and Postfertilization Infertility in Rabbits

BIOLOGY

OF REPRODUCTION

20, 93 1-937

(1979)

Sperm Fractions Responsible of Pre- and Postfertilization A.

C.

MENGE,

HELLE

Department

of

PEEGEL

Obstetrics

University

Ann

for Immunologic Induction Infertility in Rabbits

of

and

and

Michigan

Arbor,

M.

L.

RIOLO

Gynecology, Hospital,

Michigan

48109

ABSTRACT Female rabbits were isoimmunized against 0.3 M lithium diiodosalicylate (LIS) soluble extracts of washed rabbit sperm and homogenized rabbit testis. The rates of fertility, measured as percentage of corpora lutea represented by viable embryo implants at 9-13 days after breeding, for the 2 groups were 45% (P10.

Fertility

Triala

Rabbits were artificially inseminated 5-7 days after the fourth and fifth immunizing injections. Each doe was inseminated with 101 sperm in 0.25 ml of semen diluted with Hanks balanced salt solution (HBS) and given an iv. injection of 100 IU chorionic gonadotropin (hCG). Antifertility effects of sera were evaluated after artificial insemination of untreated estrous rabbits with a 0.25 ml sample containing io sperm in a 1:5 dilution of immune serum. Fertility was determined postinsemination either at 26-28 h by microscopic observation for normal appearing cleavage in ova flushed from the excised oviducts or at 9-15 days by comparing number of viable appearing implantation sites in the uterine horns with the number of corpora lutea in the ovaries. In some groups after the first insemination trial, viable conceptuses were manually ruptured to terminate the pregnancy and allow for a second fertility determination.

Embryo

Transfer

Fertilized ova (2-4 cell) were flushed from the oviducts of superovulated rabbits at 26-28 h after mating. Superovulation was induced by daily injection for 3 or 4 days of 0.5 unit of porcine follicle stimulating hormone (FSH. Sigma) suspended in 0.5 ml of 5% beeswax in sesame oil. The day after the last FSH injection, the rabbits were mated with 2 bucks and injected intravenously with 100 IU hCG. The flushing and transfer medium used was HBS with 10% rabbit normal serum (heated at 56#{176}C,30 mm) plus 100 IU of penicillin and 100 pg of streptomycin. After recovery, the ova were observed microscopically and transferred to fresh medium and then placed in a 37#{176}C incubator with a humid atmosphere of 5% CO2 in air. The recipient rabbits were anesthetized with sodium pentabarbital and the abdominal midline incised. Five or 6 fertilized ova were transferred with a minimal amount of medium by a glass micropipette into the fimbriated end of each oviduct. After transfer the

IMMUNOLOGIC

incision to

was

their

sutured

cages.

12-14

days

closed

and

the

after

animals

rate

Implantation

INFERTILITY

was

IN RABBITS

933

returned observed

at

9

transfer.

0r

a) 0.

en

“0

Statistical

‘0

‘0

a) a a)

Analysis

Significance of differences was based on Student’s test. Because differences between results of the first and second fertility trials did not differ within experimental groups, the data were pooled.

C

0 a

E

-3

3

a)

z

an

an

0

-

9 9

.0

E a)

RESULTS

a)

a

Postimplantation

fertility

insemination), rabbits

based

pregnant

lutea

on

and

(CL)

(Days

on

the the

represented

implants,

was

by

with

comparison

to

of

of

viable

a a-)

by

of

0.

C

9

im-

sperm

a)

a a)

C

3

z .0 .0

a

a

Immunization

with

tended

to

from

control the

reduce

the

immobilizing

titers

(Log

LIS

group

respectively.

the

not

signficant.

Results

of

5-8

c) C

0

.9

a) a

The

and

artificial

-

testis

a C

C

antibody

from

U

1-anna

.0

difference

agglutinating

ranged

1).

of

but was

and

2)

(Table

extract

fertility

control

sperm

group

0.

0

a

3 U

the

0

-3 .3

9

in

ci)

a)

U

appearing

extract

a) .9

corpora

depressed

LIS

9

after

proportion

number

significantly

munization

9-13

a)

a

E

9

a)

a) C

4-8,

as

C

a

insemination

U

ci)

of

untreated

rabbits

different

sera

revealed

a

compared sera

from sera

blocking 1

SE-LIS

rabbit.

used

in

rabbits.

The

3

infertile

not

rabbit

was

The

from

2 sera

sperm for

artificial antibody

unless

4

the

U

.3

.9 C

.9

a

9

C

insemi-

a)

3

0.

-

Es

a

rabbits

sera

:1)

en

a

0

tiers

the

.9 C

>‘

a pregnant

TE-LIS

a

0

and

from

for

were

did

from

a

rabbits

control

SE-LIS

of

of

fertility

were

.0 .0

from

of

at the mode decrease fertility

nation

group

pregnancy

treatment

diluted

suppression from

fertile

allowing

sperm

SE-LIS

fertility

and

rabbits

the

significant with

serum

with

en

-

.3

N-i-an

a)

N

a

E

U

0

but

0

z

0

a)

were U

unheated,

in

which

case

they

immobilized

ci,

the a

sperm

and

In

prevented

the

fertility.

second

phase

a)

of

the

study

the

a)

I-.

ci)

LIS

U

extraction

procedure

was

modified

in

a LiC12

solution

that

the

‘0

‘0

‘0

C

initial

dialysis

the

amount

was

increased.

as

was of

sperm In

antigenic

by in

the

the

of

any

the

reduction

control

U

0

>S

z

0

.9 a) N

.0 .3

C 3

implants

(Table

2).

sperm

immunized and

significant rabbits testis

against

occurred

the

immunized the

LIS

LIS-extracted

reduction

and

the

C

.0 .3

in rab-

NP.40

extract

sperm in

fertility

against

the

extract

pellet

and

a

occurred US of

0.

ci)

en

pellet NP.40

Immunization after and

the the

of double NH4

rabbits

extraction

SCN

extract

of

the

of

‘

LIS-extracted

with

. -

in

extract

with

-

cn

an

C

sperm.

.

“

of 0

.9

.9

a

bits

“0

9.0

0 a

a highly

rabbits,

a)

‘

as

9 9

washed

testis

in fertility

a

fertility

viable

of

a

injection

against

extract

to

and

of

immunized

NP.40

comparison

per

inhibition

lack

rabbits

and

significant

material

A complete

indicated

occurred

against

and did

-

-0,s

a)

a

I-

C

b

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