Conjugated linoleic acids (CLA) are mixture of positional and geometric isomers of linoleic acid with conjugated double bonds, which are unique components of ruminant lipids and have beneficial effects on human health (Pariza, 1999). The major dietary source of CLA from ruminant dairy products is c9t11CLA in the milk fat (Erin et al. , 2006). This isomer is produced by two pathways. One is produced in the rumen, as an intermediate during isomeration of linoleic acid (c10c12C18:2) by bacteria, and the another is produced in the mammary gland by Δ⁹ desaturase from vaccenic acid (t11C18:1) which is formed in the rumen during the rumen biohydrogenation (BH) of linolenic acid (c9c12c15C18:3) or c10c12C18:2 (Bauman et al. , 2000).

　Lock and Garnsworthy (2003) reported that the concentration of c9t11CLA in the cow milk varied in seasons, and Bargo et al. (2006b) reported that the variation of c9t11CLA concentration in the milk was explained about 41% by intake of c9c12c15C18:3. Other factors affecting the variation of milk c9t11CLA concentration during grazing season did not clear. The objectives of the current study were to investigate the contributions of t11C18:1 formation in the rumen and c9t11CLA desaturation in the mammary gland to the variation of c9t11CLA concentration in the grazing cow milk, effect of rumen conditions such as pH and energy supply for the rumen bacteria on t11C18:1 formation from c9c12c15C18:3, and effects of energy supplement on c9t11CLA concentration in the grazing cow milk.

　To realize these objectives the following experiments were carried out.

1. Variation of c9t11CLA concentration in cow milk, depending on feeding systems in different seasons

　A variation in c9t11CLA concentration in cow milk was investigated in different feeding systems during grazing seasons. Milk samples were collected from Holstein dairy cows, which either grazed in whole day (WG), only grazed day time (TG), or were offered by a total mixed ration (TMR) without grazing (NG), from April to December in 2005. In April, November and December, the cows in TG and WG treatments received grass silage and some concentrate, while from May to October, the cows were grazed on temperate pastures. The cows in the NG treatment received the TMR throughout the season. The major fatty acid obtained in the pastures was c9c12c15C18:3. There was no significant difference in the pastures c9c12c15C18:3 contents from May to September, but there was a significant decrease in October. However, the c9c12c15C18:3 contents obtained in the pastures were always much higher than those obtained from the TMR. The c10c12C18:2 was also the major fatty acid in the TMR, but these contents were higher in the TMR than in the pastures. There was no significant difference in the milk c9t11CLA concentrations between the three feeding systems while the cows were fed by conserved pasture in April, November and December. Although c9t11CLA concentrations were lower in the TMR, the cows grazed in fresh pastures showed significantly higher concentrations of c9t11CLA in the milk than those received only TMR. The cows in the WG treatment showed higher c9t11CLA concentrations than those in the TG treatment. In the WG and TG treatments, c9t11CLA concentrations were the highest in June, and gradually decreased (p<0.01) until October. For the NG treatment, there was no significant change in the concentrations of c9t11CLA (p>0.05) during seasons. Overall, t11C18:1 and c9t11CLA were greatly influenced by seasons, with higher variation in the WG treatment than in the TG treatment and no variation in the NG treatment.

2. Comparison of the contributions of t11C18:1 formation in the rumen and c9t11CLA syntheses in the mammary gland to the variation of c9t11CLA concentration in the grazing cow milk

　Contributions of t11C18:1 formation in the rumen and c9t11CLA syntheses in the mammary gland to the variation of c9t11CLA concentration in the grazing cows milk was investigated. This experiment was conducted from May to September in 2005 and 2006 on dairy farms in Ashoro, Hokkaido, northern Japan. Four dairy farms of Holstein cow were chosen for this experiment in each year. These farms grazed cows in whole day. Samplings of pastures and bulk milk were collected from May to September, in each farm once a month. There was no significant difference in the pastures c9c12c15C18:3 contents during the experiment, but the NDF content in the pastures increased from May to September. The pastures WSC content was the highest (P＜0.05) in May and September, and the lowest (P＜0.05) in July and August. The t11C18:1 and c9t11CLA concentrations in the milk were the highest (P＜0.05) the June and the lowest (P＜0.05) in September. The variation coefficient of desaturation index and BH index were 7.7% and 20.8%, respectively. It was demonstrated that the explanatory variables for the c9t11CLA concentration in the milk of grazing cows were BH index and milk t11C18:1 concentration, which were selected by the stepwise regression analyses. From these analyses it is suggested that the BH of unsaturated fatty acid in the rumen may be more effective factor to the c9t11CLA concentration in the milk compared with disaturation of t11C18:1 in the mammary gland.

