投喂云芝多糖对银鲫免疫机能和生理生化特性的影响
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摘要
本研究以银鲫(Carassius auratus)为试验对象，在饲料中分别添加不同浓度的云芝多糖(Coriolus versciclor polysaccharides，CVP)，研究云芝多糖对银鲫非特异性免疫机能、免疫应答能力及血液生理生化特性的影响。主要研究结果如下：
1．在研究云芝多糖对银鲫非特异性免疫机能影响的试验中，云芝多糖在饲料中的添加量分别为：0g／kg(C_0组)、0．25g／kg(C_(0．25)组)、0．5g／kg(C_(0．5)组)、1．0g／kg(C_(1．0)组)、2．0g／kg(C_(2．0)组)和4．0g／kg(C_(4．0)组)。各组分别投喂相应的饲料，于试验开始前(第0d)和试验开始后第7d、14d、21d、28d、35d、42d和56d取样检测供试鱼血液的白细胞吞噬活性、血清溶菌酶活力、血清SOD活性和补体C3、C4的含量。结果显示：饲料中添加0．25g／kg的云芝多糖，对银鲫的白细胞吞噬活性、血清溶菌酶活力、血清SOD酶活性和补体C3、C4的含量影响不显著(P>0．05)；添加0．5g／kg和1．0g／kg的云芝多糖，对供试鱼上述指标都有一定程度的提高，并当添加量为1．0g／kg时，在14d～21d内效果最显著(P<0．05)；而添加2．0g／kg和4．0g／kg的云芝多糖，上述指标在试验的后期均显著低于对照组。本研究结果说明在银鲫饲料中添加0．5g／kg～1．0g／kg的云芝多糖对银鲫的非特异性免疫机能有增强作用。
2．在研究云芝多糖对银鲫免疫应答能力影响的试验中，Ⅰ_0组、Ⅰ_(0．5)组、Ⅰ_(1.0)组和Ⅰ_(2．0)组注射灭活的嗜水气单胞菌(Aeromonas hydrophila)疫苗作为免疫组，C_0组、C_(0．5)组、C_(1．0)组和C_(2．0)组注射无菌生理盐水作为对照组。其中Ⅰ_(0．5)组与C_(0．5)组饲料中云芝多糖添加量为0．5g／kg，Ⅰ_(1．0)组与C_(1．0)组饲料中云芝多糖添加量为1．0g／kg，Ⅰ_(2．0)组与C_(2．0)组饲料中云芝多糖添加量为2．0g／kg，Ⅰ_0组与C_0组投喂基础饲料。各组分别投喂相应的饲料，于免疫前(第0d)和免疫后第7d、14d、21d、28d和35d测定银鲫的血液白细胞吞噬活性、血清溶菌酶活性、补体C_3、C_4含量、凝集抗体效价、RBC-C_(3b)受体花环率、RBC-IC花环率和攻毒后的免疫保护率。结果显示：
(1)饲料中添加0．5g／kg和1．0g／kg的云芝多糖可显著提高受免银鲫的血液白细胞吞噬活性、血清溶菌酶活性及补体C3、C4含量(P<0．05)；而饲料中添加2．0g／kg的云芝多糖在试验的后期对银鲫的上述指标起抑制作用。
(2)与对受免银鲫血液白细胞吞噬活性、血清溶菌酶活性及补体C3、C4含量的影响相似，云芝多糖也能提高非受免组银鲫的上述指标，影响趋势基本一致；但与受免组银鲫相比，非受免组银鲫的上述指标均低于相应的免疫组。
(3)饲料中添加0．5g／kg和1．0g／kg的云芝多糖在第21d和28d能显著提高银鲫的RBC-C_(3b)受体花环率和降低RBC-IC花环率；而饲料中添加2．0g／kg的云芝多糖在试验后期会降低银鲫RBC-C_(3b)受体花环率和提高RBC-IC花环率，但与对照组差异不显著(P>0．05)。
(4)云芝多糖能提高银鲫的凝集抗体效价。其中，当云芝多糖浓度为1．0g／kg，投喂时间为21d时，受免银鲫的平均凝集抗体效价最高，为597．3。
(5)投喂云芝多糖能提高银鲫经嗜水气单胞菌活菌攻毒后的存活率。其中，当云芝多糖在饲料中的添加浓度为1．0g／kg时，受免银鲫的存活率和免疫保护率最高，分别为85％和57．14。
3．在研究云芝多糖对银鲫血液生理生化特性影响的试验中，云芝多糖在饲料中的添加量分别为：0g／kg(C_0组)、0．25g／kg(C_(0．25)组)、0．5g／kg(C_(0．5)组)、1．0g／kg(C_(1．0)组)、2．0g／kg(C_(2．0)组)和4．0g／kg(C_(4．0)组)。各组分别投喂相应的饲料，于试验开始前(第0d)和试验开始后第14d、28d、42d和56d取样检测供试鱼血液的红细胞数、白细胞数、血红蛋白含量和红细胞沉降率等生理指标，以及血清血糖、胆固醇、甘油三酯、白蛋白、总蛋白、尿素氮、谷草转氨酶、谷丙转氨酶和碱性磷酸酶等生化指标。结果显示：投喂0．25g／kg的云芝多糖对银鲫的血液各个指标无显著影响(P>0．