WO2023000851A1 - 一种预防罗非鱼脂肪肝病的养殖方法 - Google Patents

一种预防罗非鱼脂肪肝病的养殖方法 Download PDF

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WO2023000851A1
WO2023000851A1 PCT/CN2022/097273 CN2022097273W WO2023000851A1 WO 2023000851 A1 WO2023000851 A1 WO 2023000851A1 CN 2022097273 W CN2022097273 W CN 2022097273W WO 2023000851 A1 WO2023000851 A1 WO 2023000851A1
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breeding
tilapia
feeding
days
fatty liver
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PCT/CN2022/097273
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French (fr)
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蔡佳
简纪常
汤菊芬
夏立群
黄瑜
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广东海洋大学
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    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K61/00—Culture of aquatic animals
    • A01K61/10—Culture of aquatic animals of fish
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A40/00—Adaptation technologies in agriculture, forestry, livestock or agroalimentary production
    • Y02A40/80—Adaptation technologies in agriculture, forestry, livestock or agroalimentary production in fisheries management
    • Y02A40/81—Aquaculture, e.g. of fish

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  • the invention belongs to the technical field of aquatic product cultivation, and in particular relates to a cultivation method for preventing fatty liver disease of tilapia.
  • Tilapia commonly known as African crucian carp and white salmon
  • Tilapia is a cultured species recommended by the Food and Agriculture Organization of the United Nations to the world.
  • Nutritional fatty liver disease caused by many factors such as feed quality and feed nutrition imbalance has become a relatively common disease, which seriously plagues the sustainable and healthy development of tilapia aquaculture.
  • Fatty liver disease can lead to slow growth of tilapia, decreased ability to resist stress, and can also cause a large number of tilapia to die in high temperature seasons.
  • Commercial fish with fatty liver lesions will cause abdominal swelling and affect the appearance quality.
  • fatty liver disease occurs in intensive farming of tilapia, it is often large-scale and comprehensive. It mainly harms high-density cultured fish species and adult fish, especially mature individuals. Although such a disease is non-infectious, its harm and consequences far exceed those of infectious diseases. In severe cases, it can cause infectious diseases and other syndromes, causing huge losses to production and dampening the enthusiasm of farmers. Therefore, how to reduce fat deposition in the liver of tilapia, increase the absorption and utilization of feed fat, so as to achieve the purpose of promoting the healthy farming of tilapia has become an urgent problem to be solved at present.
  • the method of adjusting tilapia feed formula or feed additives is mostly used to prevent tilapia fatty liver, and some also induce and adjust the exercise intensity of tilapia countercurrent swimming exercise by controlling the water flow velocity to improve tilapia fatty liver disease.
  • the technology of preventing fatty liver disease in tilapia by fasting and refeeding is no relevant report on the technology of preventing fatty liver disease in tilapia by fasting and refeeding.
  • the purpose of the present invention is to provide a kind of cultural method of preventing tilapia fatty liver disease, this method can effectively prevent and treat the nutritional fatty liver disease in the tilapia culture process.
  • the invention provides a breeding method for preventing fatty liver disease of tilapia, which comprises the following steps: putting tilapia fry in a breeding pond for feeding and breeding, performing fasting treatment after 28 to 31 days, and then feeding and breeding until harvesting.
  • the mass specification of the tilapia fry is 28-32g.
  • the stocking density of the tilapia fry is 1800-2200 fish/mu.
  • the feeding culture is to feed 3-8% of the fish body weight every day, and feed twice a day at 6:00 and 17:00.
  • the amount of the two feedings is the same.
  • the duration of the fasting treatment is 7-14 days.
  • the re-feeding method is incremental feeding.
  • the daily feeding amount of the incremental feeding is fed in increments of 1-4% of the fish body weight at each stage until the final feeding amount is 3-8%.
  • the breeding time of each stage of the incremental feeding is 4-11 days.
  • the present invention has the following beneficial effects:
  • the method of the present invention can significantly reduce the increase of liver cell lipids, and at the same time the activities of alanine aminotransferase (GPT), aspartate aminotransferase (GOT), lactate dehydrogenase (LDH) and alkaline phosphatase (AKP) are significantly reduced, achieving effective Effects on the prevention and treatment of nutritional fatty liver disease in tilapia culture.
