The Consequences of Mishandling Cryopreserved Specimens

The Consequences of Mishandling Cryopreserved Specimens Mexico Embryo Transfer Association 2012 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

The US Cattle Embryo Transfer Industry Provides an Excellent Forum and Data to Support the Assumption

No. Recovered AETA Statistics and Data Retrieval Committee for 2006 : Reported 280,000 Unfertilized and Degenerate Ova Collected 600,000 a 300,000 200,000 100,000 0 In 52% 34% 48% Non-viable 18% Total Ova Viable Embryos UFO Degenerate

Reasons for Low Fertility Nutritional stress Poor heat detection Inadequate AI skills Viral, bacterial, metabolic diseases Genetic influences Poor semen quality Mishandled frozen semen

Video 1...Semen DIC 10x Hi conc Hi Mot DVD.mpg Video...Semen Phase 10x 5 percent Mot Mishandlrd DVD.mpg

6 5 4 3 2 Series 1 Series 2 Series 3 1 0 Category 1 Category 2 Category 3 Category 4

SVES Frozen Semen Spot Test Videotape 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

20 Years Later...

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 82.1 a 61.2 a Good 67.7 b 55.7 b Fair 58.3 c 53.9 c Poor 51.8 d 33.7 d ( abcd P<0.002) (*Semen quality combined concentration, % motile, RFM, direction of movement and morphology) (Schrick et al., 2003)

Looking a little closer at the data, we noticed two distinct consistencies.

Effect of Origin of Shipment on Percent Classified Unacceptable (Beef Bulls Only) Origin of Shipment Total # of Shipments # Classified Unaccept able % Unacceptable Bull studs 426 9 2.1 Owners/others 314 25 8 SVES Data

We saw gross differences in motility between frozen straws of identical batch numbers. Shipper

We saw gross differences in motility between frozen straws of like batch numbers. Shipper

What happens to cells that are frozen with their intracellular water intact? Video...Humphrey Animation Failure to Dehydrate.mpg

The primary objective in freezing biological cells using equilibrium cryoprotection is to remove intracellular water

Commercial Freeze Medium Glycerol Ethylene Glycol

Embryo Response to Glycerol Video...Humphrey Animation Embryo Response to Glycerol.mpg

The embryo then undergoes equilibration

And is ready for slow cooling

Almost all of the Intracellular water has been removed and been replaced by 10% Glycerol.

The remaining intracellular water must be removed by slow cooling

Embryo During Slow Cooling Video...Humphrey Animation Extracellular Soln Crystallizing around emb.mpg

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 Glass Transition Temperature of Water - 130 Celsius -150 C - 190 C

Recrystallization of Ice Electron micrograph by H. T. Meryman

Electron micrographs by Dr. Harry T. Meryman Recrystallization Water vapor on a metal plate at 196 C Water cooled to 196 C on a metallic plate Plate Warmed to 96 C for 3 minutes Cooled back to 196 C Shadowed & photographed Water cooled to 196 C on a metallic plate Plate Warmed to 85 C for 30 seconds Cooled back to 196 C Shadowed and photographed Water cooled to 196 C on a metallic plate Plate Warmed to 77 C for 1.5 minutes Cooled back to 196 C Shadowed & photographed Water cooled to 196 C on a metallic plate Plate Warmed to 80 C for 5.5 minutes Cooled back to 196 C Shadowed & photographed Water cooled to 196 C on a metallic plate Plate Warmed to 70 C for 5 minutes Cooled back to 196 C Shadowed & photographed

Dr. Meryman concluded that the longer the time, or the higher the subzero temperature, the faster that the amorphous ice undergoes Recrystallization.

Recrystallization effect on embryo Video...Humphrey Animation Recrystallization Effect.mpg

Visual Effect on Frozen Semen Straw

Thawed Semen Straw Under Stereomicroscope

Semen Straw Immediately after Removal from LN2 Video...Frozen Straw Thawing.mpg Video semen straw...frozen Straw Thawing Fast Forward.mpg

Plasma Membrane Membrane Damage to Sperm Outer Acrosomal Membrane Abnormal Morphology of Bovine Spermatazoa A.D. Barth R.J. Oko

Cell damage Loss of acrosome DIC wet mounts Slide from Dick Saacke, VPI

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

Exposure Duration/Damage For a 0.5 ml frozen semen straw, it takes 3. 5 minutes in ambient air to dissolve all of the ice crystals (total thawing) For sperm motility to decease to zero requires a 50 second ambient exposure and re-entry into liquid nitrogen. Most sperm samples require a 30 second ambient exposure and re-entry before motility is noticeably depressed. The fertilizing potential of sperm is likely depressed after only brief exposures of 5 to 8 seconds or less in some conditions. Other cell types may require more or less exposures before damage is done. The nature of the cell/tissue or packaging for example.

Important Facts Dewar Temperature Gradients