Lecture 2. semem collection lecture 2 & 3

4/24/2017ANS203 SEMEN COLLECTION & EVALUATION 1
31/03/2017

Semen Collection And Evaluation

Learning Outcomes;
At the end of this lesson, each one of you should be able to;
1. List down methods of semen collection
2. Identify parts of AV
3. Remember the Advantages and disadvantages of AV,
normal values of the bull sperm, freezing methods and
packaging methods.
4. Explain the reasons of semen extension
5. Recall the advantages and disadvantages of frozen
semen.

METHODS OF SEMEN
COLLECTION
LEARNING OUTCOMES.
After the completion of this lesson, each student should be
able to;
 Recall various methods of semen collection.
Describe advantages and disadvantages of each method.
Explain semen collection by AV method and precautions.
Demonstrate the procedure to prepare artificial Vagina.

Preparatory Activities For Semen
Collection
Semen collection is like harvesting any other farm crop.
Effective harvesting of the semen involves obtaining the
maximum number of sperms of highest possible quality in
each ejaculate.
The ultimate objective is to make maximum use of
superior sires.
 Semen collection is a complex procedure involving
coordinated efforts between the animal handler and the
collector.

BULL PREPARATION
Why is it essential?
• To decrease microbial load
• To increase semen volume, sperm concentration
• To decrease reaction time
PROCEDURE
•Prepucial hairs must be clipped leaving a tuft of 2 cm all around.
•Prepucial washing with warm normal saline may be used to
decreases microbial load.
•Clean and wash the bull, especially the hind quarter 1 hour before
the semen collection.
•Ordinary washing soap and mild brush may be used.
•Special attention must be given on lower abdomen area and
prepuce.
•Paper towel or damp towel may be used to remove excess water

2. TEASER / DUMMY PREPARATION
 Teaser may be a male or a female in estrus.
 It is also cleaned the same way as for bull.
 Special attention should be given to the back and perineum
area.
3. WASHING AREA
Washing area should not be more than 20 m away from
collection area / serving area.
It should be made of rough concrete.
Floor should be slanting with drainage facility of water,
dung etc.
Adequate facility of water with reasonable pressure must
be there.

4. COLLECTION AREA
 It should be away from noisy places.
 It should not be more than 30 m away from semen
evaluation lab.
 The ground must not be slippery / muddy.
 Collection area should be fenced with strong metallic bars
to avoid escape of bull.
 There should be one slanting metal stanchion fitted with
wooden arms for the restraint of teaser.
 Sufficient space should be there for free movement of
bulls and the semen collector.

Methods of semen collection
1.Pan collection method
2.Vaginal spoon method
3.Sponge method
4.Breeder’s bag method
5.Sperm collector method
6.Artificial vagina (AV) method (most commonly used)
7.Massage method (Used for semen collection from lame
bulls and in dogs)
8.Electro ejaculation (From lame bulls)
9.Fistula method
10.Dummy females

SEMEN COLLECTION BY AV METHOD
The AV Method of collecting semen is the fastest and most
sanitary of the various methods available. It has been
proved to be the best method and is now most commonly
used.
WHY IS IT DONE?
 Semen is collected in natural status
 Semen is collected in hygienic condition
Frequency of semen collection:
 It should be done 2-3 times a week. More frequent
semen collection results in poor semen quality.

Artificial Vagina Assembly

ADVANTAGES
 Practically the whole ejaculate is collected in
uncontaminated and natural stage.
It is free from the extraneous secretions.
Sterile conditions of the apparatus ensure disease
control.
The viability of the sperm is better.
No female is needed if dummy is a success.
DISADVANTAGES
Occasionally it is difficult to get the males to serve the
artificial vagina.
The apparatus involved is slightly costly and requires
technical hands.

SEMEN EVALUATION
WHY IS IT ESSENTIAL?
 To investigate the infertility problems
 For breeding soundness evaluation of bulls
 For semen extension and processing

METHODS OF SEMEN EVALUATION
A.Physical / macroscopic
Color :creamy to milky white color
Volume:
Consistency:
pH:6.6 to 6.9
B.Microscopic
Mass motility
Individual motility
Sperm concentration
Live sperm count
Abnormal sperm count
C. Biochemical
Fructolysis index
Methylene blue reduction test
Resazurin reduction test
Rating Grade Type of wave motion
+++++ Good Very dense fast wave motions just like
sea waves
++++ Fairly good Waves are comparatively not so rapid as
above
+++ Average Slow and thin waves
++ Poor Waves absent, progressive individual
movement visible
++ Unsatisfactory Only about 20% may show
progressive scattered movements
Mass Activity

Parameters Cows
Color creamy
Volume 3-5 ml
Consistency Rough estimate of sperm
concentration
Concentration 1000-1500 millions/ml
Motility >50% with forward progression
Live sperm >50%
Total sperm abnormality <20%
Normal Values of Semen

Normal Sperm Morphology

Sperm Abnormalities
Rationale: Only morphologically normal sperm participate in fertilization
process which is prerequisite to fertilization.
Sperm abnormality is detected by Nigrosin staining technique ( Practical Class).
Many abnormalities of structure may occur ;
•In the development of spermatozoa
•In the handling: → during handling → during collection
valuable diagnostic and in assessing the potential fertility of bulls. These may be
in the head, middle piece or tail, which may interfere with progressive motility
and fertility of spermatozoa.
Classified as;
1.Primary : caused by aberration during spermatogenesis.
2.Secondary: aberrations at the subsequent developmental stages i.e. after
spermatogenesis is complete and sperm has left the seminiferous tubules.
Usually due to dysfunction of epididymis, seminal vesicles and genital diseases.

