INSTRUMENTAL INSEMINATION

OF

QUEEN BEES

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AGRICULTURE HANDBOOK NO. 390

AGRICULTURAL RESEARCH SERVICE

UNITED STATES DEPARTMENT OF AGRICULTURE

IN COOPERATION WITH

LOUISIANA STATE UNIVERSITY
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INSTRUMENTAL INSEMINATION

OF

QUEEN BEES

OTTO MACKENSEN AND KENNETH W. TUCKER

ENTOMOLOGY RESEARCH DIVISION

Agriculture Handbook No. 390

Washington, P.C. December 1970

For sale by the Superintendent of Documents, U.S. Government Printing Office

Washington D.C. 20402 — Price 25 cents
About the handbook

Control of mating

Development of instrumental insemination

CONTENTS

Pasre

1

Structure and function of the reproductive organs 2

Female reproductive organs 2

Male reproductive organs 3

Natural mating g

Equipment for instrumental insemination 7

Microscope and light Y

Carbon dioxide equipment 7

Queen holder 7

Syringe g

Sting hook and ventral hook 9

Valvefold probe and sting depressor 12

Manipulating apparatus 12

Use of carbon dioxide 12

Insemination procedure 14

Preparing the anesthetic 14

Preparing the syringe 14

Preparing the queen 15

Ejaculation of drones 15

Filling the syringe 16

Injection 16

Cleaning and sterilizing the syringe tips 17

Disease as a factor in insemination I7

Estimating the number of sperm in the spermatheca 18

Laidlaw apparatus and method 19

Age of queen for insemination 21

Procedure for various objectives 21

Storing and shipping semen 22

Maintenance of queens 23

Maintaining queens in nuclei . 23

Maintaining queens in nursery colonies 24

Rearing and maturing drones 24

Collection of drones for insemination 26

Control of parentage 27

Bibliography 27

Trade names are used in this publication solely for the purpose of

providing specific information. Mention of a trade name does not constitute a guarantee or warranty of the product by the U.S. Department

of Agriculture or an endorsement by the Department over other

products not mentioned.

IV
INSTRUMENTAL INSEMINATION

OF

QUEEN BEES

By Otto Mackensen and Kenneth W. Tucker, Research Entomologistsj

Entomology Research Division, Agricultural Research Service

ABOUT THE

This manual is intended to introduce instrumental insemination of honey bees to beekeepers, scientists, and technical workers in apiculture who want to learn the technique. A prior

knowledge of bees, bee equipment, management

procedures, and queen rearing is desirable for

the student. However, an untrained person can

master instrumental insemination and gain adequate experience in rearing queens and drones in

CONTROL

The purpose of instrumental insemination in

honey bees is to control matings. Since queens

in nature mate away from the hive and in free

flight, natural mating cannot be controlled except where one has absolute control of the

drones flying in the area in which the queen

mates. The flying drone population can be controlled only where complete isolation from other

HANDBOOK

a relatively short time, under the guidance of

one who is already experienced.

This publication brings together current information on instrumental insemination in

easily readable form. This information has been

gathered from the technical literature, the most

important of which is listed under "Bibliography," on page 27, for those who want to study

the subject in detail.

OF MATING

bees can be achieved, as on small islands and in

alpine meadows. The control of mating by confinement has been tried many times without

repeatable success. Queen and drones do not

mate within the hive, and most attempts to get

queens to mate in enclosures have failed. By instrumental insemination any desired matings

may be made.

DEVELOPMENT OF INSTRUMENTAL INSEMINATION

Instrumental insemination of queen bees did

not succeed with the first attempt ; rather it was

paced by man's understanding of the structure

and function of the reproductive systems of

queens and drones. The first attempts at insemination, more than 180 years ago, probably

failed because the mucus of the drone's ejaculate

was thought to be semen. Not until 1920 was

enough known of the reproductive organs of

bees to start the development of a workable

technique for instrumental insemination.

The techniques now employed had their beginnings in the late 1920's and early 1930's. In

these first approximations, semen from one or

two drones was taken up into a fine syringe and

then part of the semen injected into a queen's

vagina. Typical results were some partial inseminations: queens, tardy to begin laying, that

produced at least some fertilized eggs. But, occasionally, inseminations were good enough to

encourage further effort.

By the late 1930's it was recognized that the

semen was not placed deeply enough into the

queen's reproductive tract. Obstructing further

penetration is a tonguelike structure, the valvefold. When it is moved out of the way, the

syringe tip can be placed beyond it, and the

semen discharged into the median oviduct. Placing the semen beyond the vagina and into the

oviducts led to greatly improved results from

instrumental insemination, even though partial

inseminations were still too frequent, and

queens were usually tardy to begin to lay.

It was soon discovered, during the early
AGRICULTURE HANDBOOK 390, U.S. DEPT. OF AGRICULTURE

1940's, that partial inseminations could be almost eliminated by injecting a larger volume of

semen. Since that time, the semen of three to

10 drones per insemination has been used with

consistently good results.

The problem of tardy onset of laying was

solved about the same time. It was discovered

that anesthesia of queens with carbon dioxide

would hasten the onset of laying, not only of

inseminated queens, but also of virgin queens.

This discovery fits in well with insemination

technique, because carbon dioxide is used to

keep the queens still during instrumental insemination.

Along with the advances in technique, the

equipment used for instrumental insemination

was developed and improved into the two types

of apparatus now used. Early means of holding

STRUCTURE AND FUNCTION OF

A knowledge of the reproductive organs of

queens and drones is a basic requirement for

anyone learning the technique of instrumental

insemination, just as it was for those who developed the technique. The information presented here should fill this need.

The reproductive organs of bees are located in

their abdomens, along with several other organs

concerned with other bodily functions such as

digestion and breathing.

Female Reproductive Organs

The female reproductive organs are illustrated schematically in figure 1. Figure 2 shows

a posterior exterior view of the genital opening

with the sting chamber opened as in preparation for insemination.

A large part of the abdomen is occupied by

two ovaries (0), each consisting of long parallel

egg tubules. Each ovary leads posteriorly into a

lateral oviduct (LOD), which has accordion

walls permitting great expansion for temporary

storage of semen in newly mated queens and of

mature eggs in laying queens. The two oviducts

join posteriorly to form a short passage, the

median oviduct (MOD), which in turn leads into

the vagina (V). In instrumental inseminationn

the end of the syringe must pass through the

vagina and be placed in the median oviduct and

the queen still by tying her with thread to a

wooden support was supplanted by queen holders of the tubular or clamp type in which the

queen is also anesthetized. The technique of

holding the sting chamber open and the sting

from over the opening to the vagina by handheld forceps was replaced by using hooks manipulated through friction mounts or by rack

and pinion. For collecting and injecting semen,

the screw type syringe with a fixed glass tip and

a plunger extending into the tip has been modified into a disassemblable metal syringe with a

plastic tip and a plunger extending not into the

tip but pressing against a rubber diaphragm,

which activates a column of saline solution

within the tip. A probe, designed especially to

move the valvefold, has been used since the importance of the valvefold was recognized.

THE REPRODUCTIVE ORGANS

the semen discharged into the two lateral

oviducts.

Above the vagina lies the spermatheca (SP),

a thin-walled spherical structure about 1 mm. in

diameter, which is filled with a clear liquid in

virgin queens. It is covered with a network of

tracheae, which gives it a white appearance. It

is in the spermatheca that the sperm are stored

for the life of the queen. The spermathecal duct

(SPD) leads from the spermatheca to the dorsal wall of the vagina.

A tonguelike structure, the valvefold (VF),

projects into the vagina from its ventral wall.

This structure can act as a valve to close the

opening to the median oviduct when forced anteriorly against the forward wall of the vagina.

It has transverse ridges which make it recognizable at times when it is exposed during instrumental insemination.

The vagina opens through the vaginal orifice

(VO) into the anterior part of the sting chamber, the bursa copulatrix (BC), which is set off

from the remainder of the sting chamber by a

transverse fold in the ventral wall. In figure 2

this fold is stretched to form a triangle with the

sting as its base. To either side of the vaginal

orifice are two openings (BPO) that lead into

a pair of pounches, the bursal pouches (BP).

These openings can be mistaken for the vaginal
INSTRUMENTAL INSEMINATION OF QUEEN BEES

ST

LOD SPD MOD / V I BP\ ßC

VF VO BPO

FIGURE 1.—Reproductive organs of the queen in approximately their natural position in the abdomen, with left side

of vagina and bursa copulatrix cut away: BCy bursa copulatrix; BP, right bursal pouch; EPO, opening to bursal

pouch; LOD, lateral oviduct; MOD, median oviduct; O, ovary; Ä, rectum; SCH, sting chamber; SP, spermatheca;

SPD, spermathecal duct; ST, sting; V, vagina; VF, valvefold; VO, vaginal orifice.

orifice during instrumental insemination if the

queen is not properly positioned.

In figure 2 the sting chamber is held open as

in instrumental insemination by a ventral hook

(VH) placed over the last ventral plate (VP) of

the abdomenal exoskeleton and a sting hook

(STH), which fits between the bases of the

lancets of the sting (ST). Here the reproductive

tract has been compressed, the bursa copulatrix

opened wide to expose the vaginal orifice, and

the vagina collapsed so that the valvefold lies

immediately inside the vaginal orifice and

occasionally is visible without any probing.

