An essential tool for honey bee research and selection, the technique of Instrumental Insemination is presented, step by step, with specialized beekeeping practices to enhance success.
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>> Controlled mating is essential to achieve the goals of any breeding program. Honey bees present a unique challenge. The queen mates in flight with an average of 15 to 20 drones in congregating areas consisting of 10,000 to 30,000 drones from diverse genetic backgrounds. The technique of instrumental insemination provides a reliable and highly successful method to control honey bee mating. It is an essential tool for the development and maintenance of stocks selected for economically valued traits and reduce susceptibility to pests and pathogens. Instrumental insemination is also a powerful tool for research. The sequencing of the honey bee genome provides new possibilities identifying the function of specific genes and new tools such as marker selection. Molecular techniques offer new research capabilities and the ability to develop and utilize innovative selection methods. Today, we are in the infancy stages of these possibilities. Instrumental insemination also provides a means to create specific crosses beyond what occurs naturally, an advantage for research and stock improvement. For example, a single drone can be mated to one or several queens isolating and amplifying a specific trait not expressed due to the effects of mating many drones. Varying degrees of inbreeding can be created including selfing, the mating of a virgin to her own drone sons. To the other extreme, semen from hundreds of drones can be pooled to inseminate a group of queens increasing inter colony genetic diversity and uniformity among colonies, enhancing brood viability and colony fitness. Another significant advantage of instrumental termination is the ability to store semen, both short term and long term. For short term storage, semen can be held at room temperature for about two weeks and maintain good viability. This provides an efficient means to transport and minimize the risk of shipping live bees. The cryopreservation of honey bees semen enables long term storage. This provides a means for the preservation, conservation and reconstruction of valued stocks.
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In appreciation of the capability we have today, let’s take a brief look at what went into the development of this technique. It’s a fascinating history spanning 200 years of experimentation, failures, skepticism, and also critical discoveries that built the foundation for the capability we have today. In attempt to control matings, virgin queens and drones are confined to various types of enclosures from glass jars, various types of cages to large greenhouses. Attempts to confine mating included the cylindrical rotating cage with glass at either end, a dark cloth covered the bottom glass and the cage was rotated in hopes the virgin and drones would fly upward toward the light to mate. In the early 1900s, screen tents were built. J.S. David said he could mate 500 queens a day in this tent. Several rewards were offered for a method to confine mating though never claimed. In 1922, Dr. Lloyd Watson demonstrated the first successful technique of instrumental insemination as he called it. He used a hand blown glass syringe and secured the virgin with a silk thread on a wooden bed as seen here. The queen was not anaesthetized, so imagine this. He used a pair of handheld forceps to open the queen’s vaginal chamber. His success was partial. Each queen received multiple insemination over several hours, resulting in some queens laying worker brood only briefly. At the time, there were still many unknowns about queen physiology and mating behavior. Several persistent researchers contributed to increasing success in their discoveries. It wasn’t until 1944 that Dr. Harry Laidlaw described the valve fold, an invaginative [assumed spelling] flap of tissue blocking passage of semen into the oviduct. Insemination of the queen requires delivering semen directly into the median oviduct. A few years later, in 1947, Dr. Otto Makinson designed his first prototype instrument within an anaesthetizing chamber. He used carbon dioxide to anaesthetize the queen’s so she would not be struggling during the procedure. He also found that this treatment had the added benefit of simulating egg laying. Modern instruments today are based on this simple design, upgraded with micro manipulators and innovative new sting hook designs. Today, the technique of instrumental insemination is highly successful and routine. Using proper techniques, the performance and success rate of instrumentally inseminating queens is similar to that of naturally mated queens. Given a full semen dosage and provided optimal care, instrumentally inseminated queens can [inaudible]e productive colonies for several years. [Inaudible] a series of studies comparing the performance of instrumized inseminated queens and naturally mated queens dating back to the ’40s. Colony performance was evaluated in terms of honey and brood production, and longevity of queens in field colonies. Factors affecting performance of instrumentally inseminated queens were also reviewed showing the treatment of queens clearly influences results. The studies are listed by author and year followed by the number of colonies observed comparing instrumentally inseminated queens in yellow, and naturally mated queens in blue. The studies are divided into three groups, one, equal performance of instrumentally inseminated queens and naturally mated queens; two, higher performance of instrumentally inseminated queens, which is generally attributed to selection in these studies; and