Honey Bee Mating Control and Production Cost Analysis In Africanized Regions Using Instrumental Insemination

Final report for FW17-017

Project Type: Farmer/Rancher
Funds awarded in 2017: $20,000.00
Projected End Date: 10/31/2019
Grant Recipient: Jaime de Zubeldia
Region: Western
State: Arizona
Principal Investigator:
Jaime de Zubeldia
Jaime de Zubeldia
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Project Information

The Africanized honey bee hybrid possesses traits that continue to challenge beekeeper maintenance of gentle European honey bees. Since 1993 Africanization of European honey bees in the southwestern United States has severely diminished the number of hobby and commercial beekeepers (1). The arrival of disease vectoring Varroa mites has further exacerbated the loss of managed and wild European honey bee colonies.  The number of managed colonies in Arizona decreased from 55,000 to 35,000 colonies in a ten-year span (1993-2003) per USDA statistics (9). Ten years later national honey production increased 5% while Arizona’s production lagged at 3%. Since these figures account for the entire state, it is highly likely that southern Arizona faired far worse - the likelihood of Africanization is more substantial in the lower half of the state due to geographic and climate factors.
Commercial pollination frequently involves transporting colonies to warmer locations in the southern United States to ‘over-winter’ where defensive colonies reside. European colonies used for pollination are at risk of being taken over by Africanized swarms through a process called usurpation. Keeping honey bee colonies gentle increases safety, and saves time when moving and inspecting colonies for pollination
Hives that have become Africanized may not readily accept queens of a different strain. An accepted anecdotal observation of Africanized honey bees is they tend to reject requeening efforts by beekeepers, superceding European queens within the first year of introduction. This makes the act of requeening hives more time consuming and expensive as additional queens may be needed before a hive is successfully requeened.
Beekeepers in Arizona rely on a volatile supply of imported queens for requeening needs. Honey bee packages and queens need to arrive in Arizona in early to mid-March. Currently packages and queens arriving from out-of-state tend to arrive in mid-April or later, just as hot summer weather diminishes nectar and pollen resources. This contributes to a several month delay in hive development compared to other
temperate regions.
Despite rising demand for honey bees in southern Arizona queen and honey bee production by local beekeepers for the local market is almost non-existent due to these challenges.
Through partial mating control, efforts to reduce problems associated with Africanization have been successful yet limited (4). Traditionally, beekeepers maximize successful queen mating with European male honey bees (drones) by flooding a mating area with high drone populations from strategically placed hives containing European honey bees. This “open-mating” method has proven useful, but it’s not 100% successful, it’s expensive and labor intensive. Controlling the drone type and quality in an open-mating system also requires suitable space – several hundred acres or more – which is becoming difficult to find and negotiate.
Studies indicate that defensive behavior is likely paternally inherited. Therefore, controlling the source of drone semen is essential in producing a gentle honey bee (7). In a personal communication with Dr. Ernesto Guzman-Novoa, Department of Environmental Biology at University of Guelph, Ontario, he confirmed that a fully-controlled mating study using instrumental insemination to develop a gentle strain of Africanized honey bee - using only selected gentle Africanized parents - has not been attempted (11).
Honey bees found in the wild without a recent history of beekeeper influence, and that have demonstrated survivability amidst waves of pests and diseases, are said to be a “survivor” strain. Recent research and several years of observing the results of selecting from our own survivor strains of Africanized stock suggests that a gentle Africanized strain of honey bee can be further developed (5). This, while retaining their improved grooming behaviors and Varroa mite resistance. Consequently, chemical treatments to mitigate pests can be reduced or eliminated.
However, our current methods of minimizing the influence of defensive Africanized honey bees are insufficient to supply queen bees to regional beekeepers on a commercial scale, or to contract hives for crop pollination. These are two markets that we would like to participate in.
ReZoNation Farm (tradename "Sun Apiaries") would like to obtain instrumental insemination training and equipment to establish on-farm propagation of European queen replacement stock, and eliminate the influence of Africanized honey bee strains in a significant percentage of our apiaries. In addition, Sun Apiaries seeks to use instrumental insemination training and equipment to determine whether we can accelerate development of our existing gentle Africanized honey bee survivor strain. Together, these objectives may enable us to supply region-specific breeder queens needed for ongoing replacement of queens on honey bee farms

We hope to measure a significant reduction in costs due to the time savings of using instrumental insemination. We also hope to develop a production model specific to our region that could be used in beekeeping communities with similar constraints where Africanization persists in warmer parts of the country.

By training over 400 people in beekeeping techniques since 2008, Sun Apiaries has been active in encouraging the selection of gentle honey bees. Through these trainings, internet presence, and meetings with beekeeping associations, the methods and results of this work will be shared. In the future, we would like to supply queens and bees to our region, and spearhead a collaborative effort to develop gentle survivor honey bee strains in all Africanized regions.
Controlled mating using instrumental insemination could substantially improve public safety because it maximizes the distribution and likelihood of gentle drones and queens mating with unmanaged defensive colonies beyond city boundaries.
Project Objectives:
The objectives of this project are to:
1) Obtain instrumental insemination training and equipment to establish on-farm propagation of European queen replacement stock, and to minimize influence of defensive Africanized honey bees in our existing apiaries.
Measurable outcomes
a) Acquire, and secure European strain queens for breeding material by October 2017.
b) Produce instrumentally inseminated daughter queens: 48-80 by May 2018, 60 by November 1st 2018, and replicate these quantities for 2019.
c) Produce open-mated daughter queens: 30-50 by November 2017, 20 by May of 2018 and replicate these quantities for 2019.
d) Report difficulties and measures taken to maintain European queen genetics in hives.
e) Estimate number of hives lost from Varroa mite influence or other challenge.  Preserve samples for future study.
2) Use instrumental insemination training and equipment to increase the predictability of targeted inherited traits, and increase the selection speed of a gentle Africanized honey bee survivor strain.
Measurable outcomes
a) Compare characteristics between a random selection of open-mated Africanized hives, and instrumentally inseminated Africanized hives. 
b) Produce instrumentally inseminated gentle Africanized daughter queens: 10 by November 1st, 2017, 30 by November 1st 2018, and 10 by April of 2019.
c) Record the number of hives decommissioned in open-mated and instrumentally inseminated groups due to defensive temperament.
d) Record number of hives lost from Varroa mite influence or other challenge.  Preserve samples for future testing.
3) Determine cost savings generated by using instrumental insemination technology, and evaluate whether a regional honey bee improvement program and local distribution of queens and nucleus hives are feasible.
Measurable outcomes
a) Estimate time/cost differences between mating methods
b) Estimate time/cost differences resulting from temperament and disease resistance differences between European and gentle Africanized strains.
c) Evaluate reliability of inherited behavioral and production characteristics.
d) Estimate future change in operation size due to instrumental insemination use.
e) Record number of regional beekeepers educated about this project, and evaluate feasibility of a regional honey bee improvement program.

