| Rutin is the flavonoids extracted from the plant, and is widely distributed in nature. Studies have shown that rutin can be used not only in treatments but also as health care products for humans. From perspectives of digestive physiology and the physiology of lactation, the performance of dairy cows depends to a large extent on the rumen digestion and metabolism of nutrients and the development of lactating mammary gland.In this series of researches, dairy cows and Wistar rats were used as animal models to study the effects of rutin supplementation on the production performance of diary cows, internal environment, digestion and metabolism of rumen, and the development and lactation maintenance of mammary gland. Besides, the action mechanisms and using effects of rutin on dairy cows physiological nutrition and mammary gland development were explored from aspects of nutriology and physiology. The present research consists of ten experiments which were listed as follows:EXP 1. Effect of Rutin Supplementation on Lactation PerformanceThis study was designed to investigate the effects of rutin supplementation on milk yield in dairy cows, to find the optimum dosage of rutin and subsequently to offer the scientific basis for the usage of rutin in cows nutrition and feed. Twenty cows in their peak lactation were randomly divided into 4 groups and each was offered a basal diet supplemented with 0, 1.5, 3.0 or 4.5 mg rutin/kg·BW of diet. The trial lasted 11 weeks, including an adaption period of one week and a ten-week experimental period. Milk yields were recorded every other day, and milk compositions were analyzed on the 5th, 35th and 65th day. The experiment showed that, compared to the control group, the cows receiving 3.0 and 4.5 mg·rutin/kg·BW had significantly higher milk yield (P<0.05). No significant difference in milk yield was detected between the cows receiving 1.5 mg·rutin/kg·BW and the control group (P > 0.05). The milk fat content of the cows receiving 1.5 or 3.0 mg·rutin/kg·BW was lower than that of the control group (P<0.05). No significant difference in the milk fat content was observed between the cows offered 4.5 mg·rutin/kg·BW and the control group (P>0.05). The contents of milk protein, lactose and milk non-fat solid matter did not differ significantly among groups (P>0.05). Experiments indicated that dietary rutin improved the production performance of dairy cows,while the main milk compositions were not affected. Under this experimental conditions, the appropriate dosage for rutin supplementation was 3.0 mg/kg·BW diet. EXP 2. Study on the Peak of Rutin in Different Parts of the Gastrointestinal Tract in Dairy Cows4 adult cows with permanent rumen fistula and duodenal cannulae were attributed to a self control design and used to investigate the time and characteristics of peak occurrence in different parts of the gastrointestinal tract in dairy cows. Total mixed rations ( TMR ) were used as a basal diet in control period. Cows were fed with rutin one at a time per day in test period. The levels of rutin and quercetin in rumen fluid, duodenal content and blood were detected once per hour from 1 h to 8 h after feeding. The results showed that the concentration of rutin and quercetin in rumen fluid reached their peak levels at 1 h ( 3.017±0.153μg/ml ) and 2 h ( 1.148±0.153μg/ml ) respectively. The peak level in duodenal content occurred at 6h ( 2.133±0.057μg/ml and 0.214±0.004μg/ml ). Rutin had two peaks in blood at 1 h ( 0.180±0.029μg/ml )and 8 h(0.229±0.014μg/ml)respectively. So did quercetin at 1h(0.064±0.002μg/ml )and 8h(0.089±0.007μg/ml). These results indicated that feeding time and digest positions affected the peak occurrence of rutin and aglycone quercetin in the gastrointestinal tract in dairy cows.EXP 3. Effects of Rutin Supplementation on Blood Metabolites and Levels of Hormone in Dairy CowsBy investigating the changes of blood metabolites and levels of hormone in dairy cows after rutin supplementation, we try to study the effects of rutin on immune function and milk-related hormones. Ten cows in their peak lactation