05)，对机体的生理状态影响较小。持续投喂0．5g／kg～1．0g／kg的云芝多糖，短期内(14d或28d)可使银鲫血细胞数量和血红蛋白含量升高，红细胞沉降率下降；长时间投喂(42d或56d)，可使银鲫血清血糖、尿素、胆固醇和甘油三酯含量及谷草转氨酶、谷丙转氨酶活性降低，血清总蛋白和白蛋白含量及血清碱性磷酸酶活性升高；表明0．5g／kg～1．0g／kg的云芝多糖在一定程度上可改善银鲫的生理机能，提高机体的代谢强度。长时间持续投喂2．0g／kg～4．0g／kg的云芝多糖，银鲫血细胞数量、血红蛋白、血清总蛋白、白蛋白含量和碱性磷酸酶活性下降，血液红细胞沉降率、血清血糖、尿素氮、胆固醇和甘油三酯含量及谷草转氨酶、谷丙转氨酶活性升高，表明机体氧运输能力减弱，消化排泄功能减退。
The paper studied effects of Coriolus versciclor polysaccharides (CVP) on the non-specific immune function, the ability of immune response and the physiological and biochemical characteristic in blood of Carassius auratus. The main results were summarized as follows:
1. In order to study the effects of CVP on the non-specific immune function of C. auratus, the concentrations of CVP in six groups were respectively 0g/kg (group C_0), 0.25g/kg (group C_(0.25)), 0.5g/kg (group C_(0.5)), 1. 0g/kg (group C_(1.0)), 2.0g/kg (group C_(2.0)) and 4.0g/kg (group C_(4.0)). During the experiment, fishes of different groups were fed with appropriate feed separately and the phagocytic activity of leukocytes, lysozyme activity, Superoxide dismutase (SOD) activity and the content of complement C3, C4 in serum of C. auratus were tested at 0 day, 7~(th) day, 14~(th) day, 21~(st) day, 28~(th) day, 35~(th) day, 42~(th) day and 56~(th) day. The results showed that the addition of 0.25g/kg CVP in diet had no siginificant effect on the fish of phagocytic activity of leukocytes, lysozyme activity, SOD activity, the content of complement C3, C4 in serum of C. auratus (P>0.05). However, the non-specific immune function was markedly enhanced by dietary 0.5g/kg and 1.0g/kg CVP, especially, the effects of 1.0g/kg CVP were best during 14-21 days (P<0.05). When the addition was 2.0g/kg and 4.0/kg, these indexes were much lower than that in the control group at the anaphase of the experiment. And the results indicated that the non-specific immune function of C. auratus was enhanced by dietary 0.5g/kg to 1 .0g/kg CVP.
