New World Carniolan Stock Maintenance Protocol

In May 2003 Ed completed Sue Cobey’s Queen Rearing course at the University of Ohio. At the time Sue had uploaded her New World Carniolan Stock Maintenance Protocol to her web site. Sue is now at Washington State University.  I think it is important that new beekeepers see this protocol.  Sue’s current web site is




The NWC stock selection and maintenance protocol is described here for two basic purposes.

1.  To give beekeepers interested or using NWC queens an overview of the program.

2.  To provide a working model for beekeepers interested in establishing their own CPBP.

The Closed Population Breeding Porgram, CPBP is a practical and flexible breeding scheme. This involves maintenance of a large and dynamic population in which the desirable characteristics of the stock are continually selected and improved over time.

A genetically diverse Carniolan gene pool is maintained and subject to an annual evaluation program.

Controlled mating utilizing instrumental insemination maintains the purity of stock. Annually, a new generation of several hundred NWC queens are reared, instrumentally inseminated, established in full size colonies and evaluated in the field. With the challenge of parasitic mites and new pests, selection for specific traits that will contribute toward reduction of diseases and infestations are included in the selection criteria.

The selection index and evaluation methods described have been used since 1982. The success of this program is the long term commitment to an annual rigorous evaluation program and careful record keeping. The theoretical model of the Page-Laidlaw closed population breeding program predicts a 20 year life (starting with a base of 50 breeders) before signs of inbreeding become apparent. Therefore, to ensure the longevity of our program we maintain a large base population of 200 colonies at Ohio State University. This population has been expanded by Cooperating NWC Members-Producers. Occasionally we test new sources of Carniolan stock for potential inclusion into the gene pool. These sources are subject to the same rigorous evaluation process before inclusion into the gene pool. Care is taken to do this slowly and cautiously to prevent unforeseen changes in stock performance.

FIELD EVALUATION AND SELECTION PROTOCOL FOR PERFORMANCE Selection is based upon colony performance. The interaction of genetic makeup of the colony, the environmental influences and beekeeper management practices are considered. To observe genetic variability and effectively make selections based upon this requires that environment factors and management practices be as uniform as possible. All colonies are treated equally regardless of their performance. Colonies being evaluated are not equalized or boosted with the addition of brood or bees. Colonies that do not meet the high standards for performance are weeded out of the program.

Selection is based upon behavioral performance at the colony level. A colony has an intricate social structure of many subfamilies displaying a variety of traits. The specific characteristics or combination of characteristics that are responsible for high performance are not specifically and individually identified. Our goal is selection for general overall performance. Choosing the top performing colonies allows us to select for the combination of characteristics responsible for high production without the detailed and tedious measurements of the specific traits responsible.

A two phase approach to selection is used. The first phase, referred to as Pre-selection, is designed to evaluate general performance. At this stage poor and mediocre colonies are cut from the program.

The Pre-selection test uses a selection index to evaluate several traits efficiently in the field. This test is performed twice, once in the fall after colonies have become well established and a second time during the early spring to test buildup and overwintering ability. Colonies are also monitored for parasitic mite levels and tested for hygienic behavior.

Colonies scoring average or above in the Pre-selection tests qualify to enter the second phase of selection. This phase involves a weight gain test designed to determine colony industry.

The weight gain test is conducted during the late spring or summer honey flow. The top scoring colonies are then selected as breeders for the following season. Virgin queens and drones are reared from these selected colonies and crossed to establish the next generation, and the cycle is repeated.

Our evaluation and scoring methods are intended for commercial use and designed to be simple, efficient, and provide an accurate overview of general performance. In this way we can minimize labor and easily identify the top performing colonies of a large population. If this process becomes too cumbersome, it will limit the number of colonies you can evaluate and the likelihood of completing this task.


The pre-selection process consists of observing several traits at a given time and ranking each of these with a numerical point value, scoring the occurrence of each valued trait. The point values of several traits are added and the sum of the total compared among colonies.

Several traits are observed quickly and simultaneously, requiring only minutes per colony. A point value system of 1 to 5 is used, 5 being the highest value. A score is given for each trait observed and these are added to determine colony rank. Scoring is based upon comparison between colonies. Colonies are evaluated at the same time, by the same person to maintain consistency. When colonies are located in different apiaries, a yard average is used to compare scores between these. Colonies scoring average or above qualify for the second phase of evaluation.


The index for Pre-selection includes observing: brood viability, temperament, colony buildup, pollen collection and any incidence of disease. A point value of 1 to 5 is used:

5 – Excellent 4 – Good 3 – Average 2- Fair 1- Poor 0 – Unacceptable

Negative point values are given for the incidence of disease which almost always eliminates this colony from the program.



Selection for high brood viability is essential to maintain the genetic integrity of the population. An estimate of brood viability is determined by observing the solidness of the capped brood pattern. Several frames are scanned, as one may appear spotty due to nectar or pollen in the broodnest. Less than 80% brood viability is unacceptable.