were randomly divided into 2 groups and each was offered a basal diet supplemented with 0.0 and 3.0 mg rutin/kg·BW of diet. The trial was 11 weeks, including an adaption period of one week and a ten-week experimental period. The vein blood samples were analyzed on the 5th, 35th and 65th day. Test serum biochemical indexes, immune indexes and lactation-related hormones. Compared to the control group, no significant difference was observed in the serum concentration of TP, ALB and Cort (P>0.05). But rutin could reduce the content of urea nitrogen in blood (P<0.05).There was no significant difference in the serum concentration of IgG, ALT, AST, ALP and cortisol between the cows receiving 3.0 mg·rutin/kg·BW and the control group (P<0.05). The lysozyme content of the experimental group was higher than that of control group (P<0.05). The serum concentration of estrogen (E2) and progestin (P) did not differ significantly between the treatments (P>0.05), but the serum progesterone level which increased from 12.19 ng/ml on the 5th day to 13.94 ng/ml on the 65th day showed an upward trend in rutin-supplemented cows compared to control. It suggested that rutin could decrease blood urea nitrogen and increase serum lysozyme content.EXP 4. Effects of Rutin on the Ruminal Digestibility and Metabolism of Dairy CowsThe study was designed to investigate the role of rutin on the ruminal digestibility and metabolism of dairy cows, and subsequently to provide data and references to exploring the effect of rutin on ruminal fermentation. 4 adult cows with permanent rumen fistula and duodenal cannulae were attributed to a self control design and used in the investigation. TMR were used as basal diet in the control period. Cows were fed with rutin one at a time per day in the test period. The levels of pH, ammonia nitrogen ( NH3-N ), volatility fatty acid ( VFA ), the number of rumen protozoa and microbial protein ( MCP ) were detected once per hour from 1 h to 8 h after feeding. The results showed that the rumen fluid pH in the test period ( 5.92 ) was lower than that in the control period ( 6.16 ) (P<0.05). The concentration of NH3-N in the test period ( 11.03 mg/100ml ) was less than that in the control period ( 20.07 mg/100ml ) (P<0.05).Compared to the control period( 83.12 mmol/L ) , the concentration of TVFA in the test period ( 96.72 mmol/L ) was higher (P<0.05). Acetic acid in the test period ( 61.00 mmol/L ) was on a higher level compared to the control period ( 51.47 mmol/L ) (P<0.05) and so was propionic acid in the test period ( 36.35 mmol/L ) compared to the control period ( 19.95 mmol/L ) (P<0.05). There were no significant difference in both the proportion of acetic acid and propionic acid in TVFA (P<0.05). The number and protein content of rumen protozoa in the test period ( 12.54×105个/ml, 3.71 mg/ml ) were less than those in the control period ( 16.37×105个/ml , 5.14 mg/ml ) (P<0.05). In the test period the MCP ( 1.50 mg/ml ) was higher compared to the control period (0.86 mg/ml ) (P<0.05). Those results indicated that rutin could reduce the concentration of NH3-N , increase the TVFA and the concentration of acetic acid and propionic acid, lower pH level, bring down the number and the protein content of rumen protozoa, and increase MCP in the rumen.EXP 5. Study the Effect of Rutin on Activities of Digestive Enzymes in the Rumen in VitroThis study was conducted to investigate the effect of rutin on activities of digestive enzymes in the rumen of dairy cows. 5 healthy about 600 kg-weight China Holstein Cows with permanent rumen fistula were used in this study. Rutin was added into the enzyme ( solid and liquid fraction) extracted from rumen content on the dosage of 0.00 mg/ml (control group), 0.02 mg/ml, 0.04 mg/ml, 0.06 mg/ml respectively and. After mixing, the enzyme activities of protease, amylase, total dehydrogenase, filter paper enzyme,β-glucosidase, carboxymethyl cellulose (CMC), avicelase, pectinase, xylanase were measured. The results showed that compared to the control group, the enzyme activities total dehydrogenase, filter paper