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Apidologie 38 (2007) 390–410 Available online at:c INRA / DIB-AGIB / EDP Sciences, 2007 www.apidologie.orgDOI: 10.1051 / apido:2007029

Original article

Comparison studies of instrumentally inseminatedand naturally mated honey bee queens and factors aff ectingtheir performance*

Susan W. C

Ohio State University, Department of Entomology, Columbus, Ohio, USA

Received 10 August 2006 – Revised 10 March 2007 – Accepted 7 April 2007

Abstract – Instrumental insemination, a reliable method to control honey bee mating, is an essential toolfor research and stock improvement. A review of studies compare colony performance of instrumentally in-seminated queens, IIQs, and naturally mated queens, NMQs. Factors aff ecting queen performance are alsoreviewed. The collective results of the data demonstrate that the diff erent methodologies used, in the treat-ment of queens, has a significant aff ect on performance rather than the insemination procedure. Beekeepingpractices can optimize or inhibit performance. The competitive performance of IIQs is demonstrated whenqueens are given proper care. The advantage of selection and a known semen dosage can result in higherperformance levels of IIQs.

Apis mellifera / queen honey bees / instrumental insemination / colony performance

1. INTRODUCTION

Honey bee queens mate in flight with nu-merous drones from diverse genetic sources.The ability to control mating has been oneof the most challenging aspects of honey beebreeding. Attempts to mate honey bees in con-finement date back to the 1700’s and remainunsuccessful today. The technique of instru-

mental insemination, developed in 1920’s andperfected in the 1940’s and 1950’s, providesa method of complete genetic control, as re-viewed by (Cobey, 1983; Laidlaw, 1987). To-day, with improvements in instrumentation,the technique is highly repeatable and highlysuccessful.

Instrumental insemination also enables thecreation of specific crosses that do not occur

Corresponding author: Susan W. Cobey,

[email protected] adress: University of California, Davis,Dept. of Entomology, Harry Laidlaw Honey BeeRsearch Facility, one Shields Ave., Davis, CA,95616, USA.* Manuscript editor: Peter Rosenkranz

naturally, providing significant advantages toresearch and stock improvement. For exam-ple, a single drone can be mated to one or sev-eral queens, isolating and amplifying a specifictrait. Varying degrees of inbreeding can be cre-ated to produce diff erent relationships, includ-ing “selfing”; the mating of a queen to her owndrones. Specific backcrosses can be created byextracting semen from the spermatheca of one

queen to inseminate another (Harbo, 1986b).

The ability to pool and homogenized spermcells from hundreds of drones and inseminatea portion to a queen or batch of queens enablesunique mating system designs and simplifiesstock maintenance. Within a closed populationbreeding system, the eff ective breeding popu-lation size, viability and fitness are increasedusing this technique (Page and Laidlaw, 1985).

Another advantage of instrumental insemina-tion is the ability to store and ship honey beesemen. Short term storage has been perfected(Collins, 2000b). The ability to ship semen,rather than live bees, minimizes the risk of spreading pests and diseases.

Article published by EDP Sciences and available at http://www.apidologie.org

or http://dx.doi.org/10.1051/apido:2007029

http://dx.doi.org/10.1051/apido:2007029http://dx.doi.org/10.1051/apido:2007029http://dx.doi.org/10.1051/apido:2007029http://www.apidologie.org/http://dx.doi.org/10.1051/apido:2007029http://dx.doi.org/10.1051/apido:2007029http://www.apidologie.org/http://www.edpsciences.org/


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Performance of instrumentally inseminated queens 391

Over the past 50 years instrumental insem-ination has been utilized in bee research insti-tutions, worldwide, although the commercialindustry has been slow to adopt the technique.A factor in the reluctance of beekeepers totake advantage of this tool is the perceptionof poor performance of instrumentally insem-inated queens. The ambiguity in reported dif-ferences, compared to naturally mated queens,can be explained in the varying treatments andmethodologyused. The objective of this articleis to review comparison studies and the factorsaff ecting queen performance.

Comparison studies, of instrumentally in-

seminated queens (IIQs) and naturally matedqueens (NMQs), date from 1946 to the present.Various aspects of queen performance are re-viewed and compared across studies: colonyproductivity, queen longevity, onset of ovipo-sition, the number of sperm stored in the sper-matheca and rate of success. Factors aff ectingqueen performance are also reviewed: rearingconditions, mating age, treatment of queens,semen dosage and handling, pheromone devel-opment, eff ects of CO

2 treatments and envi-

ronmental conditions.

2. PERFORMANCE COMPARISONSTUDIES

The various comparison studies are sum-marized in Table I and categorized into threegroups based on their findings. Group I showsthe equal performance of IIQs and NMQs,

in six studies, Group II shows higher perfor-mance of IIQs in seven studies and Group IIIshows higher performance of NMQs in onestudy. The diff erent methodologies used in thetreatment of queens across studies are alsocompared; the age of queens at insemination,semen dosage and methods of queen introduc-tion.

2.1. Colony productivity

To properly evaluate and select coloniesfor stock improvement, queens must be capa-ble of heading productive field colonies. Sev-eral studies compared field performance of IIQs and NMQs based on honey production orweight gain and brood production (see Tab. I).

An early study by Roberts (1946) comparedthe performance of queens introduced as pack-age bees and reported higher honey produc-tion in colonies with IIQs (see Tab. I). Queenswere inseminated three times with 2.5 µL of semen and given a direct release introduction.Roberts (1946) attributed the higher perfor-mance of IIQs to selection.

In a similar study of queens introduced aspackage bees, Nelson and Laidlaw (1988) re-ported no significant diff erence between IIQsand NMQs (see Tab. I). The IIQs were insem-inated with 8 µL of semen at 6 to 10 daysold and banked (individually caged in nurs-

ery colonies) for 6 days before introduction.Queen weights were compared when the pack-ages were hived and after establishment. Ini-tially the NMQs were heavier than the IIQsand produced more brood, (see Tab. I). Thesediff erences were attributed to mating time,queen development and the fact that the NMQshad begun oviposition before shipment. Thelower initial weights and brood production of the IIQs were attributed to their not being al-

lowed to begin egg laying before shipmentfrom California to Canada in 2 lb (one kg)packages. As the season progressed, no signif-icant diff erences between the two groups wereobserved in queen weights, brood productionor honey production. During the peak sea-son brood production of the IIQs was slightlyhigher than the NMQs (see Tab. I).

Woyke and Ruttner (1976) summarized theresults of instrumental insemination statingthat most production colonies were headedby IIQs at the Apicultural Research Insti-tute in Oberursel, Germany and performedwell. They reported similar performance of IIQs and NMQs, concluding that IIQs canbe productive for 3 and 4 years. In a 1970study reviewed, colonies headed by IIQs werethe highest producers. The authors (Woykeand Ruttner, 1976) also reviewed a study byRuttner in 1961 indicating similar, althoughslightly lower mean brood and honey produc-

tion of IIQs compared to NMQs (see Tab. I).In reviews of honey production of coloniesheaded by IIQs in the Czech Republic from1961 to 1980, IIQs produced an average of 8% and 12% more than honey NMQs (Vesely,1984; Woyke, 1989c).


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Table I. Summary of Comparison Studies of IIQs and NMQs.

