which fresh and frozen sperm (dead sperm cells) are mixed in different proportions, it was determined that in the sperm samples at least 46% of the sperm cells must be viable in order for the queen bee to lay fertilized eggs for at least one season after insemination. The viability of fresh drone sperm can vary greatly from 55% to 99%. When sperm reaches the queen bee’s spermatheca, its viability decreases by 10%. It should be noted that viability indicators may vary depending on the age of the drones, their nutrition, the environment, and other factors. Also, in the queen bee’s spermatheca, sperm viability can vary; on average it is 80–98%, but with increasing queen age it can decrease. Sperm viability is determined by the permeability of the sperm cell’s outer envelope – the plasma membrane. If the sperm cells are damaged, the permeability of the cell membrane is greater than in healthy cells. Accordingly, sperm samples are stained with various substances, e.g., fluorochromes such as propidium iodine (PI), SYBR-14, Hoechst 33342, or acridine orange, which can cross the cell membranes and, upon contact with DNA, emit a red fluorescent color in damaged cells and a green or blue fluorescent color in viable cells. After cell staining, the healthy and damaged cells are counted in several fields of view under a fluorescence microscope, and their proportions are expressed as percentages. However, determining only the sperm viability indicator may provide misleading information about sperm quality. Even if a true viability indicator is obtained for the sample, there is no guarantee that the viable cells are also functional (motile) cells.

To assess sperm quality, studies also determine other parameters, but the three parameters described above are the most important among them. Sperm viability, motility, the number of cells in the queen bee’s spermatheca, and queen fertility (the proportion of worker bees) are used as benchmarks for drone sperm storage methods. The higher the drone sperm parameters and queen fertility indicators when using sperm that is frozen/unfrozen and stored for a long time, the better the sperm storage method will have been found. By improving storage methods, it will be possible to store drone sperm for long periods and, if necessary, restore genetic material by inseminating queen bees with the stored sperm and then rearing the next generation of queen bees. This is also the main goal of long-term sperm storage – to obtain fertile offspring and ensure the restoration of the gene pool.

Thus far, the main findings regarding the long-term storage of honey bee drone sperm:

• For several decades, the Harbo diluent has been the most commonly used sperm diluent for sperm freezing. 
• In the freezing process for sperm samples, substances—cryoprotectants—are used that protect sperm cells from cold shock and the formation of crystals within the cells during freezing. The substance that best fulfills this role is dimethyl sulfoxide (DMSO) at a concentration of 10% in the final sample. To reduce osmotic stress on sperm cells and the negative side effects that cryoprotectants tend to have, dialysis—or a gradual addition of the substance to the sample—is used during the freezing process. 
• In order for sperm samples to maintain high quality indicators (motility, viability), programmable freezers are used for sample freezing, and the sample is frozen from +4°C to -40°C at a rate of 3°C per minute. Afterwards, the samples are immersed and stored in liquid nitrogen (LN2) tanks at -196°C. 
• Cheaper, more mobile, and easier-to-use alternative solutions for programmable sperm freezing are being sought and developed. For example, sample freezing is carried out in liquid nitrogen vapors, or various sample packaging is used that slows down the sample freezing process. 
• Thus far, frozen sperm samples have been stored and tested after 343 days (using programmable freezing), whereas unfrozen drone sperm has been stored with good results in the temperature range from +10°C to +25°C for periods ranging from 2 weeks to as long as 439 days (using various glass capillary fillings). 
• In order to reduce the resource consumption required for storing sperm, testing sperm quality, inseminating queen bees, forming nuclei, conducting monitoring, and then performing additional determination of sperm quality indicators in the queen bee’s spermatheca in studies, relationships between the initial sperm quality indicators and the sperm quality indicators in the queen bee’s spermatheca and queen fertility are being sought. Thus far, it has been found that the strongest relationship is observed between sperm motility and the other sperm quality and queen fertility indicators, e.g., the higher the motility in the initial sperm sample, the more sperm cells have reached the queen bee’s spermatheca.

Over the last 22 years, rapid progress has been observed in the development of long-term storage methods. Innovations have been introduced with the aim of improving the quality of sperm samples and queen bee fertility indicators, and of reducing the negative impact of chemical and physical processing on sperm cells and queens. Innovations are also being sought in easy-to-use and low-cost methods that would be widely available in the beekeeping industry.

References: Smilga-Spalvina, A., Spalvins, K., & Veidenbergs, I. (2023) Review of Sustainable Cryopreservation and Above-Freezing Storage Solutions of European Honey Bee Drone Semen. Environmental and Climate Technologies, 27(1), 177-194. DOI: https://doi.org/10.2478/rtuect-2023-0014

Published in the journal "Biškopis," 3rd issue of 2023, by the Latvian Beekeepers Association.