The main objective of this experiment was to evaluate the effects of two milking intervals (8 and 16 h) on milk constituents (fat, protein, lactose, dry matter, and log10 SCC) of nineteen Istrian × Awassi × East-Friesian crossbred ewes in different milk fractions (0–25, 25–50, 50–75 and 75–100%) during the course of milking and in machine stripping (MS) milk. Furthermore, we sought to determine the effect of the two milking intervals on milking characteristics (average milk flow rate, peak milk flow rate, machine-on time, total milk yield, and milk production rate) and whether each milk constituent within each milking interval is best described by a linear, quadratic, or cubic function. Average milk flow rate and milk yield per milking decreased in the 8 h milking interval compared to the 16 h milking interval (P < 0.05). Peak milk flow rate, machine-on time, and milk production rate were not different between the two milking intervals. Overall, milk fat content, dry matter content, and log10 SCC increased in the 8 h milking interval compared to the 16 h milking interval (P < 0.05). Milk protein content did not change through the main milk fractions at either milking interval. Milk lactose content did not change through the milk fractions at the 8 h milking interval, whereas it decreased in the 75–100% and stripping milk fractions at the 16 h milking interval (P < 0.05). The 0–25% and stripping milk fractions contained the highest log10 SCC compared to all other milk fractions (P < 0.05). Changes of milk fat and dry matter content throughout milking were best described by quadratic functions, whereas milk protein content, milk lactose content, and log10 SCC were best described by different functions depending on the milking interval. These results demonstrate that milking interval influenced all milk constituents in various milk fractions during the course of ewe milking. Moreover, milking characteristics such as average milk flow and total milk yield, and the appropriate mathematical function to characterize milk constituents throughout a milking, were affected by milking interval.

Immune cells in the milk are most important in combating pathogens that invade the mammary gland. This study investigated the immune competence and viability of somatic milk cells that are already resident in milk and udders free of infection. Cells were studied in freshly removed milk to simulate conditions in the udder. Effects of incubation, cell preparation, and immunological stimulation with 0·5 μg/ml lipopolysaccharide (LPS) from Escherichia coli were analysed. Viability and differential counts of milk cells between high and low somatic cell count (SCC) quarters, and cisternal and alveolar milk with and without LPS stimulation were compared. Incubation and preparation of cells caused a cell loss which further increased with time independently of SCC and milk fraction. The viability of these cells was stable until 3 h post incubation and decreased until 6 h. Cell populations differed between both investigations, but did not change during the course of the experiment. mRNA expression of immune and apoptosis factors of the cells, measured by qPCR, did not change substantially: mRNA expression of caspase 3, Toll like receptor 4, and GM-CSF did not change, whereas the expression of the death receptor Fas/APO-1 (CD95), lactoferrin and lysozyme was decreased at 6 h. Cyclooxygenase-2 and TNF-α mRNA expression were decreased after 6 h of LPS treatment. In comparison with other studies in vivo or in vitro (in cell culture), in this study where cells are studied ex vivo (removed from the udder but kept in their natural environment, the milk) resident milk cells seem to be more vulnerable, less viable, less able to respond to stimulation, and thus less immune competent compared with cells that have freshly migrated from blood into milk after pathogen stimulation. The cell viability and differential cell count differed between high- and low-SCC milk and between cisternal and alveolar milk depending on the individual cow. In conclusion, the results support the view that for a most effective defence against invading pathogens the mammary gland is reliant on the recruitment of fresh immune cells from the blood.

The goal of the study was to evaluate the composition of leucocyte populations in different milk fractions as a basis on which to judge their possible role in the immune response in different compartments of the udder. The milk of one healthy quarter of nine dairy cows (SCC/quarter [les ]125000/ml; bacteriologically negative) was removed separately and during the course of milking divided into: cisternal milk (C), alveolar milk 0–25%, 25–50%, 50–75%, 75–100% (A25; A50; A75; A100, respectively) and residual milk (R). Each fraction was analysed for the main constituents, SCC and distribution of leucocyte populations and their viability. The content of fat increased steadily during milking and reached highest values in R. Protein and lactose increased from C to A25, decreased from A25 to A100 and reached their minimum in R. Na and Cl ion levels diminished from C to A25 and thereafter increased from A50 to R. Electrical Conductivity (EC) also decreased from C to A25 but remained similar within the alveolar samples and reached a minimum in R. SCC decreased from C to a minimum in A25 and increased subsequently to a significant maximum in R. Somatic cell viability increased throughout consecutive fractions with a maximum value in R. The ratio of cell populations in the various milk fractions showed a reverse trend of macrophages (M) and polymorphonuclear neutrophils (PMN). M values were highest in C while PMN levels increased to their maximum in the R fraction. The lymphocyte (L) fraction remained similar in C, A25, A50, A75 and R but was higher in A100. Proportions of L, PMN and M were, respectively, 9·3%, 38·2%, 52·3% in C, 10·9%, 64%, 25·1% in A25–A100 and 10·2%, 64·9%, 24·8% in R. Numbers of L, PMN and M in milk showed a similar pattern for all three cell types: high levels in C decreased to a minimum at A25 and increased steadily thereafter to their maxima in R. It is concluded that, for healthy quarters, M, the predominant cell type in C, are located near the teat canal, the main entrance of pathogens. Obviously they are the first immunological barriers for invading pathogens. In contrast, PMN are the most important population in the alveolar compartment. However, each leucocyte fraction had a higher concentration in C than in early alveolar fractions, thus indicating the crucial role of immune defence in the cisternal compartment.

Seven variables – electrical conductivity (EC), somatic cell count (SCC), N-acetyl-β-D-glucosaminidase (NAGase), lactose, protein, fat and pH – were compared in four quarter milk fractions (MF1: strict foremilk; MF2: first 12–15 ml foremilk; MF3: subsequent 40–45 ml milk; MF4: strippings) and in one cow composite milk sample (CC) per cow. The study used 142 quarters from 37 lactating cows of the German Black Pied breed. To rule out any possible effect due to management, animal physiology and analytical procedures, the collection and processing of milk samples from each cow was repeated for three consecutive days, and the means of 3-d values were used. All variables were affected significantly by milk fraction and udder health. Compared with foremilk, EC, lactose and protein levels in strippings decreased, while SCC, NAGase and fat increased. The pH of foremilk and strippings did not differ significantly in healthy or in mastitic quarters. The difference between MF1 and MF2 was significant for EC in mastitic quarters, and for SCC in healthy quarters only. In general, mastitis resulted in a significant increase in EC, SCC, NAGase and protein but in a decrease in lactose and fat contents of milk in one or more of the milk fractions studied. Comparison of cow composite milk samples from healthy and mastitic cows revealed the significance (P<0·01) of udder health for EC, SCC and lactose. Of the different parameters that can distinguish between healthy and mastitic quarters or cows, EC could be used to classify 76% of quarters and 73% of cows correctly, while the lactose content permitted correct identification of 81% of quarters and 76% of cows. NAGase and pH could be used to determine the status of 73% and 61% of quarters, respectively. In general, the correlation observed in strippings was higher than in foremilk for almost all the variables studied. Surprisingly, EC, SCC, NAGase and lactose in milk from healthy quarters of mastitic cows (with at least one mastitic quarter) differed significantly (P<0·05) from those from healthy quarters of cows with all four healthy quarters, indicating an inconsistent effect of mastitic quarters on neighbouring healthy quarters (quarter interdependence).