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This study was performed to investigate the effect of the inclusion rate of corn silage (CS) in ensiled total mixed ration (TMR) on dry matter intake (DMI), nutrient digestibility, and ruminal fermentation in Japanese Wagyu steers. Four ensiled TMRs, in which the crude protein (CP) and total digestible nutrients (TDN) levels were adjusted to be similar, were prepared with 0, 15, 30, and 45% CS, respectively. Four Japanese Wagyu steers during the fattening period were used in a feeding trial with the four dietary treatments of the ensiled TMRs in a 4 × 4 Latin square design. Increasing the inclusion rate of CS resulted in higher pH values and lower lactate production in the ensiled TMR, although all TMR silages showed good fermentative characteristics, such as low pH value and high lactate content. The DMI linearly increase when the inclusion rate of CS in the TMRs increased. There were differences in the apparent digestibilities of CP, non-fibrous carbohydrate, acid detergent fiber, and neutral detergent fiber. The TDN contents were greater in the ensiled TMRs with 15 and 30% CS. The feeding of TMRs with higher inclusion rates of CS gave rise to slower fermentation in the rumen. Corn silage should be utilizable as a TMR material, and its inclusion at approximately 30% in a TMR is a safe upper limit for Japanese Wagyu steers during the fattening period.

Objective An experiment was conducted to assess the antioxidant contents and activities of colored rice grains and to evaluate their nutritive characteristics in terms of chemical composition and in situ ruminal degradation. Methods Ten cultivars of colored rice grains (Oryza sativa L.) collected from several areas of Japan were studied, and control rice without pigment, maize, barley, and wheat grains were used as control grains. Their chemical compositions, pigment, polyphenol contents, total antioxidant capacity (TAC), and degradation characteristics were determined. Results The starch contents of the colored rice grains were in the range of 73.5% to 79.6%, similar to that of the control rice grain. The black and red rice grains contained anthocyanin (maximum: 5,045.6 μg/g) and proanthocyanidin (maximum: 3,060.6 μg/g) at high concentrations as their principal pigments, respectively. There were significantly (pl0.05) positive relationships among the pigment contents, polyphenol content, and TAC values in the colored and control rice grains, indicating that the increase in pigment contents also contributed to the increased polyphenol content and TAC values in the colored rice grains. The dry matter and starch degradation characteristics, as represented by c (fractional degradation rate of slowly degradable fraction) and by the effective degradability, of the colored rice grains and the control rice grain were ranked as follows among commonly used grains: wheatgbarley ≥ricegmaize. The colored rice grains also included the most-digestible starch, since their potential degradable fraction and actual degradability at 48 h incubation were almost 100%. Conclusion Colored rice grains have high potential to be used as antioxidant sources in addition to starch sources in ruminants.

The objective of this study was to evaluate the effect of replacing corn with brown rice (BR) on dry matter intake (DMI), milk production, blood metabolites and energy balance (EB) in cows during the periparturient period. Twenty-four nonlactating multiparous Holstein cows in late gestation were studied from 3 week before the expected calving date through 6 week after parturition. The cows were divided into one of two dietary treatment groups based on parity and the expected calving date. The treatments were a diet containing steam-flaked corn and a diet containing steam-flaked BR. Prepartum DMI and body weight (BW) and postpartum DMI, BW, milk yield, milk protein and lactose yield were not affected by the dietary treatment. However, the milk fat content and fat yield were higher ( P = 0.02 and P = 0.01) for cows fed BR than for cows fed corn. Thus, cows fed BR-based total mixed ration (TMR) tended to have more negative EB in early lactation ( P = 0.06) compared with cows fed corn-based TMR. During the prepartum period, the plasma concentrations of glucose, insulin, β-hydroxybutyrate (BHBA), nonesterified fatty acids (NEFA), aspartate transaminase (AST) and Ca were not affected by dietary treatments. On the other hand, the postpartum plasma glucose and insulin concentrations were decreased ( P P = 0.02) and the BHBA and AST concentrations were increased ( P P = 0.02) by replacing corn with BR with no dietary effect on postpartum plasma NEFA and Ca concentrations. These results show that feeding BR instead of corn does not affect DMI or milk yield, but increases milk fat production and leads to a more negative EB state in early lactation. Replacing corn with BR during the periparturient period has adverse effects on the metabolism and energy status in early lactating cows.

