sheep research and mastitis control programs has increased ... sheep milk, which has a higher fat content than ... method have not been specified for sheep milk.
Use of the Fossomatic Method to Determine Somatic Cell Counts in Sheep Milk CARLOS GONZALO, JESUS A. BARO, JUAN A. CARRIEDO, and FERMIN SAN PRIMITIVO Departamento de Produccion Animal Universidad de Leon 24071 Leon, Spain ABSTRACT
infection (13). The main sec methods (direct microscopic, Coulter counter, and Fossomatic) have been completely standardized for cow milk (3, 9, 11, 14). Recently, interest in milk cell counting in sheep research and mastitis control programs has increased. However, sec methods for sheep milk have not been widely evaluated or tested (10). Some counting methods, such as direct microscopic SCC (DMSCC) and Coulter counter, have been modified 0, 2) to adapt to sheep milk, which has a higher fat content than cow milk. The Fossomatic SCC method (FSCC) automatically processes the milk samples so that they need no prior adaptation. However, optimal conditions for use of this method have not been specified for sheep milk. Basically, the Fossomatic counter is a fluorescence microscope. The ethidium bromide dye penetrates the cell and forms a fluorescent complex with the nuclear DNA. Each cell produces an electrical pulse, which is amplified and recorded. The objectives of this study were to compare the FSCC with the DMSCC (reference method) in sheep milk and to evaluate the effect of the storage method (fresh, refrigerated, and frozen milk) and of the sample age on the sce of foremilk and strippings.
The Fossomatic method for SCC was compared with the direct microscopic method in 85 half-udder samples of sheep milk. The correlation coefficient was .986. The repeatability of the Fossomatic method showed average variation coefficients less than 5%. The carry-over effect between samples was less than .5%. The effect of the storage method (fresh milk, refrigerated at 4°C and frozen at -19°C) and the sample age were studied in 48 samples of foremilk and strippings. The storage method had a significant effect on the SCC variation. The average fresh, refrigerated, and frozen sample counts were 125,000, 110,000, and 82,000 cells/ml for foremilk and 201,000, 192,000, and 145,000 cells/ml for strippings, respectively. The effect of age on the refrigerated samples was also significant; counts were reduced by about 14% from d 1 to 7 in both types of milk. The effect of age on the frozen sample varied. These results suggest standardization of age and storage conditions of the milk samples to reduce variation of SCC. The milk must not be frozen. (Key words: sheep milk, somatic cell count, Fossomatic method) Abbreviation key: DMSCC microscopic SCC method, FSee matic SCC method.
= direct = Fosso-
INTRODUCTION
Somatic cell count in milk is a widely used method to evaluate the status of cow udder
Received June 3. 1992. Accepted August 14, 1992. 1993] Dairy Sci 76:115-119
MATERIALS AND METHODS Comparison of the FSCC and the DMSCC
Eighty-five samples of sheep milk preserved in .05% potassium dichromate were analyzed within 24 h after milking by FSCe and DMSCC. The samples were collected from half-udders and represent sec varying from 15,000 to 4 million cells/mt. For the DMSeC, the milk was heated to 40°C in a water bath and held at that tempera-
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ture for 15 min before being cooled to 20·C by careful stirring. The method used was that recommended by the International Dairy Federation (6), adapted to sheep milk by Gonzalo and Gaudioso (I). Two slides of each sample were prepared and counted. Slides were stained with May Grunwald-Giemsa (Vorqufmica, Vigo, Spain). The working factor was 1600. For the FSCC, the milk samples were heated to 40·C in a water bath and held at this temperature for 15 min. The samples were then double processed in a Fossomatic 90 (AlS N. Foss Electric, Hillen'ld, Denmark). The reagents were prepared following the manufacturer's instructions, which coincide with the method recommended by International Dairy Federation (6). Repeatability
The repeatability of the FSCC was evaluated by counting a number of subsamples from 20 milk samples with different numbers of cells (from 46,000 to 12.1 million cells/ml). The coefficients of variation for the data of each sample were calculated. In order to determine the possible carryover effect from one sample to another in the FSCC, a sample with low SCC (79,900 cells! ml) was counted, alternating with a sample with high SCC (7.88 million cells/ml). First, 15 counts were carried out with the low cell concentration sample. Then 15 counts were made alternating between high and low SCC milk. The low count sample was then counted 15 more times. Effect of the Storage Method and Sample Age
Thirty-five milliliters of foremilk and 25 ml of hand strippings (after machine removal) were obtained from 48 half-udders from a total of 24 primiparous, midlactation Churra sheep during morning milking. Potassium dichromate to a final concentration of .05% was used as a preservative for all samples. After collection, each sample of original milk (foremilk and stripping) was divided into eight aliquots (4 and 3 ml, respectively). One aliquot was used for SCC on the sampling day (fresh milk). Four aliquots were refrigerated at 4·C and Journal of Dairy Science Vol. 76. No.1. 1993
counted I, 3, 5, and 7 d after collection. The remaining three aliquots were frozen at -19·C and analyzed I, 7, and IS d after collection. All samples were counted in a Fossomatic 90 following the previously mentioned specifications. The frozen samples were thawed in a water bath at ambient temperature for 45 min before being heated at 40·C. Statistical Analysis
The statistical study of the storage method and sample age factors was made using analysis of variance according to the model Yijk
= JL
+ Mi + Dj(i) +
