a total of 14 cynomolgus macaques housed in individual cages were used for the experiments , including three males ( from china , 9 years of age , weighing 67 kg ) 
 and 11 females ( from china , 68 years of age , weighing 34 kg ) . the present study was reviewed and approved by the institutional animal care and use committee 
 of shin nippon biomedical laboratories , ltd . semen was collected from the three males by electric penile stimulation with direct current pulses as described previously . all male monkeys had a history of their semen producing pregnancy by natural mating , and the motility of the sperm in their semen was confirmed . briefly , ejaculates were collected without anesthesia in the morning , with at least a week intervening between collections . one pre - sized defibrillator gel electrode was wrapped around the base of the penis and connected to the negative lead . a 
 second gel electrode was positioned immediately behind the glans and connected to the positive lead . a slow and steady stimulation by increasing the 
 output - adjust dial induced a slight erection , engorgement of the glans , and/or elevation of the testicles into the inguinal region . typically , engorgement and 
 ejaculation occurred at 1020 v. the ejaculates were collected in clean tubes ( 15 ml high clarity polypropylene centrifuge tube , bd falcon , bedford , ma ) . the 
 seminal coagulum of three cynomolgus macaques were separately liquefied , and sperm motility in the liquid portion was evaluated under a light microscope . to 
 liquefy the sperm , an equal volume of bww medium prepared from talp - hepes medium supplemented with 4 mg / ml bovine serum albumin [ 14 , 15 ] was added to the semen , and it was kept at 37c ( 5% o2 , 5% co2 ) for 30 min in an 
 incubator ( juji field , tokyo , japan ) . the liquid portion of the semen was transferred into a 15 ml conical tube and washed with bww medium , followed by 
 centrifugation ( 112  g ) at room temperature for 7 min . the sperm were counted using a makler tm counting chamber ( sefi - medical instruments , 
 haifa , israel ) , and diluted to 50  10 cells / ml with bww medium . progressive motility of the sperm samples ( sperm moving in a forward direction ) , 
 assessed subjectively , was > 90% . each sperm suspension made from an ejaculate was used for three or four females in experiment 2 . the newly developed device is composed of a 1-ml syringe ( nipro , osaka , japan ) and a conventional outer cylinder that is fitted to the size of the vagina 
 ( fig . 1 ) so that liquefied sperm can not flow out of the device and the vagina . , females were anesthetized with 7 mg / kg of ketamine hydrochloride ( kamud drugs pvt , india ) and 1.25 mg / kg of xylazine ( nippon 
 zenyaku kogyo , tokyo , japan ) . after aseptic preparation of the perivaginal area , the outer cylinder was introduced into the vagina and placed around the 
 cervical os . the 1-ml syringe was then placed into the cylinder . in all experiments , the sperm suspension ( 50 10 cells / ml fresh sperm in 1 ml of 
 bww medium ) was placed into the syringe , and slowly injected to the female . this position was maintained for approximately 30 min until the female recovered 
 from anesthesia . in experiment 1 , to assess whether sperm can reach the oviduct , a female showing a normal menstrual cycle , and with at least two births by 
 means of vaginal delivery , was inseminated with 1 ml of sperm suspension diluted to 50  10 cells / ml with bww medium . at 24 and 48 h after ai , the 
 female was anesthetized with isofluran and the oviduct was surgically removed . the lumen of the oviduct was flushed with bww medium by using a 23-gauge needle 
 ( nipro ) attached to a 1-ml syringe and the washed medium was subjected to microscopic examination for sperm . in experiment 2 , to determine whether this ai 
 method can lead to pregnancy , ai was performed once on each of 10 females that were at pre - ovulation as determined by observing the dominant follicle using 
 laparoscopy . females treated with 50 g / head / day levonorgestrel ( d(-)-norgestrel , sigma - aldrich , st . louis , mo , usa ) for 14 days to synchronize their menstrual 
 cycles , as described previously , were subjected to ai using the newly developed device . levonorgestrel inhibits 
 follicular maturation and ovulation in cynomolgus macaques at different phases of the menstrual cycle , and the menstrual cycle restarts upon withdrawing 
 levonorgestrel administration . ai was performed as described earlier , and pregnancies were assessed at day 20 after insemination by real - time ultrasonography 
 ( sonosite titan , hitachi aloka medical , tokyo , japan ) under anesthesia using ketamine hydrochloride as described earlier . a total of 14 cynomolgus macaques housed in individual cages were used for the experiments , including three males ( from china , 9 years of age , weighing 67 kg ) 
 and 11 females ( from china , 68 years of age , weighing 34 kg ) . the present study was reviewed and approved by the institutional animal care and use committee 
