c57bl/6 mice were purchased from clea japan ( tokyo , japan ) and charles river laboratories 
 japan ( kanagawa , japan ) for use as oocyte and sperm donors .
icr mice ( clea japan ) were 
 used as recipients for the transfer of 2-cell embryos .
all animals were kept under a 12 h 
 dark - light cycle ( lights on : 07001900 h ) at a constant temperature of 22  1 c with free 
 access to food and water .
all animal experiments were approved by the animal care and use 
 committee at kumamoto university .
the sperm were collected from the vasa deferentia and caudae epididymides of 
 three - month - old male mice and were frozen via the cryotube freezing 
 method   and the mtg - straw freezing method 
 .
frozen samples were rapidly thawed by 
 transferring them from liquid nitrogen into a 37 c water bath .
half of the sperm were used for either the cryotube 
 ivf method or the mtg - straw ivf method [ 12 , 13 ] , and half of the remaining samples were used for 
 the mbcd - gsh ivf method .
the procedures for the cryotube ivf method and the mtg - straw ivf method were as described 
 by sztein et al .   and by 
 ostermeier et al . 
 respectively .
a : transfer cumulus oocytes 
 complexes ( cocs ) into a drop of card medium .
b : remove a frozen sperm sample and 
 immerse it in a water bath maintained at 37 c. c : transfer the sperm suspension into 
 a tube , and add mhtf to the tube containing the sperm suspension .
d : remove the supernatant and add fertiup mouse 
 sperm preincubation medium into the tube .
e : after pipetting gently , transfer the 
 entire sperm suspension into the drop of fertiup preincubation medium , and incubate 
 the sperm in a co2 incubator .
f : introduce the preincubated cocs into the 
 drop of sperm suspension , and incubate them in the co2 incubator .
g : 
 after 3 h , wash the oocytes three times in drops of mhtf , and then culture them in 
 the co2 incubator .
h : twenty - four hours after insemination , transfer just 
 the obtained 2-cell stage embryos to a fourth drop of mhtf .
( see 
 http://www.kyudo.co.jp/fertiup/methods.htm ) .. the oocytes used were obtained from females at 23 months of age .
females were 
 induced to superovulate via injections of 7.5 iu pmsg ( aska 
 pharmaceutical , tokyo , japan ) and 7.5 iu human chorionic gonadotropin ( aska 
 pharmaceutical ) administered 48 h apart .
intact cumulus oocyte complexes ( cocs ) were 
 released from the excised oviducts of superovulated females into a drop ( 200 l ) of card 
 medium ( kyudo , saga , japan ) covered with paraffin oil in a plastic dish ( cat.#no.#430588 , 
 corning , corning , ny , usa ) 1517 h after hcg and were preincubated for 60 min ( fig .
sperm samples frozen via 
 the cryotube freezing method and the mtg - straw freezing method were removed from the 
 liquid nitrogen and immersed in a 50 ml plastic centrifuge tube ( in a water bath 
 maintained at 37 c ) for 10 min ( fig .
after 
 thawing , the sperm suspension in a cryotube or a straw was transferred into a 1.5 ml tube 
 ( cat .
509-grd , bm equipment , tokyo , japan ) , and 1.2 ml of mhtf   was added slowly to the tube containing the sperm suspension . 

thereafter , the tube was immediately centrifuged at 300 g for 5 min ( fig .
after centrifugation , as much supernatant was removed as 
 possible , and an appropriate amount of fertiup mouse sperm preincubation medium ( kyudo ) 
 was added to the tube ( the final volume was adjusted to 100 l ) ( fig .
after pipetting gently , the entire sperm suspension ( 100 
 l ) was transferred into a drop of fertiup mouse sperm preincubation medium ( 100 l ) 
 covered with paraffin oil in a dish .
the dish was placed in an incubator ( 37 c , 5% 
 co2 in air ) for 30 min ( fig .
then , the preincubated cocs were sucked up from the drop of card medium using a tip ( cat . 
 no.#17021 - 1 , nippon genetics , tokyo , japan ) and introduced into the drop of sperm 
 suspension ( insemination ) ( fig
after 3 h , the inseminated oocytes were washed three times 
 in drops ( 80 l ) of mhtf covered with paraffin oil in a dish ( fig .
1-g ( a  c ) ) , and cultured at 37 c with 5% co2 .
seven 
 to eight hours after insemination , the formation of pronuclei was observed under a phase 
 contrast microscopy .
twenty - four hours after insemination , just the obtained 2-cell stage 
 embryos were transferred to a 4th drop of mhtf ( fig . 

