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J. Dairy Sci. 86:2583-2587
© American Dairy Science Association, 2003.

Effect of Freezing on Fossomatic Cell Counting in Ewe Milk

J. R. Martinez, C. Gonzalo, J. A. Carriedo and F. San Primitivo

Departamento de Producción Animal, Facultad de Veterinaria, Universidad de León, 24071-León, Spain


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS AND DISCUSSION
 CONCLUSIONS
 ACKNOWLEDGEMENTS
 REFERENCES
 
Using the Fossomatic method, a total of 10,072 analytical somatic cell count (SCC) observations were carried out on 4760 aliquots taken from 70 individual ewe milk samples with the objective of studying whether freezing showed significant differences of SCC in comparison with refrigeration, according to different analytical conditions. These conditions were four preservation procedures (without preservation, potassium dichromate, azidiol, and bronopol), two storage temperatures (refrigeration and freezing), five milk ages within storage (24 h postcollection in refrigeration, and 24 h, 15, 30, and 60 d postcollection in freezing), two thawing types (rapid and slow), and two analytical temperatures (40 and 60°C).

Preservation, storage, and analytical temperature, type of thawing and milk age within storage, and most of the interactions showed a significant effect on the SCC variation. On average, the SCC was lower after freezing than in refrigeration. This effect depended specifically on type of preservation and analytical temperature of milk. The SCC of milk unpreserved or preserved with bronopol or potassium dichromate, and analyzed at 40°C, was not affected by freezing; however, use of azidiol as a preservative before freezing, and heating the milk to 60°C following thawing resulted in significantly decreased SCC. Milk age had little quantitative influence on SCC of thawed milk. The type of thawing (rapid and slow) did not significantly influence SCC of milk analyzed at 40°C. As a result, when using properly handled samples, the Fossomatic method could be used to enumerate SCC in samples frozen over the 60 d.

Key Words: ewe milk • somatic cell count • Fossomatic method • frozen milk

Abbreviation key: AZ = azidiol, BR = bronopol, FT = freezing temperature, PD = potassium dichromate, RT = refrigeration temperature, WP = without preservative


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS AND DISCUSSION
 CONCLUSIONS
 ACKNOWLEDGEMENTS
 REFERENCES
 
In dairy sheep (González-Rodriguez et al., 1995; Gonzalo et al., 2002) SCC is a useful predictor of IMI and, therefore, an important component of milk with regard to aspects of quality, hygiene, and mastitis control. Accuracy of milk SCC is very important to most dairy farmers and to the dairy industry. The Fossomatic is the most widely used SCC method in milk-testing laboratories and its performance has been completely standardized for cow milk (Heald et al., 1977; IDF, 1995; Schmidt-Madsen, 1975, 1979). In ewe milk, which has a higher content of total solids than cow milk, the performance of Fossomatic method has been evaluated by some authors (Gonzalo et al., 1993), and its optimal analytical conditions (type of preservation, analytical temperature, and milk age) have recently been defined for refrigerated and stored at ambient temperature milk (Gonzalo et al., 2003).

Basically, the Fossomatic is a DNA-specific counter based on optical principle of fluorescence (IDF, 1995). 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. Automation of this process means that large numbers of samples can be analyzed per hour in milk-testing laboratories. In most cases, Fossomatic counters use fresh or preserved milk. Very few studies have been carried out on their performance with frozen milk (Barkema et al., 1997). Freezing milk could be useful in the event of a breakdown in SCC equipment, or in biological or bacteriological protocols for frozen milk samples. However, very little is known about SCC variation factors (perservative, type of thawing or analytical temperature) in thawed ewe milk (Gonzalo et al., 1993).

The objectives of this study were to assess the suitability of frozen milk for SCC determination by the Fossomatic method, using refrigerated milk as the gold standard, and to define the influence of preservation, thawing conditions, analytical temperature, and duration of freezing on the SCC.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS AND DISCUSSION
 CONCLUSIONS
 ACKNOWLEDGEMENTS
 REFERENCES
 
