A manual for the artificial insemination of queen bees

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Title:
A manual for the artificial insemination of queen bees
Physical Description:
Book
Language:
English
Creator:
Mackensen, Otto, 1904-1995
Roberts, W. C
United States -- Bureau of Entomology and Plant Quarantine
Publisher:
U.S. Dept. of Agriculture, Agricultural Research Administration, Bureau of Entomology and Plant Quarantine ( Washington, D.C )
Publication Date:

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All applicable rights reserved by the source institution and holding location.
Resource Identifier:
aleph - 030360990
oclc - 10650882
System ID:
AA00023159:00001

Full Text







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A MANUAL FOR THE ARTIFICIAL INSEMINATION OF' QUEEN BEES

By Otto Mackenser and W. C. Roberts,t/
Division of Bee Culture


INTRODUCTION

Ever since the discovery that the queen mates in the air, the con-
trol of mating has been the bee breeder's dream. A number of methods
have been devised for controlling the natural mating act, of which iso-
lation at mating stations has been the most satisfactory. It has long
been realized, however, that absolute control of mating could only be
attained through some method of artificial insemination, and many efforts
have been made in this direction with varying degrees of success.

The many early attempts at artificial insemination have been reviewed
by Nolan (5) and, although the early investigators have advanced the
method, the works which have contributed most are those of Watson (9, i0),
Nolan (E5, 6), and Laidlaw (2). By modifying the apparatus and methods
devised by these three men, the writers have been able to obtain results
far superior to any heretofore presented, and it is the purpose of this
publication to describe the apparatus and procedure in sufficient detail
so that anyone may duplicate them.2/

THE REPRODUCTIVE ORGANS

To be successful with artificial insemination one must be familiar
with certain anatomical features of the queen. Although several workers
have studied the sex organs of bees, Laidlaw (2) made the most recent
and thorough study from an artificial-insemination standpoint, and was
the first to recognize the full significance of the valvefold of the
queen in the success of the insemination process.

The tip of the abdomen of the queen is made up of an upper, or
dorsal, plate and a lower, or ventral, plate, which close at the tip
like a clam shell. The cavity that these plates enclose is called the
sting chamber. In figure 1 the tip of the abdomen is shown in proper
position for artificial insemination, with the dorsal plate (DP) and the
ventral plate (VP) drawn apart exposing the sting chamber and its various
structures, including the sting (ST) and the vaginal orifice (VO).


j/ In cooperation with the Louisiana State University and the
University of Wisconsin.

/ The insemination apparatus, exclusive of microscope and anesthe-
tic equipment, can be purchased from the Department of Economic Entomology,
King Hall, University of Wisconsin, Madison, Wis.




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Figure 2 illustrates the internal portions of the reproductive tract
with the side toward the observer removed. The dorsal and ventral plates
and the sting are not shown, but portions of the walls of the sting chamn-
ber are seen at the right. A fold across the anterior floor of the sting
chamber loosely separates a region called the bursa copulatrix from the
sting chamber proper. The vagina (V), through its vaginal orifice (VO),
and the bursal pouches (BP) open into this region. There are two bursal
pouches, their openings lying at the side of and below the vaginal orifice.
Only the left pouch is shown in figure 2. The position of their openings
in relation to the vagina, when the queen is properly mounted for insem-
ination, is shown in figure 1, BP. They are easily found with a dull
probe. Sometimes, when the queen is poorly mounted, a beginner might
mistake a bursal pouch for the vagina.

The spermathecal duct (SPD) from the spermatheca (not shown) enters
the vagina anteriorly from above. Just below the opening of this duct is
the valvefold (VW), a large tonguelike structure with transverse ridges,
which make it distinguishable from other tissues when viewed through the
vaginal opening. Its position is such that it can close the passage
between the -agina end the median oviduct (SH and K) with a valvelike
action. The paired oviducts (POV) enter the median oviduct anteriorly.
Each paired oviduct leads to an ovary (not shown). They are large fluted
structures capable of great expansion for the temporary storage of sperm
after mating and of eggs in a laying queen. In figure 2 the reproductive
tract is extended. During insemination the queen is held in such a way
that the vagina is collapsed, and the valvefold often appears to lie
just inside the vaginal opening (fig. 1, VF).

Laidlaw found the diameter of the vaginal orifice to vary from 0.65
to 0.68 mm., and the average diameter of the oviduct orifice to be 0.33 mm.
These diameters are important considerations in syringe construction.

A detailed knowledge of the anatomy of the drone reproductive organs
is not necessary for the mastery of the insemination technique. A des-
cription of their structure and function is given by Laidlaw (2). Dur-
ing natural mating the penis everts, and the reproductive fluids are
ejaculated probably more or less simultaneously with the version. The
semen, a crear-colored fluid containing the sperm, passes out first and
is followed by the mucus, which is more viscous, pure white, and coagu-
lates after ejaculation. Further details about the ejaculation process
are given under Insemination Procedure.

Queens just returned from the mating flight have been studied by
a number of investigators. According to laidlaw (2), who reviewed the
earlier work, most of the semen is found in the oviducts, some is in the
vaFirna, and some has already migrated into the spermathecal duct and
aperma+lteca. Parts of the penis are fourd in the sting chamber buried
in mucus, whicl. also extends into the folds of its walls. Since the
penis apparently does not enter the vagina, the queen probably lowers
the valvefold during the mating act to permit the semen to pass.





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In order to get the amen in its natural position by artificial
insemination, the valvefold mst be pushed ventrally to permit the point
of the syringe to pass into the median oviduct. If the syringe enters
only the mouth of the vagina, the semen presses the valvefold against
the median oviduct and is forced back around the syringe and out into
the sting chamber. The vagina is not easily distendable, but the ovi-
duots expand to take care of a large quantity of semen.

BQWIPMENT AND CONSTRUCTION OF INSTRUMENTS

Microscope and Light

The major equipment needed to perform the artifioial-insemination
operation is illustrated in figure 3, and the manipulating apparatus with
instruments in place is shown in more detail from the side of the operator
in figure 4. The binocular dissecting microscope should preferably be one
provided with a revolving nosepiece or other mechanism making adjustment
from low to high power easy. The low power should give a magnification of
about 6 diameters, and the high power about 20 diameter. A single inter-
mediate magnification can be used. When two powers are used, sperm is
taken into the syringe under low power and injected under high power. A
still higher magnification will be found useful in making and measuring
instruments* An attached lamp that always illuminates the focal point
of the microscope is a great convenience.

Carbon Dioxide Equipment

Carbon dioxide serves as an anesthetic* The equipment for its appli-
cation is illustrated in figure 3. This gas is obtained in cylinders from
wholesale growers or similar supply houses. To reduce the high pressure
in the cylinder, a regulator is provided which permits adjustment to a
delivery pressure of 4 to 6 pounds per square inch. A needle valve perm!Vte
adjustment of the flow of gas to a very fine stream. A rubber tube carries
the gas to the queen holder by way of a three-way stopcock, which permits
diversion of the stream of gas while the queen is being mounted, without
disturbing the needle-valve adjustment. Another line leads into a jar,
in which queens are given additional anesthetizations to be described
later.

The Manipulating Apparatus

The manipulating apparatus is a stand (fig& 4) on which the queen holder
.QH), the syringe (S), and holding hooks (VH and STH) are mounted in such a
way that they can be adjusted or manipulated. It is essentially the same
as the apparatus developed by Nolan (5), with modifications which make more
accurate adjustment possible. The stage (ST) is made of a piece of 1/4-inch
iron plate, 3 inches wide and 11 inches long, raised at each end by a piece
of wood thick enough to permit the microscope base to slip under the stage*
The weight of the stage gives stability to the apparatus. Two upright
3/8-inch brass rods threaded at the bottom end are screwed into the stage
at the points illustrated, and to these rods all other parts are attached.
The rod at the left of the operator is 3 inches high and the one to the
right 3 1/2 inches. These rods are 4 1/2 inches apart center to center.




