Hank s balanced salts solution

The continuous cell line of bovine mammary epithelial cells, BME-UV1, was grown to confluence in 6-well plates (Falcon Becton Dickinson Labware, Franklin Lakes, NJ) for 58 h, as described by Zavizion et al. (1996) . The cells were washed twice with Hanks' balanced salts solution (Sigma-Aldrich, St. Louis, MO). Complete medium without fetal calf serum was added before the challenge with CFS or live lactococcal cultures to avoid possible interference of the serum with the enzyme activity; the experiment was performed in duplicate.
Two L. lactis strains (LL11 and SL153) were incubated in RPMI-1640 medium for 48 h and the derived CFS were assayed with the MIC test to confirm the antibacterial activity. The CFS were then added to BME-UV1 cell-culture to a final concentration of 10% in fetal calf serum-free medium. Analogously, 30 μL of a suspension (10 4 cfu/mL) of each lactococcal live culture were added to distinct wells, and after 4, 8, 15, and 24 h of stimulation both culture medium and epithelial cells were collected and stored separately. Aliquots of 3 × 10 6 BME-UV1 cells were suspended in RNAlater (Sigma-Aldrich) and stored at -80°C until RNA extraction, whereas culture medium was frozen at -20°C.

Female crossbred pigs, 12 individuals, 95 ± 5days of age, weighing 30-35 kg were purchased 14 days before the experiments from a commercial piggery (with natural length of day) and used in the investigation. The animals were kept in a room, in which natural lighting from windows (in April) was supplemented during the day with fluorescent illumination with a light intensity of 500 lx at the level of the animals' heads. The fluorescent illumination was automatically turned on at 06:00 and turned off at 20:00. The gilts had free access to water and were fed twice daily (08:00 and 14:00) with standard food.
The gilts were slaughtered between 10:00 and 10:30 a.m. The pineal glands (4 in each experiment) were removed under sterile conditions as quickly as possible and placed in Hanks' Balanced Salts Solution (Sigma, USA) with penicillin (1000 U/ml, Sigma-Aldrich, USA), streptomycin (100 μg/ml, Sigma-Aldrich, USA) and amphotericin B (2.5 μg/ml, Sigma-Aldrich, USA).
Explanations: C -incubation in control medium, NE -incubation in medium with 10 μM of NE All experimental procedures on animals were performed in accordance with Polish and EU law. They were approved by the Local Ethics Committee for Experiments on Animals in Olsztyn.