Semen Evaluation



 

• Gross motility is examined first. 
  • Mix the semen sample with a wooden stick, as motile sperm cells will try to swim upward and dead cells will settle to the bottom. 
  • For gross motility use 2 wooden sticks to place a drop of semen on a warm slide. 
  • Do not use a cover slip and examine the cells under a 10X objective. 
  • The motility is judged by the swirling motion of the sample. 
  • The swirling pattern will definitely indicate that there are cells alive. 
  • The swirling looks like currents and eddies (like a fast motion weather map). 
  • Assessment of individual cells cannot be made as live cells will carry the dead cells. 
  • The gross motility will tell you though that the cells are alive, and if the cells are dead when you examine them for individual motility there has been a handling problem.  
  • Gross - swirl pattern in bull, ram and buck.

  

  Gross Motility

 

• Individual motility is examined next. 
  • Individual motility checks for the progressive movement of the sperm cells. 
  • Make the sample by placing a drop of diluent (saline or Na citrate) on a warm slide. 
  • Place a small amount of semen into the saline. 
  • You want about 10 cells/high power field in order to accurately estimate the number of cells that are progressively moving across the field. 
  • Then place a warm cover slip on the drop. 
  • Examine the sample under high dry (40X) power. 
  • You must examine the sample quickly as the motility changes very rapidly with heat, light, and cold.

• The motility is a very subjective measurement and is affected by many things, such as diluent (it may be old and hypertonic etc.), cold, the glassware, urine, soap, prostatic fluid, seminal pH, and ion composition.
• Computerized sperm motility computers (CASA) are available for $30-50000
  • Objective, but they probably do not tell us that much more than by eye.

    

    Individual Motility
    
    

    Sperm morphology
• Morphology is usually examined with an eosin-nigrosin (Society for Theriogenology) stain (background stain) to highlight the cells. 
  • The slide is made by painting a drop or line of stain on a warm slide, then using a wooden stick to place a small amount of semen into the stain; the semen and stain are mixed using another slide, then slowly push the second slide through the stain and across the first slide while pressing firmly down . 
  • The goal is to get a dark background, as the stain is a back round stain and is not intended to stain the cells. 
  • In fact, some cells will stain red, but this makes no difference in our evaluation. 
  • You want the cells to be close, but not overlapping. 
  The final slide should have dark and light areas that allow you to view different colored backgrounds as needed when examining the slide.

• Examine the cells under 1000X (oil) to fully assess the morphology.

• Count 100 cells and, in a practice situation, you can just differentiate normal from abnormal cells. 
  • Using eosin-nigrosin all the cells will appear flat, as if looking at your hand. In order to do a full spermiogram, differentiate the abnormalities by type. 
  • A phase contrast microscope can be used, but is rare in a practice situation. 
    • A phase contrast mount is made by placing a small amount of semen in formal-buffered saline to kill and preserve the cells. 
    • Then place a drop of the the formal-buffered saline sample onto a slide and place a cover slip over the drop. 
    • You need a phase contrast microscope to examine the specimen. In using the phase-contrast microscope, match the phase ring with the power that you are using. 
    • The phase contrast acts like a 'stain', however the cells will float by instead of lying flat. 
    • The cells now look like your hand from the top and side views.

Abnormalities  

• Abnormalities are classified as primary and secondary. 

  • Primary abnormalities are thought to arise in the testes, whereas secondary abnormalities arise in the epididymis or ejaculate. 

  • Secondary abnormalities may be just as serious primary. 

  • Primaries abnormalities decrease through transit and secondary abnormalities increase through transit.  

• Major and minor may be a better clasification. 

  • Major problems cause Early Embryonic Death or prevent fertilization. 

  • For example, an acrosome problem prevents zona entry, and a nuclear problem leads to nonfertilization or EED.  

  • Minor problems such as tail abnormalities etc. stop sperm movement, so the sperm cell cannot get to egg. 

• Some abnormalities are compensable and some are non-compensable. 

  • A non-compensable abnormality gives the animal a poor prognosis for any recovery. 

• Problems with sperm cell examinaiton. 

  • The light microscope cannot always detect abnormalities.  

  • Abnormal sperm cells indicate cellular damage, but the relationship to fertility is only circumstantial.
    
    
    
    Normal Sperm Cell above
    
    
    Abnormalities of the sperm cells include:
    

• Decapitated sperm (not pictured)- the basal plate is defective; tails move (wrap around drops); 100% cells involved; occurs in epididymis (secondary)  

• Loose heads (not pictured)- these may be increased on the first ejaculate (rusty load) because the tail attachment is frail. 

• Knobbed or flat acrosome (not pictured)- the acrosome folds over itself of the apex of the acrosome is knobbed or flattened. When 20% of the cells have acrosome problems the result may be infertility in the bull. This condition may be hereditary in Charolais, Hereford, and Holsteins.
   

• Wrinkled acrosome - this may reflect a nuclear problem which prevents zona attachment by the sperm cell. It is a rare condition.  

• Pyriform and tapered heads - the nuclear material is poorly distributed. The defect may be subtle.

• Giant or small heads - This a a nuclear problem. If the head is twice normal size the cell is a giant cell.

• Nuclear vacuoles - These distort the shape of the head.

• Diadem defect - With this you see invaginations in the nucleus, mostly by the post nuclear cap. The pit lacks DNA. The condition may be associated with stress in bulls and may come and go as stress changes.