  • GPT alanine aminotransferase
  • GAT aspartate aminotransferase
  • LDH lactate dehydrogenase
  • ADP alkaline phosphatase
  • the present invention for the first time uses short-term fasting to treat the cultured tilapia.
  • the fasting treatment can reduce the increase of fat mass in liver cells of the tilapia without affecting the yield of the tilapia, and further achieve the effect of preventing and treating fatty liver disease of the tilapia.
  • Fig. 1 Comparative example 1 and embodiment 1 Nile tilapia liver section microscopic observation diagram.
  • Fig. 2 is a microscope observation diagram of liver slices of Nile tilapia in Comparative Example 2 and Example 1.
  • Fig. 3 Changes of IL-6 expression in serum of Nile tilapia at different stages.
  • Fig. 4 Changes of IL-1 ⁇ expression in serum of Nile tilapia at different stages.
  • the invention provides a breeding method for preventing fatty liver disease of tilapia, which comprises the following steps: putting tilapia fry in a breeding pond for feeding and breeding, performing fasting treatment after 28 to 31 days, and then feeding and breeding until harvesting.
  • the aquaculture pond can optionally be an outdoor cement pond; the mass specification of the tilapia fry is preferably 28-32g, more preferably 30g.
  • the stocking density of the tilapia fry is 1800-2200 fish/mu, more preferably 2000 fish/mu.
  • the high-density culture of tilapia culture in the present invention can reduce the concentration of dissolved oxygen in water, resulting in a decrease in water quality, waste of culture resources in low-density culture, and reduced economic benefits.
  • the feeding culture is preferably feeding 3-8% of the fish body weight every day, feeding twice a day at 6:00 and 17:00; the amount of feeding twice is the same.
  • the feeding culture is more preferably 5% of the fish weight per day, and the feeding amount for both times is preferably 2.5% of the fish weight.
  • the bait of the present invention is commercialized extruded compound feed for tilapia.
  • the duration of the fasting treatment is preferably less than 21 days, more preferably 7-14 days, and even more preferably 10 days. If the fasting time of the present invention is too long, it will lead to an inflammatory reaction, and if the fasting time is too short, the effect of preventing and treating fatty liver disease of tilapia cannot be achieved.
  • the re-feeding method is incremental feeding; the daily feeding amount of the incremental feeding is preferably fed in increments of 1 to 4% of the fish body weight at each stage until the final feeding The amount is preferably 3-8%, and the daily feeding amount of the incremental feeding is more preferably fed in increments of 1-2.5% of the fish body weight at each stage, until the final feeding amount is preferably 3-5%, The daily feeding amount of the incremental feeding is further preferably fed in increments of 1.5-3% of the fish body weight at each stage, until the final feeding amount is further preferably 4-7%.
  • the incremental feeding amount of the present invention can be selected to feed 1%-2.5%-5% of the fish body weight every day, and the re-feeding bait is consistent with the bait fed before fasting.
  • the breeding time of each stage of the incremental feeding is preferably 4-11 days, more preferably 5-7 days, and even more preferably 7 days.
  • Healthy Nile tilapia fry were weighed in advance, and the initial body mass specification was 30 ⁇ 2g, and they were put into the breeding pond at a density of 2000 fish/mu. :00 o'clock and 17:00 o'clock feeding twice, the amount of feeding twice is 2.5% of fish body weight, carry out normal breeding for 30 days.
  • Healthy Nile tilapia fry were weighed in advance, and the initial body mass specification was 30 ⁇ 2g, and they were put into the breeding pond at a density of 2200 fish/mu. :00 o'clock and 17:00 o'clock feeding twice, the amount of feeding twice is 1.5% according to the fish body weight, carry out normal breeding for 30 days.
  • samples were taken from day 0, day 7, day 14, day 21, day 28, day 35 and day 42 of fasting to measure the body weight of Nile tilapia.
  • Healthy Nile tilapia fry were weighed in advance, and the initial body mass specification was 30 ⁇ 2g, and they were put into the breeding pond at a density of 2000 fish/mu. :00 o'clock and 17:00 o'clock feeding twice, twice feeding amount is 4% according to fish body weight, carry out normal breeding for 28 days.
