Hitoshi Ushijima

Japanese field vole (Microtus montebelli) is a wild-derived rodent and have unique characteristic. Thus, these species have been expected as model animal. This study was performed to develop novel superovulation procedure for Japanese field vole. First, when 30 IU pregnant mare's serum gonadotropin (PMSG) and 30 IU human chorionic gonadotropin (hCG) were administrated 48 hours apart, females showed higher response to hCG compared with three concentrations of PMSG. Second, to effectively induce ovulation on females after vaginal opening, they were mated with vasectomized male instead of hCG administration. Average number of ovulated oocytes using PMSG mating (13.9 +/- 1.9 oocytes) was higher than PMSG-hCG (control; 6.9 +/- 23 oocytes) or PMSG-hCG mating (6.8 +/- 0.8 oocytes). Finally, we attempted superovulation using GnRH agonist (GnRHa). With this treatment, we speculated that GnRHa might induce endogenous luteinizing hormone releasing to cause ovulation. Such superovulation was performed with 30 IU PMSG and different concentration of 20% polyvinylpyrrolidone-GnRHa (15, 30, 45, and 60 mu g/kg). As results, average number of ovulated oocytes was highest with 30 mu g/kg GnRHa (14.5 +/- 4.1 oocytes). The numbers of ovulated oocytes of other concentrations were 5.0 +/- 1.4 (15 mu g/kg), 12.8 +/- 2.7 (45 g/kg), and 8.8 +/- 3.7 oocytes (60 g/kg). Nuclear status of most collected oocytes was the second meiotic division (range, 94.3%-100%). These superovulation procedures will be useful for development of in vitro culture systems and assisted reproductive technologies for not only Japanese field vole but also other voles. (C) 2016 Elsevier Inc. All rights reserved.

Pentobarbital sodium (Somnopentyl) can induce surgical anesthesia with a strong hypnotic effect that causes loss of consciousness. Animals have been known to die during experimental surgery under anesthesia with Somnopentyl, causing it to be declared inadequate as a general anesthetic for single treatment. An anesthetic combination of 0.3 mg/kg medetomidine, 4.0 mg/kg midazolam and 5.0 mg/kg butorphanol (M/M/B:0.3/4/5) was reported to induce anesthesia for a duration of around 40 min in ICR mice; similar anesthetic effects were reported in both male and female BALB/c and C57BL/6J strains of mice. However, the anesthetic effects of this combination in Japanese field vole, Microtus montebelli, remain to be evaluated. In the present study, we assessed the effects of Somnopentyl and different concentrations of anesthetic combination (M/M/B:0.3/4/5, 0.23/3/3.75 or 0.15/2/2.5) in Japanese field voles, by means of anesthetic scores. We also examined effect of these anesthetics on production of offspring. Death of the animals was observed only with Somnopentyl. The anesthetic score of Somnopentyl was lower than those of the other anesthetics, although there were no significant differences in duration, body weight and frequency of respiratory among the evaluated anesthetics. Abortion rate with Somnopentyl was significantly higher than that with the M/M/B:0.23/3/3.75 combination, although there was no significant difference in the number of offspring between two. In conclusion, results of this study provide basic information for achieving appropriate anesthetic concentrations in addition to indicating a new, safe and effective surgical anesthetic for Japanese field voles.

Canine embryos (8-cell to blastocyst stages) frozen-thawed using the slow-freezing method with glycerol (four recipients) or dimethyl sulfoxide (three recipients) as a cryoprotectant and vitrified-warmed using the Cryotop method (five recipients) were surgically transferred into the unilateral uterine horn of recipient bitches. As a result, the morphology of embryos frozen-thawed using the slow freezing method was judged to be normal, but no conception occurred in any of the recipient bitches. Two of the five bitches that received transferred embryos (morula to early blastocyst stages) vitrified-warmed using the Cryotop method became pregnant and produced normal pups (1/9 embryos, 11.1% and 1/6 embryos, 17.0%). It was concluded that the Cryotop method was more appropriate for canine embryo cryopreservation than the slow-freezing method, which is used for the cryopreservation of embryos of other mammalian species.

