Evaluation of semen

Dr.K.Lalrintluanga
 Evaluation of semen
The different tests for evaluation of semen are:
1. Appearance
2. Colour
3. Volume
4. Mass motility
5. Individual motility
6. Hydrogen ion concentration ( pH )
7. Concentration or Density of spermatozoa
8. Live spermatozoa percentage ( Live sperm count ).
9. Sperm abnormalities.
10.Other tests. Viz. 1) Resistance to cold shock . 2) Fructolysis index
3) Methylene blue reduction test. 4) Resazurin reduction test
5) Millovanov’s resistance test (R-test ). 6) Catalase test.
7) Oxygen utilization test.
Appearance: Translucent few spermatozoa
Uniform and opaque high sperm conc.
curdy appearance reproductive infections.
Colour :bull semen is milky or creamy white
appearance and colour have correlation with the sperm conc.
in the semen samples.

Appearance and colour. Sperm density

Thick creamy over 2,000 million/ ml
Creamy 1,500 – 2,000 million/ ml
Thin creamy 1,000 – 1,500 million/ ml.
Milky 500 – 1,000 million / ml
Watery less than 100 million/ ml
Dark red or bloody blood
brownish colour orchitis
yellowish green if left at room temp. Ps. aeruginosa
Clots or flakes pus
 Volume:. The semen volume may be affected by:
1. Body weight/size: usually less in small size
2. Age : Semen volume increases as the age
increases up to 6 or 8 years.
3. Pre-coital stimulating and false mounting :
increases in volume if stimulated properly.
4. Frequency of semen collection or natural
service.
Normal volume of semen in different species are –
Bull - 1 – 15 ml ( 4 ml average )
Boar - 125 – 500 ml ( 250 ml)
Ram/Goat - 0.7 - 3 ml ( 1 ml )
Stallion - 30 – 250 ml ( 70 ml )
Dog - 1 – 25 ml ( 10 ml )
Man - 3 – 4 ml
Cat - 0.01 - 0.12 ml ( 0.04ml )
Cock - 0.2 – 0.5 ml
Mass motility : collective movement of spermatozoa in fresh undiluted semen at
37o C under low magnification . It indicates conc . and viability.

Characteristic of movement Numeri Descript

cal ive
value

scale
1
Extremely rapid waves and eddies. It is difficult to
trace the origin and disappearance of the waves. Nearly Very
100% of the spermatozoa are motile. good

+ ++ +
2.
The waves and eddies are comparatively not so rapid.
The swirls are observes to move towards the +++ good
extremities. Nearly about 90 % of the sperms are
motile.
3
The waves and eddies are slowly moving and are
scattered in the field. About 50-80 % of the sperm are ++ fair
motile
Individual motility: the percentage of progressively
motile sperm present in the semen sample.
Types of motility:-
1. Progressive, rectilinear motility where sperm are
moving rapidly in straight forward direction.
2. Forward circling motility due to defect in middle
piece and tail.
3. Reverse circling motility.
4. Oscillatory or pendulatory and jerky motility.
The progressively motile sperm cells of bull may
cover a distance of 100-120 micron in one second.
The individual motility rating is done as follows.
Motile cells. Descriptive value. Numerical value.
80 – 100 % Very good 5
60 – 80 % good 4

40 – 60 % fair 3
20 – 40 % poor 2
0 – 20 % very poor 1
Hydrogen ion concentration ( pH) :
pH of semen of different species are:
Bull - 6.2 – 7.5 ( 6.8 )
Ram/Buck - 6.2 - 7.0 ( 6.8 )
Dog - 6.0 - 6.8 ( 6.7 )
Boar - 7.0 – 7.8 ( 7.4 )
Stallion - 7.0 – 7.8 ( 7.4 )
Man - 7.2 – 8.0
 
 
Concentration or Density of spermatozoa:

Concentration of spermatozoa in different species are :

Bull - 300 – 2500 million/ml (average 1200million/ml)
Boar - 25 - 1000 million/ml (150 million/ml)
Stallion - 30 – 600 million/ml (120 million/ml)
Dog - 20 – 540 million/ml (125 million/ml)
Ram /Buck -1000 – 6000 million/ml (3000 million/ml)
Man - 120 – 150 million /ml
Cock - 3000 – 7000 million/ml
Live sperm count:
Cell membrane of dead sperm is permeable to
Eosin.
The dead sperm takes eosin ( pink ) while live
sperms do not take any colour.
Nigrosin provides a dark background.
Partially stained sperms are considered as dead
sperm.
Sperm abnormalities:
Most of the workers agree that semen of fertile
bulls should not have more than
4 % head abnormalities,
4-10 % midpiece abnormalities,
5 % tail abnormalities,
6 % free head and
a total of 20 % sperm abnormalities.
The abnormalities in spermatozoa may be produced from :
1. Inactive spermatozoanic tubules, present in testis of all fertile
bulls.
2. Pathological condition of testis.
3. Pathological condition of epididymis.
4. Pathological condition of accessory glands.
5. Prolong sexual rest
6. Heat stress due to hot climate.
7. Insulation of testis
8. Extreme exposure to cold and winter wind.
9. Frost bite
10.Application of cold experimentally
11.As a sequel of virus infection eg. FMD or Ephemeral fever.
12.Old age
13.Inherited
 Abnormalities of spermatozoa can be classified into :

