Lectures of Reproduction Biotechnology (AR 402)

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Oct 23, 2013 (3 years and 9 months ago)

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Lectures of Reproduction Biotechnology

(A
R
402)



(Mid Term Examination)












Biotechnology


It is the use of chemical,

physical and genetic engineering techniq
ues and
principles through the b
iological system

to produce goods for
human being
s.”

The

target is to get maximum
benefit f
r
o
m

biological system

for human beings
.

In 1980 due to increase in population,
needs and demands

scientists have to use
technique
s

to produce more from same resource
s. Biotechnology focuses on pu
re
s
ciences

like
ce
ll biology
,

genetics
,

embryology
, microbiology. It

a
l
so covers the
industrial science.

There are four ma
jor are
as of biotechnology:

Medicine, Agriculture,
I
ndustry,

&
Environment.

(1):
Medicine:

Techniques are used

for d
rug production, gene t
herapy, geneti
c testing,

&

Pharmacogenomics:

(a):
Drug

Production
:

Biotechnology has changed

the

methods of traditional drug production.

First drugs were
discovered accidentally or by hit and trial method but now it is planed.

(b):
Gene Therapy:

In gene
therapy

scientis
ts produce

growth hormones,

blood

clotting factors,
fertility

drugs

and insulin.
If closure of fallopian tube, it is sterile animal
; its treatment is possible
.

Previous method fo
r

getting
insulin

was from s
laughter house but now it is go
t

in lab

from E.
col
i

by introducing alphabacter in it.

In g
ene therapy,

patients of cancer, and
AIDS, are being treated
.

(c):
Gene Testing:

In gene testing direct exam
ination

of DNA

is carried out. It
helps in career screening
. It is

used for sex determination
. Sex different
iation occurs at 8 or 9 days particularly in
embryo transfer technique.

Eight day embryo is
having 32 cells. Embryo is sp
lit
t
ed into
four
part
s. One half is used for sex determination and remaining is used for production of
triplets.
Gene testing is also u
sed in Forensic identifying labs.

(d):
Agriculture:



To increase the crop production
.



To

increase the

resistance

against

environmental disease
s.



To

increase the nutritional quality
.



To

decrease the
dependence

of pesticides of crops




To

increase

the

appearan
ce
of

crop
s
.




To

increase the shelf life of crops.




To

produce transgenic

and hybrid

plants
.


(e):
Pharmacogenomics:

It

is reaction in
heritance character of individual toward a
drug.

Genetics of individual
affects

the

body
’s

response to

drug
. W
ith this the
y

are making tailor made medicine

and

safer vaccines
.


Branches

of Biotechnology

Bioinformatics
:

It

is the i
n
t
e
r
-
disciplinary field betw
een biological problems
and analysis of biological
data.

Blue

Biotechnology:

It deal
s

w
ith water,

aquatic and marine lif
e
.

Green Biotechnology:

It is biotechnology applied to agriculture processes e.g. techniques to produce
hybrid or

transgenic plants.

Red Biotechnology:

All medicina
l techniques are included in it e.g. designing of organisms to produce
antibiotics.

White b
iotechnology:

It is also called industrial biotechnology. This is related to industrial processes.

Reproductive Biotechnology:

Technologies or principles used to focus on reproductive system to get
maximum

output

and profit from

that

system.

Techniques

s
ta
rt from AI, embryo transfer (selection

criteria

for male and
f
emale). E.T

includes the
steps/
parameters for
selection

of embryo, evaluation of embryo,
embryo transfer and allied techniques

e.g. determination of sex.

In

vitro embryo
production

(IVEP)
:

colle
ction of oocyte and

its

maturation, semen
treatment

(capacitation)
,

then in vitro insemination of
oocyte
, fertilization, then go for
fresh
embryo

transfer
or

storage.

Cloning, s
ex determination, synchronization, disease diagnosis during
pregnancy,

treatmen
t of diseased fetus,

checking fo
r

drugs

whether
these can pass placental barrier
are also included in reproduction biotechnology.


Cultural
mandate

Series
of

trial
are

conducted on animal after
developing

technique
, then

these are
implemented on human

bein
gs
Who has allowed man to play with animal a
nd upto what
extent he should pl
ay. The study of human embryo is quite easy and also its growth in
in
-
vitro is brilliant growth.


When sex is
determined

in early l
ife, if it is undesir
ed then ther
e will be

the

pr
essure
of

abortion,
wh
y to abort that is et
h
ical
mandate
.

Early conception in heifers is only due to biotechnology

In transgenic animal
e.g. ma
re is with four teats. Why this has been done with ma
re
? W
as
it a need
?

That

come
s

in cultural
mandate
.

Hemophili
a

treatment requires

50

embryos from that diseased family to produce a
healthy
one
.

What is the f
ate of other 49
embryos?


That

comes under

cultural
mandate
.

In
Europe

human semen bank has been developed but these can not be applied in

Asi
a;
this comes

und
er cultural
mandate
.



Artificial
Insemination

“C
ollection of semen from male
,

processed,
evaluated

in lab a
nd then transferred
to

female

reproductive tract

by
artificial

means for the purpose
of

conception.


It is

most
widely

accepted technique
w
orldwide
for

the

genetic
improvement

in animals
.
For large no. of animal
s

AI

is
beneficial

at national or international level

but

f
or

a
farmer

embryo transfer and allied technique
s

are
preferable
.


History of AI

In 1322 one
of the

Arabian chief inseminated

his

mare

with semen of price winner
horse
.

First document
ed report

of

AI report
wa
s in 1780 when an Italian
physiologist

L.
Spall
n
zani

carried out AI in bitc
h

and got three
puppies

after 62 days.

In 1677
,

Le
e
u
wenhoe
k

first time observed the spermatozoa under micro
scope.

In 1899, Ivanoff, Russian considered to be

the

pioneer
of

AI in birds, equines,
cattle and
sheep.

In 1931
,

in Russia AI at mass level was introduced and

19800 cows were
inseminated at national level first time
.

In 1936 in
Denmark

first AI associatio
n w
as established and in 1938 in US
A same
association

established
.

In 1970 tremendous growth of AI and
mass breeding

occurred.

40
-
60 % of cattle
population w
as

inseminated

in USA

and while over 90 % inseminated in
Denmark
.

First

freezing of semen

reported
in England in 1949.


AI is 1
5
-
20 % in developing countries while 80 % or above i
n

developed
countries.

In Pakistan 5
-
10 % of

the

area
where AI occur
s
.
I
n

buffalo
is 5
-
10 %
and

in
cow

12
-
15 %
.

In
Pakistan

first
time

AI was introduced in 1954 when first AI c
entre was
established in CVS
, Lahore.

From 1954
-
1961, about 8461 buffalos and 4885 cow
s were insemi
nated in CVS.

In 1962 first time it was accepted at government level and being produced at
government farms.

First time in 1971 Department of Therionology wa
s established in UAF.
First
semen production
u
nit in
Q
adrabad
was also established in the same year.

Now
Qadraba
d, kheripur, Bahadernagar, and Klorek
ot semen production u
n
its are working.

There are also some private semen production units.


U
ses

and Advant
ages

of AI


(1):
It increases the efficiency of bull usage
:


In

AI

program, the

proven bull is being used
.

The
s
e

bull
s

are
on the
pedigree or progeny
record base
d
.

In a breeding
season a bull can maximum servic
e

to 100 females and that
bull is of good
qual
ity.

But

with AI

thousands

of females can be inseminated.

So
efficiency of
bulls’

usage increases.

(2):
It increases the potential for genetic selection
:


In

natural, bull serves 100
cows;

60
-
70 %
is

successful so for next selection only 60
-
70
calves are t
here
. In case of

AI selection
increases.

(3):
It decreases the inbreeding
:

In natural there is increase inbreeding and that is dangerous because lethal genes are
transmitted from generation to generation
.

With

AI inbreeding is reduced.

(4):
It
is cost eff
ective
:


To keep a bull
more expenses are required.

So if we use AI that
is economical.