Male Reproductive Organs

The male reproductive system is illustrated

schematically in side view in figure 3, with the

right-hand members of the paired structures

not shown. Its principal paired structures are

testes, vasa deferentia, and mucous glands. The

ejaculatory duct and penis are single structures.

In a young drone the testes ar.e very large,

slimy appearing organs that occupy almost the

entire upper half of the abdomen. They gradually shrink to small greenish-yellow structures

in the sexually mature done. A testis consists of

small tubules which empty into a chamber at

the end of the vas deferens. The vas deferens

has a coiled section where it joins the testis

and a long enlarged section, the seminal vesicle,

which joins and empties into the lower end of

the corresponding mucous gland. The two

mucous glands unite at their lower ends into the

narrow ejaculatory duct which leads to the

penis.

The sperm cells go through development in

the tubules of the testes and then pass into the

vesicles where they remain until ejaculated. In

the meantime the testes shrink.

The seminal vesicles have muscular walls and

are lined with secretory cells that provide nourishment for the sperm. At 3 to 4 days of age a

few sperm are already in th« seminal vesicles,

and at 4 to 5 days there are about 5 million and

this increases to 10 to 11 million at 8 days. It is

very important that drones receive proper care
AGRICULTURE HANDBOOK 390, U.S. DEPT. OF AGRICULTURE

during' this period. Sperm obtainable at any age

ai^ usable in artificial insemination; however,

it is best to wait until all sperm have had a

chance to mature. This is considered to be about

12 days of age. There seems to be no deterioration of sperm as aging continues.

The mucous glands also have muscular walls

with secretory cells. As the drone matures

sexually these glands become distended and

white from the amorphous white mucus secreted

into the lumen of the gland.

The penis is a soft membranous sack with a

FIGURE 2.--Sting chamber of queen properly opened for insemination: BPO, opening of bursal pouches; DP, dorsal

plate; QH, queen holder; ST, sting; STH, sting hook; VF, valvefold; VH, ventral hook; VO, vaginal orifice- VP

ventral plate. ' '
INSTRUMENTAL INSEMINATION OF QUEEN BEES

VAS DEFERENS SEMINAL VESICLE

MUCOUS GLAND

TEST

RECTUM

/

EJACULATORY DUCT

FIGURE 3.—Male reproductive organs in approximately their natural position in the abdomen. The right-hand members of the paired organs (testes, vasa deferentia, and mucous glands) are not shown.

number of bizarre processes and hairy areas. At

its inner end it is enlarged into the bulb and provided with a pair of hard comma-shaped brown

plates. The bulb is filled with a clear liquid

which dilutes the sperm during ejaculation.

In mating the penis turns inside out to the

outside of the body, pulling the ejaculatory duct

through itself (fig. 4). This eversión is brought

about by the simultaneous contraction of all the

muscles of the abdomen. Ejaculation takes place

during this eversión. A peristaltic contraction of

the muscles of the seminal vesicles, beginning at

their anterior ends, squeezes the sperm out

through the ejaculatory duct; then the muscles

of the mucous gland contract to push the mucus

after the sperm into the bulb. These processes

can be started by a number of artificial stimuli

such as decapitation, pressure on the abdomen,

chloroform fumes, or electric shock. When

started in this way, the eversión usually stops

at the stage illustrated in figure 4, A, With further pressure on the abdomen, which must De

applied to complete the eversión, the bulb passes

on through a narrow section of the penis with a

jerk, the points of the comma-shaped plates

appear at the very end of the everted penis, and

the semen and mucus are ejected from the bulb

to the outside (fig. 4, ß). In natural mating the

transfer of semen to the queen is thought to

take place at this stage. Under the artificial conditions of mechanical pressure on the abdomen,

the eversión often proceeds still further until

the bulb and its plates are turned completely inside out (fig. 4, C). Sometimes the internal

pressure becomes so great that the penis explodes. In the technique of inducing eversión and

ejaculation artificially, the aim is to stimulate

muscle contraction and not to force the action.

The sperm and mucus then come out in natural

sequence with minimum mixing of the two. It is

impossible to obtain a normal ejaculation by

mechanical means alone.
AGRICULTURE HANDBOOK 390, U.S. DEPT. OF AGRICULTURE

FIGURE 4.—Stages of the eversión of the drone's penis: A, Partial eversión usually encountered after initial stimulation; B, a more complete eversión usually obtained by squeezing the abdomen, with semen and mucus exposed;

and C, a fully everted penis (semen and mucus not shown).

The semen is made up of the sperm plus

liquids from the seminal vesicles and the bulb.

The sperm are in bundles, giving the semen a

cloudy or mottled appearance. This appearance

together with its cream color makes semen

easily distinguishable from the homogeneous

NATURAL

The natural act of copulation has been difficult

to study, but considerable information has been

obtained by examining queens immediately

after their return from the mating flight. The

queen evidently cooperates actively by opening

the sting chamber and lov^ering the valvefold

voluntarily. The end of the penis enters the

bursa copulatrix but not the vagina. Since the

queen is not cooperating during artiñcial insemination, it may be necessary to lower the

snow-white mucus. The semen becomes darker

and contains more sperm as the drone ages; the

best quality semen of dark-cream color, not

mixed with mucus, is obtained from drones at

least 12 days old. The quality does not seem to

deteriorate with further aging.

MATING

valvefold with a probe, to facilitate placement

of the syringe tip in the median oviduct.

When the queen returns to the hive after mating, usually both oviducts are distended with

semen, but often unequally. The anterior part of

the vagina also contains semen, and some sperm

have already reached the spermatheca. The bulb

of the penis is found in the sting chamber or

bursa copulatrix buried in mucus which also ex-
INSTRUMENTAL INSEMINATION OF QUEEN BEES

tends into the vagina, or the bulb may be

absent.

During the course of the next 6 to 7 hours

most of the sperm reach the spermatheca

through the vagina and spermathecal duct, but

some may remain in the oviducts 24 hours, especially after artificial insemination. How this

translocation takes place is not yet perfectly

understood. It has long been thought to be a

process of active migration; however, while it is

taking place, the queen frequently contracts her

abdomen and little strings of dried semen are

eliminated. From this evidence it has been assumed that the sperm is forced into the spermatheca during these contractions while the valvefold serves as an imperfect seal of the vaginal

orifice. Perhaps both processes play a part.

Whatever the procedure, it is as effective after

instrumental insemination as after natural

mating.

Naturally mated queens, on return from mating nights, contain an average of 11.6 microliters (fjih) of semen in their oviducts (maximum

28 /xl.). From this it is estimated that queens

mate with eight to nine drones on the average

(maximum: 17). If the first mating does not

satisfy, the queen will mate a second or even a

third time.

EQUIPMENT FOR INSTRUMENTAL INSEMINATION

The major equipment needed to perform the

insemination operation is illustrated in figure 5.

It consists of the manipulating apparatus (fig.

6) to which the queen holder (QH), ventral

hook (VH)y and sting hook (STH) and sijringe

(S) are attached in movable fashion, a cylinder

of carbon dioxide for use as an anesthetic, a

stereomicroscope, and a source of light. A jar in

which queens are given additional treatments

with carbon dioxide is also shown.

Microscope and Light

The microscope should be of the stereoscopic

type with a wide field. The most desirable magnifications will vary with the operator. An experienced operator may prefer to use a single

magnification of 10 to 15 diameters for both the

collection of semen and insertion of the syringe.

A beginner may wish to have also a higher magnification of about 20 diameters available for

the insertion; the experienced operator might

find 20 diameters useful with diflScult queens.

The experienced operator may be able to use an

even lower magnification (6 to 7 diameters) so

that the two operations can be performed under

a single field without moving the microscope.

Some type of illumination is essential. A

microscope lamp with a concentrated beam of

adjustable brightness is best, but any lamp that

gives sufficient light to satisfy the operator is

satisfactory. The beam should be large enough

to cover both the semen collection and injection

operations without moving the lamp, or the

lamp should be attached to the microscope so

the point of focus will always remain within the

beam.

Carbon Dioxide Equipment

Carbon dioxide serves as anesthetic. It is obtainable in cylinders under high pressure which

must be reduced to a few pounds per square inch

by a reduction valve. If taken directly from the

cylinder the gas will alternately freeze and thaw

causing an irregular flow of gas. A needle valve

permits adjustment of the flow to a very fine

stream. A rubber tube carries the gas to the

queen holder and another tube leads to a container in which queens are given additional

anesthetizations.

