prac exam repro

• Initial Assessment of Semen

  Post collection, ejaculates undergo an initial assessment for suitability in AI programs
• Semen assessment parameters


  • Colour
  • Consistency
  • Wave motion
  • Vol
  • Conc
• Cryopreservation of sperm


  The process of preserving sperm at very low temperatures
• Slow Cooling method


  1. Fill beaker with water at 30 degrees Celsius

  2. Samples are placed into water bath and placed to cool at 4 degrees for about 2 hours or until they reach 4 degrees
• Colour and consistency


  Directly related to sperm to seminal plasma ratio of ejaculate (rough estimate of conc.)
• Thawing cryopreserved semen

  1. Fill small polystyrene box with liquid nitrogen

  2. Remove pellets and straws from storage tank into polystyrene box

  3. Wear gloves, safety glasses and enclosed shoes when handling liquid nitrogen

  4. Ensure water bath is heated to 37 degrees in the lab and all receptacles are labelled appropriately

  5. Place pellet into dry glass tube and put tube into water bath for 2 minutes, shaking vigorously

  6. Not advised to thaw more than 3 pellets in a single tube

  7. Thaw straws for 1 and 1/2 minutes

  8. Dry straws, cut both ends and drain into pre warmed dry glass tube

  9. Place thawed samples from pellets and straws into 30 degree Celsius water bath for processing and assessment

  10. Conduct post sperm assessment to ensure no further damage has been done
• Pellet method


  1. Semen is frozen on dry ice at minus 79 degrees celcius

  2. Semen is plunged into liquid nitrogen at minus 196 degrees celsius

  3. Special tool used to make indentations in dry ice (light pressure used)

  4. All equipment to be cooled beforehand as well

  5. 200-250 microlitres of semen is aspirated and gently pipetted into the indentation (pippete is only depressed at first stop to avoid air bubbles being created in the pellet)

  6. 2-3 minutes for pellet to freeze (it will turn from glassy and vibrant to a duller matte solid)

  7. Frozen pellet taken from dry ice into pre-cooled forceps and plunged into liquid nitrogen for storage

  8. Pellets are stored into goblets that are labelled and are secured by placing a piece of gauze or cotton wool at the end
• A small polystyrene box is filled with liquid nitrogen to transfer samples from storage tank to laboratory
• Semen colour and consistency


  • Thick creamy = 5
  • Creamy = 4
  • Thin creamy = 3
  • Milky = 2
  • Cloudy = 1
• Pellets and straws are removed from storage tank into the polystyrene box
• Wear gloves, safety glasses and enclosed shoes when handling liquid nitrogen
• Straw Method


  1. After chilling period, a 1ml pipette tip is fixed onto pre cooled straw (1 straw hold 250 microlitres of semen, so to account for any loss of liquid, pipette is set to just over this volume)

  2. Open end of the straw is placed into sample and gently aspirated by pipette

  3. To seal open end of straw, it is dipped into PVC powder and then dipped into water to create a plug

  4. Metal rack is placed in the liquid nitrogen so top of rack is at 4 cm above it

  5. Straws laid out horizontally on rack and is laid out for about 6 mins

  6. Straws then plunged into liquid nitrogen and stored indefinitely (straws can also be stored in goblets for additional identification)
• Ensure water bath is heated to 37 degrees in the lab and all receptacles are labelled appropriately
• To begin thawing, place a pellet into a dry glass tube and place the tube into a water bath for 2 minutes, shaking vigorously to ensure even heating
• Ram ejaculates are milky or cream in colour
• Straw Method


  • Easily allows for identification of individual samples
• Not advised to thaw any more than 3 pellets in a single tube
• Semen tinged with pink


  May be contaminated with blood and should be discarded immediately
• Straws thawed similarly for 1 and 1/2 minutes
• Straws are then dried, both ends cut and drained into a pre warmed dry glass tube
• Thawed samples from pellets are straws are placed into a water bath at 30 degrees Celsius for processing and assessment
• Semen samples contaminated with urine

  Have distinct smell
• Once thawed, post sperm assessment must be made to ensure no further damage has been done to the sperm during the freezing or thawing process (check against pre-frozen parameters)
• Semen samples that are brow/gray


