Effect of stage of lactation on somatic cell count in sheep milk of Pleven Blackhead breed

Milk secretion in small ruminants present some particularities for cytoplasmatic particles which are shed in the milk together with leukocytes and epithelial cells. Therefore, a parameter like a somatic cell count is worldwide used as a measurement indicator of subclinical mastitis. The aim of the present study was to establish the change of chemical and microbiological characteristics on the composition of sheep milk, obtained from Pleven Blackhead sheep breed during different stages of lactation. It was established that the chemical characteristics of sheep milk varied widely according to the way animals were raised and fed. The obtained results described a slight increase in the values of the total concentration of minerals (up to 1%) and titratable acidity from 19 to 25°T. A decrease in the values of total solids (19.2-16.6%), milk fat (8.06.2%), and protein content (5.8-4.9%) during the stages of lactation was established. The microbiological analysis showed that somatic cell count and total bacterial count during lactation progressively decreased, probably due to the account the hygienic conditions, nutrition, environment and health status of the animals. Moreover, changes in the composition of sheep milk were directly dependent on stable, stable/pasture and pasture feeding system during lactation period.


Introduction
The agricultural sector is a primary part of the economy of Bulgaria and has a significant impact on overall social and economic development. Sheep breeding is one example of this traditional sector in Bulgarian animal husbandry. The high levels of protein, fat, and calcium by casein unit make it an excellent matrix for cheese production therefore sheep milk is mainly used for the production of high-quality cheese varieties. The composition and properties of sheep milk primarily depend on several physiological factorsbreed, variability between individual animals, stage of lactation, seasonal fluctuations, manner of raising and feeding the animals, age, various diseases, etc. (Tamime et al. 2011;Claeys et al. 2014).
The quality of raw sheep milk is determined by its chemical composition and its hygienic environment. Moreover, it is chemical composition of milk that determines this product nutritional value, technological suitability, yield and the subsequent quality of dairy products. The hygienic conditions of milk determine its two main characteristics -total bacterial count (TBC) and total somatic cell counts (SCC). The hygienic conditions of sheep milk are negatively affected by the hygiene standards and the way of milking, the health of the animals, a balanced diet beside the needs, and the physiological condition of the ewes (Idoui et al. 2010;Yilmaz et al. 2011). A high SCC in milk is an indicator of inflammation of the mammary gland (mastitis) (Sharma et al. 2011;Petzer et al. 2017), as SCC reflects the health status of the udder such as immune response of the glandular tissue to the presence of pathogenic microorganisms. There is strong evidence that high SCC values in raw milk result in number of adverse effects that are mainly related to reduction in milk`s nutritional value, quality and the cheese yield of dairy products (Halasa et al. 2007;Chen et al. 2010). According to Cinar et al. (2015) and Sharma et al. (2017) the mean values of SCC depend significantly on the origin of the milk and the lactation period. These authors reported that healthy sheep tend to have higher SCC values than those of healthy cows.
The aim of the present study was thus to determine the changes in chemical and microbiological composition of raw sheep milk, obtained from Pleven Blackhead sheep during different stages of lactation.

Materials and Methods
Raw materials. The study of raw sheep milk was carried out during the period from March to August. A total number of 600 Black-head Pleven dairy sheep were included in the study, fed in different ways (stable, stable/pasture and pasture) and bred in the region of Parvomay, Plovdiv region.
Methods. The samples were examined in the research laboratory of the Department of Milk and Dairy Products, University of Food Technologies, Plovdiv and in the laboratory of the dairy processing plant of Bor Chvor Ltd. The samples were maintained from the time of sampling until the moment of chemical and microbial analysis at temperatures between 2 and 6°C. No preservative for milk samples was used.
Chemical analysis -The percentages of milk components, including milk fat, milk protein; minerals and lactose, as well as total solids (water content, respectively), solid-non-fat, freezing point and density were determined by Lactoscan SFP Options Milk Analyzer. All analyses were performed at room temperature (24 ± 1°C); Titratable acidity (TA) of the milk samples was determined by the Thorner's method according to the Bulgarian National Standard (BNS 1111-80). To 10 mL of milk sample were added 20 mL of distilled water, and 1-3 drops of phenolphthalein (prepared at 1% in 95% ethanol). The mixture was titrated with standardized 0.1 n NaOH until the first colour change (to light-pink) persisted for 30 s. One more drop of 0.1 n NaOH was added and the final volume of 0.1 n NaOH added was noted. The titratable acidity was represented in degrees Thorner and was calculated by the following formula: where: V -the quantity of 0.1n NaOH used to titrate the milk sample, ml; Ffactor of 0.1n NaOH.
Potentiometric measurement of milk samples active acidity was done by a pH meter (model MS 2000, Microsyst, Plovdiv, Bulgaria) with a glass electrode (Sensorex, Garden Grove, USA) standardized at 20°C in the range 7.01-4.01.
Microbiological analysis -SCC was determined by ЕKOSCOPE -FPS1, produce by Bultech 2000 Ltd, Bulgaria; TBC was determined according to ISO 4833-1:2013, microbiology of the food chainhorizontal method for the enumeration of microorganisms -Part 1: Colony count at 30°C by the pour plate technique where the bulk milk samples were decimally diluted in sterilized water to perform serial dilutions of the test sample in order to achieve a colony count of between 15 and 300 colonies per plate. Then the inoculum was transferred into plates and added medium named Plate Count Agar. Inoculated plates were incubated aerobically at 30°C for 72 hours. The number of colonies was calculated according to the formula described in the standard. The results were represented as log CFU/mL. Statistical analysis -Computer processing of the results was performed by Microsoft Excel 2010 (ANOVA) with significance level p ≤ 0.05 was performed (Draper and Smith 1998). Table 1 shows the chemical composition of sheep milk obtained during different stages of lactation from Pleven Blackhead dairy sheep. From the data obtained it was evident that during lactation, the values of individual characteristics varied. For example, over the study period it was established that the fat content decreased by approximately 3.3%, and proteins by 1.0%, in contrast to the density, which was relatively law at the beginning of lactation but increased with time and eventually reached 1.037g/ml. The observation showed that the freezing point of milk was higher at the beginning of lactation, which may be explained by the higher values of total solids in milk. * The results are presented as mean value ± SD As lactation progressed, the above mentions values decreased, given the observed tendency for change noted in chemical characteristics of milk. The lactose content did not change during the study period. The obtained results correspond to those established by other authors (Panayotov et al. 2011;Angelov et al. 2015), who studied the dynamics of changes composition of sheep milk. The increased mineral content in sheep milk in the July-August period correlated with the obtained higher values of titratable acidity and the lower рН values (Figure 1). Xie and Li (2008) noted similar correlations between solids-non-fat values, titratable acidity and buffer capacity in different types of milk.