Timeline                                 Task

(2nd-3rd quarter 2017)       Select top 10% of gentle Africanized survivor queens for instrumental insemination using semen from gentle survivor Africanized drones.
(2nd-4th Quarter 2017)     Acquire instrumental insemination equipment and training. Develop data collection sheets.
(3rd quarter 2017)            Acquire commercially supplied breeder queens. Produce instrumentally inseminated European and gentle Africanized queen daughters.
(Ongoing 2017-2018)         Record time and process savings due to instrumental insemination vs open-mating. Continue monitoring for temperament, production, and Varroa mite levels.
(Ongoing 2017-2018)         Conduct outreach to producers and community stakeholders.
(Mar-Oct 2018)         Produce instrumentally inseminated European and gentle Africanized queen daughters.
(March 2018)                   Submit 1st Year Progress Report
(Ongoing 2017-2018)       Conduct outreach to producers and community stakeholders.
(Oct 2019)                       Complete Final Report.


Materials and methods:

Preparation for this project began several years ago through the continued selection of feral colonies for gentle temperament as we grew our beekeeping operation.  In March of 2017 we obtained queen cells derived from Canadian Buckfast and Italian strains from a local beekeeper with family ties to an exceptional queen producer in Florida.  These cells were used to requeen various colonies with questionable temperament, and were expected to be a source of European strain drones for planned crosses in 2018 and 2019.  Additional European queens were planned to be brought in later in 2017 and 2018 from other beekeepers.

Training for instrumental insemination was received over a period of three days during the summer of 2017 from Kelly Rausch and Adam Finkelstein of VP Queen Bees in Iva, South Carolina.  Sue Cobey (Washington), Joe Latshaw (Ohio), and Dr. Kaftanoglu (Arizona State University) were other instructors that were considered.  However, for various reasons including timing and availability, VP Queen Bees became the optimal choice.  Sue Cobey was able to provide the necessary specialized Harbo syringe, syringe tips, and other minor equipment, while mechanical engineer and beekeeper Mark Sweatman provided the instrumental insemination stand upon arriving in South Carolina.  Aside from a thorough introduction of the technical aspects of instrumentally inseminating queens, students at VP Queen Bees received insights to the selection and rearing of preferred queens and drones.  The information exchange surrounding beekeeping and breeding experiences in the Southwest compared to the Southeast United States was invaluable.


Project Coordinator with instructor Kelly Rausch of VP Queen Bees discussing queen positioning and equipment set-up.
Collected drone semen in capillary tube with drone holding cage in background
Portion of equipment needed for II. Sterile and sanitary work area is essential.


Awaiting our arrival in Arizona was a number of surplus virgin “Rockstar” queens from fellow instrumental insemination student Zach Lamas.  These were installed in a surrogate hive (queen bank) for gradual installation over the next week during the middle of June, but at that time we experienced an extreme heat wave that prevented mating flights and general hive maintenance.  Consequently very few queens from that batch were successfully introduced to new colonies.  Of the few that were introduced before becoming too old to mate, poor record keeping at that time prevented us from tracking their locations.  At least one showed exceptional qualities, but she unfortunately went missing while attempting to take a photo of her to send to Zach to confirm her origin by coloring – the frame she was on slipped and dropped a few inches onto the top of the other frames, likely sending the queen flying away from the hive.  She did not return and as a result the hive began making new queens from her eggs.  At best we may have one daughter from this queen which has since proven to be of exceptional temperament for a first generation open-mated queen.


VP Queens instructors caging queen post 2nd CO2 treatment

In early September we renewed conversations with VP Queen Bees to obtain breeder queen stock for production of European strain daughter queens for the coming year.  Adam and Kelly work directly with the Baton Rouge USDA Agricultural Research Station involved in breeding for varroa resistance using their Pol-line strain of honey bees sampled from the best commercial beekeeper hives in use.  Unfortunately, the weather in South Carolina did not allow for good production of drones in their hives to provide instrumentally inseminated queens to us by the end of 2017 breeding season.  By their recommendation we were able to locate and install another batch of 12 queens from Mike Lamb of Lambs Honey Farm.  These queens were open-mated in North Dakota and were possibly slightly related to the Pol-line strain and similar stock at VP Queen Bees.  It was our hope that we would be able to obtain instrumentally inseminated breeder queens form Adam and Kelly by the following spring of 2018 to add to our selection of breeding material.


Due to the timing of obtaining and installing the Lambs Honey Farm (LHF) queens (August 26th – September 18th) we were unable to obtain mature drones from them to collect semen for creating instrumentally inseminated queens before the end of October when queen rearing becomes difficult.  An additional limitation was that not enough drone sized combs was available in the hives containing newly installed LHF queens, and we were not prepared to supplement the entire apiary and internally feed these hives with sucrose syrup to stimulate drone comb production.  LHF mated queens would have had to produce larva by September 7th on drone size foundation for drones to be mature by the 15th of October when they would be ready to yield semen.  They require approximately 2 weeks to mature after their initial 22-24 day larva/pupal stage.  It was not until the middle of September when all LHF queens were finally installed. 

Given that fall was rapidly approaching, and that it is was unlikely we would have good success with obtaining semen for instrumental insemination in 2017, we decided the best course of action was to concentrate on producing some of the other open-mated crosses for evaluation during the upcoming year.  This involved grafting larva on three separate days, from 7 different queens, using four separate cell-starter/finisher colonies.   The grafting sessions occurred on 9/20/18, 9/24/18, and 10/10/18.  The hives selected to create daughter queens were, SWM#17-16H, #64, #87, #79, SWM#15-16H, #66, and #63.