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The mount for the queen holder is made of wood, and consists of a
queen-holder mounting block (QHMB) attached to a horizontal strip (HS).
The strip is 1/2 by 3/4 by 6 inches, with a hole bored near each end so
that it can slip up and down on the two posts and with set screws to
hold it fast at any desired level. Near the right-hand post is a hori-
zontal slot about 2 inches long. A long bolt passing through this slot
and also through the block makes it possible to adjust the block at any
desired angle* The queen holder (QR) fits into a hole in this block, and
a set screw holds it securely in place. A wide groove is out on the set-.
screw side of the hole, and a piece of leather tacked to the top of the
block fits into this groove preventing the set screw from scratching or
breaking the queen holder. It also keeps the queen holder in place by
light friction until the set screw can be tightened. The set screw works
in a hole bored just small enough for the threads to take hold.

Since the mountings for the syringe (S), the ventral hook (O). and
the sting hook (8TH) are essentially similar, a detailed description of the
syringe mounting will suffice for all three. A block of wood, the syringe
mounting block (8MB), is bored so that it will fit snugly over the post
as illustrated* On the left side of the post this block is divided by a
vertical saw out, and a 3/16in oh bolt is provided with which the separated
parts can be pulled together if necessary to increase friction on the post.
This bolt may have a wing nut, or one side of the divided block may be
bored to a smaller diameter. In any case the head of the bolt must be
flat enough not to interfere with the movement of the syringe*

On the other side of the post the syringe holder (SH) is attached to
the block* The syringe holder is simply a piece of sheet metal out and
bent into the shape of a box 1/2 by 1/2 by 1 1/2 inches. A 1/8 by 1-inoh
bolt 1/2 inch from one end holds the bottom of this box to the blook*
This bolt projects about 1/2 inch back of the blook, and a piece of coil
spring is put on under the nut so that the friction between the box and
the block can be increased or decreased by tightening or loosening the
nut* In each end of the box a hole is bored of such a size that the
syringe will slip through easily but not loosely* A curved piece of
clock mainspring (8P) slipped in between the syringe and the side of the
box holds the syringe in place. The curvature of this spring can be ad-
jusated to permit the syringe to slip in and out easily but still stay in
place when released* The ventral hook and stine hook are mounted in the
same way, except that the boxes for them are 3/8 by 3/8 by 1 1/2 inches*

It is important to keep the syringe and the hook handles clean sand
polished with oil or a hard wax to make them slip easily. If the wooden
parts are treated with wax, they will not expand and contract excessively
with changes in humidity*

Queen Holder

The queen holder (fig. 6) is a modification of the type devised by
Jans I. Hambleton and first used extensively by Nolan (E). It is a tube
slightly constricted at one end, into which a long stopper fits snugly.
The queen is made to back into the holder until her abdomen protrudes





-f-


from the small end and then is held in place by the stopper. Both parts
are made of Lucite, a transparent plastic. The stopper is also a tube,
which permits a stream of carbon dioxide to flow gently over the queen,
keeping her anesthetized during the insemination operation* The drilling
and polishing of Luoite are disouessed later under Plastic Syringe.

Dimensions are given of the holder used suooessfully by the authotse
There oan be some flexibility of dimensions, but it is well to remember
that the thoraxes of most well-developed queens range in diameter from
0*195 to 09205 inoh* The opening at the end of the holder may be from
0.18 to 0*19 inoh in diameter* The 5/16-inoh taper is adequate to permit
only the last three segments of the queen to protrude from the holder*
This taper may be obtained by stretching the plastic tube after heating,
as described under construction of the syringe tip, or by drilling with a
suooession of drills* Several small grooves should be out with a three-
oornered file on the inside of the holder at the tapered end to facilitate
the escape of gas.

The stopper diameter must be very near the diameter of the holder
(0*257 inch) to move in and out of the holder with sufficient friction to
remain firm at any plaoes If the stopper loses friction through wear, its
diameter may be increased with a light coat of plastic cement or lacquer*
Thse slight protuberance at the front end of the stopper has four addition-
&l lateral outlets (not shown in diagram)* This protuberance aids in
adjusting the queen in the holder and protects her antennzmae from being
mashed. The enlarged knob at the posterior end holds the rubber tubing
onto the stopper* The stream of carbon dioxide from the cylinder passes
through the stopper, is dispersed by the five openings at the end of the
stopper, and flows over the queen and out the end of the holder*

If facilities are not available for making the plastic queen holder,
a glass one ais easily made by drawing 9-gn glass tubing to about 4 ma.
outside disaster, finding the right inside diameter for the small ends
breaking at this point, and grinding off the edges. Since ooommeroial
glass tubing varies in size, it is necessary to select a piece of the
proper inside diameter from the 9-ane size.

The stopper is a piece of spongy paper, suoh as paper toweling,
rolled ever a match stiek, which is later pushed out to make a thick-
walled tube* If the tube is of the right six*, friction with the walls
of the glass holder keeps it in plaoe* It is made long enough to project
from the glass holder when the queen is in plaoe, and additional paper is
glued onto this *aed to make it taper* If preferred, the carbon dioxide
supply tube ear easily be slipped on and off the tapered end at each
operation, and the threewway stopoock eliminated.

Syringe

The syringe is a modification of the instruments used by other workers
in this field* It consists of a small tube inside of which a tightly
fitting plunger draws in and expels the semen. Two types of syringes are
being used*




-8-


Glass syringe. For the glass syringe (fig. 8) a glass tube is
employed as a plunger barrel. This tube will hereafter be referred to
as the glass tip or simply the tip. A brass spring wire serves as a'
plunger, and mechanical pen!il, as recommended by Nolan (.), provides
the mechanism for moving the plunger* The glass tip is attaohed to the
end of the pencil housing, and the plunger takes the place of the leads
A number of ppeoial tools and materials are needed for a conetruation
of this type.

For drawing capillary glass tubing an ordinary bunasen burner will do,
but a blast burner is a great convenience where beth gea and air are avil-
able* A gasoline blowtorch has been found very satisfactory,

A jewelers' fine-grain glass-agrinding wheel is needed for grinding
off glass tips to the right diameters Such a wheel oan easily be mounted
on a shaft and made to turn slowly by means of a belt and motor. The
lower part of the wheel should dip into a tray of water to keep it flooded
as it turns. The cooling action of the water prevents chipping of the
glasses

A special gas burner is needed for producing a fine flame for use in
forming the tapered end of the glass tips* This burner oan be a piece of
6-mm. glass tubing drawn to an inside diameter of 045 me at one end. The
burner is attached to an ordinary gas outlet, which is adjusted to produce
a flame about S mma in diameter.

A oompound microscope equipped with a micromaeter eyepiece and having
a magnification of about 60 diameters is useful in measuring the disasmeter
of the glass tips as well as other small parts of the insemination equip-
ment while under construction. Some of these measurements san be made
with an ordinary micrometer* If a compound microscope is not available,
a micrometer eyepiece can be used in the dissecting microscope with a
magnification of about 60 diameters,

A number of small tools that can be obtained from jewelers' supply
houses are needed* They include No# 6 out files of various shapes, small
flat-nose and pointed-nose pliers, and rods and triangular slips of fine-
grain Arkansas stone* The end of a rod is ground on a coarser stone to
a cone-shaped tip, rhich is used in grinding the inside edges of the
capillary tubing. These tools are also used in making the holding hooks
and vaginal probe.

A supply of No. 26 B.and S. gage wire should be on hand for making
plungers. Several kinds of wire will do, but spring brass is preferred
because it is stiff, yet solders well, and does not corrode easily*

Some mneohanical pencils are better adapted for syringe making than
others. One having a round housing and using a 4-inch lead has been found
mn'st satisfactory. This type (fig* 6) has a stationary, hollow metal core
with a slot running its entire length. The lead and lead holder slide in
this oore, and a spiral made of steel wire or a steel strip encloses
Jnmst its entire length. Projections on the lead holder extend through
'he slot into the spiral, and as the spiral lo turned the holder slides,
movin- the lead in or out of the pencil.





