**3.1. The effect of DD on the behavioral and clinical symptoms, blood plasma cortisol, and/or catecholamine level**

Duodenal distension by 40 mL of warm water resulted in a significant increase in the behavioral pain responses: motility, bleating, teeth grinding, prostration, wetting, defecation, tachycardia (from 60 ± 3 beats·min−1 to 86 ± 6.2 beats·min−1), hyperventilation (from 36.3 ± 3.6 number·min−1 to 50.3 ± 4.5 number·min−1), inhibition of reticulo-rumen contractions rate (from 6.15 ± 0.54 c × 5 min−1 in control to 1.09 ± 0.33 c × 5 min−1 during DD and to 1.35 ± 0.52 c × 5 min−1 10 min after DD (**Figure 2**; p = 0.001), from −82.2% to −78.0% during 15–20 min; a significant increase in plasma cortisol concentration from 10.51 ± 2.66 ng·mL−1 in control to 24.72 ± 8.25 ng·mL− 1 during DD and to 34.44 ± 5.46 ng·mL−1 (p ≤ 0.01) 10 min after DD (**Figure 3**); a statistically significant increase of plasma catecholamine concentration (over seven-fold increase of E from 0.34 ± 0.12 nM·L−1 in control to 2.87 ± 0.65 nM L−1, during 2 hours following the DD (**Figure 4**); 100% NE—from 1.29 ± 0.23 nM·L−1 in control to 2.32 ± 0.24 nM·L−1—120 min after DD (**Figure 4**) and 126% increase of DA from 0.93 ± 0.02 nM L−1 in the control to 2.33 ± 1.16 nM L−1, 120 min after DD) (**Figure 4**).

**Figure 2.** Influence of *i.c.v.* 1 min infusion after 10 min pretreatment of different voltage-gated calcium channels inhibitor (diltiazem, nifedipine or verapamil) in different doses (1.0 or 2.0 mg/animal) per number of reticulo-ruminal contractions (c × 5 min−1) in sheep in comparison with the group with duodenal distension, DD40 (x ± SEM, n = 6). Mean values of results obtained from the same blood collection (time point) with different superscript sign p ≤ 0.001–0.05 level in comparison to DD40 value.

**Figure 3.** Comparative analysis of 10 min premedication influence with *i.c.v.* diltiazem, nifedipine, and/or verapamil (in doses 1.0 and/or 2.0 mg/animal) and DD40 on plasma cortisol concentration in sheep, in comparison to DD40 value at p ≤ 0.001–0.05 (x ± SEM, n = 6).

c × 5 min−1 10 min after DD (**Figure 2**; p = 0.001), from −82.2% to −78.0% during 15–20 min; a significant increase in plasma cortisol concentration from 10.51 ± 2.66 ng·mL−1 in control

(**Figure 3**); a statistically significant increase of plasma catecholamine concentration (over seven-fold increase of E from 0.34 ± 0.12 nM·L−1 in control to 2.87 ± 0.65 nM L−1, during 2 hours following the DD (**Figure 4**); 100% NE—from 1.29 ± 0.23 nM·L−1 in control to 2.32 ± 0.24 nM·L−1—120 min after DD (**Figure 4**) and 126% increase of DA from 0.93 ± 0.02

**Figure 2.** Influence of *i.c.v.* 1 min infusion after 10 min pretreatment of different voltage-gated calcium channels inhibitor (diltiazem, nifedipine or verapamil) in different doses (1.0 or 2.0 mg/animal) per number of reticulo-ruminal contractions (c × 5 min−1) in sheep in comparison with the group with duodenal distension, DD40 (x ± SEM, n = 6). Mean values of results obtained from the same blood collection (time point) with different superscript sign p ≤ 0.001–0.05

**Figure 3.** Comparative analysis of 10 min premedication influence with *i.c.v.* diltiazem, nifedipine, and/or verapamil (in doses 1.0 and/or 2.0 mg/animal) and DD40 on plasma cortisol concentration in sheep, in comparison to DD40 value

nM L−1 in the control to 2.33 ± 1.16 nM L−1, 120 min after DD) (**Figure 4**).

1 during DD and to 34.44 ± 5.46 ng·mL−1 (p ≤ 0.01) 10 min after DD

to 24.72 ± 8.25 ng·mL−

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level in comparison to DD40 value.

at p ≤ 0.001–0.05 (x ± SEM, n = 6).

**Figure 4.** Comparative analysis of duodenal distension and premedication with different diltiazem, nifedipine and verapamil doses (1.0 or 2.0 mg/animal) on plasma epinephrine concentration in comparison with DD40 (x ± SEM, n = 6, p ≤ 0.001–0.05). Mean values of results obtained from the same blood collection (time point).