(5):
AI technique increase
s

the safety of animals
:

It controls the early m
ating or pregnancy. Premature

mating leads to dystokia at

the

time
of birth

which

may also le
ad to
the death of animal
.

If male is mature and heifer is young
one
,

there are chances of permanent injury to heifer

when male jumps.

This

can be
avoided by AI.

(6):
AI increases the safety of herd

man
:


In

1980
,

2
-
3 % death of herd man was due to injury
by bull

in America.

So by

using AI
it

can be avoided.

(7):
It decreases the
transmiss
ion of disease
:

Diseases transmitted by
coitu
s

l
ike tricho
moniasis, brucellosis, compyloba
ct
e
rosis. In
natural if
bull

is
infected,

female will also be infected.

But i
n

AI

after collection of semen
it

is

processed and

checked

for any pathogen. For precautionary measures, there is
ad
dition of antibiotic
s
.

(8):
AI technique helps in diagnosis o
f male infertility
:

For

evaluation of bull there is

mating
behavior

and semen quali
ty. Natural mating is good
but poor quality semen will be diagnosed later

after 5
-
6 months by pregnancy testing
. But
with

A
I

after
e
ach

collection

the semen is checked for
fertility

so that diagnosis is earlier
and reliable. So it
helps

in screening
of

mal
e diseases.

(9): I
t hel
ps to provide service at doorstep
:


Only

semen container is taken to

the

place where needed and is
easier one. So it aids in
breeding program. Semen can be spread worldwide

(10): Storage:

The good quality can be stored for years even

after

death of bull
. S
emen can be stored

for
25
-
28 years under
optimum storage conditions
.

(11):
Sex determination:

At

national herd level sexed semen is used to

control the sex
of offsprings
.

(12):
Allied techniques:

In
AI
,

transfer of embryo
helps

in

a
llied techniques,
like

embryo

transfer
, in vitro
fertilization

etc
.


(13):
Life of sire:

AI increases the sire life.

In

natural mating there is possibility of injury but in AI that is
reduced.


Drawbacks of AI



AI technique decrease
s

the efficiency of heat
detection
.

I
n natural male and
female are kept together
and heat

detection is
carried out
by bull

which is more
efficient
.
So it is recommended that teasers should be used for heat detection or
relies

on the techniques like ELISA, RIA.



I
f

wrong bull is use
d in

AI thousands o
f

females will be inseminated but in natural
that harm will be limited.



High skilled

man power is required for handl
ing
,

collection, processing,
evaluation and

insemination of semen
, and

heat detection etc.
M
issing of one step
will lead
to

the

failure of AI.



A strong sexual he
alth program is required for th
e succ
e
ss

of AI.


Why we are far
behind

in this technique
?

In Pakistan 5
-
10 % of buffalo and 12
-
15 % of cattle population is able to enjoy the
artificial insemination. We are far behind

due to


(1):
Lack of genetic potential:

We

do not have anything to show to farmer. We have the best world buffalo and Sahiwal
cat
tle. But problem is with our wor
k and policies at government

level. S
o there is no lack
of genetic potential.

(2):
Acute shor
tage of breeding B
u
lls:

The problem is acute shortage of breeding bull and this i
n

the

case of buffalo
is
1:138
and
if we in
clude the urba
n area then 1:171
.

I
n case
of

cattle
it
is 1:19
.

It shows the shortage
of bull
.

Reason

is that
we just keep females
;

6
-
12 month males are sold for meat.

(4):
Lack of trained man power:

Due to lack
of sufficient knowledge this

technique is failing in Pakistan. At government
level there must be a training program.

(5):
Lack of infrastructure:

Facility of o
ffices
,

roads, tran
sport
, system,
and
residence is less. Veterinary

service

should be provided

for 24 hours because
animal

can

be in heat at any time.

(6):
Research:

Need to work more on deep freezing and storage of semen.
When
semen preservation

is
complete
d
, the
mortality

is 40
-
45 %
.

Transportation

stress decreases it further due to

increased temperature

and jerks
. There are

no optimum storage condition
s in hospit
als.

There is also the
lack of knowledge of
storage

of semen.

(7):
Availability of liquid nitrogen:

Availabilit
y of liquid nitrogen is also a problem.
At bottom

of container

temperature
is

-
196
o
C and
at top
it is
-
124
o
C.

Temperature

rises due

to insufficient supply of liquid
nitrogen so semen quality

deteriorate
s
.

(8):
Political Influence:

In Pakistan there is
be
aurocratic
and

political interference
; good working persons are
shifted from place to place on demand.


Success of
Artificial Insemination

Two leading factors in

the

success of AI

(1):
Accurate estrus detection:

(a): It may be visual observation in case of

less number of animals.

(b): Lab methods of which hormone analysis is important one.

For detection of progesterone and estrogen we take the sample of milk, plasma and saliva
and analysis will show the same trend
.

CL
,
placenta
(
in case of pregnancy
)

and a
drenal
cortex

are the sources of progesterone.

At the time of estrus
the level of progesterone s
hould be minimum or at basal level i.e.
0.2
-
0.5
mg or in general less than 1 ng. If it is more or less than this level,

it will
negatively affect the conception
.

The level of estrogen will be maximum
.

It

is detected by
Radio

I
mmuno Assay (RIA), Enzyme Immuno Assay,

and

ELISA.

(c): Use of teaser bulls in which there is deviation of penis. With this we can also use
KMAR heat mount detector or chin ball.

(d): Use o
f androgenized female: Heifer is prepared for detection of estrus after giving
her testosterone (male hormone).

(2):
Proper time of insemination:

It is principle that animal should be inseminated before the end of estrus
.

Mare:


Maximum chances of concept
ion occur 36 hours prior to end of estrus. Ovulation usually
occurs 24
-
48 hours after the end of estrus. Estrus period is 3
-
7 days and alternate day
insemination is recommended in field condition. By using ultrasonography we can
measure the
size of follicl
es

and the no. of doses can be can reduces In one dose 200
-
500
× 10
6

is the no. of sperms that should be there.

After confirmation of estrus, we go for vaginal or Recto
-
vaginal technique in which one
hand in rectum and one hand in vagina. When it touches c
ervix rings then pass the rod.
Preference should be given to recto
-
vaginal technique because in mare cervix is very soft
and chances of contamination and infection are less.

She
-
camel:

Animal is receptive for male even if there is no follicle and that is d
ue to the fear of male.
So for insemination follicle must be observed. The length of both horn
s

is same in all
species but in camel left horn is always larger and over
90
-
95 %

of pregnancies occur in
left side. If ovulation occurs in right side, there is
trans
-
migration of ovum (chances are
more in she
-
camel, mare, and swine). Ultrasonography is the only way to detect time for
insemination by measuring the size of follicle.
It
size is 1,

1.2
,
1.3

or maximum 1.5

cm.

There is direct relationship between size

of follicle and conception. In camel size of
follicle is very important. Usually semen is deposited in the body of uterus but in case of
camel it should be deposited on the left side particularly. It is possible to work with camel
in the standing position

if properly restrained, but usually in sitting position.
Semen
deposition should be deep uterine. After detection of follicle semen should be deposited
on
particular side of ovum.

Sheep &
G
oat:

Hind quarters are elevated 10
-
15 inches in the breeding racks
. Fix the tail and vaginal
speculum (glass/steel) is used (mostly glass).

It is like a test tube opened from lower side.
Insert speculum into vagina, by using head light. Confirm that speculum is touching the
cervix. Then deposit the semen.

Bitch:


Same in

case of sheep and goat.

Buffalo and C
attle
:


In buffalo and cattle recto
-
vaginal technique is used.


Constraint

of AI



Small number of breeding bulls.



No reliable AI services



No knowledge of allied problems.


Embryo Transfer

It is also called ovum transfe
r.
In 1890, the idea of embryo transfer was given first
time. Its common use started in 1970.

“It is the collection of fertilized ovum from the donor prior to its attachment/nidation with
uterus and transfer into surrogative/recipient mother for completion

of gestation period.”

or

“Collection of embryo from donor cows & transfer to the recipient cows where they carry
till term or completion of term.”


