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Manual for the cryopreservation ofthe silkworm germ cells

Yuuji Mochida, Yoko Takemura, Hiroshi Shinbo, Yutaka Banno(Published in Sanshi-Konchu Biotec, Vol 83.)

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Extirpate ovaries
from larvaeCollect ovaries in Grace insect medium
containing cryoproptectant

Store ovaries in BICELL(container)

37℃

ＬＮ
-80℃

Keep ovaries in freezer

Store ovaries in liquid nitrogen

Transplant ovaries into the castrated
recipient larva

Thaw ovaries in water bath

Rare larvae to complete theirlife cycle

Procedure for cryopreserving the larval ovary

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ForcepsRazor blade forsurgical knife
Scissors
Sterilized petri

Eppendorf tube for freezing

Cane for ampule holding

Forceps (large-scale)Lab wares used

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Manipulation of collection and transplantation ofovaries should be done in clean benches

5Star spot

3rd instar

4th instar

5th instar

Ovaries are located immediately under the “star spots”
on the 8th laraval segment

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Sterilize body surface in the part of “star spots” with alcohol

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Make small incisions on a pair of“star spots” with a razor blade

Extirpate ovaries on each side with forcepsExtirpate the larval ovaries

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Collect the ovaries in Grace insect
mediumSuspend the collected ovaries in Grace insect
medium containing 1.5M DMSO
as cryoprotectant

9BA

Transfer the ovaries into Eppendorf tube for freezingby using forceps (A) or pipette (B)10Tissue paper
Eppendorf tube

Fix the Eppendorf tube in BICELL ( Bio freezing vessel)

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BICELLFreezer inside

Keep the BICELL (Bio freezing vessel) in -80℃ freezer for ca. 3-20hrs

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Eppendorf tube

Eppendorf tube
Cane for ampule
holding
Cane for ampuleholding

Fill box of Styrofoam with liquid nitrogen,and place cane for ampule holding in theliquid nitrogen.

Then, attach the Eppendorf tube to thecane for ampule holding in liquid nitrogenby using forceps (large-scale)

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mark

Mark the tip of the cane
to distinguish samples

Store the cane in liquid nitrogen preserving container

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Sift the cane into a 37℃ water bath to thaw the ovaries

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Discard Grace insect medium containing
1.5M-DMSO into petri (upper). Then,pour Grace insect medium into the petri
containing ovaries.

Replace the medium to remove DMSO (cryoprotectanat)

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Stain the frozen and thawed ovaries with Nile Blue SulphateOvary ofrecipientThread body
Ovary ofdonorincision
Star spot

Ovaries of donor (blue) and recipient (milky white translucent)17

①

Ovaries of
レシピエント卵巣
recipient

Frozen andthawed ovaries(donor)
凍結・融解卵巣②

Ovaries of③ レシピエント卵巣recipient

18④

Frozen and
凍結・融解卵巣thawedovaries(donor)Connect thread bodies of donor and those of recipient by coiling

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Restore the donor ovary with the two thread bodies coiled
around one another into the recipient via the original incision

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Cover the incisions with paraffin

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2 days after thetransplantation

newly moulted 5th instar
larvaeRear the larvae to complete their life cycle

pupae

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testisseminal vesicle
prostate gland
penis

Extract reproductive organs frommale mothCollect semen from seminal vesicle

Store semen with cryoprotectant inplastic artificial insemination straw

37℃

ＬＮ

-80℃

Store semen in liquid nitrogen

Thaw semen in waterbath

Keep semen in freezer
Inseminate female moth withthe frozen and thawed semen

Procedure for cryopreserving the spermatozoa

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Silicon spuit

Silicon screen
Sterilized petri(large)

Disposable cup
Cryoprotective
agent

Microdispenser
Artificial insemination straw

Small holes．
○MicroSilicon dispenser
spuit

Suction tube

Cane for straw holding
Sterilized petri(small)Forceps

Lab wares used for collecting
semen

Forceps

Lab wares used for cryopreseving
semen

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Three-waysolenoid valve

Pressure
regulator

Capillary

Micro dispenser

Nitrogen gas cylinder
Foot switch

Instrument for artificial insemination

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2nd segment from
caudal portion②①

Anchor the ventral part of the 2nd segment with forceps③

Tear up the segment④

Internal whole reproductive
organs containing seminal vesicle⑤

Caudal portion containing whole internal reproductive organs

Collect caudal portion containing whole internal reproductive organs

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Petri(small)
Paper wiper
Stereoscopic
microscopeCollected caudal portion
Grace insect medium

Petri (large)
Ice pack

Prepare the collecting reproductive organs containing
seminal vesicle

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Fasten the innermost of the caudal
portion softly with forceps

Hold the caudal portion down with the left forceps, andpull the reproductive organs softly with the right forceps
reproductive organs
containing seminal vesicle

cut here

Cut the reproductive organs
off at the tip of prostate glandprostate gland

Collect the reproductive organs

28Collected reproductive organs

Wash the dirty things, like scaly hair,attached to the reproductive organs
out in the petri filled with Grace insect
mediumWash the collected reproductive organs

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Prepare the collecting semen

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testisseminalvesicle(containingampulla)
glandula
prostatica

penis

Reproductive organs of male moth31

Spread the reproductive
organs, and collect the semenby cutting the ampulla of theseminal vesicle off withforceps.
Cut the glandula prostatica off
in the same manner to collectexpressed prostatic secretion.

Collect the semen and expressed prostatic secretion

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small holes

The pipette consist of micro dispenser,
silicon screen and silicon spuit

Collect the semen by using pipette

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Store the semen in plastic artificial
insemination straw by using suction tube

Grace Air
insectmediumMixture ofsemen andcryoprotectant

Air

Grace PVA
insectmediumStore the semen in artificial insemination straw

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Seal the both tips of the straw up with heat sealer

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Straw

StrawFreezer inside

Keep the straw in -80℃ freezer for 10 min

36Fill box of Styrofoam with liquid
nitrogen. Then, place the straw
frozen at -80℃ in the liquid nitrogen

Then, attach the straw to the canefor straw holding in liquid nitrogenby using forceps (large-scale)Store the cane in liquid
nitrogen preserving container

Store the semen in liquid nitrogen37

Cut the straw at the portion containing
air, and collect the semen in petri

Shift the straw into 37℃ water bathto thaw the semen

Thaw the semen stored in liquid nitrogen38Mix the sperm with the trypsin solution at thetime of artificial insemination

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three-waysolenoid valve○capillarypressure
regulator

nitrogengas cylinderfoot switch

Artificial insemination work

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Make the glass capillary

Micro dispenser
containing semen

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Glass
capillary

Micro dispenser
containing semenGlasscapillary

Semen

Load the semen into glass capillary

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Inject an aliquot of semen
into the bursa copulatrixof virgin female

Inject the semen into the bursa copulatrix

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Manual operation for artificial insemination by using pipette

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Egg

SeminalreceptacleBursa
copulatrix

Seminalreceptacle

Bursa
copulatrix

Glass capillary

Before injection

After injection

After oviposition

Injection of semen into the bursa copulatrix of virgin female moth45

ovariessperm

cryoprotectant

cryoprotectant

extirpationcollection

freezing

freezing

thawing

thawingtransplantation

insemination(long-term preservation in -196 ℃)

Method for obtaining offspring using cryopreserved ovaries and sperm