clinical laboratory techniques subject for undergraduate zoology students

1. MODULE 2
EXAMINATION OF SEMEN
Sub code: MLT504
Sub Name: Medical Lab Technician -1 (T)
Department: Department of MLT, SMAS
Faculty: A. Vamsi Kumar
Designation : Assistant professor

2. Course outcomes
• On completion of this course student will able
to perform & Conduct analysis of body fluids/
samples.

3. Learning outcomes
• Can recall sample collection, gross inspection
and routine microscopic analysis of semen
sample.
• To be briefly familiar with various chemical,
immunological, microbiological and function
tests of semen.

4. Contents
1. Indications of semen examination
2. Composition of semen
3. Sample collection
4. Gross examination
5. Microscopic examination
6. Chemical examination
7. Immunological assays
8. Microbiological assays
9. Sperm function tests
10.Semen cryopreservation

8. watch
• From conception to birth TED talk by Tsiras

15. Microscopic examination of Semen

17. https://www.google.com/search?q=indications+for+semen+examination&source=lnms&tbm=isch&sa
=X&ved=0ahUKEwiltpT6pYXkAhVFeH0KHXvVDAoQ_AUIESgB#imgrc=I0ws7Pjde4mN9M:

18. https://www.google.com/search?q=indications+for+semen+examination&source=lnms&tbm=isch
&sa=X&ved=0ahUKEwiltpT6pYXkAhVFeH0KHXvVDAoQ_AUIESgB#imgrc=XvppaY2bh9Y83M:

19. Composition of semen

20. A brief anatomy

33. 1. Gross examination
• Semen is examined grossly for the following
features:
1. colour,
2. Odour
3. volume,
4. viscosity,
5. reaction and
6. liquefaction.

34. 1. Colour. Normally it is whitish, grey-white or slightly
yellowish.
2. Volume. Normally, volume of semen is between 2.5 and
5 ml. The volume is slightly more in patients of infertility. The
volume does not vary with the period of abstinence.
3. Viscosity. When ejaculated, semen is fairly viscid and it
falls drop by drop.
4. Reaction. Normally, it is slightly alkaline with pH between
7 and 8.
5. Liquefaction. Liquefaction occurs because of presence of
fibrinolysin. Normally liquefaction occurs at room
temperature
within 10-30 minutes (average 20 minutes).

42. 2. Microscopic Examination
1. Motility
2. Morphology
3. Viability
4. Count

43. a. Motility test
1. Place a drop of liquefied semen on a clean glass slide.
2. Put a coverslip over it and examine it under the
microscope, first under low power and then under
high power.
3. Normally within 2 hours of ejaculation, more than
60% of spermatozoa are vigorously motile, and in 6-8
hours 25-40% are still motile.
4. If motility is less than 50%, a stain for viability such as
eosin Y with nigrosin as counterstain can be done.
Heads of non-motile sperms show red dye.

46. Abnomal Condition is called as..?

47. b. MORPHOLOGY
1. Prepare a thin smear from liquefied semen on a glass slide.
2. Stain it with any of the Romanowsky stains, Pap or H & Estain.
3. Observe at least 200 spermatozoa for any abnormality in their
morphology.
4. Normally 80% of spermatozoa are normal.
5. The abnormal forms of spermatozoa are with double head,
swollen and pointed head, double tail and rudimentery forms.
6. Also look for the presence of RBCs or WBCs, if any.
7. Computer-assisted morphologic screening is particularly useful in
samples with very low numbers of normal sperm count which may
otherwise remain undetected.

51. Abnormal morphology condition is
called as……?

54. c. Sperm Vitality test

55. Sperm Viability
• Sperm Viability
• Decreased sperm viability may be suspected
when a specimen has a normal sperm
concentration with markedly decreased
motility.
• Viability is evaluated by mixing the specimen
with an eosin-nigrosin stain, preparing a
smear, and counting the live sperms.

