SEMEN EXAMINATION

Submitted By : Vasant R. Parmar. M.V.Sc ( Gynaecology) Reg. No.: 04-00317-07E mail; vasantvet@gmail.com

Introduction• Semen is collected for examination of breeding

soundness, infertility, Artificial insemination and Parasitic diseases.

• Semen quality of the first ejaculate after a long period of sexual rest may have a lowered motility and an increased no of dead spermatozoa.

• Semen quality in many rams and a few bulls may be decreased during the hot summer months.

• Season of the year had a significant effect on semen quality in the stallions with the best quality of semen produced late in spring or early summer and the poorest quality semen in the winter months

1. While handling semen samples avoid exposure to water, changes in pH and temperature.

2. All laboratory equipments (microscope slides, cover slips, pipettes) stains and diluents should be warmed at 37°C.

3. Equipment should be clean and dry. Room should be warm and dust free.

4. Examine the semen ejaculate for volume, gross appearance, wave motion, microscopic motility, spermatozoa morphology and concentration, ratio of live to dead and presence of other materials.

5. For record note age, history, breed, other abnormalities if any and methods of semen collection (massage, artificial vagina or electroejaculation)

Important features of semen evaluation:

Semen is subjected to the evaluation of the following aspects:

(A)Physical characters (Macroscopic Characters)

(B) Physiological and Biochemical studies

(C) Microscopic Evaluation

(D)Examination for genital infections

(A) Physical characters (Macroscopic Characters)

1. Colour :• Creamy• Milky• Watery• Cloudy

Bull & Ram Concentrated Milky or creamy white & opaque

Stallion, Boar & Dog

Less Concentrated Pearl white to grey and translucent

Brownish color Blood Pigment Orchitis

Light yelllow color Riboflavin secreted by accessory glands

Yellowish – Green Presence of Pseudomonas aeruginosa

Clots and Flakes Presence of Pus

Semen samples from a bull (left) and dog (right), showing differences in opacity and concentration

Blood in a canine ejaculate (hemospermia)

Color of Semen Bulls & Rams (per cmm)

Thick Creamy 25,00,000

Creamy 20,00,000

Light Creamy 10,00,000

Milky 5,00,000

Cloudy, watery, translucent 1,00,000

Almost clear, transparent, watery

Less than 50,000

Sperm cell Concentration and Semen Color

in Bulls and Rams

(Gunn et al., 1942)

2. Volume :

Normal volume of semen in different animals

Bull - 4 (1-15) ml

Stallion -70 (30-250) ml

Ram/Buck - 1 (0.7-3.0) ml

Boar - 250 (125-500) ml

Dog - 10 (1-25)

Cat - 0.04 (0.01-0.12) ml

(Roberts, 1971)

1. Sperm free watery secretion

2. Sperm rich secretion

3. Sperm poor fraction

Ejaculate consist of 3 portions :

In Tom, Bull, Buck and ram – all three fractions are collected

In stallion, Dog & Boar third secretion is voluminous than first two fractions may be collected.

Physiological and Biochemical studies

• Immediately after collection take a small drop of semen and examine under low power (10x)

1. Mass motility

Scales Criteria for semen 5 (100%) Excellent motility, extremely rapid swirls

and eddies

4 (90%)Very good motility, waves and eddies observed

3 (75-85%)Good motility, slow formation of waves and eddies

2 (50-75%)Fair motility, vigorous movement but no waves or eddies

1 (<50%)Poor motility, weak and oscillatory movement of spermatozoa

0 No motility(Herman and Madden, 1953)

Individual motility:

Take a small drop of semen on warmed glass slide put a cover slip and estimation under 40x

Scales Criteria for semen 5 Straight and rapid displacement of spermatozoa

4 Rapid displacement, some sperm cells with straight trajectory, others with circular trajectory

3 Spermatozoa follow curvilinear displacement with no trembling movement

2 Slow displacement, trembling, disorganized movement, some spermatozoa move more rapidly

1 Very slow (or no) displacement, trembling of spermatozoa, tail oscillation

0 No displacement of sperm cells

A progressively motile sperm swims forward in an essentially straight line, whereas a non-progressively motile sperm swims, but with an abnormal path, such as in tight circles.

• Take one ml of semen in test tube• Put in a beaker containing ice (0°C) for 10 min• Determine motility and live sperm percentage• Compare the values before and after cold shock• Estimate storage ability and freezability of

spermatozoa

Cold shock resistance test:

Hydrogen ion concentration:

In bulls, rams and dogs About 6.7In Stallions, Boars and cats About 7.4

In Bulls pH of 7.0 or higher seen in incomplete ejaculation and in pathological or inflammatory condition

Methylene blue reduction time test:

Hydrogen ions are released during sperm metabolism.

These ions reduce the methylene blue to leucomethylene.

The liberation of hydrogen ions is due to dehydrogenase enzyme present in active sperm.