Each grafting session involved grafting 33 larva, around 12 hours old, for a total of 264 larva.  From this approximately 136 virgin queens were produced and banked for later mating.  In addition, during the first week of October, 24 four-frame mating nucs (the units used to allow virgin queens to open-mate) were created from strong colonies to facilitate mating of these queens.  Mated queens were collected 2-3 weeks later at which time another round of virgin queens were installed for open-mating.  This cycle was much quicker than normal since the season was rapidly running out and virgin queens were rapidly aging in queen banks - virgin queens should not be banked for longer than about 18-20 days (including 5 days post-cell emergence) before their potential for mating success decreases greatly.  A number of virgin queens had aged too much before they could be installed due to limitations in our work schedule, and we purposefully held back a batch of virgin queens to practice instrumental insemination techniques. 


Frame of drawn queen cells
Yard of mating nucs.
Protected queen cells in incubator


Despite these challenges approximately 50-60 additional hives were created during the months of September-November for the purposes of this trial.


During the winter of 2017/2018 a revised list of planned crosses were as follows:


  • E x E (II): European queen crossed with European drone semen using instrumental insemination.
  • gA x E (II): Gentle Africanized queen crossed with European drone semen using instrumental insemination.
  • E x gA (II): European queen crossed with gentle Africanized drone semen using instrumental insemination.
  • E x uA (OM): European queen crossed with unknown Africanized drone semen using standard open-mating method.
  • gA x gA (II): Gentle Africanized queen crossed with gentle Africanized drone semen using instrumental insemination.
  • gA x uA (OM): Gentle Africanized queen crossed with unknown Africanized drone semen using standard open-mating method.


As a result, since Combination #6 has been the method our operation has been built on up to this point, it served as a type of control in that there has been little control over the drone source.


Combination #2 and #3 would have attempted to assess or confirm the likelihood that aggressive temperament traits are paternally inherited through drones.  However, these were dependent on weather patterns that would efficiently produce optimal numbers of drones/semen.


Combinations #4 and #6 are to be assessed through 2018 in order to assess population build-up and temperament which are important characteristics to measure. 

When talking about selection using assumed strains of honey bee we should make an important distinction.  When we refer to European or Africanized honey bees (EHB or AHB), "neither can correctly be called race, subspecies, stock, or line representative."  This is due to a lack of precise laboratory verified genotypic and ancestral history which is unavailable to most beekeepers when selecting strains of honey bees at the macro- or apiary-level.  "The term "geographical type" or "ecotype" is more accurate.  These terms would indicate that the bees studied showed characteristics typical of temperate or tropically (A.m. scutellata, [aka, bees from southern Africa]) adapted bees." (Rinderer, 1985).  The indiscriminate use of the term "Africanized" is common in everyday conversation absent of actual genetic analysis.  While the probability of the infiltration of scutellata derived traits (specifically those assumed to confer mating advantages and unpredictable temperament) is very high in regions with a higher propensity to have aggressive feral colonies about, using the term indiscriminately tends to sway constructive conversation away from a focus on breeding and husbandry to one that encourages a general fear of all bees. 

Nevertheless, in keeping with already established historical common name nomenclature used in the scientific literature, and for ease of understanding and notation within this report, the term "Africanized" will be used to describe all feral honey bees both in our region, and in our managed colonies originally selected from feral bees.  Feral honey bees are most correctly defined as those not managed by a beekeeper in hives with movable framed combs that facilitate inspection and maintenance by a beekeeper.


Selection Criteria

Seven characteristics have been identified for possible data collection.  These are Temperament (T), Comb Stability (C), Brood Area (B), Brood Pattern (P), Mite Load (M), and Hording/Honey Production (H)


Stinging behavior, which is so often quantified in past studies as the number of stings per unit time, has been left out of this analysis.  In our experience, counting stings is a redundant measure of defensive behavior, it is already strongly correlated to temperament and comb stability, and it is highly influenced by other variables such as weather, food availability, and beekeeping skill.  Stinging behavior is also less likely to be measured continuously in the field as part of everyday agricultural performance since excessively defensive hives are quickly eliminated in an agricultural setting for economic and safety reasons.


Temperament is a subjective measure of overall behavior and workability at any given time during the inspection, but especially when the hive is first opened and as combs are pulled from the brood area.  It is expected that temperament will be strongly correlated to comb stability.  In terms of data collection in the field, our record sheet has been modified to the following grading scheme:  1=very gentle, 2=mostly calm and workable, 3= difficult to work and unpredictable, 4=viscous, unmanageable.


Comb stability is a subjective measure of how well bees remain still or calm on the combs, rather than vacating the brood combs in a hurried or nervous manner by running, parading, dripping off the bottoms of the frames, or bearding on the outer top rim of the box. In terms of data collection in the field, our record sheet has been modified to the following grading scheme:  1=very calm, 2=active yet covering brood, 3=nervous and leaving brood, 4=dripping off frames or parading along walls.    


A word on hoarding of nectar and hoarding of pollen as related to a hive’s efficiency in pollinating crops, and frequently referred to in other studies: In an agricultural setting, the grading of individual frames by the beekeeper to quantify the amount of honey and pollen is prohibitively time consuming and expensive in terms of labor.  Frequently, the condition of a colony can be assessed without the need to inspect all frames.  For the purposes of this project, our data will conform to existing management techniques that involves confining the queen and brood combs below a queen excluder in the bottom ten frame deep brood box while moving frames of honey above the queen excluder during peak nectar flow.  In some cases empty frames containing foundation are exchanged for frames of capped brood in the brood chamber to stave off swarming, encourage the building of new combs, and provide additional queen laying space if needed.  Honey is then harvested from the boxes above the single brood chamber just prior to summer dearth when supplemental feeding would begin to maintain hive population at normal levels of brood rearing.


Because poor performing queens are not kept for very long after it’s noticed that hive populations are less than optimal, it would not make economic sense to continue monitoring honey, pollen, and brood production of all hives regardless of performance.  This is one reason why we have chosen not to assess honey production of all hives continuously.  It is also our experience that hoarding of nectar and pollen is already highly correlated to brood rearing or the area of comb being used to rear brood.  This is especially true for the harvesting and storing of pollen which is depleted and renewed in a matter of a few days depending on environmental conditions.  Since we will already be attempting to quantify brood rearing, this is yet another reason not to assess hording on a continuous basis.  Rather, hives that have progressed enough in population to produce a crop of honey, and which have proven to be useful in terms of the other chosen characteristics, will be generally assessed for honey production either at harvest times, or more likely by weighing hives to obtain an average weight gain over a short period of time.  This could also be done without taking the extra time to open the hive or having to count the honey content on each frame.  It has been found that average daily weight gain is strongly correlated to overall honey production and hording.  In terms of data collection in the field, our record sheet has been modified with a field to accept colony weight or number of combs of honey. 