-9-


A number of changes must bo- made to adapt a pencil to its new use.
The friction that keeps the lead at the adjusted level must be eliminat-
ed. Usually the pencil must be taken apart to accomplish this. Since
the friction is caused in different ways in different makes of pencils,
the exact procedure cannot be described. The point of the pencil is
sawed or filed off sufficiently to let the lead holder project. If the
housing is hexagonal, it should be replaced with a round tube, preferably
of solderable material. The lead holder should be filed to a smaller dia-
meter to allow more play between it and the core, or it can be replaced
entirely with a piece of wire bent and filed into shape. "hen the pencil
is reassembled the spiral should turn without friction.

For the plunger a piece of 26 B. and S. gage brass spring wire is
selected which has no kinks to prevent a tight fit. Any gentle bend
resulting from the wire being wound on a spool may be straightened by
pulling the wire between thumb and forefinger a number of times, but
sharp kinks are difficult to remove and should be avoided. One end of
the wire is ground off squarely, and the burred edges are polished.
Then after the wire has been cut to the proper length, the other end is
soldered into the hollow end of the lead holder. The length of the plung-
er will depend upon the construction of the pencil and on the length of
the completed glass tip. It should be slightly longer than the tip, and
long enough not to slip out of its upper end when withdrawn as far into
the pencil as possible. If there is still some bend in the plunger, it
should be attached so that the back of the arch will rub against one of
the sides of the core. Such a position will prevent the arch from push-
ing the end of the glass tip against the spiral and possibly breaking
it, and at the same time will prevent the end of the plunger from slip-
ping out of the core groove when drawn into the pencil while no glass
tip is present.

To make a good glass tip requires some practice and the exercise
of proper care. The first step is to draw out a supply of capillary
tubing. Ordinary 6-mm. soft-glass tubing has been found very satisfac-
tory for this purpose. Long sections of fairly uniform bore can best
be obtained by heating a section of tubing about 1 inch long, drawing
slowly at first until the center of the section is of about the right
diameter, and then drawing more rapidly to a length of 3 to 4 feet.
'With 26 gage (0.41-mm.) wire the inside diameter is tested until a
piece of nearly uniform bore about 60 mm. long is found which fits
closely. The glass is broken at the point where the end of the wire
fitted most tightly, and this end is made the inside end; the larger
end becomes the point of the syringe. The inner edge of the small end
is ground under water by twirling on the pointed Arkansas stone, so that
there will be no sharp edge to scratch the plunger and gradually decrease
its diameter. The plunger should not be inserted from this end until
the glass tip is completed and ready for mounting, and then only under
the microscope, because the glass is easily broken if the plunger is not
inserted perfectly straight.




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The large end of the tip is drawn to a smaller diameter to form the
point of the syringe* To do this heat a spot near the end in the small
gas flaae and draw the ends to a diameter of somewhat less than 0.27 Sa
Sometimes two or three attempts must be made before a satisfactory product
is obtained. The drawn section is then scratched with the sharp edge of
a stone, broken off at the proper point, and ground back to an outside
diameter of 0,27 rome The outside edge is ground off lightly on the grind-
ing wheel and the inside edge by spinning between thumb and forefinger on
the pointed stone under water, or with a drop of water covering the point.
The point is then polished until it appears perfectly smooth when magnified
about 60 times, by twirling the tip on the end of a soft piece of wood
rubbed with Jewelers' rouge, or by holding it against such a piece of wood
mounted on the end of a motor shaft. The completed tip should to 35 to
50 me long. To attach it to the pencil, put it over the plunger and draw
it into the pencil until it sticks out about 26 nam. then glue it into
place with an aoetone-soluble cement su*h as is used to build model air-
planeso This oemeat is easily dissolved when it becomes necessary to
replace the tip.

The proper diameters at the point of the glass tip are of utmost
importance. The outside diameter must be small enough to permit passage
into the oviduot, and yet the inside diameter must be large enough to per-
mit semen to be taken up easily and quickly* The outside diameter should
not be over 0S mm., and 0.27 m-e is usually satisfactory. The inside dia-
meter should not be less than 0*15 =* These limits differ slightly from
the dimensions recommended for the plastic syringe*

When made as described with 26-gage wire (0.41 mmo), the glass tip
has a oapaoity of 1 Rm43 for every 7*6 mm, of length. If a 27-gage
(0o36 mm.) plunger is used, the capacity is 1 nam. for every 9.8 mm. of
length. Good instruments have been made with both sizes. Tips of smaller
diameter are too delicate and have too small a capacity to be practical.
Larger ones are more difficult to finish at the point with a large enough
inside diameter and a small enough outside diameter.

Plastic syringe. The plastic syringe is made of Lucite and brass.
Most of itw parts must be made on a lathe. The Lucite tip is superior to
glass because it is not easily broken, sharp edges are eliminated, and it
can be machined and drawn to the desired diameter and bore. Threaded re-
placeable tips can be readily changed and are easily cleaned. Lucite does
not have the surface hardness of glass, and consequently will wear slowly.
It softens when heated. Grease and oil can be removed with hexane,
naphtha, methanol, or mild soap and water.

The machining qualities of Luoite and brass are similar. Since this
plastic is a poor conductor of heat, it is necessary to prevent overheating
when drilling, cutting, or polishing. Drills should be lubricated with
mineral oil or an oil solution C2 percent of soluble oil in water). Fre-
quent removal of chips is necessary when deep holes are drilled, and the
holes are filled with the oil solution at each removal of the drill.
Sharp drills and proper lubrication give uniform and polished inside walls
to the tip, which result in good suction without a lubricant*






-11-


All the syringe parts are shown in figure 7, and the structural
details of the tips and plunger are given in figure 8. Sinoe the measure-
ments and details of construction of the housing, turning screw, couplings,
and supplemental rod are of secondary importance, they will not be described,
Their approximate size can be ascertained from the picture. The univArsal
joint between the turning screw and the supplemental rod allows the metal
plunger to move within the tip without rotating. Lack of rotation reduces
wear and gives better suction.

The tip is made from a Lucite rod of 0.1875-inch diameter* This rod
is placed in the machine and drilled to a depth of about 1 inch with a
No. 72 (0.025 inch) drill. A smaller drill-No.79 (0.0145 inch)-provided
with a long shank is then used to drill an additional 0.1 inch in the
bottom of the 0*025-inch hole. The outside diameter is then turned down
to 0.164 inch, and the end threaded for a distance of about 0.25 inch with
an 8-32 threading die* Before the rod is removed from the machine, its
diameter between the threaded portion and the end of the drilled portion
can be reduced to the approximate size of the main portion of the tip
(0,086 inch).

The end of the tip containing the small hole is then rotated on an
eleotrlo soldering iron until it becomes soft enough to stretch. This
Oel-inch section with the 0.0145-inch bore is then stretched to a length
of 0*2 to 0.25 inch and, while still held taut, is hardened in cold water.
If the rod is properly heated, the 0.025-inch hole is not distorted. The
0.0145-inch hole becomes a gradual taper from that diameter down to less
than 0.008 inch. The end of the tip is then filed off square, and the
outside diameter reduced to 0*012 inch. No. 2 out files can be used to
reduce the diameter to near size, but the polishing should be done with
9/0 production finishing paper. The final polish may be obtained by rub-
bing with moistened cigar ash.

The plunger can be made from brass spring wire of 0.025-inoh dia-
meter* It must be straight and uniformly round without snarp edges.
The butt end is soldered into a 0.0625-inch brass rod which has been
threaded to screw into the supplemental rod of the syringe.