  • the Nile tilapia in the pond were fed in increments of 2%-4%-7% of the fish body weight, and the breeding time of each feeding stage was 10 days until harvest .
  • the breeding program follows the method described in S1 in Example 1, and the culture is normal until harvest.
  • the culture method of S1, S2 is the same as embodiment 1.
  • Nile tilapia were fed 5% of their body weight in ponds until harvest.
  • samples were taken from the 28th, 35th, and 42nd days of fasting, and liver tissue sections were stained.
  • ns no significant difference (P>0.05), s means significant difference (P ⁇ 0.05)
  • Example 1 Observe and compare the similarities and differences of the livers in Example 1 and Comparative Examples 1 and 2 through liver slices. Specific steps are as follows:
  • Paraffin-embedded section After transparent with xylene, embed in paraffin, cool and solidify, and cut 5 ⁇ m sections with a microtome;
  • the expression of the inflammatory regulator Hsp70 was detected by fluorescent quantitative PCR.
  • the specific steps include: designing specific primers according to the conserved region of the Nile tilapia sequence, using the ⁇ -actin gene as an internal reference, and analyzing the inflammatory factors IL-6 and IL- The relative expression level of 1 ⁇ , the results are shown in Figure 3 and Figure 4.
  • kits for detecting GPT, GOT, LDH and AKP were used to detect the activity changes of GPT, GOT, LDH and AKP in the serum of Nile tilapia.
  • the kits were all purchased from Nanjing Jiancheng Bioengineering Institute.
  • ns no significant difference (P>0.05), s means significant difference (P ⁇ 0.05)

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  • Life Sciences & Earth Sciences (AREA)
  • Environmental Sciences (AREA)
  • Marine Sciences & Fisheries (AREA)
  • Zoology (AREA)
  • Animal Husbandry (AREA)
  • Biodiversity & Conservation Biology (AREA)