To maximize income from providing agricultural pollination services it’s desirable to maintain high levels of population and brood consistently throughout the year so that populations are maximized come February for the grading of hives. The fecundity of a queen also tends to be assessed by the brood pattern and the number of frames containing brood at various stages of development.  Particularly prolific queens are identified on a continuous basis by a simple count of the percentage of frames in the brood box being used to rear young.  The challenge is to ensure queens always have space to lay eggs post peak nectar flow when they are confined to the single brood chamber containing a maximum of ten combs.  Again, it’s not always necessary to pull all frames containing brood to assess the condition of the queen.  Management style may also limit the number of frames being used by the hive for brood rearing, which tends to vary between 5 and 12 depending on the time of year unless the brood nest has been artificially combined or expanded. In terms of data collection in the field, our record sheet has been modified with a field to accept a value measuring the number of brood combs in use at any given time.


Of side interest is brood Pattern.  In the case of actual field inspections during day-to-day operations, it is a subjective indicator of the effects of environmental variables on the hive, and condition of the queen.  Although brood pattern is greatly influenced by numerous unpredictable variables, it is useful when deciding which queens should be selected as queen mothers for successive generations throughout the course of the year.  Brood Pattern should be assessed over a longer period of months for stability rather while striving to keep hive resources plentiful as available natural forage resources varies with the weather.


While T and C are traits representing the degree of calm temperament, and B and H are the trait related to resource productivity, mite resistance is a trait that could be associated with overall fitness.  Care should be taken not to dilute the capacity of the Modified Selection Index to determine the best hives for subsequent generations by selecting for too many traits. Being judicious and minimizing the number of desirable traits to be evaluated is especially important for small operations that don’t have the available labor outside of regular hive maintenance for extensive data collection and analysis. Establishing too many traits for monitoring, while increasingly time consuming and expensive, presents the risk of giving too much weight to traits that may already be very highly correlated with others.  For example, in the traits evaluated during a study of Ontario beekeepers, temperament and calmness on the combs were quantified separately (VanEngelsdorp, Otis, 2000).  It’s unclear how these are differentiated in the study, but we would consider them to be almost one in the same. While we have included both of these traits in this study also, we are taking care to define each separately, and we feel that including both characteristics gives a higher priority and finer adjustment to the importance of gentle behavior in Africanized regions.  In addition, the trait of resistance to varroa mite through various types of hygienic behavior – also noted in the study - may be very strongly correlated to resistance to other diseases since we regard resistance to varroa mite (a primary vector of, or precursor to other diseases) as a measure of general fitness against disease (Page and Guzman-Novoa, 1997).  In terms of data collection in the field, our record sheet has been modified with a field to accept the number of mites found per ½ cup of bees (approximately 300).


For the purposes of monitoring the most important characteristics in a commercial setting this report focuses on Temperament, Comb Stability, Brood production, and to some extent brood Pattern.


Other factors such as time of day, weather, presence of swarm cells, and queenlessness, are recorded so that any patterns due to these conditions among a number of hives can be noted.  However, they are recorded as a matter of everyday maintenance activity as a general note, and will be incidental to this study.  To the extent possible these conditions are considered to remain relatively constant, yet some variability of these factors is inevitable.  Every effort is being made to avoid losing queens due to swarming or other events since queenlessness is a condition requiring elimination of the hive from the study. 

The number of inspections are limited to the regular time point intervals (TP#) associated with realistic agricultural management activities related to queen rearing and mating:


1)         TP1 - Initial diagnosis of queenrightness (successful mating) – 7 to 10 days post release from queen cages for open mated virgins, or 3 to 4 days after instrumentally inseminated queens are released from push-in cages.

2)         TP2 - 7-14 days after placing a mated or inseminated queen into hives being requeened to ensure queenrightness and laying pattern.

3)         TP3 – 14-21 days after TP2 to assess needed expansion of brood area, resource availability/storage, and to prevent swarming.

Additional general inspections will occur to prevent swarming (i.e. every 9-10 days through swarm season), to assess resource availability during times of dearth, or to take measures to mitigate other issues such as overpopulation (i.e. splitting).



The relative importance of the aforementioned traits will be prioritized by assigning a weight factor, V, as follows:

T: VT = 0.25

C: VC = 0.20

B: VB = 0.18

M: VM = 0.20

H: VH = 0.17

Total: 1.0 or 100%

From the data recorded during hive inspections, a z-score for each trait is extrapolated for each genetic family of bees using the following equation:


Z = (T - L) / SL


where T is the average value of a given trait of a genetic family line or sample, L is the average of a given trait for all families of bees, and SL (also classically denoted as σ) is the standard deviation of a given trait for all hives in a family line.

Since smaller numbers will indicate the more desirable behavioral phenotype score for all traits except for B and H, they are multiplied by -1.  Normalizing all traits among various genetic families or lines by assigning a z-score enables us to compare them even though they may have been measured and recorded using different units (i.e. pounds of honey vs. number of stings), and it gives us a sense of the distance away from the mean trait value, or peak in a normal distribution of those values.

Finally, some traits are usually considered to be more important than others in a breeding program.  In which case an economic value or weighting factor, V, as mentioned previously, can be applied to each trait’s z-score.  Summing the product of all the z-scores and their corresponding economic values provides a selection index number, I, for a given family line.  Like z-scores this is a number that eliminates units and puts all sample subjects on an equal numerical footing so they can be compared and classified quickly.  Given that the field of genetics specific to honey bees is young, there are still no agreed upon empirical values for the genetic correlation of various honey bee traits, and the likelihood of their heritability.  Therefore, a modified selection index has been recommended in which heritability of all traits is assumed to be equal, and the genetic correlation between traits is non-existent or zero (14. Rinderer, 1986, 13. VanEnglesdorp, Dennis and Otis, Gard, 2001).  The simplified equation for a modified selection index is:


Imod = ∑ Zi · Vi  for n traits, or alternatively,  Imod = ZV+ ZV+ ZV+... ZVn


Estimating Sample Size

Minimizing the variance typically requires the highest sample size possible.  However, sample sizes are also constrained by available resources and labor.  Especially in smaller agricultural enterprises as previously mentioned.  In an effort to understand what our limited labor and resources dictate with regard to sample size, we conducted sample size estimations that includes various planning values (targeted standard deviations based on experience and past research values) for each trait, and evaluated the margin of error as a percentage of the planning value while keeping the number of hives between 12 and 20 in each family line or sample.  Establishing this range of hive quantity within each family line requires between 72 and 120 total hives, and provides a margin of error that ranges from 22-28% of the planned standard deviation.  Although it would be ideal to test thousands of hives to obtain much smaller margins of error we feel that these odds are realistic given the economics, while still providing a solid direction for selection over time.  In reality, most beekeeping operations have few other options. To put this issue into perspective, to reduce the expected margin of error from 0.071 to 0.020 we would have to increase the total number of test colonies from 120 to over 1,470.