Holding Hooks

The function of the holding hooks is to hold the sting chamber open
and the sting back out of the way during the operation. The ventral hook
(figs# 1 and 4, VHII; fig. 9) fits over the ventral plate* It is constructed
of a piece of No. 24 gage brass wire slightly flattened at the end, and
bent as illustrated in figure 9. The distance from the bottom of the U to
the tip is 2.5 mmi, and the distance between the inside walls of the U is
0*37 mm. The tip of the ventral plate fits into the U-shaped portion so
that the plate remains in a vertical position as it is pulled ventrally
by the hook,

The dorsal or sting hook (figs. 1 and 4, STE; fig. 9) is desired to
pull the entire sting mechanism dorsally* JA similar instrument was used
by Laidlaw (2). It has an enlargement at the end, which fits into the





-12-


triangular area between the basal portions of the sting lancets, and
extends underneath them (fig. l)e Figure 9 gives'a side view of this
instrument at A and a top view at B from the angle indicated by the arrow
in A. The enlarged tip is 0.77 mm. wide* The thinnest part of the stem
is 0*08 mm. when viewed from above and 0*17 mm. when viewed from the aide.
The stem is bent to fit the queen parts. This instrument is made from
wire about 0.92 mm. in diameter, and filed down to the proper shape with
jewelers' files. The enlarged tip represents the end of the wire filed
off at an angle.

All rough edges of both the ventral and sting hook are polished first
with a fine stone and then with jewelers' rouge. Both hooks are soldered
to the ends of brass rods 3/16 inch in diameter and 5 inches long, whioh
serve as handles and fit into the sheet-metal boxes.

Other Equipment

Other items of equipment needed for the operation are a pair of for-
ceps, a sting depressor, a dish of water for cleaning the syringe, a vagi-
nal probe, and a container for treating drones with chloroform.

The sting depressor is simply a dull-pointed dissecting needle.

The probe (fig* 9) is an instrument used to push the valvefold down-
ward. It is a piece of No* 24 gage brass wire bent at right angles 4 Nm.
from one end* This end is flattened to a thickness of 0*13 mm, on a plane
with the main stem of the instrument and polished down to a width of about
0.19 mm. The probe is bent slightly near the tip, so that the main part
of the wire does not obstruct the view when the tip is inserted into the
vagina.

Drones are made to ejaculate in a 1-by-4-inch glass vial containing
paper soaked in chloroform wadded in the bottom. A stopper fastened to
the table so that the vial can be put onto it in a horizontal position
is a great time saver.

INSEMINATION PROCEDURE

Adjusting the Flow of Carbon Dioxide

The end of the carbon dioxide supply tube is iinersed in water and
the flow of gas adjusted to a slow bubbling. It should not be faster than
Jis necessary to keep the queen completely anesthetized. Experience will
soon show just how much this should be. The tube is then passed through
the queenholder mounting block from below, the stopper attached, and the
three-way stopcock in the supply line turned, to prevent gas from passing
through the stopper while the queen is being mounted.

Preparing the Syringe

To function properly the tip of the glass syringe must be filled
with water, which acts as a lubricant and increases the auction of the
plunger. Water is first drawn into the tip by withdrawing the plunger*






-13--


Then, while a finger is held against the end, the plunger is pushed out
again until the air around it has been entirely replaced by water* A
5-amn column of water is left at the end of the plunger.

The plastic syringe can be prepared in much the same way as the
glass syringe when a lubricant seems necessary Ordinarily the water
will be present after the rinsing operation described later. The plunger
should be pushed out as far as it will go.

Tap water has been found satisfactory, but in some localities distilled
water may be better. Physiological salt solution must not be used with the
glass syringe, because it cannot be thoroughly rinsed and, when the water
dries up, the salt residue freezes the plunger in the tip and corrodes
the syringe parts.

Preparing the Queen

The queen is made to walk into a tube similar in size and construc-
tion to the queen holder When she reaches the partly closed end she
begins backing up, and if the queen holder is quickly put in place she
usually backs into it* The stopper is pushed in after her until her
abdomen protrudes from the small end of the holder and she cannot move
about readily. For best results only about the last three segments of
the abdomen should protrude*

The three-way stopcock is turned so that carbon dioxide will flow
through the queen holder* The queen breathes heavily for a few minutes
and then gradually becomes quiet. If her abdomen expands abnormally,
the gas is being forced into her abdominal air sacs, an indication that
the flow is too strong. Unless very severe this action seems to do no
harm, although it makes insemination difficult@ The holder is withdrawn
into its mounting block by pulling on the carbon dioxide supply tube,
and tightened in place by the set screw. The dorsal part of the queen
should be at the right of the operator* The queen holder should be about
30 from vertical, with the upper end leaning to the right (fig. 4)*

The queen is usually quiet by the time she is completely mounted,
and the holding hooks can be put in place. This is done under low-power
magnification* First one hook and then the other is inserted into the
sting chamber, and the abdominal plates are 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 the sting hook is left in this position to prevent unnecessary
drying of the delicate tissues while the syringe is being loaded.

Filling the Syringe

The microscope is withdrawn slightly and the syringe placed in its
box, with care not to break the tip. This is done with greatest safety
by depressing the spring with forceps while the syringe is being inserted.
The microscope and syringe are then so adjusted that the end of the syringe
is in focus.





-14-


A drone is anesthetized by droppinghim in the vial already described.
His abdomen contracts and usually the penis partially everts, as illus-
trated in figure 10, A. He is then made to evert more completely by
being squeezed between thumb and forefinger. There is great variation
in the degree of version, the distribution of semen and mucus, and the
amount of semen ejaculated, Eversion usually stops at the stage illus-
trated in figure 10, B, which is about two-thirds complete* As the pro-
cess of version proceeds, the cream-colored semen passes out first fol-
lowed by the thicker white mucus* If the version process can be stopped
at just the right point, a drop of pure semen will be found at the tip
with all the mucus left inside, When this ideal situation exists it is
very easy to take up the semen. Usually, however, at least some of the
mucus comes out after the semen, and the two are distributed on the penis
in various arrangements. Often the semen is spread so thinly over the
mucus that it is difficult to take up.

It is best to see that an ample supply of drones is available and to
use only those that evert and ejaculate most satisfactorily* This is
especially important when individual matings are being made, so that the
maximum amount of semen is obtained from the single drone used* Movement
of sperm causes the semen to spread in a thin layer over. the mucus and to
mix with it until too thick to be taken into the syringe easily. This
mixing also takes place while the drone is partially everted. It is,
therefore, important that drones be utilized as soon as possible after
anesthetization. Without abdominal contraction semen is rarely obtainable,
but when the abdomen contracts without partial version the version can
often be completed by pressure, and a good amount of semen obtained.

The ejaculated drone is brought near the tip of the syringe with the
left hand and the plunger withdrawn slightly to make an air bubble. The
surface of the semen is then made to touch the point of the syringe at
about a 45-degree angle. If the syringe is raised slightly after contact
has been made, the semen will adhere to it and flow toward it as the plung-
er is withdrawn. This procedure helps to avoid the mucus, which is more
viscous and will not flow so easily as semen. Mucus is too thick to pass
through the end of the syringe easily and will stop the passage of semen.
lIhen this happens, the plunger is pushed out until the passage is clear-
ed and then the.taking of semen is continued. By moving the syringe
about, the mucus ban be skimmed of practically all its semen covering.
Semen is taken from as many drones as necessary to fill the syringe to
the desired point.
!
As the syringe is filled, it will be noticed that the suction pulls
some of the water from around the plunger, thus increasing the amount
above the air bubble. Later, as the plunger is pushed out during injec-
tion, this water passes back around the plunger. This action will be
negligible with a well-made tip. When the plunger is poorly fitted,
however, it is sometimes withdrawn as far as it will go before the
desired amount of semen has been drawn up. In this case the plunger can
be pushed down again while a finger is being held over the end. Such a
tip is a time consumer and should be replaced as soon as possible. Some
water should remain between the end of the plunger and the air bubble to
pass back around the plunger as the semen is injected; otherwise the air
bubble, or even some of the semen, may pass around the plunger.