Principal :

Method:

1. Prepare methylene blue solution by dissolving 50 mg of methylene blue in 100 ml of citrate buffer

2. Dilute 0.2 ml of semen with 0.8 ml of egg yolk citrate dilutor in a 10 ml vial and mix.

3. Add 0.1 ml of methylene blue solution and mix.

4. Seal the mixture by layering with liquid paraffin, 1.0 centimeter thick.

5. Observe the time required for the sample to lose its blue color.

The semen which bleaches the blue color within 9 minutes is considered to be of good quality.

Resazurin reduction:

Resazurin, a hydrogen acceptor which changes color upon reduction replaces methylene blue in the above test

Microscopic Evaluation

Sperm concentration:Five Methods:

1. Direct sperm count of semen by hemocytometer

2. Use of Spectrophotometer 3. Macroscopically by consistency

or density of semen 4. Opacity tube method by

comparing with standard opacity solution

5. Compare PCV after centrifugation vs Hemocytometer

1. Thoroughly mix specimen and dilute 1:20 with diluent. (To obtain this dilution, dilute 50 uL of liquefied semen with 950 uL of diluent)

2. Thoroughly mix diluted specimen and allow a drop to into each side of the hemocytometer covered with a coverglass.

3. Allow chamber to stand for about 5 minutes in a humid container to prevent drying. During this period, the cells settle and can be more easily counted.

4. After cells have settled, place chamber under phase contrast microscope (preferably), using 40x

Procedure:

Calculation:

1 primary square – 1mm length & 1mm width = 1 sq. mm area

Total 8o squares are counted So,

Surface area of 80 tertiary squares

80/400 × 1 sq. mm = 1/5 sq. mm

Height betn counting chamber & coverslip – 0.1 mmSo, cubic volume of 80 squares 1/5×1/10 = 1/50 cmm

No. of sperms (N) present 1/50 cmm of diluted semen

So, No. of sperm in 1 cmm diluted semen = N×50

Semen is diluted 1:200 times and 1ml = 1000 cmm

No. of sperms in 1 ml of undiluted semen

= N×50×200×1000

= N × 107

• Photometers such as the Photometer SDM5are in common use.

• A sample of raw semen is diluted in a cuvette with a predetermined volume of diluent and analyzed with the photometer.

• This analysis takes about 30 seconds.

• The volume, progressive motility, number of sperm per dose and the volume per dose is entered.

• The number of doses to be frozen and the final extender volume will be calculated.

Electronic Counting

CASA System

• Systems such as Sperm Vision use computer programmed digital analysis of microscope fields of moving sperm.

• Several characteristics of motility are quantified, and sperm concentration is also determined.

• These systems require appropriate dilution of semen samples before filling of one or more commercially supplied disposable counting chambers with a depth of 20 microns.

• A counting chamber is placed under a microscope and up to 7 microscope fields are analyzed in just seconds per field.

• Data can be stored and provided on customized printed forms.

• Sperm cells are translucent when observed with bright field microscopy; therefore, phase contrast microscopy or the use of sperm stains are needed for analysis of sperm morphology.

• Eosin-nigrosin stain is commonly used as a "live/dead" stain because in addition to providing background-contrast for sperm cells with the nigrosin component.

• Sperm membrane penetration by eosin, or lack thereof, is an indicator of sperm membrane integrity and thus of sperm viability.

Sperm Morphology Evaluation

• Put a glass slide on a warming plate (37°C) for 30 - 60 seconds.

• Put a 5 - 6 mm droplet of eosin-nigrosin stain at one end of the glass slide.

• Put a droplet of semen beside the droplet of stain

• The droplet’s size depends on the density of the semen

Technique:

• Mix the stain and the semen on the slide.

• Spread the mixture slowly on the slide from one end to the other using the edge of another glass slide.

• Dry the smear quickly by blowing air over it.

• Perform the sperm morphology evaluation at 1000 x magnification using immersion oil, counting at least 100 sperm per sample.

• If a high number of abnormalities are observed, a count of 300 or more sperm will give a more accurate differential count.

Other staining techniques

1. Toluidine blue stain

2. Eosin-opal blue stain

3. Eosin fast green stain

4. Eosin-Aniline blue stain

5. Rose bengal stain

Double headed sperm Misshapen head along with 4 normal sperm

Elongated head Pyriform (pear-shaped) head and bent, abnormal midpiece

Proximal droplet Distal droplet

Detached head Coiled tail

Examination of semen for Genital Infections

Microorganism like Mycobacterium tuberculosis, Brucella abortus, Corynebacterium pyogenes, Staphylococci and Streptococci can be demonstrated by culture methods.

Campylobacter fetus :

Primary cultures must be incubated under 10% CO2 or in a gas mixture containing not more than 5% oxygen.

The commonly employed method for diagnosis of vibriosis is to test presence of antibodies for C. fetus in the vagina mucus

Brucellosis:Agglutination test on seminal plasma can be conducted for detection of antibodies against Brucella abortus by the conventional tube method

Viral infectious agents:

Estimated by tissue culture technique

Trichomonas fetus:

Detected by microscopic observation of moving protozoa.

Also cultures from preputial washings can be obtained.