ANDROLOGY

LECTURE-3
 Semen Collection in Bulls
Introduction to Semen Collection
• Proper and clean collection of semen is critical in artificial insemination.
• Key goals: collect microbe-free semen with spermatozoa of good vigour,
resistance, and livability.
• Quality of semen depends on the bull and the collection method.

Semen Collection Methods in Bulls

1. Collection from Vagina
2. Ampullary Massage Technique
3. Electroejaculation Method
4. Artificial Vagina (AV) Method
 Method Advantages Disadvantages
Poor semen quality, risk of
Vaginal Method Simple, low-cost method
contamination.

Useful for bulls with low Poor concentration, requires

Ampullary Massage
libido or physical incapacity skill, possible inflammation.

Useful for bulls with low Painful, lower semen quality,

Electroejaculation
libido or unable to mount higher contamination.

Requires skilled personnel,

Best quality semen, most
Artificial Vagina (AV) can be affected by
natural simulation
temperature.
 Parts of Artificial Vagina (AV)

PART DESCRIPTION

Rubber Hose/Cylinder Outer tube of the AV (varies in size based on species).

Inserted inside the rubber cylinder to form a smooth
Inner Liner
surface.

Director Cone Guides semen into the collection vial.

Collection Vial Holds the collected semen.
Insulation Bag Prevents semen shock due to temperature variations.
 Artificial Vagina Artificial Vagina Artificial Vagina
Species
Hose Liner Cone

Length: 24 cm
Cattle Length: 35 cm Length: 54 cm (Upper: 5.0 cm,
Lower: 1.5-2.0 cm)

Buffalo Length: 30 cm Length: 54 cm -

Stallion Length: 54 cm Length: 30 cm -

Sheep Length: 20 cm Length: 30 cm -

Goat Length: 15 cm Length: 30 cm -

 Semen Collection in Stallion:
[Link] Method:
The stallion mounts a mare, and a rubber tube is used to collect semen from the vagina into
a sterile container.
[Link]-End Sample:
Similar to the vaginal method, with semen collected into a sterile bottle.
[Link]/Breeder’s Bag:
A sterilized rubber bag covers the erect penis. After ejaculation, semen is transferred to a
pre-warmed sterile tube for processing.
[Link] Vagina (AV):
The most effective method, using specially designed models like Missouri or Colorado. AVs
are adjusted for temperature (45-48°C) and pressure to ensure semen quality while
maintaining hygiene. Semen collection is optimized at the stallion’s Daily Sperm Output
(DSO), stable after 7-10 days of regular collection.
Semen Collection in Boars:
1. Gloved Hand Method
2. Artificial Vagina:
a. Length: 12.5 cm, diameter: 4.5 cm
[Link] liner diameter – 40 cm
c. Temperature of AV: 45-50°C
[Link] continues for 15 mins
e. Sperm rich fraction present in 3rd or 4th minute
3. Electro-ejaculation method:
a. Electric current (12-20 V, 5-10 sec interval) passed
through rectum
[Link] is heavily contaminated
Semen Collection in Dogs:
1. Digital pressure/massage
a. Ejaculation in three fractions:
i. First fraction – clear fluid with few sperms
[Link] fraction – Actual semen
[Link] fraction – Only prostatic fluid
[Link] fractions collected in different digital cups