The equation used for evaluating realistic sample sizes is:


n = [(z/ 2) 2 (SL2)] /  E 2


where n is the sample size, za is a commonly used confidence interval of 1.96 which corresponds to a confidence level of 95%, SL is the planning value or desired standard deviation, and E is the margin of error.

These equations have been incorporated into spreadsheet format for efficient calculation, and for tracking of all family lines as hives are created and assessed. 


Cost Production Analysis vs. Mating Scheme

The other major item being tracked is the time and effort necessary to complete various tasks in the different queen production/mating scenarios: instrumental insemination vs. open (natural) mating.  A spreadsheet with a breakdown of tasks within each of these scenarios is being developed to assign the time necessary to complete them so they can be summed and compared.  It’s also important to compare perceived rates of success to the effort required for each scenario along with the pro's and con's involved with each method and family of honey bee produced.

In general, not accounting for management difficulties due to prevalence of aggressive colonies, and assuming all other tasks remain equal, the major differences between mating regimes in terms of the number of necessary trips per queen produced, and time a specific amount of equipment is occupied is:


Instrumental Insemination

  • Drone propagation and management trips (~ 3 total): 1) assess, feed and possibly treat drone production colonies, 2) isolate/corral drone comb for easy access, 3) capture drones and collection of drone semen (20-30 drones per queen).
  • Trips to produce II queen (~ 5 total): 1) collect from bank, CO2 treatment, place identification number on thorax, 2) install in nuc after treatment 3) 2nd treatment CO2, 4) confirm laying after 5 days, 5) remove for sale or use in another hive.
  • Pro: Lower number of mating nuc equipment necessary - time needed per mating nuc is less
  • Pro: No mating flight time required = more queens per insemination/evaluation cycle
  • Con: Very high level of skill/organization and time is required for drone production and semen collection.
  • Con: Double the trips required to access hives compared to open-mating scenario.



  • Drone colony management trips (~1 total): 1) assess, feed and ensure drone comb being drawn in preferred colonies.
  • Trips to produce open-mated queens (~ 3 total): 1) collect from bank and install in nuc 2) check 10 days after install of mature virgin 3) remove for sale or use in another hive.
  • Con: Higher number of mating nucs necessary to mate each queen, due to more time needed per queen to occupy a mating nuc.
  • Con: 4 days of mating flight time increases queen loss by ~20-25% due to predation or other cause.
  • Pro: Much less time spent to manage drone populations, since no need to collect semen.
  • Pro: 50% less trips to hives compared to instrumental insemination.


The need to collect drone semen and manage for drone populations at a higher level of accuracy, adds a third degree of difficulty to the II scheme, in that, drone/semen collection has to be more accurately timed with queen rearing/maturity, and mating nucleus availability/population.  In the open-mating scheme, one is able to take advantage of “free” drone semen in the environment, and in terms of timing there are only two main elements to coordinate – queen rearing/maturity, and mating nucleus availability/population.  However, if the need for marketing purposes is predictability of temperament through drone semen quality – to be able to provide gentle temperament with some measurable degree of certainty to a customer – then the drone semen readily available in the southern Arizona environment would be seen as less than “free”, or in fact, a liability.  That is, unless a measurable degree of certainty that first generation open-mated European or gentle Africanized queens are comparable in temperament and overall performance to a strain open-mated in non-Africanized regions.


Worth noting is the added level of difficulty in mastering the insemination of queens, of which the number possible can be limited by how plentiful the drone population is in drone rearing colonies.  Especially during times of the year when colonies are not in an optimal condition to rear them.  European strain queens are generally regarded as difficult to keep alive, and not as vigorous, when speaking to experienced beekeepers in Africanized regions.  Using instrumental insemination tools to show that first generation open-mated queens in Africanized regions by-and-large have acceptable temperament, while being substantially more hardy and productive, may prove the technology an essential tool.


Lastly, an attempt needs to be made to put a value on managing apiaries largely headed by gentle queens vs. apiaries with a higher percentage of more defensive and more unpredictable bees.  If the results of this trial show definitive value and substantial differences between the family lines produced, it could have an influence on future business management decisions.  Since temperament of hives is key to long-term management this evaluation helps beekeepers to assess other operational aspects besides mating, such as swarm management, honey harvest, and any aspect having to do with transporting honey bees for pollination purposes.








Research results and discussion:


European vs. Regionally-Adapted Hybrid Stock

While hybrids (in the generic sense) are technically a good choice for production due to heterosis (hybrid vigor) true honey bee hybrids created from inbred lines do not reliably transfer their genetic advantage in the next generations, and are said to produce mediocre second-generation bees. In addition, inbred parents are necessary to produce a true hybrid, and honey bees are difficult to keep alive and healthy since the more inbred the colony the more their population diminishes – workers do not let inbred larva survive to adulthood.

Our business model demonstrates that above the 200 colony level, a 2.5 FTE of labor would be required, and a 1.5 FTE seemed manageable for a 100-200 colony level. Given our experience, in terms of labor at a 1.5-2.0 FTE, we did not feel we could reliably maintain inbred lines for a true hybrid product while simultaneously maintaining a semi-closed breeding population for developing specific strains. In addition, the amount of time needed to create new queens each year to replace failing queens in addition to maintaining inbred lines, would be just as much or less than that of maintaining breeders onsite through instrumental insemination.

