Viability of vitrified mouse embryos using various cryoprotectant mixtures

THERIOGENOLOGY

VIABILITY OF VITRIFIED MOUSE USING VARIOUS CRYOPROTECTANT H. Ishimorit

Y. Takahashi

EMBRYOS MIXTURES

and H. Kanagawa

Department of Theriogenology Faculty of Veterinary Medicine Hokkaido University, Sapporo 060, Japan Received

for

publication:

Accepted:

February 27, 1991 October 29, 1991

ABSTRACT Mouse morulae and blastocysts were cryopreserved by vitrification using six types of solutions. Each solution was composed of two types of cryoprotectants. glycerol(GL) + ethylene GL + dimethyl sulfoxide(DMSO), glycol(EG). GL + propylene glycol(PG), EG + PG. EG + DMSO, and PG + DMSO at an each cryoprotectant concentration of 25% v/v. Embryos were exposed to each type of vitrification solutions, which had been diluted 50% in PBS, for 5 then for another 5 minutes at 4 "C. minutes at room temperature, The embryos were loaded into straws containing vitrification solution at 4°C and plunged into liquid nitrogen within 30 seconds. After warming in water at OC and following one-step dilution of the cryoprotectant in 0.5 M sucrose+PBS, the embryos were cultured in vitro. The survival rates of morulae were 51, 16, 78, 44, 79 and 50%. respectively, for the six solutions. The survival rates of the morulae using GL + DMSO and EG + DMSO were significantly higher than those of the other solutions (P
words:

mouse

embryo,

vitrification,

cryoprotectant

mixture

INTRODUCTION Rall

and

Fahy

(1)

first

reported

vitrification

Acknowledgments The authors thank Drs, Conrad0 A. Valdez reviewing this manuscript. 1 Present address : Embryo Transplantation Milk Products Co., Ltd., Uenae Tomakomai,

FEBRUARY 1992 VOL. 37 NO. 2

and Antonio

as

a new

A. Rayos

for

Laboratory, Snow Brand Hokkaido 059-13. Japan.

481

THERIOGENOLOGY approach for the preservation of embryos. Since then, many researchers have studied cryopreservation of embryos by vitrification. Some effective vitrification solutions such as mixtures of glycerol and propylene glycol (2). polyethylene glycol and glycerol or propylene glycol (3), and ethylene glycol, ficoll and sucrose (4) have been reported. Scheffen et a1.(2) showed high in vitro survival rates of compacted mouse morulae and early blastocysts vitrified in a mixture of glycerol and propylene glycol. Massip et al.(S) reported the solution to be effective for bovine morulae and early blastocysts. Both their vitrification solution and method were simple; however, the survival rates for mouse and bovine blastocysts were lower in comparison with those for morulae. Other researchers also have reported similar findings using the same solution (6) and another vitrification solution (7, 8). Embryos at the morula and blastocyst stages are used most A vitrification solution frequently in bovine embryo transfer. same equilibration and applicable at both stages using the vitrification conditions is therefore desirable. There have been no reports regarding the comparison of different cryoprotectant mixtures used for the vitrification of embryos. This study was the in vitro viability of undertaken to investigate and compare morula and blastocyst stage embryos using different cryoprotectant mixtures. MATERIALS

AND

METHODS

Embryos Female ICR mice (4 to 7 weeks old) were induced to superovulate by intraperitoneal injections of 5 IU PMSG and 5 Ill hCG 48 hours apart. They were mated with male ICR mice except in the embryo transfer experiment, for which C57BL/6 males were used. Morulae or blastocysts were flushed from the oviducts and uteri at 70 to 72 hours or 90 to 92 hours after hCG treatment using Dulbecco's with 0.4mg/ml bovine phosphate buffered saline (PBS) supplemented serum albumin. Vitrification

Solution

Six types of vitrification solutions were used, each consisting of a mixture of two types of permeating cryoprotectants in PBS: glycerol(GL) + ethylene glycol(EG), GL + propylene glycol(PG), GL + dimethyl sulfoxide(DMSO), EG + PG, EG + DMSO, and PG + DMSO. The concentration of each cryoprotectant was 25% v/v. All solutions in the straws remained transparent during cooling and warming, which is one indication that the solutions vitrified (4). Equilibration Each

