**2.5. Statistical analysis**

in 0.9% NaCl solution) in a dose of 0.25 mg in the first, 0.5 in the second, 1.0 mg in the third or 2.0 mg *in toto* (6.25, 12.5, 25.0 or 50.0 μg kg−1 B.W.) in 4 weeks and then the registration was

In the third group of sheep (n = 6), after 30 min of control registration of the rumen motility, a rubber balloon (10 cm long) was inserted into the duodenum *via* the duodenal fistula. After placing the balloon in the jejunum, soon after the 2nd blood collection (0 time), the balloon was filled with 40 mL of warm water (DD40) and the distension was maintained for 5 min [28]. Then, the recording of ruminal contractions was continued for 60–90 min. Ten minutes before DD40, each animal received *i.c.v.* infusion of 100 μL of solvents for the drugs tested

In the fourth group of sheep (n = 6), after 30 min of control registration of the rumen motility, a rubber balloon (10 cm long) was inserted into the duodenum and 30 min after the animals received the 100 μL *i.c.v.* infusion of diltiazem, nifedipine (in 20% DMSO solution) or verapamil at a dose of 0.25 mg in the first, 0.5 mg in the second, 1.0 in the third or 2.0 mg *in toto* in the 4th week (the same mode it was used for diltiazem, nifedipine and verapamil experimentation). After 10 min of the diltiazem, nifedipine or verapamil, 1-minute infusion duodenum was distended for 5 min with the balloon containing 40 mL of water (DD40) at body temperature.

Experimental procedure lasted for 10 months. The doses of 1.0 and 2.0 mg diltiazem, nifedipine

The ruminal contractions were analyzed using the electronic tensometric recorder PIT 212 (COMT, Bialystok, Poland). The analysis of mechanograms and calculations of results were performed similarly as in a case of electromyographic recording [32]. The number of the rumen motor activity was determined by the frequency on mechanograms, with 5 min intervals before

Blood samples for the analysis of CA estimation were collected from the jugular external vein (according to a scheme described above—**Figure 1**). Blood samples were placed in 10 mL test tubes containing reduced glutathione (0.05 mM). The plastic tubes were maintained on ice, and after the centrifugation, plasma was stored at −80°C, until the beginning of the analytical process. The detection of CA levels was performed by radioimmunoassay using REA kits (CATECHOLA, Czech Republic). The sensitivity of this method was for E, 0.37, for NE, 0.53, and for DA, 0.85 nM L−1. The intra serial error for E was of 3%, for NE 4.2%, and for DA 6.1%,

Cortisol levels were detected by radioimmunoassay (RIA), according to previous experiments [18]. The mean intra and inter assay of the method was of 9.5 pg, for a sample of 10 μL (ORION DIAGNOSTICA, Espoo). Δmax concentration for each hormone was the difference between the

After the 5 min distension was over, the recording was continued for 60–90 min.

or verapamil *in toto* were effective in premedication contra DD40 only.

**2.3. The estimation of blood cortisol and catecholamine (CA) levels**

whereas the error among the series was of 4.2, 7.4, and 6.6%, respectively.

basal concentration and the highest concentration measured.

maintained for the next 90 min.

230 Pain Relief - From Analgesics to Alternative Therapies

(**Figure 1**).

**2.2. Mechanography**

and after the DD40.

Statistical significance of the results was carried out through the comparison of control values with those obtained after mechanical distension (duodenal distension—DD40), as well as after VGCCIs premedication and concurrent DD40, using a multifactorial analysis of variance (ANOVA). The statistical significance of the results was detected with a post hoc Tukey-Kramer test; the results are shown as a mean ± SEM. A p value, less than 0.05 was considered statistically significant in all tests.

The researches were performed according to the rules of the Principles of Laboratory Animal Care (NIH publication no. 86-23, revised 1985), as well as the specific national laws on protection of animal (National Law for Animals Protection – 1997, Dz. U.23 XI; Permission of 3rd Local Ethical Commission No. 9/2001 issued 11.01.2001).