3. Effect of pH on ruminal biohydrogenation and intermediates of linolenic acid in vitro

　An in vitro experiment was conducted to investigate the effect of incubation pH on BH of 　c9c12c15C18:3. The pH of the incubation liquids was adjusted to 5.54 (P5), 6.12 (P6), 6.65 (P65), and 　7.04 (P7). Thereafter, 200mg of linolenic acid reagent and 2 g of freeze dried perennial ryegrass 　( Lolium perenne L.) were incubated for 8 h in 100ml of incubation liquid (rumen fluid: buffer = 1:1). The total VFA concentration significantly (P＜0.05) increased with an increasing incubation pH. The contents of c10c12C18:2 during the incubation were higher (P＜0.01) in the P5 treatment and lower in the P7 treatment, but did not differ between the P6 and P65 treatments. The contents of c9c12c15C18:3 significantly (P＜0.01) decreased from the P5 to the P7 treatment, but the contents of C18:0 significantly (P＜0.01) increased from the P5 to the P7 treatment. The BH of c9c12c15C18:3 increased with increasing pH of the incubation liquid. However, the highest BH level of c10c12C18:2 was below 12% in the present study and the BH of c10c12C18:2 varied by 6% between the treatments. The t11C18:1 content during the incubation was the highest (P＜0.001) in the P65 treatment and the lowest (P＜0.01) in the P5 treatment. The c9t11CLA content was the highest in P6 treatment but the t10c12CLA content was the highest in the P65 treatment. The experiment result suggests that the highest formation of t11C18:1 from C18:3 may occurr when ruminal pH was above 6.5.

4. Effect of fructose addition into incubation on ruminal biohydrogenation and intermediates of linolenic acid in vitro

An in vitro experiment was conducted to investigate the effects of fructose addition into incubation on BH of c9c12c15C18:3. The buffer liquid was prepared by the method of McDougall, and the fructose was added into the buffer liquids 0.0 g/L (F0), 0.5 g/L (F5), 1.0g/L (F10). Thereafter, 200mg of linolenic acid reagent and 2 g of freeze dried perennial ryegrass were incubated for 8 h in 100ml of incubation liquid (rumen fluid: buffer = 1:1). The acetic, propionate and total VFA concentrations were the highest (P＜0.05) in the F10 treatment and the lowest in the F0 treatments. The content of c10c12C18:2 was unaffected by the fructose addition. The content of c9c12c15C18:3 was the lowest (P＜0.05) in the F10 treatment than other treatments, and there was no significant difference between F0 and F5 treatments. The BH of c10c12C18:2 was unaffected by the fructose addition and these values were 13%, 14% and 13%, respectively. However, the BH of c9c12c15C18:3 in the F10 was treatment 35%, and it the highest (P＜0.05) among the treatments. The content of t11C18:1 in F10 after the incubation was the highest (P＜0.05) among the treatments. However, the content of c9t11CLA in the incubation liquid did not differ among the treatments. These results suggested that the formation of t11C18:1 in the rumen may be increase with increasing pasture WSC contents.

5. Enhancement of c9t11CLA concentration in the milk of grazing cows

　This study was carried out to determine the effects of potato pulp silage (PPS) as a diet supplement on the composition of milk fatty acids in grazing dairy cows in comparison to a barley grain supplement. Eight Holstein dairy cows at late lactation were fed by grazing based diets supplemented with 150 g/kg of PPS as a substitute for barley, and grazed in 4.3 ha swards, mainly containing orchardgrass for 56 days. Total DM and pasture DM intake and milk yields, as well as milk composition, did not differ between the treatments. Also, the c9c12c15C18:3 intake did not differ (329 g/day vs. 323 g/day DM). The t11C18:1concentration in the blood was higher in cows fed by PPS (2.4 g/100 g FA) compared to those fed by barley (1.9 g/100 g FA). There was no significant difference between the treatments in the c9t11CLA concentration in the blood. The short-chain and medium-chain fatty acids in the milk also did not differ between the treatments. The t11C18:1 concentration in the milk was higher in cows fed by PPS (2.32 g/100 g FA) compared to those fed by barley (1.84 g/100 g FA), and c9t11CLA was higher in cows fed by PPS (1.25 g/100 g FA) than those fed by barley (0.95 g/100 g FA). Milk c9t11CLA concentration was positively correlated to blood t11C18:1 concentration. These results suggest that an increase in milk c9t11CLA concentration may relate to an increase of the formation of t11C18:1 concentration in the rumen and it flow into the blood by Δ⁹ desaturase in mammary glands in the PPS treatment compared to the barley treatment.