Queen Holder

The queen holder (fig. 7) consists of an outer

transparent plastic tube 1^4 inches long and an

inner stopper (also a tube) of the same length

to which the carbon dioxide supply tube is attached. The stopper is made of two parts that

screw together permitting the insertion of a felt

washer. This washer provides friction to keep

the stopper in place and prevents leakage of

carbon dioxide back around the stopper. The

friction is adjustable to a limited extent. Screwing the parts together more tightly tends to increase the diameter of the felt washer thus

increasing the friction with the tube. The queen

is induced to back into this tube and when her

abdomen begins to protrude she is secured with

the stopper. The carbon dioxide flows through
8 AGRICULTURE HANDBOOK 390, U.S. DEPT. OF AGRICULTURE

FIGURE 5.—Complete insemination equipment, showing manipulating apparatus under microscope with queen in

place ready for injection of semen, jar for giving additional carbon dioxide treatments, and carbon dioxide cylinder

with pressure regulator attached.

the hole in the stopper and bathes the queen as

it passes out of the queen holder.

Syringe

Various types of syringes have been used

during the development of the insemination

technique. The Mackensen syringe (fig. 7) is the

one most commonly used today. The barrel and

plunger are made of stainless steel. The plunger

is constructed in two parts so that when the

screw plunger turns, the diaphragm plunger

does not turn nor bore into the diaphragm. The

diaphragm is made of rubber one-sixteenth inch

thick. The tip and adapter are made of clear

plastic. At its point the tip has an inside diameter of 0.005 inch and an outside diameter of

0.009 inch. This is about as small as the outside

diameter can be made and still permit an inside

diameter large enough for reasonably easy

semen collection. Just inside the point, the inside diameter gradually enlarges for a distance

of five thirty-seconds inch and then continues at

a uniform bore of 0.031 inch. The tip is graduated in microliters and has a capacity of 10.

With the 5/16-inch base, still larger capacities

can be obtained by increasing the diameter of

the uniform bore.

In operation the tip and adapter socket are

filled with physiological salt solution, the tip is

screwed into the adapter against the rubber

diaphragm, and the adapter is then screwed into

the syringe barrel socket. When the sci-ew

plunger is advanced, it causes the diaphragm

plunger to push the diaphragm into the cavity at

the base of the tip thus forcing the saline solution out. The semen is then taken in by retracting the screw plunger. Thus, the saline solution

acts as a liquid plunger.

Originally the piece between the tip and barrel

was the same diameter as the barrel and was
INSTRUMENTAL INSEMINATION OF QUEEN BEES

simply an extension that could be omitted. When

the tip was screwed directly into the barrel a

1/2-inch-long diaphragm plunger was used.

When it was found that the old style tips with

1/4-inch-diameter base did not have the capacity

desired by modern workers, the base was enlarged to five-sixteenths of an inch and the connecting piece correspondingly. Thus, this piece

became an adapter.

The adapter is a great convenience when sterilization is required as in a series of individual

matings or to prevent the spread of disease.

Several adapters and tips can be prepared, used

one by one, and all cleaned and sterilized at the

same time.

Sting Hook and Ventral Hook

Two hooks are used to hold the sting chamber

open and the sting out of the way during the

operation. Each hook is secured to a long

handle, which slides within its hook holder. The

critical parts of these instruments are shown

enlarged in figure 8. The ventral hook is

mounted to the left of the operator and is little

more than a bent wire that hooks over the ventral plate. The sting hook is mounted to the

right of the operator. It has an enlargement at

the end which fits into the triangular area between tlie sting lancets and extends underneath

them (fig. 2). It is used to pull the sting dorsally exposing the vaginal orifice. These hooks

may vary considerably in dimensions and shape,

and different workers in instrumental insemination have their preferences. One should be prepared to make these hooks out of spring temper

brass wire. Suitable tools for this purpose are

number six cut files of round, oval, and flat

shapes, obtainable from any jeweler.

FIGURE 6.—Manipulating apparatus viewed from operator's side: QH, queen holder; S, syringe; STH, sting hook;

and VH, ventral hook.
10 AGRICULTURE HANDBOOK 390, U.S. DEPT. OF AGRICULTURE

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INSTRUMENTAL INSEMINATION OP QUEEN BEES 11

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12 AGRICULTURE HANDBOOK 390, U.S. DEPT. OF AGRICULTURE

Valvefold Probe and Stîng Depressor

The valvefold probe (fig. 8) is used to press

the valvefold ventrally to make way for the

point of the syringe on its way to the oviduct.

There are two 45° bends on a plane with the

main stem of the instrument instead of one 90°

bend to minimize obstructing the view. The last

0.020 inch is bent counterclockwise about 30°

when viewed toward the handle. The sting depressor is simply a needle that is used to depress the sting while the sting hook is placed.

These instruments can be mounted in pin vices

or other suitable short handles.

Manipulating Apparatus

A good manipulating apparatus will not only

support the parts already described but will hold

the syringe and holding hooks so that their

functioning ends can be moved in any direction

smoothly and easily and yet remain exactly

where placed when released. The apparatus illustrated in figure 6 fulfills these requirements,

and the position of its parts can be reversed for

a left-handed operator.

It has a heavy base to which are attached the

mounting for the queen holder in the center and

%-inch posts at either side 41/2 inches apart.

The left post supports the ventral hook mounting and the right post, the syringe mounting

(above) and the sting hook mounting (below).

The queen holder mounting block can be

rotated in a vertical plane or moved from side to

side. After it is once adjusted according to the

wishes of the operator it is tightly secured. It is

provided with a spring so that the queen holder

can be simply and quickly snapped in place and

moved up or down or revolved for accurate positioning while the tension of the spring holds the

queen holder in place. We suggest that the queen

holder block be adjusted so that the queen

holder leans 30° from vertical and makes a

10° angle with the syringe about as illustrated

in figure 6 and changes be made later as

desired.

Since the mountings for the syringe and holding hooks are similar, only the mounting of the

ventral hook (fig. 9) will be described. The hook

handle passes through holes in each end of a

sheet metal holder. These holes are rounded at

the bottom and inverted V-shaped at the top. A

flat spring pushes the handle upward into thé

inverted V. The tension of the spring can be adjusted so that the hook handle can be slid in and

out with just the right amount of friction. The

hook holder is seated in a tapered depression in

its support block so that it can rotate in a vertical

plane. It extends through the block as a bolt and

is held in place by a spring washer and two nuts.

The degree of friction between the hook holder

and its seat is controlled by the tension of the

spring, which is adjusted by the inner nut. The

outer nut locks the inner nut in place. The support block consists of two parts which clamp

onto the upright post permitting rotation in a

horizontal plane and adjustment up or down on

the post. The parts are held together by two

bolts with coiled springs under the nuts. The

amount of friction with the post is controlled by

adjusting the tension of the springs. This arrangement makes it possible to move the end of

the hook in any direction quickly and easily and

have it remain exactly in the position it was in

when released. A split collar under the block is

tightened around the post to hold the block at

the desired level.

An insemination apparatus similar to the one

just described is available commercially.

USE OF CARBON DIOXIDE

Carbon dioxide not only serves as an anesthetic, but also stimulates queens to begin laying. Proper treatment with this gas causes

virgin queens, as well as instrumentally inseminated queens to begin laying practically as soon

as naturally mated queens, whereas without

such treatment only about 20 percent start

earlier than 30 days after emergence.

At least two anesthetizations are necessary

and a third may be given as a margin of safety

without apparent harm. They may or may not

be accompanied by insemination. An anesthetic

treatment without insemination is given by

placing the caged queen for 10 minutes in a convenient container through which carbon dioxide
INSTRUMENTAL INSEMINATION OF QUEEN BEES 13

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is flowing (fig. 5). This gas is heavier than air,

and as it fills the container from the bottom,

the air is forced out through an escape tube at

the top, insuring a high concentration. (Any

wide-mouth container with a loosely fitted lid

will suffice for carbon dioxide treatments.) Oviposition starts 2 to 6 days after the second

treatment and a day or two later than would be

the case after natural mating. Although treatments given as early as the second or third day

are effective, they do not cause laying to start

earl-er than the normal age. Since exposure during insemination may be too short or the anesthetization incomplete, it is recommended that

two treatments be given after a single insemination and one treatment after two inseminations. These exposures to carbon dioxide appear

to have no ill effect, but since anesthesia has

been shown to change the behavior of worker

bees, overexposure should be avoided. The minimum effective dose in time or concentration

has not been determined.

INSEMINATION PROCEDURE

The best way to learn the insemination technique is to study under an experienced operator.

A microscope arrangement permitting the student and the instructor to observe the same

image is ideal.

Since an instructor may not be available, the

procedure is here described in sufficient detail

for the reader to learn the technique on his own.

The description applies specifically to the apparatus that has just been described. The reader

can adapt it to any other equipment he might be

using.

The student's task will be made easier by the

following suggestions:

1. Work with queens reared from several different breeder queens. Daughters of some

breeders will be easier to inseminate that those

of others. Try queens of different races if available.

2. Use queens reared under the most favorable conditions because large queens are easier

to inseminate than small ones.

3. Use virgin queens that have had the free

run of the nucleus and that are at least 7 days

old. Such queens have less feces in their rectums than queens maintained any other way. A

distended rectum pushes the reproductive tract

ventrally making it more difficult to hold back

the sting and to find the entrance to the median oviduct with the syringe tip.

