Breeding tests of chimaeric male mice which were produced with the embryonic stem cells (F1/1) carrying the neomycin phosphotransferase (neo) gene indicated chimaerism in their germ lines. Fifty to 100% of fertile chimaeric mice derived from four F1/1 cell lines carrying the neo gene were germ line chimaeras and germ lines in most of them were taken possession of derivations of F1/1 cells completely. Southern blot hybridization or polymerase chain reaction analysis of genomic DNAs confirmed that F1 and F2 generation of germ line chimaeric mice were transmitted with the neo gene. Genetic analysis of offspring obtained by mating F2 transgenic mice with ICR mice identified homozygous mice. These results suggest that F1/1 cells can be utilized for the production of transgenic mouse.

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Superovulation was induced in mature nulliparous Japanese voles (Microtus montebelli) with PMSG as a follicle stimulating factor and hCG as an ovulation inducing agent. PMSG was injected at priming dose levels of 25, 50, 75 and 100 IU respectively, and the dose of hCG used as the ovulator was injected additionally at a high or low level for each dose of PMSG. The results obtained are as follows:
1. When voles were treated with an additional injection of 5 or 10 IU of hCG after pretreatment with 25 IU of PMSG, the average number of ovulated ova was 5.4 and 3.4 respectively, almost the same as the 2.3 of the intact group.
2. The average number of ovulated ova was 6.8, when 50 IU of PMSG and 10 IU of hCG were administered, but when 50 IU of PMSG and 20 IU of hCG were injected the average number of ovulated ova increased to 12.5. There was no significant difference in the average number of ova between the groups given 50 IU of PMSG and the other groups.
3. When 75 IU of PMSG and 15 IU of hCG were administered, the count was 12.4. The largest average number of ovulated ova in this experiment was 14.0, when 75 IU of PMSG and 30 IU of hCG were injected. The average number of ovulated ova was significantly larger in the group with the additional injection of 30 IU of hCG pretreated with 75 IU of PMSG than in the group receiving the injection of 25 IU of PMSG or in the intact group (P<0.05).
4. When the dose of PMSG was increased to 100 IU, an average of 11.1 ova were obtained after additional treatment with 20 IU of hCG and 11.0 ova after additional treatment with 40 IU of hCG, indicating a decrease in the response to more than 75 IU PMSG.
5. Further experiments are necessary to increase the number of ovulations and to decrease the variation in the response to gonadotropin treatment.

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Summary. Twenty six of in vivo embryos were collected from 7 Holstein cows and 1 Japanese black cows by single or superovulated flushing. Thirty one of in vitro embryos were produced using in vitro fertilization techniques using Japanese black oocytes aspilated from ovaries obtained local slaughter house. These embryos were suspended in 1.8 M ethylene glycol (EG) supplemented with 10% superovulated cows serum (scs) at 25 C and held 10 min, then embryos were loaded into 0.25 ml plastic straws, placed into an alcohol bath freezer at 0°C, cooled from 0°C to -7°C at 1°C/min, seeded, held for 10 min, cooled at 0.3 C/min or 0.5°C/min to -30°C. Straws were then plunged and stored in liquid nitrogen. After a 20 day storage period in liquid nitrogen, the cryopreserved straws were placed in air for 6 sec and plunged into 30°C water bath for thawing and transfered immediately into syncronized recipients. The pregnancy rates of 0.3°C/min [90% (9/10)] were significantly higher (P<0.01) than those of 0.5 C/min [12.5% (2/16)] for cooling rates in vivo embryos. And the pregnancy rates of in vitro frozen embryos were 63% (12/19).

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Summary. Blood samples were taken from 22 stallions at hourly intervals for a 24-h period in all seasons, and plasma testosterone (T) and dihydrotestosterone (DHT) were measured by RIA. The stallions aged 11-17 (11-17 year old group) showed a biphasic diurnal variation in T in all seasons. The first peak appeared in the daytime and the second peak, at night. The time of the first peak was correlated with sunrise (r=0.69; P<0.05). DHT remained low and no peaks could be detected. For stallions aged 20-21 (20-21 year old group) no distinct biphasic shifts in T could be detected. The 24-h mean concentrations of T and DHT in the 11-17 year old group were highest in the summer and lowest in the fall (T, P<0.01; DHT, P<0.05), but that of T in the 20-21 year old group was highest in the spring and lowest in the fall (P<0.05). When T 24-h mean concentrations of the groups in the same season were compared, significant differences were noted for the summer (P<0.05), but not in any other season. It is concluded that in stallions, diurnal variation and 24-h mean concentration of T are affected by age and season.

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Clinical signs, changes in blood characteristics, plasma estrogen and progesterone levels were investigated during the period of the 7 days before expulsion to the 3 days after expulsion in 5 cows having twins. Values obtained were compared with those of 8 cows with single birth. An average of gestation length of the cows with twins was 8 days shorter than those of the cows with single birth. At expulsion, proper handling at the dystocia was needed in 2/5 twinnings. Retained placenta occurred in all twin calvings, although in 3 cases either of the 2 placentas was discharged within 12 h after the expulsion. In many twinnings, the rectal temperature did not decrease below 38.5C before the expulsion, and increased above 39.6C after expulsion. Ligamentum sacrotuberale latum relaxated at expulsion did not fully resume to the former condition after the expulsion. Maximum values of glucose concentration before and after the expulsion in twinnings were lower than those at the same periods in the single birth. The ratios of segmentcytes to lymphocytes after the expulsion and numbers of eosinophils before and after expulsion were lower in the cows with twins. In some twinnings, a decrease in estrogen levels was slower and a slight increase in progesterone levels was observed after expulsion.

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An attempt was made to develop a new type of uterine cervix dilator for safety and efficient embryo collection in cows.
The characteristics of the designed dilator is that it was a plastic catheter with a tip having a silicon rubber balloon, of which pressure was controlled by putting air from a plastic syringe connected to the catheter.
The ballooned dilator was applied to the cervical dilation of the cows, in combination with a rectal manipulation assisting its entry into the canal. The insertion of the dilater with repeated expansion of the balloon permitted the dilation of the tight cervical fold-neck as follows.
(1) In all of 5 cows mainly consisted of multiparous ones, the cervical dilation by the ballooned was completed within 24 minutes, and no sign of bleeding due to the injury on cervical was noted.
(2) In 8 multiparous cows, the cervical canal dilation by this apparatus was successfully achieved within 1.5 minutes, without exception. The subsequent embryo collection from these animals was also successful.
(3) The successful dilation in the cervical canal was obtained by this dilator in 5 out of 8 cows of which cervical canals had extremely twisted or partially closed due to multitime treatments in previous embryo collections. The subsequent embryo collection succeeded in these 5 animals. These results indicate that this simple dilator is useful as a cervical dilator for embryo collection as well as for embryo tranfer in cows.

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