I) Classification on the basis of causative reasons:

a) Primary abnormalities: The sperm abnormalities develop in the testis during

spermatogenesis.
1. Pyriform head or pear shaped head
2. Tapering head
3. Narrow head
4. Dwarf head
5. Giant head or, Macrocephalic head
6. Small head or microcephalic head
7. Loose normal head
8. Tightly coiled tail ( dag defect)
9. Double head
10.Double midpiece
11.Abaxial attachment
12.Knobbed sperm
13.Diadem defect
14.Proximal protoplasmic droplets.
 
b) Secondary abnormalities: The sperm
abnormalities develop during post-
spermatogenesis when the sperm leaves the
testis and traverse through the epididymis.
Eg. Detached normal head,
Distal protoplasmic droplets,
detached galea capitis,
bent tail.
c) Tertiary abnormalities: develop due to
damage of the spermatozoa during or after
ejaculation. It includes improper handling of
semen and various abnormal factors like
sudden change of temperature. Excessive
agitation, drops of water or urine or
antiseptics in semen.
eg. coiled tail,
acrosomal abnormalities(broken or
ruptured acrosome),
bent tail etc.
II) Classification on the basis of affected portion of spermatozoa:

• Under this classification the abnormal sperm

can be divided into:-
– Sperm head abnormalities (Spermatozoal caput
anomalies):
– Sperm body/midpiece abnormalities
(Spermatozoal corpus anomalies).
– Sperm tail abnormalities(Spermatozoal cauda
anomalies).
 Sperm head abnormalities are:
• Micro-cephalic head
• Macro-cephalic head
• Double head
• Elongated or narrow head
• Pyriform or Pear-shaped
• Round short head
• Irregular head
• Knobbed sperm
• Pouch formation or invagination of nuclear
enveloped (Diadem defect).
Sperm body/midpiece abnormalities:
• Swollen neck.
• Kinked neck
• Naked or Filiform neck
• Abaxial attachment
• Swollen mid piece.
• Double mid piece
• Coiled mid piece
• Kinked mid piece.
• Corkscrew mid piece.
• Loose or free mid piece
• Bent mid piece.
 Sperm tail abnormalities:
Tightly coiled tail (Dag defect).
Double tails
Absent tail
Shortened tail
Coiled tail
Bent tail
Broken tail.
Blom’s Classification
In 1972 Blom proposed a new classification of sperm abnormalities as major and
minor sperm defects. This is the latest type of classification and now it is universally accepted
as standard.
 

Major sperm defects :

1.Underdeveloped
2.Double form
3.Acrosome defect ( Knobbed sperm )
4.Decapitated sperm.
5.Diadem defect or Pouch formation.
6.Pear shaped head
7.Narrow at the base
8.Abnormal contour
9.Small abnormal head
10.Free abnormal head
11.Corkscrew defect
12.Other midpiece defect
13.Proximal droplets
14.Pseudodroplets defect
15.Strongly folded or coiled tail ( Dag defect )
 
Minor sperm defects :
1. Narrow head
2. Small, normal head
3. Giant head and short broad head
4. Free head (normal) i.e. Free loose head
5. Detached acrosomal caps
6. Abaxial implantation of midpiece
7. Distal droplets
8. Simple bent or coiled tail
9. Terminally coiled tail
10.Other defects such as : Medusa formation,
Prepucial cells, R.B.C., and W.B.C.
Knobbed sperm : accentrically place thickening of the
acrosome caused by an inherited autosomal
recessive sex linked related to defective
spermatogenesis involving the golgi apparatus

Diadem defect : dark necklace along the anterior border of

the postnuclear cap and result from the
invagination of the nuclear membrane due to
disturbed spermatogenesis.
Decapitated sperm : defect is associated with an ultrastructural
abnormality in the neck or implantation
region of the spermatozoa. The sperm, due to this
ultrastructural abnormality in the neck region,
disintegrate into head and tail in the caput epididymis.

Dag defect : mainpiece is strongly coiled over the midpiece.

This defect is found associated with elevated
level of Zinc in the spermatozoa as well as in seminal
plasma. the motility is only 10 – 20 %. Such bulls are
invariably infertile/ sterile.
Corkscrew defect : It is the defect of mid piece due to an
irregular distribution of the mitochondrial
sheath. The midpiece appears as a
corkscrew.

