School Papers

EMBRYO frozen semen for the past several decades.

EMBRYO TRANSFER IN CATTLE 

Artificial
Insemination has allowed genetic process to be achieved relatively,
quickly through the widespread and efficient use of frozen semen for the
past several decades. Genetic programmes were limited to male side for
genetic contribution because cows could relatively produce only one calf
in one year. Today due to the advancement of embryo transfer technique
cows can produce many offspring every year.Selection
of donorsSynchronization
of estrousInduction
of superovulationEmbryo
collectionEvaluation
of embryosSelection
of recipientsTransfer
of embryosEmbryo
transfer techniques have been extensively used in cattle and more
sporadically in Sheep, Goats and Pigs.

SELECTION OF DONORS

following
are the criteria for selection of donor,Donor
should be of age between three years and ten years.Donor
should be free from genetic diseases and conformational abnormalities.They
should exhibit regular oestrus cycle.Superior
production traits of economic importance.Previous
record of sound reproductive performance including successive fertility
and artificial insemination.

INDUCTION OF SUPEROVULATION 

Superovulation
means the induction of multiple ovulations by application of exogenous
hormones (PMSG – Pregnant Mare Serum gonadotrophins, FSH- Follicle
Stimulating hormone, HMG – Human Menopausal Gonadotrophins) in the early
follicular or in the luteal phase of the oestrus cycle in order to collect
large number of fertilized eggs. Most frequenctly PMSG or FSH is used.PMSG
– one single injection (2000 to 3000 IU) as the substance has a very long
half life time.FSH
– Multiple injections of FSH (35-50 mg) twice daily for four days.48
and 60 hours after beginning the gondaotrophin treatment, Prostaglandins
(PG) are administered to induce oestrus. Inseminations are performed at
oestrus two days later. Embryos are recovered 8 days after insemination.

SCHEDULE FOR SUPEROVULATION IN CATTLE

·        
 

In
the bovine, embryos are recovered by non surgical methods.Special
catheters(Foley’s catheters) are introduced via the cerevix into the
uterine horn and embryos are flushed with 250 – 300ml flushing medium.Recovery
rate is influenced byThe
position of the embryos in the uterusThe
flushing methodThe
time of recoveryOvarian
responseEmbryo
viabilityIn
other farm animals, Sheep, Pigs and Goats embryo recovery is performed by
surgical methods. Disadvantage of the surgical method is the number of
repetitions is limited by the occurrence of adhesions.After
recovering embryos from the flushing medium their further developmental
capacity has to be evaluated. Embryo viability can be evaluated by various
methods.

Morphological evaluation

Excellent embryos: Embryos
in the appropriate developmental stage with a perfect morphology.Good embryos: Embryos
in the appropriate stage of development with slight morphological
deviations. Eg. Minor damage of the zona pellucida and excluded
blastomeres or vesicles in the perivitelline space.Degenerated and / or retarded embryos: Embryos in the
appropriate developmental stage with major morphological deviations
(degenerated embryos)Unfertilized
ova.

Staining methods

Embryo
viability can be evaluated by various vital staining and fluorescence
techniques.

SELECTION OF RECIPIENTS 

Recipients
are selected based on theNormal
physiological and health conditions.The
reproductive status.Lack
of any reproductive disorders.Compatibility
to the donor with respect to size of the foetus.Oestrus
synchronization.In
bovines, heifers and young cows are best suited as recipients.

TRANSFER OF EMBRYOS

Recently
special catheters have been developed to perform non – surgical transfers
in cattle.It
is important to transfer the embryo in to tip of the uterine horn without
damaging the endometrium.For
a successful embryo transfer, the most important is embryo quality. It is
very important to transfer the embryo ipsilateral, that means into the
horn bearing the corpus luteum.

CRYOPRESERVATION OF EMBRYOS 

Cryopreservation
of embryos is an essential part of embryo transfer programme and it allows
worldwide shipment of embryos.Reliable
freezing methods have been developed for bovine and sheep embryos.An
optimal method for bovine embryos includes a one step addition of 1.4M
glycerol as cryoprotectant, a 20 minutes equilibration period and 0.25 ml
straws as embryo containers, slow cooling down to -35°C and subsequent
plunging to liquid nitrogen (-196°C). Embryos are thawed by placing the
straws directly into warm water.

APPLICATIONS OF EMBRYO TRANSFER 

Exploitation
of female reproductive capacity (more offspring from valuable donors).Significant
facilitation of import and export for valuable genetic material.Development
of new breeding concepts.Gene
conservation by freezing techniques.Twin
production (Embryo splitting).Introduction
of new genes into closed herds.Manipulation
of embryos.Gene
transfer.