#### **3.2. The influence of VGCCIs premedication on the behavioral changes, clinical symptoms, rumen motility, plasma cortisol, and catecholamine level in animals with/without DD**

*I.C.V.* infusion of 1 min diltiazem, nifedipine or verapamil in doses of 0.25, 0.50, 1.0 or 2.0 mg *in toto*, did not have any significant influence on the behavioral and clinical symptoms (**Table 1**), rumen contractions count (**Figure 2**), cortisol (**Figure 3**), and CA's level in blood plasma (**Figures 4**–**6**).

**Figure 5.** Comparative analysis of duodenal distension and premedication with different diltiazem, nifedipine and verapamil doses (1.0 or 2.0 mg/animal) on plasma norepinephrine concentration in comparison with DD40 (x ± SEM, n = 6, p ≤ 0.001–0.05). Mean values of results obtained from the same blood collection (time point).

 **Figure 6.** Comparative analysis of duodenal distension and premedication with different diltiazem, nifedipine and ve- rapamil doses (1.0 or 2.0 mg/animal) on plasma dopamine concentration in comparison with DD40 (x ± SEM, n = 6, p ≤ 0.001–0.05). Mean values of results obtained from the same blood collection (time point).

 *I.C.V.* infusion of diltiazem, nifedipine, and verapamil (1.0 or 2.0 mg *in toto*), 10 min before the DD inhibited and/or completely attenuated the beginning of clinical symptoms of jejunal nociception, provoked by duodenal distension (**Table 1**). In control animals before premedication, intense acceleration of cardiac beats was observed (mean from 70 to 102 beats·min−1 ) and in the animals treated with diltiazem or verapamil, it was decreased to 63–86 beats·min−1 ; after nifedipine premedication, the DD caused an increased cardiac frequency from 68 to 90 beats·min−1. Respiration frequency was 50 and 34·min−1 , respectively.


 Value are mean ± SEM of 6 sheep, and indicate significant difference corresponding from control group. ẍ ± SEM, n = 6. \*p ≤ 0.001–0.05.

 **Table 2.** Frequency of ruminal contraction of the five groups (control, DD40, diltiazem + DD40, nifedipine + DD40, verapamil + DD40) during the course of the experiments.

 VGCCIs premedication caused that inhibition of rumen frequency after 5 min DD, decreasing from average 6.12 ± 0.40 to 5.00 ± 0.61, in 5 min, and 4.00 ± 0.38 c × 5min−1 , in 10 min, after DD, but not from 6.15 ± 0.54 in control to 1.09 ± 0.33 c × 5 min−1 after DD (**Table 2**, **Figure 2**). Plasma cortisol concentration changed from 10.83 ± 1.19 in control to 11.95 ± 1.25, during (NS), and 9.53 ± 1.36 (NS) 30 min, after DD, but not increased from 10.51 ± 2.66 in control to 24.72 ± 8.25, during DD, and 34.44 ± 5.46 (+227.7%) 10 min, after DD (p˂0.001, **Figure 3**). Diltiazem pre- medication caused that increase of plasma CA concentration after 5 min DD, decreased E from average +606.41% in control to +23.02%, during 120 min after DD (**Figure 4**); NE from +120.95% in control to −21%, during 120 min after DD (**Figure 5**) and DA from +124.2% in control to −24,8%, during 120 min after diltiazem premedication (**Figure 6**).

 Nifedipine *i.c.v.* premedication caused that inhibition of rumen frequency after 5 min DD, decreased from average 5.82 ± 0.45 to 1.89 ± 0.81 (−69%) in 5 min only, during DD, and 5.54 ± 0.23 (−5.2%) 10 min, after DD, but not from 6.15 in control to 1.78 c × 5 min−1 (−71.%), after DD (**Figure 2**). Nifedipine premedication diminished the increase in plasma cortisol concentration from 11.81 ± 1.13 ng L−1 (NS) in control to 10.25 ± 1.65 ng · L−1 (NS) in 10.00 and to 11.85 ng L−1 120 min after DD (**Figure 3**). Premedication by 1 min *i.c.v.* nifedipine infusion caused that increase of plasma catecholamine concentration, after 5 min DD, statistically significantly decreased from average 0.34 to 2.98 nM L−1 (+767.8%) in DD and from 0.91 in control to 1.08 nM L−1 (+23%) to E, from +98.5 to +23.7%, to NE, and from +124.2 in control to 61.3% to DA, during 120 min after DD (**Figures 4**–**6**).

 ẍ ± SEM, n = 6. \*p ≤ 0.001–0.05.

verapamil + DD40) during the course of the experiments.