Lenvatinib (an inhibitor of vascular endothelial growth factor (GF) receptors 1–3, fibroblast GF receptors 1–4, platelet-derived GF receptor α, rearranged during transfection, and stem cell factor receptor) was non-inferior to sorafenib in a phase 3 (REFLECT) trial of advanced hepatocellular carcinoma. This study examined the efficacy and safety of lenvatinib in a real-world setting. This was a retrospective, multicenter, observational study. Inclusion and exclusion criteria were based on the phase 3 trial, and participants were observed for at least 12 weeks. Therapeutic effect was determined using the modified Response Evaluation Criteria In Solid Tumors (m-RECIST) at the 8th week. Patients received oral lenvatinib 12 mg/day (body weight > 60 kg) or 8 mg/day (body weight

The effects of the substitution of brown rice (Oryza sativa L.; BR) for corn (Zea mays L.) in ensiled total mixed ration (TMR) that had a high proportion of grain on feed intake, lactation performance, ruminal fermentation, digestion, and N utilization were evaluated. Nine multiparous Holstein cows (51 ± 9 d in milk) were used in a replicated 3 × 3 Latin square design with 3 dietary treatments: a diet containing 0, 20, or 40% steam-flaked BR and 40, 20, or 0% steam-flaked corn (dry matter basis). Cows were fed ad libitum an ensiled TMR consisting of 40.7% alfalfa silage, 11.8% grass silage, 7.1% soybean meal, and 40.0% steam-flaked grain (dry matter basis). The ensiled TMR was prepared by baling fresh TMR, and then sealed by a bale wrapper and stored outdoors at 5 to 30 °C for over 6 mo. Dry matter intake and milk yield were lower for cows fed 40% BR than for cows fed 40% corn. The ruminal pH and total volatile fatty acid concentrations were not affected by dietary treatment. The ruminal ammonia-N concentration decreased as the percentage of BR in the diets was elevated. The proportion of acetate decreased, and that of propionate and butyrate increased with the increasing levels of BR. Plasma urea-N concentrations was lower and glucose and insulin concentrations were higher for cows fed 40% BR than for cows fed 40% corn. The whole-tract apparent digestibility of dry matter, organic matter, and starch increased, and the digestibility of neutral detergent fiber and acid detergent fiber decreased with the increasing BR level in the diet, with no dietary effect on crude protein digestion. As a proportion of N intake, the urinary N excretion was lower and the retention of N was higher for cows fed 40% BR than for cows fed 40% corn, with no dietary effect observed on N secretion in milk and fecal N excretion. These results show that substituting BR for corn decreases urinary N losses and improves N utilization, but causes adverse effects on milk production when cows are fed high-grain diets at 40% of dietary dry matter.

Mastitis is most critical disease in dairy cows and causes huge cost in the dairy industry. To prevent and treat it, it is important to understand the mechanisms of immune function in the mammary gland. Innate immunity is non-specific acute-response immune function. Some components of innate immunity in the mammary gland are found, e.g. Lingual Antimicrobial Peptide (LAP), Lactoferrin (LF). These components are found to be localized in the alveolar epithelium of mammary gland. LAP belongs to the β-defensin family, and plays a crucial role in killing a large variety of microorganisms. LF belongs to an iron-binding glycoprotein and has antibacterial activity. It is reported that LF has been localized immunohistochemically in mammary epithelial cells of lactating cows. Our previous study revealed that secretion of LAP into milk proceeded to that of LF after lipopolysaccharide (LPS) injection into the mammary gland. From this result, it is hypothesized that immunohistochemistry probably shows positive to either LF or LAP but not both in the alveolus vs epithelium in the mammary gland. Therefore, the aim of the present study is to investigate the immunolocalization of LAP and LF in the same bovine mammary tissue. Bovine mammary tissues were collected in the slaughterhouse and were fixed with neutralized formalin immediately. Paraffin sections (2-um thickness) were processed with antigen retrieval treatment followed by blocking with casein milk. Sections were cultured with LF antibody or LAP antibody. Immunoreaction products were visualized by incubation with a DAB. LAP and LF were localized in the cytoplasm of epithelial cell of alveolus. In some cases, LAP and LF were seen clearly in the same alveoli of section. In other cases, some epithelial cells were stained only LAP, but not LF, and other epithelial cells of alveolus were stained only LF, but not LAP. These results suggest the possibility that LAP and LF are differentially synthesized in the alveolar epithelium and may support our previous findings that their secretion occurs at the different time course.

The objective of this study was to evaluate the effect of substituting brown rice grain for corn grain in total mixed ration (TMR) silage containing food by-products on the milk production, whole-tract nutrient digestibility and nitrogen balance in dairy cows. Six multiparous Holstein cows were used in a crossover design with two dietary treatments: a diet containing 30.9% steam-flaked corn (corn TMR) or 30.9% steam-flaked brown rice (rice TMR) with wet soybean curd residue and wet soy sauce cake. Dietary treatment did not affect the dry matter intake, milk yield and compositions in dairy cows. The dry matter and starch digestibility were higher, and the neutral detergent fiber digestibility was lower for rice TMR than for corn TMR. The urinary nitrogen (N) excretion as a proportion of the N intake was lower for rice TMR than for corn TMR with no dietary effect on N secretion in milk and fecal N excretion. These results indicated that the replacement of corn with brown rice in TMR silage relatively reduced urinary N loss without adverse effects on feed intake and milk production, when food by-products such as soybean curd residue were included in the TMR silage as dietary crude protein sources.