2. In the test to study the effects of CVP on the ability of immune response of C. auratus, the influences were determined by the phagocytic activity of leukocytes, lysozyme activity in serum, the content of serum complement C3, C4, the red blood cell C_(3b) receptors rosette forming rate (RBC-C_(3b)RR), the red blood cell immunocomplex rosette forming rate (RBC-ICR), agglutinating antibody titer in the serum and relative percent survival (RPS) by challenging with Aeromonas hydrophila. Fishes were divided into 8 groups, and fishes in group C_0, group C_(0.5), group C_(1.0) and group C_(2.0) were injected with physiological saline and in group I_0, group I_(0.5), group I_(1.0) and group I_(2.0) they were all inoculated with formalin killed A. hydrophila The concentrations of CVP in group C_(0.5) and group I_(0.5) was 0.5g/kg, which in group C_(1.0) and groupI_(1.0) was 1.0g/kg, which in group C_(2.0) and groupI_(2.0) was 2.0g/kg, and the feed of group C_0 and group I_0 didn't contain CVP. The immune indexes were tested on the 0 day, 7~(th) day, 14~(th) day, 21~(st) day, 28~(th) day, and 35~(th) day of the experiment and the RPS was also recorded after challeng with A. hydrophila. The results showed that:
(1) The phagocytic activity of leukocytes, lysozyme activity and the content of serum complement C3, C4 in serum of the immunized C. auratus were markedly enhanced by dietary 0.5g/kg and 1.0g/kg CVP (P<0.05). Howere, these indexes were restrained by dietary 2.0g/kg CVP at the anaphase of the experiment.
(2) CVP could improve the phagocytic activity of leukocytes, lysozyme activity and the content of serum complement C3, C4 in serum of non-immunized C. auratus, and the trend is the same as the effects of CVP on immunized C. auratus. But these indexes of non-immunized C. auratus is lower than that were immunized.
(3) RBC-C_(3b)RR of C.auratus was markedly improved and RBC-ICR was markedly debased by dietary 0.5g/kg and 1.0g/kg CVP at 21~(st) day and 28~(th) day. RBC-C_(3b)RR of C. auratus was debased and RBC-ICR was improved by dietary 2.0g/kg CVP, but there were no significant differences in RBC-C_(3b)RR and RBC-ICR among control group and other testing group (P>0.05).
(4) CVP could improve agglutinating antibody titer of C. auratus. The agglutinating antibody titer of immunized C. auratus was highest by dietary 1.0g/kg CVP at 21~(th) day, and the highest agglutinating antibody titer was 597.3
(5) CVP could improve the survival rate of C. auratus after challenged with A. hydrophila. Meantime, the survival rate and RPS of immunized C. auratus was highest by dietary 1 .0g/kg CVP, and they was respectively 85%, 57.14.
3. In order to study the effects of CVP on the physiological and biochemical characteristic in blood of C. auratus, the concentrations of CVP in six groups were respectively 0g/kg (group C_0), 0.25g/kg (group C_(0.25)), 0.5g/kg (group C_(0.5)), 1.0g/kg (group C_(1.0)), 2.0g/kg (group C_(2.0)) and 4.0g/kg (group C_(4.0)). Some physiological parameters such as the number of red blood cell, he number of white blood cell, the content of hemoglobin, sedimentation rate of red blood cell and some biochemical parameters such as the concent of Glucose, Cholesterol, Triglycerides, Total protein, Albumin, Urea and the activity of Glutamate-oxaloacetate transaminase (GOT), the activity of Glutamate-pyruvate transaminase (GPT) and the activity of Alkaline phosphatase(ALP) in the serum were assayed at 0 day, 14~(th) day, 28~(th) day, 42~(th) day and 56~(th) day. The results showed that: When the concentrations of CVP was 0.25g/kg, there were no significant differences in the physiological parameters and biochemical parameters of C. auratus, and it had less effects on physiological function of C. auratus. 0.5g/kg~1.0g/kg CVP of dietary could markedly elevated the number of red blood cell, white blood cell, the content of hemoglobin and reduced the sedimentation rate of red blood for 14 days or 28 days. When the experiment continued for more than 42 days or 56 days, the concent of Glucose, Urea, Cholesterol, Triglycerides and the activity of GOT and GPT in the serum declined, but the concent of Total protein, Albumin and the activity of ALP in the serum increased. It indicated that 0.5g/kg-1.0g/kg CVP could improve the physiological function and metabolism intensity of C. auratus. However, when the fishes were fed with 2.0g/kg-4.0g/kg CVP for long time, the number of red blood cell, white blood cell, the content of hemoglobin, the concent of Total protein, Albumin and the activity of ALP in the serum of C. auratus declined, the sedimentation rate of red blood, the concent of Glucose, Urea, Cholesterol, Triglycerides and the activity of GOT and GPT in the serum increased, indicating weakened oxygen transportation, absorption and excretion.