three, higher performance of naturally mated queens. Looking at the treatment of queens instrumentally inseminated queens, group one and two, are combined together and these studies compared to the group three. The instrumentally inseminated queens in group one or two were inseminated between five and ten days post emergence, with the semen dosage of 7.5 to 12 microliters. Queens in group one and two were introduced into colonies by direct release, or some banked for a few days up to a week. Greek three instrumentally inseminated queens were treated differently. These were inseminated two to three weeks post emergence past their prime mating age. They were inseminated with two smaller semen doses totaling 5.4 microliters, and banked for an additional two to three weeks before introduction. The treatment of queens in this study are known facts to reduce sperm migration and the performance of instrumentally inseminated queens. [Music]. These quotes by the leading honey bee scientists of the time emphasize the importance of the treatment of queens during this procedure. Collectively, they state, “instrumentally inseminated queens can perform as well as naturally mated queens in field colonies provided they are given proper care. Attention to these details will improve the performance and longevity of instrumentally inseminated queens. Proper beekeeping practices are important to ensure these queens are capable of heading productive field colonies.
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The care of instrumentally inseminating queens is critical to their success. A major factor in queen longevity is a number of spermatozoa stored in the spermatheca. A full semen dosage for instrumental insemination is 8 to 12 microliters. Sperm migration into the spermatheca is influenced by the treatment of queen’s condition you can manage and optimize. After mating, the queen is very active, well attended by nurse bees and kept at brood nest temperature. Active movement and warm enhance migration of spermatozoa into the spermatheca, which takes about 50 hours. Providing conditions as natural as possible is important during this critical time. For best results, a direct release introduction after the insemination procedure is preferable over holding queens in banks. This takes planning an extra labour to establish virgin queens in [inaudible] colony before the insemination procedure and then returning her for a direct released introduction. Mating age is also crucial. Virgin queens normally take mating flights during the first week post emergence. Virgin queens confined by poor weather over 14 days mate with fewer drones and store less sperm in the spermathecas. Queens caged and banked beyond their mating age will store less sperm. Also, banked queens may be subject to injury by aggressive bees which can result in premature [inaudible]. Instrumentally inseminated queens are more difficult to introduce and variable in their initiation of egg laying. Mating stimulates rapid and major changes in queen behavior, physiology, and gene expression. Recent research has shown there are some differences between instrumentally inseminated queens and naturally mated queens. These includes variances in their pheromone profiles, ovary activation and [inaudible] levels. For these reasons, queen introductions need to be given extra care and attention. In my experience once instrumentally inseminated queens are established in field colonies, these differences equalize. Given a full semen load and proper care, instrumentally inseminated queens are capable of having productive colonies. Some live several years and outperform naturally mated queens. Knowledge of advanced beekeeping practices and attention to detail are required for success. The [inaudible] of queens and drones specifically for insemination purposes requires some expertise. The pre and post insemination care of queens is especially important. We’ll come back to this. First, let’s look at what equipment is needed.
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Instruments are specialized. Of the options available, these vary in quality and lack standardization. Quality can make the difference in the ease of learning and high repeatability of use. Precision and accuracy of fine movements and a wide range of flexibility and adjustments are key. The basic requirements are an instrument base supporting the syringe; a set of hooks and a queen holder assembly with a source of carbon dioxide. A [inaudible] microscope with a compatible standard and code light is also needed. For demonstration purposes, we are using a standard slate instrument with a Harboe syringe. The instrument consists of a sturdy, heavy base with support posts that are adjusted along the base and a micro manipulated syringe drive that swings forward and backwards. The [inaudible] assembly provides smooth and precision control of hook movements, the ventral hook and the sting hook. The queen holder assembly, also adjustable, delivers carbon dioxide. Sanitation is important, so before and after use clean the instrument and your working area with alcohol wipes. Correct instrument alignment is also important. The syringe and queen holder angle must be aligned. The set of hooks are set above the queen holder level and slightly above the queen’s abdomen. The Harboe large syringe capacity simplifies the collection, handling and storage, and shipment of semen. It also increases efficiency and provides easy and precise measurements of semen volume. Detachable capillary tubes provide unlimited capacity in the collection and storage of semen. The glass tip has the advantage of a flexible attachment and also is easily detachable. Here, I’ll show you the assembly as proper setup will avoid problems later.