The bees should be calm and gentle regardless of colony size and conditions. Temperament is noted during all manipulations, as this will help eliminate a reaction to any disturbance that may have unknowingly occurred before evaluations.


An estimate of population size is made by counting the number of frames covered with bees. A comparison of colonies for early spring buildup is very telling. Poor buildup is often an indicator of other problems, such as tracheal mites.


A scan of pollen stores, noting the amount and placement of pollen is taken. A rainbow of pollen surrounding the brood is most desirable.


Several frames of brood are scanned for any evidence of disease, such as PMS, AFB, evidence of Nosema, etc. Any disease or abnormality is noted and almost always eliminates this colony from the program. Minor incidences are given a negative point value, so that to survive the scoring process this colony must be exceptional in other areas.


Color is used as an indicator. The traditional dark color is well established in the NWC population. Color of the queens will range from almost uniformly dark to stripes with dark dots down the dorsal (top) abdomen. The ventral abdomen (belly) is generally lighter in color. The drones should be uniformly dark with tan to grayish thoracic hairs.


This is an example of how the NWC breeder colonies are scored. We will follow 10 colonies through the evaluation process.

 This is the fall Pre-Selection score sheet for breeder colony B96. There are a possible 20 points, 5 for each trait, of which this colony scored 15. Note that B96 is given a negative point value for chalkbrood (CHB). The incidence of any disease is of concern and is weighted heavily or the colony simply eliminated from the program. B96 will be evaluated again in spring, with a careful eye out for CHB. As an example, we will follow her score throughout the evaluation process.


The NWC breeding population at OSU has not been treated for tracheal mites. Without knowing the

mechanisms of resistance, it has been assumed that choosing the top performers would include these traits. We did experience heavy losses in the early 1990’s when mite levels of untreated colonies approached 50%. Levels have dramatically declined annually since 1994. Early spring mite loads of untreated, overwintered colonies

have remained low, averaging between 2% and 3% from 1994 to 1998. In 1999 levels averaged 7%, a slight increase, though a noticeable reduction in performance was not detected. We are continuing to monitor mite levels to ensure this trait is maintained.


Mite samples are taken from the hive entrance and inner cover to increase the likelihood of sampling older infested bees. We use a”bee vac”, a small hand vacuum cleaner modified with PVC pipe and an attached sample jar. Samples of over 50 bees are collected from each colony and frozen for later dissection. The trachea of 50 bees per colony are examined and the mite prevalence determined.

Note that timing is critical in sampling. Spring samples must be taken very early in spring, with the start of brood rearing. A large emergence of host young bees will dilute tracheal mite levels. Fall sampling is done when the weather turns cold and brood rearing is minimal.

Selection Score sheet

This score sheet of 10 potential breeder colonies represents the fall and spring pre-selection scores, plus tracheal mite (T.mite) data and two tests for hygienic behavior (hyg). Breeder B96 scored 15 and 14 in her Pre- selection tests, giving her a score of 29 of 40 points. The incidence of chalkbrood was not observed in her

spring test, though is noted in the comment section. Also noted here are specific traits of particular colonies, the high brood viability (BV+) of breeder R8, the runniness of G10, high pollen hoarding ( P+) of B97 and the two colonies testing hygienic, B98 and R3.

Colonies scoring below 35 of 40 points (20 fall plus 20 spring) which includes B96, G10, and R7 have been culled from the program at this stage. For this reason, they have not been tested for hygienic behavior. A hygienic colony that does not perform well in the field will not have commercial value. Note that G10 CJ is an experimental queen in which tracheal mites, runniness and a low score are noted. This colony does not meet NWC standards and therefore will no longer be considered for inclusion in the program. Marginal colonies are culled at this stage.



Hygienic behavior has been shown to reduce the incidence of disease and Varroa mites. For this reason, this specific test has recently been added to our selection criteria. Colonies are first tested for performance during the Pre-selection phase. Those scoring average or above are tested for hygienic behavior. Of the 10 colonies tested here, only 2 consistently display this trait.

This is a common trait found in about 10 % of European honey bee populations. It is controlled by two recessive genes, one for uncapping brood and one for removal. Because this is a specific trait, colonies should first be selected for general performance to ensure their productivity.

Colonies must be tested twice for hygienic behavior because results can be variable due to colony conditions, nectar flows and environmental influences. During a honey flow or when there is a large emergence of young nurse bees, colonies will appear more hygienic. Only colonies consistently displaying this trait are considered hygienic.

A frozen brood assay is used. We use the liquid nitrogen method, as this simplifies some steps. The cut comb method and liquid nitrogen methods are described here. Both systems can be problematic, so attention must be paid to the following procedures.



1.  Cut a two inch by two and a half inch section of sealed brood out of a comb. This is about 100 cells on one side. Cut sections individually from each colony or cut up a whole frame of brood and use the sections to test several colonies. A template is helpful to mix and match sections.