1．在研究云芝多糖对银鲫非特异性免疫机能影响的试验中，云芝多糖在饲料中的添加量分别为：0g／kg(C_0组)、0．25g／kg(C_(0．25)组)、0．5g／kg(C_(0．5)组)、1．0g／kg(C_(1．0)组)、2．0g／kg(C_(2．0)组)和4．0g／kg(C_(4．0)组)。各组分别投喂相应的饲料，于试验开始前(第0d)和试验开始后第7d、14d、21d、28d、35d、42d和56d取样检测供试鱼血液的白细胞吞噬活性、血清溶菌酶活力、血清SOD活性和补体C3、C4的含量。结果显示：饲料中添加0．25g／kg的云芝多糖，对银鲫的白细胞吞噬活性、血清溶菌酶活力、血清SOD酶活性和补体C3、C4的含量影响不显著(P>0．05)；添加0．5g／kg和1．0g／kg的云芝多糖，对供试鱼上述指标都有一定程度的提高，并当添加量为1．0g／kg时，在14d～21d内效果最显著(P<0．05)；而添加2．0g／kg和4．0g／kg的云芝多糖，上述指标在试验的后期均显著低于对照组。本研究结果说明在银鲫饲料中添加0．5g／kg～1．0g／kg的云芝多糖对银鲫的非特异性免疫机能有增强作用。
2．在研究云芝多糖对银鲫免疫应答能力影响的试验中，Ⅰ_0组、Ⅰ_(0．5)组、Ⅰ_(1.0)组和Ⅰ_(2．0)组注射灭活的嗜水气单胞菌(Aeromonas hydrophila)疫苗作为免疫组，C_0组、C_(0．5)组、C_(1．0)组和C_(2．0)组注射无菌生理盐水作为对照组。其中Ⅰ_(0．5)组与C_(0．5)组饲料中云芝多糖添加量为0．5g／kg，Ⅰ_(1．0)组与C_(1．0)组饲料中云芝多糖添加量为1．0g／kg，Ⅰ_(2．0)组与C_(2．0)组饲料中云芝多糖添加量为2．0g／kg，Ⅰ_0组与C_0组投喂基础饲料。各组分别投喂相应的饲料，于免疫前(第0d)和免疫后第7d、14d、21d、28d和35d测定银鲫的血液白细胞吞噬活性、血清溶菌酶活性、补体C_3、C_4含量、凝集抗体效价、RBC-C_(3b)受体花环率、RBC-IC花环率和攻毒后的免疫保护率。结果显示：
(1)饲料中添加0．5g／kg和1．0g／kg的云芝多糖可显著提高受免银鲫的血液白细胞吞噬活性、血清溶菌酶活性及补体C3、C4含量(P<0．05)；而饲料中添加2．0g／kg的云芝多糖在试验的后期对银鲫的上述指标起抑制作用。
(2)与对受免银鲫血液白细胞吞噬活性、血清溶菌酶活性及补体C3、C4含量的影响相似，云芝多糖也能提高非受免组银鲫的上述指标，影响趋势基本一致；但与受免组银鲫相比，非受免组银鲫的上述指标均低于相应的免疫组。
(3)饲料中添加0．5g／kg和1．0g／kg的云芝多糖在第21d和28d能显著提高银鲫的RBC-C_(3b)受体花环率和降低RBC-IC花环率；而饲料中添加2．0g／kg的云芝多糖在试验后期会降低银鲫RBC-C_(3b)受体花环率和提高RBC-IC花环率，但与对照组差异不显著(P>0．05)。
(4)云芝多糖能提高银鲫的凝集抗体效价。其中，当云芝多糖浓度为1．0g／kg，投喂时间为21d时，受免银鲫的平均凝集抗体效价最高，为597．3。
(5)投喂云芝多糖能提高银鲫经嗜水气单胞菌活菌攻毒后的存活率。其中，当云芝多糖在饲料中的添加浓度为1．0g／kg时，受免银鲫的存活率和免疫保护率最高，分别为85％和57．14。
3．在研究云芝多糖对银鲫血液生理生化特性影响的试验中，云芝多糖在饲料中的添加量分别为：0g／kg(C_0组)、0．25g／kg(C_(0．25)组)、0．5g／kg(C_(0．5)组)、1．0g／kg(C_(1．0)组)、2．0g／kg(C_(2．0)组)和4．0g／kg(C_(4．0)组)。各组分别投喂相应的饲料，于试验开始前(第0d)和试验开始后第14d、28d、42d和56d取样检测供试鱼血液的红细胞数、白细胞数、血红蛋白含量和红细胞沉降率等生理指标，以及血清血糖、胆固醇、甘油三酯、白蛋白、总蛋白、尿素氮、谷草转氨酶、谷丙转氨酶和碱性磷酸酶等生化指标。结果显示：投喂0．25g／kg的云芝多糖对银鲫的血液各个指标无显著影响(P>0．05)，对机体的生理状态影响较小。持续投喂0．5g／kg～1．0g／kg的云芝多糖，短期内(14d或28d)可使银鲫血细胞数量和血红蛋白含量升高，红细胞沉降率下降；长时间投喂(42d或56d)，可使银鲫血清血糖、尿素、胆固醇和甘油三酯含量及谷草转氨酶、谷丙转氨酶活性降低，血清总蛋白和白蛋白含量及血清碱性磷酸酶活性升高；表明0．5g／kg～1．0g／kg的云芝多糖在一定程度上可改善银鲫的生理机能，提高机体的代谢强度。长时间持续投喂2．0g／kg～4．0g／kg的云芝多糖，银鲫血细胞数量、血红蛋白、血清总蛋白、白蛋白含量和碱性磷酸酶活性下降，血液红细胞沉降率、血清血糖、尿素氮、胆固醇和甘油三酯含量及谷草转氨酶、谷丙转氨酶活性升高，表明机体氧运输能力减弱，消化排泄功能减退。
