Factors affecting the concentration of sphingomyelin in bovine milk

Sphingomyelin is a phospholipid located in the outer leaflet of the plasma membrane of most cells and is a component of the milk fat globule membrane. Sphingomyelin and its digestion products participate in several antiproliferative pathways that may suppress oncogenesis. Although milk and dairy products are important sources of sphingomyelin in the human diet, little is known about factors that influence sphingomyelin concentrations in milk fat or whether concentrations can be modified via the nutrition of cows. Sphingomyelin concentrations were determined in milk from Holstein and Jersey cows matched for parity and stage of lactation. Sphingomyelin was more concentrated in milk fat from Holstein cows than in milk fat from Jersey cows (1,044 vs. 839 μg/g of fat). Concentrations in whole milk did not differ because of greater milk fat content for milk from Jerseys. Differences between breeds may be related to the greater fat globule size in milk from Jerseys. Sphingomyelin content in whole milk increased with increasing days in milk because of associated increases in milk fat content. Regardless of breed, primiparous cows had greater amounts of stearic acid and less palmitic acid in sphingomyelin than did older cows. The sphingomyelin concentration in milk fat of cows in a commercial Jersey herd was lower for cows in their fourth or greater parity. Sphingomyelin content in whole milk was greater for cows in late lactation because of greater milk fat content. Feed restriction of multiparous Holstein cows to 37% of ad libitum dry matter intake increased milk fat content but did not affect milk sphingomyelin content or milk fat globule size. Supplementation of the diet with 4% soybean oil did not affect milk composition, sphingomyelin content, or milk fat globule size. Milk was sampled seasonally from 7 herds throughout Illinois during a 2-yr period. Sphingomyelin concentration in milk fat was greatest during summer and least during winter, but whole milk concentrations did not vary across seasons. We conclude that 1) sphingomyelin content of milk fat is greater in milk from Holsteins than that from Jerseys, 2) sphingomyelin content in whole milk increases with stage of lactation, and 3) sphingomyelin content of milk fat is greater during summer. However, efforts to produce milk with a greater sphingomyelin content through altering management and nutrition are unlikely to be successful.     

牛乳中乳铁蛋白的分离纯化研究

牛乳经酸沉淀除酪蛋白后,通过超滤、离子交换法分离纯化得到乳铁蛋白,利用考马斯亮蓝、SDS-PAGE电泳定性定量检测。结果表明:每毫升牛乳能提取乳铁蛋白0.22mg,纯度为95%。     

Lactoperoxidase activity in milk is correlated with somatic cell count in dairy cows

Lactoperoxidase (LPO) is a milk protein with antimicrobial function. The present study was undertaken to examine the correlation between LPO activity and somatic cell count (SCC) in milk to use LPO activity as an indicator of mastitis. Composite milk of 36 cows and quarter milk of 3 cows were collected once per week from 0 to 300 d postpartum and twice per day for 1 wk, respectively. For the measurement of LPO activity, milk was mixed with tetramethylbenzidine solution and incubated at 37°C for 30 min, followed by the measurement of optical density. When only milk with low SCC (132 ± 12 × 10³ cells/mL) was used, a significant decrease in LPO activity was detected in primiparous cows from 0 to 4 mo postpartum. Lactoperoxidase activities of primiparous cows in mo 1, 2, and 3 postpartum were significantly higher than those in multiparous cows. When composite milk was divided based on LPO activity, the SCC was significantly higher in the groups with LPO activity >5 and from 3 to 3.9 U/mL in the second- and fourth-parity cows, respectively, compared with the group with LPO activity <2 U/mL. Extremely high SCC were found in the ≥fifth-parity cows, even in low-LPO activity groups. In the case of quarter milk, higher LPO activity was associated with increased SCC in all 3 cows. The percentage of quarter milk samples with high SCC (4,062 ± 415 × 10³ cells/mL) increased with an increase in the LPO activity. The percentage of quarter milk samples with high SCC was 50.0 to 100% in the milk with LPO activity ≥5 U/mL. These results indicate that the correlation of LPO activity to the SCC in bovine milk may point to the potential use of the former as an indicator of SCC.     