A total of 70 individual ewe milk samples (250 ml/ewe), ranging between 15 x 103 and 6500 x 103 cells/ml, were divided into 68 3-ml aliquots, which, in turn, were divided into four groups according to the preservative used: 17 without preservative (WP), 17 with potassium dichromate (PD) (0. 1 g/100 ml), 17 with azidiol (AZ) (0.024 g sodium azide/100 ml) and 17 with bronopol (BR) (0.05 g/100 ml). As shown in the schematic diagram in Table 1Go, one aliquot out of each group was stored at refrigeration temperature (RT) (4°C) and the other 16 were stored at freezing temperature (FT) (-20°C). The refrigerated aliquots were analyzed at 40 and 60°C, 24 h postcollection, and were used as reference. Aliquots were heated successively in a water bath increasing water temperature from 40 to 60°C after the first analysis, according to Gonzalo et al. (2003); so only one aliquot/preservation type was used (Table 1Go). The 16 frozen ones were analyzed by Fossomatic method 24 h, 15, 30, and 60 d postcollection, at a rate of four aliquots/age. Two types of thawing were used for each age: slow and rapid. Slow thawing consisted of keeping two aliquots in a nonshaking water bath at 18°C for 45 min. One of them was then heated to 40°C and the other to 60°C, both for 20 min, before being analytically determined. The other two aliquots were subjected to rapid thawing by placing them in nonshaking water baths at 40 and 60°C, respectively. The SCC analyses started 20 min later. All the SCC analyses were in duplicate using a Fossomatic 90 (A/S N Foss Electric, Hilleroød, Denmark) and following the previously described method (Gonzalo et al., 1993, 2003; IDF, 1995). The total number of SCC analytical observations came to 10,072.


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  		Table 1. Study design from 70 ewe milk samples (68 aliquots/ewe sample) using a schematic diagram.
 
Statistical Analysis
The statistical study was carried out following the GLM procedure of SAS (SAS, 1992). In the model used for this study, the effect of ewe was random and the others were fixed:


where:

Yijklmr=dependent variable logSCC;

µ=mean;

Ei=ewe effect (70 levels);

Sj=storage temperature effect (two levels: RT and FT);

Pk=preservation effect (four levels: WP, PD, AZ and BR);

Tl=analytical temperature effect (two levels: 40 and 60°C);

SPjk=storage x preservation interaction;

STjl=storage x analytical temperature interaction;

PTkl=preservation x analytical temperature interaction;

SPTjkl=storage x preservation x analytical temperature interaction;

Am(j)=effect of milk age within storage. Ages considered were 24 h at RT and 24 h, 15, 30 and 60 d at FT;

Dn(j)=effect of thawing type within storage. Two types of thawing were considered: rapid and slow;

PAkm(j)=preservation x age interaction within storage;

PDkn(j)=preservation x thawing type interaction within storage;

TAlm(j)=analytical temperature x age interaction within storage;

TDln(j)=analytical temperature x thawing type interaction within storage;

ADmn(j)=age x thawing type interaction within storage;

PADkmn(j)=preservation x age x thawing type interaction within storage;

TADlmn(j)=analytical temperature x age x thawing type interaction within storage;

TPAlkm(j)=analytical temperature x preservation x age interaction within storage;

TPDlkn(j)=analytical temperature x preservation x thawing type interaction within storage; and

eijklmnr=random residual.


   RESULTS AND DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS AND DISCUSSION
CONCLUSIONS
 ACKNOWLEDGEMENTS
 REFERENCES
 
Log10SCC mean of analytical observations was 5.39 ± 0.005. Table 2Go shows the analyses of variance for the factors affecting logSCC. Ewe, storage temperature, preservation, analytical temperature, age of milk within storage, type of thawing within storage, and most of the interactions of the previously mentioned effects contributed significantly to the SCC variation.


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  		Table 2. Analysis of variance of the variation factors studied for logSCC variable.
 
Regardless of storage factor, preservation and analytical temperature were important logSCC variation factors (F = 698 and 1014, respectively), as well as interaction of both (Table 2Go). The highest values were obtained for BR preserved milk (5.52 ± 0.005) and the lowest ones for AZ preserved milk (5.34 ± 0.005), coinciding with a recent study on RT stored ewe milk (Gonzalo et al., 2003). However, the analytical temperature of 60°C caused a significant decrease (P < 0.001) in log SCC (5.42 ± 0.003) in comparison with that of 40°C (5.48 ± 0.003), contrasting with results obtained by other authors in refrigerated cow (Miller et al., 1986) and ewe milk (Gonzalo et al., 2003). Biological significance of this discrepancy will later be explained after analyzing interaction of temperature with other effects.