In the dairy in Japan, farms that secure successor cattle by introducing heifers are increasing with the expansion of herd scale. However, the price of heifers is rising remarkably, so in this study, we have focused on dairy management in Tochigi prefecture and clarified the factors to decide what action to select for securing successor cattle and the needs for public rearing pasture centers of users and nonusers with CS analysis and analyzed its potential for the expansion. As a result of the analysis, the following has become clear ; ①71.7% of the farms secure successor cattle by self-producing, and 31.4% of those outsource it to public rearing pasture centers. ②outsourcing successor cattle supports the dairy management in the aspects of their stall capacity and labor forces. ③public rearing pasture centers utilize the artificial insemination by using sex sorting semen and the technology of embryo transfer and produce successor cattle effectively and highly profitable heifer. So it has been suggested that they are advisable to secure successor cattle according to the expansion of herd scale.

酪農経営は，その経済性と持続性を追求しつつ，具体的な目標と計画をもって日々経営活動を行っている。安定した育成牛頭数の確保は初産牛を増やし，更新率や淘汰率を高めることにより，安定した生乳生産を実現させる。同時に，優良な後継牛の確保は，牛群全体の能力を向上・斉一化させることとなる。しかし，後継牛の育成は，生乳生産に結びつくまでの時間と労力，育成費用，及び育成牛の資産価値などが必ずしも明確でないことから疎かにされがちである。さらに，都府県の酪農経営は，飼養頭数規模を拡大する傾向にあることからも，効率的，かつ低価格の優良後継牛の自家確保が喫緊の課題となっている。そこで，本稿は，自家生産された育成牛の初産分娩までの生産費(以下，育成費用とする)を算出し，その経営経済性を検証する。併せて，優良後継牛の能力的評価額を明らかにし，初産牛能力水準の向上が，酪農経営に及ぼす経済効果を検討する。

酪農経営の収益性を規定する要因の1つに繁殖技術があげられるが，その経済的価値は酪農家に要され営農の場で利用された時に初めて生じる．本研究は性判別受精卵の利用を希望する酪農家層の経営特性および技術普及に必要な諸条件を明らかにすることを目的に，アンケートと聞き取り調査を実施した．これらの結果から性判別受精卵を「利用したい」，「既に利用している」と回答した酪農家は①多頭飼養，②後継者を確保，③性判別精液または受精卵移植の利用率が高い特性を有していた．したがって「性判別受精卵の利用に積極的な酪農家層」は「今後も酪農を永続させるため優良な後継牛の確保や牛群改良を志向し，先進技術の導入や利用に積極的な酪農家層」と推察される．また①繁殖技術の向上，②受精卵の価値に適した価格の設定，③酪農経営への収益性を明確化，④各種研究機関や育成牧場などによる利用推進の働きかけが利用普及に必要な諸要件として示唆された．

This study was designed to evaluate the effect of estrus synchronization treatments on recipient heifers for embryo transfer (ET). Holstein heifers were separated into the following three groups: (i) an administration of 50 mu g GnRH (gonadotropin-releasing hormone) analog was given to heifers at a random stage of the estrus cycle, followed 7 days later by two administrations of 7.5 mg prostaglandin F2 alfa analog (PG) as control; (ii) another administration of 100 mu g GnRH was given to the control group at 48 h after the administration of PG as the second GnRH group; and (iii) an administration of 0.75 mg estradiol benzoate (E2) was given to the control group at 24 h after the administration of PG as the E2 group. Each method caused estrus synchronization. Fresh embryos were nonsurgically transferred into the suitable recipients that had a functional corpora lutea (CL) 7 days after estrus. The E2 group showed a significantly higher (P < 0.01) rate of estrus synchronization (98.9%) at 13 days after PG administration and the final pregnancy rate of the E2 group (50.6%) was also significantly higher than the other groups (37.1%, P < 0.05 and 30.9%, P < 0.01, respectively). These findings demonstrate that E2 administration 24 h after PG protocol is effective for estrus synchronization of Holstein heifers, thus improving the productivity of ET.