enzyme,β-glucosidase, CMC, avicelase in experimental groups significantly differed in both liquid and solid fraction ( P<0.05 ). The enzyme activities of protease were lower than control group ( P<0.05 ). There were no significant difference in the activities about amylase, pectinase and xylanase( P>0.05). In all groups, the enzyme activities of solid fraction were significantly higher than that of liquid faction. It suggested that rutin could enhance the enzyme activities of filter paper enzyme,β-glucosidase, CMC, avicelase in solid and liquid fraction in the rumen of dairy cows. The value of the enzyme activities in solid fraction was higher than that in liquid fraction. EXP 6. Study the Effect of Rutin on the Enzyme Activities of Bovine Colostrum in VitroThe enzyme ( the bioactive substances ) secreted into colostrum by dairy cows was used as an indicator to analyze and evaluate the action of rutin on cow mammary cells. The colostrum samples of five healthy Chinese Holstein cows was collected the day after delivery. Rutin was added into the samples on the dosage of 0(control),10-7,10-6,10-5,10-(4mol/L ). After mixing, the enzyme activities ofβ-N-acetylglucosaminidase ( NAG ), alkaline phosphatase ( ALP ), lactoperoxidase ( LP ),γ-glutamyl transpeptidase (γ-GT ) were measured. Results showed that the enzyme activites of NAG in 10-6,10-5,10-4(mol/L )groups ( 7.61,6.92,6.43 U/L respectively)were lower than that in the control group 9.74(U/L)(P<0.05). No significant difference was observed between 10-7(mol/L)group (8.17 U/L) and the control group (P>0.05). The enzyme activities of ALP did not differ significantly among the treatments (P>0.05). The enzyme activities of LP in 10-7,10-6,10-5,10-4(mol/L)groups ( 58.30,65.00,72.94,77.03 U/ml respectively) were higher than the control group ( 48.89 U/ml )(P<0.05). The enzyme activities ofγ-GT in 10-7,10-6(mol/L)groups (11613.49,11371.16U/L respectively)were higher than 0(mol/L)(10248.84U/L)(P<0.05), but there were no significant differences in 10-5,10-4(mol/L)groups 10036.74,9542.33U/L) ompared to the control group(P<0.05). This result suggested that, under such experimental conditions, rutin could improve the enzyme activities of LP andγ-GT, inhibit the enzyme activities of NAG, and exert no significant effect on the enzyme activities of ALP.EXP 7. Effect of dietary rutin on laction performance in ratsThe objective of this study was to investigate the regulatory effect of rutin on lactation of rats by examining the effects of rutin on milk yields, endocrine hormones and mammary organ indexes of rats. Eighteen pregnant Wistar rats were randomly assigned to 3 treatments: control, gastric infusion of rutin at 60 mg/kg·BW per day, and intramuscular injection of estradiol 60μg/kg·BW weekly from the fourth lactation day for two weeks with the same basal diet. The milk yield of rats, body weight gain of the neonatal rats, mammary gland index, the level of estrogen (E2), progesterone (P), prolactin (PRL) and growth hormone (GH) in plasma and gland tissues were measured. The results shown that the milk yield of the rats receiving rutin ( 7.35 g/h ) was higher than that of the control group ( 5.73 g/h ) (P<0.05), but no obvious difference was observed when compared to the estradiol group ( 8.22 g/h ) (P>0.05). The body weight gain of the neonatal rats in the rutin group had no distinct difference with that in the estradiol group (P<0.05), but these two groups were higher than the control group (P>0.05). The level of E2, PRL and GH in plasma and gland tissues of the rats in the rutin group was higher than that in the control group (P<0.05), but less than that in the estradiol group (P<0.05), and the differences of P level among groups were not distinct (P>0.05). The indexes of mammary gland of the rats in the estradiol and rutin group were higher than that in the control group (P<0.05). Those results indicated that, under such experimental conditions, dietary rutin could promote mammary gland to secrete milk , significantly improve the milk yield of female rats, affect to some extent the development of the