How Mishandling Frozen Semen can Lead to Unexplained Breeding Failure What You and Your Staff Need to Know

Transcription

1 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

2 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

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

6 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 Med Circular Tight Yes Good Low >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

7 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)

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

10 What happens to water molecules when they are frozen?

11 The primary objective in freezing biological cells using slow cooling is to remove the intracellular water

12 Commercial Freeze Medium Glycerol Ethylene Glycol

13 Cryomicrophotograph of oocyte during crystallization

14 Bovine Embryos are slow cooled to 30 Celsius

15 Plunged into liquid nitrogen bath

16 Plastic straws are then placed into goblets and canes

17 The cells are now safely preserved in liquid nitrogen or vapor for an indefinite period of time.

18 Glass Transition Temperature of Water

19 -70 C -100 C -130 C Glass Transition Temperature of Water Celsius -150 C C

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

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

22 Damaged acrosomes = enzyme leakage = fertilization failure

23 Important Fact

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25 The duration and degree of the exposure above C determines the extent of the damage done to cryopreserved cells

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28 From the time it s collected,

29 Processed,

30 Frozen,

31 Stored,

32 Shipped,

33 And ultimately thawed for insemination...

34 Many opportunities exist for accidental thermal exposure, especially after it reaches the farm or breeding center...

35 The list of personnel on a farm, ranch, or breeding center that routinely handle frozen semen samples is long...

36 The list of personnel on a farm, ranch, or breeding center that routinely handle frozen semen samples is long...

37 The list of personnel on a farm, ranch, or breeding center that routinely handle frozen semen samples is long...

38 The list of personnel on a farm, ranch, or breeding center that routinely handle frozen semen samples is long...

39 The list of personnel on a farm, ranch, or breeding center that routinely handle frozen semen samples is long...

40 The list of personnel on a farm, ranch, or breeding center that routinely handle frozen semen samples is long...

41 The list of personnel on a farm, ranch, or breeding center that routinely handle frozen semen samples is long...

42 The list of personnel on a farm, ranch, or breeding center that routinely handle frozen semen samples is long...

43 The list of personnel on a farm, ranch, or breeding center that routinely handle frozen semen samples is long...

44 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?

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48 Major Objective When Handling Frozen Semen and Embryos Keep the internal temperature of straws below 130 Celsius at all times

49 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

50 Essential tools for manipulating straws of frozen semen Towel clamps Forceps Hemostats Liquid N bath Eye protection

51 How many farms, or ranches have an organized educational curriculum in place for handling and storing frozen semen and embryos?

52 Challenge is educating a lot of folks who don t think they need educating!

53 But damage occurs 130 degrees C below the freezing point of water DIC wet mounts

54 The bottom line 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

55 DVD Training Video All Staff Should Watch