fertilization rates were calculated as the total number of 2-cell 
 embryos divided by the total number of inseminated oocytes , multiplied by 100 .
in addition , gem ( c57bl/6 background ) sperm frozen via the 
 mtg - straw freezing method , which were sent to our center from the jackson laboratory 
 ( usa ) , were thawed and used for ivf in the same manner .
a portion of the 2-cell embryos 
 were transferred into the oviducts of icr females ( 10 embryos / oviduct ) on the day a 
 vaginal plug was found ( day 1 of pseudopregnancy ) using the method of embryo transfer 
 through the wall of the oviduct .
a : transfer cumulus oocytes 
 complexes ( cocs ) into a drop of card medium .
b : remove a frozen sperm sample and 
 immerse it in a water bath maintained at 37 c. c : transfer the sperm suspension into 
 a tube , and add mhtf to the tube containing the sperm suspension . thereafter , 
 immediately centrifuge the tube .
d : remove the supernatant and add fertiup mouse 
 sperm preincubation medium into the tube .
e : after pipetting gently , transfer the 
 entire sperm suspension into the drop of fertiup preincubation medium , and incubate 
 the sperm in a co2 incubator .
f : introduce the preincubated cocs into the 
 drop of sperm suspension , and incubate them in the co2 incubator .
g : 
 after 3 h , wash the oocytes three times in drops of mhtf , and then culture them in 
 the co2 incubator .
h : twenty - four hours after insemination , transfer just 
 the obtained 2-cell stage embryos to a fourth drop of mhtf .
statistical analysis was performed using the graphpad prism version 5.0 software 
 ( graphpad , san diego , ca , usa ) .
group results were compared using the student s 
 t - test after arcsine transformation of the percentage data ; p<0.05 
 was considered significant .
c57bl/6 mice were purchased from clea japan ( tokyo , japan ) and charles river laboratories 
 japan ( kanagawa , japan ) for use as oocyte and sperm donors .
icr mice ( clea japan ) were 
 used as recipients for the transfer of 2-cell embryos .
all animals were kept under a 12 h 
 dark - light cycle ( lights on : 07001900 h ) at a constant temperature of 22  1 c with free 
 access to food and water .
all animal experiments were approved by the animal care and use 
 committee at kumamoto university .
the sperm were collected from the vasa deferentia and caudae epididymides of 
 three - month - old male mice and were frozen via the cryotube freezing 
 method   and the mtg - straw freezing method 
 .
frozen samples were rapidly thawed by 
 transferring them from liquid nitrogen into a 37 c water bath .
half of the sperm were used for either the cryotube 
 ivf method or the mtg - straw ivf method [ 12 , 13 ] , and half of the remaining samples were used for 
 the mbcd - gsh ivf method .
the procedures for the cryotube ivf method and the mtg - straw ivf method were as described 
 by sztein et al .   and by 
 ostermeier et al . 
 respectively .
a : transfer cumulus oocytes 
 complexes ( cocs ) into a drop of card medium .
b : remove a frozen sperm sample and 
 immerse it in a water bath maintained at 37 c. c : transfer the sperm suspension into 
 a tube , and add mhtf to the tube containing the sperm suspension . thereafter , 
 immediately centrifuge the tube .
d : remove the supernatant and add fertiup mouse 
 sperm preincubation medium into the tube .
e : after pipetting gently , transfer the 
 entire sperm suspension into the drop of fertiup preincubation medium , and incubate 
 the sperm in a co2 incubator .
f : introduce the preincubated cocs into the 
 drop of sperm suspension , and incubate them in the co2 incubator .
g : 
 after 3 h , wash the oocytes three times in drops of mhtf , and then culture them in 
 the co2 incubator .
h : twenty - four hours after insemination , transfer just 
 the obtained 2-cell stage embryos to a fourth drop of mhtf .
( see 
 http://www.kyudo.co.jp/fertiup/methods.htm ) .. the oocytes used were obtained from females at 23 months of age .
females were 
 induced to superovulate via injections of 7.5 iu pmsg ( aska 
 pharmaceutical , tokyo , japan ) and 7.5 iu human chorionic gonadotropin ( aska 
 pharmaceutical ) administered 48 h apart .
intact cumulus oocyte complexes ( cocs ) were 
 released from the excised oviducts of superovulated females into a drop ( 200 l ) of card 
 medium ( kyudo , saga , japan ) covered with paraffin oil in a plastic dish ( cat.#no.#430588 , 
 corning , corning , ny , usa ) 1517 h after hcg and were preincubated for 60 min ( fig .
sperm samples frozen via 
 the cryotube freezing method and the mtg - straw freezing method were removed from the 
 liquid nitrogen and immersed in a 50 ml plastic centrifuge tube ( in a water bath 
 maintained at 37 c ) for 10 min ( fig .
after 
 thawing , the sperm suspension in a cryotube or a straw was transferred into a 1.5 ml tube 
 ( cat .
