superovulation protocol for

Improving C

umulus-O

ocyte-Com

plex Yield in a Superovulation Protocol for Perom

yscusm

iceR

achael Labitt 1,2

and YukselA

gca1

1De

pa

rtme

nt o

f Ve

terin

ary P

ath

ob

iolo

gy, C

olle

ge

of V

ete

rina

ry Me

dicin

e, U

nive

rsity of M

issou

ri, Co

lum

bia

, MO

2Co

lleg

e o

f Ve

terin

ary M

ed

icine

, Co

rne

ll Un

iversity, Ith

aca

, NY

•T

he Perom

yscusgenus of m

ice, or deer m

ice, are used in research on evolution, gene im

printing, monogam

y, and environm

ental toxicology.•

Preserving and resurrecting strain variants or

generating new transgenic lines is im

possible w

ith current protocols, and superovulation protocols for M

usm

ice are ineffective.•

Previous superovulation studies in

Perom

yscushave yielded sim

ilar numbers of

cumulus-oocyte-com

plexes (CO

Cs) to that

which a fem

ale would naturally ovulate, or

require cumbersom

e in vitro maturation

techniques. 1

•T

he purpose of the present study was to

optimize an efficient, scalable superovulation

protocol for deer mice.

•S

ince the timing of P

eromyscus

follicular w

aves is unknown, w

e hypothesized that a sustained release of F

SH

via osmotic pum

p w

ould increase CO

C yield com

pared to previously published injection m

ethods.

t=56 hrsIn

du

ceo

vula

tion

Co

un

t o

varia

n

follicle

s

Su

bcu

tan

eo

us im

pla

nt:

5 mg FSH

viao

smo

tic pu

mp

5 IU PM

SG

intra

pe

riton

ea

lly

Perom

yscusm

aniculatusF

em

ale

s ag

es 11

-25

we

eks

Implant group

Control group

t=0 hrsIn

du

cefo

llicula

rd

eve

lop

me

nt

Animals

t=71 hrsO

vidu

cth

arve

st

Co

un

t CO

Cs

5 IU H

CG

5 IU LH

10 IU LH

AB

CD

Figure 1. Pum

p effect on ovarian folliculogenesis.O

varies were harvested at 56 hours from

Perom

yscusaged 11-15 w

eeks that had rece

ived

5 IU

PM

SG

I.P. (n=1

0) o

r an o

smotic pum

p conta

ining 5

mg F

SH

(n=9). (A)T

he number of follicles per ovary are sim

ilar between

groups (p=0.92). (B

) Two ovaries from

Perom

yscusthat received F

SH

via osmotic pum

p showing m

ature follicles, which w

ere larger and m

ore uniform than the control P

MS

G group (data not show

n). (C) and

(D)

Oocyte and zona

pellucida(arrow

) are visible within follicles of

control PM

SG

group (C) o

r FS

H pum

p trea

ted

(D) m

ice.

AB

Figure 2. Morphology of C

OC

s. CO

Cs w

ithin the oviduct (A)or after

dissection into media (B

). CO

Cs form

an elongated structure in the oviduct that is rem

oved by an action similar to “squeezing a tube of

toothpaste”, unlike the clustered clutch produced by Mus

mice. T

his suggests that superovulation is not a sim

ultaneous event in P

eromyscus.

Figure 3.O

ptimizatio

n of CO

C yield. F

SH

implant follow

ed by 10 IU LH

yields more C

OC

s. (A)C

OC

yield by treatment group (n=

15). (B)

CO

C yield stratified by age group (n=

5). Perom

yscus17-20 w

eeks old treated with F

SH

osmotic pum

p and 10 IU LH

yield the most oocytes,

significantly higher than the conditions previously published, 12-14 week old m

ice treated with 5 IU

PM

SG

and 5 IU H

CG

.(C) S

uperovulation success rate, stratified by age group (n=

5). Success w

as defined as the percent of mice that produced ≥6 C

OC

s and therefore more than

one standard deviation above our colony’s average litter size of 4.2+/-1.5 pups.

AB

BC

•M

any unovulatedfollicles

remained and ovulation did not

appear synchronous; higher yields m

ay be achieved by w

aiting longer before CO

C

collection.•

We did not test the 13-15 w

eek tim

e point, which m

ay be the m

ost effective age. •

This efficient, practical m

ethod of superovulation is the first step tow

ard eventual embryo

transfer protocols or transgenic m

anipulation of Perom

yscus.

Methods

Results

Introduction

Conclusions

1.

Ch

oi JK

, He

X (2

01

3) In Vitro

Ma

tura

tion

of C

um

ulu

s-Oo

cyte

Co

mp

lexe

s for E

fficien

t Isola

tion

of O

ocyte

s from

Ou

tbre

d D

ee

r M

ice. P

Lo

SO

NE

8(2

): e5

61

58

. do

i:10

.13

71

/jou

rna

l.po

ne

.00

56

15

8

References

Ka

itlin E

pp

erso

n fo

r hu

sba

nd

ry sup

po

rt. Re

sea

rch G

ran

t: NIH

-Mu

tan

t Mo

use

R

eso

urce

Ce

nte

r. Stu

de

nt S

up

po

rt: Gla

xoS

mith

Klin

e, A

me

rican

So

ciety fo

r L

ab

ora

tory A

nim

al P

ractitio

ne

rs Fo

un

da

tion

, IDE

XX

-Bio

Re

sea

rch

Acknowledgem

ents

New

protocolPreviously publishedconditions

Animal age

17-20 weeks

12-14 weeks

First hormone

5 mg F

SH

via subcutaneous osm

otic pump im

plant5

IU P

MS

G

Second hormone

10 IU LH

i.p., t=56 hours

5 IU H

CG

, t=56 hours

CO

C collection

t=71

hours (needs optim

ization)t=

71 hours

Average CO

C yield

9 (range 5-15)4.2 (range 0-5)

Superovulation success rate

80%0%

Statistical analysis: Data are plotted as m

ean +

/-S

D. H

istograms w

ere plotted (not shown) to

determine that data w

as non-parametric.

Dichotom

ous data was analyzed by M

ann-W

hitney test (Fig. 1A

, Fi. 3B

) with B

onferronicorrection for m

ultiple tests (Fig. 3B

). P

olychotomous

data was analyzed by K

ruskal-W

allis test (Fig. 3A

). * p<0.05.

New

efficient conditions for superovulation of Peromyscus

mice