  • Fodder In General (AREA)
  • Feed For Specific Animals (AREA)

Abstract

本发明提供了一种预防罗非鱼脂肪肝病的养殖方法，所述养殖方法包括：将罗非鱼苗投放于养殖池塘中投喂养殖，28～31天后进行断食处理，然后再投喂养殖至收获。本发明通过该养殖方法可显著降低肝细胞脂质增加，同时谷丙转氨酶(GPT)、谷草转氨酶(GOT)、乳酸脱氢酶(LDH)、碱性磷酸酶(AKP)的活性显著降低，进而达到有效防治罗非鱼养殖过程中的营养性脂肪肝病的效果。

Description

一种预防罗非鱼脂肪肝病的养殖方法
本申请要求于2021年07月21日提交中国专利局、申请号为202110824299.2、发明名称为“一种预防罗非鱼脂肪肝病的养殖方法”的中国专利申请的优先权，其全部内容通过引用结合在本申请中。
技术领域
本发明属于水产品养殖技术领域，尤其涉及一种预防罗非鱼脂肪肝病的养殖方法。
背景技术
罗非鱼俗称非洲鲫鱼、白色三文鱼，是联合国粮农组织向全世界推荐的养殖品种。近年来，随着罗非鱼养殖面积的扩大和养殖密度的提高，罗非鱼的病害问题日趋严重。由于饲料品质、饲料营养不平衡等诸多因素引发的营养性脂肪肝病成为一种较为常见的疾病，严重困扰罗非鱼养殖业持续、健康发展。脂肪肝病可导致罗非鱼生长缓慢，抗应激能力下降，在高温季节还会引起罗非鱼大量死亡。发生脂肪肝病变的商品鱼，会引起腹部膨大影响外观品质。
罗非鱼集约化养殖中一旦发生脂肪肝病，往往是大规模和全面性的。其主要危害高密度养殖的鱼种、成鱼，尤其成熟个体更为严重。虽然这样的疾病无传染性，但它的危害和后果远远超过传染疾病，严重时可引发传染性疾病与其他综合症，给生产造成巨大的损失，挫伤养殖户的积极性。因此，如何降低罗非鱼肝脏脂肪沉积，增加饲料脂肪吸收与利用，从而达到促进罗非鱼健康养殖的目的成为当前迫切需要解决的问题。现有技术多采用调整罗非鱼饲料配方或饲料添加剂的方式预防罗非鱼脂肪肝，有的还通过控制水流速度诱导和调节罗非鱼逆流游泳锻炼的运动强度改善罗非鱼脂肪肝病。而通过断食处理再投喂方式预防罗非鱼脂肪肝病的技术未见相关报道。
发明内容
有鉴于此，本发明的目的在于提供一种预防罗非鱼脂肪肝病的养殖方 法，该方法可有效防治罗非鱼养殖过程中的营养性脂肪肝病。
为了实现上述发明目的，本发明提供了以下技术方案：
本发明提供了一种预防罗非鱼脂肪肝病的养殖方法，包括以下步骤：将罗非鱼苗投放于养殖池塘中投喂养殖，28～31天后进行断食处理，然后再投喂养殖至收获。
优选的，所述罗非鱼苗质量规格为28～32g。
优选的，所述罗非鱼苗投放密度为1800～2200尾/亩。
优选的，所述投喂养殖为每天按鱼体重的3～8％投饵料，每天于6:00点和17:00点投饵料两次。
优选的，所述投饵料两次的量相同。
优选的，所述断食处理的时间为7～14天。
优选的，所述再投喂方式为递增式投喂。
进一步优选的，所述递增式投喂的日投饵量每个阶段按鱼体重的1～4％递增量投喂，直到最终投喂量为3～8％。
进一步优选的，所述递增式投喂每个阶段的养殖时间为4～11天。
与现有技术相比，本发明具有如下有益效果：
本发明所述方法能够显著降低肝细胞脂质增加，同时谷丙转氨酶(GPT)、谷草转氨酶(GOT)、乳酸脱氢酶(LDH)、碱性磷酸酶(AKP)的活性显著降低，达到有效防治罗非鱼养殖过程中的营养性脂肪肝病的效果。
本发明首次通过短期断食处理养殖罗非鱼，该断食处理在不影响罗非鱼产量的前提下，达到降低罗非鱼肝细胞中脂质量的增加，进而达到防治罗非鱼脂肪肝病的效果。
说明书附图
图1对比例1与实施例1中尼罗罗非鱼肝脏切片显微镜观察图。
图2对比例2与实施例1中尼罗罗非鱼肝脏切片显微镜观察图。
图3不同阶段尼罗罗非鱼血清中IL-6表达量变化情况。
图4不同阶段尼罗罗非鱼血清中IL-1β表达量变化情况。
具体实施方式
下面结合实施例和附图对本发明进一步说明。
本发明提供了一种预防罗非鱼脂肪肝病的养殖方法，包括以下步骤：将罗非鱼苗投放于养殖池塘中投喂养殖，28～31天后进行断食处理，然后再投喂养殖至收获。
在本发明中，所述养殖池塘可选的为室外水泥池；所述罗非鱼苗质量规格优选为28～32g，更优选为30g。
在本发明中，所述罗非鱼苗投放密度为1800～2200尾/亩，更优选为2000尾/亩。本发明罗非鱼养殖中高密度养殖可使水中溶氧浓度低，造成水质量下降，低密度养殖浪费养殖资源，经济效益降低。