2. AV method
a. Length of AV – 19-20 cm, Diameter – 5 cm
[Link]: 40-42°C
Semen Evaluation
Semen Evaluation and Quality Assessment for Artificial Insemination
Introduction to Semen Evaluation
• Semen evaluation is not the sole indicator of bull fertility.
• Suitability for artificial insemination depends on multiple semen
characteristics.
• Semen evaluation provides insight into sexual function and fertility potential.
• Semen cannot be evaluated based on a single trait—multiple factors must be
assessed.
• Semen tests are essential for assessing semen quality before use.
• Semen: A suspension of spermatozoa in seminal plasma, discharged during
mating.
Composition of Semen
• Spermatozoa:
o Produced in the testes and stored in the epididymis (10% of total
semen volume).
• Seminal Plasma:
o A mixture of secretions from seminal vesicles, Cowper’s glands,
prostate, ampullae, and epididymis.
o Seminal vesicle secretions constitute 55% of total semen volume.
o Nourishes sperm cells during ejaculation.
Factors Affecting Semen Characteristics
• Breed of the bull.
• Frequency of semen collection.
• Age and health of the bull.
• Seasonal effects (e.g., heat stress).
• Sexual excitement before collection.
• Handling and collection techniques.
Semen Quality and Fertility
• Factors like aging, cold shock, and preservation can deteriorate
semen quality.
• Energy sources in spermatozoa limit their lifespan.
• Semen examination helps diagnose genital or testicular conditions.
• Semen quality is a key indicator of male fertility.
• Semen quality variation is minimal between ejaculates of the same
bull.
Semen Storage and Handling
• After collection, place semen in a water bath at 37°C.
• Avoid overheating, rapid chilling, or shaking semen.
• Prevent sunlight exposure to maintain semen vitality.
Precautions to Maintain Semen Quality
1. Cleanliness: Ensure AV (artificial vagina) and collection containers
are free of contaminants (e.g., alcohol, petroleum jelly, antiseptics).
2. Pre-Collection: Keep dirt and urine out of the artificial vagina.
3. Post-Collection: Place semen in a 37°C water bath immediately.
4. Temperature Control: Avoid overheating or rapid cooling.
5. Avoid Excessive Shaking: Prevent damage to spermatozoa
MACROSCOPIC SEMEN EVALUATION
• Parameters:
o Volume
o Colour
o Consistency/Density
o Presence of Foreign Materials
o Gross Motility
• Volume Determination: Measured immediately after collection.
• Factors Affecting Volume:
o Species
o Individual variation
o Age, body size, and reproductive health
o Frequency of service
• Increased Volume: Seen with age up to 6-8 years.
• Decreased Volume:
o Young males Note:
o Overused males • Aspermia: No sperm
o Incomplete ejaculation • Hypospermia: Reduced sperm volume
o Bilateral seminal vesiculitis • Hyperspermia: Increased sperm volume
 Semen Volume by Species:
Species Average Volume (ml) Range (ml)
Bull 4 1-15 (2-8)
Stallion 70 30-250
Ram/Buck 1 0.7-3
Boar 250 125-400
Dog 10 1.25-30
Cat 0.04 0.01-0.12
Fowl 0.75 0.25-2.0
Man 2-6 -
Elephant 50-100 -
COLOUR OF SEMEN
• Normal Colour:
o Bull/Buck: Milky white, creamy, opaque.
o Buffalo: Whitish (lighter than bull semen).
o Stallion/Boar/Dog: Pearly white to grey, translucent.
• Deviations:
o Yellow: Normal in some bulls (due to riboflavin).
o Brownish: Orchitis (blood pigments).
o Dark Red/Pink: Hemorrhage.
o Yellow-Green: Pseudomonas aeruginosa infection.
o Light Brown: Dung contamination.
o Dull White: Increased spermatogenic cells.
o Yellow: Presence of urine.
• VISCOSITY AND DENSITY
• Viscosity: Depends on sperm concentration.
• High concentration → Creamy, viscous.
• Low concentration → Watery, less viscous.
• Specific Gravity of Bull Semen: 1.036 (positive
correlation with sperm concentration). Colour Density Grade
• Density Grading: Creamy DDDD
• Pathological Conditions Affecting Milky DDD
Consistency: Thin Milky DD
• Epididymitis: Less milky semen. Translucent D
• Catarrhal Conditions: Thick viscous semen. Watery O
PRESENCE OF FOREIGN MATERIALS
• Sources of Foreign Materials:
o Animal: Dung, pus, urine, hair, dust.
o Environment: Sand, bedding, insects.
AV (Artificial Vagina): Water, lubricant jelly, dusting
powder.
 GROSS MOTILITY:
• Assessment: A drop of semen placed on a Grade Description
Percentage of
warm slide and observed with naked eye for Active Sperm

wave motion. 5 Very vigorous, rapid 90-100%

* Wavy Motion: Indicates live sperm. waves, extreme
eddies

Grading Based on Gross Motility: 4 Vigorous, rapid 70-80%

waves, abrupt eddies
• Acceptable Grades: 4 and 5.
3 Rapid movement, 50-60%
• Mass Activity: Collective movement or slow waves
wave motion of sperm.
2 Oscillatory or rotary 30-40%
movement