-15-


The function of the air bubble is to prevent the mixing of semen
and water and to mark the boundary between them so that diluted semen will
not be injected. As injection proceeds semen adheres to the side walls
and mixes with the water which follows, so that in the absence of an air
bubble it is difficult to distinguish between pure and diluted semen.
dhen a series of inseminations is made without the syringe being cleaned,
as is possible with the plastic syringe, the air bubble is superfluous.

Injection

As soon as the syringe is filled, the point is moistened to prevent
clogging by drying of semen and to lubricate it. The syringe is moved
into position over the queen and the microscope pushed forward so that
the queen is in the field and the magnification changed to high power.
The sting hook is then drawn dorsally until the sting chamber appears as
in figure i. This act stretches the loose membranes so that the vaginal
orifice, and often the valvefold, are visible* It also stretches the
dorsal wall of the vagina so that the syringe can slide along it into the
median oviduct,

With the left hand the probe is inserted into the dorsal part of the
vagina and the valvefold pushed veutrally, until the point of the syringe
has passed beyond. Then, as the syringe is pushed in farther, the probe
is removed. The syringe should be inserted no farther beyond the valve-
fold than is necessary for satisfactory insemination (about 1 to 1.5 mm.
The plastic tip can be inserted deeper than the glass tip on account of
its more gradually tapering point. If it appears that the syringe carries
tissue in with it as if caught on the end of the syringe, then the point
has not hit the, median oviduct and should be withdrawn and reinserted,
possibly after some readjustment of the holding hooks.

'With the syringe in place the plunger is slowly pushed downward.
If the semen moves down the barrel easily and does not leak out around the
point, then the syringe is in proper position and the injection can proceed
rapidly. If the glass syringe is used, great care must be exercised that
the plunger never reaches the cone-shaped point. The plunger of the plas-
tic syringe, on the other hand, can be pushed to the end of the larger
bore without danger of breakage.

The syringe is now withdrawn from the queen, and then from its box,
the spring being depressed with forceps so that the syringe slips out
easily without danger of the point breaking. Removal of the queen from
her holder completes the operation.

Cleaning the Syringe

The glass syringe should be cleaned immediately, by alternately draw-
ing in and expelling fresh water until the liquid inside the syringe is
relatively clear. When the plunger does not fit closely, a little semen
left in the liquid may help to increase suction.

LBRARY
STATES p BOARD




-16-


Regardless of the care exercised, a coating gradually accumulates
on the inside walls of the syringe, and finally interferes with the
passage of semen. This coating can be removed with a piece of wire
0.002 inch in diameter slightly bent at the end. Flakes that are too
large to pass through easily can usually be removed by drawing back the
plunger slowly so that water passes around and behind them and then push-
ing out very suddenly so that they will be carried with the stream of
water Several repetitions of this procedure will usually clear the
barrel. The )la3s tip can be cleaned by dipping in a hot solution of an
alkali such as sodium hydroxide or common lye, which dissolves the depos-
its; however, the lalkali must be thoroughly rinsed out before the syringe
can be used acain. "hen using alkali it is best to withdraw the plunger
15 to 20 mm. and leave the tip full of air, then to draw in a small quan-
tity of the hot alkali. Later the alkali can be forced out and the rins-
inz done without any contact with the plunger.

The plastic syringe need be cleaned only when it is to be stored or
when the plunger becomes too tight. Cleaning is simple. The tip and plunger
are unscrewed, and water is drawn in and out with the plunger with such
force that usually all obstructions are easily rinsed out.

Sterilizing the Syringe

a.;n a quick change is made from one genetic type of sperm to another,
it is necessary to sterilize the syringe. The glass syringe is sterilized
by rinsing it thoroughly and then dipping it in boiling water deeply
enough to submerge part of the pencil as well as the exposed part of the
glass tip, so that the inside end of the tip also becomes heated. If several
syringes are available, a fresh one can be employed for each kind of drone
used in a single day. The plastic tip is sterilized by quickly drawing
in and expelling 95 per cent ethyl alcohol, and then thoroughly rinsing
with water immediately, because Lucite is slowly soluble in alcohol.

ESTIY.TIING THEi NUMBER OF SPERLMS

One criterion of successful insemination is the number of sperms that
reach the spermatheca. A fairly good estimate can be made by the following
method:

Items of equipment needed include a counting chamber such as is used
to count blood corpuscles, a 50-cc. burette, a medicine-dropper pipette, a
small glass dish, a pair of sharp-pointed forceps, and a dissecting needle.
The pipette should have an inside diameter of about 1 mm. at the tip. For
the glass dish an individual saltcellar almost as deep as wide, having a
capacity a little over 10 cc., and having a rounded bottom somewhat smaller
than the diameter of the rim, has been found to be just the right size and
shape.

To dissect out the spermatheca one must tear off the last segment of
the abdomen by grasping, the last ventral plate with forceps. The spermatheca
is usually found imbedded in the tissue inside this removed segment. It
is a sphere about 1 mm. in diameter tnd appears rough and white because of
the network of tracheae which ocverS it completely. ;'.hen the tracheae are





-17-


removed, the spermatheca is found to be smooth and transparent in a
virgin queen and milky-white to cloudy-cream in mated queens, depending
on the degree of insemination.

The spermatheca is placed in the dish and 1 cco of tap water added
from the burette* Then the spermatheca is broken with the sharp-pointed
forceps and needle, the sperm teased out, and the empty skin removed The
sperm is dispersed by alternately drawing the water into, and expelling
it from, the pipette about twenty times, or until all lumps have disappeared.
Then 9 cc* of water is added to make a total of 10 co. and the sperm again
thoroughly dispersed with the pipette. A drop of this mixture is quickly
placed in the counting chamber. Vnhen the sperm number seems to be small,
a dilution to only 5 co or less can be used* The sperms are counted
under a compound microscope with a magnification of 60 diameters, and
against a dark field, which makes the sperms stand out as white filaments.
From the number counted in a certain volume the number in 10 cc. is calcu-
latede

The sperms usually appear as almost headless, slightly curved, fila-
ments about 0.27 mm. long, but they may be coiled or looped into various
shapes, sueh as small circles, spheres, or like the numerals 6 and 8.

It is important that the dispersion be continued no longer than is
absolutely necessary and that the drop be placed in the counting chamber
quickly before any great amount of settling or adhesion of sperm can take
place. It is also important that the pipette be kept cleaa to prevent
lumps of sperm from sticking to its walls* In some localities a physiolog-
ical salt solution may be more satisfactory than tap water. Distilled water
is not recommended.

There is, of course, considerable chance of error in making counts
from such a small sample. Nevertheless, this method is certainly more
accurate than judging by appearance of the spermatheoa and the viscosity of
the contents, as has been done by previous investigators. Greater accuracy
can be obtained by counting a larger sample# All sperm counts given in
this manual were made by diluting individual matings to 5 cc. and other
matings to 10 coo. and counting the sperms in 0.8 mm.3

The number of sperms in the seminal vesicles of drones can be estimated
by the same methods A window is carefully out into the dorsal wall of the
abdomen and the seminal vesicles are very gently out off where they join
the mucus glands. If this is not done very carefully, the muscles of the
seminal vesicles will contract and some of the sperm will be lost. After
being placed in water, 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 seminal vesicle to pieces*

THE USE OF CARBON DIOXIDE

Carbon dioxide not only serves as an anesthetic, but also stimulates
queens to begin laying (Mackensen 4). Proper treatment with this gas
causes virgin as well as 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.