Differential leukocyte count method for bovine low somatic cell count milk

Whereas many differential leukocyte count methods for high somatic cell count (SCC) milk from mastitic cows are available, only a few have been developed for low SCC milk. We have developed a flow cytometric differential leukocyte count method for low SCC milk. The procedure consists of 1) 1.5 ml of diluted milk sample (30%, vol/vol dilution with PBS), 2) centrifugation, 3) leukocyte labeling with SYTO 13 and 4) flow cytometric analysis. Four major leukocyte populations can be clearly identified in the green fluorescence-side scatter dot plot: lymphocytes and monocytes (LM), polymorphonuclear neutrophils (PMN), mature macrophages (Mphi), and cells with apoptotic features based on chromatin condensation and nuclear fragmentation. The optimal processing temperature was 20 degrees C. Significant differences among samples with similar differential leukocyte counts were found. Storage of milk samples during 2 d at 7 degrees C had no effect on differential leukocyte count. Using the new method, differential leukocyte count was performed in low SCC milk samples from cows in early, mid, and late lactation. In accordance with previous studies, PMN and Mphi percentages were lower and LM percentages were higher in early lactation than in the other stages of lactation. The percentage of cells with apoptotic features was higher in early lactation than in mid and late lactation. In conclusion, a rapid, simple, accurate, and reproducible standard procedure was developed to determine the differential leukocyte count (Mphi, PMN, LM, and cells with apoptotic features) of bovine low SCC milk.     

Proteomic and peptidomic study of proteolysis in quarter milk after infusion with lipoteichoic acid from Staphylococcus aureus

Mastitic milk is associated with increased bovine protease activity, such as that from plasmin and somatic cell enzymes, which cause proteolysis of the caseins and may reduce cheese yield and quality. The aim of this work was to characterize the peptide profile resulting from proteolysis in a model mastitis system and to identify the proteases responsible. One quarter of each of 2 cows (A and B) was infused with lipoteichoic acid from Staphylococcus aureus. The somatic cell counts of the infused quarters reached a peak 6 h after infusion, whereas plasmin activity of those quarters also increased, reaching a peak after 48 and 12 h for cow A and B, respectively. Urea-polyacrylamide gel electrophoretograms of milk samples of cow A and B obtained at different time points after infusion and incubated for up to 7 d showed almost full hydrolysis of β- and α_S1-casein during incubation of milk samples at peak somatic cell counts, with that of β-casein being faster than that of α_S1-casein. Two-dimensional gel electrophoretograms of milk 6 h after infusion with the toxin confirmed hydrolysis of β- and α_S1-casein and the appearance of lower-molecular-weight products. Peptides were subsequently separated by reversed-phase HPLC and handmade nanoscale C₁₈ columns, and identified by matrix-assisted laser desorption/ionization time-of-flight tandem mass spectrometry. Twenty different peptides were identified and shown to originate from α_s1- and β-casein. Plasmin, cathepsin B and D, elastase, and amino- and carboxypeptidases were suggested as possible responsible proteases based on the peptide cleavage sites. The presumptive activity of amino- and carboxypeptidases is surprising and may indicate the activity of cathepsin H, which has not been reported in milk previously.     

Effect of bovine lactoferrin addition to milk in yogurt manufacturing

The aim of this work was to study the effect of milk supplementation with lactoferrin of different iron saturation on the manufacturing and characteristics of yogurt. Bovine lactoferrin was added at concentrations of 0.5, 1, and 2 mg/mL in the holo (iron saturated) and apo (without iron) forms. Some physicochemical properties, such as pH, concentration of lactic acid, and texture of supplemented yogurts, were determined throughout the shelf-life period storage (28 d) at 4°C. We also evaluated the stability of lactoferrin in supplemented yogurt throughout the storage time. The supplementation of milk with bovine lactoferrin did not greatly affect the physical properties of the yogurt, though apo-lactoferrin slightly delayed the decrease of pH. This could be attributed to the partial inhibition observed on the growth of Streptococcus thermophilus. The integrity and immunoreactive concentration of lactoferrin, determined by Western blotting and noncompetitive ELISA, respectively, remained constant throughout the shelf life of yogurt.     