When embryo is embedded in endometrium of uterus is called as nidation. This
term is used in mare and porc
ine.
But in buffalo, cattle, sheep, goat there are specific
areas/ cotyledons where attachment of embryo occurs.


The cow from which we get the embryo is called as donor. The cow in which we
transfer embryo is called recipient.
I
n the offspring 50% charact
ers come from mother
and 50 % from father. In AI, semen is inseminated in different cows. The genetic
potential of male is distributed which is same for all cows but different cows have
different potential. In embryo transfer genetic potential of female is

being distributed.
Every cow produces one egg at the time of estrus. Donor cows are induced to produce
more than one egg at the time of estrus, inseminate these eggs and fertilized
eggs/embryos are taken from donor. At the same time recipient cows are als
o prepared
which have low genetic potential and one embryo is placed in each cow. In this way more
than one calf can be obtained from one donor.

If donor cow has high potential of production, we do not let it to become pregnant
and after two months we get
eight eggs. In this way we get 40 embryos in a year (5 times
a year). If 60 % is birth rate then we can get 24 calves from donor cow in a year
otherwise only one calf in a year.

The fertilization site in bovine is ampullary isthmus junction
in animal
.

The

reason is that this portion contains more ciliary epithelium, increased secretion, and
increased suitable environment for fertilization. In mare only fertilized ovum descends
down in uterus but in

bovine ovum stays for 2
-
4 days

and then descends down
. In
bitch
this duration is 9
-
10 days.
When it descends down, remains in wandering position in
uterus for some days and gets nutrition from uterine m1ilk and then gets attachment.


Advantages

of Embryo Transfer



It improves the genetic potential.



It increases th
e productivity



It increases the economic benefit



It increases the disease resistance



It increases the no. of calves in life time



It reduces the generation interval.



It increases the selection intensity.



Import and export is easier because it is done in
the form of embryo transfer in
which transportation is easier, genetics is diversified, and cost is decreased.

The study of physiology and pathology of ovario
-
uterine pituitary hypothalamic was the
main purpose of ET but later it became commercialized
.



Steps

of

Embryo Transfer

a.

Selection of donor

b.

Selection o
f recipient

c.

Synchronization of donor

and recepient

d.

Superovulation of donor

e.

Insemination of donor

f.

Collection of embryos

g.

Evaluation

of
e
mbryos

h.

Transfer or storage of embryos.


Selection of Donor



It shoul
d be
from known fertile blood line i
.
e. we

must have pedigree
record

and donor should have calved at least once a year so selection of
heifer donor is not required
.



It s
hould have calved once earlier so heifer as

a

donor is not
required.



Donor

should have

known calving

history

or
easy calving

history.




It should be r
egula
r

cyclic
animal.



It should be disease free animal e.g. T.B, brucellosis



It s
hould be vaccinated
animal
.



It should be high producing, reproduction wise normal, and three years of
age without

any disorder.


Advantages

of Embryo Transfer


(1):
Genetic Improvement:

E.T is best for increasing genetic potential fo
r
female while AI is f
or

male. Out
of

8
-
10
embryos,
6
-
8 are transferable and
3
-
4
pregnancies

occur.

If

repeated five times a year
from
15
-
20 calves

will be produced
.

So donor

cow
with good potential will produce
15
-
20
calves a year.


(2):
Selection Intensity:

ET will increase the
selection intensity
among the calves. Research says that if a

cow
gives six calves in a year then selection inten
sity becomes double.

(3):
Planed Mating:

We

can plan to select the b
ull with high
genetic

potential to mate with the cow which is
going to give 8
-
10 calves.

(4):
Genetic
T
esting:

Commonly it is
done to
null out

the deleterious genes.

Cows

that are carrier

and mated
with a carrier bull
,

offspring
will be

affected
. S
o such bull
s

are rejected for AI.


Super ovulation

Production
of

more than one egg by the use of hor
mones or drugs

is called super
ovulation.
PMSG and FSH

are the hormones used for super ovulation
. FSH is short
acting and PMSG is long acting
. Repeated

FSH injections have to be given, w
hile PMGS
is given at on
c
e. At day 10 of
estrus

cycle

CL is present.
So give
5 mg a.m. and 5 mg
p.m
.

S
imilarly
FSH is given 5 mg a.m. and 5 mg p.m.

on day 11
, 5 mg a.
m. and 5 mg
p.m
.

along with PG
F2α

on day 12
,
and

5 mg a.m. and 5 mg p.m
. on day 13.

In this way

FSH

is
given for four days tw
o

times a day I/M
.
On d
ay 14
,

donor will be in heat and

there will be

ovulation
of
more then one egg
s
.

PMSG

is given:

Day 10


2500 IU
I/M

Day 11


nothing

Day 1
2


FSH + PMG2
α


Day 13


nothing

Day 14


super

ovulation

Day 8
-
12 or
day 12
-
15 c
an

a
l
so be scheduled.


Synchronization

The reproductive cycle of donor and recipient should be at the same level.









d1




d
11



d14



d24



d26

d28

d35

Donor:


------------------
----

-------

------
--------
-----

-
---

------

----
-----------

------




PG
-
1



PG
-
2






Heat


PMSG
PG
-
3
H
eat

C
ollect embryo








d1

d
11


d14



d25


d28


d35

Recipient:

------------------
----

-------

------
--------
------
------

------

----
-----------

------




PG
-
1



PG
-
2

Heat


PG
-
3
Heat
transfer

embryo









(AI: at day 28 a.m. and day 29 a.m. & p.m.)




Synchronization

(By some other teacher)

We

have 100 cows

which are no
n

pregnant

and we have to bring them in heat in a
small given period.

Synchronization means to
b
ring

the even
t
s

together
. W
e

synchronize
all
these cows
.
Out

of 100
, on palpating

20 are already in
estrus,

20 are in proestrus

that
may come in heat after two days
, 15 are in post

es
trus they are just in ovulation, and

30
are in diestrus. Other 10
-
15 are in
anestrous.

In bovine function of PG
F
2
α

is only to
r
e
gress the CL.
This purpose can be use
d

any
where.









d1
d6
d11


d14

d17


Estrus:






----------

----------

------

-
-
-
-----








CL



PG
-
2

Heat



d1


d3

d
9

d11

d14


d
17


Proestrus: ∙
----

----------
--

----

---
-
--

----
--






Heat





CL

PG
-
2
Heat




d1


d
4




d11


d14

d15



Post
estrus: ∙
----
-
--

--------
--
--
----

---
-
--

--








CL




PG
-
2
Heat




d1

d3



d
9

d11

d14


d20

D
i
estrus
:




-----

-
-
----------
--

----

---
-
--

----
------







PG
-
2


Hea
t



CL

PG
-
2

Heat




Prostaglandin giving is wastage to all animals which do not have CL.



Animal in e
strus

do

not have any CL. 6 days later there will be CL. T
h
is CL will
persist for 11
-
1
7 days.



Proestrous anim
a
l come
s

in estrus a
fter

one
or two

days
. They do not have
C
L. So
no response of PG on day t
hird. She is in heat
.

O
n day 9
,

CL will present which
remain for day 20.



Post
oestrus animal was in estrus two day
s

earlier.

If
s
he was in he
at on 30
th

December. On day four CL will form which stays in ovary till day 15
.



In diestrus animal has functional CL.
Give

PG on first day, on day 3 they com in
heat.
They will develop CL on
ovary on day 9. 6 d
ays w
ill be taken by CL to
develop. On day 20

CL will persist.



An
estroue do not respond to any one.

At that time
wastage of first dose of PG in those

animal
s

which do not have CL
. So it is
effective in those which have CL. As

diestrus which
h
as CL.

On day 11 we
g
ive second short on second

short all
come in heat on day
1
4
.

One

injection of PG
costs

300 rupees. So for 100 cows it costs 60,000 rupees.

Programming:

Super ovulation is one of the major problem because all donors do not
be
have equally
.
I
ndividual variation is so much

Al
ways put more t
han tw
o donors into the program

as several
recip
i
ents
. On cow
produce
s

78
viable

embryos

in one collection
.