4. Select a humid day if possible. In a dry

atmosphere the exposed tissue of the queen will

dry quickly making it more difficult to insert

the syringe tip. Also, the semen may dry at the

end of the syringe tip clogging the passage before it can be inserted or before semen can be

taken from another drone. It may be necessary

to moisten these parts occasionally. An experienced operator will work so fast that he will have

little difficulty, but a beginner may find these

inconveniences very disturbing.

5. Practice the semen collection and injection procedures separately using water or saline

solution instead of semen for the injection procedure.

Preparing the Anesthetic

The reduction valve of the carbon dioxide

cylinder should be adjusted to a delivery pressure of about 5 pounds per square inch and the

flow of gas to the queen holder stopper adjusted

to a very small stream. It should be just enough

to keep the queen quiet. The flow can best be

judged by dipping the stopper in water.

Preparing the Syringe

The syringe is prepared by filling the

syringe tip with a physiological salt solution to

serve as a liquid plunger. A 0.9 percent sodium

chloride solution is satisfactory. An antibiotic

may be added to help prevent infection. A plastic squeeze bottle of the type used for dispensing liquids in laboratories is an aid in forcing

the liquid into the tip. The socket of the adapter

with diaphragm in place is then filled with the

saline solution and the tip screwed into it

firmly against the diaphragm. Be careful to

exclude air because it tends to nullify the purpose of the liquid plunger. The extension is then

screwed into the metal syringe body. By advancing the plunger, sufficient liquid is forced

out to permit taking in the desired amount of

semen when the plunger is retracted.
INSTRUMENTAL INSEMINATION OF QUEEN BEES 15

The syringe is placed in the syringe holder

and swung around until the tip is away from

the operator so that it will not interfere with

the mounting of the queen.

Preparing the Queen

The queen is made to walk into a tube similar

to the queen holder. When she reaches the constricted end she backs up, and if the queen

holder is quickly put in place, she usually backs

in readily. As soon as she reaches the constricted end of the queen holder, the stopper

through which carbon dioxide is flowing is

quickly pushed in after her (before she has a

chance to move forward again). To be properly

positioned the last three visible segments of the

abdomen should protrude, and the hind legs

should remain in the queen holder. A beginner

may wish to insert a 3-way stopcock in the carbon dioxide supply tube so the gas can be diverted while the queen is placed. (If the queen's

abdomen expands abnormally, the gas is being

forced into her abdominal air sacs, an indication

that the flow is too strong.) The queen holder is

then put in place on the manipulating apparatus and adjusted so that the dorsal surface of

the queen's body is to the right.

As soon as the queen is quiet, the sting chamber can be opened with the holding hooks, with

the ventral hook to the left and sting hook to

the right. The hooks are manipulated more or

less simultaneously, but usually first the ventral hook and then the sting hook are inserted

into the sting chamber and abdominal plates

pulled apart. With the left hand, the sting

depressor is used to hold the sting down while

the sting hook is placed in the triangular area

between the bases of the sting lancets, and left

in this position to prevent unnecessary drying

of the delicate tissues while the syringe is being

loaded.

Ejaculation of Drones

Drones vary greatly in the ease with which

they are induced to ejaculate and in the amount

of semen delivered, especially when one is dealing with inbred drones. Some have no semen;

others may not evert and ejaculate properly

when the stimulation is artificial; still others

may evert so violently that the semen is projected and lost or the penis explodes.

The method commonly used to induce eversión

and ejaculation is the following: The drone is

decapitated and, if this does not cause a partial

or complete eversión, he is grasped by the

thorax with the thumb and forefinger of the left

hand with the drone's ventral surface up. Then,

while the anterior part of the thorax is squeezed

with the left hand, the dorsal part of the abdomen is teased or repeatedly squeezed lightly

with the thumb and forefinger of the right hand.

This action usually brings about contraction of

the abdominal muscles and a partial or sometimes a more or less complete eversión and

ejaculation (fig. 4). If eversión is only partial

(fig. 4, A), the abdomen is squeezed progressively from the anterior dorsal region to the

posterior ventral part to continue the eversión

by force until the semen is extruded (fig. 4, B or

4, C). Semen is rarely obtainable without abdominal contraction, but when the abdomen contracts without partial eversión, the eversión can

often be completed by pressure and a good

amount of semen obtained.

The amount of semen and mucus varies. The

cream-colored semen comes out first followed

by the thicker white mucus. Sometimes only

semen is ejected, but usually at least some mucus comes out after the semen, and the two are

distributed on the penis in various arrangements. Movement of sperm causes the semen to

spread in a thin layer over the mucus, making it

progressively more diflScult to collect. Also, delay after the first stage of eversión makes the

semen more difficult to collect, possibly due to a

mixing of semen and mucus. It is, therefore, important to complete the whole procedure as

quickly as possible.

Sometimes drones evert and ejaculate better

if they are first induced to exercise by holding

them by the legs with forceps while they attempt

to fly or by permitting them to fly against a

windowpane. Sometimes caging free-flying

drones for a day or two improves eversión and

ejaculation. Perhaps these methods increase the

volume inside the abdomen by forcing air into

the air sacs or by increasing the accumulation

of feces; thus the contraction of the abdominal

muscles is more effective in everting the penis.
16 AGRICULTURE HANDBOOK 390, U.S. DEPT. OF AGRICULTURE

Sometimes exposure to chloroform fumes is effective when all other methods fail; in fact, it is

quite reliable in causing at least a partial eversión; however, an experienced operator will

usually find this method too time-consuming.

Filling the Syringe

This operation is done under the microscope

with the syringe tip swung a few inches toward

the operator and the microscope moved correspondingly. The ejaculated drone is brought

near the syringe tip with the left hand. The

plunger of the syringe is withdrawn slightly to

provide an air space (as small as practical—onehalf microliter) between saline solution and

semen and to faciliate measuring the amount of

semen. The surface of the semen is then made to

touch the point of the syringe at about a 45°

angle. If the drone is pulled away from the

syringe slightly without breaking contact, the

semen will continue to adhere to the syringe and

will flow toward it as the plunger is withdrawn.

This procedure helps the operator to avoid taking up the mucus which is too thick to pass into

the syringe tip and will stop the passage of

semen. If mucus clogs the tip, the plunger is

pushed out until the passage is cleared ; then the

taking of semen is resumed. By moving the

drone about in relation to the syringe tip, the

mucus can be skimmed of practically all its

semen covering. Semen is taken from as many

drones as necessary, and when the syringe tip is

filled to the desired point a small amount of air

or physiological salt solution is drawn in to prevent sealing of the end of the syringe tip by

drying of semen. This air or salt solution can be

injected with the semen without harm. The average yield of semen per drone is about 1 /¿I. The

beginner should take up semen very slowly; this

is very important. Speed will come with experience.

Injection

The microscope is again positioned over the

queen and the magnification changed to a higher

power if desired. The sting hook is drawn dorsally until the sting chamber appears as in figure 2. Applying a 3 percent aqueous solution of

Fast Green stain while the queen is in this position will help the beginner to recognize the

vaginal orifice and possibly the valvefold.

The syringe tip is then posed above the

vaginal opening. With the left hand the valvefold probe is inserted into the dorsal part of the

vagina and the valvefold is pushed ventrally

until the point of the syringe has passed beyond

(fig. 10). Then the probe is removed as the

syringe is pushed in farther (about 1.5 mm.). If

the surrounding tissue begins to move before

the syringe has reached this depth, it is probably not in the oviduct. Sometimes the syringe

needs to be moved ventrally slightly. The probe

and syringe should be inserted only as far as

necessary. Beginners have a tendency to insert

these instruments too deeply.

Carefully attempt to inject the semen. If the

column of semen does not begin to move immediately and the air between semen and saline

solution begins to compress, the syringe is not

in the median oviduct and must be withdrawn

for another attempt. Some adjustments may be

necessary. The dorsoventral centerline of the

queen's body should be perfectly in line with the

ventral and sting hooks, and the syringe should

be inserted exactly in this centerline. If these

precautions are observed, the syringe should

enter the oviduct without difficulty. When the

semen begins to move properly without any

leakage around the point, the injection can proceed rapidly. Sometimes leakage can be stopped

by inserting the syringe a little deeper, but if

leakage continues, stop the operation and try

again another day. When the injection is completed, the syringe is withdrawn and the queen

removed from the queen holder.

Throughout the operation always steady the

arms against the edge of the table and the

hands and fingers against parts of the apparatus. Some operators object to use of the probe

because it cannot be held steady enough to prevent injury to the queen. When we use the

probe, we hold it steady by leaning the forefinger against the queen holder mounting.

If the probe is omitted, insert the syringe

along the dorsal wall of the vagina; then move

the syringe ventrally to push the valvefold aside,

and then inward again. Queens of some strains

are easily inseminated without the probe.

Some operators load the syringe first while

others prepare the queen first. The order is a

matter of personal preference.
INSTRUMENTAL INSEMINATION OF QUEEN BEES 17

VALVEFOLD PROBE—

\ VAGINA

VALVEFOLD

MEDIAN OVIDUCT

B

FIGURE 10.—Manner of pushing the valvefold aside and inserting the syringe. (Redrawn from Laidlaw and Eckert,

1962, by permission of The Regents of the University of California.)