482

type

and

Cooling

of vitrification

solution

was

diluted

by 50%

in PBS

FEBRUARY 1992 VOL. 37 NO. 2

THERIOGENOLOGY and was prepared for equilibration before vitrification. Embryos were equilibrated in a 50% diluted vitrification solution for 5 minutes at room temperature (22 to 24 "C) and for another 5 minutes 20-k 1 at 4°C , at which time they were pipetted into a vitrification solution column into 0.25 ml French straws (IMV, Within 30 seconds the straws were heat-sealed and L'Aigle. France). They were stored for periods between immersed into liquid nitrogen. 1 hour and 1 week. Warming,

Dilution

and

In Vitro

Culture

The vitrified samples were warmed in water at 0°C seconds. The contents of each straw were expelled into M sucrose+PBS. The embryos were held for 5 minutes then were transferred to fresh PBS. Recovered embryos in Ml6 solution (9) and were cultured in 0.1-1111 drops paraffin oil in 5% CO, in air at 37 "C. The survival embryos was assessed by their ability to develop to blastocyst stage following 48 hours (morula) (blastocyst) of culture. In Vivo

for 15 to 20 2.5 ml of 0.5 at 37 "C and were washed of Ml6 under rate of the the expanded or 24 hours

Survival

Morulae and blastocysts vitrified in a mixture of EG and DMSO were transferred to Day-3 pseudopregnant recipient females immediately after warming and dilution. Five to seven embryos were transferred to each uterine horn. In addition, fresh embryos were transferred as controls. The recipients were autopsied and examined on Day 18 of pregnancy to record the number of live fetuses. Statistical Data

Analyses were

analyzed

by the

x'

test.

RESULTS The survival rates of morulae vitrified in GL + DMSO and in EG + DMSO were significantly higher than those of the other solutions (PCO.01). On the other hand, the survival rates of blastocysts using GL + EG and EG + DMSO were significantly higher than those of the other solutions (PcO.05). The mixture of EG and DMSO gave the highest in vitro survival rates (79%) for both morulae and blastocysts, as shown in Table 1. Results after the transfer of vitrified embryos are shown in Table 2. The in viva development rates of vitrified morulae and blastocysts were 34 and 50%. respectively. These rates were not significantly different from those of the fresh control embryos (36 and 49%). DISCUSSION

The

FEBRUARY

primary

advantages

1992 VOL. 37 NO. 2

of

the

vitrification

method

for

the

483

THERIOGENOLOGY Table

1. In vitro survival after vitrification

Type of solution

GL GL GL EG EG PG

+ + + + + +

of mouse morulae and blastocysts in six types of vitrification solutions

No.(%) of morulae vitrified developed

EG PG DMSO PG DMSO DMSO

59 6'2 60 61 63 60

No.(%) of blastocysts vitrified developed

30(5$ lO(16) 47(78)c 27(44)a 50(79)c 30c50ia

a5 85 85 a4 89 89

61(72)d 25(29); 47(55)f 39(46)d 70179)f 41(46)

GL : Glycerol, EG : Ethylene gtycol. PG : Propylene glycol. DMSO : Dimethyl sulfoxide. Data were pooled for 7-10 replicates. Values with different superscripts within the same columns significantly different (a, b, c: P~0.01; d, e. f: P~0.05).

Table

2.

In viva development of mouse morulae and blastocysts vitrified in a mixture of ethylene glycol and dimethyl sulfoxide No.of

recipients

used

pregnant

Stage

of embryo

are

Treatments

No.of embryos transferred total to pregnant recipient

No. (X) of normal fetusesa

Morula

vitrified control

9 5

6 3

118 68

79 42

27 (34) 15 (36)

Blastocyst

vitrified control

8 5

6 2

96 68

71 28

35 (49) 14 (50)

a Total number to recipients

of live fetuses / total which became pregnant.

number

of embryos

transferred

cryopreservation of embryos are that freezing equipment is not necessary and the time required for cooling is reduced. However, vitrification solutions are known to be embryo-toxic at room temperature (1, 10). In our present study, embryos were equilibrated in each vitrification solution that was diluted by 50% for 5 minutes at room temperature and for another 5 minutes at 4 "C; then the embryos were exposed to the vitrification solution at 4°C for a short period (30 seconds). The mixture most effective blastocysts by conditions. The

494

of ethylene glycol and dimethyl sulfoxide was the for the cryopreservation of mouse morulae and vitrification under the present experimental in viva development of embryos vitrified in this

FEBRUARY 1992 VOL. 37 NO. 2

THERIOGENOLOGY solution in

was

the

previous by Kasai of

the

mentioned

workers et a1.(4)

embryos.