 of shin nippon biomedical laboratories , ltd . semen was collected from the three males by electric penile stimulation with direct current pulses as described previously . all male monkeys had a history of their semen producing pregnancy by natural mating , and the motility of the sperm in their semen was confirmed . briefly , ejaculates were collected without anesthesia in the morning , with at least a week intervening between collections . one pre - sized defibrillator gel electrode was wrapped around the base of the penis and connected to the negative lead . a 
 second gel electrode was positioned immediately behind the glans and connected to the positive lead . a slow and steady stimulation by increasing the 
 output - adjust dial induced a slight erection , engorgement of the glans , and/or elevation of the testicles into the inguinal region . typically , engorgement and 
 ejaculation occurred at 1020 v. the ejaculates were collected in clean tubes ( 15 ml high clarity polypropylene centrifuge tube , bd falcon , bedford , ma ) . the 
 seminal coagulum of three cynomolgus macaques were separately liquefied , and sperm motility in the liquid portion was evaluated under a light microscope . to 
 liquefy the sperm , an equal volume of bww medium prepared from talp - hepes medium supplemented with 4 mg / ml bovine serum albumin [ 14 , 15 ] was added to the semen , and it was kept at 37c ( 5% o2 , 5% co2 ) for 30 min in an 
 incubator ( juji field , tokyo , japan ) . the liquid portion of the semen was transferred into a 15 ml conical tube and washed with bww medium , followed by 
 centrifugation ( 112  g ) at room temperature for 7 min . the sperm were counted using a makler tm counting chamber ( sefi - medical instruments , 
 haifa , israel ) , and diluted to 50  10 cells / ml with bww medium . progressive motility of the sperm samples ( sperm moving in a forward direction ) , 
 assessed subjectively , was > 90% . each sperm suspension made from an ejaculate was used for three or four females in experiment 2 . the newly developed device is composed of a 1-ml syringe ( nipro , osaka , japan ) and a conventional outer cylinder that is fitted to the size of the vagina 
 ( fig . 1 ) so that liquefied sperm can not flow out of the device and the vagina . , females were anesthetized with 7 mg / kg of ketamine hydrochloride ( kamud drugs pvt , india ) and 1.25 mg / kg of xylazine ( nippon 
 zenyaku kogyo , tokyo , japan ) . after aseptic preparation of the perivaginal area , the outer cylinder was introduced into the vagina and placed around the 
 cervical os . the 1-ml syringe was then placed into the cylinder . in all experiments , the sperm suspension ( 50 10 cells / ml fresh sperm in 1 ml of 
 bww medium ) was placed into the syringe , and slowly injected to the female . this position was maintained for approximately 30 min until the female recovered 
 from anesthesia . in experiment 1 , to assess whether sperm can reach the oviduct , a female showing a normal menstrual cycle , and with at least two births by 
 means of vaginal delivery , was inseminated with 1 ml of sperm suspension diluted to 50  10 cells / ml with bww medium . at 24 and 48 h after ai , the 
 female was anesthetized with isofluran and the oviduct was surgically removed . the lumen of the oviduct was flushed with bww medium by using a 23-gauge needle 
 ( nipro ) attached to a 1-ml syringe and the washed medium was subjected to microscopic examination for sperm . in experiment 2 , to determine whether this ai 
 method can lead to pregnancy , ai was performed once on each of 10 females that were at pre - ovulation as determined by observing the dominant follicle using 
 laparoscopy . females treated with 50 g / head / day levonorgestrel ( d(-)-norgestrel , sigma - aldrich , st . louis , mo , usa ) for 14 days to synchronize their menstrual 
 cycles , as described previously , were subjected to ai using the newly developed device . levonorgestrel inhibits 
 follicular maturation and ovulation in cynomolgus macaques at different phases of the menstrual cycle , and the menstrual cycle restarts upon withdrawing 
 levonorgestrel administration . ai was performed as described earlier , and pregnancies were assessed at day 20 after insemination by real - time ultrasonography 
 ( sonosite titan , hitachi aloka medical , tokyo , japan ) under anesthesia using ketamine hydrochloride as described earlier .
in cynomolgus macaques , an important animal species for biomedical research , efficient reproduction has been hampered partly due to the difficulties of 
 artificial insemination ( ai ) using straw tubes developed for humans or farm animals , because cynomolgus macaques have a complex cervical canal structure . in 
 this study , taking into consideration the unique structure of the macaque cervical canal , we developed a novel device for ai , comprised of a syringe and an 
 outer cylinder . at 24 and 48 
 h after using this device to inject semen into one female , viable sperm were observed in the oviduct where the sperm meets the 
 oocytes . 
 we then attempted ai using this new device on 10 females that were at pre - ovulation , and pregnancy was successful in three animals ( 30% pregnancy 
 rate ) . 
 these results show that the newly developed device can be used for ai in cynomolgus macaques .