Palpate

all

the
recipient
s
. Grade them also. Best embryo with best CL contain
in
g

ovary
.
Also grade
ovary

like R
1

i.e. right ovary has grade one CL similar
ly
L
2
.


Non Surgical

Collection

of Embryo

Until 1975, embryos were collected surgically. After anesthesia (GA or LA),
entering the uterus and collect embryos. It was very difficult to collect from donors at
farm. The cost of collection was too much. Anoth
e
r problem was some damage lead

to
future adhesion in reproduction tract.

People tried

to collect without surgery.

Non surgical method

is

preferable now

because there is
no damage or very little
damage that provide
s

repeatability for t
he don
o
r

to donate em
bryos and

made collection

at farm level

possible.

Disadvantages:

We can only

collect the embryo when it enters the uterus.

But

with surgery we
can collect embryo
s

w
hile they are in oviduct. In bovines we collect embryos with 6
-
8
day
s

after the onset of est
rus.

I
f before this

time

collection is made
,

embryo will be in

the

oviduct and
beyond this time the embry
o

start
s

hatching

and cytoplasm will sta
rt coming
out and collection b
ecomes difficult and embryo sta
r
t striking with endometrium.

Procedure of
collect
ion
:

Place the donor in crushes, wash the perennial region. Evacuate rectum from feces
and evaluate no. of CL on both ovaries that tell the no. of embryos
. W
ash with luke warm
water
. O
l
der

cow
s

suck air in rectum and uterus
,

so
very

old cows

are not used b
ut if they
are g
ood
we

can

use

them.

Apply

a bally band that would create a positive pressure
instead

of negative pressure.

Apply epidural anesthesia
to

decrease

the

movement
s

in

cow. Collection is done
with th
e help of catheter. Th
r
ee kinds
of

catheter ar
e used most
;

commonly used is
Foley’s Catheter. It could be two
ways

or three way
s, and used in h
uman

side.

It is easily
available

and in
expensive also. It i
s soft in nature and threading of

uterus
becomes difficult (i.e. fixation of uterus
).

Cervix

is clo
sed during diestrus.

In

surgery we
cl
amp the uterus horns and throw fluid inside and suck it again which will

contain
embryos.

In non surgical collection we have to fix catheter in

uterus horn in a method that
embryos can not pass out of horn into uterus a
nd
portion is

blocked complete
ly.

Two methods:

Continuous flow method:

There is less loss of fluid but there are
a
lot of tubes

that

we have to

handle
. It
may

cause contamination.

Interrupted

syringe method:

This is less
expensive

method. All
equipment

is

disposable.

When

catheter
s

are used, they are sterilized either with ethylene oxide or by color
sterilization method.

These agents a
re harmful for embryos.

With e
thylene

oxide
it should
be sterilized
one

week before collection and put it in air so that h
armful material is
decreased. If cold chain is

to be

used then use N.S.

Embryo collection
is done in diestrus when cervix

is
closed

but still it is
penetrat
able. A steel rod known as cervix dilator is used.

A
ll

things sterilize
.

Pass it
slowly that will lo
o
sen

the

rings of cervix
, take out

dilator and pass the catheter.

Catheter
s

are not hard enough because they are of rubber
. T
o create stiffness in catheter
we pass s
t
eel rod in
i
t

and after passing

we direct it toward one of the horn
s
. Tip of
c
athete
r

has
opening and

an

area wher
e there is balloon i.e. deflator
.


E
nter N.S so that balloon is
flatted when the balloon is
bigger

enough to fill the
lumen of the uterus the catheter is fixed now.

N
ow

fill the uterus from
the
opening inside the catheter
,

the fluid

when sta
r
t
s

filling
in uterus, mov
e the top of horn where the embryos would be and take out the fluid
which contain
s

embryo
s.

In

first attempt
o
f filling fluid it contains

85 % of embryos. No.
of plates

are marked.
Immediately after the collection, you ha
ve to shift the materi
a
l in
lab and then search of embryo.


Searching

Square

shaped
Petri
plates

are used. S
earch with stereomicroscope at 10X.

Criteria for Evaluation:



Continuity of zona pellucida

(zona is double layered and it
s diameter is 12
-
15
µ
m
)
.



The

arrange
ment of blastomeres in the zona:

they

should be arranged in circular
fashion and no extension of cells from the zona.



Blastomeres should be of
uniform

size.



Presence of
degenerative

area
: If

degeneration
present

it will
appear as b
lack area.
It is
in %age.



Presence of vacuole.


By following the above mentioned criteria we can evaluate the embryos in all grade:

Excellent



A
:

ideal, spherical, symmetrical with uniform blastomeres,
no
degenerative

area and no vacuole formation.

Good


B
:


F
ew
e
xtrusi
on of blastomere
s
, irregular sh
a
pe, one or two
vacuoles

F
air



C
:


E
xtruded blastomeres, vacuole formation with 19 %
degenerative area.

Poor



D
:


Numerous extruded blast
omeres, more variation in size,
more vacuoles, degeneration upto 25 %.

Unfertilized
:



T
here will be no cell and just spots present.

For fresh embryo transfer, then you can use upto C grade embryo but if after
freezing then you can use only upto B grade embryo. The searching of embryo is done at
10 X but evaluation is done at 50
-
100 X ma
gnification.


Insemination of
Donor

Insemination of donor in standing heat. Inseminate two or three times after twelve
hours and each time go for double dose.



Transfer of embryo

Washing:

First step before transfer is washing. It is necessary because whe
n we collect
uterine material, it als
o

contains mucous, blood etc. So to pro
te
ct embryo from
deleterious effect of that we wash

it.

5 ml embryo washing medium

is taken

in

three Petri
dishes. Embryo is put in each

one for five minutes.


Loading of embryo:

T
he straw is used for deep freezing
. Straw

of

semen can be used for loading of
embryo

Sterilize

it with ultraviolet rays or by ethylene oxide gas
.

Attach straw with
tuberculin syringe for
the picking of

embryo.
Then
wash

it with pure water 2
-
3 times but
tha
t w
a
ter should not touch PVP plug.

Suck th
e

embryo washing medium 2
-
3 cm followed by air
column
and then second
medium

column

larger than first

one

i.e. 2.5
-
3.5 cm
. T
hen again co
l
umn
of

medium of 2
-
3 cm and again column of air of

0.5

cm and then there is o
pen space. Embryo

should be
loaded in the centre of middle

column of

medium
.

It
is

helpful in freezing.



Transfer:

Embryo transfer gun is used for transfer
.

Pull
p
lunger of E.T gun back
.

Put the straw
in
side from the side of co
tton plu
g.

Then cover with another straw. But keep in mind that

the margin of straw which is inside the gun and the margin

of straw

which is outside the
gun should be at same level

because if one drop remain may be embryo is present in it.

Then cover
it wi
th an
other oute
r

straw
which

will

protect the corner from a
ny debr
is in
tract.



U
se 1
-
2 ml of epidural

anesthesia

and go for examination of ovary to check on
which ovary the CL is present. Transfer of the embryo should be
transferred into

ipsilateral horn
(horn containing CL)
.



Ideal sit
e

for the depositio
n or transfer is 5
-
10 cm above
f
r
om bifurcation
.




Firstly you have to straighten the horn as tonicity is present at

the

day eight of
embryo.


After tra
nsfer that recipient should be k
ept in observation for
the next estrous cycle. If
it d
oes not come in estrous then go

ultrasonography for pregnancy confirmation.


Factors Affecting Superovulatio
n Response

(1):
Type of hormone:

FSH
-
P (collected from porcine)

PMSG

(pregnant mare serum gonadotropin)

ECG (equine
chorionic gonadotropin)

HMG

(human menopausal gonadotropin)

Nature

or origin

of hormone

(2):
S
t
age of estrous cycle:

Good response if do
ne in diestrus.

(3):
Presence
of

dominate follicle:

If at the time of

injection
,

presence of follicle larger than

size

1
0 mm; s
ize
will badly
affect the superovulation response.