Conclusion

　The pasture c9c12c15C18:3 and c10c12C18:2 did not differ during the grazing seasons, but the NDF and WSC contents varied during grazing seasons. The concentration of t11C18:1 and c9t11CLA in the milk of grazing cows greatly varied during the grazing seasons. There was no relationship between pastures c9c12c15C18:3 content and c9t11CLA concentration in the milk of grazing cows, but the negative relationship between pasture NDF content and BH index was found. It was demonstrated that formation of t11C18:1 from c9c12c15C18:3 in the rumen was more effective factor to affect c9t11CLA concentration in the milk of grazing cows when compared with disaturation activity in the mammary gland by the stepwise regression analyses.

　It is suggested that the variation of pasture NDF and WSC contents during grazing season may alter ruminal pH, and the BH of UFA in the rumen. The incubation liquid pH strongly affected to the BH of c9c12c15C18:3 which increased with increasing ruminal pH and decreased with decreasing of ruminal pH. The highest formation of t11C18:1 from c9c12c15C18:3 occurred ruminal pH was about 6.5. It is suggested that the lower ruminal pH may inhibit the first step of BH of c9c12c15C18:3 and the higher ruminal pH enhance BH of c9c12c15C18:3 to C18:0. The BH of c9c12c15C18:3 and formation of t11C18:1 were higher in the 1g/L fructose addition into incubation liquid compared with in the 0.5g/L fructose addition or without addition into incubation liquid. It is suggested that the formation of t11C18:1from c9c12c15C18:3 in the rumen may be increased with increasing pastures WSC content.

　The concentration of t11C18:1 in the blood and c9t11CLA in the milk were higher in grazing cows fed by potato pulp silage compared to cows fed by barley grain. It is suggested that an increase of t11C18:1 concentration in the blood may relate to consistent ruminal pH by PPS starch than barley grain. An increase in the formation of t11C18:1 from c9c12c15C18:3 in the rumen, and the increased c9t11CLA concentration in the milk is relate to an increase of t11C18:1 concentration in the blood by Δ⁹ desaturase in mammary gland which is due to the PPS supplement.

These results suggests that the variation of c9t11CLA in the milk of grazing cows during grazing seasons may relate to pasture nutrient contents, which may alter during seasons and these variation may affect the formation of t11C18:1 in the rumen. It is suggested that the c9t11CLA concentration in the milk of grazing cows may enhanced by the supply of the carbohydrate which may give consistent ruminal pH for grazing cows.

（和訳）

1．飼養形態と季節の違いが牛乳中共役リノール酸（CLA）濃度の変動に及ぼす影響

　飼養形態と季節の違いが牛乳中の共役リノール酸（CLA）濃度に及ぼす影響を明らかにするため昼夜放牧（WG） 、昼間放牧（TG）、混合飼料給与（放牧なし）（NG）実施されている酪農家を4件ずつを選び、4月から12月にかけて月1回牧草、 給与飼料、バルク乳を採取し、それぞれの脂肪酸組成を調べた。 調査始まった4月にはWG、TG酪農家でも放牧はしないで泌乳牛に牧草サイレージを給与していた。 5月から10月にかけてWG及びTG酪農家では泌乳牛を放牧していた。 11からは再び放牧を止め、牧草サイレージを給与していた。 混合飼料給与区では通年保存飼料をTMRとして給与していた。 放牧草中のc9c12c15C18:3含量は混合飼料のc9c12c15C18:3含量より多かったが放牧季節の間に放牧草中C18:3の含量は有意差はなかった。 牛乳中のt11C18:1とc9t11CLA濃度は保存飼料給与していた4月に各区の間有意差はなかったが、 放牧開始した5月からWG区とTG区では増加し6月に最も高くなった。 その後、10月にかけてだんだん低下した。 一方、NG区では牛乳中のt11C18:1とc9t11CLA濃度は試験期間として変わりはなかった。 放牧が終了した11月と12月に牛乳中のt11C18:1とc9t11CLA濃度は各区の間有意差はなくなった。
　この研究では牛乳中t11C18:1とc9t11CLA濃度の変動はWG区において最も多かった。
　　牛乳中のt11C18:1とc9t11CLA濃度の変動は放牧草に関与していると考えた。