The paper studied effects of Coriolus versciclor polysaccharides (CVP) on the non-specific immune function, the ability of immune response and the physiological and biochemical characteristic in blood of Carassius auratus. The main results were summarized as follows:
1. In order to study the effects of CVP on the non-specific immune function of C. auratus, the concentrations of CVP in six groups were respectively 0g/kg (group C_0), 0.25g/kg (group C_(0.25)), 0.5g/kg (group C_(0.5)), 1. 0g/kg (group C_(1.0)), 2.0g/kg (group C_(2.0)) and 4.0g/kg (group C_(4.0)). During the experiment, fishes of different groups were fed with appropriate feed separately and the phagocytic activity of leukocytes, lysozyme activity, Superoxide dismutase (SOD) activity and the content of complement C3, C4 in serum of C. auratus were tested at 0 day, 7~(th) day, 14~(th) day, 21~(st) day, 28~(th) day, 35~(th) day, 42~(th) day and 56~(th) day. The results showed that the addition of 0.25g/kg CVP in diet had no siginificant effect on the fish of phagocytic activity of leukocytes, lysozyme activity, SOD activity, the content of complement C3, C4 in serum of C. auratus (P>0.05). However, the non-specific immune function was markedly enhanced by dietary 0.5g/kg and 1.0g/kg CVP, especially, the effects of 1.0g/kg CVP were best during 14-21 days (P<0.05). When the addition was 2.0g/kg and 4.0/kg, these indexes were much lower than that in the control group at the anaphase of the experiment. And the results indicated that the non-specific immune function of C. auratus was enhanced by dietary 0.5g/kg to 1 .0g/kg CVP.
2. In the test to study the effects of CVP on the ability of immune response of C. auratus, the influences were determined by the phagocytic activity of leukocytes, lysozyme activity in serum, the content of serum complement C3, C4, the red blood cell C_(3b) receptors rosette forming rate (RBC-C_(3b)RR), the red blood cell immunocomplex rosette forming rate (RBC-ICR), agglutinating antibody titer in the serum and relative percent survival (RPS) by challenging with Aeromonas hydrophila. Fishes were divided into 8 groups, and fishes in group C_0, group C_(0.5), group C_(1.0) and group C_(2.0) were injected with physiological saline and in group I_0, group I_(0.5), group I_(1.0) and group I_(2.0) they were all inoculated with formalin killed A. hydrophila The concentrations of CVP in group C_(0.5) and group I_(0.5) was 0.5g/kg, which in group C_(1.0) and groupI_(1.0) was 1.0g/kg, which in group C_(2.0) and groupI_(2.0) was 2.0g/kg, and the feed of group C_0 and group I_0 didn't contain CVP. The immune indexes were tested on the 0 day, 7~(th) day, 14~(th) day, 21~(st) day, 28~(th) day, and 35~(th) day of the experiment and the RPS was also recorded after challeng with A. hydrophila. The results showed that:
(1) The phagocytic activity of leukocytes, lysozyme activity and the content of serum complement C3, C4 in serum of the immunized C. auratus were markedly enhanced by dietary 0.5g/kg and 1.0g/kg CVP (P<0.05). Howere, these indexes were restrained by dietary 2.0g/kg CVP at the anaphase of the experiment.
(2) CVP could improve the phagocytic activity of leukocytes, lysozyme activity and the content of serum complement C3, C4 in serum of non-immunized C. auratus, and the trend is the same as the effects of CVP on immunized C. auratus. But these indexes of non-immunized C. auratus is lower than that were immunized.
(3) RBC-C_(3b)RR of C.auratus was markedly improved and RBC-ICR was markedly debased by dietary 0.5g/kg and 1.0g/kg CVP at 21~(st) day and 28~(th) day. RBC-C_(3b)RR of C. auratus was debased and RBC-ICR was improved by dietary 2.0g/kg CVP, but there were no significant differences in RBC-C_(3b)RR and RBC-ICR among control group and other testing group (P>0.05).
(4) CVP could improve agglutinating antibody titer of C. auratus. The agglutinating antibody titer of immunized C. auratus was highest by dietary 1.0g/kg CVP at 21~(th) day, and the highest agglutinating antibody titer was 597.3
(5) CVP could improve the survival rate of C. auratus after challenged with A. hydrophila. Meantime, the survival rate and RPS of immunized C. auratus was highest by dietary 1 .0g/kg CVP, and they was respectively 85%, 57.14.
3. In order to study the effects of CVP on the physiological and biochemical characteristic in blood of C. auratus, the concentrations of CVP in six groups were respectively 0g/kg (group C_0), 0.25g/kg (group C_(0.25)), 0.5g/kg (group C_(0.5)), 1.0g/kg (group C_(1.0)), 2.0g/kg (group C_(2.0)) and 4.0g/kg (group C_(4.0)). Some physiological parameters such as the number of red blood cell, he number of white blood cell, the content of hemoglobin, sedimentation rate of red blood cell and some biochemical parameters such as the concent of Glucose, Cholesterol, Triglycerides, Total protein, Albumin, Urea and the activity of Glutamate-oxaloacetate transaminase (GOT), the activity of Glutamate-pyruvate transaminase (GPT) and the activity of Alkaline phosphatase(ALP) in the serum were assayed at 0 day, 14~(th) day, 28~(th) day, 42~(th) day and 56~(th) day. The results showed that: When the concentrations of CVP was 0.25g/kg, there were no significant differences in the physiological parameters and biochemical parameters of C. auratus, and it had less effects on physiological function of C. auratus. 0.5g/kg~1.0g/kg CVP of dietary could markedly elevated the number of red blood cell, white blood cell, the content of hemoglobin and reduced the sedimentation rate of red blood for 14 days or 28 days. When the experiment continued for more than 42 days or 56 days, the concent of Glucose, Urea, Cholesterol, Triglycerides and the activity of GOT and GPT in the serum declined, but the concent of Total protein, Albumin and the activity of ALP in the serum increased. It indicated that 0.5g/kg-1.0g/kg CVP could improve the physiological function and metabolism intensity of C. auratus. However, when the fishes were fed with 2.0g/kg-4.0g/kg CVP for long time, the number of red blood cell, white blood cell, the content of hemoglobin, the concent of Total protein, Albumin and the activity of ALP in the serum of C. auratus declined, the sedimentation rate of red blood, the concent of Glucose, Urea, Cholesterol, Triglycerides and the activity of GOT and GPT in the serum increased, indicating weakened oxygen transportation, absorption and excretion.
引文
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