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First sterilize the parts. Flush with alcohol and rinse with distilled water. Fill the syringe completely with saline [inaudible]. Assemble and check and make sure there are no air bubbles. Attach the tygon tubing over the needle head and fill this with saline using the micrometer. Attach the small piece of connector tubing and fill this with saline. Fill the capillary tube with saline and connect avoiding air bubbles. Pull this through the protective glass barrel, past the small end. Attach the connector to the tip. Attach the tip to the capillary tube then twist this to fit snugly in the barrel. Expel enough saline to make space to collect semen. Check for any air bubbles in the line as this will make the syringe spongy in response. The micrometer has a 200 microlitre capacity with accurate calibrations. Each division on the sleeve corresponds to one microliter about one drone’s semen volume. Each microliter is calibrated in divisions of 0.2 microliters for very fine work. One complete rotation of the sleeve and each division on the main barrel delivers 10 microliters. You can also use the simple plunger type syringe shown here. The tip and semen storage tube are one piece with a capacity of about 50 microliters. Fill the syringe with saline.
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Secure the tip with a piece of 5 millimiter silicone tubing pushing this past the end. This forms a seal. Assemble and expel enough saline to make space to collect semen. In addition, have these supplies available.
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As emphasized earlier, the success of instrumental insemination is also dependent upon beekeeping skills, which we will review here. Each step of the procedure from the rearing; to care for virgins and drones; to the establishment of laying queens and colonies must be given attention. For evaluation and selection purposes, these queen need to have productive colonies and thrive to be used as breeders. Proper beekeeping practices will ensure their productivity and longevity. Queen rearing conditions must be optimal. Rearing conditions influence queen quality including the number of ovarioles [assumed spelling] in the ovaries, size of the spermatheca and number of spermatozoa stored. Drone rearing requires equal attention and is demanding on colonies. Nutrition is important as developing drone larvae also requires a specialized, rich, high protein diet. A plentiful supply of healthy, mature drones from the desired select sources requires attention. Drones are seasonally produced, have a high rate of attrition, and are sensitive to stressors. Take care to minimize pests, diseases and pesticide residues as these factors reduce drone numbers as well as the viability and quality of sperm. Honey bees naturally mate seasonally and during a small window of time. This is also important for instrumental insemination. The optimal age of virgin queens for insemination is 6 to 8 days post emergence, with a range of 5 to 12 days. Drones, which are slower to develop and mature, peak in maturity at three weeks post emergence. Drones will yield semen at about two weeks old although this is less dense and mixes more readily with mucus, so more difficult to collect. Semen from older drones, past their peak, is viable although this has to be attributed to plugging the oviducts especially when queens are confined after insemination. Follow the natural seasonal rhythms of honey bee mating habits when rearing conditions are optimal. Late season, the task becomes more challenging and quality control more difficult. As mentioned, it is optimal to establish each virgin queen in her own nucleus colony before insemination, and release her directly after the procedure to allow free movement to remote sperm migration. This nursery colony holds emerging queen cells. For convenience, virgins can be aged in nursery colonies, though limit the time as much as possible. Use queen less nursery colonies with a high ratio of young nurse bees supplied with open brood and feed. Remove the cell cups of newly emerged virgin queens as some crawl back into the cells and die. As their pheromones develop and virgins approach mating age, they may be subject to injury by aggressive worker bees resulting in tubed tarsal pads and tarsal claws, missing parts of legs, etc. For this reason it is important to properly maintain nursery colonies and bank virgin queens for the least amount of time possible. A queen’s healthy tarsal pad is pearly white and plump. Discoloration indicates injury and this may prevent the production of footprint pheromone which functions to inhibit the construction of queen cells. The queen’s ability to hold on to the comb is also a function of the tarsal claws and pads. To have mature drones readily available, I usually collect these from several breeder yards and bank them overnight for the next day’s semen collection. Well fed cell [inaudible] and nursery colonies are a good place to hold cages of mature drones. Note that normal, queen ripe colonies tend not to take good care of mature, caged drones. Drones drift heavily between colonies. To identify the genetic origin and age of drones, these can be confined and matured in nursery colonies or marked and allowed free flight. I prefer marking drones as confined drones are messy at collection. Newly merged drones are easily marked with a paint pen.