2．反芻胃内での水素添加指標と乳腺での不飽和化酵素の放牧牛乳中CLA濃度の変動に及ぼす役割

　反芻胃内での不飽和脂肪酸への水素添加指標と乳腺での不飽和化酵素の活動が放牧されているホルスタイン朱比泌乳牛の 乳中CLA濃度の季節変動に及ぼす役割を検討するため、 2005，2006年5月から9月にかけて昼夜放牧されている牛のバルク乳の脂肪酸組成ならびにCLA濃度を調べた。 調査期間中、毎月に1回、放牧草とバルク乳を採取し、放牧草ではNDF、WSCと脂肪酸含量を分析した。 バルク乳については脂肪酸分析を行った。 放牧草のNDF含量とWSC含量は放牧季節によって変化した（P＜0.05）が、 放牧草中のc9c12c15C18:3の含量は各月の間に有意差がみられなかった。 乳脂肪中のt11C18:1 とc9t11CLA濃度は共に春に高く秋に最も低くなり、変動係数はそれぞれ22.6％、19.5％であった。 反芻胃内での水素添加指標の季節によって変化し、変動係数は20.8％になった。 これに対して乳腺での不飽和化酵素指標は季節によって変化したが変動係数は7.7％と小さかった。 重回帰解分析の結果反芻胃内での水素添加指標と乳中のt11C18:1濃度が乳脂肪中c9t11CLA濃度の説明変数として選択し、 乳中のc9t11CLA濃度の季節変動は42.14％程度の説明出来た。 これらの結果から放牧されている泌乳牛の乳脂肪中のCLA濃度の季節変動には乳腺での不飽和化酵素よりも 反芻胃内での水素添加の方が強く関与していると考えられた。

3．培養液中pHの違いがリノレン酸の水素添加に及ぼす影響

　放牧された牛の乳中のC18:1とCLA濃度は放牧草からのC18:3の摂取量だけなく、 放牧草の栄養成分の季節変化に伴う反芻胃内環境（pH）変化によりC18:3への水素添加が影響を受けると考えれ、 培養液pHの違いがC18:3の水素添加について検討した。 培養液のpHを5.64、6.23、6.75、7.05で調整したP5区、P6区、P65区、P7区での培養液（反芻胃液：人工唾液＝50：50ml）に リノレン酸試薬200mgと凍結乾燥したペレニアルライグラス2gを加え8時間にかけて培養した。 培養後のc10c12C18:2の濃度はP5区で最も高く、P7区で最も低くなった。 培養後のc9c12c15C18:3の濃度は培養液pHの増加に伴い低下した。 培養液pHの上昇によりc9c12c15C18:3への水素添加率は増加した。 c10c12C18:2への水素添加率はpHの影響を受けたが、いずれの区も水素添加率は12％以下であった。 培養後のt11C18:1の濃度はP65区で最も高くなり、P5区で低くなった。 培養後のC18:0の濃度は培養液pHの上昇に伴い増加した。 これらの結果からc9c12c15C18:3からt11C18:1の転換率はpHを6.5前後で高くなり、 t11C18:1からC18:0への転換率はpHの上昇に伴い増加すと考えられた。

4．培養液中へのフルコトス添加がリノレン酸の水素添加に及ぼす影響

　培養液中へのフルコトス添加がC18:3の水素添加に及ぼす影響について検討した。 培養液にフルコトスを0.0mg/L、0.5g/L、1.0g/Lを添加したF0区、F5区、F10区での培養液（反芻胃液：人工唾液＝50：50ml）に リノレン酸試薬200mgと凍結乾燥したペレニアルライグラス2gを加え8時間にかけて培養した。 培養後の酢酸、プロピオン酸、総VFA濃度はF10区において高かったが、F0区とF5区の間に差はみられなかった。 培養後のc10c12C18:2濃度はフルコトス添加により変わりはなかった。 培養後のc9c12c15C18:3濃度はF10区において最も低く（P＜0.05）なった。 培養後のc10c12C18:2の水素添加率は各区の間に差がみられなかった。 培養後のc9c12c15C18:3の水素添加率はF10区において最も高かった（P＜0.05）が、F0区とF5区の間には差がみられなかった。 培養液中t11C18:1濃度はF10区においては高かった（P＜0.05）が、F0区とF5区においては変わりはなかった。 培養後のc9t11CLA濃度はフルコトス添加によって変化はなかった。 これらの結果から反芻胃内でt11C18:1合成は放牧草のWSC含量の変動より変化すると考えられる。