After noting the differences in spring colony population increases and disease resistance among the strains, maintaining pure European stock did not seem like a viable option compared to what was possible otherwise when using a regional hybrid. Since we still had to institute the same safety protocols given our proximity to feral AHB colonies throughout the region, our hope of maintaining consistently docile colonies of primarily European heritage at a profitable level did not seem realistic. By the same token, sufficient levels of docile behavior among most colonies seemed to be possible when using open-mated colonies selected for good temperament over a long-period of time. Otherwise, we'd also have to resort to some other mite control through mechanical or chemical means which would not prove to be as economical compared to what we are used to - the natural varroa fighting ability of AHB.


Pure European vs. Open-Mated Stock

After noting the differences in spring colony population increases and disease resistance among the strains, maintaining pure European stock did not seem like a viable option compared to what was possible otherwise when using open-mated stock. Since we still had to institute the same safety protocols given our proximity to feral AHB colonies throughout the region, our hope of maintaining consistently docile colonies of primarily European heritage at a profitable level did not seem realistic. It was consistently noticed that early spring brood build-up of the AHB colonies exceeded that of the European colonies by a substantial margin, and in terms of filling pollination contracts that is an advantage. By the same token, sufficient levels of docile behavior among most colonies seemed to be possible when using open-mated colonies selected for good temperament over a long-period of time. In addition, we'd also have to resort to some other mite control through mechanical or chemical means which would not prove to be as economical compared to what we are used to - the natural varroa fighting ability of AHB.

After sorting out the sufficiently usable data we were able to classify hives into four groups based on level of selection pressure as follows:

Old Line: These hives were identified as having a recorded temperament over the longest period of time in our apiaries, and/or those that for one reason or another have been chosen to rear queen mothers for future generations. These are the hives that have survived the test of time and have proven worthy of future selection.

European: These were hives that were brought in specifically to establish a baseline of ideal temperament with no Africanized influence, and to compare productivity.

1st Generation Cross: These were hives that were created from mostly 1st generation open-mated queens created from the European group, and a some queens created from the best of the Old Line.

Lowest Selection Pressure: These were the hives that were subject to the least amount of selection pressure over time, and that for some reason - either due to high productivity or lack of time to cull out - have remained in our apiaries.

After comparing the average values of temperament and comb stability of each group against the level of productivity (brood area) it was clear that while the European group maintained the best temperament overall, the 1st Generation Cross placed second in terms of temperament while beating the European grouping by 15% in terms of productivity (see attached data).

Summary of data:


Temperament (least = best)

European = 2.0

1st Gen Cross = 3.42

Old Line = 3.77

Low Selection Pressure = 4.33


Productivity (least = worst)

Low Selection Pressure = 9.72

1st Gen Cross = 8.90

Old Line = 8.12

European = 7.56


Raw Data By Number - Final

Compiled Data - Final

Sample Size Reasoning Calcs - Final


Use of II - Viability

Based on what we knew at the beginning of the project - which also became apparent from the II training in South Carolina - we decided that the extent of using II would be to maintain limited E x gA and gA x gA parents (also called breeder stock). These breeders would then be used to produce first generation crosses through open-mating, rather than attempt to use II directly to compete with the production efficiency of naturally mated queens in an open-mating system.

With that said, it's my opinion that instrumental insemination would not have added a reliable enough savings to our overall process - at least enough to be used on a continual basis due to the added challenge of ensuring adequate drone populations throughout the year, and doubling the number of trips into the breeding hives. However, a very seasonal use of instrumental insemination could be handy to maintain preferred regionally-based queen stock, but maybe not enough to justify the cost and effort to learn the technique rather than partner with those who chose to specialize in providing instrumentally inseminated stock. A community-based effort to maintain optimal regionally adapted stock and sharing of equipment might be another solution. While there would be significant cost-sharing advantages with respect to equipment and materials, the learning curve remains steep.


Physical Effects of Climate and Random Environmental Constraints

Additional European strain queens were scheduled to be acquired in early spring 2018 from VP Queen Bees as long as weather events in South Carolina did not interfere with the viability of those strains – notably hurricane Irma, and below average freezing winter temperatures.  During the winter of 2017/2018 much warmer than average weather occurred in southern Arizona, which caused the bees to continue rearing brood.  While this might be considered to be a benefit, it could also prevent a natural cleansing of mites due to a break in the brood cycle that normally occurs when temperatures are lower. Varroa mite depends on a continuous brood cycle to breed. Honey bees also go through limited food stores much quicker during warmer winter weather when natural nectar and pollen availability are low.  During this period the bees did not cease to consume open-fed pollen substitute in the form of BeePro from Mann Lake Ltd.

Not only is this an incomplete supplement if some amount of natural pollen is not being foraged, it also increases costs. Only time will tell if natural resistance or hygienic behavior will keep mite levels low during early spring build-up in spite of this extreme climate event.


Economic Effects Based On Climate and Random Environmental Constraints

By February/March of 2018 it became apparent that instrumental insemination using viable drone semen collected from our own stock would not be possible given both weather constraints and unexpected loss of colonies due to vandalism.

Unfortunately, due to the poor 2017/2018 La Nina winter, and early 2018 poisoning issues that killed about 60-75% of our colonies around the San Xavier Coop Farm, we experienced a severe reduction in planned income for 2018 estimated at around 80%. Given that we also increased the size of our family by one in March 2018, without income in 2018, it would have been detrimental on many levels to continue our business in 2019 – our future success in 2019 depended largely on our financial success in 2018. Moreover, without saved income from 2018 no labor would be hired in 2019 and success in spring 2020 would be unlikely after a solid year of working at 1.0 FTE. Since the odds of having a perfect year in terms of weather and physical stamina was very low – not to mention the effects on family life due to over work - we made the difficult decision to begin the process of closing our business and apiaries after collecting enough data from our colonies to complete this project.

One of the goals of the project was to rigidly quantify the economics of various mating schemes under real-world farming conditions. During the project it became clear that a rigid recording of time against each task would be unrealistic, as the tasks of instrumental insemination proceeding in concert with all of the other general hive maintenance duties would prove too time consuming and costly to manage. In addition, during our attempt to recover over the summer from the losses experienced during the 2017/2018 winter-spring season, and because I had to discontinue paying for part-time labor after April 2018, there was not enough time to record the economics portion of the project in detail. Normally plentiful swarming of local feral colonies greatly supplemented our income during the spring by answering contract calls to relocate them. The number of opportunities for that work was almost non-existent since local feral populations either died by early spring, or were not strong enough to swarm. Lastly, it was apparent early on that instrumental insemination would likely, at most be used to develop and maintain local breeders in low numbers and in a very seasonal manner rather than attempting to inseminate large numbers of queens throughout the year.