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Drones have a high rate of attrition and are highly susceptible to stressors. Anticipate a low recovery rate of mature drones, about 25% to 40%. Of the mature drones recovered, not all will yield usable semen. Therefore I recommend rearing three to four times the number needed to assure a plentiful supply. In preparation for insemination, I recommend giving virgin queens a brief CO2 treatment the day before the procedure. Queens require two carbon dioxide treatments to simulate egg laying. One is given during the examination procedure; the second day, a before or after the insemination. It is optimal to give a treatment prior to insemination as this tends to initiate egg laying faster and does not interrupt the semen migration. The CO2 treatment can be given to caged virgins in nucleus colonies or to a frame of virgins in a bank. Let’s get started. We’ll go through the insemination procedure step by step including some troubleshooting to help you avoid common mistakes. We will collect semen first, then inseminate the queens.
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A flight box provides easy access to drones. A screen top with the light above stimulates the drones, keeps them warm and aids in final selection for color and activity. Drones must be healthy and sexually mature to obtain semen. Drones from different genetic sources can be mixed in the flight case. Here each cage represents drones collected from a different apiary of select breeders. Genetic diversity is important to maintain the integrity of a breeding program. Be sure to have a good supply of drones, at minimum, twice the number needed. During semen collection, some drones will not yield semen. Others cannot be used due to contamination. Some drones will revert when simply handled. The eversion is an explosive process and semen can be contaminated. If the semen touches your finger or drone body parts, discard. Contamination will cause infection in the queen. The semen collection process is tedious. With practice you will gain efficiency. It’s especially important to maintain sanitary conditions as the drones tend to defecate. Have a paper towel soaked in alcohol handy. Latex gloves are recommended as the feces causes skin irritation. To expose semen, [inaudible] the endophallus of the drone is a two step process, the partial eversion and the full eversion. This is the partial eversion. At this stage the abdomen contracts with haemostatic pressure. A pair of horn like structures appear, the cornua. Their orange yellow color indicates the drone is mature. If the drone is immature, the cornua are clear, lacking color, and the abdomen remains soft. Discard and try another drone. The full eversion, the second step exposes semen. This is obtained by applying additional pressure to the partial eversion. The semen is a marble, creamy color resting on a bed of pearly white mucus. The layers are distinguishable by color and viscosity. There is some variation with age of the drone. To gain efficiency in collection, recognize drone maturity as each is grasped. If they’re mature, discard and try another. To obtain the partial eversion, grasp the drone by the head and thorax, dorsally and ventrally. To simulate, apply pressure to the thorax. Dependent upon the drone, the pressure required will vary from slight to crushing the head and thorax. During this stage, take care not to apply pressure to the abdomen as this will damage the endophallus and prevent the full eversion. During partial eversion, the abdominal muscles contract making the abdomen hard to detach and the cornua will appear. At this stage, notice the contraction of the abdomen and the orange yellow cornua, indicating the drone is mature. Hesitate a few seconds before completing the eversion. During the partial eversions, drones often defecate. Take care to hold the drones away from your face and away from your insemination equipment during the process. Wipe with alcohol soaked towel as needed. Take care not to contaminate the semen, hooks or syringe tip. The semen is a rich medium for bacteria growth. To complete the second step of the eversion to expose semen, additional pressure is applied to the abdomen. With a partial eversion obtained, grasp the drone with your thumb and forefinger at the interior base of the abdomen. [Inaudible] your two fingers along the sides of the abdomen squeezing in a strong steady motion to the tip. The endophallus is everted and semen exposed. This pressure forces the endophallus inside out to expose the semen. It is important to apply the pressure starting at the base of the abdomen and work towards the tip otherwise the endophallus can be damaged and will not evert. For efficiency and to have semen ready for collection, use one hand for each of the two steps. With the right hand, grab the drone and obtain the partial eversion. With the left hand, complete the eversion and position the drone under the microscope. The left hand is now free to control the syringe for semen collection. You will go through many drones. Keep them warm with a light above and fed with a piece of candy or honeycomb inside the cage. I wipe a little honey on top of the mesh cage for the drones to self feed. Here’s another drone. Not all drones will yield. This one is immature. The abdomen is soft, and the cornua lack color. Discard and pick another drone. This drone appears mature, indicated by the color of the cornua, but yields no semen. Discard and pick another drone. A mature drone with the full eversion and exposed semen, hold the drone downwards to prevent the endophallus from flipping back upon your finger causing contamination. The everted endophallus can be floppy at this stage. Avoid transferring between hands after the eversion is complete. Excessive handling usually results in contamination of semen. Once the semen is exposed, this should be collected immediately to prevent drying and to minimize the chance of contamination.