thymus, and excert E2-like effects.EXP 8. Effects of Rutin on the Development of Mammary Gland and Levels of Relevant Hormones and Hormonal Receptors of Pubertal Female RatsThe objective of this study was to investigate the regulatory effect of rutin on mammary glands of unmarried rats by studying the plasma level of endocrine hormones, the expression of hormonal receptors and the changes in the histomorphology of mammary tissues. Twenty four intact virgin rats were randomly assigned to three treatments: 1) gastric infusion of 2 ml normal saline per mouse per day (control (Con)); 2) gastric infusion of 60 mg rutin/kg·BW per day (Rutin (Rut)); and 3) intramuscular injection of 60μg estradiol/kg·BW weekly (Estradiol (Est)). The trial lasted two weeks. The concentration of estrogen (E2), progesterone (P), prolactin (PRL) and growth hormone (GH) in plasma and gland tissues was determined, and the expression of estrogen receptors (ER), progesterone receptors (PR), prolactin receptors (PRLR) and growth hormone receptors (GHR) in gland tissues was measured. The histomorphology of mammary tissues was also monitored. Results showed that the concentration of E2 and PRL in plasma and mammary tissues was ranked as Est>Rut>Con, and the differences were distinct (P<0.05), the concentration of P in plasma and mammary tissues in Rut group was higher than that in Con group(P<0.05), and there was no difference between Rut and Est groups(P>0.05).The concentration of GH in Est grouo was higher than that in Rut group, so was Rut group compared to Con group, and the difference was not significant(P>0.05). The expression of ER, PR, PRLR and GHR in mammary tissues was ranked as Est>Rut>Con (P<0.05). The diameter of mammary acinar cells and lumen was higher (P<0.05) in Rut group than that in Con group, but lower than that in Est group (P>0.05). These results suggested that, under such experimental conditions, rutin could improve the plasma concentration of E2 and P in pubertal female rats, promote PRL release, and up-regulate the expression of ER, PR, PRLE and GHR, exert E2-like effects.EXP 9. Experiment Study of Rutin on Mammary Gland Development in Ovariectomized Virgin RatsThe objective of this study was to realize the estrogen-like effect of rutin on promoting mammary gland development by examining the levels of endocrine hormones , the expression of hormonal receptor and the changes in the histomorphology of mammary tissues of ovariectomized virgin rats. Thirty-two ovariectomized virgin rats were randomly assigned to four treatments: sham, gastric infusion of 2 ml normal saline per mouse and per day; Ova, gastric infusion of 2 ml normal saline per mouse and per day; Ova+Rut, gastric infusion of 60 mg rutin/kg·BW per day; Ova+Est, intramuscular injection of 60μg estradiol/kg·BW weekly. The trial lasted two weeks. The samples of blood, gland tissues, thymuses and spleens were collected for quantitative analysis. The level of estrogen (E2), prolactin (PRL) and growth hormone (GH) in plasma and gland tissues were detected and the expression of estrogen receptor (ER), prolactin receptor (PRLR) and growth hormone receptor (GHR) in gland tissues were detected. Histomorphology of development about mammary gland were observed. The results showed that, the level of E2 in plasma in Ova+Rut and Ova+Est were higher than that in Ova (P<0.05),and the difference between these two groups was not significant (P> 0.05), Ova+Rut was lower than Sham (P<0.05), Ova+Est and Sham had no distinct difference(P>0.05). The level of E2 in gland tissues showed the same trend among different treatment groups. The order of the PRL in plasma and gland tissues was Ova0.05), the difference between Ova+Est and Sham was not significant either (P>0.05). The expression of ER, PRLR, GHR in gland tissues in Ova, Ova+Rut, Ova+Est and Sham increased in turn (P<0.05), and showed the same trend. The observation of histomorphology showed that the development of gland tissue in Ova+Rut, Ova+Est and Sham was better than that in Ova (P<0.05). The results suggested that under the experimental conditions, rutin