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Summary in Japanese
夏季の暑熱ストレスによる乳牛の受胎率低下の原因は多岐にわたる。これまで
の研究では、卵子の成熟および受精の過程、8 細胞期以前の初期胚などが高温に感受性
であり、これらの時期の卵子および初期胚への暑熱負荷によって早期胚死滅が起こるこ
とが主な原因とされてきた。一方、桑実胚期以降の胚は高温に対して抵抗性であり、こ
れらの胚を子宮内へ移植する胚移植は、夏場の受胎率向上に有効であることが明らかに
されている。しかし、暑熱ストレスは子宮の機能異常を引き起こすことで、胚移植によ
る受胎率も低下させる可能性が考えられる。また、人工授精による受胎率の低下は、涼
しくなる秋季においても継続する。これは夏季の暑熱ストレスによって発育途中の小さ
な卵胞内の卵子がダメージを受けているためであると考えられる。したがって、本研究
では、第 1 章において暑熱ストレスによる乳牛の子宮内膜機能への影響について、第 2
章においては小卵胞内の卵子の発育および発生能への影響について評価した。
牛では子宮内膜における上皮成長因子 (Epidermal growth factor: EGF) 濃度は受胎性
および子宮内膜機能の指標とされ、その発情周期中の変化が消失すると、早期胚死滅が
増加して受胎性が低下する。子宮内膜 EGF 濃度異常の発生には、リピートブリーダー
牛や高泌乳牛において共通してみられる、血中の卵巣ホルモン濃度の変化が関わってい
ることが示されている。また、この血中卵巣ホルモン濃度の異常は、暑熱ストレスを受
けている牛においても同様にみられる変化である。そこで第 1 章では、子宮内膜の EGF
濃度異常が暑熱ストレスによる受胎率の低下に関与しているか調べるために、ホルスタ
イン種泌乳牛において、暑熱環境下における EGF 濃度異常の発生頻度と胚移植後の受
胎率の関係について調べた。北海道および九州において飼養されているホルスタイン種
泌乳牛 365 頭を用い、6-9 月 (暑熱期、北海道: 90 頭、九州: 121 頭) および 10-1 月 (対
照期、北海道: 86 頭、九州: 68 頭) において試験を行った。発情後 3 日目に子宮内膜組
織を採取し、組織中の EGF 濃度を測定した。その結果、北海道および九州いずれの地
域においても、EGF 濃度異常を示す牛の割合は、10-1 月に比べて 6-9 月の方が高かった
(P < 0.05)。10-1 月と比較した 6-9 月の EGF 濃度異常を示す牛の割合は、北海道および
九州において、それぞれ 2 倍および 3 倍に増加した。試験期間を通じた (6-1 月) EGF 濃
度異常を示す牛の割合は、北海道 (26.1%) と比較して九州 (34.9%) において高い傾向
にあった (P = 0.07)。次に、6-9 月において九州地方のホルスタイン種泌乳牛 79 頭を用
い、発情日および発情後 3 日目の直腸温度による EGF 濃度異常の発生率への影響を調
べた。試験牛の一部 (67 頭) には、受胎性を評価するために、発情後 7 日目に胚移植を
行った。発情後 3 日目の直腸温度にかかわらず、発情日の直腸温度が高い (≥ 39.5℃) 牛
において、EGF 濃度異常の発生頻度は高く (64.1 vs. 30.0%、P < 0.05)、胚移植による受
胎率は低かった (26.7 vs. 51.4%、P < 0.05)。これらの結果から、発情日の暑熱ストレス
によって引き起こされる子宮内膜 EGF 発現の異常が、暑熱期における乳牛の受胎率低
下の一因となることが示された。
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夏季の暑熱ストレスによる乳牛の受胎率低下が、涼しくなる秋季にも持続することは、
夏季の暑熱ストレスによる小卵胞内の卵子へのダメージにより、卵子の発生能が低下す
ることが原因であると推測されている。小卵胞の中でも、初期胞状卵胞 (直径 0.3-1 mm)
の時期は、卵子が発生能を獲得する上で重要な時期であるが、暑熱ストレスによる初期
胞状卵胞の発育および卵胞内卵子の品質への影響を調べた報告はない。そこで第 2 章で
は、初期胞状卵胞 (直径 0.5-1 mm) に由来する卵子-卵丘-顆粒層細胞複合体 (oocytecumulus-granulosa complexes: OCGCs) の体外発育培養系 (in vitro growth: IVG) を用いて、
暑熱期の乳牛の日内体温変化を模した条件を設定しその影響を調べた。OCGCs を牛の
正常な体温に近い 38.5℃で培養する対照群と、暑熱環境下の乳牛の体温変化を模した温
度条件 (38.5℃: 5 h, 39.5℃: 5 h, 40.5℃: 5 h, 39.5℃: 9 h) で培養する暑熱群に分け、12 日
間の IVG に供し、暑熱負荷が卵子の発育、発生能、および卵子発生能と関連がある指標
(顆粒層細胞のステロイドホルモン産生能、卵子の酸化ストレス状態、および卵子と卵
丘細胞との細胞間結合) に及ぼす影響を調べた。培養前後の卵子直径の増加は、対照群
と比べて暑熱群の方が小さかった (P < 0.05)。卵子の核成熟率、受精後の卵割率、顆粒
層細胞のステロイドホルモン産生能、卵子中の活性酸素種量および卵子と卵丘細胞との
細胞間結合の程度には、群間で差がみられなかった。一方、胚盤胞発生率は対照群
(27.7%) と 比 べ て 暑 熱 群 (0.0%) で 低 く 、 卵 子 中 の 還 元 型 グ ル タ チ オ ン (reduced
glutathione: GSH) 量も暑熱群において低かった (P < 0.05)。また、GSH の合成を促進す
るシステインの培地への添加が、IVG において暑熱負荷を受けている卵子の発育、発生
能および GSH 量に及ぼす効果を調べたところ、システインの添加によって卵子中の
GSH 量が増加し、卵子の発育と胚盤胞への発生率 (27.9 vs. 6.1%) も改善された (P <
0.05)。これらの結果から、夏季の暑熱ストレスが卵子中の GSH 量を減少させることに
よって、初期胞状卵胞中の卵子の発育および発生能を低下させ、冷涼な秋季においても
受胎率を低下させる可能性が考えられた。
本研究において、夏季の暑熱ストレスが乳牛において子宮内膜機能の異常を引き起こ
し、夏場の受胎率改善に最も有効とされる胚移植による受胎率も低下させることを示し
た。さらに、暑熱ストレスは卵子中の GSH の枯渇を介して、小卵胞中における卵子の
発育および発生能を低減させることを示した。夏季と秋季における乳牛の繁殖成績は、
子宮内膜機能と小卵胞の発育に着目した対策や治療法によってさらに改善できる可能
性がある。
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