Diagnosis of corpus luteum (CL) function by rectal palpation (RP) has been widely used for recipient selection of embryo transfer (ET), a technology essential for genetic improvements in cattle. To examine the accuracy of RP diagnosis method, the relationship between RP-based CL function and reproductive performance was compared in this study. In Experiment 1, CL of Holstein heifers on day 7 after estrus was classified into functional or hypoplastic by RP, and the results were compared with ultrasonographic (US) images and plasma progesterone (P4) levels. As a result, heifers with functional CL judged by RP had a mean maximum CL diameter of 20.1 +/- 3.1 mm on US and a mean P4 concentration of 8.1 +/- 2.3 ng/mL. These values were significantly greater than those of heifers with hypoplastic CL (12.4 +/- 5.4 mm, 4.0 +/- 2.8 ng/mL) (P < 0.001). In Experiment 2, the length of the estrus cycle was examined between functional CL and hypoplastic CL. The rate of heifers with a normal estrus cycle length with 1825 days was significantly lower with hypoplastic CL than with functional CL (16/24 vs. 43/46, P < 0.01). In Experiment 3, 543 inseminated heifers were similarly classified by CL function by RP 7 days after estrus. The heifers with functional CL showed higher pregnancy rate compared with the heifers with hypoplastic CL (75.2 vs. 47.9%, P < 0.0001). Finally, the CL function of 66 heifers was examined by RP on day 7 post-estrus, and ET was performed in 49 (74.2%) heifers with functional CL. As a result, 27 (55.1%) of them became pregnant. Taken together, these results reconfirm that RP on day 7 after estrus is useful for selection of heifers with functional CL.

Bovine in vitro fertilized (IVF) embryos were cultured in either CR1aa (CR1) or TCM-199 (TCM) medium and compared for their daily development based on the number of cleavage divisions (CDN), as calculated from the total cell numbers of the embryos. Embryos with advanced developmental stage and higher morphological quality were selected for use in the experiment. The relation between the CDN and the number of embryonic days after IVF in both groups showed a linear correlation no significant difference (P &gt 0.05) was found between the two groups. However, CDN on days 3 and 8 after IVF in the CR1 group did not increase, which suggests that transient developmental arrests occurred at these stages. In contrast, embryos in the TCM group showed a transient evelopmental arrest 3 d after IVF, but CDN increased regularly with age in days at later stages of culture with 100 M mercaptoethanol. A significant difference was found between the regression lines of the two groups during 59 d after IVF (P &lt 0.001). Consequently, numerical analysis of embryonic development in terms of CDN enabled objective evaluation of the developmental progress of bovine IVF embryos.

Quantification based on cleavage division (CD) of bovine preimplantation embryos facilitates quantitative analyses of embryonic developmental processes because CD occurs roughly once each day for all blastomeres for up to at least 9 days after ovulation. Therefore, embryonic morphological changes during this period were classified according to CD number. In this study, embryos collected from superovulated donors 0-9 days after ovulation were first classified morphologically into 14 conventional developmental stages. The total cell numbers (TCN) of embryos were measured using the air-dry method. The respective CD numbers of the embryos were then determined using logarithmic transformation of the TCN. The CD numbers of embryos were increased 0-10th with 11 stages. The 0th CD corresponded to 1-cell stage embryos the 1st CD corresponded to 2-cell stage embryos the 2nd CD corresponded to 3-4-cell stage embryos the 3rd CD corresponded to 5-8-cell stage embryos the 4th CD corresponded to 9-16-cell stage embryos, the 5th CD corresponded to morulae (17-32-cell stage embryos) and the 6th CD corresponded to the compact morulae. Furthermore, the 7th CD included early blastocysts to blastocysts. The 8th CD included expanded, collapsed and hatching blastocysts. The 9th CD included hatched blastocysts. The 10th CD included expanding-hatched blastocysts. The relationship between the CD number and the morphological characteristics of the bovine embryos 0-9 days after ovulation was expressed using a linear equation, and this revealed a high degree of correlation (y=0.98 x-0.96, r=0.99). These results suggest that morphological changes of bovine embryos can be classified accurately using an 11-stage classification system based on the number of cleavages.