Some anthocyanins have been reported to exhibit antioxidant activity and to enhance antioxidant enzyme activity. The aim of this study was to determine the fermentative quality of purple rice, which is abundant in anthocyanin, and its effects on digestibility, ruminal fermentation and oxidative status markers in sheep. The whole crop silages of purple and control rice were prepared in drum silos after the materials were chopped and lactic acid bacteria was inoculated. These were followed by a feeding trial using eight wethers in a 2 × 2 crossover design. Both the purple and control rice silages showed excellent fermentative qualities. Although the purple rice silage contained abundant anthocyanin, it had inadequate nutritional values in comparison to the control rice silage, including higher levels of fiber fractions, lower nutrient digestibility and lower production of ruminal volatile fatty acids. The feeding of the purple rice silages led to an elevation in plasma activity of superoxide dismutase, which is an important antioxidant enzyme. The results of this study suggest that purple rice may become a source of anthocyanin to improve oxidative status in ruminants. To achieve that, however, a new variety of purple rice with good nutritional value is needed for practical use as a ruminant feed.

Anthocyanins are pigments found widely in plants. They have been reported to possess antioxidant activity and an enhancing effect on superoxide dismutase (SOD) activity in monogastric animals. Thus, anthocyanins could also affect the status of the antioxidative defense system in ruminants. This study aimed to investigate the effect of the supplementation of purple pigment from anthocyanin-rich corn on blood antioxidant activities and oxidation resistance in sheep. Twelve sheep were used in a crossover design with two dietary treatments: a diet with purple corn pigment (pigment treatment) and one without (control). The pigment was added to the diets at an anthocyanin concentration of 0.5%. Blood and urine samples were collected to determine the markers of oxidative status. A portion of the plasma was used for an oxidation resistance test, in which plasma was incubated with an oxidizer at 37 °C for 0, 4 and 8 h, and then the degree of oxidation was measured. The pigment addition to the diet caused a significant increase in the SOD activity of the plasma, although the total antioxidant capacity and glutathione concentration in the plasma of both treatment groups were similar. In the oxidation resistance test, the oxidation in the plasma from the pigment treatment group was significantly suppressed compared to that in the control group at 4 and 8 h after the onset of the incubation. These findings indicate that the intake of purple pigment from anthocyanin-rich corn increases the SOD activity and the potential of the antioxidant activity of plasma in sheep.

Nine multiparous Holstein cows were used in a replicated 3 × 3 Latin square design to determine the effects of substituting corn grain with brown rice (BR) grain in total mixed ration (TMR) silage on milk yield, ruminal fermentation and nitrogen (N) balance. The TMR silages were made from the ensiling of TMR containing (dry matter basis) 50.1% forage in rice silage and corn silage combination, and 49.9% concentrate. The grain portion of the diets contained 31.2% steam-flaked corn, 31.2% steam-flaked BR or an equal mixture of corn and BR. Dietary treatments did not affect dry matter intake, milk yield and milk fat, protein and lactose yields. The ruminal pH and total volatile fatty acid concentrations were not affected by dietary treatment. The urinary N excretion decreased linearly (P < 0.01) in response to increased levels of BR, with no dietary effect on N intake, N secretion in milk and fecal N excretion. Our results indicate that steam-flaked BR is a suitable replacement for steam-flaked corn in dairy cow diets, and that it can be included in rations to a level of at least 31.2% of dry matter without adverse effects on milk production, when cows were fed rice silage and corn silage-based diets.

Anthocyanin in purple corn (Zea mays L.) has been reported to show several functional and biological attributes, displaying antioxidant, antiobesity and antidiabetic effects in monogastric animals. The objective of this study was to investigate the effect of feeding anthocyanin-rich corn (Zea mays L., Choko C922) silage on digestibility, milk production and plasma enzyme activities in lactating dairy cows. The cows were fed diets based on the control corn or the anthocyanin-rich corn silage (AR treatment) in a crossover design. The anthocyanin-rich corn silage-based diet had a lower starch content, nutrient digestibility and total digestible nutrients content when compared to the control diet. The milk yield, lactose and solids-not-fat contents in the AR-treatment cows were lower than in the control cows. The feeding of the anthocyanin-rich corn silage led to a reduction in aspartate aminotransferase (AST) activity and an increase in superoxide dismutase (SOD) activity in the plasma. These data suggest that the anthocyanin-rich corn has a lowering effect on AST activity with concomitant enhancement of SOD activity in lactating dairy cows. However, a new variety of anthocyanin-rich corn with good nutritional value is needed for practical use as a ruminant feed.