MODULE-19: IN VITRO FERTILIZATION, SEXING,
MICROMANIPULATION AND ANIMAL CLONING 

Learning objectives

The learner will learn about

In
vitro fertilizationSteps
in in vitro fertilizationSexing
of EmbryosMicromanipulationAnimal
Cloning 

IN VITRO FERTILISATION (IVF) 

Embryo
transfer is not used widely because of its cost, technical difficulty and
the limited supply of embryos available from super ovulated donors. These
limitations would be removed if we could fertilize in vitro,
the thousands of oocytes that are present in a female’s ovaries.Breeding
programmes would select for female genetics as easily as for male
genetics. The genetic influence of a male would be increased further since
the amount of semen required for fertilization in vitro is
fraction of the amount needed for Artificial insemination.Fertilization
consists essentially the fusion of two cells, the oocytes from the female
and spermatozoon from the male to form single cell, the Zygote.IVF
is generally quite successful, resulting in about 70-80% of fertilized
eggs. The practical difficulties arise in sourcing eggs for fertilization
and in the development of fertilized zygotes to term.IVF
involves three steps namelyOocyte
recoveryOocyte
maturation in vitroIn
vitro fertilization
of matured oocytes

OOCYTE RECOVERY 

In the second (follicular)
phase of the oestrus cycle, a number of ovarian follicles (20 or so)
grow and become filled with fluid. The fluid -filled space is called the
antrum and such follicles are known as ‘antral follicles’ (or Graafian
follicles).In the normal course of
events just one of these matures and ruptures, releasing the egg for
fertilization.Super ovulation occurs when
many more antral follicles are stimulated to mature by injection of
gonadotrophins.Pre- ovulatory follicles lie against
the surface of the ovary and quite large (about 15 mm in cattle, 8 mm in
sheep and pigs).Laparoscopy surgery can be
used to recover oocytes from these follicles, allowing them to be
matured and fertilized in vitro.A number of eggs can be
collected in this way from superovulated donors. The technical demands
and cost of surgical procedure of oocyte recovery is high.

IN VITRO CULTURE AND MATURATION OF OOCYTES

Oocytes
for in vitro fertilization are obtained from the oviducts, follicles and
surface of ovary after ovulation either from live animals by flushing some
medium or from slaughtered animals. The immature oocyte is collected from
the ovary and cultured in vitro to induce maturation.Cow
ovaries are collected from the slaughter house and maintained in phosphate
buffered saline at 30-32°C for transportation to the laboratory. Following
aspiration of follicles, the oocytes are then observed under a dissecting
microscope.The
oocytes are graded according to their morphology, washed twice with HEPES
buffer and once with BMOC-3 (Branchat and Olivhant medium for oocyte
collection). After washing, 5-10 oocytes are transferred to 0.2 ml of
BMOC-3 drops under paraffin oil at 37°C with 5% CO2 in 95%
air for 12-32 hrs.

Oocyte maturation in vitro

Large
numbers of oocytes are allowed to mature under in vitro conditions.

 

IN VITRO FERTILIZATION IN FARM ANIMALS 

The in
vitro matured oocytes are mixed with the capacitated sperms in a
petridish and incubated at 37ºC with 5 % CO2.After
24 hours incubation, the cumulus mass is removed manually and examined for
fertilzation indicated by the presence of 2 pronuclei. Further the
fertilized oocytes were incubated for division.The
morula stage embryos (16 cell stage) are transferred to recipient animals
for further development of embryos.Among
farm animals, invitro fertilization has been achieved
in cattle, pig and goat; but the number of new born animals were
limited.

SEXING OF EMBRYOS

·        
Embryo sexing in animals has the advantage to increase the milk
yield or to increase meat production. The production of preferred sex is done
either by sexing the sperm or by sexing the embryos. In case of sexing sperm,
it could be used both in artificial insemination as well as in the in vitro
production of embryos whereas the sexing of embryos could be done either in
vivo produced or in vitro produced embryos prior to their transfer to recipient
animals.

METHODS

Sexing the sperm

This is done by using flow cytometer attached with a sorter. In
this method, the sperm is stained with a stain – Hoechst 33342 and then sex
specific sperms are separated by using the flow cytometer’s sorter. This method
has an accuracy of 90 % sexing sperms. Commercially it is used in cattle.

Embryo sexing

Y-specific DNA probes

Sexual
differentiation of mammalian embryos is determined by the presence or
absence of genetic elements located on the Y-chromosome. Molecular genetic
techniques allow the detection of specific DNA sequences in interphase
cells.Sex
determination with Y-specific probes involves the identification and
cloning of a probe (a DNA fragment with a nucleic acid sequence found only
on the Y-chromosome) and the use of this probe to diagnose the presence or
absence of Y-chromosomal DNA in a sample of embryonic cells.

PCR

Using
polymerase chain reaction (PCR) to amplify a Y-chromosome-specific repeat
sequence, DYZI, that is present 500-8000 times on the Y chromosome. The
presence of this amplified target sequence indicates the presence of the
Y-specific DNA sequence in the sample DNA and, hence, indicates that the
animal is male.A
disadvantage of this technique is that the absence of the amplified target
sequence may be due either to the absence of the sequence in the sample
DNA or to a failed reaction,

PCR-RFLP

Second
method involves PCR-based genotyping. In brief, a pseudo-autosomal region
(common to both X and Y chromosomes) present in both X and Y chromosomes
is amplified. These homologous regions are called ZFY (in the Y
chromosome) and ZFX (in the X chromosome).The
amplified product is then digested with restriction enzymes that take
advantage of restriction enzyme fragment length polymorphisms between ZFY
and ZFX. The digested DNA is separated via electrophoresis. The ZFX and
the ZFY DNA are identified by their different digestion patterns.

 

ADVANTAGES AND DISADVANTAGES OF MICROINJETION

Advantages 

The
amount of DNA delivered per cell is not limited by the technique and can
be 
optimized. This improves the chance for integrative transformationThe
delivery is precise, again increasing the chance of integrative
transformation.The
small structures can be injected containing only a few cells and with high
regeneration potential.Since
it is a direct physical approach, it is host-range­ independent.

Disadvantages 

Injection
can cause damage that affects embryonic survival and can result in quite
high mortalities.Only
one cell is targeted per injection