  Cannot be used (possible infection in reproductive tract) therefore discarded
• Wave motion


  10 microlitre drop of undiluted semen is placed on a warmed slide under a phase contrast microscope
• Wave motion scores


  • Dense rapidly moving waves = 5 (90% or more sperm are motile)
  • Dense vigorous waves but not as much as 5 = 4 (70-85% of sperm are active)
  • Slower smaller waves = 3 (45-60 percent of sperm are motile)
  • No waves with some movement = 2 (20-40% of motile sperm)
  • 10% of sperm showing any activity = 1
• Scores of 3 or below are not suitable for freezing
• Sample with all sperm motionless = 0</b>
• Samples with scores of 4 and 5 are good quality for freezing
• Sperm concentration


  Calculated on spectrophotometer or haemocytometer
• If using spectrophotometer, 3 measurements should be taken for accuracy
• Sperm Handling and Assessment

  1. Turn on microscope and make sure heat stages have been warmed to approx. 37 degrees

  2. Prewarm glass slides and coverslips by placing them on heat stage, prevent sperm from being cold shocked

  3. Swing in 10x objective, and match phase ring to objective (matching phase is phase 1 for 10x)

  4. Adjust lenses to suit your eyes

  5. Place 10 microlitre drop on slide and cover with coverslip

  6. Transfer slide to microscope stage and use x and y knobs to bring sample into field of view

  7. Take out right eyepiece and insert centring telescope

  8. Light ring (phase ring) dark ring (phase plate)

  9. Adjust dial on focusing telescope to bring light and dark rings into focus

  10. Adjust screws at rear of condenser to align the light ring over the dark ring

  11. Replace w/eyepiece

  12. Close left eye, looking through right eyepiece only, focus the sample

  13. Then looking through left eyepiece, focus again
• Haemocytometer concentrations


  1. 2 counting chambers w/microscopic grid

  2. 3% saline is used prior to loading sample to immobilise sperm and preserve them for later counting

  3. Must be dilute enough to count individual sperm (by factor of 400 suitable for ram)

  4. Mix sample thoroughly before counting to evenly distribute sperm

  5. Fixe coverslip over counting chambers by first moistening either side to prevent movement

  6. Load into haemocytometer and place into humid chamber to let sample settle for 3-5 mins

  7. 25 larger squares with 16 smaller squares in each

  8. Only need to count sperm in 5 larger squares (count gained from 4 corner squares and centre square)

  9. Swing in 20x or 40x objective to count sperm

  10. All sperm including loose heads within the smaller squares are counted

  11. Sperm falling in centre of triple ruled line are only counted if located on upper or left hand side of square

  12. Sperm on lower and right hand side of square in those lines are discounted

  13. Use cell counter, add counts together before progressing to 2nd counting chamber

  14. If less than 10 sperm are counted in first square, all 25 squared need to be counted to reduce variability
• Calculating Sperm Conc


  Number of sperm/mL = average of total count on both sides x 5 x dilution factor x 10 000 (vol of haemocytometer)
• Cryobiology


  Study of the effect of subzero temperatures on biological materials
• Why cryopreserve?


  • Increasing longevity of sample
  • Food storage
  • Research applications - stores stocks of stem cells to keep research consistent between different stem cell and tissue culture lines
  • Agricultural/medical applications - preservation of tissues or cells like sperm, oocytes, embryos
• Freezing


  Conversion of water to ice, prevents chemical reactions from occurring, cells become quiescent
• Cell membranes


  • Lipid bilayer interspersed with proteins and carbohydrates
  • Semi-permeable, regulates movement of ions, molecules, solutes and water in and out of cell
  • Damage causes cell death
• Damage to cell membranes


  • Physical ice damage
  • Oxidative stress from lipid peroxidation
  • Changes to fluidity
• Successful cryopreservation


  1. Minimise damage from dehydration

  2. Minimise damage due to ice formation

  3. Contamination of water (e.g. from salt addition), DECREASES freezing/thawing point

  4. Reduce production of reactive oxygen species (ROS)

  5. Addition of antioxidants

  6. Minimise changes to membrane fluidity

  7. Addition of supplementary cholesterol (or other FAs)