STUDIES COLONY PREFORMANCELONGEVITY

TREATMENT

INTRO. METHODPERFORMANCE No. of QUEENS HONEY PROD. BROOD PROD. OF IIQsAuthor, Year IIQ NMQ IIQ NMQ IIQ NMQ IIQ NMQ AGE / II SemenGROUP IEQUAL PERFORMANCEP ritsch & Bi enefeld, 2002 1105 1114 37.9 kg 38.0 kg

1656 2025 37.4 kg 37.0 kgGerula, 1999 85 45 45.3 kg 50.0 kg 242.35 dm2 221.2 dm2

Cobey, 1998 14 12 109.8 kg 114.6 kg 8.8 Fr 8.6 Fr 18 mo 18 mo 5 d 8 µL DR127.9 kg 142.4 kg 10.4 Fr 10.7 FrVesely, 1984 672 1483 8% more 1yr.50% 1yr.58%

2yr.27% 2yr.15%Nelson & Laidlaw, 1988 19 20 80 kg 70 kg 2074 cm2 2303 cm2 6–10 d 8 µL Bnk 6 d / Pkg

4145 cm2 3998 cm2

Konopacka, 1987 276 285 1st.yr-3.8 r* 1st.yr-4.0 r* 1yr-94% 1yr-98%3rd.yr-3.4 r* 3rd.yr-3.8 r* 2yr-54% 2yr-87%

GROUP IIIIQs HIGHER PERFORMANCETajabadi et al. 2005 5 10 7.8 kg 7.0 kg 3757 cm2 2757 cm2 6–7 d 8 µL Bnk 10 d / nucsČermák, 2004 612 137 21.3 kg 19.4 kg 7–8 d 12 µL DR / nucs

233 50 23.4 mo 21.5 mo 7–8 d 12 µL DR / nucsSzalai, 1995 24 24 Slct 22 kg 17.8 kg 1011 egg / day 735 egg / day

24 Unslct 12.3 kg 631 egg / dayBoigenzahn & Pechhacker, 1993 186 399 Slct 20.5 kg 19.0 kg

46 Unslct 17.9 kgWilde, 1987 16 9 7.0 kg 4.6 kg 18 963 cells 18 343 cells 2yr. II=NM 7–10 d 8 µL Bnk 8-10 d

23 10 15.4 kg 11.8 kg 34 413 cells 21 817 cells Bnk 8–10 dWoyke & Ruttner, 1976 15 72 54 kg 39 kgRoberts, 1946 65 43 95.3 kg 52.6 kg 3 × 2.5 µL DR / PkgGROUP IIINMQs HIGHER PERFORMANCEHarbo & Szabo, 1984 59 59 42.3 kg 75 kg 1840.0 cm2 2782.5 cm2 1st.yr.31% 1st.yr.58% 2–3 wk 2 × 2.7 µL Bnk 2–3wk / Col

Legend: Slct is selected; Unslct is unselected; mo is month;wk is week; d is days; DR is direct release; bnk is bank; Pkg is package bees; nucs is nucleus colony;r is a rating system of 1-4.


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Harbo and Szabo (1984) reported signifi-cantly lower brood and honey production, andreduced survival rates of IIQs (see Tab. I). Thetreatment of queens in this study varied signifi-cantly from other studies. Queens were insem-inated past their receptive mating age, givenseveral small semen doses and banked beforeand after introduction.

In Poland, Konopacka (1987) measuredbrood production in colonies using a ratingsystem; a low of 1 to a high of 4. During thefirst and third year of production the IIQs andNMQs averaged similar ratings (see Tab. I).

Cobey (1998) recorded total colony weights

and measured brood areas during the activeseason and found no significant diff erences be-tween the IIQs and NMQs over two seasons ina study in Ohio, USA (see Tab. I). Colonieswere managed as production hives, and di-vided in spring for swarm control. IIQs wereinseminated at 5 days old with 8 µL. of semenand introduced directly into nucleus colonies.

In a study conducted in Iran, Tajabadi et al.(2005) reported no significant diff erences in

and brood production of IIQs and NMQs,measured at five points in time over the sea-son. The IIQ group produced more honey (seeTab. I). The IIQs were given 8 µL of semen at 6to 7 days old, a second CO2 treatment two daysafter insemination and banked for 10 days be-fore introduction into nucleus colonies. A highloss of IIQs at introduction was attributed to aninsufficient introduction time.

Two studies compared three groups; un-selected NMQs, selected NMQs and se-lected IIQs, showing higher productivity inthe selected groups, regardless of the methodof insemination. Boigenzahn and Pechhacker(1993) reported that IIQs in Austria producedthe most honey, followed by the NMQs matedin isolated mating stations with select drones,followed by the NMQs mated in non-isolatedapiaries with unselected drones (see Tab. I).

Szalai (1995) measured honey productionand egg laying rates of unselected NMQs, se-

lected NMQs and selected IIQs, and reportedsimilar findings in Hungary. The selected IIQswere the top performers, followed by theselected NMQs, followed by the unselectedNMQs (see Tab. I). The authors (Boigenzahnand Pechhacker, 1993; Szalai, 1995) attribute

the higher performance of the selected groupsto selection and the ability to control mating.

Over a three year period in Poland, Gerula(1999) observed the performance of IIQs andNMQs of Apis mellifera caucasica and A. m.carnica and their hybrids. No significant dif-ferences were found between the IIQs andNMQs (see Tab. I). Although, slight diff er-ences observed between the stocks were at-tributed to diff erences in the breeding strainsand flow conditions. Caucasian colonies wereslightly more productive than the Carniolancolonies.

In another Polish study, Wilde (1987) mea-

sured the performance of colonies headed byIIQs subject to varying conditions, over a twoyear period and reported higher production of IIQs compared to NMQs (see Tab. I). Queenswere inseminated at 7 to 10 days old with 8 µLof semen and banked up to 10 days or given adirect release introduction.

More recently, several large data sets col-lected by bee research institutions that rou-tinely use instrumental insemination for stock

improvement programs, report minor or nosignificant diff erences in the performance of IIQs and NMQs. Pritsch and Bienefeld (2002)evaluated honey yields of colonies at the Cen-tral Breeding Evaluation Program in Germany,and report equal or higher yields in coloniesheaded by IIQs (see Tab. I).

In the Czech Republic, also reporting ona large data set, Čermák (2004) found simi-lar results, that IIQs had slightly higher honeyyields compared to NMQs (see Tab. I). The

IIQs were inseminated at the age of 6 to 7 dayswith 8 µL of semen and banked for 10 days be-fore release into nucleus colonies.

The majority of studies observed similar orhigher performance levels of colonies headedby IIQs. The methodology of pre and post in-semination care of queens, compared in Ta-ble I, was similar among the studies in Group1 and II. Queens were inseminated at a youngage, 5 to 10 days old, given semen doses of

7.5 to 12 µL, and introduced directly or withinseveral days post insemination into small nu-cleus colonies or package bees.

The treatment of queens varied significantlyin the Group III, Harbo and Szabo (1984)study, reporting less competitive performance


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394 S.W. Cobey

of IIQs (see Tab. I). In particular, queens wereinseminated at an older age, 2 to 3 weeks,and caged in nursery colonies for another 2to 3 weeks before introduction. Queens weregiven two small semen doses of 2.7 µL Queenswere caged and shipped at the start of egglaying, and introduced into large colonies orpackage bees.