Genetic variability of lactoferrin content estimated by mid-infrared spectrometry in bovine milk

The effects of lactoferrin (LF) on the immune system have already been shown by many studies. Unfortunately, the current methods used to measure LF levels in milk do not permit the study of the genetic variability of lactoferrin or the performance of routine genetic evaluations. The first aim of this research was to derive a calibration equation permitting the prediction of LF in milk by mid-infrared spectrometry (MIR). The calibration with partial least squares on 69 samples showed a ratio of standard error of cross-validation to standard deviation equal to 1.98. Based on this value, the calibration equation was used to establish an LF indicator trait (predicted LF; pLF) on a large number of milk samples (n = 7,690). A subsequent study of its variability was conducted, which confirmed that stage of lactation and lactation number influence the overall pLF level. Small differences in mean pLF among 7 dairy breeds were also observed. The pLF content of Jersey milk was significantly higher than that in Holstein milk. Therefore, the choice of breed could change the expected LF level. Heritability estimated for pLF was 19.7%. The genetic and phenotypic correlations between somatic cell score and pLF were 0.04 and 0.26, respectively. As somatic cell score increases in presence of mastitis, this observation seems to indicate that pLF, or a function of observed pLF, compared with expected LF might have potential as an indicator of mastitis. The negative genetic correlation (−0.36) between milk yield and pLF could indicate an undesirable effect of selection for high milk production on the overall LF level.     

The analysis of milk components and pathogenic bacteria isolated from bovine raw milk in Korea

Bovine mastitis can be diagnosed by abnormalities in milk components and somatic cell count (SCC), as well as by clinical signs. We examined raw milk in Korea by analyzing SCC, milk urea nitrogen (MUN), and the percentages of milk components (milk fat, protein, and lactose). The associations between SCC or MUN and other milk components were investigated, as well as the relationships between the bacterial species isolated from milk. Somatic cell counts, MUN, and the percentages of milk fat, protein, and lactose were analyzed in 30,019 raw milk samples collected from 2003 to 2006. The regression coefficients of natural logarithmic-transformed SCC (SCCt) on milk fat (−0.0149), lactose (−0.8910), and MUN (−0.0096), and those of MUN on milk fat (−0.3125), protein (−0.8012), and SCCt (−0.0671) were negative, whereas the regression coefficient of SCCt on protein was positive (0.3023). When the data were categorized by the presence or absence of bacterial infection in raw milk, SCCt was negatively associated with milk fat (−0.0172), protein (−0.2693), and lactose (−0.4108). The SCCt values were significantly affected by bacterial species. In particular, 104 milk samples infected with Staphylococcus aureus had the highest SCCt (1.67) compared with milk containing other mastitis-causing bacteria: coagulase-negative staphylococci (n = 755, 1.50), coagulase-positive staphylococci (except Staphylococcus aureus; n = 77, 1.59), Streptococcus spp. (Streptococcus dysgalactiae, n = 37; Streptococcus uberis, n = 12, 0.83), Enterococcus spp. (n = 46, 1.04), Escherichia coli (n = 705, 1.56), Pseudomonas spp. (n = 456, 1.59), and yeast (n = 189, 1.52). These results show that high SCC and MUN negatively affect milk components and that a statistical approach associating SCC, MUN, and milk components by bacterial infection can explain the patterns among them. Bacterial species present in raw milk are an important influence on SCC in Korea.     

Changes of protein content and its fractions in bovine milk from different breeds subject to somatic cell count

Global milk production is undeniably dominated by 2 dairy breeds recognized worldwide: Holstein-Friesian and Jersey. A third breed, Simmental, serves as a dual-purpose breed. The objective of the present research was to establish potential changes in the fractional components of bovine milk protein (mainly whey) in relation to the health status of a dairy cow's mammary glands, which is closely determined by somatic cell count (SCC). The milk of 3 breeds was studied: Polish Holstein-Friesian (Black and Red-White varieties), Simmental, and Jersey. The cows were housed in freestall barns and fed according to the total mixed ration feeding system for both winter and summer periods. Milk samples were collected individually from each cow twice a year, in the winter and summer seasons. A total of 1,822 milk samples were evaluated (946 in winter and 876 in summer). The milk was examined for SCC, crude protein, casein, and whey fraction proteins (α-lactalbumin, β-lactoglobulin, lactoferrin, BSA, and lysozyme). The research material for each breed was split into 4 groups based on SCC (group I: ≤100,000 cells/mL; group II: 101,000 to 400,000 cells/mL; group III: 401,000 to 500,000 cells/mL; and group IV: 501,000 to 1,000,000 cells/mL). It was found that an increase of SCC promulgated a progressive decline in the daily yield of milk, which was significantly true for the Polish Holstein-Friesian. The level of crude protein decreased slightly as SCC increased, and casein concentration (r = −0.591) also followed this trend of decline. Elevation of SCC produced a decrease of major albumins (i.e., α-lactalbumin and β-lactoglobulin). However, SCC increase induced an increase in the immunoactive proteins (lactoferrin and lysozyme) as well as BSA. The interactions of a breed with increased SCC, which can be measured based on the BSA content of the milk, has indicated various levels of susceptibility to the increase in different breeds. This is confirmed by different values of correlation coefficients for these relationships: 0.71 in the Holstein-Friesian, 0.58 in Simmental, and 0.47 in the Jersey cows. Holstein-Friesian cows are more sensitive to mammary gland infections causing a greater decline of their daily milk yields, which, in turn, is reflected in an increase of the negative value of the correlation coefficients between SCC and milk efficiency (−0.24). In the other 2 breeds, the correlations were also negative, but substantially lower (−0.12 and −0.15).     