-18-


At least two anesthetizations of 10 minutes duration spaced a day
apart are necessary to start egg laying. These may or may not be accom-
panied by insemination, An anesthetic treatment without insemination is
given by placing the caged queen in a convenient container through which
carbon dioxide is flowing (fig. 3)o If the queen is to begin laying at
the normal age (8 to 11 days after emergence), the second treatment must
be completed before the seventh day, and preferably before the sixth day,
because she begins laying 2 to 6 days after the second treatment. Although
treatments given as early as the second and third day are effective, they
do not cause laying to start earlier than the normal age*


PROCEDURES FOR VARIOUS TIPS Or NATIMCS AND RESULTS
TO BE EXPECTED


Inseminations should be made from the fourth to the tenth day after
emergence. They have often been made earlier than this (second and third
day), but at times with poor results* At the other extreme, queens can be
inseminated at any age if they have not begun laying, but if not made to
start soon after the tenth day, they will be balled and mistreated by the
worker bees, and the percentage and quality of the laying queens will be
reduced.

As much as 10 mm.3 of semen has been given in one operation with suc-
cess, but because the percentage of sperm that reaches the spermatheca de-
clines as the load increases, it is better to give several small insemina-
tions than a single large one. Except for individual matings, a load of
2.5 to 4.0 nmm.3 has been found very satisfactory. Results are also less
variable when several insemination are made than when an equal amount of
semen is given in a single insemination It is best to allow 2 days between
inseminations for clearing of semen from the reproductive tract, although
often insemination on successive days have been quite successful. The
operation should be performed as rapidly as possible to avoid unnecessary
exposure of the sperm.

Individual Matings

Often it is desirable to mate each queen with a single drone* In this
case it is important that the maximum possible number of sperms be obtained
from the single drone used. Therefore, well-matured, well-nourished drones
should be available, and only those used that seem to ejaculate perfectly
and to yield a large amount of semen heavy with sperm. The best drones
yield nearly 1 mm#0 of semen. Individual matings are usually made only in
genetic experiments in which it is imperative that the sperm come from but
a single drone. Therefore, if a series of such matings is to be made, the
syringe must be sterilized between operations to prevent contamination with
sperm previously used. In individual matings the percentage of laying
queens is usually high, and most of them will produce some worker offspring.
However, so few sperms reach the spermatheca that the supply does not last
long, and such matings are only made when absolutely necessary. One addi-
tional carbon dioxide treatment must be given to start egg laying.








One Insemination With Sperm From Several Drones


When neither individual mating nor loag prcdu-tivet life is desired,
several drones from the sam source can be utiae:, m'kAnth ooe injection con-
sisting of at least 2.5 mm. of semen. Vlith this procedure it is almost
certain that none of the queens will begin laying as partial or complete
drohe layers, and if kept in nuclei most of LIaurn wll la & season with-
out becoming drone layers. One additional carbon dioxide treatment is
necessary to start egg laying.

Two or More Inseminations

To fill the spermatheca sufficiently to carry a queen through a season
in a large colony, two or more inseminations must be made, each consisting
of 2,5 mm.3 of semen. An additional carbon dioxide treatment is not neces-
sary. Hundreds of queens mated two or three times have performed as well
as naturally mated queens*

A still greater number of sperms can be made to reach the spermatheoa
by giving three or four insemination with 2*5 mm.3 of semen. They are
given on alternate days beginning on the fourth day or, in the case of four
insemination, they may be started the third day to reduce the likelihood
of interference with the beginning of oviposition.

Considerable variation can be expected in the percentage of laying
queens obtained. In one group of 95 queens inseminated three times, 83 per
cent started laying; however, results as low as 60 percent can be expected
occasionally. In general, the more inseminations made the more chances of
injury and consequent loss. Practically all queens that survive to laying
age will begin laying.

Table 1 compares the number of sperms in various artificially inseminated
and naturally mated queens. These data are taken from several experiments.
Note that the coefficient of variation decreases as the number of insemina-
tions is increased, and that it is lower in some of the inseminated queens
than in those naturally mated. Note also that the minimum in the group
inseminated three times is higher than the minimum in the naturally mated
queens, and that the average and maximum in the group inseminated four
times are nearly as great as the corresponding figures for the naturally
mated queens.

The performance of artificially inseminated queens compares very well
with that of naturally mated queens. In 1945 queens inseminated artificially
three times were compared with queens of identical parentage mated naturally
at an isolated mating station. Surplus honey produced was about the same
for the two types of matings, as already reported by Roberts (7) Brood
production and brood quality were not significantly different, but survival
to the end of the season was somewhat lower in the artificially inseminated
group.





-20-


Table 1.- A bompazison between the number of sperm in the spermatheoa
of naturally mated queens and artificially inseminated queens
inseminated as indicated.
Number of sperms in
Number of spermatheca (millions) Coefficient
.. .Treatment queens Average Range .of variation

-P-rtificial insemination:

Semen from one drone 17 0.87 0*22 2*24 61

Semen from many drones
(2.5 mm.)

Onoe 11 2.97 1.28 4.41 56
Twice 9 4.11 2.05 5.66 29
Three times 10 4*85 3.71 5.80 16
Four times 11 56.052 466 6*79 10

Natural mating 33 5.73 3.*34 7.36 18


THE ABSOLUTE CONTROL OF PARENTAGE

The purpose of artificial insemination is the control of parentage,
and the virgin queens and drones used are reared from eggs laid by selected
breeder queens which are already mated. The virgins are reared from fertil-
ized eggs and inherit from both the breeder queen and the drone with which
3he mated, whereas the drones develop from unfertilized eggs and inherit
only from the breeder queen. These are the desired breeding individuals,
but occasionally other types of individuals appear in the hive, with
which they might be confused. These types are (1) drones and queens
reared from unfertilized eggs of laying workers and (2) queens reared
from the unfertilized eggs of the breeder queen. Only the drones are a
serious menace to pure mating, but all types will be discussed because
they must be considered in the interpretation of genetic experiments.

The number of off-type drones and queens depends largely upon the
number of laying workers in the hive which varies considerably with race,
season, and hive conditions. Queenlessness for any length of time will
cause some workers to start laying, and they may continue to lay after
the colony has been made queen right. Laying workers are apparently more
common in early spring, when the hive is populated mainly with old, over-
-:intered bees. They are very common among the Cape bees of South Africa
(Jack 1), but in the bees of the United States and the European races
they are relatively rare.

Under conditions where they could be positively identified, 13 off-
4ype drones were found to emerge among about 5000 drones of the right type
roared in a full-sited colony, which is at the rate of 0.26 per cent. Drones
have been reared under controlled conditions for a number of seasonsand
although the percentage of off-type drones has usually not been determined
by actual count, it is estimated not to have exceeded 0.26 per cent. Often





-21-


none were found among several thousand drones. They can be recognized
and avoided if the breeder queen is introduced to a colony of bees of a
distinguishable color. For example, if a queen of a yellow strain is
introduced to bees of a black strain, her sons and daughters will be
yellow and the sons of worker bees will be black.

If a selected breeder queen is supported entirely by her own worker
daughters, it is not so important to guard against the off-type individuals,
because all descend from the breeder queen and her mate, and therefore repre-
sent only the desired germ plasm. Often, however, it is unpractical to
wait for a queen to produce her own supporting population.

The other types of undesirable individuals, queens reared from unfer-
tilized eggs of either queen or worker, are called impaternate because they
have no male parent. These queens are quite common in some races. Among
Cape bees of South Africa laying workers develop readily and produce female
offspring in abundance when the hive becomes queenless (Jack 1). In other
races they have been considered to be rare or not to occur at all, but
Mackensen (3) has proved their occurrence in three American strains of bees
representing the Caucasian and Italian races. He found female progeny in
the brood of 21 out of 50 virgin queens tested. Of the larvae grafted
from one queen producing a high percentage of female offspring, 0O85 per
cent developed into queens. Impaternate females also occur in the related
insect Habrobracon juglandis Ashm., and Speicher (8) has presented evidence
that they develop from diploid eggs which arise from patches of tetraploid
tissue in the ovaries.

