How Mishandling Frozen Semen

can Lead to Unexplained

Breeding Failure

What You and Your Staff Need to

Know

NCBA January 2011

Brad Stroud, DVM

Stroud Veterinary Embryo Services

Weatherford, Texas

Objectives of Presentation

• Define an industry problem

• Briefly review the science of freezing cells

• Review the science of thermal exposure damage

• Show exposures commonly seen with frozen semen

• Illustrate proper handling techniques

• Tips for Dewar management

• Debate solutions for handling problems

Reasons for Low Fertility

• Nutritional stress

• Poor heat detection

• Inadequate AI skills

• Viral, bacterial, metabolic diseases

• Genetic influences

• Poor semen quality

• Mishandled frozen semen

SVES Frozen Semen Spot TestVideotape____ TC

: : :00

Sire ID:___________________ Stud ID_______ Batch #_____________

Reg#: _________________ Owner:______________ Date:_______

Dewar ID_______________ Canister_______ Cane Code__________

Flush Quality Rating: __Excellent __Good __Fair __Poor __Unacceptable

Conc % Motile RFM Direction Spiral __Morphology

__ High __0 __<5 __Rapid __Linear __ No __Excellent

__ Med __5-10 __11-20 __Med __Circular Tight __Yes __Good

__ Low __20-30 __>30 __Slow __Circular Med __Fair

Shipped___Stud ___ Owner ___Spastic __Circular Large __Poor

Extender___Milk ___ Other __Twitching __Asymmetrical __Heads

Notes: __Midpieces

Evaluator________ __Tails

This test is subjective, and may not be an accurate assessment

of this semen's ability to fertilize ova. It is however, a relatively

reliable pre-insemination screening exam. Brad Stroud, DVM

Effect of semen quality on fertilization and embryo

quality in superovulated cattle (227 bulls)

Semen

Quality*

% Fertilized

(n = 9,732)

% Excellent

Embryos (n = 4,035)

Excellent 82.1a 61.2a

Good 67.7b 55.7b

Fair 58.3c 53.9c

Poor 51.8d 33.7d

(Schrick et al., 2003)(abcdP<0.002)

(*Semen quality combined concentration, % motile, RFM,

direction of movement and morphology)

Origin of

Shipment

Total # of

Shipments

# Classified

Unaccept

able

%

Unacceptable

Bull studs 426 9 2.1

Owners/others 314 25 8

SVES Data

Effect of Origin of Shipment on Percent

Classified Unacceptable (Beef Bulls

Only)

What happens to water molecules

when they are frozen?

The primary objective in freezing

biological cells using slow

cooling is to remove the

intracellular water

Commercial Freeze Medium

Ethylene Glycol

Glycerol

Cryomicrophotograph

of oocyte during

crystallization

Bovine Embryos

are slow cooled

to – 30 Celsius

Plunged into liquid

nitrogen bath

Plastic straws are then placed into

goblets and canes

The cells are now safely preserved in liquid

nitrogen or vapor for an indefinite period of

time .

Glass Transition Temperature of

Water

-70 C

-100 C

-130 C

-150 C

- 190 C

Glass Transition

Temperature of

Water

- 130°Celsius

Membrane Damage to

SpermPlasma

Membrane

Outer

Acrosomal

Membrane

Abnormal Morphology of

Bovine Spermatazoa

A.D. Barth

R.J. Oko

Cell damage

Loss of acrosome

Slide from Dick Saacke, VPI

DIC wet mounts

Damaged acrosomes

= enzyme leakage

= fertilization failure

Important Fact

The duration and degree

of the exposure above

- 130°C determines the

extent of the damage done

to cryopreserved cells

From the time it’s collected,

Processed,

Frozen,

Stored,

Shipped,

And ultimately thawed for

insemination...

Many opportunities exist for

accidental thermal exposure,

especially after it reaches the

farm or breeding center...

The list of

personnel on a

farm, ranch, or

breeding center

that routinely

handle frozen

semen samples

is long...

The list of

personnel on a

farm, ranch, or

breeding center

that routinely

handle frozen

semen samples

is long...

The list of

personnel on a

farm, ranch, or

breeding center

that routinely

handle frozen

semen samples

is long...

The list of

personnel on a

farm, ranch, or

breeding center

that routinely

handle frozen

semen samples

is long...

The list of

personnel on a

farm, ranch, or

breeding center

that routinely

handle frozen

semen samples

is long...

The list of

personnel on a

farm, ranch, or

breeding center

that routinely

handle frozen

semen samples

is long...

The list of

personnel on a

farm, ranch, or

breeding center

that routinely

handle frozen

semen samples

is long...

The list of

personnel on a

farm, ranch, or

breeding center

that routinely

handle frozen

semen samples

is long...

The list of

personnel on a

farm, ranch, or

breeding center

that routinely

handle frozen

semen samples

is long...

Thermal exposure opportunities are

aplenty.

• Receiving a shipment

• Searching for samples to

thaw

• Thawing samples

• Preparing a dry shipper

• Preparing shipments

• Taking inventory

• Breaking a cane!!!!

• Dewar to Dewar transfer

• Cattle sales

• Storage conditions

• Who’s responsible?

– Courier

– Farm secretary

– Owner

– Farm manager

– Animal handlers

– Staff support

– Lab staff

– Veterinarian

– Embryologist

– Others?

Major Objective When Handling

Frozen Semen and Embryos

Keep the internal temperature

of straws below

– 130°Celsius at all times

8 Second Rule

•Canes or canisters should not be exposed any longer than 8 seconds in the neck of a Dewar.

•Remember, it takes 10 to 12 seconds for the internal temperature of a straw of frozen semen to reach – 100 C in the neck of a medium sized ranch Dewar half full in LN2

•Recrystallization begins at – 130 C

Essential tools for

manipulating straws of frozen

semen• Towel clamps

• Forceps

• Hemostats

• Liquid N bath

• Eye protection

• How many farms, or ranches have

an organized educational

curriculum in place for handling

and storing frozen semen and

embryos?

Challenge is educating a lot of folks

who don’t think they need

educating!

But damage occurs 130 degrees C

below the freezing point of water

DIC wet mounts

The bottom line...

1. The fate of reproductive and medical patients

is in the hands of numerous potential handlers

of cryopreserved cells and tissues.

2. Many, if not most, have never been formally

educated in the art and science of handling

frozen cells.

3. Education is the key to solving the problem

4. More work should be done to show the effects

of thermal exposures on various cell types

DVD Training Video

All Staff Should

Watch

www.frozendvd.com