乳铁蛋白的功能特性及其在婴儿配方奶粉中的应用

介绍了母乳中乳铁蛋白的功能特性，及其应用在婴儿配方奶粉中对非母乳喂养婴儿营养的重要性，国外有关乳铁蛋白在婴幼儿配方奶粉中的应用情况。探讨了乳铁蛋白在婴儿配方奶粉中应用的研究过程，包括有关配方设计依据、使用的原料、生产工艺流程、检验方法、检验结果等。     

牛乳体细胞数与乳蛋白含量相关性的研究

对呼和浩特郊区一牧场30头荷斯坦乳牛进行6个月单个采样，共得427个有效样本，检测乳样中体细胞数、酪蛋白（包括α-酪蛋白、β-酪蛋白、κ-酪蛋白）、乳清蛋白、总蛋白、游离氨基氮、酪蛋白／总蛋白和乳清蛋白／总蛋白。结果表明，乳中总蛋白、游离氨基氮含量及乳清蛋白／总蛋白与SCC呈显著正相关；酪蛋白／总蛋白与SCC呈显著负相关；乳清蛋白含量与SCC呈极显著正相关。酪蛋白、α-酪蛋白／酪蛋白、β-酪蛋白／酪蛋白、κ-酪蛋白／酪蛋白与SCC的相关性不显著。酪蛋白含量与总蛋白含量呈极显著的正相关，与游离氨基氮含量、乳清蛋白／总蛋白呈极显著的负相关。乳清蛋白含量与游离氨基氮、总蛋白含量呈极显著正相关。     

Genetic parameters for yield traits of cows treated or not treated with bovine somatotropin

The objective of this study was to estimate genetic correlations between yield traits of cows treated with bovine somatotropin (bST) and the same yield traits of untreated cows. Lactation records from registered Holstein cows were divided by parity into 3 data sets: 1, 2, and 3 through 5. Approximately 10% of the records in each data set were from cows treated with bST. The numbers of records of treated and untreated cows in the data sets were 4,337 and 48,765; 3,730 and 37,796; and 3,645 and 33,957. Two-trait animal models (records for cows treated or not treated) were used to estimate genetic parameters for milk production traits and somatic cell score (SCS). Estimates of heritability for milk yield for records of treated and untreated cows for the 3 data sets were 0.13, 0.16, and 0.09, and 0.18, 0.18, and 0.14, respectively, with estimates of repeatability of 0.50 and 0.41 for data set 3. Estimates of heritability for fat yield for records of treated and untreated cows were 0.31, 0.16, and 0.12, and 0.27, 0.21, and 0.16. Estimates of repeatability were 0.50 and 0.43 for data set 3. Heritability estimates for protein yield for records of treated and untreated cows were 0.13, 0.17, and 0.12, and 0.20, 0.23, and 0.16, with estimates of repeatability of 0.52 and 0.47. Estimates of heritability for SCS for treated and untreated cows were 0.08, 0.15, and 0.13, and 0.11, 0.13, and 0.13 with repeatability estimates of 0.52 and 0.45. Estimates of genetic correlations between milk yields with and without bST treatment in lactations 1, 2, and 3 to 5 were all 0.99. Estimates of genetic correlations for fat and protein yields were 0.96 for all data sets. Estimates for SCS were 0.99. Estimates of genetic correlations between records of treated and untreated cows were large enough to conclude that records of treated and untreated cows could be considered to be one trait, with treatment as a fixed effect to account for differences in means.     