Cleaning and Sterilizing the Syringe Tips

The best way to clean and sterilize the

syringe tips is to flush them with water and

soak them in ordinary household bleach (active

ingredient: sodium hypochlorite) used full

strength. Soaking overnight in this material

loosens adhering semen so the tips can be

flushed clean the following day. This material

also sterilizes and does not affect the plastic. A

fine wire, 0.005 inch or less in diameter, should

be available in case the syringe tip does become

clogged. The wire should be cut off squarely

and have no sharp edges to damage the soft

plastic.

Ordinarily, aseptic methods are not necesssary. When changing from one type of sperm to

another, the syringe tip can be cleaned and any

adhering sperm killed by rinsing thoroughly

with water. Precautions to be taken when paralysis or septicemia are present in the apiary are

outlined next.

DISEASE AS A FACTOR IN INSEMINATION

There are two adult bee diseases to contend

with in instrumental insemination—septicemia

and paralysis. Neither one is considered a very

serious disease under ordinary apiary management, but in instrumental insemination they

can cause disastrous losses if not controlled.

A queen that has contracted septicemia

during insemination will die a day or two later

and soon show the typical symptom of this disease: dismemberment of the body parts. If this

bacterial disease is suspected but the symptoms

are not yet evident, hold the queen a day under

humid conditions. If the queen's legs and wings

are then not pulled off easily (practically fall

off), then the trouble is not the type of septicemia common in the United States. Other septicemias that do not cause dismemberment have

been reported in Europe and would probably

also be spread by insemination.

Septicemia is spread by instruments that

come in close contact with the queen: syringe,

sting hook, and valvefold probe. Contaminated

ventral hook, queen holder, and hands are not

likely sources of infection. The semen of drones

dying of septicemia seems to be uncontaminated.
18 AGRICULTURE HANDBOOK 390, U.S. DEPT. OF AGRICULTURE

Paralysis is caused by a virus. It kills less

rapidly than septicemia. Infected queens returned to their nuclei after insemination may

simply not lay and disapper after a few days.

Others gradually become sluggish and bloated

with clear body fluid. In this condition they will

live for several days on the comb or on the bottom board. Some will lay a few eggs before they

become sick.

These diseases can usually be avoided by

using free-flying drones of any age or conñned

drones as soon as they are sexually mature or at

least within 3 weeks after emergence. Whenever they have appeared, we have controlled

these diseases by instituting sterilization procedures. Between each operation or after every

third operation, the plastic syringe tip was sterilized with sodium hypochlorite, and the probe

and sting hook were sterilized with 70 percent

alcohol.

Antibiotics have been used by a number of

workers and can be used routinely. At the Bee

Research Laboratory, Tucson, Ariz., Terramycin is used. A stock solution is made up with 2.5

grams Terramycin (poultry formula—6.25

grams activity in one-quarter pound) in 100

cc. physiological sodium chloride solution and

refrigerated. The stock solution is diluted with

eight parts of the salt solution as needed. In

loading the syringe with semen, a microliter of

the Terramycin solution both precedes and follows the semen. All this is injected, and, as the

syringe is withdrawn, an additional microliter

of the Terramycin solution is placed at the

opening of the reproductive tract before removing the sting and ventral hooks. The syringe is

used continuously without cleaning. In a similar

method, we substituted 0.25 percent streptomycin sulfate for the Terramycin. At the Bee Management Investigations Laboratory at Madison,

Wis., streptomycin sulfate (0.5 grams per cc.)

is applied with a brush to the hooks and loaded

syringe just prior to insertion of the syringe.

These methods control septicemia. In the case

of paralysis, sterilization procedures may be

necessary because it is a virus disease.

ESTIMATING THE NUMBER OF

The success of an instrumental insemination

can be measured by the number of sperm reaching the spermatheca. To dissect out the spermatheca for examination, the last visible segment

of the abdomen is torn off by grasping the last

ventral plate with forceps. The spermatheca is

usually found embedded in tissue inside this removed segment. It is a sphere about 1 mm. in

diameter and appears rough and white because

of the net work of tracheae which covers it completely. The spermatheca is lifted onto a finger

and the tracheae removed by rolling with the

forceps. The spermatheca itself is smooth and

transparent in a virgin queen; and in mated

queens it varies from translucent through

milky-white to cloudy and a dark cream color

depending on the number of sperm present.

With up to 0.5 million sperm it ranges from

clear to opaque milky. When the number

reaches 0.8 to 1.0 million it becomes slightly

cloudy. At 1.5 million it is cloudy and light

cream colored. As the number increases it becomes dark-cream colored.

A more accurate estimate can be made by

counting the number of sperm in a sample taken

SPERM IN THE SPERMATHECA

from the diluted contents in the following manner.

Equipment needed includes a countingchamber slide, a 10-cc. pipette, a medicine-dropper pipette, a small dish holding a little more

than 10 cc, a pair of forceps, and a dissecting

needle.

The spermatheca is placed in the dish, and 1

cc. of 0.9 percent sodium chloride solution

added. Then the spermatheca is broken with the

forceps and needle, the sperm teased out, and

the empty skin removed. The sperm is dispersed

by alternately drawing the saline solution into

and expelling it from the medicine-dropper

pipette until all lumps have disappeared. Then

9 cc. of tapwater is added to make a total of 10

cc. and the sperm again thoroughly dispersed

with the medicine-dropper pipette. A drop of

this mixture is quickly placed in the counting

chamber.

In the saline solution the sperm appear as

headless filaments about 0.25 mm. long and are

easily dispersed. The tapwater kills them, but

before they die most of them coil up into various shapes such as small circles or configura-
INSTRUMENTAL INSEMINATION OF QUEEN BEES 19

tions resembling the numerals 6 and 8. In these

forms they are more easily counted than when

relaxed. They are most easily seen and counted

under a phase-contrast microscope or one with

a dark field. The number in 10 cc. is calculated

from the number counted in a given volume.

The number of sperm in the seminal vesicles

of drones can be estimated by the same method.

A window is carefully cut into the dorsal wall of

the abdomen and the seminal vesicles very

gently cut off where they join the mucous

glands. If this is not done very carefully, the

muscles of the seminal vesicles will contract

prematurely and some of the sperm will be lost.

After being placed in the saline, the muscles are

made to contract by pricking and mashing, and

what sperm is not forced out in this way is released by tearing the vesicles to pieces.

LAIDLAW APPARATUS AND METHOD

The Laidlaw apparatus for instrumental insemination (fig. 11) differs from that of Roberts and Mackensen in the way the queen is

held and in the way the movement of the holding hooks and syringe is controlled by means of

racks and pinions. The track on which the

syringe is advanced is fixed, so that the queen

is positioned beneath the syringe by moving the

whole queen manipulator which holds the

queen. The operation of instrumental insemination is quite similar to that with the Roberts

and Mackensen apparatus.

The Laidlaw apparatus consists of two parts,

a queen manipulator and a syringe manipulator.

The queen manipulator has a heavy circular

base whose bottom surface is smooth and slides

easily on the glass stage of a microscope for

positioning the queen in relation to the syringe.

The remainder of the queen manipulator is connected to the base by a ball and socket joint,

which is tightened to a fixed position of optimal alinement of the queen to the syringe. The

holding hooks are mounted by chucks on vertical racks and pinions, which in turn are

mounted on horizontal racks and pinions to

either side of the centrally located queen holder,

with the sting hook to the right and the ventral hook to the left when the control knobs

face the operator. The queen holder consists of

two vertical surfaces, the left one fixed and the

right one moveable. Pieces of foam rubber glued

to each of these surfaces from the pads between

which the queen is clamped. The moveable surface can be moved back and forth by a rack and

pinion. The fixed surface is recessed into a

block, so as to make a closed anesthetization

chamber about the queen when she is clamped

in position. Carbon dioxide gas is directed toward the queen's thoracic spiracles from ports

located in either side wall of the chamber.

The syringe manipulator consists of a rack

and pinion tract to which the syringe is clamped

and permits movement up and down at the adjusted angle. The syringe manipulator is

mounted from a post attached to the front of

the microscope stage. The attachment from the

post to the syringe manipulator includes joints

by which the syringe manipulator may be adjusted to the correct angle in relation to the

queen, but tightened securely once this adjustment is made.

The approximate alinement for the Laidlaw

apparatus is with the queen manipulator tilted

about 10° to the right of vertical, and the

syringe manipulator about 20° to the right of

vertical. The syringe tip should enter the

queen's reproductive tract directed slightly towards the queen's ventrum about 10° from the

queen's long axis. When the queen is positioned

for insemination, the syringe and queen manipulator should be alined across the stage of the

microscope in a line with the queen's dorsum

towards about 1 o'clock and ventrum towards

about 7 o'clock.

Because the alinement of the Laidlaw apparatus differs from that of the Roberts and Mackensen apparatus, the construction of holding

hooks and of the valvefold probe differs.

Detailed plans for the Laidlaw insemination

apparatus are available from Dr. Harry H.

Laidlaw, Department of Entomology, University of California, Davis, Calif. 95616.