Harbo (unpubl. data) recently repeated thisstudy, changing the pre and post inseminationtreatment of queens. Preliminary results sug-gest findings similar to the majority of stud-ies reviewed above. The diff erent methodologyused in the initial study may have contributed

to the reduced performance of IIQs. The af-fects of the diff erent treatments are discussedin Section 3.

2.2. Queen longevity

The lifespan of the queen must be sufficientto allow time for selection and to ensure breed-ing stock is available for propagation. The pro-cess of evaluation requires field selection at the

colony level, over time. Several studies com-pared queen longevity.Vesely (1984) presented a large data set

on the longevity of IIQs and NMQs recordedfrom 1961 to 1980 at the Bee Research In-stitute Dol in the Czech Republic. Survivalof IIQs was slightly lower in the first year,and decreased in the second year, comparedto the NMQs (see Tab. I). Another data set of queen longevity, reported by (Čermák, 2004),also from the Czech Republic, observed higherrates of survival for both IIQs and NMQs (seeTab. I).

Konopacka (1987) observed the survivalrates of IIQs and NMQs over a 12 year pe-riod in Poland. She reported no diff erences inthe first year, and a higher rate of survival of NMQs in the second year (see Tab. I). The IIQswere given diff erent treatments and high mor-tality of queens due to infection was observedin both groups. The reduced survival of IIQs

in the second year was attributed to excessiveCO2 treatments, two exposures of 10 minutes.Several other studies report no statisti-

cally significant diff erences in the longevityof IIQs and NMQs. Cobey (1998) observedthat both IIQs and NMQs survived an average

of 18 months in production field colonies inOhio, USA. Woyke (1989b) cited a study inPoland by Wilde in 1986, stating both groupshad equal survival rates over a two year period(see Tab. I).

In contrast, Harbo and Szabo (1984) re-ported a significantly lower survival rate of IIQs, compared to NMQs after one year (seeTab. I). A major diff erence in this study wasthe treatment of queens. Queens were insem-inated past their prime receptive mating age,held in banks for several weeks and givensmall semen doses. IIQs stored less spermcells, starting with a mean of 3.2 million

sperm cells, compared to the 5.5 million storedby NMQs. The NMQs had more sperm cellsstored after one year, a mean of 4 million, thanthe number stored initially by the IIQs. Thelow initial sperm cell counts of IIQs in thisstudy may have contributed to their lower rateof survival.

A major component of queen longevity isthe number of sperm cells stored in the sper-matheca. Queens that cannot maintain a large

brood nest during the active season are of-ten prematurely superseded. Many factors in-fluence sperm migration into the spermathecaand the number of sperm cells stored, these arediscussed in more detail in Section 3.

2.3. Onset of oviposition and rateof success

The criteria for the success of instrumentalinsemination are often based upon the onsetof oviposition and queen survivorship to pro-duction of worker brood. Several studies havepresented extensive data concerning these as-pects. Most studies report similar rates amongIIQs and NMQs. There is speculation that thelack of a mating flight may delay queen de-velopment, although the data suggests this isinsignificant. The influence of seasonal andhive conditions plays a more significant rolein physiological developmental diff erences be-

tween the two groups.Woyke and Ruttner (1976) state that ahigher success rate of IIQs, over natural mat-ing, can be achieved with proper insemina-tion techniques. The authors refer to a reportby Mackensen who recorded a 92.5% success


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rate of 759 queens inseminated from 1964 to1966. They also report high success rates inGermany. Of the 1480 queens inseminated atthe Apicultural Research Institute in Kirchhain76.8% began laying in 1972 and 83.5% be-gan laying in 1973. Moritz and Kühnert (1984)also report a success rate of 90% of the 3440IIQs produced from 1972 to 1983 in Germany.Over a 5 year period of an insemination pro-gram supporting a breeding project in Aus-tralia, Kühnert et al. (1989) report a 85% suc-cess rate.

Otten et al. (1998) observed the influenceof both queen age and time of day of insemi-

nation on the success rate to oviposition of 522IIQs in Germany. Queens inseminated 10 dayspost emergence had a rate of 95% success,compared to 82% success rate of 7 day oldqueens and an 80% success rate of 13 day oldqueens. The authors suggest physiological fac-tors change with the age of the queens and alsoduring the time of day queens are inseminated.Afternoon inseminations were found to be op-timal, 92% of queens inseminated between

2PM and 3PM began oviposition as comparedto only 7% inseminated in the morning be-tween 8000h and 9000h.

Onset of oviposition, the time between in-semination and the start of egg laying, is gen-erally considered to be somewhat slower andvary more widely among IIQs compared toNMQs. During the peak season, the majorityof NMQs begin egg laying within 2 to 4 dayspost mating. However, Szabo et al. (1987) re-ported that the onset of oviposition of 1396

NMQs ranged from 4 to 22 days after emer-gence, with a mean of 10 days in Alberta,Canada.

Wilde (1994b) observed queens over a threeyear period and reported that of 59 NMQs thestart of oviposition ranged from 7 to 26 dayspost emergence and of 77 IIQs the range wasfrom was 10 to 37 days post emergence. Theconditions queens were kept influenced re-sults. The IIQs kept with worker bees laid

sooner and stored more sperm cells than thosecaged without bees.In another study Wilde and Bobrzecki

(1994) observed 79 IIQs and 53 NMQs overa five year period and reported some interest-ing results. The NMQs produced slightly more

brood and began laying earlier, although thosebeginning earlier, 11.5 days post emergence,produced more brood than those beginninglater, 16.2 days. The opposite results were ob-served among the IIQs, those that beganovipo-sition later produced more brood than IIQswhich began earlier.

An extensive data set of 1212 IIQs, reportedby Prabucki et al. (1987) from the Bee Re-search Institute in Poland, observed the startof oviposition of queens inseminated at diff er-ent ages, from 6 to 16 days post emergence.Queens were given one semen dose of 8 µL or2 doses of 4 µL. The majority of both groups

started egg laying within 14 days, althoughhigher mortality was observed in the group in-seminated twice. Queens inseminated when 10to 12 days old began egg laying in 6 to 8 days.Queen inseminated at 6 to 9 days old beganlaying in 10 to 13 days. Queens inseminated at14 to 16 days old began laying in 11 days. Theauthors suggest seasonal eff ects and the con-ditions queens where kept also influence theonset of egg laying.

Skowronek et al. (2002)reported on anotherlarge data set of 1289 IIQs in Poland, observedover a six year period. The start of oviposi-tion varied from 3 to 36 days post insemina-tion. Queens inseminated when 5 to 12 daysold started the earliest. The majority of IIQs,67.7%, began oviposition within 6 to 10 daysof insemination, 90.4 % within 15 days and2.9% began within 5 days. The authors alsostate that seasonal conditions are a contribut-ing factor for determining the onset of egg lay-

ing.In addition, Skowronek et al. (2002) ob-

served that the type of brood in colonies in-fluences the onset of oviposition. Queens es-tablished with capped and emerging brood be-gan laying sooner than queens established incolonies with eggs and young brood. Queensintroduced during a nectar flow, warm weatherand low relative humidity also started layingsooner. Cold nights and humidity above 90%

delayed the start of oviposition.A study by Chuda-Mickiewicz et al. (2003)confirms the observation that the onset of oviposition is influenced by the treatment of IIQs. They observed 79 IIQs and reported thatqueens confined to small boxes with small


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drones and store fewer sperm cells. Zamar-liki and Morse (1962) reported that queensprevented from mating for one month did notmate at all or stored fewer sperm cells. To en-sure queen quality, commercial producers rou-tinely discard queens that have not started lay-ing within three weeks due to poor weatherconditions.