The frequency with which impaternate daughter queens of laying workers
are likely to be encountered in rearing queens by the usual methods has
not been determined. If the percentage of impaternate females developing
from unfertilized eggs of queens. (0.85 per cent) can also be taken as the
percentage of laying worker eggs that develop into females and the rate
of occurrence of off-type drones (0926 per cent) is taken as the percentage
of laying worker eggs in the hive, the expected rate of occurrence of
queens of this type can be calculated to be 0.000022 per cent, or 1 in
4*5 million. Obviously they are so infrequent that precautions need not
be taken to avoid them. If it should be desirable, the method described
for off-type drones could be used.

Impaternate queens developing from eggs of the breeder queen inherit
only from her. In ordinary breeding work there is no way of recognizing
and avoiding them. They probably do not occur among the larvae grafted at
all because fertilization would lead to triploidy and probably death, and
the larvae ordinarily used for grafting develop from fertilized eggs.

CARE OF QUEENS

To provide the most natural conditions queens are not kept in nursery
cages prior to insemination, as recommended by some workers on artificial
insemination, but introduced as cells and maintained in nuclei until they
begin laying, Excluders are placed over the hive entrances to prevent the
loss of queens by mating flights* As soon as possible after emergence,





-22-


7hile the qu -r re still r1 sizable -s newly emerged, one wing is
eli_-..*- to n-event flight and the rthiorax is marked with a spot of color,
A .-._r called trLib--iao mixed with the desired pigment is suitable au
&-- A solutc. of celluloid in acetone is also a satisfac-
...- vehicle for t'r.c pi :_-nt.

..The q.-':L tken iretly f.rom the nucleus and returned immediately
..er .- l.t:rj, whi.la she i si'l completely motionless from the effect
of c.rbor ji:c to ball her than if she is active. In warm weather she is dropped into
the nucleus, but in cool weather she is left in the cage in which she is
transported. 71,1-: case is opened and slipped between the top bars of the
frames*

.LR.IN'} AND CARE OF DROONES

It is not always casy to obtain large numbers of drones of the
desired : rentU-,e During the spring they can usually be obtained in
abundutce romn tny queen, because they are naturally produced at this sea-
son, but later in the year it is sometimes almost impossible to obtain
any drones at all from some queens. Sometimes, even though the queen will
lay in drone comb, the workers refuse to rear larvae to maturity. "hen a
heavy flow is on, the drone .nmb is filled with fresh nectar so that the
queen does not have a chance to lkiy drone eggs.

-ro.es are most likely to be obtained out of season if conditions
similar to those existing during the swarming season are created. Pollen
and sugar sirup can be fed if necessary, and a crowded condition created
either by adding bees and brood or by reducing the size of the hive.
Vshen the nectar flow is heavy, empty combs can be provided to help keep
the drone comb clear. If drone combs are extracted gently, the thin nec-
tar can be removed without destroying eggs and young larvae that might be
present in some of the cells.

i- sure wav to obtain drones is to rear then from drone-laying queens,
which can be produced by treatln- virgin queens with carbon dioxide, as
alreijy described. These virgins would, of course, be daughters of the
selected broaler queen.

The follo.- ing routine has been used successfully for a number of years
to rear and maintain lar-c numbers of drones of controlled parentage in
yellow strains;

A five-fre nucleus is used as a drone-rearing colony. It is made
up with three combs of brood r-o- black selected Caucasian stock, a comb
of honey, and a drone comb, which is placed in the middle of the nucleus.
Extra i.p-.len is provided by sprin.lin: dry pollen pellets into the combs
and sorydyi.r with water. The yellow breeder queen is introduced with a
ph-in e and released 2 or 3 day9 later, depending on how well she is
accepted. 'hr- nucleus is stocked with 3 1/2 pounds of black bees added
from a screen cage which covers the top of the nucleus hive* They are
forced to ,o th: o', an e-,ludor, which sifts out drones as they leave
Othe oe ,ih-:. bee are -e-. n'ifinild for 2 days. Pollen is added oooa-
sially, nd th nucleus is sometimes fed very lightly to help stimulate
the "'" :':"









As soon as the first drone comb is well filled, another drone comb
is substituted for one of the brood combs. This is usually about a week
or 10 days after the queen has commenced laying. After another week or
10 days the first drone comb is removed to a drone nursery colony and re-
placed with a third drone comb. In this way sometimes three or four drone
combs can be filled within a month after laying starts.

Special precautions are taken to prevent strange yellow bees, which
might develop into laying workers, from drifting into the drone-rearing
colony. The colony is established at least 100 yards away from the queen-
yard and frequent examinations are made to find and kill any yellow bees
that might have drifted in. If other drone-rearing colonies are kept
near by, they are removed before yellow bees begin to fly from them.

The drone nursery colony is placed in or very near the queen yard,
so that drones from it are conveniently available for insemination. It
is kept separated from other bees as much as possible to prevent drifting
in of yellow bees. A 1-story 10-frame standard hive is provided with 5 or
6 combs of brood and 4 pounds of bees of black stock added from a screen
cage through an excluder, together with some honey and a great excess of
pollen* The bees are confined for 2 days. A screen top is placed under
the cover, so that ventilation can be provided by lifting the cover slight-
ly when the excluder at the entrance becomes crowded with drones trying to
get out. The bees rear their own queen, which remains a virgin because
she cannot get out of the hive to mate. Her presence prevents the develop-
ment of laying workers. More brood is added later to provide a continual
supply of young bees.

If the nursery colony is so situated that yellow bees drift in, the
drone combs are removed after 24 days, and all other drone brood is killed
at 10-day intervals. These precautions are taken to prevent sons of lay-
ing workers from maturing, and are unnecessary when the entire worker
population is of a body color distinguishable from the drones being nursed
by the colony.

All manipulations of the nursery colony are made early in the morning
while drones are not normally flying. Often it is necessary to work the
colony in a portable screen cage to prevent stray drones from drifting in.
After long confinement or when the presence of excluders has kept them
out of the hives during the night, drones will often fly in the morning.

The screening of bees for rearing and nursery colonies serves two
purposes. It eliminates superfluous black drones which would crowd entrances
unnecessarily and also prevents the accidental addition of a queen. For
these reasons the procedure has become a matter of routine not only in
stocking these colonies but also in stocking nuclei.

At the age of 8 days drones become sexually mature and try to leave
the hive* At this age the maximum number of sperms has accumulated in the
seminal vesicles, but the drones often do not ejaculate well until they have
been trying to get out for a number of days. The feces that accumulate








du-ing coninement seem to be an aid rather than a hindrance. Its pre-
sence seems to increase the pressure inside the abdomen when the muscles
cotract, assuring good version of the penis and ejaculation* It has
been noticed that drones whose flight has not been restricted by confine-
ment to the hive will ejaculate better and in greater numbers after they
have been kept in a cage for a day or two with workers, sugar sirup, and
pollen. Drones do not live so long when confined as when they are able
to fly naturally. In the nursery colonies some of.them die soon after
they reach the age of 25 days, and few reach 35 days.

Three L'n:'ortant requirements of the drone nursery colony are ample
bees, ample pollen,and queenlessness. Drones need the care of worker bees.
V'hen kept in cages and provided only with pollen, water, and sugar syrup,
they soon die, but when about twice their number of worker bees are added,
they live to sexual maturity and contain a good quantity of usable semen.
*rien pollen is omitted drones live for some time, but very few sperms
develop. ' are present; they live longer and mature better in queenless colonies,
in 1944 the young drones in two queen-right nursery colonies were noticed
massed on the bottom board with not a single one on the combs* After the
queens had been removed long enough for the bees to realize their queenless-
ness, all the drones were found on the combs apparently being well oared
for.