While weather extremes were a primary cause for eliminating certain aspects of our original project goals, we were pleased that we were able make some management conclusions that would prove profitable and essential to others in the future. The latest climate models maintained by the International Research Institute for Climate and Society (IRI) were helpful in making end of season decisions (12).

A three-month rolling average of likelihood that an El Nino or La Nina weather event will occur during fall through early spring proved to be fairly accurate for our region. In essence, if an El Nino event is strongly predicted for the fall/winter of a given year then Southern Arizona can expect higher than average rainfall and slightly cooler temperatures. Conversely, if a La Nina event is strongly predicted moving into fall/winter then lower than average rainfall and slightly warmer temperatures can be expected. This has to do with high pressure systems being forced out or tending to hang over our region depending on the fluctuation of Pacific ocean average surface water temperatures around the equator. Unfortunately, we did not learn of these specific weather models until late spring 2018 when we were concerned about ongoing observations. However, based on our observations of poorer than average hive conditions, and the anecdotal observations of other beekeepers from Southern California to Eastern New Mexico (all indicating failing hives and very low swarming rates – especially of feral colonies), we made the decision to implement the following decisions that would determine colony over-wintering success and in turn, future profitability:

El Nino Probability High: If an increase in total colony numbers is desired consider splitting all double-deep colonies in the fall to overwinter. Conditions for maintaining strong colonies for almond pollination should remain favorable, and the likelihood is high that good forage through winter will be available and a strong spring bloom will occur due to high stored soil moisture.

La Nina Probability High: Avoid splitting hives in the fall to increase colony numbers. Leave as strong doubles and consolidate weaker colonies as much as possible. The probability that lower rainfall and warmer than average temperatures is high due to high pressure systems remaining over the region causing a shortage in winter/spring pollen. This dynamic also tends to kill off weaker colonies or delay colony build-up in the spring due to premature consumption of stored colony resources and lower availability of winter/spring pollen. This is especially true and more pronounced in feral colonies that don’t rely on beekeepers. Expect feed costs to be higher to keep hives in optimal condition for early spring almond pollination.

As predicted by these models the 2019 spring season experienced the best conditions since approximately 1975 by some accounts due to record and consistent rainfall. But without a significant number of years to compare to, and beyond the scope of this project, it would be hard to determine what minimal level of seasonal rainfall would result in a profitable production of drone semen. I suspect that if we continued instrumental insemination into 2019, we may have experienced an adequate year, and I would guess that one year in 6 or 7 years would yield conditions good enough to make significant management of drones for instrumental insemination profitable in a commercial beekeeping setting.


Out Of Season Queen Rearing

Although possible to mate queens during the month of October, failure rate is high due to low availability of drones or a predominance of infertile drones. A high rate of laying workers in mating colonies was observed as if the queens never properly developed. This seems contradictory since a drone laying pattern was not observed, but regardless, conditions for successful mating during the fall, in a manner that is economically possible is not considered feasible. However, good mating results were had approximately 2-3 weeks after the first major monsoon activity and continued as long as consistent natural pollen flow and nectar was available. Good to excellent mating conditions were had from mid-July to September during 2018 within 1 mile of a substantial stand of cotton producing fields.


Mitigation of Vicious Temperament

A large amount of time is disproportionately spent in AHB regions to mitigate the occasional hive that proves too vicious. After significant selection over a period of years, and depending on proximity to a concentration of beekeepers in urban areas vs. rural areas where there are comparatively fewer beekeepers maintaining pure European stock, there still exists a proportion of hives that inadvertently express a high degree of unpredictable and vicious behavior. In our experience, this hovers anywhere from around 5% to 15% depending on how conscientious the beekeeper. Given that time is always a limiting factor in any operation, dealing with this proportion of hives in a humane yet profitable way has yet to be figured out to our liking.

To date, most of our mitigation has involved splitting these hives to repurpose for requeening and increasing the number of colonies. However, this still proves to be a stressful and fairly risky process, and an adequate amount of space (preferably around 600 ft minimum) must be maintained around the hive in question to eliminate stinging incidents. This process tends to incite other nearby hives to react defensively to some extent. In larger operations, more economical methods in terms of time and risk need to be developed to eliminate whole colonies from the general population that prove an unmanageable liability.

Solutions need to be developed to humanely euthanize an entire hive, at any time of year (excluding daylight hours) in a chemical-free way that does not require opening or moving the hive, and so that the combs can be repurposed to other hives as fast as possible. A method to segregate and euthanize only the aggressive portion of older-forager bees from younger non-flying nurse bees might be a way to make a once vicious hive ready for splitting into nucleus colonies. Perhaps a lethal dose of CO2 applied to bees collected by vacuum into a sealed chamber, followed by freezing, would represent a fairly benign way to segregate and euthanize the fraction of aggressive flying bees while conserving the brood and honey combs, and a good proportion of nurse bees that tend not to leave the brood combs. A porous funnel over the hive entrance could be formed in a way to lead to the vacuum opening of a device already commonly used by experienced beekeepers that conduct live-removal of feral colonies from urban areas.  For maximum effect this would have to occur at sundown once all bees have returned from the field or before sunrise, before any bees have left to forage. Splitting the hive would need to occur immediately afterward to reduce the possibility of robbing the day after, or collapsing combs due to heat buildup from a reduced population.

Aside from this, the complete euthanizing of all bees within a hive would have to employ a quick method of sealing the entire hive in plastic after closing off the entrance just before administering the gas from a small hole in the rear of the hive similar to some methods of applying oxalic acid vapor to treat for mites (note that sealing a hive in plastic is not part of any oxalic acid treatment to date). Care would need to be taken so that death does not occur due to overheating which would also cause collapse of honey combs and cause a much larger problem of honey robbing by other colonies.

If easy and quick methods of euthanizing the most vicious hives could be developed, beekeepers could focus on breeding and maintaining the balance while conserving their strength and peace of mind.