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Prepare the syringe for semen collection. Collect an airspace in the tip to separate the saline column from the semen to be collected. Each [inaudible] yields about one microliter of semen. There’s the creamy marbled, cafe au lait color of the semen distinguished from the underlying layer of pearly white mucus. Also notice the semen is more fluid compared to the viscous and sticky mucus. Take care to avoid collecting mucus. This will plug the syringe tip and also plug the queen. Sometimes the semen and mucus are separated during the explosive eversion process and sometimes the semen ends up on your finger, contaminated and unusable. This is normal and the reason to have a plentiful supply of drones. The semen must be skimmed off the mucus layer. The consistency and quantity of semen varies dependent on age, nutrition and care. The semen can appear as a dense and compact clump or as several thin, stringy threads spread over the endophallus. The semen of young drones tend to be thin and lighter in color. The semen of older drones tend to be dense and darker in color. Bring the syringe tip into focus under the microscope. If using an angle tip, check the angle is positioned downward to provide more surface area for collection. Be sure an airspace separates the semen and saline column. With a Harboe syringe, I usually collect two airspaces. The second is insurance the airspace is not lost and the extra bubble of saline collects residual semen from the up and down movements. If the airspace is lost, mixing results in dilution of semen and inaccurate measurement of the semen dosage. To start a collection from the first row, precede with about half a microliter of saline in the tip. Bring the tip and semen into focus. Expel the strap of saline onto the semen to make contact. Skim the semen off the mucous layer. Move lightly over the thin surface of the semen to avoid mucus. The semen is fluid and it will separate readily from the thick, sticky mucus if the tip is not placed into this underlying layer. If resistance is felt, the tip is in the mucus. Try to avoid this. Semen from numerous drones is collected. For each semen load, expel a small amount of semen from the tip to make contact and skim this into the syringe. Avoid collection of air bubbles during this process. If the tip becomes sticky or to prevent the tip for drying between drones, collect a small drop of saline, half a microliters. During the collection process, avoid adding extra saline and diluting the semen. Repeat this procedure until the desired amount of semen is obtained. The column of semen should be solid without air bubbles and uniform in color. Light and dark areas indicate excess collection of saline, making measurement of the semen volume inaccurate. The time it takes to collect semen is largely dependent upon the quality of the drones and the skill of the inseminator. Concentrate on proper techniques with care to maintain sanitary conditions and avoid mucus. With practice and experience, efficiency is gained. During semen collection, if a small bit of mucus is detected in the tip, this can be teased out. First pull the semen column safely back into the syringe while isolating the mucus plug in the tip. Collect and expel a few microliters of saline in the tip, working this back and forth to loosen the mucus in the tip. Expel this with the extra saline if possible. Early recognition of a small mucus plug will avoid problems later. If the mucus plug cannot be dislodged from the tip, the tip can be removed and back flushed. Again, pull the semen column safely back into the syringe before removing the tip so it is not lost. The syringe should be highly responsive to fine movements. Do not keep turning the micrometer if there is no response as buildup of pressure will blow the line. An unresponsive syringe is usually due to a mucus plug lodged in the taper of the tip. Remove the tip and back flush to expel the mucus. An unresponsive syringe can also be due to excessive air in the line causing the semen column movement to be spongy or jumping aboard collection of air during the semen collection process resulting from losing contact with the semen or problems with mucus. Also check all connections to ensure air has been expelled in the line during setup. If the problem persists, check for a leak in the line at all connection sites including the needle head. With experience, these problems can be avoided.
