Dr. G.K.

Jalu
Asst.Director Of A.H.
Regional Artificial Insemination Center
Rajkot
 AI in mares was initiated with Liquid semen

 More number of mares can be inseminated

 Short duration. (room or refrigerator temp.)

 Long term storage is not possible, viability maintained

for only 48 hours.

 Frozen - potential advantages - Long term storage is

necessary.
Merits of Cryopreservation & A.I

 Genetic improvement in equines.

 Increasing the numbers of females covered per stallion.

 Control of transmission of diseases

 Decreased stress and risk to mare.

 Minimize transport costs.

 Allowing the covering of injured mares, precluded from natural

covering.

 Uses of injured stallion.

Halts the metabolic processes of spermatozoa and
allowing indefinite storage without loss of fertility.

Polge et al. (1949) made cryopeservation possible and

stored at -196°C in liquid nitrogen for indefinite
period without losing its fertilizing ability.

Discovery of cryoprotectant properties of glycerol was

a boon in the development of cryopreservation
technique.
Damages :

Internal ice crystal formation (Fast freezing)

Increase in solute concentration (Slow freezing)

Changes in plasma membrane permeability to

calcium.

Leakages of important enzymes and cellular

materials to external environment.
Various steps in semen freezing:

 Collection of semen : A.V. over a mare well in estrous or

dummy

 The temperature of A.V. should be maintained near about

42-45C.

 The female should be restrained properly during collection

to avoid any type of injury to stallion/handler

 The tail should be properly covered by bandage.

 Gel removal

 Quality assessment (pH - Volume - Sperm

concentration – Initial motility, progressive motility,
live & dead sperm count and sperm abnormality)
 Dilution of gel free semen at 1:1 with primary semen
extender (i) Glucose 0.15 g
(ii) Sod.Citrate dehydrate 2.60 g
(iii) disidium EDTA 0.37 g
(iv) Sod.Bicarbonate 0.12 g
(v) Streptomycine 0.10 g
(vi) Benzyl Penicilin 0.10 g
(vii) double D.W. Up to 100 ml
 Centrifuge at 1500-2000 rpm for 3-5 minutes in refrigerated centrifuge
to get the sperm pellet in the bottom.
 Remove supernatant plasma carefully without disturbing the sperm
pellet and resuspend the pellet in freezing media (secondary extender).
 A (i) Glucose 6.00 g
(ii) Sod.Citrate dehydrate 0.37 g
(iii) disidium EDTA 0.37 g
(iv) Sod.Bicarbonate 0.12 g
(v) Streptomycine 0.10 g
(vi) Benzyl Penicilin 0.10 g
(vii) double D.W. Up to 100 ml
B (i) Lactose 11.00 g
(ii) Streptomycine 0.08 g
(iii) Benzyl Penicilin 0.08 g
(iv) Double D.W. Up to 100 ml

Solu.-A 25 ml + Solu.B 50 ml + 20 ml Egg yolk

Centrifuged at 3000 rpm at 10’c for 30 minute,collect
clear supernatant fluid and add Glycerol 3 % of total
volume
 Add secondary extender to an extent so that 100x10 6
progressively motile sperm per ml and insemination dose
should be maintained about 400 – 600x 106 per mare.

Keep the freezing media mixed semen at 5oC for 2 hrs

Marking of Straws: Mark the name of the stallion, place

and date of freezing

Filling of straws: Machine filling/Manual filling

 Loading of straws in programmable cryo-freezer

 Cryo-freezer having controlled cooling rate as per the

following protocol.

 Cooling of semen @ 0.3 C/ min from 18 C to 5 C.

Hold for 10 minute at 5C.

 Cooling @ 10 C/ min from 5C to -15C.

 Cooling @ 19 C/min from –15C to –100 C. Hold for

10 minutes

 Plunge the straws into LN2 container for preservation

at- 196C for longer period.
Thawing and Artificial Insemination (AI):

 37C for 1 min in water bath and evaluate for post thaw
semen quality.

 The optimum time for AI: 12 hours before ovulation or 6 hr

within ovulation

 Normal insemination dose is 4-5 ml containing 400-600

million sperms (8-10 straws).
When should be semen evaluated?

 Routinely each year before the breeding season

 When lowered fertility is suspected

 When abnormal sexual behaviour is seen

 If a pathogenic infection is suspected

 Before sale, or sale of liquid or frozen semen

 For semen preservation and artificial insemination

Macroscopic Examination:

 Volume
Range 30-250 ml

 gross appearance
milky white to creamy

 Osmolarity - 290 and 310 m Osm

 Seminal fluid pH - 6.9 to 7.7

Microscopic Examination
 sperm concentration - Normal 100-250 million/ml
Season, ejaculation frequency, testicular size, breed, age , environmental
factors etc.

 Initial motility – Progessive motility is good indication for viable

spermatozoa– 60 -90 %

 PTM – 35-50% Stallion, 40-60% Jack stallion

 Morphology- Longevity- expected survival time for the spermatozoa within

the female tract

 Live/Dead – eosin-nigrosin stain, Fertile stallion will normally show less than
10-15% of any single abnormality
 Cytology
 Hemospermia or genital tract infections
Haematoxylin-eosin stain or Wright’s
 1500 leukocytes per ml and ideally
no erythrocytes
 Microbiology
Bacteriology, virology etc., to cheek out infected semen

Functional tests.
 Assess the functional integrity rather than physical integrity of the
spermatozoa
 biochemical analysis, membrane integrity test,
 Flow cytometery, Computer assisted semen analysis(CASA)
 Filtration assay, Hypo-osmotic sperm swelling test (HOS Test),
 In vitro capacitation and acrosome reaction test
 Sperm egg interaction or sperm egg binding test etc.
Fluorescent stains
 Carboxyfluorescein diacetate (CFDA) and propidium iodide
(PI) or calcein AM and ethidium homodimer, can be used to
assess cell viability.
 Hoechst 33258, ethidium bromide (EB) and SYBR14
 FITC - green fluorescence over the acrosomal cap, while
acrosome-reacting spermatozoa showed a patchy disrupted
image of fluorescence.

 Mitochondrial activity can by evaluated by Rhodamine 123

JC-1 accurately reflects changes in mitochondrial membrane

potential.
water will enter the spermatozoon in an attempt to
attain osmotic equilibrium and increases the volume of
the cell, plasma membrane bulges. The influx of water
only occurs in the tail region and creates different types
of curls.
Ability of the sperm to undergo acrosome reaction and
bind to the plasma membrane of the oocyte

Evaluate sperm motility, zona binding and penetration,

sperm capacitation, and the acrosome reaction
 Ability to induce the acrosome reaction, in vitro, has
proven to be a useful tool to evaluate sperm function, as
well as being a monitor of an essential step for in vitro
fertilization
 Modification of the composition of the sperm membrane
lipids that occur during capacitation and acrosome
reaction
 Chromatin integrity status may be assessed with acridine
orange test and observed in fluorescence microscopy

 chromatin structure in which the DNA in situ is resistant

to denaturation, whereas the DNA of spermatozoa with an
abnormal chromatin structure is susceptible to
denaturation in situ.
 In vitro penetration of zona-free hamster oocytes provides
information about the capability of sperm that have
already undergone the acrosome reaction to penetrate the
oocyte. Multiple spermatozoa can penetrate the
heterologous hamster oocyte .

In vitro fertilization (IVF)

 IVF has proven to be a reliable test for sperm quality and
fertilizing capacity in human fertility clinics, but being an
invasive and expensive procedure, it cannot be routinely
used as a semen evaluation test