 **Figure 6.** Comparative analysis of duodenal distension and premedication with different diltiazem, nifedipine and ve-rapamil doses (1.0 or 2.0 mg/animal) on plasma dopamine concentration in comparison with DD40 (x ± SEM, n = 6,

 *I.C.V.* infusion of diltiazem, nifedipine, and verapamil (1.0 or 2.0 mg *in toto*), 10 min before the DD inhibited and/or completely attenuated the beginning of clinical symptoms of jejunal nociception, provoked by duodenal distension (**Table 1**). In control animals before premedication, intense acceleration of cardiac beats was observed (mean from 70 to 102 beats·min−1 ) and in the animals treated with diltiazem or verapamil, it was decreased to 63–86 beats·min−1 ; after nifedipine premedication, the DD caused an increased cardiac frequency from 68 to 90

**Drugs Control 5 min 10 min 15 min 20 min 25 min 30 min** 0.9% NaCl 6.45 ± 0.75 6.12 ± 0.28 6.00 ± 0.25 6.80 ± 0.33 6.10 ± 0.60 6.35 ± 0.15 6.11 ± 0.43 DD40 6.15 ± 0.54 1.09 ± 0.33\* 1.78 ± 0.49\* 1.35 ± 0.52\* 2.20 ± 0.31\* 0.61 ± 0.12\* 4.91 ± 0.75 Diltiazem + DD40 5.00 ± 0.61\* 4.00 ± 0.38\* 3.80 ± 0.60\* 4.23 ± 0.36\* 5.14 ± 0.42 4.82 ± 0.64 6.12 ± 0.40 Nifedipine + DD40 5.82 ± 0.45 1.89 ± 0.81\* 5.54 ± 0.23 4.88 ± 0.62 5.12 ± 0.74 6.11 ± 1.11 5.59 ± 1.22 Verapamil + DD40 6.12 ± 0.89 5.33 ± 0.51 5.75 ± 0.11 5.05 ± 0.80 4.97 ± 0.65 5.55 ± 1.02 5.85 ± 0.61

Value are mean ± SEM of 6 sheep, and indicate significant difference corresponding from control group.

**Table 2.** Frequency of ruminal contraction of the five groups (control, DD40, diltiazem + DD40, nifedipine + DD40,

 VGCCIs premedication caused that inhibition of rumen frequency after 5 min DD, decreasing from average 6.12 ± 0.40 to 5.00 ± 0.61, in 5 min, and 4.00 ± 0.38 c × 5min−1 , in 10 min, after DD,

p ≤ 0.001–0.05). Mean values of results obtained from the same blood collection (time point).

 Pain Relief - From Analgesics to Alternative Therapies Pain Relief - From Analgesics to Alternative Therapies

beats·min−1. Respiration frequency was 50 and 34·min−1 , respectively.

 Verapamil *i.c.v.* premedication by 1 min infusion caused that inhibition rumen frequency after 5 min DD, decreased from 6.12 ± 0.89 to 5.38 ± 0.53 (−12.1%) during 30 min after DD and by average 44.7% in comparison to DD only (**Table 2**). In the same time, verapamil premedication caused that increase of plasma cortisol concentration after 5 min DD, decreased from average 11.53 ± 0.98 in control to 7.91 ± 1.58 (−36.1%; p˂0.05) during DD and 11.39 ± 1.48 ng·L−1 (NS) during 120 min after DD and by −153% (p˂0.001) in comparison to DD group (**Table 3**).


 **Table 3.** Comparative analysis *i.c.v.* diltiazem, nifedipine, or verapamil (in the doses 2 mg *in toto*) premedication influence and DD40 on plasma cortisol concentration changes in sheep.

 Verapamil *i.c.v.* premedication caused that plasma epinephrine after 5 min DD increased, but nonsignificantly from average 1.19 ± 0.29 in control to 1.36 ± 0.59 (+14.7%), during 120 min after DD, but not from 0.34 ± 0.12 in control to 2.99 ± 0.81 nM L−1 , e.g., +767.8% (p˂0.001), during 120 min after DD (**Table 4**). Decrease of epinephrine plasma concentration by verapamil premedication was 753.08%.

Verapamil *i.c.v.* premedication caused that increase of plasma norepinephrine concentration after 5 min DD, increased from average 1.29 ± 0.22 in control to 1.58 ± 0.84 nM L−1, during 120 min after DD, but not from 1.29 ± 0.23 in control to 2.50 ± 0.42 nM L−1 (+98.56%), during 120 min after DD. Decrease in norepinephrine concentration by verapamil premedication was 75.71%.


**Table 4.** Comparative analysis *i.c.v.* diltiazem, nifedipine, and verapamil (in the dose of 1.0 or 2.0 mg *in toto*) premedication influence and DD40 on concentration epinephrine, norepinephrine and dopamine plasma level changes in sheep in comparison to the control values (ẍ ± SEM; n = 6).

*I.C.V.* verapamil premedication caused that plasma dopamine concentration after 5 min DD increased from 0.92 ± 0.04 in control to 1.52 ± 0.13 nM L−1, during 120 min after DD (+65.04%), but not from 0.93 ± 0.02 in control to 2.33 ± 1.16, during 120 min after DD (+126.3%; p˂0.001, **Table 4**). Decrease in dopamine concentration by verapamil premedication was 61.26% (p˂0.01).