Pseudodroplets defect : Characterised by rounded or elongated thickening on

the midpiece.

Medusa formation : A portion of ciliated epithelial cells from the

efferent ducts of the testis. They are deeply
staining cells about the size of the head of
sperm with 10-30 filiform, or long brush- like
projection or cilia.
Protoplasmic (Cytoplasmic) droplet:
This is composed of residual
cytoplasm and disintegrating Golgi apparatus. It may be:-

Proximal droplet: The droplet may be sometime retained in the

neck region when it is known as Proximal
droplet. This is indicative of immature spermatozoa.
This type of spermatozoa is considered abnormal and
infertile.

Distal droplet : It may be also retained after migration to

annulus, where it is called Distal droplet.
Sperm with distal droplets are considered normal and
fertile.
Resistance to cold shock :to test the semen samples for varying
degree of resistance of spermatozoa against cold shock. It may be
likely that the semen having more spermatozoa with resistance
against cold shock live longer when preserved and may predict high
fertility.
Method :
1. Take small quantity ( 0.25 – 0.5 ml ) of undiluted semen in a small
tube.
2. Immerse the tube in crushed ice ( sudden change of temperature
from 370 C to 00 C ) and keep it for 10 minutes.
3. Remove the tube and thaw at 300– 37o C
4. Prepare slide in eosin-nigrosin stain for live and dead count.
Recovery of large number of live spermatozoa would mean better
resistance against cold shock.
 
Methylene Blue Reduction ( M.B.R. ) test :
More are the number of hydrogen ions liberated , less is the time taken by
methylene blue for reduction and to change its blue colour. The reduction
time is directly related to the motility and the concentration of the
spermatozoa in the semen sample.

Method : 1. Add 0.2 ml of freshly collected semen in a small test tube

containing 0.8 ml EYC dilutor .
2. Add 0.2ml of methylene blue solution in the above diluted semen
and mix the content.
3. Seal the mixture by covering with a 1-1.5 cm. thick layer of liquid
paraffin or mineral oil to ensure anaerobic condition.
4. Place the tube in a water bath at 46.5o C
Note the time taken for the disappearance of the blue colour.
 
A good quality semen would reduced the methylene blue in only 3 – 5
minutes. Average samples, would require about 9 minutes and poor samples
would require more than 9 minutes.
Fructolysis index : It is described as the amount
of fructose (mg) utilised by 109 spermatozoa in 1
hour at 37oC. Fructolysis has a direct correlation
with the metabolic activity of the spermatozoa.
In normal bovine semen the rate is
1.2-2.9 mg of fructose/hour.
 Millovanov’s Resistance Test (R- test ): R-test is denoted as the ml of 1 %
sodium chloride solution required to stop the progressive motility of all the
spermatozoa in 0.02 ml of semen.
Method : 1) Take 0.02 ml of freshly collected semen in 200 ml capacity
flask.
2) Add 10 ml of 1 % sodium chloride solution in several steps and
after each addition examine a drop of mixture under
microscope till progressive motility of all the spermatozoa is ceased.
3) Calculate R-value ( Resistance value ) as below :
 
R-value = ml. of Nacl2 solution required to cease the progressive motility.
0.02
 
Good quality semen would have R-value not less than 5000 i.e.
progressive motility would cease after adding more than 100 ml of 1 % Sod.
Chloride solution.
Catalase test :
to detect increase in the catalase enzyme in the presence of pus and blood and
the bacterial contamination in the semen.
Resazurin Reduction Test : Resazurin reduction test is employed to evaluate
the metabolic activity of semen based on the dehydrogenase activity of the
spermatozoa.
Method :
1. Add 0.2 ml of freshly collected undiluted semen in a small test tube.
2. Add 0.1 ml Resazurin solution in the above undiluted semen and mix the
contents ( to prepare Resazurin solution 5.5 mg of Resazurin is disolved in
100 ml distilled water).
3. Cover the mixture with 1 cm thick layer of liquid parrafin or mineral oil to
ensure anaerobic condition.
4. Incubate the contents at 45o C.
5. Look for changes in colour i.e. blue to purple and from purple to colourless.
6. In good quality samples colour changes from:

Blue to pink or purple in 1 minute and pink to colourless in 3-4 minutes.

The average samples would take a time of about 5 minutes for second end point.
Oxygen utilization test :The measurement of
oxygen uptake is related to the biochemical
changes taking place in whole semen. If the
spermatozoa are very active, they utilized more
oxygen per unit of time.
Objective test to evaluate fertility of spermatozoa:
1. Cervical mucus penetration test(CMPT)
2. Hypo-osmotic swelling test (HOS Test):
3. Penetration of zona free hamster egg as a test
to assess fertilization ability of spermatozoa
and study sperm chromosome: