Effects of different dietary levels of Piperine supplementation on growth, feed utilization, intestinal morphology, innate immunity and IGF-I expression in olive flounder (Paralichthys olivaceus) and red sea bream (Pagrus major)

Abstract

This study was carried out in two sets of experiments to evaluate the effects of dietary supplementation of piperine and black pepper for red sea bream (Pagrus major) (Exp-I) and olive flounder (Paralichthys olivaceus) (Exp-II). In this study, fish growth performance, feed utilization, diet digestibility, gene expression level of liver insulin-like growth factor I (IGF-I), intestinal histology, innate immunity and disease resistance were examined. In Exp I, a fish meal based diet was regarded as a control diet and five other experimental diets were formulated by dietary inclusion of piperine at different inclusion levels of 0.025, 0.05, 0.1 and 0.2% (T1,T2,T3 and T4, respectively) or black pepper at a level of 0.1% (T5). Triplicate groups of fish (initial mean body weight, 7.6 ± 0.1 g) were fed one of the experimental diets to apparent satiation for 13 weeks. At the end of the feeding trial, all the treatment groups exhibited significantly higher growth compared to the control group. Specific growth rate (SGR) and protein efficiency ratio (PER) have followed similar trends to weight gain. Feed conversion ratio was significantly reduced with piperine and pepper supplementation while 0.05% piperine supplemented group showed the best performance. Plasma hemoglobin level was increased significantly, while hematocrit, glucose and protein levels were increased numerically by piperine. Total cholesterol level in plasma was significantly reduced with piperine supplementation in diets. Analysis of innate immunity parameters revealed significant enhancements with the supplementation. The highest Superoxide dismutase (SOD) value was observed in T2 group and the highest lysozyme and total immunoglobulin were observed in T5 group while all treatment groups showed significantly higher immune response for myeloperoxidase (MPO), SOD, total immunoglobulin, lysozyme and antiprotease activities compared to the control group. Results of whole-body composition showed significant reduction in dry matter of fish fed piperine compared to the control group. Ash content was significantly increased with the supplementation except for T5 group. The highest protein content was found in T2 group. Liver Insulin like growth factor-I (IGF-I) mRNA level was influenced by dietary inclusion of piperine and pepper even though it was not significant. Conditional factor and goblet cell count increased significantly with the dietary supplementation and the highest values were observed in T1 group. Also, villi length increased significantly in piperine supplemented groups in which all the treatment groups showed significantly higher values compared to the control while T1 has shown the highest. In Exp II, a fish meal based diet was regarded as a control diet and six other experimental diets were formulated by dietary inclusion of piperine at different inclusion levels of 0.025, 0.05, 0.075, 0.1 and 0.2% (T1,T2,T3,T4 and T5 respectively) or black pepper at a level of 0.1% (T6). Triplicate groups of fish (initial mean body weight, 27.6 ± 0.4 g) were fed one of the experimental diets to apparent satiation for 8 weeks. At the end of the feeding trial, growth performance and feed utilization of the fish groups were significantly improved with dietary supplementation of piperine. Weight gain and specific growth rate were observed the highest in T2 group. Lowest feed conversion ratio was given by T4 group even though it was not affected significantly. All treatment groups showed significantly higher PER compared to the control while T3 group showed the highest. T6 group showed the highest Immunoglobulin and antiprotease levels whereas T4 represented the highest lysozyme activity. T5 exhibited the highest NBT value among the treatments. Other immunity parameters were increased with the dietary supplementation but were not significantly affected. Significantly higher whole-body protein level was observed in T2 group compared with the control group while dry matter and lipid levels were found to be the highest in T1 group. T1, T2, T3 and T6 groups showed significantly higher dry matter digestibility compared to the control group while T2 was the best. T1 and T2 groups showed significantly higher protein digestibility than other treatments. Liver IGF-I mRNA expression level was increased significantly with the dietary supplementation of piperine. During the E. tarda challenge test, all treatment groups showed higher survival rates compared to the control group. T1, T2 and T6 groups showed the highest survival rate. Conditional factor of fish was increased by the dietary treatment numerically while villi length and goblet cell count were increased significantly. All piperine supplemented groups exhibited significantly higher villi length values compared to the control while T2 group was the highest. In conclusions, dietary piperine supplementation could improve the growth rate of red sea bream and olive flounder. Dietary piperine supplementation might assist the digestion and absorption of dietary nutrients to both fishes and thereby increase the diet utilization efficiency. Dietary piperine supplementation could improve the innate immunity of the fishes and increase disease resistance. Dietary piperine supplementation would have beneficial effects on the length and surface area of villi, and on the goblet cells which help to excrete mucosa, enzymes and hormones to facilitate a better digestibility in both fishes. Further studies are necessary for the IGF-1 expression even though piperine showed some positive effects in both fishes. Therefore, optimum level of piperine in diets would be approximately 0.05% for both red sea bream and olive flounder.

양어사료 내 piperine과 black pepper 가루의 첨가 효능을 평가하기 위해 참돔(실험-I)과 넙치(실험-II) 치어를 대상으로 총 2개의 사양실험이 수행되었다. 실험 목적을 달성하기 위해 어류의 성장률, 사료효율, 외관상소화율, 비특이적 면역력, 장 조직 변화, IGF-I 유전자발현 및 공격실험에 의한 질병저항성을 조사하였다. 실험-I에서는 어분을 기초로 한 대조구(control), piperine을 각각 0.025, 0.05, 0.1, 0.2% (T1, T2, T3, T4) 첨가한 실험구 및 black pepper를 0.1% (T5) 첨가한 실험구로 총 6개의 실험사료로 구성하였다. 참돔 치어(초기평균무게: 7.6 ± 0.1 g)는 총 3반복구로 배치되었으며 1일 3회에 나누어 13주 동안 만복공급 하였다. 13주간 실시된 사양실험 결과, 사료전환효율에서 사료 내 piperine이 0.05% 이상 첨가된 실험구(T2, T3, T4)가 대조구에 비해 유의적으로 낮았다. 비특이적 면역 분석 결과, SOD에서 모든 첨가실험구가 대조구에 비해 유의적으로 높은 값을 보였다. NBT를 제외한 MPO, SOD, total immunoglobulin, lysozyme, antiprotease 분석결과, piperine 첨가 실험구가 대조구에 비해 유의적으로 높은 결과 값을 보였다. 참돔의 형태학적 및 장 조직학적 변화 결과, 비만도(CF)는 첨가실험구들이 대조구와 비교하여 유의적으로 높았으며, T1실험구가 가장 높은 값을 보였다. 또한, 융모길이(VL)는 사료 내 piperine을 첨가한 실험구(T1, T2, T3)가 대조구에 비해 유의적으로 높은 값을 보였다. 배상세포(GC)의 수에서도 piperine 첨가실험구가 대조구와 비교하여 높은 경향을 보였다. 간 내 IGF-I mRNA분석 결과에서는 실험구간에 유의적인 차이는 없었지만, 저농도의 piperine을 첨가한 실험구에서 대조구에 비해 증가하는 경향을 보였다. 실험-II는 실험-I과 동일하게 어분을 기초로 한 대조구(control), piperine을 각각 0.025, 0.05, 0.075, 0.1, 0.2% (T1, T2, T3, T4, T5) 첨가한 사료구 및 black pepper를 0.1% (T6) 첨가한 사료실험구로 총 7개의 실험사료로 구성되었다. 넙치 치어(초기평균무게: 27.6 ± 0.4 g)는 총 3반복구로 배치되었으며 1일 3회에 나누어 8주 동안 실험사료를 만복공급 하였다. 8주간 사양실험이 종료 된 후 성장지표와 사료효율 측정결과 유의적인 차이는 없었으나, 성장율과 일간성장율에서 T2실험구가 가장 높은 값을 보였으며, 사료전환효율에서 T4실험구가 가장 낮은 값을 보였다. Total immunoglobulin, lysozyme 활성 분석 결과, 모든 실험구에서 대조구와 비교하여 유의적으로 높은 값을 보였다. T6실험구는 Ig분석에서, T4실험구는 lysozyme 활성 분석에서 가장 높은 결과를 보였다. 외관상소화율 분석결과, 건물소화율은 첨가실험구에서 유의적으로 높은 값을 보였다. T1, T2, T3, T6실험구는 대조구와 비교하여 유의적으로 높은 소화율 값을 보였으며, T2실험구가 가장 높은 소화율 값을 보였다. 단백질소화율 결과, T1, T2실험구가 다른 실험구에 비교하여 유의적으로 높은 값을 보였다. 간 IGF-I mRNA 분석 결과, 첨가실험구에서 증가하는 경향을 보였지만 유의적인 차이는 없었다. E.tarda를 이용한 공격실험 결과, 모든 실험구에서 대조구와 비교하여 높은 생존율을 보였으며, T1, T2, T6실험구가 가장 높은 생존율을 보였다. 비만도 측정 결과 수치상으로 증가하는 경향을 보였으며, 융모길이 측정 결과, piperine첨가실험구가 대조구와 비교하여 유의적으로 높은 값을 보였다. 결론적으로, 사료 내 적당량의 piperine 첨가는 참돔과 넙치의 성장을 촉진시킬 수 있으며, 사료 내 영양소의 소화, 흡수를 도와 사료의 이용효율을 높일 수 있을 것으로 보인다. 또한 참돔과 넙치의 비특이적 면역력을 높일 수 있으며, 소장 내 융모의 길이와 표면적을 높이고 배상세포의 증식에도 긍정적인 영향을 미칠 것으로 사료된다. 참돔과 넙치 사료 내 piperine 적정 첨가함량은 약 0.05% 내외가 될 것으로 추측된다. Piperine은 IGF-I의 유전자 발현에도 긍정적인 영향이 있을 것으로 추측되지만 보충 연구가 요구된다. 또한 양어사료 내 piperine의 첨가효과와 piperine의 체내 이용 및 대사에 관한 보다 많은 보충연구가 요구된다.