With the Laidlaw apparatus, the operation of

instrumental insemination consists of the same

steps as with the Roberts and Mackensen apparatus, but the details of how some of these

steps are accomplished differ. The major differences are the following:
20 AGRICULTURE HANDBOOK 390, U.S. DEPT. OF AGRICULTURE

FIGURE 11.—Laidlaw insemination apparatus with Mackensen syringe. (Courtesy H. H. Laidlaw.)
INSTRUMENTAL INSEMINATION OF QUEEN BEES 21

Placing the queen into the queen holder is

done with the queen already anesthetized, by

holding the queen in one's hand along with a

tube delivering carbon dioxide. The motionless

queen is then grasped gently with one's left

hand by the anterior part of her abdomen and

suspended, head downward and dorsum to the

right, into the opened queen holder, so that her

dorsoventral body axis is in a straight line with

the holding hooks and with her thorax opposite

the foam rubber pads. The queen holder is then

closed by turning the control knob with one's

right hand until the queen is firmly clamped

into position. Placing the queen into the queen

holder is done while the queen manipulator is

on the laboratory table to the left of the microscope; the action is easily done without magnification.

With the queen manipulator placed onto the

stage of the microscope, the sting chamber is

opened and kept spread open with the sting depressed with a pair of fine jeweler's forceps

held in one's left hand. The sting hook and then

the ventral hook are positioned by turning the

appropriate control knobs for the racks and

pinions, after which the forceps are withdrawn.

The queen is oriented in relation to the syringe by sliding the queen manipulator on the

glass microscope stage. Since the syringe can

be moved only upward and downward, and cannot be tilted or moved from side to side, even

the final adjustment to get the syringe tip in

position for insertion is made by moving the

queen manipulator.

Inserting the syringe tip into the median oviduct with the Laidlaw apparatus differs only

slightly from that operation with the Roberts

and Mackensen apparatus. In moving the valvefold, the operator moves it toward 7 o'clock,

rather than toward 9 o'clock. The syringe is advanced and withdrawn by turning the control

knob for the rack and pinion on the syringe

manipulator. Also, since the queen is nearly vertical, the valvefold is seen more readily, and one

can often see through the vagina to the opening

of the median oviduct when the valvefold is

moved.

AGE OF QUEEN FOR INSEMINATION

Whenever possible, inseminations should be

made at the normal mating age of the queen,

around the seventh to 10th day after emergence. We have inseminated queens successfully in quantity as early as the fourth day.

They can be inseminated at any later age so

long as they have not started laying. The minimal interval between two inseminations should

be 1 day, and a 2-day interval will permit a more

complete clearing of the reproductive tract

when very large doses of semen are given. In

this connection, oviposition may start as early

as 2 days after the second exposure to carbon

dioxide. There is some evidence that when development is retarded by cool weather, inseminations are more successful at 10 to 12 days

than earlier.

PROCEDURE FOR VARIOUS OBJECTIVES

The number of inseminations and volume of

semen will depend upon the objective of the inseminator and the time and effort he is willing

to expend. Usually only one or two inseminations are made. The efficiency, that is the percentage of sperm reaching the spermatheca, decreases as the amount of semen injected

increases. After insemination with 2 to 20

microliters of semen, the percentage of sperm

reaching the spermatheca will range from about

15 to 4, respectively. Results will vary with the

stock used.

In bee breeding and genetic studies it is often

necessary to inseminate a queen with semen

from a single drone. For best results with such

individual matings, well-nourished drones

reared in large cells in relatively new drone

comb should be available and only those utilized

that ejaculate perfectly and yield a large quantity (1.25 to 1.5 fjil,) of dark cream-colored semen. Results will depend upon the stock used.

One might expect about 10 to 15 percent of

queens to be partial or complete "drone layers"

immediately and more to become "drone layers"

later. Some may lay fertilized eggs two seasons
22 AGRICULTURE HANDBOOK 390, U.S. DEPT. OF AGRICULTURE

in a nucleus. The spermathecae of 17 queens

killed soon after insemination contained an

average of 0.87 million sperm (range: 0.22 to

2.24), about one-sixth the number found in

naturally mated queens.

If a queen is expected to produce fertilized

eggs for a few months only and individual mating is not required, an insemination with semen

from three to four drones (3 to 4 /¿I.) will be sufficient. Average spermathecal sperm counts of

2.9 and 3.6 million have been reported after insemination with 4 fih

If a queen is to head a large colony for a year

or more, it is logical to attempt to duplicate the

number of sperm a queen receives in a completely satisfying natural mating. On the average, naturally mated queens receive about 5

million sperm in their spermathecae. Experiments have shown that queens that have no desire to make further mating nights after their

first mating contain an average of 5.3 million

sperm in their spermathecae. Further experiments have shown that queens do not mate

naturally after instrumental insemination with

8 microliters semen, but do fly out for further

mating if given smaller amounts. The number

of sperm reaching the spermatheca with a dose

of 8 /¿I. semen has been reported as 3.16 and 5.4

million in different experiments. Although the

number of sperm reaching the spermatheca increases as the dose increases, diminishing efficiency makes it hardly worth while to give more

than 8 /xl. of semen in a single insemination.

Also, with higher amounts the danger of injury

is increased. In some experiments a high death

rate was obtained with single inseminations of

12 fú; in others, 18 /xl was the maximum given

without injury.

If a certain quantity of semen is given in two

inseminations of equal amounts instead of in

one insemination, the number of sperm reaching the spermatheca will average about 15 percent greater. (Increases of 1 to 41 percent have

been reported.) Another advantage is that variability will be lower. These advantages must,

however, be weighed against the additional

effort involved and the additional chance of injury during the second insemination.

The percentage of laying queens obtained will

vary with stock used, but an experienced operator should be able to obtain 90 percent laying

queens after either one or two inseminations

during the most favorable part of the season if

the stock is vigorous and disease is absent. Properly inseminated queens have been shown to

perform as satisfactorily in brood and honey

production as naturally mated queens.

Queens that are already laying can also be

inseminated, but egg production must first be

reduced. The technique is most commonly used

in mating a queen with her own drones to attain

a high rate of inbreeding. The virgin queen is

first induced to lay by treating with carbon

dioxide. When her drones are mature she is

caged away from her colony with a few attendant bees for about 4 days to stop or retard egg

development. Then she is inseminated as usual

but with small amounts (3 /xl. or less) of semen

to avoid congestion in the oviducts in case some

eggs are still being produced.

STORING AND SHIPPING SEMEN

Semen can be shipped long distances for use

in instrumental insemination. Successful inseminations have been made with semen shipped

between the United States and Europe and between the United States and Brazil.

The technique is as follows: The semen of

many drones (30 to 40) is placed in a 2 mm.

diameter capillary tube, a syringe load at a time,

and centrifuged just enough to force the semen

down to the closed end and eliminate air spaces.

Mucus must be scrupulously avoided. The open

end is then sealed over a flame 5 to 8 mm. above

the semen while care is exercised not to overheat the semen and at the same time leaving a

minimum air space. Properly protected, such

tubes of semen can be sent by airmail in an ordinary letter envelope. To use the semen, the

tube is broken in such a way that the first 1 to

2 mm. of semen next to the air space is discarded and the remainder of the semen is taken into

a syringe as usual.

By similar methods, but with antibiotics

added (streptomycin sulfate or tetracycline hydrochloride), some of the sperm has remained

capable of fertilization for 16 to 18 weeks.
INSTRUMENTAL INSEMINATION OF QUEEN BEES 23

MAINTENANCE OF QUEENS

Queen rearing will not be dealt with here except to emphasize that large queens are more

easily inseminated than smaller ones and that if

one spends the effort involved in instrumental

insemination one should start with the best. For

queen rearing methods the reader is referred to

the many books that have been written on the

subject.

There are several ways to maintain queens

for instrumental insemination. Some methods,

which the reader can modify to suit his convenience, are described below:

Maintaining Queens in Nuclei

A method we use is as follows: Mature queen

cells are placed in vials (fig. 12, A) and kept in

an incubator at 35° C. until emergence. The

neck of the vials is just large enough to receive

the cells, but not the wooden cell bases to which

they are attached. In the bottom of the vials is

placed a 14-inch layer of coarse sawdust or fine

wood shavings, to absorb moisture, and a small

pellet of queen candy. As the queens emerge a

wing is clipped on each and a color mark placed

on the dorsum of the thorax for identification

of mating groups. They are then confined to

holding cages (fig. 12, ß) and placed individually in the center of the brood nest of previously prepared nucleus colonies. The nuclei are

"baby" frame size (9Vè-inch top bar; 614,-inch

depth) and when first established, are composed of a comb each of honey and brood plus

two empty combs and % to 1 pound of bees.

Before adding them to the nuclei, the bees are

sifted through a queen excluder and all drone

brood is killed to eliminate drones that would

otherwise clog the excluders, which must be kept

over the entrances until the queen starts laying.

The brood is examined between the seventh and

10th day after the nuclei were made up, and all

emergency queen cells are destroyed. The

queens are taken to the laboratory in their

cages, several at a time in convenient numbers

for each insemination or carbon dioxide treatment. After the last treatment, each queen is

returned to her nucleus by simply dropping her

into the cluster of bees while still anesthetized,

or the cage is opened and placed into the nucleus

>*2*.