To determine the optimal age of queens forinstrumental insemination, Woyke and Jasin-ski (1976) inseminated 237 queens, from 1 to47 days old with 8 µL of semen. Queens in-seminated at 5 to 10 days had similar num-bers of sperm cells stored in their spermatheca

and similar survival rates compared to NMQs.Queens inseminated at younger and older agesstored significantly fewer sperm cells. Youngqueens, inseminated when 1 to 3 days old, hadhigh mortality. Jasnousek (1987) reported thatqueens inseminated at 7 to 9 days old beganlaying the soonest.

Seasonal eff ects influence the timing of nat-ural mating and oviposition. Spring rearedqueens tend to mate more efficiently and be-

gin oviposition sooner compared to fall rearedqueens. Moritz and Kühnert (1984) report thatthe start of oviposition, for both NMQs andIIQs, is delayed late in season. They observed3440 IIQs produced from 1972 to 1983 inGermany and found that spring queens be-gan laying 5.7 days post insemination, andqueens inseminated late in the season began in14.3 days. Jhajj et al. (1992) confirmed thesefindings, stating that queens mate about 5 dayssooner early in the season, and take longer to

begin egg laying, up to 11 days, late in the sea-son.

I have also observed that both IIQs andNMQs reared in the fall season can take upto 3 to 4 weeks to begin laying. Seasonal ef-fects contribute to delayed oviposition and arenot solely based on a lack of available drones.Genetic diff erences also play a role, althoughGuler and Alpay (2005) reported that environ-mental conditions have greater eff ects than the

genotype.

3.3. Eff ects of banking queens

Banking, the practice of confining queensto individual cages and holding these together

in a queenless nursery colony, provides con-venience. A large number of queens can beheld until the proper age for insemination andheld after the procedure until new coloniesare prepared for their introduction. This al-lows flexible scheduling and is labor efficientalthough, does not provide optimal conditionsfor queens.

In a series of studies, Woyke (1988, 1989a)observed that virgin queens confined to cagesin nursery colonies and inseminated past theirreceptive mating age store less sperm, are de-layed in the onset of oviposition and are subject to injury from aggressive worker bees.The practice of banking queens was also ob-served to negatively eff ect queen weight andattractiveness. Szabo and Townsend (1974)observed that heavier queens are more attrac-tive. Confined to cages, queens may not re-ceive adequate nutrition. A protein rich diet isessential for egg development and lack of suf-ficient feeding of protein may contribute to de-layed onset of oviposition. Queens confined tocages after insemination and later introduced

into individual colonies are slower to beginegg laying and store fewer sperm cells (Wilde,1994b). This may also delay pheromonedevel-opment in queens.

Wilde (1994a,b) also observed the eff ect of diff erent conditions in which queens are keptbefore and after insemination; free in nucleiand caged, with or without attendant bees, andcompared these to NMQs in nuclei. The IIQskept with worker bees laid sooner and stored

more sperm cells than those caged withoutbees. During the second season, queens thathad been kept in boxes with 200 worker beesproduced more brood, although the diff erenceswere not significant.

Newly mated queens are very active, run-ning on the comb often bending their ab-domens, which promotes sperm migration.The movement of sperm cells from theoviducts into the spermatheca is a com-

plex process, involving muscular action of the queen and sperm motility (Ruttner andKoeniger, 1971). Active, free movement of thequeen and attendance by workers immediatelyafter insemination helps clear the oviducts of semen and increases the efficiency of sperm


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cell migration into the spermatheca (Woyke,1979).

During mating flights, queens obtain large

quantities of semen from numerous drones.Only about 10% of sperm cells collected inthe lateral oviducts migrate into the spermath-eca and are stored (Koeniger, 1986). Con-finement of queens after insemination reducessperm migration into the spermatheca. Twosets of data of IIQs given 8 µL and caged,stored 2.9 and 2.5 million sperm cells com-pared to 4.2 million and 4.4 million spermcells stored by IIQs allowed free movementamong bees and held in an incubator (Woykeand Jasinski, 1973, 1976). Woyke (1979) re-ported that of a total of 96 IIQs inseminatedwith 8 µL, queens allowed free movementand access to worker bees stored 5.3 mil-lion sperm cells while queens screened with-out bees stored 3 million sperm cells. Woyke(1989b) also reported that IIQs restricted tosmall push-in cages made of excluder materialstored 1.8 million sperm cells, compared to5.2 million sperm cells stored by IIQs allowed

free movement and released directly after in-semination The author states that the qualityof care given to queens during the critical stageof sperm migration can produce better resultsthan a second insemination.

Queens caged after insemination tend toretain semen in their lateral oviducts whichcan be harmful and sometimes fatal (Vesely,1970). Woyke and Jasinski (1978) observedsemen in the oviducts of queens after they be-

gan laying, under certain conditions. Queenstend to retain semen in their oviducts when:established with small populations unable tomaintain brood nest temperatures; inseminatedwith preserved semen or with semen col-lected from older drones, and when insemi-nated late in the season (Vesely, 1970; Woykeand Jasinski, 1978, 1980, 1985; Woyke, 1979,1983, 1989a).

Interestingly, Rhodes and Somerville

(2003) reported that 11.5% of NMQs com-mercially produced in Australia, observed upto the age of 31 days, had semen residue intheir oviducts. They also reported that thiscould be a factor of small nucleus colonypopulations and poor drone quality. In this

study, sperm cell counts were low in bothdrones and the spermatheca of queens.

Queens caged and banked often suff er frominjuries by aggressive workers bees. Work-ers bite the arolium (tarsal pads), tarsi, legs,and sometimes the antennae and wings of caged queens (Jasinski, 1987; Woyke, 1988).Of 354 queens stored for one week in queen-less colonies, 60% were injured. Queens withdamaged or missing arolium do not depositsufficient foot print pheromones that inhibitqueen cell construction and may contributeto supersedure (Lensky and Slabezky, 1981).Furthermore, queens with injured tarsi or legs

tend to fall off brood comb when colonies areexamined.

A study by Gerinsz and Bienkowska (2002)of 225 IIQs and NMQs showed that 55% of queens in both groups sustained injuries dur-ing routine handling. The injury level and su-persedure rates were higher among IIQs, al-though injury did not seem to significantlyaff ect egg laying rates. The rate of superse-dure did increase with the extent of injuries;

damaged arolia verses a paralyzed or missingleg. The authors emphasize that the diff erentmethods of handling of queens, for examplethe number of times queens come into contactwith unfamiliar bees, determines injury levels.

Woyke (1989b) showed that reducing thetime virgin queens are caged before insemi-nation, from 10 to 4 days, reduced injury lev-els from 54% to 0%. The practice of bank-ing queens is a valuable management tool,although the duration of the banking period

should be minimized. Provision and mainte-nance of proper nursery colony conditions isalso an important factor in preventing queeninjuries that may reduce queen performanceand increase supersedure rates.