The storage effect (RT and FT) showed high statistical significance (F = 1012; P < 0.001); logSCC was significantly higher (P < 0.001) at RT (5.52 ± 0.004) than FT (5.37 ± 0.001), in agreement with results of other cow (Barkema et al., 1997) and ewe (Gonzalo et al., 1993) milk studies. Interactions of storage with the previous effects were significant too (P < 0.001): storage x analytical temperature (F = 1050), storage x preservation (F = 152), and storage x preservation x analytical temperature (F = 24). Therefore, storage factor must be analyzed considering type of preservation and analytical temperature at the same time.

Age and type of thawing, both within storage, were also significant. Age within storage (F = 4.5; P < 0.01) showed little quantitative importance (Table 3Go); least squares means of logSCC in frozen milk were 5.37 on d 1, 15, and 60, and 5.38 on d 30 postcollection. Small variations of logSCC over time were also found by Barkema et al. (1997) in thawed cow milk in accordance with our results. However, type of thawing showed high statistical significance (F = 108; P < 0.001); rapid thawing showed higher values (5.39 ± 0.002) than slow thawing (5.35 ± 0.002). Milk is a colloidal suspension and so these differences of SCC could be due to the different effect of thawing conditions on aqueous and fat phases of milk. Because SCC is more associated with the fat fraction, SCC results may get skewed due to nonhomogeneous or mixed samples. In addition, thawing also showed significant interactions with preservation (F = 5.9; P < 0.001) and especially with analytical temperature (F = 132; P > 0.001) within storage. Thus, whereas at 40°C there were no significant differences (P > 0.05) of logSCC means between refrigeration (5.48), rapid (5.48), and slow (5.48) thawing, such means significantly differed (P < 0.001) at 60°C (5.57, 5.30, and 5.23, respectively) (Table 4Go).


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  		Table 3. Least squares means of logSCC by age of freezing.
 

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  		Table 4. Least squares means of logSCC by analytical temperature x thawing type interaction within storage.
 
Finally, type of thawing x preservation x analytical temperature interaction within storage was highly significant (F = 9.5; P < 0.001) and enabled the previously mentioned effects to be studied more suitably, considering for this aim the least squares means that are shown on Table 5Go. To compare these means, it is necessary to bear in mind that SCC of milk BR preserved RT stored and analyzed at 40°C must be considered as a reference value. In fact, Gonzalo et al. (2003) found that these conditions elicit the most accurate SCC compared with the reference microscopic method (IDF, 1995). Thus, at 40°C, logSCC of BR preserved milk was independent (P > 0.05) of storage temperature and type of thawing (5.53, 5.55, and 5.54 for refrigeration and rapid and slow thawing, respectively). At such temperature (40°C) thawed WP or PD-preserved milk also showed logSCC values (5.51 to 5.54) similar (P > 0.05) to those of RT-stored BR preserved milk (Table 5Go). However, SCC noticeably decreased (P < 0.001) after thawing in AZ preserved milk (5.31 and 5.33 after rapid and slow thawing, respectively) (Table 5Go). The log SCC of refrigerated milk analyzed at 60°C (5.55 to 5.99) were higher (P < 0.001) than those found at 40°C (5.44 to 5.53), coinciding with results obtained by Gonzalo et al. (2003) for ewe milk, probably because higher temperatures favor better penetration of the ethidium bromide dye in the cell, or because it disperses the fat in ewe milk better. However, log SCC decreased very significantly (P < 0.001) in milk stored at FT and analyzed at 60°C (4.99 to 5.45) (Table 5Go). The sum of the effects of thawing and heating to 60°C could have too traumatic an effect on the cells and affect their capacity to fix the ethidium bromide dye, or lead to samples in which aqueous and fat phases were nonhomogeneous. At this temperature, rapid thawing showed significantly higher (P < 0.05) logSCC (5.09 to 5.45) than slow thawing (4.99 to 5.39), whereas at 40°C no statistical differences were recorded between both types of thawing (5.31 to 5.55 for rapid thawing and 5.33 a 5.54 for slow thawing). The lowest values of logSCC corresponded to AZ preserved milk analyzed at 60°C after thawing (5.09 and 4.99) (Table 5Go).