59. d. Sperm count
 This is done in Neubauer’s (haemacytometer) chamber using a WBC pipette.
 Draw liquefied semen in WBC pipette up to mark 0.5 and then draw the diluting fluid up to mark 11.
 The composition of diluting fluid is as under:
 Sodium bicarbonate 5 gm
 Formalin (neutral) 1 ml
 Distilled water 100 ml
 After mixing it properly, charge the Neubauer’s chamber.
 Allow the spermatozoa to settle down in 2 minutes.
 Examine under microscope and count the number of spermatozoa in two large peripheral squares (used
for TLC counting) and multiply the number by 1 lakh (100,000) which gives number of spermatozoa per
millilitre:
 In 1 × 1 × 0.1 μl volume,
 number of spermatozoa = n × 10
 But dilution factor is 10
 Number of spermatozoa per μl = n × 10 × 10
 or Number of spermatozoa per ml = n × 10 × 10 × 1000
 = n × 100,000
 i.e. n × 1 lakh
 (where n is the average number of spermatozoa counted in two squares).
 Normal value = > 60 million/ml
 Abnormal value = < 20 million/ml.

62. 3. Chemical Examination
• 1. Fructose test
• 2. Acid phosphatase test

63. FRUCTOSE TEST
• This test determines androgen deficiency or ejaculatory
obstruction to semen; the level of seminal fructose is low in
both these conditions. Normal seminal fructose level is
• 150-600 mg/dl.
• Fructose is measured qualitatively by Resorcinol test.
• Procedure
• Take 5 ml of dilute HCl in a test tube.
• Add 1 ml of semen.
• Add 5 mg of resorcinol.
• Boil.
• Interpretation. Appearance of red colour indicates
• presence of fructose which can be measured by
spectrophotometer.

64. ACID PHOSPHATASE TEST
• This test is used for seminal stain and on
vaginal aspirate in medicolegal cases.
• Normally semen has 2500 KA units/ml of acid
phosphatase.

65. 4. Immunological Assays
• The presence of sperm antibody binding to
head or tail antigens is considered specific for
immunologic infertility.
• The antibodies are usually of immunoglobulin
A (IgA) or IgG, and rarely of IgM class.
• These are detected by direct or indirect mixed
agglutination reaction tests. (MAR test)

66. Antisperm Antibodies
Antisperm antibodies can be present in both men and
women. They may be detected in semen, cervical mucosa, or
serum and are considered a possible cause of infertility. It is
not unusual for both partners to demonstrate antibodies,
although male antisperm antibodies are more frequently
encountered.
Under normal conditions, the blood-testes barrier separates
sperm from the male immune system. When this barrier
is disrupted, as can occur following surgery, vasectomy reversal
(vasovasostomy), trauma, and infection, the antigens on
the sperm produce an immune response that damages the
sperm. The damaged sperm may cause the production of antibodies
in the female partner.8

70. 5. Microbiological Assays
• Genital tract infections by bacteria, yeast and
sexually transmitted diseases may have
significant adverse effect on male infertility.
• If the concentration of bacteria exceeds 1000
CFUs per ml, the colonies should be identified
and tested for antibiotic sensitivity.

71. 6. Sperm Function Tests
• Defective sperm function may affect various fertilising functions.
Most importantly, it includes transport of sperm in the male and
female reproductive tracts, which is responsible for fertilisation
activities such as specific zona binding, penetration and formation
of male pronucleus. A list of common sperm function tests is as
under:
1. Sperm penetration assay: to test the success of penetration of
egg by the spermatozoa
2. Hypoosmotic swelling test: to test the membrane integrity of the
sperm
3. Cervical mucus penetration test: to test the relative ability of motile
sperms to pass through cervical mucus of the
• partner collected at midcycle.

73. 7. Semen Cryopreservation
• Cryopreservation or semen banking is
indicated in the
following conditions:
• 1. For assisted reproduction
• 2. For donor insemination
• 3. For men undergoing vasectomy
• 4. In men before starting cancer therapy
• 5. In life threatening jobs (e.g. military service)

74. References
1. Harshamohan practical pathology, 4th edition
2. Strasinger Body fluids and urine analysis, 6th edition
3. https://www.slideshare.net/morrisjawahar/semen-analysis-by-
drrenukadevi
4. https://www.slideshare.net/RaviJain7/semen-analysis
5. https://www.slideshare.net/drvipinDrvipinsharma/semen-
analysis-91057379
6. https://www.slideshare.net/pathologybasics/semen-analysis-
29140526?from_action=save
7. https://www.slideshare.net/drfurquan/semen-analysis-59490363
8. https://www.slideshare.net/danish29/semen-analysis-61887227
9. https://www.slideshare.net/WafaAlAhmed/semen-analysis-
powerpoint