2.  Freeze the brood sections for 24 hrs., not less.

3.  Place the brood section back in the comb while it is still frozen. Estimate the number of sealed cells to get an idea of how many you begin with, though don’t count the edge cells, especially those not completely intact. The bees usually clean these out very quickly initially.

4.  After 48 hours, estimate the percentage of cells that have been cleaned out. For our purposes a visual estimate is sufficient. Select colonies that clean out more than 80% of the cells in 48 hrs. Give these colonies a second test. Those testing with over 80% removal on both tests are considered hygienic


When using the cut comb method, a good score can sometimes be due to some irregularity the brood section. A comb section that is broken or smashed, or not set flush in the brood frame or too thawed out with sunken cappings will cause questionable readings. In these cases the bees tend to clean out the cells fast and this gives a false reading.


If you have access to N2, this method is a labor and time saver and eliminates cutting the comb.

1.  A soup can with the top and bottom cut out works well. Use a 3″ diameter can which is about 111 brood cells. Sharpen one end of the can so this cuts easily into the comb. A piece of 3″ diameter dryer duct cut into 6 ” lengths can also be used.

2.  Ream the can into the comb over newly capped brood.

3.  Pour 8 oz of N2 into the cylinder and wait for this to thaw (5 min.). Note, if an insufficient amount is used the brood will not be killed and will emerge. It is best to repeat this process to ensure the brood is killed.

After 48 hours, estimate the percentage of cells that have been cleaned out. For our purposes a visual estimate is sufficient. Select colonies that clean out more than 80% of the cells in 48 hrs. Give these colonies a second test. Colonies that have uncapped and removed over 80% of the dead cells within 48 hrs. in two tests are considered hygienic. As you increase the frequency of this trait, your criteria can become more strict, example:

85% removal over 24hrs.


When using liquid nitrogen method choose young, newly capped cells. The older pupae are very hardy and can survive severe, short term chill.


The second phase of selection is the weight gain test, indicating colony industry. Colonies scoring average or above based upon the fall and spring Pre-selection tests are given the weight gain test. Colonies are tested during the honey flow in late spring or summer dependant upon the flow conditions. Short term weight gain during the flow is correlated to colony performance over the entire season. This will provide a good estimate of colony industry.

Colonies are initially weighed at the start of a honey flow. A week or few days into the flow, colonies are again weighed. Estimate of colony weight is easily obtained by lassoing the bottom cleat of one side of the colony and tipping it upward with a scale attached. The other side is than weighed in the same manner and the two sides added together to give the total weight of the colony. A scale attached to a bee boom can also be used. The percent weight gain and total weight gain are calculated.









Of the original 10 colonies, only 7 have gone into the second phase of evaluation. Colonies B96, G10 and R7 have been culled.



The final colony score-sheet ranks potential breeder colonies making it easy to choose the top performers. Note that selection for specific traits, tracheal mite resistance and hygienic behavior are also noted. A higher percentage of drones from the Breeders scoring high in these areas are used to increase these traits in the entire gene pool.







For simplification of record keeping to determine colony rank the following formula is used.

Pre-selection Score X Percent Wt Gain + Mite Score + Hygienic Score = Rank

For example B97 scored 39 Pre-selection points of a potential of 40 and had a weight gain of 54%.

39 X 54 = 21.1 Her score for T. mite resistance was 10 and for hygienic behavior was 0. These scores are added

39 X 54 = 21.1 + 10 + 0 = 31.1. Here final rank is 31.1

Colonies with a rank of over 30 points are selected as breeders to establish the next generation. Of the 10 colonies tested here, 7 made it to the weight gain test, and of these 4 have been selected. Colony headed by Queen R2 which had a score of 27.9 is close to the cut off point and may get a second look. This is where the comment section can have value in making a decision.

The annual cycle of establishing and evaluating a new generation is continued. From the breeders selected, virgin queens will be reared and inseminated to a pool of drones from these same colonies. Using this system, a large gene pool is essential. If you are using this system and have a population smaller than 50 genetically diverse breeding colonies, you will need to make some modifications to avoid inbreeding. The value of a breeding program is in the long term selection and demonstration of the heritability of traits you are selecting in the population.




Scoring is based upon the comparison of colony performance. To observe genetic differences, queens of similar age must be established in colonies of equal size in the same environment and managed in the same manner.

If breeders are established in different yards environment influences will vary between these yards and scores cannot be adequately compared. In this situation, calculate a yard average and choose the top performing colonies of each yard, rather than comparing these across the board.


The scoring system presented here is based upon a simple selection index with values of 1 to 5. A specific trait of concern can be weighted more heavily to give this priority attention. For example, if there is a concern that the stock is becoming more defensive, a point value of 1 to 10 can be used to increase the selection pressure for this trait. Remember that your main focus is high overall performance. Concentrated attention on a specific trait may eliminate the complexity of traits that make colonies productive.