  1. Juliana Rangel, Department of Entomology, Texas A&M University, “Africanized Honey Bee Biology”, National Honey Show video, 2015, min 18:30, https://www.youtube.com/watch?v=rDLXFChK61g
  2. Juliana Rangel, Department of Entomology, Texas A&M University, “Africanization of honeybee pop in S. Texas, does a decade make a difference?”, Ecology and Evolution 2016; 6(7): 2158–21692016
  3. Randy Oliver, “Understanding Colony Buildup and Decline – Part 4 – The Linear Growth Phase”, American Bee Journal, May 2015.
  4. Ernesto Guzman-Novoa, Department of Entomology, University of California - Davis, CA, “Selective Breeding of Honey Bees in Africanized Areas”, Journal of Economic Entomology, Vol. 92, no.3, 1999.
  5. Bert Rivera-Marchand, Department of Biology, University of Puerto Rico, “Gentle Africanized bees on an oceanic island”, Evolutionary Applications ISSN 1752-45712012, 2012
  6. Alberto Galindo-Cardona, Department of Crops and AgroEnvironmental Sciences, University of Puerto Rico, “Genetic structure of the gentle Africanized honey bee population (gAHB) in Puerto Rico”, BMC Genetics 2013, 14:65 http://www.biomedcentral.com/1471-2156/14/652013
  7. Ernesto Guzman-Novoa, Department of Environmental Biology, Univ. of Guelph, “Paternal Effects on the Defensive Behavior of Honey Bees”, Journal of Heredity 2005:96(4):376–380
  8. E. Rinderer, H.H. Laidlaw, “Bee Genetics and Breeding”, Northern Bee Books, 2013.
  9. 1993 AZ Number of Colonies (USDA) https://quickstats.nass.usda.gov/data/printable/26A77FB0-13D9-3BAA-BFDC-D008494EE5FD
  10. 2003 AZ Number of Colonies (USDA) https://quickstats.nass.usda.gov/data/printable/05693FA0-87C1-37AA-9AD9-FD1F03CACACE
  11.  Personal email communication: August 2, 2016, Inquiry by J. de Zubeldia, ReZoNation Farm “…Question: Has there been any formal research using controlled matings among Africanized stock to support the hypothesis that a gentle strain of AHB (termed gAHB by some) can be reproduced consistently?...”.  Reply on August 4, 2016 by Dr. Guzman-Novoa, “…Answer: No attempts have been made with 100% mating control, because that requires either isolation in mating yards (islands, deserts, high mountains) or instrumental insemination of queens.”….”
  12. The International Research Institute of Climate and Society’s probabilistic seasonal climate forecast product is based on a re-calibration of model output from the U.S. National Oceanographic and Atmospheric Administration (NOAA)’s North American Multi-Model Ensemble Project (NMME) https://iri.columbia.edu/our-expertise/climate/forecasts/
  13. VanEngelsdorp, Dennis & Otis, Gard. (2001). Application of a Modified Selection Index for Honey Bees (Hymenoptera: Apidae). Journal of economic entomology. 93. 1606-12. 10.1603/0022-0493-93.6.1606.
  14. Rinderer. (1985).  A Comparison of Africanized and European Drones:  Weights, Mucus Gland and Seminal Vesicle Weights, and Counts of Spermatazoa.
  15. Page, RE., and Guzman-Novoa, E. (1997) The Genetic Basis of Disease Resistance.  In: Honey Bee Pests, Predators, and Diseases. Morse, R.A., Flottum K., (Eds) Medina OH: AI Root Co. pp.469-492.
Participation Summary
2 Producers participating in research

Research Outcomes

5 New working collaborations

Education and Outreach

10 Consultations
3 Curricula, factsheets or educational tools
6 On-farm demonstrations
2 Published press articles, newsletters
5 Webinars / talks / presentations
2 Workshop field days

Participation Summary:

100 Farmers participated
Education and outreach methods and analyses:

To date, five talks have been given to introduce this SARE Project and it’s objectives.  These are being recorded separately elsewhere.  Talks included a farming group on the Hopi reservation near Flagstaff, the president of the Northern Arizona Organic Beekeeping Organization, the Southern Arizona Beekeepers Association, Pima County Office of Sustainability, and the first annual Arizona Honey Bee Festival in Tempe.

We were instrumental in compiling an educational resource for identifying feral Africanized colonies, and situations in which they might be relocated successfully vs. situations where the likelihood of successful live relocation is low.  That resource can be found here (http://southernazbeekeepers.org/resources/honey-bee-safety-africanization/)

These are blank record sheets developed for this grant:

Hive Increase and Requeen Record

Daily Inspection Record 2018

Total Hive Inventory Sheets 2018

Queen Cell Building Record

Harvest & Processing Log


Other general information:

Pesticide-free Honeybee Extermination

Swarm Capture How-To

Managing Heat

Honey Bee Loss Report - San Xavier Coop Farm 032818

12 Farmers intend/plan to change their practice(s)
8 Farmers changed or adopted a practice

Education and Outreach Outcomes

Recommendations for education and outreach:

Difficulties of recording data and affordable methods to automate data recovery into an electronic form via open source software solutions should be developed for sideline level beekeepers is necessary.  It’s important to eliminate manual recording with pen and paper if efficient record keeping and analysis is going to occur.

Honey produced is more an indicator of environmental characteristics, it’s time-consuming to measure, and strength of a hive as measured by number of frames containing bees are more reliable indicators of future productivity given ever-changing levels of forage year on year dependent on rainfall variability.  Historically, attempts to tie honey production to a particular strain were more indicative of the strains ability to efficiently produce a healthy population to take advantage of plentiful resource availability.

As unpalatable as it is to suggest that whole hive euthanization be implemented as a means of dealing with excessively vicious hives, based on our experience this is already something that is occurring in certain instances and is sometimes necessary.  However, in a production setting a more efficient method of doing so while preserving comb resources free of chemical contamination is necessary.

Interesting would be a comparison of "gentle" Africanized stock vs. European stock when it comes to the business of pollination and all the steps involved in accomplishing that monumental year-long endeavor.

12 Producers reported gaining knowledge, attitude, skills and/or awareness as a result of the project
Key changes:
  • Awareness of Africanized and hybrid breeding and production qualities.

  • Awareness of the importance of breeding and ongoing assessment/maintenance of queen pedigree in Africanized regions.

  • Awareness of risk and ways to modify handling and placement of hives in more human populated settings, and around other farm animal husbandry projects.

  • Awareness of the effects of heat on hives in warmer regions where Africanization is prevalent.

  • Ongoing awareness of seasonal effects on honey bee breeding and overwintering.

Any opinions, findings, conclusions, or recommendations expressed in this publication are those of the author(s) and do not necessarily reflect the view of the U.S. Department of Agriculture or SARE.