could increase the level of E2 in ovariectomized virgin rats, promote pituitary PRL and GH release, up-regulated the expression level of ER, PRLE and GHR.EXP 10. Effect of Dietary Rutin on the Expression of Hormonal Receptor in Lactating RatsThe objective of this study was to observe effects of rutin on mammary gland development, endocrine hormone level and hormonal receptor expression in lactation rats. Eighteen Wistar rats were randomly assigned to three treatments: control (Lac-Con), gastric infusion of 2 ml normal saline per mouse and per day. Rutin (Lac-Rut), gastric infusion of 60 mg rutin/kg·BW per day. Estradiol (Lac-Est), intramuscular injection of 60μg estradiol/kg·BW weekly. The experiment lasted for two weeks from the fourth lactation day with the same basal diet. The expression of estrogen receptor (ER), progesterone receptor (PR), prolactin receptor (PRLR) and growth hormone receptor (GHR) in gland tissues were measured. Histomorphology of mammary gland development were observed. The results showed that the expression of ER, PR, PRLR and GHR in gland tissues was Lac-Est > Lac-Rut > Lac-Con (P<0.05). The diameter of mammary gland alveolus showed that Lac-Est and Lac-Rut were higher than Lac-Con (P<0.05), there was no significant difference between Lac-Est and Lac-Rut (P>0.05). The results indicated that, under such experimental conditions, rutin had the function of inducing the expression of ER, PR, PRLE and GHR. To sum up, in this dissertation, we systematically studied the effect of rutin on production performance of dairy cows, the regulating action of rutin on rumen digestion and metabolism, and the influence of rutin on the immune function of the mammary gland and the organism. We also explored the role of rutin on mammary gland development and the maintenance of lactating as well as the mechanisms of related endocrine system. The following main conclusions could be drawn according to the experimental results, analysis and discussions. Under the study conditions rutin had the functions as follows:The peak time of rutin and its metabolite– quercetin in the rumen fluid occurred at 1h and 2 h after feeding respectively, in the duodenal content the peak time both occurred at 6 h, and in the blood there were two peaks which both occurred at 1h and 8 h. Dietary rutin added on the dosage of 3.0 mg/kg·BW could obviously promote the milk yield and no significant difference was observed in the milk composition. Dietary rutin could improve rumen fermentation and the nitrogen metabolism, promote the nitrogen metabolism and utilization, bring down the number and the protein content of rumen protozoa to some extent, and increase MCP in the rumen. Dietary rutin could enhance the enzyme activities of total dehydrogenase, filter paper enzyme,β-glucosidase, CMC, avicelase in solid and liquid fraction in the rumen of dairy cows, but had no regulatory role on the immune function and hormone level. It could also increase serum lysozyme content to some degree, and decrease blood urea nitrogen level. Rutin could improve the enzyme activities of LP andγ-GT, inhibit the enzyme activities of NAG, and exert no significant effect on the enzyme activities of ALP in the colostrum of dairy cows. In the experiments using rats as animal model, rutin could promote mammary glands of pregnant rats to secrete milk, have some effect on the development of the rat's thymus, and excert E2-like effects. Rutin could also increase the level of E2 and P in pubertal female rats, promote PRL release, and up-regulate the expression of ER, PR, PRLE and GHR, promote the development of mammary glands, and exert E2-like effects; Rutin could increase the level of E2 in ovariectomized virgin rats, promote pituitary PRL and GH release, up-regulated the expression level of ER, PRLE and GHR; It could also increase the level of E2 in ovariectomized virgin rats, promote pituitary PRL and GH release, up-regulated the expression level of ER, PRLE and GHR, and have the function of inducing the expression of ER, PR, PRLE and GHR.
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