Primary Sperm abnormalities brought about during spermatogenesis may
include in;
1. Head: mega, micro, pyriform elongated, double, and abnormal, detached.
2.Middle piece: abaxial attachment, thick short and
3.Tail: double, stumpy, ring form
Primary sperm Abnormalities
Normal Decapitated Macro cephalic Micro cephalic & Stump Tail Pyriform
Round and Double TailPyriform Round head micro cephalic

 Secondary abnormalities could be due to dysfunction of epididymis, seminal
vesicles and genital diseases. include normal detached heads, loose heads,
detached, coiled mid piece, presence of cytoplasmic droplet.
Secondary sperm Abnormalities
Bent Tail Coiled Tail Folded tail Double Tail

Semen extension
to extend its volume so that a large number of females can
be inseminated from one ejaculate having minimum
number of spermatozoa to achieve fertilization.
 It is the dilution of semen with nutrients to increase the
life of sperm and volume of semen.
Basic components of an extender
1.Egg yolk : Membrane stabilizer, cryoprotectant
2.Buffer : Provides buffering capacity to semen
3.Antibiotics : To check microbial growth
Chilled semen the extenders used are:
•Egg yolk-citrate extender ………………..: Cow bull and buffalo bull
•Tris-yolk extender……………: Buffalo bull

For DFS the extenders used are:-196
• Egg yolk-citrate-glycerol extender : Cow and buffalo bull
• Tris-yolk-glycerol extender : Buffalo bull
Antibiotics added to the extender
• Penicillin – 1000 IU/ml
• Streptomycin – 1000 μg/ml
Composition of citrate buffer;
Sodium citrate dihydrate :2.9 g
Penicillin G sodium :1 lac Unit
Dihydrostreptomycin sulphate :100 mg
DDW :100 ml
Composition of Tris buffer;
Tris buffer :3.028 g
Citric acid monohydrate :1.675 g
Fructose (Anhydrous) :1.250 g
Penicillin G sodium :1 lac Unit
Dihydrostreptomycin sulphate :100 mg
DDW :100 ml

Basic qualities of an ideal extender:
 Should maintain osmotic pressure
 Should maintain PH
 Should have Buffering capacity
 Should provide nutrition
 Should protect sperm against cold shock
 Non-toxic, easy to prepare and easily available
 Compatible with a antibiotics
 Should permit clear sperm picture under microscope.
 Should not render cleaning of glassware or semen containers difficult.
Basic contents of an extender:
 Egg yolk – membrane stabilizer, cryoprotectant
 Buffer – buffering capacity to semen
 Antibiotics – check microbial growth

Semen Preservation or Methods of Freezing
1. Refrigeration Method at 4-6 ° C
2. Ultra low temperature at bovine sperm frozen at ‫97־‬C temp. in dry ice
or Liquid Nitrogen at -196 degree.
3. semen at room temp at 18-25 degree Celsius.
Advantages of frozen semen:
Selective mating.
Early progeny testing.
Maximum utilization of sires.
Long storage.
Reduced transportation cost.
Disease control.
Feeding and management cost of bulls reduced.
Progeny even after death of the bull.
No wastage of semen.
No need of sterilization of AI gun.
No fear of breakage of AI gun.

Disadvantages of frozen semen
 Semen from about 10 to 20 per cent of bulls will not
withstand freezing. These are often bulls of low fertility.
The ampouling, freezing and storage equipment are very
costly.
 In the freezing process about 40 per cent spermatozoa are
killed. So increased number of spermatozoa per
insemination are required.
 If proper bull health is not maintained, frozen semen has
great potential for the spread of viral and bacterial diseases.

Packing Methods For Freezing
The straw method was introduced by scientist of
Denmark during 1940 for packing of liquid semen.
Adler (1960) first frozen the semen packed in straws by
using liquid nitrogen vapor.
This technique was later modified by Cassou in 1965
which was called as “medium straws”.
This was in use for a period of time. Later Cassou in 1968
brought further improvement by reducing diameter for
better freezing which is called as “mini straws”.
Most of the straws recently used were called as “French
straws” - made of poly vinyl chloride.

 Glass ampoules (Van Demark and Kinney, 1954).
 German straws (Simmet, 1972- Mini tubes-Landshut
method – 65 mm x 2.8 mm – 0.25 ml capacity)
S.No. Type of straw Length Diameter Volume
1 French
medium
135 mm 2.8 mm 0.5 ml
2 French mini 135 mm 2.0 mm 0.25 ml
3 German straw 65 mm 2.8 mm 0.25 ml
The dimensions of French and German straws are as follows
Freezing technique: French (Cassou) technique is used in these days

 The French straws have two ends – factory seal end
and laboratory seal end. In this the factory seal end
was formed in the manufacturers themselves.
 The laboratory seal end will be open when it is
purchased. The laboratory seal has to be created in
the semen processing laboratory after filling the
straw with diluted semen before freezing.
 Factory seal has two cotton plugs with PVA powder in-between them.
This seal is formed by while manufacturing semen straw at factory.
 Laboratory seal - formed with PVA powder or sealing machine after
filling of semen. This seal is formed at semen processing laboratory.

Thawing of frozen semen:
 Warm water (35 - 40C temp.) 30 sec. is the best.
Storage and shipment of semen:
 Once semen is stored at lower temp., there should
never be change in the temp. of stored semen.
 Semen should also be transferred to other places at
the same temp. at which it is stored.
 The vial containing chilled semen should be wrapped
with paper before it is placed in thermos having ice,
to avoid cold shock to sperms coming directly in
contact with the ice in the thermos.
 Frozen semen should always be transported in the
liquid nitrogen containers.

Video ( Semen collection to AI)

Lecture 2. semem collection lecture 2 & 3

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