1 Stationary, weak 10-20%

movements

0 Immotile sperm 0%
 INDIVIDUAL MOTILITY
• Assessment: Movement of individual sperm
under a phase contrast microscope after dilution
(physiological saline, 3% sodium citrate, Tris
buffer, or Tris-egg yolk extender). Grade Progressive
• Progressive Motility: Rapid forward Motility (%)
movement (key factor for fertility).
1 90-100%
• Types of Movement:
• Circular: Cold shock. 2 70-80%
• Reverse: Movement in reverse direction. 3 50-60%
• Oscillatory: Jerky movement. 4 30-40%
Grading of Progressive Motility:
5 0-20%
• Ideal Motility: 70% progressive motility.
• Effect of Temperature: Cold or heat shock can
reduce motility.
 Estimation of Sperm Concentration

Importance of Estimating Sperm Concentration:

•Determines the number of females that can be inseminated.
•Aids in diluting fresh semen and ensures sufficient concentration in frozen semen
samples.

Methods of Estimating Sperm Concentration

[Link] Examination
[Link] Volume Method
[Link]
[Link]
[Link] Tubes
[Link] Assisted Semen Analyzer (CASA)
[Link] Method
Visual Examination:
[Link] on the appearance of semen (e.g., creamy, cloudy).
[Link] reliable for medium/low concentration samples.
[Link] in bulls and rams.

Cell Volume Method:

[Link] separates sperm from seminal plasma.
[Link] packed sperm volume.
[Link] is limited due to interference from other materials.

Colorimeter:
1. Measures optical density of semen samples.
2. Converts light transmission into sperm concentration.
3. Requires initial standardization and monthly calibration.
• Photometer
• Advanced form of colorimeter.

• Displays concentration, dilution rate, and number of doses.

• Needs calibration every 2 weeks with haemocytometer.

Opacity Tubes
oBrown's opacity tubes used to estimate concentration.
o Dilute semen with formal saline (1:100) and compare with opacity
tubes.
o Formula for concentration:
▪ Concentration (sperm/cmm) = Opacity tube number × dilution
rate × 100 × 5
Computer Assisted Semen Analyzer (CASA)
oMost advanced method, but expensive and not commonly used.

Haemocytometer Method
oGold standard for accuracy.
o Requires skill, and is time-consuming, not suitable for large
sample processing.
 NOMENCLATURE FOR SPERM CONCENTRATION:

Terminology Explanation
Normozoospermia Normal sperm concentration
Oligozoospermia Reduced sperm concentration
Polyzoospermia Increased sperm concentration
Azoospermia Zero sperm concentration
• Eosin-nigrosin staining:
Eosin stains dead sperm
pink (membrane intact in
live sperm).
• Nigrosin: Background
stain.
• Rose Bengal Stain: Used for
more detailed morphology
assessment
Interpretation of Spermiogram:
• Up to 20% abnormalities are acceptable in bulls
(7.5% major, 12.5% minor).
• Hereditary defects: <5% allowed.
• Specific minor abnormalities: <10% allowed.
• Abnormalities >15% major or >30% total may
indicate poor breeding potential.
BIOCHEMICAL TESTS:
o pH: Bull semen = 6.8, Stallion = 7.4
o Methylene Blue Reduction Test (MBRT): 3-5 min = Good, >9 min = Poor
o Fructolysis: Higher fructolysis = better semen quality
o Pyruvate Utilization: Increased oxygen uptake = high fertility
o GOT: Presence indicates sperm damage (post-thawing)
o Hyaluronidase: Indicates acrosomal damage
o Resazurin Test: Good semen reduces resazurin quickly (pink in 1 min)
o Buffering Capacity: Better semen resists pH changes
o Phosphatase Test: High levels correlate with better quality semen
o Millovanov’s Test: R > 5000 indicates good motility resistance
Note: Glutamate Oxaloacetate Transaminase. This enzyme is also known as Aspartate Aminotransferase
(AST) : used as a marker for cellular damage
SEMEN PRESERVATION METHODS
Semen preservation is critical for artificial
insemination (AI) programs in cattle and buffalo. The
preservation method depends on the temperature of
storage, which includes:
[Link] Temperature Preservation (18–25°C)
[Link] (4–6°C)
[Link]-Low Temperature Preservation (-79°C to -
196°C)
 Extender Composition Duration of Fertility (%)
Preservation

