| 1. Analysis of cis-9, tran-11-CLA in milk fat by capillary gas chromatographyA capillary gas chromatography (GC) methodology was developed. Flame ionization detector (FID) was equipped on GC for analyzing cis-9, trans-11-CLA in milk fat. Lipid in milk fat was extracted with Hexane: Isopropanol (V/V, 3:2) and methylated with methanol-NaOMe. Conjugated linoleic acid (cis-9, trans-11-CLA) was separated and quantified using gas chromatography. Retention time of the peaks was applied for qualitative analysis. External standard method was used for quantitative analysis. The recovery ratio of cis-9, trans-ll-CLA was 100.14%. The relative standard deviation of precision was 3.77%. This method presented advantage of high precision, high sensitivity, small sample volume, and simple pretreatment. It was also significant for determining other fatty acids content in milk and milk products.2. Survey and investigation of fatty acid profile and CLA content in milk fat2.1 Effect of lactation stage on CLA content in milk fat from dairy cows Milk fat from dairy cows is the main source of conjugated linoleic acid (CLA), which is a functional food component with health benefits. Holstein cows (n=60) were fed the same diet and milk samples were taken on the same day. The objective of the survey was to examine CLA content in milk fat. The result showed that the range of CLA content was from 2.2 to 7.98 mg/g fatty acid. CLA content in milk fat varied over threefold among individuals. Parity in milk had no obvious relationship with the individual variation of CLA content, but different lactaion stage in milk had a little influence on CLA variation. 2.2 Effect of seasons on CLA content in milk fat from dairy cowsMilk samples of Holstein cow (n=30) were taken from Nanjing in spring (March ) and summer (June) .The objective of this survey was to investigate the alteration of fatty acid profile in dairy-cows' milk in different seasons, with particular emphasis on CLA. The variation of fatty acid profile in milk fat of dairy cows was attributed to seasonal change of CLA. The CLA content in milk fat in summer (11.9mg/g fatty acid) was significantly higher( P<0.05 )than in March (5.1mg/g fatty acid), and the former concentration in milk fat doubled. The component of diet varied with different season, which indicated that the content of conjugated linoleic acid in milk fat could be enhanced by dietary intake of fresh grass in summer.2.3 Comparision of fatty acid composition of between goat and cow milk fat Fatty acid profile of goat and cow milk was compared. Goat milk samples (n=20) were collected and fatty acid composition in milk fat was analyzed. The results showed that in comparison with cow milk, goat milk fat was significantly rich in C8:0 and C10:0. However, cow milk was significantly rich in C4:0,C18:0,C18:1,CLA. The investigation indicated that the regulation of lipid metabolism and mammary cell function were different between goat and cow. Goat milk was rich in short-, medium-chain fatty acids which had nutritional and medical benefits to human health.3. Manipulation of CLA content in milk fat of goat by sunflower oil, Hainanmycin and calcium pyruvate and its involved mechanism3.1 Effect of sunflower oil, Hainanmycin and calcium pyruvate supplementation onCLA content in milk fat of goatsEight lactating goats were fed a diet supplemented with sunflower oil, Hainanmycin and calcium pyruvate respectively by 4x4 Latin square design, each period lasting for two weeks. The result showed that the percentage of milk fat supplemented with sunflower oil (P<0.05) or calcium pyruvate (P<0.05) was significantly lower than control. Sunflower oil had no effect on milk fat percentage and milk protein. The content of CLA in milk fat was significantly increased supplemented with sunflower oil (P<0.05), Hainanmycin (P<0.05) and calcium pyruvate (P<0.05).3.2 Effect of sunflower oil, Hainanmycin and calcium pyruvate on rumen digestionand metabolism in goats3.2.1 Effect of calcuim pyruvate on rumen digestion and metabolism of goat in vitro Rumen fluid from 3 goats were incubated in anaerobic media for 8h in vitro with hay (75%), concentrate (25%) as the substrate. Calcium pyruvate was added to incubation bottles at 0, 0.1, 0.2, 0.4, 0.8 to 1.6g/L dose, respectively. The result showed that TVFA was