3.4. Factors aff ecting the numberof sperm stored in the spermathecaof queens

The queen must store a sufficient numberof sperm cells to maintain a populous colonyover her lifetime. The number of sperm cellsstored in the spermatheca is a major factordetermining queen longevity. An insufficient


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number, less that 4 million sperm cells, resultsin premature supersedure (Harizanis and Gary,1984; Woyke, 1989c).

Although, Wilde (1994b) states that queenswhich stored a range of 3 to 5 million spermcells did not aff ect colony productivity.

IIQs are considered to have a tendency tostore fewer sperm cells compared to NMQs,although the numbers range widely in bothgroups. Generally, IIQs store 3 to 6 millionsperm cells and NMQs store 4 to 7 millionsperm cells (Mackensen and Roberts, 1948;Woyke, 1962; Harbo, 1986b). Mackensen(1964) reported that NMQs store an average of

5.73 million sperm cells with a range of 3.34to 7.36 million. Woyke (1962) reported similarfindings, sperm cell counts averaged 5.34 mil-lion with a wider range, 0.69 to 7.92 millionsperm cells. Wilde (1994b) reported a range of 4.1 to 5.9 million sperm cells stored by NMQsand a range of 2.1 to 4.8 million stored by IIQs.However, queens commercially produced andmated naturally in the United States and NewZealand, were reported to store an average

of 4 to 5 million sperm cells (Harizanis andGary, 1984; Van Eaton, 1986; Severson andErickson, 1989).

The efficiency of sperm migration is dosedependent. However, the treatment of queensis a major factor influencing the efficiency of sperm migration into the spermatheca. Mostsperm cells enter the spermatheca in the firstfew hours after insemination, a critical periodfor the queen. The conditions queens are subject to during this period aff ect sperm migra-

tion.IIQs are routinely introduced into small nu-

cleus colonies for the purpose of efficiencyand to increase acceptance. Activity, tempera-ture and hive conditions can enhance or inhibitsperm migration. In a series of experiments,Woyke and Jasinski (1973) observed the af-fect of worker populations on the efficiency of sperm migration in IIQs. They demonstratedthat queens kept at broodnest temperature,

34 ◦

C, stored more sperm cells than those heldat 24 ◦C. An increase in the population in-creased the broodnest temperature and the ef-ficiency of sperm migration.

Small populations of attendant bees andthe consequential low brood-nest temperatures

also reduced the rate of success and delayedthe onset of oviposition of IIQs (Woyke andJasinski, 1980, 1982, 1985, 1990). In a studyof 160 IIQs, inseminated with 8 µL of se-men and introduced into nucleus colonies witha varying number of attendant bees, Woykeand Jasinski (1980) found that queens withmore than 100 worker bees had clear oviductstwo days after insemination and most queenskept with fewer than 100 workers still had se-men in their oviducts after two days. To pro-duce queens that store a sufficient number of sperm cells, Woyke and Jasinski (1982) rec-ommend a population of at least 350 worker

bees is necessary for outdoor nuclei.Queens introduced into larger bee clus-

ters sizes are maintained at higher and moreconstant temperatures, and start egg layingsooner. Woyke and Jasinski (1990) observed70 queens inseminated with 8 µL of semen,7 to 8 days old and established with varyingnumbers of attendant bees. Queens establishedwith 150, 350 and 750 attendant worker beesstarted egg laying in 15, 13.5 and 11.5 days

respectively, with a range of 8 to 24 days.Doubling the number of bees attending thequeen stimulated oviposition nearly a day ear-lier. When established with 9500 worker bees,queens started laying much earlier, in 5 to9 days, averaging 7 days. An increase in theworker population increased the cluster tem-perature contributing to faster initiation of egglaying. Oviposition was accelerated by 1 to2 days with a temperature increase of 1 ◦C.

The size of the colony that new IIQs are

introduced into not only aff ects the numberof sperm cells stored, it also aff ects the rateof acceptance and success. Otten et al. (1998)also reported on the success rate of establish-ing queens in diff erent size populations andshowed results were best when queens wereintroduced into larger nucleus colonies. Theauthors reported that queens introduced intoweak colonies of 200 worker bees had a re-duced success rate: 70% and a 12% queen loss.

In colonies with 1200 workers, nearly 95% of IIQs began oviposition with less than a 1%loss.

Gontarz et al. (2005) observed diff erencesin the thermal conditions of queens held incages with or without attendant bees and


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400 S.W. Cobey

the aff ect on the efficiency of sperm stor-age. Queens re-introduced to cages establishedwith attendant bees after insemination had el-evated temperatures, sometimes above 35 ◦Cwithin the first two hours. The temperatureswere lower when the workers were newly in-troduced, or the queens were virgins. The au-thors suggest that bees caged with the queenbefore insemination had accepted the queenand adjusted to confinement. Queens held atthe higher temperatures stored more spermcells.

The treatment of virgin queens before in-semination appears to play a minor role in per-

formance compared to post insemination treat-ment, provided queens are inseminated duringtheir optimal receptive period. Wilde (1987)studied the performance of IIQs in which thevirgin queens were subject to varying pre- in-semination conditions and found no significantdiff erences in performance levels compared toNMQs. Three groups of IIQs were observed:virgin queens established in nucleus colonieswith excluded entrances; virgin queens cagedwith 200 attendant workers bees; and vir-gin queens caged individually in queenlesscolonies. The IIQs were given six-minute CO2treatments at 5 to 6 days old and inseminatedwith 8 µL of semen at 7 to 10 days old.

3.5. Semen dosage and sperm quality

The mating frequency of NMQs and thenumber of sperm cells stored in their sper-matheca is highly variable. Among drones,including those from the same colony, thenumber and viability of sperm is also highlyvariable (Lodesani et al., 2004). As discussed,environmental conditions as well as the preand post insemination treatment of queens caninhibit or enhance the number of sperm cellsstored. The dosage, quality and handling of se-men are also contributing factors.

To obtain results similar to natural mating,the standard semen dosage for instrumental in-

semination is 8 to 12 µL (Mackensen, 1964;Woyke, 1989c; Vesely, unpubl. data). A slightincrease can be gained by multiple insemina-tions of small doses. Smaller semen doses mi-grate faster (Bolten and Harbo, 1982). For thisreason, Mackensen (1964) recommended two

inseminations of 3 µL and Harbo (1986a) rec-ommended 2 or 3 inseminations of 2 to 4 µLof semen, given a day apart. However, Woyke(1989c) stated that the diff erence between sin-gle and multiple inseminations, of up to 8 µL,were not statistically diff erent. Multiple in-seminations require extra labor and handlingof queens, and may increase the risk of injuryand infection. Currently, the standard practiceis to give each queen one large semen dose.

The efficiency of sperm migration is dosedependent. Larger semen doses, 4 to 8 µL,generally migrate into the spermatheca overa 40 hour period. Small doses of semen mi-

grate faster, 1 to 2 µL in 4 to 8 hours, andare less aff ected by conditions (Woyke, 1983;Woyke and Jasinski, 1985). Although, Harbo(1986a) reported variability in the number of sperm cells stored by IIQs inseminated un-der similar condition and that uniformity in-creased with larger semen doses and multipleinseminations.

Conditions that cause retention of semenin the oviducts can aff ect queen performance.

Vesely (1970) found that queens inseminatedwith 6 µL of semen and caged after the pro-cedure often retain semen in their oviducts.Results greatly improved when queens wereallowed free movement in colonies, well at-tended by worker bees, and maintained atbrood nest temperatures, as discussed above.