Application of a new portable microscopic somatic cell counter with disposable plastic chip for milk analysis

The somatic cell count (SCC) is one of the international standards for monitoring milk quality, and it is a useful indicator of mastitis. The current reference method for determining the SCC in raw milk is direct microscopic analysis, but this method requires well-trained staff to maintain its accuracy and reproducibility. To overcome these inconveniences, we developed a portable system (the C-reader system) that utilizes the capillary flow of a microfluidic chamber by surface modification of the hydrophilicity. The microfluidic technology of disposable microchips allows for low consumption of reagents, and a combination of ready-to-use reagents makes the daily work easier. The repeatability test of the C-reader using 10 composite bovine milk samples satisfied the recommended values for SCC equipment. In addition, an acceptable accuracy level of the natural logarithmic-transformed SCC [ln(SCC/1,000): ± 0.059 to 0.112] was achieved using composite raw milk samples and various somatic cell standard solutions from the American Eastern Laboratory and the Korean National Veterinary Research and Quarantine Service. After testing 875 composite milk samples, the C-reader showed a high correlation coefficient (R² = 0.935 to 0.964) and a low mean difference value in log-transformed SCC (−0.088 to 0.004) compared with 3 automatic commercialized somatic cell counters (Fossomatic 4000, Somacount 150, and Somascope). In conclusion, the C-reader system is a new, easy-to-use automatic on-farm method with acceptable repeatability and accuracy for measuring SCC in large dairies and smaller laboratories.     

Considerable variation in the concentration of osteopontin in human milk, bovine milk, and infant formulas

Osteopontin (OPN) is a multifunctional bioactive protein that is implicated in numerous biological processes such as bone remodeling, inhibition of ectopic calcification, and cellular adhesion and migration, as well as several immune functions. Osteopontin has cytokine-like properties and is a key factor in the initiation of T helper 1 immune responses. Osteopontin is present in most tissues and body fluids, with the highest concentrations being found in milk. In the present study, ELISA for human and bovine milk OPN were developed and OPN concentration in human breast milk, bovine milk, and infant formulas was measured and compared. The OPN concentration in human milk was measured to approximately 138 mg/L, which corresponds to 2.1% (wt/wt) of the total protein in human breast milk. This is considerably higher than the corresponding OPN concentrations in bovine milk (∼18 mg/L) and infant formulas (∼9 mg/L). Moreover, bovine milk OPN is shown to induce the expression of the T helper 1 cytokine IL-12 in cultured human lamina propria mononuclear cells isolated from intestinal biopsies. Finally, the OPN concentration in plasma samples from umbilical cords, 3-mo-old infants, and pregnant and nonpregnant adults was measured. The OPN level in plasma from 3-mo-old infants and umbilical cords was found to be 7 to 10 times higher than in adults. Thus, the high levels of OPN in milk and infant plasma suggest that OPN is important to infants and that ingested milk OPN is likely to induce cytokine production in neonate intestinal immune cells.     

FossMatic5000检测牛乳体细胞

讨论了体细胞数与乳房炎的关系及体细胞数对牛乳成分及产奶量损失的影响。主要介绍了FossoMadc 5000检测方法，该方法方便快捷适用于大型牧场监测牛群健康状况。     

Importance of the sampled milk fraction for the prediction of total quarter somatic cell count