increased from 10.9 to 12.01 mmol/L(P<0.05), acetate from 5.46 to 6.37 mmol/L (P<0.05), the ratio of acetate to propionate from 1.86 to 2.3 (P<0.05), MCP concentration from 30.16 to 94.49 mg/L(P<0.01) and NH3-N was decreased. It suggested that calcuim pyruvate could improve rumen fermentation.3.2.2 Effect of sunflower oil, Hainanmycin and calcium pyruvate on rumen digestion and metabolism in goatsEight goats installed with permanent rumen fistulae were used by 4X4 Latin square design, each period lasting for 2 weeks, to investigate the effect of sunflower oil, Hainanmycin and calcium pyruvate supplementation on rumen digestion and metabolism. Compared with control, supplemented with sunflower oil and Hainanmycin, the A/P value was decreased by 15.07% (P<0.01) and 10.04% (P<0.05), respectively; the concentration of NH3-N was reduced by 24.39% (P<0.05) and 11.17%, respectively. The content of TVFA (P<0.05) and MCP (P<0.05) were increased by sunflower oil supplementation. The A/P ratio (P<0.05) and TVFA content (P<0.05) were increased by calcium pyruvate. The results suggest that sunflower oil, Hainanmycin and calcium pyruvate supplementation can improve rumen fermentation. The percentage of propionate in rumen was increased by sunflower oil and Hainanmycin supplementation and dietary protein degradation in rumen was reduced. Acetate was increased significantly by calcium pyruvate supplementation.3.2.3 Effect of sunflower oil, Hainanmycin and calcium pyruvate on TVA and CLA concentration in rumen fluid of goatsEight goats installed with permanent rumen fistulae were used by 4 X 4 Latin square design, each period lasting for 2 weeks, to determine the effect of sunflower oil, Hainanmycin and calcium pyruvate supplementation on TVA and CLA concentration in rumen fluid. The result showed that sunflower oil, Hainanmycin and calcium pyruvate supplementation led to obvious increase in C16:0, C18:0, C18:l, TVA and CLA content and order was as follows: Sunflower oils > Hainanmycin > Calcium pyruvate >control. Strong positive correlationship was observed between cis-9, trans-11 CLA and trans-11, C18:1 (TVA) (r=0.57, P<0.01). 3.3 Regulation of goat mammary gland function by sunflower oil, Hainanmycin andcalcium pyruvate supplementation and involved molecular mechanism3.3.1 Effect of sunflower oil, Hainanmycin and calcium pyruvate on hormones related to lipid metabolism and biochemical parametersEight lactating goats were fed a diet supplemented with sunflower oil, Hainanmycin and calcium pyruvate, respectively, by 4×4 Latin square design, each period lasting for 2 weeks. Insulin, glucagon. leptin, glucose, triglyceride (TG). cholesterol (TC), low density lipoprotein (LDL), high density lipoprotein (HDL) parameters in serum were measured. TG and TC were increased by 25% (P<0.05) and 15.07% (P<0.05) respectively, supplemented with sunflower oil; TG and LDL were reduced by 9.44% (P<0.05) and 17.24% (P<0.05) respectively, supplemented with Hainanmycin; leptin and glucagon were decreased by 25.32% (P<0.05) and 13.12% (P<0.05), respectively and the ratio of insulin to glucagon was increased (P<0.05) supplemented with calcium pyruvate.3.3.2 Effect of sunflower oil, Hainanmycin and calcium pyruvate supplementation on SCD,ACC,FAS and LPL gene expression of mammary gland in lactating goatsEight lactating goats were fed a diet supplemented with sunflower oil, Hainanmycin and calcium pyruvate, respectively, by 4x4 Latin square design, each period lasting for 2 weeks. The objective was to investigate the change of SCD, ACC. FAS and LPL mRNA expression of mammary gland by RT-PCR with 18S rRNA as internal standard. The result showed that compared with control group, SCD mRNA gene expression was significantly decreased (P<0.05) while LPL was significantly increased (P<0.05) supplemented with of sunflower oil. SCD, FAS and LPL mRNA expression were remarkably increased supplemented with Hainanmycin (P<0.05). SCD and LPL mRNA expression were markedly increased supplemented with calcium pyruvate (P<0.05) while ACC mRNA expression was significantly decreased (P<0.05). Additionally, gene expression of ACC was decreased by 9.45%, 16.54% respectively in sunflower oil and Hainanmycin supplementation group, but no significant differences were found.
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