Figure 1. Change of titratable acidity and pH of sheep milk at different stages of lactation
Similar results were reported from Kandeel et al. (2019) who suggested that a decrease in milk pH was primarily due to an increase in the difference between the concentrations of the main strong cation (sodium) and the main strong anions in milk (chloride and soluble protein) which resulted in an increase in the difference in strong ions. El-Tahawy and El-Far (2010) and Moslehishad et al. (2010) likewise showed a higher percentage of total solids content in milk with an increase in SCC levels.
Figures 2 and 3 present our results for the microbiological characteristics TBC and SCC during the studied period. From the collected results it was evident that the quality of hygiene and animal husbandry improved during the lactation period when comparing the transition from stable to stable/pasture and pasture sheep breeding. A decrease in the total bacterial count and somatic cell count was established during lactation, as a result of free grazing. According to Regulation (EC) No. 853, 2004 of the European Parliament, which treats specific hygiene limits for raw milks TBC values should not exceed 1.0 x10 5 CFU/mL for raw cow`s milk and 1.5 x 10 6 CFU/mL for raw milk from species other than cows respectively. In raw sheep milk, both microbiological analyses served as indicators of the health status of sheep mammary gland.

Figure 2. TBC in sheep milk during stages of lactation
Data observed at Figure 2 showed that the TВC values in the March-May period ranged between 6.2 to 6.0 log CFU/mL, however in June-August period, the TBC decreased and reached values between 5.9 to 5.8 log CFU/mL. The average TВC value for sheep milk, during the reported period, was 5.95 log CFU/mL. These results correlated with the results of other authors (Alexopoulos et al. 2011;Merlin Junior et al. 2015) who concluded that the TBC values limit was sometimes exceeded in milk samples. In a study by Muehlherr et al. (2003) the authors reported TВC values of 4.70 log CFU/mL (min. 2.00 log CFU/mL and max. 8.64 log CFU/mL) in small ruminants. According to Klimešová et al. (2017), the development curves of the relationships between TCM and SCC in bulk milk over a period of months may be associated with the dynamics of lactation during the seasonal reproduction cycle of sheep. This result was similar to that noted for small ruminants, in which there was often simultaneous progress within a calendar season, and different from cows reared mostly independently of the seasonal reproduction cycle. Figure 3 presents the change in the number of SCC in sheep milk during lactation. The obtained data showed that at the beginning of the lactation period, the number of somatic cells was relatively high at 1 310 000 cells/ml. As lactation progressed, a gradual decrease in the values was established, and by the end of the lactation period the values decreased by approximately 700 000 cells/ml. A decrease in somatic cell count was also reported by Paape et al. (2007). The data set indicated that milk SCC increased with increasing parity and the stage of lactation for goats and cows but not for sheep. Similar results were reported by Othmane et al. (2002) who noted the non-significant effects of lactation stage and parity on milk SCC. The effect of parity and lactation stage on milk yield, composition and quality of organic sheep milk were studied by Králíčková et al. (2012). They found a significant and positive correlation between the parity and SCC; however, a significant but negative correlation was observed between the stage of lactation and SCC. According to Olechnowicz et al. (2010) the SCC in milk samples from udder halves and milk composition depended on the level of SCC recorded for halves of the udder. SCC in milk from one or both halves of udders exceeding 250 x 10 3 cells/ml resulted in a statistically significant (P<0.01) decrease in the daily milk production of ewes, by approx. 15.89 and 30.22%, respectively. The authors indicated that the analysis of variance also showed a significant effect of parity and lactation stage of ewes on SCC and milk composition from udder halves below 250 x 10 3 cells/ml.

Conclusions
Our study on the chemical and microbiological composition of raw Pleven Blackhead sheep milk showed a clear tendency towards changes over time.
A constant tendency towards as decrease in the main chemical characteristics (total solids, SNF, protein and fat content) was observed during lactation. At the same time, there was an increase in the values of minerals, water content, density and titratable acidity. The decrease in the total bacterial count and somatic cell count in milk during different lactation periods was most likely due to the good health of the animals along with the application of good hygienic practices in breeding and feeding. In addition, increase in SCC values causes a decrease in milk yield and affects milk composition, which leads to reduced cheese making aptitude. However, our future research direction is to use this milk to produce a White brine cheese and Kashkaval cheese and to investigate the impact of SCC values on the changes in quality characteristics of these cheese during ripening and storage period.