SV MARY

By modification of the equipment and methods of earlier workers on
artificial insemination of queen bees, the technique has been improved suf-
ficiently to make its use practical in bee breeding as well as in genetic
studies. In this manual the important features of the anatomy of the sex
organs, the construction of special equipment, and the insemination proce-
dure are described* The essentials of the method are as follows:

(1) The semen is taken from the everted penis after the drone has
been placed in chloroform fumes to induce partial version and the abdomen
squeezed to continue the version until the semen has appeared at the end
of the penis.

(2) The semen is deposited in the oviducts in natural mating. To
do this it is necessary to push aside a tonguelike projection, from the
ventral wall of the reproductive tract, called the valvefold, so that the
point of the syringe can roach the median oviduct opening,

(3) The syringe tapers at the end so that its point is small enough
to enter the median oviduct, while the main Larrel is large enough to have
a practical capacity.

(4) At least two inseminations are given at 2-day intervals to approx-
imate the normal number of sperm in the spermatheca.

(5) Carbon dioxide is used as an anesthetic. Phis gas not only
relaxes the queens, but also stimulates them to begin laying. Two anesthe-






-25-


tizations of 10 minutes' duration spaced 1 or 2 days apart will cause
virgin as well as inseminated queens to begin laying promptly; whereas
without this treatment few begin earlier than 30 days after emergence.

Individual matings can be made, but so few sperms (0.87 million)
reach the spermatheca that most queens stop laying fertilized eggs within
2 or 3 months. If 2&5 mm.3 of semen, taken from several drones, is given,
sufficient sperms (2,97 million) reach the spermatheca to last a season
if the queen's laying is restricted by keeping her in a nucleus hive.
Two inseminations of this size provide sufficient sperm (4.11 million)
to assure fertilized eggs for a season in a full-size colony. With four
inseminations of this size almost as many sperms (5.52 million) reach
the spermatheca as are found in naturally mated queens (5.73 million).

Sperm numbers are estimated by dispersing the sperms of the sperma-
theca or of the seminal vesicles of the drone in a given volume of water,
counting the number of sperms in a measured sample in a counting-chamber
slide, and calculating the number in the total volume.

Queens to be inseminated are introduced as cells and confined to the
hive by queen excluders. Soon after emergence the virgins ar3 marked and
their wings clipped. They are returned to the nucleus immediately after
insemination while they are still completely anesthetized.

When drones are reared from a selected breeder queen, sometimes as
high as 0.26 per cent of the drones obtained develop from the unfertilized
eggs of laying workers. These off-type drones can be avoided if the
breeder queen is first introduced to a colony stocked with worker bees
having a body color distinct from that of the breeder queen, so that the
drones produced by the worker bees can be recognized and discarded. Virgin
queens are also known to develop from unfertilized eggs of laying workers,
but are so rare that they need not be considered seriously by the bee
breeder.

Drones can usually be produced by creating conditions similar to
those existing at swarming time. Crowding of bees, reducing the laying
space for the queen, and feeding of sugar sirup and pollen are some of the
steps often necessary. A sure way to obtain drones is to produce drone-
laying queens by carbon dioxide treatment. Drones in large numbers are
best cared for in queenless colonies, confined by queen excluders, with
brood, bees, pollen, and honey added as needed.





-26-


LITERATURE CITED

(1) Jack, Rupert W.
1917. Parthenogenesis amongst the workers of the Cape honey-bees
Mr. G. W. Onions' experiments. Roy. int. Soo*, London,
Trans. 1916s 396-403, illus.

(2) Laidlaw, H. H., Jr.
1944. Artificial insemination of the queen bee (Apis mellifera L.)s
Morphological basis and results. Jour. Morph. 74s 429-4C5,
illus.

(3) Mackensen, Otto
1943a The occurrence of parthenogenetic females in some strains
of honeybees. Jour. Boon* Ent. 36t 465-467.

(4) ______
1947. Effect of carbon dioxide on initial oviposition of artifi-
cially inseminated and virgin queen bees. Jour. Econ. Ent.
40: 344-349.

(5) Nolan, W. J.
1932* Breeding the honeybee under controlled conditions. U. S.
Dept. -4r. Tech. Bul. 326, 49 pp., illus.

(6) _____...
1937. Improved apparatus for inseminating queen bees by the
Watson method. Jour. Econe Ent, 30: 700-705, illus,

(7) Roberts, N. C.
1946. The performance of the queen bee. Amer. Bee Jour. 86s
185-186, 211, illus.

(8) Speicher, Kathryn Go, and B. R. Speicher*
1938. Diploids from unfertilized eggs in Habrobracon. Biol. Bul.
74: 247-252, illus.

(9) Aatson, Lloyd Re
1927. Controlled mating of queenbees. 50 pp., illus.
Hamilton, Ill.

(10) ____________
1929. New contributions to the technique of instrumental insemina-
tion of queen bees. Jour. Lcon. Znm. 22s 944-954, illus.





-27-


Figure 1,.-View of sting chamber of queen properly opened for insemination:
BP, Opening of bursal pouches; DP, dorsal plate; dH, queen holder; ST.
sting; ST sting hoof; VF, valvefold; VH, ventral hook; 0, vaginal
orifice; VP, ventral plate.





-28-


Figure 2.-Reproductive tract of queen daudad of the ovandes, extended,
with side removed to show relation of valvefold to median oviduct and
vagina: BP, Bursal pouch; K, keel of median oviduct; Z.L, paired ovi-
duct; S, shelf of median oviduct; M. sting membrane; SPD, spermathe-
cal duct; V, vagina; VF valvefold; VO, vaginal orifice; VP vaginal
passage. (rm jdlaw





























Figure 3.-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 cyl-
irnder with pressure regulator attached.
















--SH


SMB-ri



IQ-


VH

* -. **-,''._ ,, ,i'^ p


--SP


STH


ST


'cE
a


Figure 4--Manipulating apparatus viewed from operator's side, showing mounting
of queen holder, syringe, and holding hooks: HS, Horizontal strip; Q, queen;
H, queen holder; M, queen-holder mounting block; S, syringe; SH., syringe
holder; SMB., syringe mounting block; SP, spring; ST,, stage; STH., sting hook;
VH ventral hook.



J. -4 -


I-- INS. DOIA. 257'


'-OUT.DIA 3.62*


QUEEN HOLDER


- DIA, 081


STOPPER


Figure 5.--Structural details of the queen holder.


IN& oIA .1875--


144 -MEM"


t' ..a ":' '. :'. -1 *'i--






,LEAD HOLDER
< I


,GLASS TIP
/ /PLUNGER
/ /
/ / [^ ^

C E M E N T' / J, *

A


v II %I
\ \
\ SPIRAL.,
CORE,.
A


N 4-


C


Figure 6.-The glae syringe: A, Longitudinal section showing modifications of the zaechanical Pencil;
B, end of glass tip enlarged; Z, cross section at point indicated.


I
!lI ii
...... ----


0


LEAD


'^r13






-31-


Figure 7.-Parts of plastic syringe. (From top to bottom): Turning
screw with supplemental rod, two Lucite tips, two plungers, and
housing.


LUCITE TIP


,025" DIA ..... ...)
0-- -INS DIA .008"
.014 5"DA,.-- -
OUTS DIA O12"
---------25" ---------

END OF TIP


PLUNGER


Figure 8.-Structural details of the Lucite tip and its plunger.




-32-


VAGINAL


PROBE


VENTRAL HOOK


T
0.19 MM.


k -- 8MM..--


AI

STING


0.17MM/


HOOK


-j- 0.77MM.
0.08MM:


Figure 9.-Structural details of vaginal probe, ventral hook side view,
and sting hook with A, side view, and B, top view from the direction
indicated by the arrow X. The arrow also marks the point where the end
begins to enlarge.





-33-


A


Figure lO.--Stages of the version of drone's penis: A, partial version
usually encountered after anesthetization with chloroform; B, a more
complete version usually obtained by squeezing the abdomen, with semen
and mucus exposed.














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UNIVERSITY OF FLORIDA

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