The age of the drones is also a factor of semen retention. Woyke and Jasinski (1978)reported that queens inseminated with semenfrom older drones tend to retain semen intheir oviducts, which can cause the death of queens. Queens inseminated with 8 µL of se-men from drones over four weeks old had se-men residue in their oviducts more often thanqueens inseminated with drones two to threeweeks old. Drone semen changes in viscosityand color with age. From young drones, lessthan 2 weeks old, the semen is fluid and light incolor. The semen of drones older than 2 weeksis darker and more viscous.

Another factor aff ecting retention of semenin the oviducts and the number of sperm cellsstored in the spermatheca is the size of theinsemination tip. Bienkowska and Panasiuk(2006) observed 246 IIQs, in which 72.3%,inseminated with smaller tips, 0.16 mm in


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diameter, had cleared oviducts 48 hours af-ter insemination. Of queens inseminated withlarger tips, 0.19 mm in diameter, 50.4% hadcleared oviducts. Queens inseminated with thesmaller tips stored more sperm cells, averaging3.3 million, compared to queens inseminatedwith the larger tips, averaging 2.6 million.

The use of smaller tips, for the collec-tion process, are more sensitive to the semenquality of drones (Bienkowska and Panasiuk,2006). The viscous semen of older drones canbe sticky and more difficult to collect. Themore fluid semen of young drones readilymixes with mucus which can cause plugs in

the smaller tips.How drones are held until maturity can also

aff ect semen collection. Drones tend to driftbetween colonies, therefore to ensure the iden-tification of stocks drones are often confinedinside colonies until mature. This practice re-sults in the buildup of feces in drones, increas-ing the risk of contamination and can causeskin irritation.

3.6. Aff ects of semen handlingand storage

Sperm cells remain viable in the queen’sspermatheca for several years. The spermathe-cal fluid contains proteins, sugars and antiox-idants, and the surrounding tracheal net pro-vides oxygen (Koeniger, 1986; Phiancharoenet al., 2004). The physical and chemical prop-erties of diluents; pH, osmolarity and nutri-ents, and the handling methods can enhanceor reduce sperm cell survival (Verma, 1978;Moritz, 1984; Harbo, 1986b; Collins, 2000a).

The diluent used is more critical when se-men is diluted, mixed or held in storage. ATris buff er solution with a pH of 8.5, whichcontains sugars, amino acids and an antibiotic,is recommended (Moritz, 1984). Nutrient richgreen coconut water also produces good re-sults (Almeida and Soares, 2002). Further re-search is needed to formulate a diluent that

promotes sperm quality and health.Honey bee semen can be held in sealed cap-illary tubes for several weeks at room temper-ature with good success. Almeida and Soares(2002) observed no significant mortality of se-men diluted with green coconut water and held

for two weeks. Locke and Peng (1993) re-ported an initial viability of 87% dropped to78% after 6 weeks of storage. Collins (2000b)reported 70% to 80% viability of semen di-luted with Tris buff er and held at room tem-perature, 25 ◦C, or in a refrigerator at 12 ◦Cfor up to 6 weeks. Skowronek and Konopacka(1983) reported similar findings of semen heldat 12 ◦C for 3 and 6 weeks.

Temperature is an important factor for invitro, short term storage of semen. The opti-mal temperature is 21 ◦C, with a range of 13to 25 ◦C (Harbo and Williams, 1987; Lockeand Peng, 1993; Konopacka and Bienkowska,

1995). Temperatures below10 ◦C or above32 ◦C, and exposure to sunlight result in poorsuccess (Taber and Blum, 1960; Harbo andWilliams, 1987).

The practice of mixing semen from nu-merous drones, mechanically or by centrifu-gation is advantageous for breeding purposes.Sperm tails are long, fragile and processing re-duces viability (Collins, 2003, unpubl. data).Several studies have shown that queens in-

seminated with diluted, mixed and reconsti-tuted semen, were slower to begin ovipo-sition and stored fewer sperm cells com-pared to queens inseminated with fresh, wholesemen (Kaftanoglu and Peng, 1980, 1982;Moritz, 1983; Skowronek and Konopacka,1983). Lodesani et al. (2004) reported that bystirring and pipetting, rather than centrifuging,the mixing of semen was more efficient and re-liable, although queens must be given two 8 µLinseminations of a 1:1 semen / diluent ratio.

Although, queens inseminated with mixedsemen may store fewer sperm cells, theyare capable of heading productive colonies.Collins (2000a) and Lodesani et al. (2004)showed that sperm cells are selected forquality during migration into the spermath-eca. Queens inseminated with 50% live and50% dead sperm cells produced viable layingqueens (Collins, 2000a). Motility, previouslyassumed as a characteristic to measure semen

quality, is not an accurate indicator (Collins,2006).Several studies have shown that survival

rates of queen inseminated with mixed semenwere similar to queens inseminated with wholesemen (Kühnert et al., 1989; Harbo, 1990). In


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402 S.W. Cobey

the Kühnert et al. study semen was diluted,centrifuged and reconstituted and in the Harbostudy semen was diluted 1:1 and mechanicallystirred. Yanke (unpublished data) states thatof the 150 to 250 queens produced annuallyover a fifteen year period and inseminated withdiluted, centrifuged and reconstituted semen,many queens are productive for a year, and afew up to three years.

The long term storage of semen using cyro-preservation techniques is possible, althoughis still in the experimental stage. Advanceshave been made with storage in liquid ni-trogen using various cyroprotectants, such as

DMSO, (dimethyl sulfoxide) and glycerol,with DMSO providing the best results (Harbo,1979a). The critical variables are the rate of freezing and thawing and the diluent used.Harbo (1979b) reported queens inseminatedwith semen stored in liquid nitrogen producedworker progeny, although many queens pro-duced more drone than worker brood and therate of eggs hatch was lower, compared tothe controls. Evidence genetic damage may

have occurred in future generations was alsoreported (Harbo, 1981). Further research isneeded develop techniques for the long termstorage of honey bee germplasm.

3.7. Eff ects of carbon dioxide treatments

Carbon dioxide treatments, used to anes-thetize the queen during the inseminationprocedure, also have the beneficial aff ect of inducing egg laying. CO2 treatments stimu-late the neurosecretory production of juvenilehormone, which contributes to the initiating of oviposition (Mackensen, 1947).

Two CO2 treatments stimulate young IIQsto begin oviposition in a similar time periodas NMQs. One treatment is given during theprocedure; another is given either before or af-ter the insemination. The timing and dosageof CO2 treatments are variable in practice and

may influence queen performance.Mackensen (1947) reported that IIQs lack-ing a second CO2 treatment have a delayed on-set of egg laying of 40 to 60 days. Although,Woyke (1966) reported that IIQs started egglaying in 8 to 12 days without a second CO2

treatment. He attributes this to the older ageof queens at insemination, which were 8 to12 days as compared to 3 to 7 days in theMackensen study. Harbo (1986b) also statesthat older virgin queens, banked for 2 monthsbefore insemination, do not need a second CO2treatment to induce egg laying. Banked virginqueens of this age often lay eggs in their cages.

The eff ects of carbon dioxide treatmentson queens are not fully understood. Workerbees exposed to CO2 have reduced longevity(Austin, 1955) and change their behavior(Skowronek and Jaycox, 1974). Exposure of virgin queens to CO2 treatments is known

to inhibit queen mating flights, cause an ini-tial weight loss and reduce or delay queenpheromone production (Harbo, 1986b; Woykeet al., 1995).