A C

FIGURE 12.—Containers used in queen handling: A, Vial used for queen emergence in an incubator; B, cage for

holding queens individually in nuclei; and C, cage used for queen emergence in an incubator and for emergence

or storage in nursery colonies.
24 AGRICULTURE HANDBOOK 390, U.S. DEPT. OF AGRICULTURE

to be removed later when the queen has revived.

In cool weather the cage is opened and placed

within the cluster. Nuclei in which queens are

lost are removed from the queen yard. Only

nuclei from which laying queens have been recently removed are reused.

If the queens are permitted to run loose in the

nuclei, it is best to put the queen cells directly

into the nuclei and clip and mark the queens as

soon as they emerge while they are still recognizable as newly emerged queens. This method

requires spending some time finding the

queens, and there is always the risk of a stray

queen being mistaken for the desired- one. An

advantage is that the virgin queens destroy any

emergency queen cells that have been started.

The nucleus hives we use have room for 11

combs. We start the nuclei with four combs and

add more as needed. We find the extra space

convenient during insemination and carbon

dioxide treatment. By adding supers these nuclei can be permitted to develop into small

colonies suitable for wintering breeder queens.

Maintaining Queens in Nursery Colonies

When queens are maintained in nursery colonies, the mature queen cells are put into individual cages (fig. 12, C). The cages are placed

into holding frames, which are put into a strong

queenless colony. Only virgin queens are kept in

this colony. The virgin queens emerge into the

cages and are fed by worker bees through the

mesh of the cages. When about a week old, the

virgins are taken from the nursery and inseminated. After insemination, the queens are each

caged with a few worker bees and kept in another nursery colony for at least 2 days. A day

after the nuclei to which they will be introduced

have been made up, the inseminated queens are

given a treatment with carbon dioxide and introduced into nuclei. These queens should be

introduced so that they are not released until

they are ready to lay.

The nursery for the virgins and that for the

inseminated queens are made up and maintained

similarly to queenless cell builder colonies.

There should be an abundance of bees, honey

and pollen, and two combs of unsealed brood

renewed each week. The caged queens should

face the unsealed brood. The nursery should be

fed sugar syrup and pollen in the absence of

natural nectar and pollen flows.

The advantage of maintaining queens in nursery colonies is that the queens are quickly

located for inseminations and carbon dioxide

treatments. The main disadvantage is that the

queens may have excessive accumulation of

feces in their rectums and be difficult to inseminate. Also, loss of queens during introduction

is a problem with this method.

REARING AND MATURING DRONES

Inducing a colony to produce drones is not

always easy. Young queens are often more reluctant to lay drone eggs than older ones.

There is considerable variability among stocks

and individual queens in the ease with which

drone eggs are obtained. Early in the season

drones can usually be obtained in abundance

from any queen because they are naturally

produced at this time of year in preparation for

swarming. Later in the season and during pollen dearth periods, colonies are often very reluctant to produce drones. At such times it may

be necessary to stimulate them by feeding pollen

and syrup with pollen added, and creating a

crowded condition by adding bees or reducing

the size of the hive. At times when the nectar

flow is extra heavy the bees will fill the drone

comb with nectar. At such times the queen can

be restricted to one brood chamber below an

excluder and dark empty combs placed above.

By extracting the drone comb gently, the thin

nectar can be removed without destroying eggs

and young larvae that might be present in some

of the cells, thus giving the queen a greater

opportunity to lay in the drone comb. The queen

can be forced to lay on a drone comb by confining her to the comb within a queen excluder

cage; however, one might be disappointed to

find that the queen has deposited only fertilized

eggs. It is best to create conditions that make

the queen desire to deposit unfertilized eggs.

The importance of pollen for rearing and

maturing drones cannot be overemphasized.

While an abundance of pollen stored in combs

often seems to suffice, some operators insist on

adding more pollen in the form of moist pollen
INSTRUMENTAL INSEMINATION OF QUEEN BEES 25

cake (without nonpollen extenders), or 25 to 50

percent pollen in sugar syrup. Pollen syrup is

better sloshed daily over the top of the frames,

where it will be consumed quickly, than fed in

feeders, where it may spoil.

After emergence, drones can be matured in

various types of nursery colonies or in cages.

The simplest method is to set the drone producing colony in a location sufficiently isolated

from other colonies to prevent undesired drones

from drifting in and permitting the drones to

emerge in this colony and fly freely.

In situations where some drifting is likely to

occur, the drone comb can be caged in an excluder cage permitting the drones to emerge in

the drone producing colony or in an incubator,

marked as they emerge, and permitted to fly

from their original colony or from any convenient colony. The drones can be sprayed with

lacquer or enamel from a pressure can using a

splatter head in place of the usual spray head.

By using different colors a number of kinds of

drones can be held in the same colony. Such

drones, if caught as they return from the field,

will be free of feces and therefore more pleasant to handle than confined drones and less

likely to carry disease.

A method we use to mature drones in large

numbers is as follows: A drone nursery colony

is made up when the drones are'nearly ready to

emerge. A standard Langstroth two-story hive

is used. Excluders are placed just above the bottom board and between the two hive bodies. The

colony is made up with two or three combs of

worker brood (all stages), two or three combs

containing honey and abundant pollen, and 4 to

5 pounds of bees. The combs, including the

comb of drone brood, are placed in the lower

body. Unwanted drone brood in the worker

brood combs is killed at time of installation and

10 days later when young drone brood that was

overlooked is old enough to be easily seen.

Other combs are added later if needed. The bees

are released from a screen cage in the second

body and, as they pass through the excluder to

the combs below, the unwanted drones among

these bees remain above the excluder and are

lifted oflf the next day with the upper hive body

and destroyed. An auger hole in the hive body

wall above the entrance, 1 inch in diameter,

covered with excluder, permits taking drones

into small cages during the afternoons for use

in insemination.

The bees rear their own queen which remains

virgin because she cannot escape through the

queen excluders to leave the hive to mate. Her

presence prevents the development of laying

workers. When the drones have been utilized,

the colony is disposed of or the excluder removed for the virgin to mate and establish a

colony.

During the afternoons the drones attempting

to leave the hive crowd the forward part of the

excluder leaving the rear part for the worker

bees to pass through thus preventing clogging

of the entrance. All manipulations are made

early in the morning while drones are not normally flying. The three most important requirements of the drone nursery colony are ample

young bees, ample pollen^ and absence of a laying queen.

As described, such a nursery colony will take

good care of about 1,500 drones—^the number

that might emerge from half of a standard

Langstroth comb. A larger colony should be

established for a larger number. When the desired number of drones has emerged, the drone

comb can be removed to restrict the age span,

or the drone comb can be moved to a new unit

every few days so that all the drones in a given

unit will mature at about the same time. For

best results, confined drones should be used

soon after they mature (between 12 and 20 days

old) although they can be used as early as sperm

can be obtained.

The above method is especially useful when

large numbers of drones are needed. When only

a few drones of each of several kinds are needed, it may be more convenient to mature them

in cages in a nursery colony like the one desscribed for queen storage. In fact, drones and

virgin queens can be kept in the same colony.

A drone maturing cage (fig. 13, B) has a screen

which slides out ; the other side is covered with

excluder material. The cages are placed in the

colony in holding frames with the excluder facing a comb of unsealed brood. The drones can

be emerged from drone brood in cages in an incubator and transferred to the storage cages

soon after emergence, or newly emerged drones

can be collected from the breeder colony.
26 AGRICULTURE HANDBOOK 390, U.S. DEPT. OF AGRICULTURE

'1

:i: ...,.».JR.. .;

,*JM» tSBSa <MM»» Sii

BS» MM^4ÍpÉin ««•«

»•' «tasiB MUM» asBi'

Ti

•I

FIGURE 13.—A, Drone collecting cage; and B, drone maturing cage.

COLLECTION OF DRONES FOR INSEMINATION

In collecting drones for insemination, we

make use of a screen cage 4 by 4 by IV2 inches

(fig. 13,4) with a 1-inch hole that can be

closed with a rubber stopper. When this cage is

held over the hole in the front of the drone nursery colony, a large number of drones can be

collected in a few minutes as they seek exit in

the afternoons. If they are not to be used immediately, worker bees are added through a

funnel, the cage is laid on its side in the laboratory, and a gravity feeder containing sugar syrup is placed on the screen. These cages of

drones can also be temporarily stored between

brood combs in a queenless colony or above a

queen excluder in a queen-right colony. Drones

can be kept this way overnight or for a day or

two.

For use in insemination, these cages of

drones, with or without worker bees, are placed

in a larger screen cage next to the insemination

apparatus. The larger cage is 7 by 7 by 12

inches with a door at one end through which the

drones can be taken one by one by reaching into

the cage. The other end is covered with a queen

excluder and opens to the outside of the building. Both drones and worker bees are attracted

to the outside light, and the worker bees gradually leave through the excluder.

In bad weather or in the mornings when

drones are not attempting to leave the hive,

they can be taken from the combs, but young

drones must be avoided. The older drones are

usually found on the side combs in the lower

part of the hive. When drones are confined to a

nursery colony but not caged, the older drones

can be easily distinguished. The drones gradually rub off the hair on the upper surface of

the thorax just behind the head as they try to

leave the hive through the excluder material.