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To inseminate the queen, semen is inserted directly into the median oviduct of the queen. This requires bypassing the valve fold, a flap of invaginated tissue blocking passage. In this diagram, notice the position of the valve fold oviducts, spermatheca and poison sac. Proper positioning and opening of the vaginal chamber are critical to bypassing the valve fold, which is not readily visible. Queens vary physically especially between subspecies. To gain proficiency, practice, give attention to details, and notice these distinctions. Adjust the CO2 rate to a slow, continuous bubble. During the insemination procedure, the queen is placed in the queen holder tube supplied with CO2. To position the queen, place her head first into the backup tube. Hold the backup tube flush against the tapered queen holding tube. The queen will walk backwards into the tapered tube. Place the queen over the CO2 valve in the instrument. The queen will quickly become motionless. The queen should fit loosely in the tube with the terminal segments of her abdomen protruding from the tapered end of the tube. Position the tube so that the dorsal, top side of the queen is to the right and the ventral side, underbelly, is to the left. Two hooks are used to open the vaginal chamber. The ventral hook on the left simply functions as a stabilizer. The sting hook on the right is used to lift the sting structure to expose the vaginal cavity. Techniques to position hooks vary depending upon how open or closed the abdomen is held and the type of sting hook used. Both hooks can be used simultaneously to open the cavity and expose the sting. Or if held tightly, the ventral hook can be initially used followed by the sting hook. In the final positioning, the queen and syringe must be carefully aligned. There are several choices in sting hook designs. Each has a different feel though the basic technique is the same. Here we will demonstrate the forceps hook, the perforated sting hook, and the classic sting hook. For ease of manipulation, place your fingers close to the hooks on the inside of the holding posts. This provides better control. Your hands should rest comfortably and be steady. The forceps are mounted on a handle with push button control. The tip of the sting is grasped, then lifted to the right following proper alignment. The ventral hook can be used to help expose the sting. With the sting secure, the ventral hook is brought back to stabilize the abdomen. The sting is grasped at the terminal in avoiding the fleshy filaments surrounding this. Lift the sting and move this to the right exposing the vaginal cavity. Avoid grasping too much of the sting or pulling the sting too far. This requires a gentle and light touch. Lifting the sting deep into the vaginal cavity, increasing the ease of bypassing the valve fold. The perforated sink is designed with a hole to thread the sting. The ventral hook can be pulled slightly to the left to help expose the sting. Bring the perforated hook over and lower to thread the sting. Take the hook to the base of the sting. So secure this, and lift the sting securely up and slightly to the right. Reposition the ventral hook so the queen is properly aligned. If the sting is difficult to thread, the ventral hook can be used to study the sting. Once threaded, reposition the ventral hook. Securely thread the sting and lift. The classic stinger hook has a flared, spoon like shape. Both hooks can be used simultaneously to open this vaginal cavity. These can also be crossed if needed. The sting hook is brought forward and lowered against the base of the sting and placed to fit between the sting lancets. The hook fits in the triangular area of the base of the sting and slightly underneath the sting structure. The sting structure is then lifted. There’s several ways to open the vaginal chamber of the queen depending upon the type of hooks being used and how loosely or tightly the queen’s abdomen is held. Both hooks can be used simultaneously. If the queen is difficult to open, the ventral hook may be initially inserted followed by the sting hook. The hooks can also be crossed and pushed past each other to open the cavity. A common mistake is to pull the queen too far to either side as this will distort the alignment making the assimilation difficult. The ventral hook function simply to stabilize the abdomen and to not be pulled to the left. Placement of hooks should comfortably align the abdomen with the syringe. The sting structure must be lifted up. A common mistake is to push downward placing pressure on the poison sac, distorting the alignment and can cause release of venom. This is more common with use of the perforated sting hook and the classic sting hooks. A descended rectum will make the insemination procedure more difficult and increase the risk of contamination. This can be a problem when queens have been banked. Notice this when the queen is placed in the instrument. If descended, remove the queen and place her on a paper towel to defecate. Another queen can be inseminated during the wait. To inseminate the queen, the syringe tip is used to lift the valve fold and semen injected directly into the median oviduct. This requires a slight zigzag movement. The valve fold is not readily visible. When the hooks are properly positioned, you can see a V of tissue, which defines the location of the valve fold. Locate the V of tissue. Position the tip above and dorsal to the apex of the V, not directly over this. Insert