5. 放牧されている牛乳中CLA濃度の増加

　実験3ではC18:3からt11C18:1の転換率は反芻胃pHを6.5前後で高くなることが報告されている。 放牧飼養季節において乳生産量を増加するため大麦などデンプンを併給飼料として給与している。 大麦デンプンはジャガイモ粕サイレージ（PPS）のデンプンに比べ反芻胃内での分解速度速い、反芻胃pHが低下し易いといわれている。 本研究では放牧飼養時における併給飼料のデンプン源の違い牛乳中の脂肪酸組成に及ぼす影響を検討するため、 デンプン源として反芻胃内で分解速度が異なるPPSと大麦を泌乳牛へ給与し、 牛乳中の脂肪酸組成を調べた。 ホルスタイン種泌乳牛8頭を用い、TDN要求量の15％を大麦から給与した大麦区とPPSを給与したPPS区の２群に分けた。 放牧地はオーチャードグラス主体混播草地4.3haで、56日間昼夜連続放牧した。 乾物摂取量、草地からの乾物摂取量、乳生産量及び乳成分はPPS区と大麦区の間に差はなかった。 c9c12c15C18:3摂取量もPPS区と大麦区の間に差はなかったが（328.8 vs. 322.9 g/ 日 DM、P=0.85）。 血液中のt11C C18:1濃度はPPS区で大麦区比べ多かった（2.4 vs. 1.9g/100g）。 乳中短鎖、中鎖脂肪酸濃度はは両区の間に差が認められなかったがt11C18:1の濃度はPPS区で大麦区に 比べて増加し（2.32 vs. 1.84 g/100g 脂肪酸、P＜0.05）、 牛乳中c9t11CLA割合はPPS区で大麦区に比べ多かった（1.25 vs. 0.95 g/100g 脂肪酸、p＜0.05）。 血液中のt11C C18:1濃度と乳中c9t11CLA濃度との間に正の相関が見られた。 これからのことから大麦に比べPPS区で乳中t11C18:1とc9t11CLAの割合が高かった理由としてPPSを泌乳牛へ給与した場合、 大麦に比べ反芻胃内でのpHが安定し、反芻胃胃内でc9c12c15C18:3からt11C18:1転換率が高かったと考えられた。

結　論 (Conclusion in Japanese)

　放牧草中に含まれるc9c12c15C18:3とc10c12C18:2の含量は放牧季節により変動はなかったが、 NDFとWSC含量は放牧季節により変動した。 放牧された泌乳牛の乳中t11C18:1とc9t11CLA濃度は放牧季節により大きく変動した。 放牧草のc9c12c15C18:3含量と乳中c9t11CLA濃度との間に相関関係が見られなかったが、 放牧草のNDF含量と乳中BH指標との間に負の相関が見られた。 反芻胃内でc9c12c15C18:3からt11C18:1の合成は乳腺での不飽和化活性に比べ強く影響している要因であることを重回帰解分析により明らかにした。

　放牧草のNDFとWSC含量の季節変動は反芻胃内のpHや反芻胃内微生物のエネルギー源を変化させ反芻胃内での 不飽和脂肪酸の水素添加に影響すると考えた。 培養液pHはc9c12c15C18:3の水素添加に強く影響した。 培養液pHの増加によりc9c12c15C18:3の水素添加が増加し、培養液pHの低下により低下した。 c9c12c15C18:3からt11C18:1の合成は培養液pHを6.5に調整した場合最も高かった。 これらの結果から反芻胃内pH低い場合反芻胃内でc9c12c15C18:3の水素添加が抑制し、 反芻胃内pH高い場合c9c12c15C18:3の水素がC18:0まで進みと考えた。 培養後、c9c12c15C18:3からt11C18:1の合成は培養液にフルコトスを1.0g/L添加し場合0.5g/L添加や無添加に比べ多かった。 この結果から反芻胃内c9c12c15C18:3からt11C18:1の合成は放牧草中のWSC含量の増加に伴い増加すると考えた。

　放牧された泌乳牛の血液中t11C18:1と乳中c9t11CLA濃度はジャガイモ粕サイレージを併給飼料として 泌乳牛へ給与した場合大麦給与に比べ多かった。 この結果はジャガイモ粕サイレージデンプンが大麦デンプンに比べ反芻胃に安定したpHを与え 反芻胃内での9c12c15C18:3からt11C18:1の合成が増加し、血液に流れ乳腺で不飽和化活性によりc9t11CLAが合成したためと考えた。

以上の研究結果から放牧飼養時において乳中のc9t11CLA濃度の季節変動は放牧草の栄養成分の変動により 反芻胃でc9c12c15C18:3からt11C18:1の水素添加の変動であり、 放牧飼養時に併給飼料の種類により乳中c9t11CLA濃度を増加させることが出来ると考えられる。