The total cell numbers (TCNs) of bovine embryos collected from superovulated donors (VIVO embryos) were counted 0-9 d after ovulation to quantify the developmental process. Using numerical analysis of embryo development, we also compared the developmental process of VIVO embryos, in vitro-fertilized (IVF) embryos and nuclear transfer (NT) embryos obtained from enucleated oocytes and blastomere nuclei. The TCNs of embryos were measured using the air-dry method. Cleavage divisions (CD) of the embryos were obtained using logarithmic transformation of the TCN. The TCN of the VIVO embryos increased significantly (P<0.001) with time. The relationship between the CD of the VIVO embryos at 0-9 d after ovulation and age in days was described by a linear equation with a high correlation (y=1.03x+0.16, r=0.99), showing that CD occurs about once each day for all blastomeres. However, compared to the VIVO embryos, the TCN of the IVF embryos did not increase from 3-4 d nor after 7 d; the TCN of the NT embryos did not increase after 7 d (P>0.05). The results suggest a delay in development at these developmental stages. The slopes of regression lines of the IVF and NT embryos were significantly (P<0.001) smaller, indicating that quantification of the developmental process of VIVO embryos according to TCN and CD would be useful as criteria for numerical evaluation of the developmental process of bovine in vitro produced embryos.

An attempt was made to develop a new type of uterine cervix dilator for safety and efficient embryo collection in cows.<BR>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.<BR>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.<BR>(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.<BR>(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.<BR>(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.

This study was conducted to examine the possibility of performing nuclear transplanta-tion among different developmental stage embryos in cattle. The embryos, which were recovered nonsurgically from the uteri of superovulated cattle on day 5 to 8, were used as nuclear donors. The unfertilized eggs matured in vitro were used for recipient cytoplasm after removal of chromosomes at the second meiotic metaphase. Each enucleated egg was fused with a blastomere from donor embryos by electrofusion. The developmental ability of reconstituted eggs were examined by in vitro culture method.<BR>The proportions of the enucleated eggs, which fused successfully with a blastomere from the 8-cell and the 16-cell stage embryos, were significantly higher (67/79, 102/121) than those obtained with a blastomere from the 32-cell or more advanced stage embryos (32-cell, 73/109; compact morula, 15/42; blastocyst, 0/56). Eleven and 20% of the reconstituted eggs fused with a blastomere from the 8-cell and 16-cell stage embryos developed in vitro to the 8-16-cell stage. None or very few reconstituted from the 32-cell or more advanced stage embryos developed to the 8-16-cell stage.

A blastomere from 2- and 8-cell embryos was fused with the cytoplasm of the enucleated unfertilized eggs by electrofusion to examine the developmental ability. The proportion of enucleated eggs fused with blastomere from 2-cell and 8-cell embryos was 76(38/50) and 51% (48/94). The reconstituted eggs fused with blastomere from 2-cell embryos developed to blastocysts (18%), but none of reconstituted eggs fused with 8-cell blastomere developed to blastocysts.

The present, study was undertaken to examine suitable conditions of electric stimuli for fusion ofa blastomere from 8-16-cell rabbit embryo with an enucleated oocyte and for the development of reconstituted eggs in vitro. The proportions of eggs fused (88%) and reconstituted eggs developed to morula to blastocyst in vitro were high when a direct current of 125V/mm was given for 100μsec twice. However, only one young(1%)was obtained after transfer of 85 reconstituted eggs to 8 recipient rabbits. Considering the developmental process of reconstituted eggs in vitro, it was estimated that nuclear reprogramming occured in these eggs.