Semen Preservation

CONTENTS

• Semen Collection

• Semen Evaluation

• Semen Extension

• Semen Preservation Techniques

Semen Collection Methods in
Different Species
1. BOVINE

AV

Electro ejaculation

Ampulla massage

Vaginal sponges

Epididymalsperm retrieval
2. Equine

AV

Pharmacologically induced
ejaculation

Use of a condom

Manual manipulation of the

penis
3. Ovine and caprine

Use of AV

Eelectroejaculation

Epididymalsperm retrieval
4. Swine

Gloved hand

Pharmacological agents
5. Canine

Digital manipulation

Eelectroejaculation

Pharmacological agents
6. Feline

AV

Eelectroejaculation

Urethral catheterization
6. Man

Masturbation

Use of condom

Coitus interruptus

Epididymal sperm retrieval

Penile vibratory
stimulationandEelectroejaculation
Semen Evaluation
 Macroscopic
 Color

 Volume

 pH
 Viscosity

 Liquefaction (human)
 Microscopic
 Motility

 Concentration

 Morphology
Bull

Parameter Normal Values

Semen color Watery to creamy
Ejaculate volume 5 ml (range 1-15 ml)
pH 6.5-7.2
1200 million/ml (range 300-2500
Sperm concentration
million/ml)
Motility Greater than 70%
Morphology Greater than 80%
Stallion

Parameter Normal Values

Semen color Milky white
Ejaculate volume 70 ml (range 30-250 ml)
pH 7.2-7.7
120 million/ml (range 30-600
Sperm concentration
million/ml)
Motility Greater than 60%
Morphology Greater than 60%
Ram & Buck

Parameter Normal Values

Semen color Watery to creamy
Ejaculate volume 1-2 ml
pH 7.0-7.2
Sperm concentration 1-3 billion/ml
Motility Greater than 80%
Morphology Greater than 85%
Dog

Parameter Normal Values

Semen color Cloudy/milky
Ejaculate volume 1-6 ml (without prostatic fraction)
pH 6.3-7.0
Sperm concentration 100-500 million/ml
Motility Greater than 70%
Morphology Greater than 80%
Tom

Parameter Normal Values

Semen color Creamy white
Ejaculate volume 250 ul
pH 6.6-7.8
Sperm concentration 20-50 million per ejaculate
Motility Greater than 70%
Morphology Greater than 60%
Boar

Parameter Normal Values

Semen color Milky colour
Ejaculate volume 100-500 ml
pH 6.8-7.6
Sperm concentration 50-100 million/ml
Motility Greater than 70%
Morphology Greater than 85%
Man

Parameter Normal Values

Semen color Whitish grey
Ejaculate volume 2-5 ml
pH 7.2-7.8
Sperm concentration 15-200 million/ml
Motility Greater than 60%
Morphology Greater than 85%
Liquefaction time 30 min
Semen Extension
Why we do dilution/extension of semen

 Better storage (avoid head agglutination)

 Maintain the fertility for long time

 Increase the number of inseminations

Required Parameters for Semen Dilution

Volume
Motility
Concentration
Which preservation method is used
 Ambient temperature (20-35 ºC)

 Refrigeration (4 ºC, 15 ºC)

 Dry Ice (-79 ºC)

 Liquid oxygen (-180 ºC)

 Liquid nitrogen (-196 ºC)

 Vitrification

 Freeze-drying
Ambient temperature (20-35 ºC)

 Basic media (isotonic with normal pH)

 Citratebased
 Phosphate based
 Tris
 Normal saline
 Nutrients should be…………….
Refrigeration (4ºC/15ºC)

 Basic media supplemented with

Egg yolk
Milk protein
Lecithin
Lipoproteins
Packaging
Freezing (-79, -180 or -196 ºC)

Dilution
is performed by two
methods:
1. One-step dilution
2. Two step dilution
1. One step dilution

 Extension of semen with basic media

supplemented cold shock protectants (egg yolk)
and glycerol
 Cooled the diluted semen upto 4 ºC
 Equilibration at 4 ºC for 2hrs
 Freezing above LN vapors
 Storage in LN
2. Two step dilution
 Preparation of two extenders
1. Basic media supplemented cold shock protectants (egg
yolk) without glycerol
2. Basic media supplemented cold shock protectants (egg
yolk) and double ratio of glycerol
 Extension of semen with basic media supplemented cold
shock protectants (egg yolk)
 Cooled the diluted semen upto 4 ºC
 Addition of extender 2 in previously extended semen
 Equilibration at 4 ºC for 2hrs
 Freezing above LN vapors
 Storage in LN
FREEZE DRYING

Semen Semen Pellet

centrifugation
collection evaluation resuspension

Tris–
HCl,NaClEDTAdil
uent

Packaging Immersion in LN2

Freeze-drying Rehydration in
0.053 mbar & -68 C Storage at 4 C
incryotubes(50ul) for 5 min pure water
0.018 mbar & 20 C
SPERM VITRIFICATION

PRINCIPLE
 Ultra
quick freezing of a small sample
volume
 Direct
contact with N2 → prevents ice
formation
 Physical process of solidification → no
crystallizes, becomes viscous, it passes from
liquid to solid state, similar to glass
Method

Pellet
Semen Semen Dilution of
centrifugation resuspension
collection evaluation semen (1:2)
(100 million/ml)

Dilution with CPA Immersion in LN2 Thawing in WM

25ulsemen:50ul
extender
 Cryoloop method  Grid method
THANKS