To estimate the response to hormone replacement therapy (HRT) by bone metabolic markers, 36 patients with postmenopausal osteoporosis or osteopenia were studied to assess the correlation between percent baseline changes in lumbar bone mineral density (BMD) after 12 months and those in various bone metabolic markers after 3, 6, and 12 months of HRT. All the patients were treated with 0.625 mg of conjugated estrogen and 2.5 mg of medroxyprogesterone per day and continued for 12 months. BMD was significantly increased up to 4.19 +/- 0.87% after 6 months and 4.93 +/- 1.27% after 12 months of HRT (p = 0.0001 by analysis of variance). In accordance with this, changes in the levels of osteocalcin (p = 0.041), alkaline phosphatase (p = 0.0001), N-terminal osteocalcin (p = 0.0001), urinary excretion of pyridinoline/Cr (p = 0.0001), and deoxypyridinoline/Cr (p = 0.0001) were significantly decreased, respectively. Among these bone metabolic markers, only the change in the serum N-terminal osteocalcin at 3 months (r = 0.557, p = 0.0022), at 6 months (r = 0.470, p = 0.0184), and at 12 months (r = 0.545, p = 0.0061) significantly correlated with the change in BMD 12 months after HRT. The elution profiles of immunoreactive osteocalcin-related molecules in serum fractionated by reverse-phase high performance liquid chromatography revealed that the N-terminal fragment as well as the intact osteocalcin molecule decreased after 3 months of HRT. These results demonstrate that N-terminal osteocalcin is a suitable predictor for estimating good responders to HRT in postmenopausal women.

The fermentative quality and quantitative change in anthocyanin of anthocyanin-rich corn (Zea mays L.) during storage and in vitro ruminal fermentation were studied. The anthocyanin-rich corn silages in bag silo, drum silo and round bale had good fermentative qualities, such as low pH ( 5% DM) and butyric acid-free, and its quality was maintained for more than 370 d. The amount of anthocyanin in the anthocyanin-rich corn decreased after ensiling by about 45% (from 3.34 to 1.88 mg/g DM), but stayed constant after day 60. The in vitro incubation of the anthocyanin-rich corn with ruminal fluid revealed little degradation of anthocyanin. These results indicate that the anthocyanin had no negative effect on silage fermentation, and the anthocyanin-rich corn silage is utilizable for practical use as a feedstuff. Our results also demonstrate alteration of the anthocyanin content during storage, and show that anthocyanin-rich corn is a suitable antioxidant source for ruminants because of the high stability of the anthocyanin in ruminal fluid.

An experiment was conducted to examine feed intake, apparent digestibility, nitrogen balance, ruminal fermentation and blood components of wethers fed diets containing increasing levels of wet green tea grounds (WGTG). The experimental design was a 4 x 4 Latin square with four wethers in four 15-day periods. Wethers were allowed access to diets ad libitum, and allotted to one of four treatments in which WGTG replaced 0% (no WGTG added, CTG), 5% (low level, LBG), 10% (medium level, MTG), and 15% (high level, HTG) of total mixed ration (TMR) dry matter (DM) as wet brewers grains (WBG). All TMR silages were ensiled for 120 days and, irrespective of the WGTG addition, they were well preserved with a high lactic acid content, low pH and ammonia-N contents. There were no differences among treatments in feed intake, with the exception of ether extract intake (P = 0.032). Digestibilities for LTG and MTG treatments were not different from CTG. However, the organic matter, crude protein, acid detergent fiber and energy digestibilities for HTG treatment were lower than the CTG (P < 0.05). As the level of WGTG increased, nitrogen intake did not differ, but fecal nitrogen increased (P = 0.002), while urinary nitrogen decreased (P < 0.001). No differences among treatments were found in pH level and volatile fatty acids concentrations. However, the ruminal ammonia-N of the HTG silage was lower than for the CTG silage at all times (P < 0.05). Increasing concentrations of WGTG in the TMR silage decreased (P = 0.019) plasma urea nitrogen content. Therefore, the possible mixing proportion of WGTG for TMR silages can be 10% of the diet DM.