(4):
Difference in age of animal:

S
uper ovulation is poor in
young

and old age.

(5): Health of the donor

(6):
Nutrition:


15
-
20 days before treatment shift into good nutrition plan.

(7):
R
esponse is

good in dairy breed as compared to beef breed.

(8);
Superovulation Failure:

All
parameters for selection of dono
r was OK but even then response was not achieved
,

it
is superovulaion failure and it occur
s

due to antibodies response
that

neutralize

the FSH,
.

If no. of embryos is more it will negatively effect the quality of embryo.

(9):
Embryo collection medium:

Supplemented

with 1 mg/ml of streptomycin and 1000 IU/ ml of penicillin plus
with calf serum (1
-
2 %), because it produces better result but it shou
ld be an inactivated
calf serum. If you keep it at 56 oC for 30
minute

it
will

inactivate the serum.

Serum is required as:

It provides nutritive value.

If not provided with
calf serum
, the
embryo stick
s

with the glass Petri plate.

Why inactivated:

The

fibr
inogen
will

be broken down by denaturing and so it will
not trap the embryo
.
But

culturing of embryo can be done by 10
-
20 % donor s
e
rum (estrus cow serum)


Storage of embryo

Short Term Storage:

For 1
-
2 days, short storage is required if recipient not ready
.

Procedure:

5 ml of DPB
S in it put embryo and
cover it with alum
inium foil and place it in a beaker
and
shift

the beaker in refrigerator at 4
-
5 0C.

Long term storage:


It comes under deep freezing after loading of straw go for the sealing of straw.
Automa
tic embryo freezing chambers are
available.

You need to add cryoprotective
agents.
10 % glycerol

is used.







Seeding:

F
o
r straw you have to
give the po
i
nt for formation of ic
e.

Freezing start
ed from the end
other than cotton p
lug.

Thawing:

Thawing is done at 37
-
38
0
C @ 19
-
15 second
.

In
semen straw is directly loaded in AI
gun but

in the case of embryo the viability

of e
mbryo

is checked under microscope before
the transfer.

For freezing morula or compact morula stage is the best
.


Surgical collection and Transfer of Embryo

First of
al
l

surgical technique was

developed. First it was don
e on the s
laug
hter house
material.

For surgery

there should be

fa
sting

from twelve hours
.

Then give general anesthesia and
g
o for laprotomy from f
lank approach or through mid line approach.
Mid
line approach
near udder

and preferred. When
uterus

is exposed, the last one third (distal part) of uterus
is
b
locked but
not with the sharp
objects

like a
rtery forceps, special instrument available
fo
r

this

a
nd then at anterior part of uterus make a small incision and infuse 30
-
100 ml of
medium.
Then

puncture from mi
dd
le
a
nd collect the collection medi
u
m and no need to
filter it. Transfer this C.V in the anterior of the uterus (in lumen of uterus)

Drawbacks:



A
dhesion
, parametrirts,

perimetrirts



For how many times we can go for leprtotmy?



Injury occurs.


Factors Affecting the Success of E.T



Specie of animal



Drug type



Breeding season
: non breeding season is better for recipient because in non
breeding season due
to high prolectin there is negative effect on activity of
pituitary gland.



Day of collection



Recipient status



Quality of embryo


AI
vs.

E.T


Artificial

Insemination



E
mbryo
T
ransfer



In this technique exploitation of
male potential



The entire national her
d based
improvement. AI is the best.



Less expensive



Less expertise required



It
is

the best to provide facility at
the door step
of

farmer.



Exploitation of potential is lower in
AI.



Exploitation of male and female
potential



Improvement of potential
at limit
ed
sc
ale or individual level
.



More expensive.



Nee
d expertise in a series of steps.



N
ot

preferred for o
ut reach program




More exploitation o
f

potential.
















Lectures of Reproduction Biotechnology

(A
R
402)



(Final)








Synchronization of Est
rus

Synchronization means to happen at the same time or to move at same speed.

This technique is being in practice since 1940. In Reproduction Biotechnology its effect
is to save the time and labour or to get maximum profit in given time period.

Applicatio
ns:

o

It helps to get maximum benefit.

o

To get proper time of insemination.

o

It is used for reproduction management of herd at individual level or at herd level.

o

Induction of parturition we also synchronize. In large farms that could be done to
overcome partu
rition; all animals parturate at same time with difference of 1
-
2
days.

o


Preparation of animal for competition. For example for milk production we
prepare animal for specific period of time by synchronization.

o

Help in breeding program specially beef produc
tion program.

o

If there is short breeding season then we can go for synchronization to get
maximum benefit.


Methods of Synchronization

There are two methods:

o

Hormonal method

o

Non hormonal method


Non Hormonal

If you are going to use non hormonal method then

it should be done 20
-
30 days prior to
commencement of breeding season. It includes the techniques as:


Introduction of Male in a Herd:

Ratio of male and female is 1:20 or 1:25 in case of buffalo and 1:30 in case of sheep and
goat.

If you enter male one mo
nth prior to breeding season then all females will show
heat. It i
s due to
pheromones
secreted in urine, feaces, sweat and saliva

of male.

The
s
e
are chemicals and act as hormones.

By smelling theses females are affected.


Provision of Artificial Light:

Par
ticularly it works in the mare. The mare is a long day breeder. It requires extra light.
We provide 15
-

16 hours extra light per day for the onset of heat.


Environmental Stress:

In pigs by the environment stress e.g. transport
pig

for long distance (100 m
iles) that will
onset estrous.


N
utrition:

By improving nutrition we may get help.


Hormonal Methods

There are three types of drugs used:

o

Prostaglandins

o

Progesterone

o

Gonadotr
op
in releasing hormone

They may be used as single or in combination


Prostaglandin
s

Synthetic or natural preparations of prostaglandins are available:

In synthetic: cloprost
enol

is active ingredient

(conc. of agent is
250
µg/ml).
2 cc

or 500
µg is used.

Routes are
I/M
, I/V, or into CL.

Prostaglandin hormone works in cyclic animals hav
ing CL on the ovary.

PG will lyse the CL (on day 16) and progesterone level will go down and feed back
mechanism stimulates anterior pituitary and FSH will be secreted. In case of plain ovary
use other hormones.

Prostaglandin

and progesterone can be given

in combination. Give progesterone for
10
-
15

days and then sudden drop in it and give prostaglandin.

There are 10 types of prostaglandins but luteolytic activity is only by PGF

.

Basic
precursor of PGF


is arachidonic acid. It is present in the form of f
at granules. At day 16
level of estrogen starts increasing, lysis of granules occurs by prostaglandin synthetase
and PGF


is produced. It causes the lysis of CL. Before 16
-
17 day estrogen is not present
upto that level to cause to lysis of granules. Cyclo
pertano perhydro phenanthine is the
basic ring for all prostaglandins.

Cloprostenol can be used through I/M, I/V, intrauterine or direct injection of in CL (using
specific needle and syringe).

For I/M dose will be hither but for others dose is low.

2 ml o
r 500 µg is dose of I/M

1 cc is dose for intrauterine (1 ml + 9 cc of dextrose solution or normal saline) and given
in the epsilateral horn.

1 ml + 9 cc of dextrose solution or normal saline

for I/V

0.5 ml for CL

This hormone can be used in case of buffalo
, cattle sheep, goat, bitch, and mare. We can
use it blindly in cattle, buffalo, sheep and goat because there is at least no harmful effect.

2 cc is dose for cattle, buffalo (I/M)

0.3 cc is dose for sheep and goat (I/M)

In bitch 2 ml through s/c. Recommend
ed rout in bitch is s/c.

1 cc or 2 cc (in divided dose) for mare. 2cc is for thoroughbred animals.

In mare it should be used very carefully. It is highly reactive and can cause abortion in
pregnant. It causes contraction of smooth muscles.

The mare should

be off feed from at last 4
-
5 hours before giving prostaglandin hormone
because it causes the increase in intestinal movement. If stomach is filled, it will cause
sever colic and death may occur. There is sweating, painting and staggering gait.