the tip half a millimeter, moving the tip slightly ventrally increasing the incline of the tip to lift the valve fold. Insert the tip another quarter to half a millimeter ventrally into the median oviduct bypassing the valve fold. The movement is a slight zigzag. Properly positioned, the tip will easily slip into the median oviduct without movement of the surrounding tissue. If tissue moves with the tip, the valve fold has not been bypassed and semen may backflow when delivered. As the valve fold is not readily visible, you need to acquire a feel for this procedure. Queens vary physically, so this takes some practice. Let’s do another queen. Collect a drop of saline, half a microliter, to proceed the next insemination. This keeps the tip moist and can be used to test if the valve fold has been bypassed. Position the queen, inject the drop of saline, and if there is no backup proceed to eject the semen. Take care to measure the amount of semen delivered. The standard dose is 8 to 12 microliters per queen. After insertion of the semen, withdraw the tip. There should be no or minimal leakage of seeing. A small amount of leakage may be due to pressure in the syringe. If semen gushes out, the valve fold has not been bypassed. After insemination, release the hooks and move these to the back of the instrument out of the way. Collect a small drop of saline in the tip to prevent drying and to proceed the next queen. Gently move the queen from the holding tube. Queens can be easily clipped and marked while anaesthetized. As queens recover from the CO2, offer a small drop of honey. Return queens to their nucleus colonies immediately after insemination. Broodness, temperature, and attendance by nurse bees is critical to sperm migration. Establish a routine to increase efficiency. The insemination procedure goes very quickly, seconds per queen. This is also important to minimize CO2 exposure. Practice is essential to gain proficiency. Semen leakage can occur for several reasons. Most often the valve fold has not been bypassed and the semen deliverable will back flow. If the tip has not completely bypassed the valve fold, some leakage will occur. If the tip is inserted on top of the valve fold, tissue will move with the tip. The tip must initially be inserted more dorsally and lifted slightly to pass the valve fold in a zigzag motion. Difficulty bypassing the valve fold is often due to improper positioning of the hooks and/or poor alignment. Check if the hooks are pulled too far to one side. Are the queen and syringe in alignment? Is the sting hook pushed down instead of lifted up? If you still have trouble, remove and reposition the hooks. Often a minor adjustment can be the difference.
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Established each virgin queen in a nucleus colony before insemination, and release her directly after the procedure is optimal. Free movement and attendance by nurse bees enhances sperm migration. Virgins can be introduced into nuclei as queen cells or soon after emergence for convenience, caged virgins. If queen cells are not caged, place an excluder over the entrance. Remove the virgin briefly before the insemination procedure. The queen in this [inaudible] has been recently inseminated and now we will release her. The longer her time away, the more important it is to take care in the reintroduction. I like to spray the bees with a scented sugar water as a distraction. Also note, queens can we returned to the nooks while still under CO2. It’s good to have an assistant runner for this job as it’s time sensitive. In general, the queen’s behavior will often determine her acceptance. If defensive, she usually is not accepted. In this case, I’ll cage her for a few days, or give a candy plug, delayed release. Take care to check the nooks for queen cells. A rogue virgin can cause this situation. Place a queen excluder over the entrance to prevent mating flights. Mating flights are inhibited by the presence of semen in the oviducts and CO2 treatments, though this precaution is recommended. A pushing cage can also be used especially when a direct release is not possible. Place the cage of emerging brood so young bees will emerge to attend the queen. Confine the queen for three to five days. If eggs are not observed when releasing the queen, exclude the entrance into laying. Queens in these nooks were inseminated a week ago and should be laying. Let’s take a look. Here she is looking plump with queen like behavior, and yes, she has eggs. As soon as eggs are observed, the excluder can be removed, usually between 4 to 10 days. Let’s look at another. There she is running and displaying virgin like behavior. No eggs are observed, so I’ll give her a few extra days, leaving the excluder in place. The start of egg laying is more variable among instrumentally inseminated queens. They often take a few days longer compared to naturally mated queens. There are also seasonal variations. Banking queens is sometimes necessary. Here are some instrumentally inseminated queens I did not have time to set up in nucleus colonies. The nursery colony is queen less, made up of young bees and well fed. Open brood is supplied, placed next to the frame of queens. These queens will be given a pushing cage introduction. In nursery colonies, be sure never to mix queens of different ages or different reproductive status. Pheromone levels vary between these resulting in different levels of care. Also note, bees related to the cage queen will take better care versus unrelated bees.