The older the drones are, the larger this polished area will be.
ü^^

INSTRUMENTAL INSEMINATION OF QUEEN BEES 27

CONTROL OF

When rearing daughter queens or drones

from a selected breeder colony, one must take all

possible precautions to assure that the queens

and drones are reared from the breeder's eggs,

not from any other source.

In rearing queens, the young worker larvae

grafted into the cell cups must be those originating from the breeder queen. Adequate assurance of this is that the breeder queen can be

positively identified, that she is present at the

time, and that no other laying queen is present.

Alternate breeders should be available in case

the first choice is lost or superseded.

In rearing drones, care must be taken to

avoid the use of drones reared from eggs laid

by laying workers of a foreign source which

may have drifted in from other colonies or

which may have been added to maintain the

breeder colony or drone nursery.

The use of drones from laying workers can

largely be prevented by the following managePARENTAGE

ment practices applied to breeder queen colonies and drone nursery colonies:

1. Whenever possible use bees that are

daughters of the breeder queen.

2. If unrelated bees are used, avoid taking

bees from colonies that have been queenless recently because such bees are more likely to become laying workers.

3. When utilizing drones and queens in insemination, discard any that are not typical of

the stock.

4. Use drones from a drone nursery colony

within 23 days of the time it was made up, or

destroy any drone brood that could be developing from laying workers' eggs. A drone nursery

colony may develop some laying workers easily

if it should accidentally become queenless.

5. Whenever practical, use bees of a distinguishable color when stocking breeding colonies

or drone nursery colonies, so that queen and

drone progeny of the worker bees are easily

recognizable.

BIBLIOGRAPHY

(1) BISHOP, G. H.

1920. FERTILIZATION IN THE HONEY BEE. I. THE

MALE SEXUAL ORGANS : THEIR HISTOLOGICAL

STRUCTURE AND PHYSIOLOGICAL FUNCTIONING. Jour. Expt. Zool. 31: 225-265.

(The structure and function of the

drone's reproductive organs.)

(2)

1920. FERTILIZATION IN THE HONEY BEE. II.

DISPOSAL OF THE SEXUAL FLUIDS IN THE

ORGANS OF THE FEMALE. Jour. Expt.

Zool. 31: 267-286. (A study of natural

insemination.)

(3) CAMARGO, J. M. F. DE and GONCALVES, L. S.

1968. NOTE ON TECHNIQUES FOR INSTRUMENTAL

INSEMINATION OF QUEEN HONEYBEES.

Jour. Apic. Res. 7: 157-161. (Staining

the vaginal orifice with fast green; a

manipulator for the valvefold probe.)

(4) FRESNAYE, J.

1966. INFLUENCE DES VARIATIONS DE L'AGE DE

MARURITE SEXUALLE CHEZ LES REINES

D'ABEILLES (APIS MELLIFICA) FECONDEES

PAR INSEMINATION ARTIFICIELLE. Ann.

Abeille 9: 237-242. (Sexual maturity

reached later by queens during cool

weather.)

(5) LAIDLAW, H. H.

1944. ARTIFICIAL INSEMINATION OF THE QUEEN

BEE (APIS MELLIFERA L.) : MORPHOLOGICAL

BASIS AND RESULTS. Jour. Morph. 74:

426-465. (The importance to insemination of the reproductive organs of queens

and drones; the importance of the queen's

valvefold; new equipment for insemination including the sting hook and valvefold probe; better insemination than earlier techniques.)

(6)

1949. DEVELOPMENT OF PRECISION INSTRUMENTS

FOR ARTIFICIAL INSEMINATION OF QUEEN

BEES. Jour. Econ. Ent. 42: 254-261.

(History of equipment for instrumental

insemination; description of Laidlaw apparatus and its use.)

(7)

1953. AN ANESTHETIZATION CHAMBER FOR THE

ARTIFICIAL INSEMINATION OF QUEEN BEES.

Jour. Econ. Ent. 46: 167-168. (Laidlaw

queen holder redesigned to include anesthetization chamber.)
28 AGRICULTURE HANDBOOK 390, U.S. DEPT. OF AGRICULTURE

(8)

(9)

LAIDLAW, H. H.

1954. BEEKEEPING MANAGEMENT FOR THE BEE

BREEDER. Amer. Bee Jour. 94: 92-95.

(Rearing drones, maturing drones, and

queens in nursery colonies.)

(17)

(10)

(11)

1958. ORGANIZATION AND OPERATION OF A BEE

BREEDING PROGRAM Tenth Internatl. Cong.

Ent. Proc. (1956) 4: 1067-1078. (Scheduling and production of inseminated

queens; testing of breeding stock.)

and ECKERT, J. E.

1962. QUEEN REARING. Univ. Calif. Press, Berkeley and Los Angeles. 165 pp., illus.

(Rearing and care of queens; bee breeding; instrumental insemination.)

MACKENSEN, O.

1947. EFFECT OF CARBON DIOXIDE ON INITIAL OVIPOSITION OF ARTIFICIALLY INSEMINATED

AND VIRGIN QUEEN BEES. Jour. Econ. Ent.

40: 344-349. (Carbon dioxide hastens onset of laying in queens not naturally

mated.)

(12)

(13)

1948. A NEW SYRINGE FOR THE ARTIFICIAL INSEMINATION OF QUEEN BEES. Amer. Bee

Jour. 88: 412. (First design of Mackensen syringe.)

(14)

1954. SOME IMPROVEMENTS IN METHODS AND

SYRINGE DESIGN IN ARTIFICIAL INSEMINATION OF QUEEN BEES. Jour. Econ. Ent. 47:

765-768. (Directions for the construction

of the improved Mackensen syringe, and

the management of queens and drones

used in insemination.)

(15)

1955. EXPERIMENTS IN THE TECHNIQUE OF ARTIFICIAL INSEMINATION OF QUEEN BEES.

Jour. Econ. Ent. 48: 418-421. (The sexual maturity of drones and queens; variations in the treatment of queens and

sperm used in instrumental insemination.)

(16)

1964. RELATION OF SEMEN VOLUME TO SUCCESS IN

ARTIFICIAL INSEMINATION OF QUEEN HONEY BEES. Jour. Econ. Ent. 57: 581-583.

(The number of sperm which get into the

spermatheca from semen loads of differing sizes.)

and ROBERTS, W. C.

1948. A MANUAL FOR THE ARTIFICIAL INSEMINATION OF QUEEN BEES. U.S. Bur. Ent. and

Plant Quar. Et-250, 33 pp. (Techniques

and equipment for artificial insemination and the care of queens and

drones.)

(18)

(19)

NOLAN, W. J.

1932. BREEDING THE HONEY BEE UNDER CONTROLLED CONDITIONS. U.S. Dept. Agr.

Tech. Bui. 326, 49 pp. (Early techniques

and equipment for instrumental insemination.)

PooLE, H. K. and TABER, STEPHEN, HI

1969. A METHOD OF IN VITRO STORAGE OF HONEYBEE SEMEN. Amer. Bee J. 109: 420-421.

ROBERTS, W. C.

1946. THE PERFORMANCE OF THE QUEEN BEE.

Amer. Bee Jour. 86: 185-186, 211. (Comparison of honey yields between colonies

with naturally mated and instrumentally

inseminated queens.)

(20)

1947.

(21)

(22)

(23)

(24)

(25)

(26)

A SYRINGE FOR ARTIFICIAL INSEMINATION

OF HONEY BEES. Jour. Econ. Ent. 40:

445-446. (First syringe with a removable

plastic tip.)

RUTTNER, F.

1956. THE MATING OF THE HONEY BEE. Bee

World 37: 2-15, 23-24. (A review of

knowledge of natural mating.)

ed.

1970. THE INSTRUMENTAL INSEMINATION OF THE

QUEEN BEE. Apimondia Publishing House,

Bucharest, Romania. 78 pp. (A thorough

treatment of the subject including anatomy and physiology of reproduction and

genetic aspects.)

SNODGRASS, R. E.

1956. ANATOMY OF THE HONEY BEE. Comstock:

Ithaca. 334 pp. (Includes the anatomy of

the reproductive systems of queens and

drones.)

TABER, S., HI

1961. SUCCESSFUL SHIPMENT OF HONEY BEE SEMEN. Bee World 42: 173-176. (Describes

preparation for shipment.)

WATSON, L. R.

1927. CONTROLLED MATING OF QUEEN BEES. American Bee Journal, Hamilton, 111., 50 pp.

(Early techniques and apparatus for instrumental insemination.)

WOYKE, J.

1960. (NATURAL AND ARTIFICIAL INSEMINATION

OF QUEEN HONEY BEES) Pszczel. Zes. Nauk.

4:183-275. (In Polish.) (Comparison of

natural and instrumental insemination;

adequacy of insemination.)

(27)

1964. CAUSES OF REPEATED MATING FLIGHTS BY

QUEEN HONEYBEES. Jour. Apic. Res. 3:

17-23. (Poor inseminations lead to further

mating flights in natural insemination.)

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