The effects of the lengths of growth of round bale whole-crop corn silage on its nutritive value, the methane production, chewing time, and ruminal fermentation were evaluated in three Holstein steers (599.3 ± 31.5 kg). Corn forages were harvested, baled and wrapped on the same day at 129 (D129), 118 (D118) and 107 (D107) days from planting. All steers were fed the three corn silages along with urea at maintenance energy requirements during three 14-day periods in a 3 × 3 Latin square design. A balance trial was conducted to investigate whole tract digestibility by the collection of total feces and urine over a 7-day test period. For 2 days of the test period, a head hood-type respiration chamber was used to measure methane production. Ruminal fluid was collected immediately before and 3 h after, the morning feeding on the last day of the test period. There was no spoilage in any of the silages (n = 9). The average fresh weight of the bales was 326.4 ± 10.7 kg. The DM content of the corn silage was highest in D118 and lowest in D129 (P

The aim of this research was to investigate the effect of peppermint feeding on dry matter intake, nutrient digestibility, ruminal fermentation and milk production in early lactating cows. Four Holstein cows were offered a diet with 5% dried peppermint and four Holstein cows remained on a diet without 5% of dried peppermint on a dry matter basis. The addition of peppermint to feed did not affect dry matter intake, although the eating time of feed was increased by mixing the feed with peppermint. There were no significant differences in the nutrient digestibilities between the two treatments. The ruminal ammonia and volatile fatty acids concentrations were similar in the two treatments, however, peppermint ingestion by cows led to a decrease in ruminal pH. The lowered pH value was within the stable pH condition range. No significant differences in the treatments were observed in milk production or milk composition except for the milk fat content. These results suggest that feeding peppermint to early lactating cows had little effect on their dry matter intake, nutrient digestibility, ruminal fermentation and milk production.

The effects of green tea (Camellia sinensis) waste silage and supplemental polyethylene glycol (PEG) on rumen fermentation and blood components were studied in cattle. Six Holstein steers were fed three diets in a 3x3 Latin square design, replicated twice. One diet was a control with no added silage, and the other two diets were supplemented (20% of the dry matter) with green tea waste silage either with (PEG) or without PEG (tea). Most of the fermentation parameters including major volatile fatty acids (VFA) were not affected by the diet treatments. The concentrations of high density lipoprotein cholesterol in the PEG group and urea nitrogen in the tea and PEG groups were greater than those in the control before morning feeding. The plasma 6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid equivalent concentration was not different before morning feeding, but 3 h after morning feeding, its concentrations in both the tea and PEG groups were higher than in the control. Although the concentration of plasma vitamin A in the animals was not affected by feeding green tea waste silage, the concentrations of plasma vitamin E were significantly higher in the tea and PEG groups than in the control, both before and 3 h after morning feeding. The results from the present study suggest that feeding diets containing 20% of the dietary dry matter as green tea waste silage to Holstein steers has no negative impact on their ruminal fermentation, and increases their plasma antioxidative activity and concentration of vitamin E.

茶系飲料製造残渣の飼料特性を明らかにする研究の一環として，乳酸菌および繊維分解酵素添加緑茶飲料製造残渣サイレージの乾物（DM）および粗タンパク質（CP）のウシのルーメン内における分解性を，数種飼料と比較検討した．緑茶飲料製造残渣サイレージのルーメン内における消失率は高く，DMおよびCPの消失率は浸漬24時間でそれぞれ82.0％と91.7％であった．また，DMおよびCPのルーメン内分解パラメータでは，易分解性区分割合a（％）が31.5と21.5，難分解性区分割合b（％）が55.0と75.7，b区分の分解速度定数c（/h）が0.089と0.092と推定された．ルーメン内通過速度定数（/h）を0.05と仮定し，ルーメン内分解パラメータから計算されたDMの有効分解率は66.8％であり，大豆粕，豆腐粕およびアルファルファヘイキューブと差がなかった．また，CPの有効分解率は70.6％であり，大豆粕と差が認められなかった．さらに，両分解率ともビール粕とビートパルプより有意に高かった（P＜0.01）．これらの結果から，緑茶飲料製造残渣を乳酸菌および繊維分解酵素添加サイレージに調製した場合，そのルーメン内における消失速度は速く，CPの有効分解率は大豆粕並みであると考えられた．

1.無給与牛と比較して,レモングラス給与牛,クローブ給与牛はラジカル捕捉活性および総ポリフェノール量が有意に増加した.増加の程度は給与量が多いほど大きかった.2.ハーブ牛乳のラジカル捕捉活性とポリフェノール量の間には高い相関が見られた.また,ポリフェノールがラジカル捕捉活性に大きく寄与していた.3.レモングラス給与牛ではシトラールが,クローブ給与牛ではオイゲノールがそれぞれ乳中に検出されたが,これらの精油成分はラジカル捕捉活性にほとんど寄与していなかった.