7. and

has

been

suggested

from

dilution

in

dimethyl vitrification cryopratectants

ethylene rapid

over also

0.5

be

effective

serious

mixture

al.(lO)

of

for

show.

solution exposure

glycerol

the

In

their

There

two

injury

and

compared to

permeable Urano

et

sulfoxide at

to

a1.(15)

protect the time

the of

glycol

and

ethylene

during

propylene not

embryos

our

warming

and

has

been

The method suggested

(2,

developmental Zwalmen et blastocysts procedure

cause The cells

dilution

equilibration blastocysts

al.

stage (17) after Massip

with

of embryos obta. ined in vitrification et a1.(18).

earlier

applied

present

and blastocyst ethylene glycol

of study

every

embryos

has

If

successfully sulfoxide

FEBRUARY 1992 VOL. 37 NO. 2

80 minutes vitrification present

low

et of study

equilibration

was lower concentration permeable

than (13).

cryoprotectant

been

reported

morulae

conventional and in

when

into

one

that

to

the

et

freezing. survival

the method,

al. with of

of

simpler

and

mouse

embryos

(15) each

Van Der bovine

method

stage

lower

equilibration however,

vitrification

vitrified under the

be

vitrification

Urano differ

viva

modifying modified

become

demonstrates

stages are and dimethyl

to

of

(16) were used. cryoprotectants

in vitro

short

vitrification

our

a

too

our

Zwalmen

in

highly the

developmental will

20

was

Der

in

time.

by This

stages. to

of

and of

“C

composition

although

lo-minute

4

between

to (2,

rate

Van

exposure

the

20%).

fast freezing effectiveness

at of

shown

vitrification

survival

concentration particular,

In

embryos

low

rate, and intracellular

in

a

or the

cryopreservation The

vs

by

mixture

difference 10)

permeation

of

a

30-second

adequate

6-8) that

successfully

a

(12.5%

in

survival optimal

(2,

eight-cell

of

succeed

also

embryos

findings

to

survival with

a

glycol

of this

the

cryoprotectant solutions.

glycol

within

compared

in as

to a low to reach

reports

lead

propylene

resulted

study,

was

previous

solution, i.e., their equilibration

not

highly 14).

pressure

cryopreservation

at 4°C led was needed

conditions.

of

control

low

permeate the embryo to time exposure (30 seconds). efflux from the embryonic

osmotic

but this mixture 5, 6, 10). The 30-second exposure study. for good embryo survival,

may

development

M sucrose+PBS. The

and

similar

fresh

was

Therefore,

may easily a short may easily

of

rate

also

it is possible technique.

is

dimethyl

found

survival

viva

fetuses

by

rates

was

7, 12), transfer

osmotic

freezing.

vitro in

(13,

reported

the

those

cell

and of

conventional

sulfoxide

causing

in

live

the

glycol

in

The

sulfoxide

change

than

from

morulae

rates

solution

10).

4. the

vitrified

to

The same

of

that

dimethyl

the

lower

a1.(6). this

studies (2, by refining

and

that

embryo

was

(6, 7, different

reported

glycol

without

et in

of

similar

it

proportion

with that of previous improve this proportion It

but

previous reports not significantly the

rate was

11). Valdez

vitrified

Although,

ethylene

survival

solution

(2,

blastocysts

to that of rates were

The

confirmed.

above

embryos,

more

in same

did can

at

be the

practical. the

morula

a mixture equilibration

of

485

THERIOGENOLOGY

The in viva development and dilution conditions. vitrified using this solution was also confirmed.

of

the

embryos

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