In camel tr
eatment is same.

In mare and camel equimate is used. Wait for 30 minutes after giving injection.

Bitch or mare should be at clinic to inject the PG because in field it is difficult to cover if
problem occurs.

PG is used in case of open cervix Pyometra. D
ilute with lukewarm water and give 1 ml
through intrauterine route. Never use in close cervix because it may rupture due to
contraction.

There is no specific anti
-
dot still for it.

Prostaglandin

is

drug of choice in habitual retention of placenta. It shou
ld be given within
one hour of calving.

Natural product is lutalyse 5 mg/ml (IM). Usually 4
-
5 ml or 20
-
25 mg of drug is used in
cattle and buffalo through I/M route. 2 ml through intrauterine.

Synthetic products are estromate or cyclomate

Estrumate, cyclom
ate are cloprostenol 250 µg/ml

Equimat: instead of cloprostenol there is floprostenol as active ingredient and specially
used in she
-
camel and mare but also used in cattle and buffalo.


Programs for Synchronization

There are 4 programs for synchronization

on herd level. At individual level go for rectal
palpation, judge CL, if persistent then give PG to make animal in heat.

Response time for PG is within 48
-
120 hours and average 72 hours.


One must be careful after injection; within 5 days animal shows h
eat (for proper heat
detection and time of insemination).


Program A

o

Single injection with 5 day breeding program

o

You will palpate all the animals, confirmation of CL

o

Injection of PGF


(natural 2 cc and synthetic 4
-
5 cc)

o

Confirmation of estrous

o

AI (12 hou
rs after standing estrus).


Program B

o

Single injection with 10 day breeding program

o

Should be implemented at the starts of breeding season

o

In first 5 days of breeding season animals show the natural estrus; they will be
inseminated.

o

Animals not showing est
rus in first 5 days will not be inseminated and they are
given single injection of PGF

.

o

Confirmation of estrus

o

A.I (12 hours after standing estrus).


Program C:

o

Double infection with 10 day breeding program

o

Inject PGF


on day 1 to all animals

o

For next 5 days detection of estrus and proper insemination

o

PGF


(second injection) to those wh
ich did not show estrus in first 5 days

o

In next 5 days detection of estrus and proper insemination


Program D:

2 injections within 5 day program

PGF


injection to all animals in herd to be synchronized and then do not go for detection
of estrus in next 5
days and without A.I give second injection to animal at the interval of
11
-
14 days (14 days preferred) from the first injection

Detection of estrous and confirmation within 5 days of second injection and AI

Accuracy of this program is above 80 %. It is mos
tly used. It is best method


Key
P
oints:

o

The animal should be shifted to high plan of nutrition before going for
synchronization. If body score 5
-
6, it is more beneficial.

o

One can go for synchronization after 40
-
50 days of calving.

o

There should be accurate

estrus detection method.

o

Herd should be cyclic one (key point for PGF

)


o

You must be careful upto 120 hours after giving injection.


Limitations for
U
se of
PGF2α
:

o

Can be used only in cyclic animals

o

Not effective one in proestrus or immediate metestrus

o

Not effective upto 7 days post estrus

Be careful about the time of insemination. It should be 12 hours after the confirmation of
standing estrus.


Progesterone

Other major hormone which can be used is progesterone

It should be used 15
-
20 days before the on
set of
breeding season


It can be thorough I/M, s/c, or oral.

Progesterone Releasing Intravaginal Device (PRID) and Control Internal Drug Releasing
(CIDR) placed in vagina.

With progesterone basic principle is; first you have to maintain luteal activity o
f animal.
You have to continue this for 10
-
12 days and after 10
-
12 days suddenly remove the
source of progesterone. Due to sudden removal there will be negative feed back, anterior
pituitary will be stimulated and there will be release of FSH. Possibility
of estrus is 5
-
7
days after the removal of source.

It is used for synchronization. Practice is 25
-
30 days before season of breeding to get
maximum benefit.

It is used for:

o

Synchronization

o

Suppress the ovarian activity

o

This hormone is used for the treatment

of abortion particularly if cause of
abortion is insufficiency of progesterone due to inferior CL. Abortion occurs in
first pregnancy month. (At day 30
-
35 mucous with small conceptus shows
abortion due to low level of progesterone). Progesterone should be

continued until
CL starts producing progesterone.

o

In synchronization used in mostly in all species.

For suppression of estrus activity it is specially used in bitch, cat and mare.

o

To avoid conception long acting progesterone is given.

o

In concentration o
f shows it is used to avoid estrus. We have to stop the ovarian
activity for this purpose dose is 0.3 mg/Kg.

o

Similarly in milking compartments it is also used because estrus cause decrease in
milk production.


This can be used by I/M, s/c, oral (aldosteron
e, melogestrol). Oral dose is 0.5
mg/herd/day.

Mix these drugs with vegetable oil and that oil is mixed in concentrate and given for 10
days. When you stop; within next 5
-
7 days following animal will show estrus.

40
-
100 mg (d 10) I/M.

After embryo transfer

in she
-
camel particularly progesterone is recommended.

Synchromate B s/c drug

Intravaginal: PRID,CIDR

Sponge: 40 mg progesterone in sheep and goat. 1.9 g in cattle and buffalo. Take care of
urination. Special speculum is used. Place near cervix. Mare and
cattle sponge comes out
with urine. So in
vagina
horizontal matrix suture is applied with umbilical tape. Gel is
used to lubricate sponge, oil is not used. Lubricate it from side. Keep it for 7
-
10 days.
Each time sterilize speculum when speculum is used in

large number of animals. Never
use oral preparation for lubrication.

CIDR is coil provided with flexible steel rim
.

Hormones ECG and PMSG are used for induction of estrus. After removal of sponge you
give ECG 300
-
400 IU through I/M in animal that increase
s the fecundity rate.

If after progesterone treatment you are to inseminate animal, inseminate 2 times. HCG is
also recommended in mare with progesterone to get in time insemination.

In
M
are:

Variation in ovulation rate if use progesterone in mare

D 0



PGF



D 6
-
7



HCG (I/M) (to control ovulation time)

D 14



PGF



D 22



AI or mating

If you use natural source it can cause BSE.


D 0
-
10



progesterone

D 7



PGF2alpha

D 15
-
16



HCG

Most of animals respond to first program.

Progesterone is never re
commended in bitch and cat due to change in physiology.


Photoperiodicity in Mare:

Mare needs extra light for estrus. If you provide it with light of 16 hours you can induce
estrus even when there is not breeding season.

Animal in room of 12 × 12 feet, str
ingent light is provided for 16 hours just for 10
-
15
days. Mare shows estrus. In Europe practice it in winter season. In case of racing where
cost of one day foal is in million so they adopt this technique.



GnR
H

It is for induction of estrus. Gonadotropi
n and gonadotropin like hormones stimulate the
release of FSH and LH.

ECG, PMSG: these are gonadotrophin like hormones

Delayed ovulation: it is used

Cystic ovary: It is good treatment.

Receptal is GnRH preparation containing FSH and LH and also helps in
-
ti
me ovulation.
It is more effective if ovary contains follicle of 5 mm or above at injection time.
If no
follicle it is better to repeat injection after 7 days.

In the case of plain ovary we can use GnRH and progesterone if no other choice is
present.

For
induction of ovulation increase the dosage. 100
-
150 µg I/M.

For ovulation 200
-
250
µ
g I/M or I/V


Estrogen

Estrogen is not good choice for synchronization. This is for estrus induction. It does not
help in formation of follicle. It only stimulates epitheliu
m of the reproductive system.
High dose may produce the behavioral signs
and some mucous
. If ovary has mature
follicle then it may help in ovulation.

It is used for induction of estrus but still not good choice.

10
-
20
mg (1
-
2 cc of stilbestrol)

I/M

This is

given in combination with progesterone or dexamethasone for induction of
parturition.

Adverse effect in case of heat.

It may lead to mastitis.