ウシにおける緑茶飲料製造残渣サイレージの栄養価を求めると共に窒素およびエネルギー出納に及ぼすポリエチレングルコール（PEG）の影響を調べた．チモシー乾草，圧ぺんトウモロコシと大豆粕を組成とした基礎飼料のみを給与（対照区），乾物（DM）比で基礎飼料の20％を緑茶飲料製造残渣サイレージに置換（茶殻区）およびPEGを茶殻区の飼料中タンパク質量の1/5となるように添加（PEG区）する3試験区を設けた．各区にホルスタイン種去勢ウシ2頭ずつを割り当て，3×3のラテン方格法による消化試験を実施した．間接方法により求められた緑茶飲料製造残渣サイレージのDM中可消化養分総量（TDN），可消化エネルギー（DE）および代謝エネルギー（ME）は，それぞれ69.8％，13.0MJ/kgと10.5MJ/kgであった．PEG区では，茶殻区に比べ粗脂肪（EE）の消化率が有意に減少した（P＜0.01）ものの，粗タンパク質（CP）の消化率が有意に上昇した（P＜0.05）．PEGに添加により，糞への窒素排出量は有意に減少した（P＜0.05）が，尿中への窒素排出量が増加したため，体内窒素蓄積量は増加しなかった．以上の結果から，緑茶飲料製造残渣サイレージのウシでの栄養価が明らかとなり，PEGの添加により緑茶飲料製造残渣サイレージに由来するタンニンとタンパク質の結合が解離する可能性が示唆された．

緑茶飲料製造残渣サイレージの給与が泌乳牛の採食量，飼料消化率，ルーメン発酵，血液性状および乳生産に及ぼす影響について検討した．緑茶飲料製造残渣サイレージを飼料乾物中に10％含む飼料（茶殻区）または含まない飼料（対照区）を給与する2試験区を設け，各区に乾乳直前のホルスタイン種乳牛4頭ずつを割り当て，馴致期を7日間，予備期を7日間および本試験期を8日間とする消化試験を実施した．採食量および乾物消化率に両区間で有意差を認めなかったが，粗タンパク質（CP）の消化率は茶殻区で低い傾向を示した．ルーメン内容液のpH，プロトゾア数および総揮発性脂肪酸（VFA）濃度に区間差はなかったが，酪酸のモル比率とアンモニア態窒素濃度は茶殻区で有意に低かった（P＜0.05）．血漿中のグルコースとコレステロール濃度に区間差はみられなかったが，茶殻区で尿素態窒素濃度が有意に低く（P＜0.05），トリグリセリド濃度が高い傾向を示した．乳量（対照区25.1kg ; 茶殻区24.5kg），乳脂率（対照区4.2％ ; 茶殻区4.1％）および乳タンパク質率（対照区3.6％ ; 茶殻区3.5％）に区間差はなかったが，乳糖率（対照区4.4％ ; 茶殻区3.9％）は茶殻区で有意に低かった（P＜0.05）．以上の結果から，緑茶飲料製造残渣サイレージは，乳量25kg前後の泌乳牛に対し飼料乾物中に10％程度の給与ならば，ルーメン発酵，血液性状，乳量および乳成分に大きな影響を与えずに利用できる可能性が示唆された．

We describe an immuno-polymerase chain reaction (immuno-PCR) assay for the detection of human angiotensinogen using identical first and second polyclonal antibodies. The reporter DNA was initially generated by PCR amplification using a biotinylated primer, and was bound with streptavidin to biotinylated second antibody. Human recombinant angiotensinogen sandwiched by antibodies was detected by amplifying the reporter DNA using PCR. To reduce the effect of nonspecific amplification, the optimal concentrations of streptavidin and DNA label were determined to be 0.1 mg/l and 0.5 ng/l, respectively. The detection limit of the immuno-PCR assay was 0.1 ng/l, an approximately 2.5 ×10 5 -fold improvement compared with a conventional enzyme-linked immunosorbent assay. These results indicate that a highly sensitive immuno-PCR for human angiotensinogen can be developed even with identical first and second polyclonal antibodies.

As a novel method for measuring bone resorption, an immunoassay system for human N-terminal osteocalcin (N-OC) was developed to determine if osteocalcin molecules are released from bone degraded by osteoclasts in vitro. The assay system employed a monoclonal antibody to the first 20 N-terminal residues of osteocalcin as the solid phase and polyclonal antibodies against these same residues as an enzyme conjugate. This assay system could detect the N-terminal portion of osteocalcin formed during the degradation of the human osteocalcin molecule by trypsin or cathepsin D. Osteoclasts were isolated from human alveolar bone and cultured on human bone slices; the osteocalcin content in the media from these cultures was measured by this N-OC assay method. The N-terminal fragment of osteocalcin was the major form of osteocalcin released during osteoclastic bone resorption along with small amounts of intact osteocalcin. Interleukin-1 (IL-1β) and interleukin-6 (IL-6), stimulators of osteoclastic bone resorption, increased the N-OC level by 1.5 fold compared with the level of N-OC in osteoclast cultures in the absence of stimulators. In contrast, treatment of the cultures with calcitonin or an inhibitor of cathepsin D (E-64), both of which inhibit osteoclastic bone resorption, decreased the amount of N-OC in the culture supernatants. These results suggest that the N-terminal fragment of osteocalcin is released during osteoclastic bone resorption and that its level may serve as an index of bone resorption in vitro.