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When learning the technique of instrumental insemination, a good test for check your success is to observe the spermatheca. The queen must be sacrificed. Bank the queens for two days after insemination. To evaluate, look at vigor. If lethargic, this indicates infection. Dissect the spermatheca. The color and density indicate how much sperm has migrated into the spermatheca. The spermatheca of a virgin is crystal clear. The spermatheca of a mated queen is a marble, creamy color, the same color as semen. If the spermatheca contains only a small amount of sperm, the appearance is cloudy as seen here. While learning the technique, this is a good test to see how well your queens are inseminated. To dissect the spermatheca, grasp the last segment of the abdomen with your fingernails or a pair of forceps and pull. Embedded in the tissue of this terminal segment is a round, white sphere with a diameter of about one millimeter. The spermatheca teases out from the tissues. The spermatheca initially appear white and rough covered with a network of trachea. The tracheal net is removed by rolling the spermatheca gently between your fingers. As you see here, the queen has been successfully inseminated. Let’s look at another. This one is clear, a virgin.
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Another significant advantage of instrumental insemination is the ability to store semen both short term and long term. For short term storage, semen can be held at room temperature for about two weeks and maintain good viability. This provides an efficient means to export and minimize the risk of shipping live bees. The detachable capillary tube of the Harboe syringe allows for easy storage and shipping of semen. For short term storage, the optimal temperature is about 55 degrees or 13 degrees centigrade. Avoid temperature fluctuations, sunlight, and do not refrigerate. For short term storage, draw the semen completely into the capillary tube. Remove the tip. Disconnect the semen filled capillary tube from the syringe. Place the petrolatum plug into one end of the tube with a sterilized spatula. To place the plug on the other end, reconnect the two to remove the column with semen and place the petrolatum plug in this end of the tube.
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Store the capillary tube of semen in a protective case. Good sanitation is especially important when working with large quantities of semen. Take extra care to avoid contamination during the semen collection process. Upon receiving the tube of semen, this is easily reconnected to the syringe for insemination. Properly sterilize and prepare the syringe in advance. The section of the capillary tube with the plug can be cut or left in place to function as a spacer. Attach the tube of semen to the syringe without the tip. Remove the petrolatum plug on the tip end of the tube before the tip is attached. Attach the tip and collect a bubble of fresh saline to precede the first insemination. Cryopreservation techniques for the long term storage of honey bees semen enable the preservation of valued stocks and the ability to do selection across time for breeding purposes. This also enables the conservation and reconstruction of threatened subspecies and their various ecotypes. Current cryopreservation techniques are adequate to recover stock although result in colonies that are not field productive. There’s some damage to sperm during the process. Researchers worldwide are working to improve these techniques. The next video in the series will show this procedure. Another specialized technique is to homogenize semen. This is currently in the experimental stage as solution and mechanical mixing cause some damage reducing the number of viable sperm. The tails of spermatozoa are very long and fragile. Here you can see the live sperm cells stained green and the dead cells stained red.
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Instrumental insemination is a powerful tool for research and breeding purposes. To gain proficiency in the technique requires practice, attention to detail, and maintaining sanitary conditions. This also requires advanced beekeeping skills to assure quality control and a high rate of success in establishing queens in productive colonies. Concentrate your efforts to become proficient in each step of the procedure. The techniques presented here are designed to help you master this valuable skill to support your research and goals for bee breeding and honey bee conservation.
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