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  		Table 5. Least squares means of logSCC by preservation x analytical temperature x thawing type interaction within storage.
 
Therefore, the previous results allow us to state that samples frozen up to 60 d postcollection but handled properly and analyzed at 40°C gave similar SCC to the BR refrigerated reference.


   CONCLUSIONS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS AND DISCUSSION
 CONCLUSIONS
ACKNOWLEDGEMENTS
 REFERENCES
 
The FT storage of BR or PD preserved milk offered ideal conditions for cellular integrity. On the other hand, AZ preservation was definitely not advised for freezing milk to be used in SCC analysis. The optimum analytical temperature after thawing was 40°C, as SCC decreased significantly at 60°C. Sample age was of less quantitative importance on SCC; logSCC means remained relatively constant over the 60 d of this study. Rapid or slow thawing did not significantly influence SCC of milk analyzed at 40°C. As a result, from a practical point of view, WP milk, and BR- or PD-preserved milk before freezing and analyzed at 40°C after rapid or slow thawing elicited SCC values similar to those of refrigerated milk at 24 h.


   ACKNOWLEDGEMENTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS AND DISCUSSION
 CONCLUSIONS
 ACKNOWLEDGEMENTS
REFERENCES
 
This paper was developed within projects: FAIR1-CT95-0881, financed by the Commission of the European Communities (Brussels, Belgium), and UE96-0037, financed by the Comisión Interministerial de Ciencia y Tecnologia (Madrid, Spain).

Corresponding author:
C. Gonzalo; e-mail:
dp2cga{at}unileon.es.

Received for publication December 12, 2002. Accepted for publication January 8, 2003.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS AND DISCUSSION
 CONCLUSIONS
 ACKNOWLEDGEMENTS
 REFERENCES
 


Barkema, H. W., J. Van Der Schans, Y. H. Schukken, A. L. W. De Gee, T. J. G. M. Lam, and G. Benedictus. 1997. Effect of freezing on somatic cell count of quarter milk samples as determined by a Fossomatic electronic cell counter. J. Dairy Sci. 80:422–426.[Abstract]

González-Rodriguez, M. C., C. Gonzalo, F. San Primitivo, and P. Cármenes. 1995. Relationship between somatic cell count and intramammary infection of the half udder in dairy ewes. J. Dairy Sci. 78:2753–2759.[Abstract]

Gonzalo, C., A. Ariznabarreta, J. A. Carriedo, and F. San Primtivo. 2002. Mammary pathogens and their relationship with somatic cell count and milk yield losses in dairy ewes. J. Dairy Sci. 85:1460–1467.[Abstract]

Gonzalo, C., J. A. Baro, J. A. Carriedo, and F. San Primitivo. 1993. Use of the Fossomatic method to determine somatic cell counts in sheep milk. J. Dairy Sci. 76:115–119.[Abstract]

Gonzalo, C., J. R. Martinez, J. A. Carriedo, and F. San Primitivo. 2003. Fossomatic cell-counting on ewe milk: comparison with direct microscopy and study of variation factors. J. Dairy Sci. 86:138–145.[Abstract/Free Full Text]

Heald, C. W., G. M. Jones, S. C. Nickerson, W. N. Patterson, and W. E. Vinson. 1977. Preliminary evaluation of the Fossomatic somatic cell counter for analysis of individual cow samples in a central testing laboratory. J. Food Prot. 40:523–526.

International Dairy Federation. 1995. Enumeration of somatic cells. FIL-IDF Standard no. 148A, Brussels, Belgium.

Miller, R. H., M. J. Paape, and J. C. Acton. 1986. Comparison of milk somatic cell counts by Coulter and Fossomatic counters. J. Dairy Sci. 69:1942–1946.

SAS (1992). SAS/STAT User’s Guide, Version 6, 10th ed. SAS Institute Inc., Cary, NC.

Schalm, O W., E. J. Carroll, and N. C. Jain. 1971. Bovine Mastitis. Lea & Febiger, Philadelphia, USA.

Schmidt-Madsen, P. 1975. Fluoro-opto-electronic cell-counting on milk. J. Dairy Res. 42:227–239.

Schmidt-Madsen, P. 1979. Influence of storage and preservation of milk samples on microscopic and Fossomatic somatic cell counts. Nord. Vet.-Med. 31:449–454.


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