牛島 仁

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.

わが国の酪農は，飼養頭数規模の拡大にともない，乳牛初妊牛の導入によって後継牛を確保する経営体が増加している．しかし，北海道を主とする初妊牛価格は高騰していることから，安定した後継牛確保は喫緊の課題である．そこで本研究は，栃木県の家族労働力を主とする酪農経営に焦点を充て，後継牛確保の選択行動を決定付ける要因と公共育成牧場に対する利用者・非利用者の預託ニーズをCS分析によって明らかにし，公共育成牧場の発展可能性を検討した．分析の結果，①酪農経営の71.7％は後継牛を自家育成し，31.4％が公共育成牧場の預託制度を利用していること，②育成牛の預託は，牛舎収容力と労働力の側面で酪農経営を補完していること，③公共育成牧場は，受精卵移植や性選別技術を活用し，効率的な後継牛生産と収益性の高い乳牛初妊牛を産出していることが明らかになった．育成牛の預託は，飼養頭数規模の拡大に応じた後継牛確保の得策であることが示唆された．

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.

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.

安全で操作の容易な牛用子宮頸管拡張器を開発した.構造は,シリコンカテーテルの先端にシリコンラバー製バルーン,後方に空気圧入弁と注射筒を付け,金属製芯棒で補強したものである.頸管拡張操作は,拡張器を外子宮口に挿入し,直腸から指と掌で頸管をほぼ直線に保持する.拡張器の先端を第1ヒダまで進め,バルーンを膨張させ内腔を拡張し,さらにバルーン後部を圧迫してヒダの間隙を充分拡張した後,バルーン内の空気を排出してこの間隙を通過し第2ヒダまで進める.以下この操作を繰返して,内子宮口に達する.本器は,頸管拡張を柔軟なシリコンラバーの接触でおこない,拡張されたヒダ間隙を直線的に通過させるため,金属製拡張棒で可通過部位を探る場合に比べて,頸管壁に与える損傷の程度は小さく,操作も単純である.<BR>19頭を供試して頸管拡張を試みたところ,未経産•経産を問わず頸管が正常な12頭では1.5~4分で操作を終り,引き続く採胚の成績も良好であった.頸管が癒着した7頭では,5頭は1~5分で終了したが,2頭は不可能であった.また癒着牛2頭,正常牛1頭に,子宮還流液に少量の血液の混入を認めた.その原因は術者の不注意による子宮体への損傷であると推察される.以上の成績から,本器は,牛胚の採取,移植に有用であると考えられる.

発育段階が異なる牛胚細胞をドナー核として核移植を試みた.ドナー核には人工授精後5~8日目に子宮から採取した胚の割球を,レシピエント細胞質には体外成熟した牛除核未受精卵を用いた.ドナー核とレシピエント卵との電気融合を行い,得られた核移植由来再構築胚の発生能を調べた.8および16細胞期胚の割球融合成功率は67/79(85%)および102/121(84%)であった.これに対し,32細胞期,コンパクト桑実胚割球および胚盤胞内細胞塊(ICM)細胞核との融合成功率は73/109(67%),15/42(36%)および0/56(0%)であり,8~16細胞期胚の割球融合成功率に比べて有意に低下した.8~16細胞期胚割球由来再構築胚を4日間体外培養したところ11~20%が8~16細胞期に発生した.これに対して32細胞期およびコンパクト桑実胚割球由来再構築胚の8~16細胞期への発生率は0~3%であり,8~16細胞期胚実割球由来再構築胚に比べて有意に低かった.

交流電流を併用した電気融合法を用いてマウス除核未受精卵細胞質と2および8細胞期胚割球の融合を行った。除核未受精卵細胞質と2および8細胞期胚割球との融合率は,76%(38/50)および51%(48/94)であった。2細胞期胚割球由来再構築胚38個のうち7個(18%)が胚盤胞に発生したが,8細胞期胚割球宙来の再構築胚では胚盤胞への発生はみられなかった。

本研究では,ウサギ8～16細胞期胚割球と除核未受精卵の電気的融合条件ならびに再構築胚の発生条件について検討した.その結果,125V/mmの直流パルスを100μsec,2回通電すると融合率(88%)ならびに体外における発生率(63%)が最も高いことが判明した.また,電気刺激後16～18時間培養した再構築胚85個を8羽の受卵雌に移植したところ5羽で妊娠が成立したが,得られた産子はわずかに1羽(1%)であった.また,再構築胚の経時的発生過程からみて,移植核が受精時の状態になっていると考えられた.