In normal estrus there is low milk production i.e. retention of milk in teat. It is due to
increase level of es
trogen because it is smooth muscle contractor. Continuous contraction
of udder muscles leads to the exhausting and total milk production will be low.

It may lead to cystic ovary.

Basic principle must be clear about the hormone. Never use blindly. Very cle
ar about
target.


Invitro Embryo Production (
IVEP
)

Production of embryo in laboratory. It is used as an alternate of E.T

Uses:

o

For research purpose

o

We can get maximum genetic potential of animal. It can be collected by ovum
pick up manner or from slaughter
ed animal. If animal of increased genetic
potential die you can get ovaries and utilize it.

o

It can be used for beef production program.

o

It can be used for treatment purpose. If infertility due to fallopian tube defects
then you can use that animal by ovum
pick up manner (for treatment purposes)



Steps
I
nvolved


Collection of O
vary

Ovaries are collected form ovary production unit (OPU) or form slaughter house. You
need normal saline 0.9 % or phosphate buffered saline (PBS) and it should be
supplemented with

antibiotics i.e. penicillin 100 IU/ml and streptomycin 1 mg/ml and
kept at 25
o
C.

After slaughtering ovaries should be collected from carcass immediately and directly.

You have to remove all the extra ovarian tissue with scissors and in thermos only ovari
es
should be transferred.

Before shifting of ovaries it is better to rinse ovaries with fluid 2
-
3 times.

Transportation of ovary from collection site to lab should be maximally completed within
5 hours.


Processing
o
f Ovary in Lab

Ovaries washed with 70 %
ethanol

Followed with 2
-
3 washing/rinsing with N.S.

Place ovaries in beaker with water bath at 30
o
C.


Collection of
O
ocyte

There are three methods:

o

Aspiration Method

o

Puncture method

o

Dissection

You have to go for one which is established to be best. Metho
ds of collection has direct
link with no. and quality of oocytes.


Aspiration Method:


Pick up ovaries one by one form beaker kept at 30
o
C. Never collect oocyte from larger
follicle because that will contain more fibrin and will interfere with process. 5
-
8 mm
range is best. Medium for oocyte collection is modified tyrode lactate medium (MTLM)
or TL
-
Hepes and should be supplemented with 10% EBS (estrus buffalo serum) or ECS
(estrus cow serum) + antibiotics and this medium should have pH of 7.1. Serum added
should be inactivated.

This medium should be kept in CO
2

incubator before two hours of use with 5 % CO
2

and
95 % humidity and at 38.5
o
C. In aspiration method the best one is 5
-
10 cc syringe and 18
guage needle. It means you have to aspirate oocyte from vi
sible follicle. Before aspiration
suck 1
-
1.5 ml of oocyte collection medium in syringe. Then collect the oocyte form the
follicle. Shift it to Petri dish containing 5
-
6 cc oocyte collection medium. Possibly with
single puncture collect form all visible ooc
ytes.


Puncture Method:

You can puncture each follicle and collect oocyte.


Dissection:

Thin slices of ovary are cut with the knife and put in Petri dish with the help of artery
forceps one by one and rinse it. Dissection method gives the more number of o
ocyte and
good quality. So it is the best method because we can collect more oocytes.

Classification of
O
ocyte

Criteria for selection of oocyte is:

o

Presence of cumulus oophorus cells around the ovum. There should be 4
-
5 or 5
-
6
cell layers.

o

The compactness

of layer with each other and around the ovum

o

Spread of cytoplasm in the ovum and firm granulation.


On these basis oocyte are graded as

1.

A grade: oocyte having 5
-
6 layers of cumulus oophorus cells, compact
layers and even spread of cytoplasm.

2.

B grade: part
ially surrounded by cumulus cell layers (2
-
3)

3.

C grade: denuded oocyte (oocyte without cumulus cell).

4.

D grade: something happened to cells and there is formation of mesh like
structure

Usually Grade A and B oocytes are selected.

Ovum Pick up Technique:

Just

give epidural
anesthesia. 200
-
300 cc is given.
By this process less than 8 mm oocytes
are

collected by the help of
leptoscopy.

Intravaginal tube is passed, pump is used

to suck,

needle is attached material comes in tube. Tube is used for evaluation.


IVM

(
Invitro
M
aturation of
O
ocyte)

.

There are two types of medium used:

o

TCM
-
199 (tissue culture medium)

o

Ham’s


F
12

For preparation of culture medium sterilize Petri plate. Medium is sterilized by filtration
(pore size 0.2 µ). Filter is attached with syringe.

For culturing of oocyte we can use two
types of Petri dishes; glass or plastic Petri dish. Glass Petri dish is preferred because in
plastic Petri dish deterioration increases with increases with time in humid environment
at 38
o
C. There are two types of c
ulture drops. Larger culture drop of 500
µ
l and smaller
culture
drop
of
100

µ
l. Most of the time smaller one is preferred. Because when oocyte
are cultured and placed in incubator, toxic products accumulate due to metabolism which
affect environment. This
is less in case of small drop. Larger one is usually placed in the
centre of Petri dish and smaller one is placed at four corners. Usually micro dispenser is
used fo
r preparation of drops.

If larger one, we can place 80
-
100 oocytes. If smaller one, we can

place usually 10
-
15
oocytes (in some books 20 oocytes). Petri dish should be sterilized. Four culture droplets
are prepared at corners. Whole Petri dish is covered with mineral oil. In each droplet
there are 10
-
15 oocytes. After this Petri dish is shifted

to the CO
2

incubator.

Mean Culture Conditions For Maturation:

Temperature: in some labs 38

o
C, in some 39
o
C but usually 38.5
o
C

Humidity: 98
-
100 %

CO
2

concentration: 2%

Upto
morulla

stage in fertilization CO
2

5% because more humidity.

Keep in incubator

for 20 hours. Then go for assessment of oocyte.


Assessment of Oocyte

Criteria

for assessment:

o

Expansion of cumulus oophorus complex

o

Extrusion of polar body


Maturation of oocyte is of

two types:

o

Cytoplasmic maturation

o

Nuclear maturation.

Both are requ
ired by oocyte to be fertilized. Presence of polar body indicates nuclear
maturation.

For staining;

f
ixing of oocyte with acetic acid

and s
taining with 1 % aceto
orecin

to check
nuclear maturation.

When it matures there is one polar body in perivetaline sp
ace. After fertilization there are
two polar bodies.

For maturation cultural medium is supplemented with cells (granulosa and cumulus) and
hormones (FSH, LH) to increase efficiency of that to increase the maturation rate.
Mineral oil is used to provide som
e nutrition and check abnormality of oocyte. It is called
co culture
technique
.

BOEC:

Bovine oviductal epithelial cells. These are added to culture medium to enhance the
development of ovum. These

epithelial cells will be 3 million per culture drop of cul
ture
media.

Mineral oil maintains integrity of culture drop and culture cells and to avoid evaporation.
It is no way dangerous for embryo. It

is also added as adjutant in vaccine


Preparation of
S
permatozoa

There are three methods:

Percol Density Method:

T
ake 3 ml of 90 % percol at the bottom of test tube. On upper side of this 1.5 ml of percol
mixed with 1.5 ml of sperm tyro

lactate solution (Sp. TL). At the top semen is deposited.
This is centrifuged at
400
G for 30 minutes at room temperature. Supernatan
t is
discarded; there is formation of sperm
pellet
at bottom. In this you will add sp TL upto 5
ml. Again centrifuge upto 400 G for 10 minute. Again supernatant is discarded and again
formation of sperm pallet. It is mixed in a tyro albumin lactate pyruvat
e (TALP).


Sodium Citrate

Method
:

0.25 ml of semen is mixed with 4.75 ml of
2.9
% sodium citrate and centrifuge at 300 G
for 10 minutes. Supernatant is discarded and pellet is mixed with 3 ml of 2.9 % sodium
citrate. Again centrifuge at 300G for
10
minute
s and pellet formation is mixed with

3 ml

TALP.