Illini Variable Sodium citrate, Sodium bicarbonate, 3–6 days 76% (non-return
Temperature (IVT) Diluent Glucose, Potassium chloride, basis)
Sulphanilamide

Cornell University Sodium bicarbonate, Sodium citrate, 3–6 days 70–80%

Extender (CUE) Potassium chloride, Glucose, Glycine,
Catalase, Citric acid

Milovanov's Method Potassium dihydrogen phosphate, Sodium 3–6 days -

(Carbonate-Phosphate citrate, Glucose, Penicillin G sodium,
Method) Streptomycin sulfate, Sulphanilamide

Coconut Milk Extender Sodium citrate, Penicillin G sodium, 7 days -

(CME) Dihydrostreptomycin, Polymixine B,
Catalase, Mycostatin, Egg yolk, Coconut
milk
 Extenders for • 2. Preservation at Refrigeration Temperature (4–6°C)
Common method before frozen semen technology.
Refrigeration •

Duration: Semen can be used for insemination up to 72

Preservation:
•
hours.

Extender Composition Duration of Preservation Fertility (%)

Egg Yolk Phosphate Extender Disodium hydrogen phosphate, 72–96 hours Good fertility in field tests
(EYP) Potassium dihydrogen phosphate,
Egg yolk

Egg Yolk Citrate Extender (EYC) Sodium citrate dihydrate, Distilled 72 hours Clear semen, widely used for bull
water, Egg yolk and buffalo semen

Caprogen Sodium citrate, Glucose, Glycine, 4–5 days -

Glycerol, Egg yolk

Kampschmidt (Egg Yolk Glucose Dextrose, Sodium bicarbonate, 72–96 hours 45% fertility for Zebu, 40% for
Bicarbonate Extender) Sulphamethazine, Egg yolk buffaloes

D2 Dilutor Sodium bicarbonate, Glucose, 4–6 days 45% fertility for Zebu, 40% for
Fructose, Egg yolk buffaloes
 3. Ultra-Low Temperature Preservation (-79°C to -196°C)
Modern method: Freezing semen for long-term storage.
Extenders for Ultra-Low Temperature Preservation:
Duration of
Extender Composition Preservation Fertility (%)
Tris-Egg Yolk-Glycerol Tris, Citric acid, Fructose, Long-term -
Diluent Glycerol, Penicillin G sodium,
Dihydrostreptomycin, Egg yolk
Sodium Citrate Sodium citrate, Penicillin G Long-term -
Extender sodium, Dihydrostreptomycin, Egg
yolk, Glycerol
Milk Extender (For Boiled milk, Sulphanilamide, Long-term -
Freezing) Penicillin G sodium,
Dihydrostreptomycin
Lactose-Egg Yolk Lactose, Egg yolk Long-term Better survival with higher
Extender molecular weight sugars
Important Points about Extenders
• Illini Variable Temperature (IVT) Diluent: Uses CO₂ to reduce sperm
metabolism.
• Cornell University Extender (CUE): CO₂ produced from glycine
breakdown, no need for external CO₂.
• Caprogen: Developed for 5°C storage, contains catalase to prevent
hydrogen peroxide buildup.
• Milk Extender: Heated milk and egg yolk protect sperm from cold
shock and extend semen lifespan.
• Tris-egg yolk-glycerol diluent: Ideal for freezing sperm at ultra-low
temperatures, widely used in advanced countries.
Other Important Extenders
• D5 Dilutor: Used for buffalo semen, provides
good preservation.
• Glycine Dilutor: Not suitable for buffalo semen,
affects sperm morphology.