A monoclonal antibody (10B) against the N-terminal sequence of human osteocalcin was selected to characterize its epitope and species specificity. The cross-reactivity of 10B with human and rat osteocalcin demonstrated that the reactivities of 10B with both human and rat osteocalcins were very similar. The pin technology method was used to determine the epitope and clearly demonstrated that the epitope recognized by 10B was localized to residues 12–16, the sequence of which is identical in rat and human osteocalcin molecules. This monoclonal antibody was found to be useful for designing region-specific sandwich immunoassay systems for human and rat N-terminal osteocalcin (N-OC) molecules using rabbit anti-hN20 and anti-rN20 polyclonal antibodies, respectively. The osteocalcin levels in serum determined by this N-OC method were stable during prolonged storage at 25°C and the method could be usefully applied in the development of immunoassay systems for many osteocalcin molecules from many other species.

Interleukin-4 (IL-4) potently inhibits bone resorption by preventing the differentiation of osteoclast precursors to osteoclasts. To elucidate the role of IL-4 in bone formation, we studied the effects of human IL-4 on human osteoblast-like cells obtained from trabecular bone, which showed increased osteocalcin production in response to 1,25-(OH)2D3 in more than 10 passages. IL-4 stimulated the proliferation of osteoblast-like cells in a concentration-dependent manner, showing the minimal and maximal stimulatory effects at 10 pg/ml and 100-1000 pg/ml, respectively. IL-4 also stimulated the expression of alkaline phosphatase mRNA (1.7-fold) and the enzyme activity to the same extent at 10-100 pg/ml. Furthermore, IL-4 stimulated collagen type I mRNA expression in human osteoblast-like cells. The cytokine did not affect osteocalcin production in a short culture period (3 days). These in vitro findings suggest that IL-4, a bone-resorption-inhibitory cytokine produced by activated T cells in bone marrow, may exert an anabolic effect on osteoblast-like cells in trabecular bone through a paracrine mechanism.

Direct immunoperoxidase technique using human monoclonal antibody (C7) against a p65 antigen of cytomegalovirus (CMV) has been utilized to detect CMV antigen-positive leukocytes in the peripheral blood (CMV antigenemia). This technique was evaluated for enumeration of CMV antigen-positive leukocytes. The parameters included stability of the reagents, reproducibility of the results and quantitativity of the detection. The horseradish peroxidase (HRR)-labeled F(ab')2 fractions of C7 antibody were found to be superior to the equivalent Fab' fractions, because the former was much more stable than the latter. Enumeration of the CMV antigen-positive leukocytes were very reproducible and quantitative. The detection limit was one CMV antigen-positive cell per 50,000 leukocytes. Thus, this technique is reliable and practical to detect the CMV antigenemia, and it will contribute to early diagnosis of CMV diseases and initiation of antiviral therapy.

We studied diagnostic significance of measuring the plasma levels of heparin cofactor II (HC II) and thrombin-heparin cofactor II complex (T-HC II) in patients with disseminated intravascular coagulation (DIC). The plasma levels of HC II, T-HC II and other factors—thrombin-antithrombin III complex (TAT), antithrombin III (AT III), α2-plasmin inhibitor-plasmin complex (PIC), FDP-E, protein C (PC), protein C inhibitor (PCI), and fibrinogen—were measured in 9 patients with DIC and 31 control subjects. HC II activity was measured by a chromogenic assay. HC II antigen was measured by immunoelectrophoresis. T-HC II was measured by enzyme immunoassay. HC II activity was correlated with its antigen. In the patients with DIC, HC II was correlated with AT III, PC, PCI and fibrinogen. T-HC II increased in all patients with DIC (vs control value, p

Lingual antimicrobial peptide (LAP), one of the β-defensins in bovines, and lactoferrin (LF) are synthesized in mammary epithelium and have bactericidal and bacteriostatic functions. However, it is not known whether they have similar expression patterns. Therefore, the present study was undertaken to compare (1) immunolocalization of LAP and LF in the mammary gland and (2) changes in concentration of these two components in milk after lipopolysaccharide (LPS) challenge. Bovine mammary tissues without LPS challenge were collected and their sections were immunostained with antibodies to LAP or LF. Milk from our previous study was collected every hour up to 12h and twice daily from d 1 to 7 after LPS challenge (the day of infusion was considered as d 0). These milk samples were measured for LAP but not LF in our previous report. Therefore, concentration of LF was measured by enzyme immunoassay in the present study. Epithelial cells of some alveoli showed immunopositive reaction for LF, but negative for LAP. Conversely, some alveoli were LAP positive in their epithelial cells but LF negative. Many alveoli had immunoreactions for neither LAP nor LF. The concentration of LAP in milk was elevated significantly at 3h after LPS infusion compared with pre-infusion values and remained at a high level until 12h. However, LF concentration in milk remained low at d 0 and increased at d 2. These results suggest that LAP and LF were mostly differentially localized in the alveolar epithelium in mammary glands. The different spatial expressions between them may be associated with their different temporal expression mechanisms.