Skowronek (1976) reported that two dayold queens, anaesthetized with CO2 for 10 to30 minutes, delayed their mating flights from9 to 25 days, compared to untreated queenswhich flew on the 6th day post emergence.Treated queens also took fewer flights and

stored fewer sperm cells.The inhibition of mating flights by queensinseminated at diff erent ages, given variedCO2 treatments was also explored by Woykeet al. (1995). In a series of studies the au-thors demonstrated that two inseminations ortwo CO2 treatments prevented mating whenqueens were allowed free flight. The age of queens at insemination was a contributing fac-tor. As the age of queens, insemination with8 µL was increased from 6 to 14 days, the per-centage of natural mating decreased from 69%to 29%. Also, queens did not mate naturallywhen treated with CO2 two days before insem-ination with of 8 µL, and when inseminatedtwice with 4 µL of semen. Of queens insem-inated with of 8 µL. of semen and given twoCO2 treatments two days after insemination,14% mated naturally (Woyke et al., 2001). Of queens inseminated with one dose of 8 µL of semen and not given a second CO2 treatment,

61% mated naturally (Woyke et al., 1995).Harbo and Szabo (1984) reported CO2treatments cause an initial weight loss inqueens, both IIQs and NMQs, 3 to 9 days af-ter the start of egg laying. NMQs were heavierand laid more eggs per day than IIQs. Nelson


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and Laidlaw (1988) also reported weight lossof IIQs at the start of egg laying, NMQs aver-aged 190 mg and IIQs averaged 174 mg. How-ever, after 2 months, both groups of queenswere similar in weight and brood production.They attributed the initial diff erence in weightto the fact that IIQs had not begun egg layingwhen introduced, whereas the NMQs had beenlaying before introduction into colonies.

Jasnousek (1987) reported that reducingthe CO2 treatments from 10 to 1 minute hadno eff ect on the initiation of oviposition of queens. However, Konopacka (1991) statedthat queens given two, 10 minute treatments

laid sooner, in 7 to 8 days, compared to queensgiven two, 3 minute treatments, which beganto lay in 15 days. These diff erences may be dueto other factors such as queen age and seasonalconditions.

Konopacka (1987) attributed a reducedlifespan of IIQs to excessive exposure to CO2yet this was only observed after the thirdyear of production. Queens given two strong,ten minute CO2 treatments lived shorter lives

compared to those given a weak, incompleteanesthetization of less than one minute inwhich queen abdominal movement continued.

Several studies suggest practical recom-mendations. Fischer (1990) showed that mi-gration of sperm cells is more efficient inqueens given a CO2 treatment the day beforeinsemination, rather than the day after the pro-cedure. Sperm cell migration takes about twodays and CO2 exposure during this time mayinterrupt or slow migration and chill queens.

CO2 treatment before insemination also in-hibits natural mating flights and the need to ex-clude nucleus entrances (Woyke et al., 2001).Ebadi and Gary (1980) showed that CO2 treat-ments diluted with air induced earlier oviposi-tion of IIQs and suggest the use of a 75% CO2.mixture.

3.8. Pheromone development and queen

acceptance

The queen undergoes many changes inphysiology and behavior: from a newlyemerged virgin, to mating age, mating, eggmaturation and production. The composition

of queen pheromone changes dramatically be-tween virgin and mated queens. Quantitativeand qualitative changes depend on queen sta-tus, and are unique to each individual (Apoe-gait and Skirkevicius, 1995). These complexchemical blends communicate reproductivestatus of the queen, maintain social cohesion,elicit retinue response and influence the behav-ior and physiology of worker bees. Pheromonedevelopment may be delayed in IIQs com-pared to NMQs and explain why acceptanceof IIQs can sometimes be more difficult.

Workers bees are less attentive to young,virgin queens compared to laying queens. The

presence, concentrations and proportions of queen mandibular gland pheromone (QMP)vary with age, reproductive status, geneticmakeup and seasonal changes (Pankiw, 2004)QMP produced by young, pre mating age vir-gin queens 1 to 2 days old, is diff erent fromthat of virgin queens of mating age, 4 to 7 days,and diff erent from QMP produced by layingqueens. Mature, mated queens have the high-est levels of QMP compared to newly mated

and virgin queens (Slessor et al., 1988, 1990).Pankiw et al. (1996) found that young, un-mated queens produce lower quantity and dif-ferent proportionof components of QMP com-pared to mated queens. Drone laying queenshave been shown to have intermediate quan-tities compared to mated and virgin queens.Pheromone production is complex and notonly a function of age.

Richard et al. (2005) suggests that howwell queens have mated also may aff ect diff er-

ences among pheromone profiles. In prelimi-nary studies multiple mated queens attracteda larger retinue response than single matedqueens.

Another source of queen pheromone, thetergal gland secretions (TGS) localized on thedorsal surface of the abdomen, plays a rolein worker-queen interactions. Workers in thequeen’s court actively lick and palpate the dor-sal abdomen of the queen. The blend of TGS

and QMP maintains and stabilizes retinue be-havior and communicates to the workers thatthe queen has mated (Slessor et al., 1988).

TGS are found on mature queens and varywith age. Young virgin queens do not produceTGS until the mating age of 5 to 10 days old.


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results. All of these aspects can be controlledand optimized, with proper beekeeping prac-tices, to enhance the performance of IIQs.

Some initial diff erences may be observed inthe establishment of IIQs. Mating flights andthe act of mating may stimulate physiologi-cal development that may be slower in IIQscompared to NMQs. These diff erences are mi-nor, and can be minimized with the properinsemination procedures and proper treatmentof queens. These diff erences can be observedduring the introduction of queens and becomeinsignificant as queens are established and lay-ing. An understanding and implementation of

beekeeping practices that promote efficacy of IIQs, as discussed in this review, will producecompetitive performance in such queens, com-pared to NMQs.

In the majority of studies, the advantageof controlled mating demonstrates that theability to select for valued traits can resultin higher performance of IIQs compared toNMQs. IIQs have also been known to surviveseveral years and out live NMQs. Yanke (un-

publ. data) observed, during years of beekeep-ing in the northern extreme of New Zealand,that IIQs are more consistent and often livelonger than NMQs, especially when weatherconditions during the mating period are lessthan optimal. This confirms my own experi-ence in the annual production of several hun-dred IIQs for a breeding program from 1981 tothe present.

Instrumental insemination off ers the advan-tage of mating queens more uniformly. Areview of commercial queen production inAustralia reported NMQs had a wide range of sperm counts (Rhodes and Somerville, 2003).Queens mated late in the season, had lowsperm counts and began laying with approx-imately 2 million sperm cells stored, whilethe average number is 5 to 6 million. Duringthe peak season, 42% of drones had less than0.5 million sperm, the authors (Rhodes andSomerville, 2003) suggest insufficient drones

and low sperm counts may be factors. Spermcell counts of drones are generally 7 to 11 mil-lion per individual (Mackensen, 1955; Ruttnerand Tryasko, 1976; Woyke, 1989c).

The insemination of queens with a knownand uniform semen dosage eliminates the wide

range of mating frequency by NMQs. It hasbeen speculated that queens continue to takemating flights until a sufficient amount of semen is stored. Although, Tarpy and Page(2000) state that there is no control over mat-ing frequency and suggest that the number of available drones, weather and seasonal condi-tions are the major influences.