• Coconut Milk Extender (CME): Can store semen

for 7 days, but limited by coconut milk
availability.
 Method/Extender Preservation Duration Fertility
Temperature
IVT Diluent Room temperature 3–6 days 76%
(18–25°C)
CUE Room temperature 3–6 days 70–80%
(20–24°C)
Milovanov's Room temperature 3–6 days -
Coconut Milk Room temperature 7 days -
Fertility and EYP (Egg Yolk
Phosphate)
4–6°C 72–96 hours Good fertility

Duration of EYC (Egg Yolk

Citrate)
4–6°C 72 hours Good fertility

Storage Summary Caprogen

D2 Dilutor
4–6°C
4–6°C
4–5 days
4–6 days
-
40–45%
Tris-Egg Yolk- Ultra-low temp (- Long-term -
Glycerol 79°C to -196°C)
Milk Extender Ultra-low temp (- Long-term -
79°C to -196°C)
In conclusion:
• Room temperature preservation: Generally used for
short-term storage, effective in resource-limited areas.
• Refrigeration: Suitable for 72-hour preservation, widely
used.
• Ultra-low temperature: Ideal for long-term storage and
international semen transport.
 PACKING SYSTEMS OF FROZEN SEMEN

Ampoule Method
o Developed by Macpherson (1954) and Vandemark & Merits:
Kinney (1954) o Contamination avoided

o Semen volume: 0.5-1.0 ml o Identification possible (bull number, etc.)

o Ampoules sealed over flame, leaving 0.5 ml air space Demerits:

o Lower freezability and fertility
o Freezing process:
o Space-consuming in storage
▪ Initial cooling in alcohol or acetone bath at 5°C
o 8-10% semen loss during thawing
▪ Rate of cooling: 1-2°C/min to -15°C
o Glass catheter use for AI has drawbacks
▪ More CO₂ to reach -79°C at 4-5°C/min
o Stored in solid CO₂ (-79°C) or liquid nitrogen (-196°C)
o Thawing: Ampoule in warm water, cut, semen drawn
into catheter
 Pellet Method
o Developed by Nagase and Niwa (1963) Thawing:
o Thawed in 3% sodium citrate solution with
o Semen volume: 0.1-0.2 ml
1.5% fructose at 20°C
o Freezing: Semen diluted with lactose,
Merits:
glycerol, egg yolk
o Economical
o Media for dilution:
o Less storage space required
▪ Lactose: 75.3 ml
Demerits:
▪ Glycerol: 4.7 ml o Identification difficult
▪ Egg yolk: 20 ml o Risk of contamination during storage
o Cooling on solid CO₂ (-79°C) o Pellets may break during handling, leading
o Stored in liquid nitrogen to sperm loss
o Moderate freezability
o Tedious thawing process
• Straw Technique
• Developed in Denmark (1940)

• First frozen by Adler (1960) using liquid nitrogen vapour

• Cassou modified it in 1965 (medium straws), then in 1968 (mini

straws)
• "French straws" made of polyvinyl chloride (PVC) commonly used
• Types of Straws:
• Sealed with steel, glass, or plastic Type of Straw Length Diameter Volume
beads (German straw)
(mm) (mm) (ml)
• Identification possible by color,
bull ID, breed, etc.
• Merits: French medium 135 2.8 0.5
• Rapid thawing due to increased
surface area
• Better sperm survival
• Easier AI (smaller diameter) French mini 135 2.0 0.25
• Efficient delivery into uterus
German straw 65 2.8 0.25
• Easier identification (color-coded)
• Reduced space and easier storage
• Demerits:
• Sealing can be difficult
• Use of glass catheters has drawbacks