Four independent antibodies (6A, 5F, 2H and 2F) to Glutathione S-transferase pi (GST-pi) were selected to characterize their epitopes. Amino acid analysis of 5.6 K and 7.4 K tryptic peptides appeared to suggest that the epitope recognized by antibodies 2H and 2F is located in the N-terminal 44 peptides of GST-pi, and that of 6A and 5F is located in the C-terminal 69 peptides. Reactivities of antibodies 6A and 5F with two synthetic peptides indicated that 6A recognized an epitope in the C-terminal hydrophilic fragment 176Leu-209Gln, and could be distinguished from 5F which recognized an epitope in the 141Thr-176Leu hydrophobic fragment. The differential immuno-reactivity of antibodies 6A and 5F with GST-pi itself, was characterized by the particularly high reactivity of 6A and almost no reactivity of 5F with the natural conformation of GST-pi in solution. This may be explained by differences in the hydropathic natures of their epitopes. The 6A antibody was useful for immunodetection of GST-pi in circulation, while 5F was found to be most suitable for histochemical staining of tumor tissue.

Free radical telomerization of vinylene carbonate with bromoform (and carbon tetrabromide) afforded two isomeric telomers, dibromomethylene-4, 4'-bis (5-bromo-1, 3-dioxolan-2-one) (5a, b) as two fold addition products in addition to the 1 : 1 adducts. On the similar treatment, this type of products (7a, b 9a, b) was obtained from 5-substituted-4-tribromomethyl-1, 3-dioxolan-2-ones. A sequence of reactions involving methanolysis and reductive photolysis to give methyl 5-methoxypyranosides permitted the stereochemical assignment of 5a and b as dl (anti)-and meso (syn)-configurations, respectively.

A quantitative assay of IgA class circulating immune complexes (IgA-CIC) by a solid phase anti-C3 enzyme immunoassay (anti-C3 EIA) is described. A stable and reproducible standard for determination of IgA-CIC was prepared successfully by chemical binding of complement C3 to human serum IgA. Two of 27 sera from patients with systemic lupus erythematosus (SLE), however, contained high concentrations of IgA class anti-F(ab′) 2 antibodies that caused false positive results when the F(ab′) 2 of anti-C3 was used for EIA. Solid phase Facb of anti-C3 was found to eliminate the false positive results caused by IgA class anti-F(ab′) 2 and IgA class rheumatoid factor. Good reproducibility and recovery were observed with this Facb anti-C3 EIA using the IgA-C3, a stable standard material, and so this method should be useful clinically in elucidating the role of IgA-CIC.

A simple, improved immunoassay for the determination of human surfactant protein A (SP-A) in human amniotic fluid was developed. The immunoreaction with a monoclonal antibody PC6-immobilized plastic bead, peroxidase-labeled monoclonal antibody PE10 and amniotic fluid sample diluting in the buffer containing 0.6% sodium dodecyl sulfate/2% Triton X-100, was carried out simultaneously at 45 degrees C for 30 min in test tubes. After washing the bead with 2% skimmilk in phosphate buffered saline containing 1% Triton X-100, the peroxidase reaction was developed by adding the substrate reagent and the absorbance was measured. The amniotic fluid obtained at full term was used as a standard instead of purified SP-A, because of the stability of the antigenicity. This immunoassay method was used to measure SP-A in 69 samples of amniotic fluid from 22 to 41 weeks of gestation. The result indicated that the SP-A values obtained by the present immunoassay can be used for predicting the fetal lung maturity. This simplified monoclonal immunoassay completed the measurement within 1 hr, and so it could be used routinely in clinical laboratory.

Free radical telomerization of vinylene carbonate with bromoform (and carbon tetrabromide) afforded two isomeric telomers, dibromomethylene-4, 4'-bis (5-bromo-1, 3-dioxolan-2-one) (5a, b) as two fold addition products in addition to the 1 : 1 adducts. On the similar treatment, this type of products (7a, b 9a, b) was obtained from 5-substituted-4-tribromomethyl-1, 3-dioxolan-2-ones. A sequence of reactions involving methanolysis and reductive photolysis to give methyl 5-methoxypyranosides permitted the stereochemical assignment of 5a and b as dl (anti)-and meso (syn)-configurations, respectively.