The ability to increase genetic diversitywithin a colony, beyond what is possiblethrough natural mating, is an advantage of in-strumental insemination. The impressive ar-ray of traits and flexibility in behavior pat-terns displayed by honey bees are due to their

intra-colony genetic variability. Genetic diver-sity enhances colony fitness, enabling honeybees to exploit and survive in wide ecologi-cal ranges, survive extreme climatic conditionsand resist pests and diseases. Several studieshave shown that adaptability, productivity andsurvivability tend to be greater in out-crossedstocks (Fuchs and Schade, 1994; Palmer andOldroyd, 2000; Tarpy and Page, 2002).

Another advantage of instrumental insem-

ination is the ability to isolate a specific traitand eliminate the complexity of genetic back-ground that masks the expression of a particu-lar characteristic. With the sequencing of thehoney bee genome, this will become an in-creasingly valuable research technique.

Applied to stock improvement, instrumen-tal insemination provides an essential tool todevelop commercial stocks that have bettersurvival in the presence of parasitic mitesand disease, as well as enhance traits suchas productivity, good temperament and over-wintering ability.

The data presented in Table I overwhelm-ingly demonstrates that instrumental insemi-nation is a reliable and practical technique.However, beekeeping practices designed to re-duce labor, increase efficiency and provideconvenience in scheduling, often provide sub-optimal conditions for queen development andsperm storage in the spermatheca. The re-

ported lower performance levels of IIQs canprobably be attributed to such factors. Withthe ability to control mating, IIQs generallyhave been selected for superior genotypes,which may mask the possible disadvantagesof instrumental insemination concerning their


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406 S.W. Cobey

performance. The unfounded reputation forpoor performance of IIQs has been difficult todispel, although the scientific community hasrepeatedly demonstrated their equal and some-times higher performance compared to NMQs.

ACKNOWLEDGEMENTS

I thank Susan Fisher and Timothy Lawrence forcritical review of this manuscript.

Études comparatives sur des reines d’abeilles

après insémination artificielle ou accouplementnaturel et facteurs agissant sur leurs perfor-mances.

Apis mellifera / reine d’abeilles / insémination ar-tificielle / performance de la colonie

Zusammenfassung – Vergleichende Untersu-chungen an instrumentell besamten und natür-lich begatteten Honigbienenköniginnen und zuFaktoren, die deren Leistungsfähigkeit beein-

flussen. Die instrumentelle Besamung ist eine zu-verlässige Methode, um die Paarung bei Honigbie-nen zu kontrollieren. Sie bietet damit ein unver-zichtbares Verfahren für die Forschung und die Bie-nenzucht. Dieses Review der Forschungsarbeitenvon 1946 bis heute vergleicht die Leistung von Bie-nenvölkern mit instrumentell besamten Königinnen(IIQs) und natürlich begatteten Königinnen (NM-Qs) sowie Faktoren, von denen die Leistungsfähig-keit der Königinnen beeinflusst wird.In den Studien wurden verschiedene Leistungspara-meter der Königinnen verglichen: Produktivität desBienenvolkes, Lebensdauer der Königin und Auf-bewahrung der Spermien. In Tabelle I sind die Er-gebnisse dieser Studien in Gruppen zusammenge-fasst: Gruppe I mit 6 Untersuchungen zeigt gleicheLeistungsfähigkeit von IIQs und NMQs; Gruppe IImit 7 Untersuchungen zeigt eine höhere Leistungbei IIQs; Gruppe III enthält eine Studie mit höhererLeistung bei NMQs.Eine detaillierte Analyse dieser Studien zeigt ein-deutig, dass die Leistungsfähigkeit der Königinsignifikant von der Durchführung der Besamungabhängt. Die Ergebnisse in Gruppe III können dem-nach auf unterschiedliche Behandlung der Königin-

nen zurückgeführt werden. Die IIQs in den Grup-pen I und II wurden in einem Alter zwischen 5 und12 Tagen mit einer Samenmenge von 8–12 µL be-samt. Diese Königinnen wurden in Jungvölker oder„package bees“ eingeweiselt, ohne zuvor über län-gere Zeit gekäfigt in weisellosen Völkern aufbe-wahrt worden zu sein. In der Gruppe III wurden die

Königinnen dagegen in einem Alter von 2–3 Wo-chen mit einer relativ geringen Samenmenge (zwei-mal 2,7 µL) besamt und zusätzlich über 2–3 Wo-chen in anderen Völkern aufbewahrt, bevor sie in

größere Bienenvölker oder „package bees“ einge-weiselt wurden.Die geringe Anzahl an Spermien sowie die gerin-gere Produktivität und Überlebensraten der IIQsin Gruppe III kann ebenfalls mit der verwendetenMethode bei der Besamung erklärt werden. WennKöniginnen vor dem Zeitpunkt ihrer ersten auf-nahmefähigen Paarung besamt werden, speichernsie weniger Samen. Das Käfigen nach der Besa-mung reduziert ebenfalls die Speicherfähigkeit fürdie Spermien und führt zudem häufig zu Verletzun-gen der Königin durch aggressive Arbeiterinnen.Bienenköniginnen durchlaufen dramatische physio-logische Veränderungen während der Vorbereitungzur Eilage. Viele Faktoren beeinflussen diese Verän-derungen und damit auch die Leistungsfähigkeit derKönigin. NMQs, die sich frühzeitig paaren, sich freibewegen können und gut von Arbeitsbienen ver-sorgt werden, paaren sich mit mehr Drohnen undspeichern mehr Spermien.Es wurden einige geringe Unterschiede zwischenIIQs und NMQs beobachtet. So können die höhe-re Variationsbreite beim Beginn der Eiablage sowieeine langsamere Produktion des Königinnenphero-mons das Einweiseln von IIQs erschweren. Diese

Unterschiede minimieren sich aber bei einer gutenimkerlichen Praxis.Andere vom Imker zu verantwortende Faktoren wiedie Art der künstlichen Besamung und die Behand-lung des Samens beeinflussen ebenfalls die späte-re Leistungsfähigkeit der Königin. Die imkerlichePraxis kann somit die Leistungsfähigkeit der Köni-gin verbessern oder verschlechtern. Es konnte aberklar gezeigt werden, dass bei guter imkerlicher Pra-xis die Leistungsfähigkeit von IIQs und NMQs ver-gleichbar sind. Dieses Review soll den Imkern Ver-trauen in die instrumentelle Besamung geben unddarüber hinaus zeigen, welche methodischen De-

tails dabei die Leistungsfähigkeit der Königinnenverbessern können.

Apis mellifera / Honigbienenkönigin / künstlicheBesamung / Leistungsfähigkeit des Bienenvolkes

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conut water and diff erent tissue culture media forin vitro honey bee semen storage ( Apis mellifera),Interciencia 27, 317–321.

Apoegait V., Skirkevicius A. (1995) Quantitative andqualitative composition of extracts from virginand mated honey bee queens ( Apis mellifera L),Pheromones 5, 23–36.


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Austin G.H. (1955) Eff ects of carbon dioxide anesthe-sia on bee behavior and expectation of life, BeeWorld 36, 45–47.

Boigenzahn C., Pechhacker H. (1993) Über die Art der

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