Swim up Method:

2 mediums can be used

o

TALP

o

Calcium free
tyrode

medium

pH of medium is adjusted at 7.3 or 7.4 and it is incubated for 2 hours before its use in
CO
2

incubator (CO
2

concentration 5 % and tempe
rature 38.5
o
C)

For processing 0.25 ml of semen mixed with 1.5 ml of medium (any one of above
mentioned) and kept for 30 minutes under the CO
2

incubator. Then centrifuge at 100 G
for 10 minutes. Supernatant is discarded and 100
µ
l at the bottom is mixed w
ith 1 ml of
medium. Then this preparation is kept at room temperature for 5 minutes. Then 3 ml of
medium is again added in this mixture and again centrifuge at 100 G for 10 minutes.
Supernatant is discarded and 100
µ
l at the bottom is dissolved with hepari
n (It is added
with rate of 21.87 IU/ml of medium). After addition it is kept in CO
2

incubator for 15
minutes. After that you have to determine the spermatozoa concentration.


Oocyte Insemination

For insemination of oocyte mature oocytes are washed 2
-
3 tim
es with
fertilized medium
i.e. TALP.

Then the culture drops pf 100 µl are prepared. Then under microscope the
mature oocytes are shifted to culture drop.

This medium should be ke
pt 2 hours before shifting of oocyte in CO
2

incubator. Then
sperms are added a
t the rate of 1 × 10
6
/ml and kept in incubator for 24 hours and after 24
hours go for assessment of fertilization rate. Upto here medium was TALP.

Culturing:

Now you have to continue with fertilized oocyte and culturing of it upto
5
-
6 day to
continue upto

morulla and compact morulla stage. Drops of TCM
-
199 are poured and
fertilized oocyte is shifted in them and incubated. You have to change at least half of
medium after every 24 hours. Continue upto compact morulla stage or upto early
blasctocyst stage
. 100

µl is added with out damaging oocytes. Now you can go for
storage of embryo or transfer of embryo.

The 32 cells (blastomeres) are not required for development of normal calf. Only 4 cells
are required for it. So you can split the
embryo into 2 or
4

halves
.

One half can be used fo
r

determination of sex and other

three
hal
ves can

be used fo
r
pr
oduction

of tripl
ets.

Embryo in morulla or compact morulla stage are more safe for freezing as compared to
blastocyst stage.


Invitro Fertilization (IVF)

IVET or IVF
has applied on 9 species including humans. Method developed for IVF for
one specie can not be applied on other specie as such. In human it is used for treatment of
infertility for both male and female. Major problem is block of fallopian tube. On male
side

problem is within sperms.




Capacitation:

Major problem is with the capacitation of spermatozoa. Time of capacitation for different
species is form 30 minutes to 10 hours. 9
-
10 hours are needed for the capacitation of
rabbit sperm.

There are protein mol
ecules which cover spermatozoa during capacitation. Protein is
destabilized which help them in acrosomal reaction. After fertilization there is rest of
growth at two cell stage and at 8 cell stage and this is known as 8 or 2 cell blockage.

Now scientists
are working how to stop this blockage. In humans IVF technique is only
used in case of infallibly. 40 % infertility in females is due to blockage of fallopian tube
and if less concentration of sperm number.


Genetic engineering

It is modification in struct
ure of RNA or DNA. It is broad term. It is any directed change
in genetic make up of a population. Any livestock farmer selects a bull and introduce in
his farm to increase milk production. It will change genetic make up.


Anima Pharming

It is related wit
h pharmacology. Change in composition by changing the nucleus or gene
part to produce immunity against specific problem.


Xenogenous Fertilization

The placing of gametes of one specie in a reproductive tract of other specie and when
embryo developed to
morulla stage it is collected and transferred to surrogate mother.


Parthenogenesis

There are two functions of sperm: Transfer of genetic material and growth of cells.

Parthenogenesis is stimulation of growth without sperm. It is possible by chemicals or
e
lectric stimulants. Now people are using it in case of cloning.


Cloning

It is creation or production of 100% true genetic copy of an individual. It is being
produced in case of animals and plants. This technique is in practice since 1963.

There are two so
urces of cloning on animal side:


Embryo cells:

If we are able to separate blastomeres.


SCNT

(somatic cell nuclear transfer)
:

Unfertilized oocyte is taken and enucleated by microsurgery and one blastomere is
implanted and electric shock is given and then
placed in culture.

Famous result is of Dolly sheep and this sheep was cloned actually. And first mammal
which was produced by scientists was Dolly sheep. Dolly sheep was produced at 277
th

attempt. It means 276 attempts were failed.

This sheep was born in J
uly 5
th

1996 ad died on 14
th

February 2003 (6.5 years).

This experiment done in Roslin Institute Edinburgh Scotland by Ian Wilmut and Keith
Capbell and their colleagues.

In this a somatic cell from mammary glands of the sheep was collected having a whit fa
ce
and the unfertilized oocyte was collected from black face sheep. Oocyte was enucleated
through microsurgery. Nucleus removed from somatic cell of mammary glands was
transferred in that oocyte and electric shock was given and cultured under CO
2

incubator
.
Then collected in blastocyst stage and transferred to surrogate mother and on 5
th

July
Dolly was born with white face.

Life was 6.5 years. Dolly produced 6 lambs; first time single, second time twins, third
time triplets. First lamb was born on April 199
8. Dolly developed arthritis of hind limb
and treated for that in 2002 with anti inflammatory drugs. Dolly was euthanised on 14
February 2003 due to lung cancer. The sheep from which somatic cell was taken was also
died of lung cancer. Average age of sheep

is 12
-
13 years and Dolly was died at age of 6.5
years. Dolly was short legged due to aging. Some say it dies at early age because donor
of this sheep was also died at 6 year due to same disease.

It was thought that it was not 100 % clone because mitochond
ria carry 1 % genetic
information that was different. Other confusion was what was the reason of development
of cancer. There was prevalence of disease in flock from which donor was selected.

There are some ethical problems in use but researchers are usin
g it for diagnosis of
familial diseases.


Transgenic Animals

Transgenic animals are those animals which carry foreign gene which has been inserted
in its genome. Gene is inserted by using DNA reproductive biotechnology or by using
recombinant DNA methodo
logy. It also contains other sequences that enable DNA to be
expressed in animal. Polio virus infects human but not mice. If special receptors are
inserted in mice, it also undergoes polio.

There are three methods:

Embryonic stem cell method:

Preparation o
f DNA: particular gene isolated, inject it into DNA Vector which will be
used as carrier. Culture it in embryonic stem cell. Then inject it in inner cell mass of
blastocyst of mouse. Then transfer it to foster mother uterus. We will get heterogenous
mouse
which are then crossed to get homogenous.

Pro
-
nuclear Method:

Prepare DNA vector with gene. Take freshly fertilized egg before pro
-
nucleus
development. Inject pro
-
nucleus with DNA. When the pro
-
nuclei have fused to form the
diploid zygote nucleus, allow th
e zygote to divide by mitosis to form a 2 cell embryo.

Retrovirus mediated gene transfer:

Retrovirus could be used as vector or plasmid of E. coli.


Uses of Transgenic Animals:

In medical research to identify specific function in complex system.

In toxicol
ogy to see response of animal to particular toxin

In molecular biology

Mammalian development genetics

In pharmacology


Mosaicism and Chimerism:

These are those animals which carry more than one distinct population of cells.

In Mosaics population of cell
arise form single zygote while in Chimeras population arise
form two different zygotes.

Cytogenetic Mosaics:

for example in human some of the cell be 46, XX and some 47, XXX. In Turner’s
syndrome there is X chromosome monosomy (46, XO). If population has h
aving such
cells animal die before birth.

X chromosome Mosaic:

All mammals have these. e.g. cat. Particularly gene on X chromosome for orange color in
coat. Ausomal gene produce black patches on cat. Male with orange and black. female
will have OO (orange)
, oo (black) or Oo (mixture of orange and black). In this case one
X is active other is inactive.

Chimerism:

e.g. freemartin in cattle. Animal produce by chrmerism is baby geep using sheep and goat
embryo. There are features of both goat and sheep.