This study investigated the changes in somatic cell counts (SCC) in different fractions of milk, with special emphasis on the foremilk and cisternal milk fractions. Therefore, in Experiment 1, quarter milk samples were defined as strict foremilk (F), cisternal milk (C), first 400 g of alveolar milk (A1), and the remaining alveolar milk (A2). Experiment 2 included 6 foremilk fractions (F1 to F6), consisting of one hand-stripped milk jet each, and the remaining cisternal milk plus the entire alveolar milk (RM). In Experiment 1, changes during milking indicated the importance of the sampled milk fraction for measuring SCC because the decrease in the first 3 fractions (F, C, and A1) was enormous in milk with high total quarter SCC. The decline in SCC from F to C was 50% and was 80% from C to A1. Total quarter SCC presented a value of approximately 20% of SCC in F or 35% of SCC in C. Changes in milk with low or very low SCC were marginal during milking. Fractions F and C showed significant differences in SCC among different total SCC concentrations. These differences disappeared with the alveolar fractions A1 and A2. In Experiment 2, a more detailed investigation of foremilk fractions supported the findings of Experiment 1. A significant decline in the foremilk fractions even of F1 to F6 was observed in high-SCC milk at concentrations >350 × 10³ cells/mL. Although one of these foremilk fractions presented only 0.1 to 0.2% of the total milk, the SCC was 2- to 3-fold greater than the total quarter milk SCC. Because the trait of interest (SCC) was measured directly by using the DeLaval cell counter (DCC), the quality of measurement was tested. Statistically interesting factors (repeatability, recovery rate, and potential matrix effects of milk) proved that the DCC is a useful tool for identifying the SCC of milk samples, and thus of grading udder health status. Generally, the DCC provides reliable results, but one must consider that SCC even in strict foremilk can differ dramatically from SCC in the total cisternal fraction, and thus also from SCC in the alveolar fraction.     

Genetic analysis of lactoferrin content in bovine milk

Bovine lactoferrin (LF) is mainly present in milk and shows important physiological and biological functions. The aim of this study was to estimate the heritability and correlation values of LF content in bovine milk with different economic traits as milk yield (MY), fat and protein percentages, and somatic cell score (SCS). Variance components of the studied traits were estimated by REML using a multiple-trait mixed model. The obtained heritability (0.22) for LF content predicted using mid-infrared spectrometry (pLF) suggested the possibility of animal selection based on the increase of LF content in milk. The phenotypic and genetic correlation values calculated between pLF and SCS were moderate (0.31 and 0.24, respectively). Furthermore, a preliminary study of bovine LF gene polymorphism effects was performed on the same production traits. By PCR, all exons of the LF gene were amplified and then sequenced. Three new polymorphisms were detected in exon 2, exon 11, and intron 8. We examined the effects of LF gene polymorphisms of exons 2, 4, 9, 11, and 15, and intron 8 on pLF, MY, fat and protein percentages, and SCS. The different observed effects did not reach a significant level probably because of the characteristics of the studied population. However, the results were promising, and LF may be a potential indicator of mastitis. Further studies are necessary to evaluate the effect of genetic selection based on LF content on the improvement of mastitis resistance.     

体细胞数对乳的影响及其控制措施

分析了乳中体细胞升高的原因，阐述了高体细胞数对乳的组成和乳制品加工的影响，并简要的说明了降低体细胞数的控制措施。     

Correlation between standard plate count and somatic cell count milk quality results for Wisconsin dairy producers

The objective of this study was to determine if a correlation exists between standard plate count (SPC) and somatic cell count (SCC) monthly reported results for Wisconsin dairy producers. Such a correlation may indicate that Wisconsin producers effectively controlling sanitation and milk temperature (reflected in low SPC) also have implemented good herd health management practices (reflected in low SCC). The SPC and SCC results for all grade A and B dairy producers who submitted results to the Wisconsin Department of Agriculture, Trade, and Consumer Protection, in each month of 2012 were analyzed. Grade A producer SPC results were less dispersed than grade B producer SPC results. Regression analysis showed a highly significant correlation between SPC and SCC, but the R² value was very small (0.02–0.03), suggesting that many other factors, besides SCC, influence SPC. Average SCC (across 12 mo) for grade A and B producers decreased with an increase in the number of monthly SPC results (out of 12) that were ≤25,000 cfu/mL. A chi-squared test of independence showed that the proportion of monthly SCC results >250,000 cells/mL varied significantly depending on whether the corresponding SPC result was ≤25,000 or >25,000 cfu/mL. This significant difference occurred in all months of 2012 for grade A and B producers. The results suggest that a generally consistent level of skill exists across dairy production practices affecting SPC and SCC.     

牛乳体细胞数的检测方法

讨论了体细胞数与乳腺炎的关系以及体细胞数对牛乳成分及产奶量损失的影响。主要介绍了4种常用的体细胞数的检测方法，即加利福尼亚细胞数测定法(CMT)，威斯康辛乳腺炎试验(WMT)，电子体细胞计数法(DHI)和直接镜检法(CMSCC)。