509-grd , bm equipment , tokyo , japan ) , and 1.2 ml of mhtf   was added slowly to the tube containing the sperm suspension . 

thereafter , the tube was immediately centrifuged at 300 g for 5 min ( fig .
after centrifugation , as much supernatant was removed as 
 possible , and an appropriate amount of fertiup mouse sperm preincubation medium ( kyudo ) 
 was added to the tube ( the final volume was adjusted to 100 l ) ( fig .
after pipetting gently , the entire sperm suspension ( 100 
 l ) was transferred into a drop of fertiup mouse sperm preincubation medium ( 100 l ) 
 covered with paraffin oil in a dish .
the dish was placed in an incubator ( 37 c , 5% 
 co2 in air ) for 30 min ( fig .
then , the preincubated cocs were sucked up from the drop of card medium using a tip ( cat . 
 no.#17021 - 1 , nippon genetics , tokyo , japan ) and introduced into the drop of sperm 
 suspension ( insemination ) ( fig .
after 3 h , the inseminated oocytes were washed three times 
 in drops ( 80 l ) of mhtf covered with paraffin oil in a dish ( fig .
1-g ( a  c ) ) , and cultured at 37 c with 5% co2 .
seven 
 to eight hours after insemination , the formation of pronuclei was observed under a phase 
 contrast microscopy .
twenty - four hours after insemination , just the obtained 2-cell stage 
 embryos were transferred to a 4th drop of mhtf ( fig . 

fertilization rates were calculated as the total number of 2-cell 
 embryos divided by the total number of inseminated oocytes , multiplied by 100 .
in addition , gem ( c57bl/6 background ) sperm frozen via the 
 mtg - straw freezing method , which were sent to our center from the jackson laboratory 
 ( usa ) , were thawed and used for ivf in the same manner .
a portion of the 2-cell embryos 
 were transferred into the oviducts of icr females ( 10 embryos / oviduct ) on the day a 
 vaginal plug was found ( day 1 of pseudopregnancy ) using the method of embryo transfer 
 through the wall of the oviduct .
a : transfer cumulus oocytes 
 complexes ( cocs ) into a drop of card medium .
b : remove a frozen sperm sample and 
 immerse it in a water bath maintained at 37 c. c : transfer the sperm suspension into 
 a tube , and add mhtf to the tube containing the sperm suspension .
d : remove the supernatant and add fertiup mouse 
 sperm preincubation medium into the tube .
e : after pipetting gently , transfer the 
 entire sperm suspension into the drop of fertiup preincubation medium , and incubate 
 the sperm in a co2 incubator .
f : introduce the preincubated cocs into the 
 drop of sperm suspension , and incubate them in the co2 incubator .
g : 
 after 3 h , wash the oocytes three times in drops of mhtf , and then culture them in 
 the co2 incubator .
h : twenty - four hours after insemination , transfer just 
 the obtained 2-cell stage embryos to a fourth drop of mhtf .
statistical analysis was performed using the graphpad prism version 5.0 software 
 ( graphpad , san diego , ca , usa ) .
group results were compared using the student s 
 t - test after arcsine transformation of the percentage data ; p<0.05 
 was considered significant .
   sperm cryopreservation has been widely adopted for maintenance of the genetically 
 engineered mouse ( gem ) . 
 the cryopreserved sperm are being exchanged among many institutes 
 worldwide 
 . however , the recipients are not always able to obtain high fertilization rates 
 with the frozen sperm shipped from senders . in this study , we cryopreserved mouse sperm 
 via various methods and performed in vitro 
 fertilization ( ivf ) in which the combination of methyl - beta - cyclodextrin for sperm 
 preincubation and reduced glutathione for insemination was used ( the mbcd - gsh ivf ) . in 
 addition 
 , frozen sperm sent from the jackson laboratory ( usa ) were thawed and used for ivf 
 in the same manner . 
 the fertilization rates of both the sperm cryopreserved 
 via the methods applied in some countries and the cryopreserved gem 
 sperm improved when used with the mbcd - gsh ivf method . 
 therefore , we strongly believe that 
 the mbcd - gsh ivf method brings about relatively high fertilization rates with any strain 
 of frozen mouse sperm . 
Methods
Animals
Sperm freezing and thawing
IVF
Statistical analysis