• Dense proximal droplets - This arises in the epididymis and indicates maturation problem.
• Stump tail defect (not pictured) - this is an axonemal problem. It looks like a cytoplasmic drop and has a poor prognosis. The incidence increases with age. Midpiece defects - You see lumps on the midpiece that can be confused with proximal drops. Coiled mainpiece - The mainpiece is coiled within the plasma membrane.

• Dag defect - This is a sterilizing defect that occurs in the epididymis so is it is actually a secondary abnormality, but it is a major defect. The condition is inherited and the axoneme is disrupted (fibrils and helix). You see split, shattered, or fractured midpiece. The tail may coil and the motility is is low.
• Coiled midpiece (not pictured). This is an epididymal defect, but is a major defect. The midpiece problems (not Dag).
   
• Abaxial midpiece - the implantation fossa is defective. You may see abaxial or double midbpieces; There is generally a low incidence and fertility is not affected; (normal in stallion).

• Coiled mainpiece - The mainpiece is coiled within the plasma membrane.

• Teratospermia - the entire cell is degenerative.
   

• Bent tails - the bend in the tail may include a droplet which may be in the membrane.

• Physiologic (distal )droplet - some consider this a minor defect, but in fact it may be a major defect. These cells do not freeze well because the water in droplet crystalizes and ruptures the cell membrane.

Temporal progression of sperm abnormalities after 4 days of scrotal insulation.

Days after scrotal insulation	Sperm abnormalities in the ejaculate	Days return to normal
7-11	Distal midpiece refles, Proximal Droplets	40
11-15	Miochondrial Sheaths, Detached heads	35
18	Knobbed acrosomes	25
20	Nuclear Vacuoles	22
22	Pyriform heads	20
23	coiled principal piece	19
42	normal	0

• In bulls and rams, the sperm count is estimated by measuring the scrotal circumference. The scrotal circumference measurement is used because electroejaculation does not give a physiologic sample that can be reliably counted. 
• To count the concentration of semen sample a hemacytometer can be used. 
  • The hemacytometer is loaded with a 1:100 dilution of semen (see below how to make a 1:100 dilution, 
  • a hemacytometer coverslip is placed over the chambers
  • the chambers are filled  
  • the sample allowed to settle, 
  • all the sperm heads in the middle big square (the square with 25 smaller squares (....and another 16 squares within each of these 25) within the triple lines) are counted. 
  • The number of sperm heads counted in a single chamber is multiplied by 10⁶ to give the concentration of cells/cc. 
  • Both chambers of the hemacytometer should be counted and the numbers should not differ by more that 10%. 
  • The coverslips are expensive so do not break them or dispose of them!
  • Sound confusing...look at the diagram.
    

The hemacytometer grid has 9 large squares. The central square has triple lines around it. Inside the triple lines are 25 smaller squares (also bounded by triple lines). Within each of the 25 squares are 16 squares (only one of the 25 cells is illustrated having the 16 smaller squares. This is illustrated below, however the triple lines are only shown on the outside edges of the middle big square. 

Here is an actual picture of the grid of the 25 squares in the middle big square. They are all bounded by triple lines. Count every sperm head bounded by triple lines.

Here is a picture of one of the 25 squares with the 16 squares inside.

For example, if each of the dots represented a sperm head, the count on the grid below would be 13 x 10⁶ cells/ml.

• The 1:100 dilution can be made using a Unopette diluter or a hand dilution. 
• The Unopette is easier, but sometimes the cells will clump, so an accurate count cannot be made.
  •  If using the Unopette, first puncture the bottle with the sharp point, then draw up the semen via capillary action into the capillary tube. 
  • The bottle is then squeezed and the capillary tube inserted into the bottle and the semen sucked into the bottle. 
  • You can turn the capillary tube over and use it as an applicator to load the hemacytometer chambers.
  • We no longer use the Unopette at LSU

• A hand dilution is made by:
  •  diluting 1 part semen with 9 parts formal-buffered saline to make a 1:10 dilution, 
  • then taking 1 part of the 1:10 dilution and adding 9 parts of formal-buffered saline to make a 1:100 dilution. 
  • This is a little more time consuming, but the cells tend not to clump, so you get a more accurate sperm count.
      

• A spectrophotometer can be used to count stallion semen (and possibly dog semen if the correct software is used). 
  • Basically, the machine measures the amount of light that passed through a sample and calculates the concentration of cells that the density reflects. 
  • Using our machine:
    • A blank tube is loaded with 3.42 ml of formal-buffered saline 
    • The blank is inserted into the machine with the clear sides on the left and right. 
    • The machine passes a beam of light through the clear sample and determines that 100% of the light is passing through. 
    • Then, 180 µl of semen is added to the tube, the tube mixed and reinserted into the machine. 
    • The sperm concentration is then calculated based on the percent of light that is transmitted through the sample. 

    • Any dirt, blood or other contamination it the sample may adversely affect the amount of light transmitted and result in an erroneous concentration reading.

Total sperm numbers.  

• Multiply the concentration X the volume to give the total number of cells in the ejaculate.
• Take that number and multiply by the percent progressively motile cells to get the total number of progressively motile cells. 
• Take that number and multiply by the percent normal cells to get the total number of normal, motile cells.
•  For example: 100 cc X 50million cells/cc = 5 billion cells 5 billion cells X 50% motility = 2.5 billion motile cells 2.5 billion motile cells X 50% normal = 1.25 billion normal, motile cells.
• Total sperm numbers are used in stallions and dogs, but not in bulls, rams or bucks.

contributed by Bruce E Eilts and modified on 1 November 2004 

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