在本发明中，所述投喂养殖优选为每天按鱼体重的3～8％投饵料，每天于6:00点和17:00点投饵料两次；所述投饵料两次的量相同。所述投喂养殖更优选为每天按鱼体重的5％投饵料，所述两次投饵量均优选为按鱼体重的2.5％。本发明所述饵料为商品化的罗非鱼膨化配合饲料。
在本发明中，所述断食处理的时间优选为小于21天，更优选为7～14天，进一步优选为10天。本发明所述断食时间过长会导致炎症反应，断食时间过短达不到防治罗非鱼脂肪肝病的效果。
在本发明中，所述再投喂方式为递增式投喂；所述递增式投喂的日投饵量每个阶段优选为按鱼体重的1～4％递增量投喂，直到最终投喂量优选为3～8％，所述递增式投喂的日投饵量每个阶段更优选为按鱼体重的1～2.5％递增量投喂，直到最终投喂量优选为3～5％，所述递增式投喂的日投饵量每个阶段进一步优选为按鱼体重的1.5～3％递增量投喂，直到最终投喂量进一步优选为4～7％。本发明所述递增式投喂量可选的为每天按鱼体重的1％-2.5％-5％投饵料，所述再投喂饵料与断食前投喂的饵料一致。
在本发明中，所述递增式投喂每个阶段的养殖时间优选为4～11天，更优选为5～7天，进一步优选为7天。
下面结合实施例对本发明提供的技术方案进行详细的说明，但是不能把它们理解为对本发明保护范围的限定。
实施例1
养殖地点：室外水泥养殖池塘
S1.鱼苗的前期养殖
将健康尼罗罗非鱼鱼苗预先经过称重，初始体质量规格为30±2g，按2000尾/亩的密度投放至养殖池塘中，日投饵量为按鱼体重的5％，每天于6:00点和17:00点投料两次，两次投料量均为按鱼体重的2.5％，进行正常养殖30天。
S2.短期断食养殖
正常养殖30天后，对池塘中的尼罗罗非鱼进行21天断食处理。
S3.再投喂养殖
21天断食处理后，对池塘中的尼罗罗非鱼按鱼体重的1％-2.5％-5％递增式的投喂量投喂，每个投喂阶段的养殖时间为7天，直到收获。
在养殖过程中，从断食开始的0天、7天、14天、21天，28天、35天、42天采样，测尼罗罗非鱼体重，肝脏组织切片染色，检测血清中谷丙转氨酶(GPT)、谷草转氨酶(GOT)、乳酸脱氢酶(LDH)、碱性磷酸酶(AKP)活性，以及检测血清中炎症因子IL-6与IL-1β的表达情况。
实施例2
养殖地点：室外水泥养殖池塘
S1.鱼苗的前期养殖
将健康尼罗罗非鱼鱼苗预先经过称重，初始体质量规格为30±2g，按2200尾/亩的密度投放至养殖池塘中，日投饵量为按鱼体重的3％，每天于6:00点和17:00点投料两次，两次投料量均为按鱼体重的1.5％，进行正常养殖30天。
S2.短期断食养殖
正常养殖30天后，对池塘中的尼罗罗非鱼进行7天断食处理。
S3.再投喂养殖
7天断食处理后，对池塘中的尼罗罗非鱼按鱼体重的1％-2.5％-5％递增式的投喂量投喂，每个投喂阶段的养殖时间为12天，直到收获。
在养殖过程中，从断食开始的0天、7天、14天、21天，28天、35天、42天采样，测尼罗罗非鱼体重。
实施例3
养殖地点：室外水泥养殖池塘
S1.鱼苗的前期养殖
将健康尼罗罗非鱼鱼苗预先经过称重，初始体质量规格为30±2g，按2000尾/亩的密度投放至养殖池塘中，日投饵量为按鱼体重的8％，每天于6:00点和17:00点投料两次，两次投料量均为按鱼体重的4％，进行正常养殖28天。
S2.短期断食养殖
正常养殖28天后，对池塘中的尼罗罗非鱼进行14天断食处理。
S3.再投喂养殖
14天断食处理后，对池塘中的尼罗罗非鱼按鱼体重的2％-4％-7％递增式的投喂量投喂，每个投喂阶段的养殖时间为10天，直到收获。
采样、检测同实施例2。
对比例1
养殖地点：室外水泥养殖池塘
养殖方案按照实施例1中S1所述方法，正常养殖直到收获。
采样、检测同实施例1。
对比例2
养殖地点：室外水泥养殖池塘
S1、S2的养殖方法同实施例1。
S3.再投喂养殖
21天断食处理后，对池塘中的尼罗罗非鱼按鱼体重的5％的投喂量投喂，直到收获。
在养殖过程中，从断食开始的28天、35天、42天采样，进行肝脏组织切片染色。
表1各养殖阶段尼罗罗非鱼体重检测结果
Figure PCTCN2022097273-appb-000001
Figure PCTCN2022097273-appb-000002
注：ns表示没有显著性差异(P＞0.05)，s表示有显著性差异(P＜0.05)
实施例4
1、组织切片与染色
通过肝脏切片观察比较实施例1和对比例1、2中肝脏的异同。具体步骤如下：
(1)取肝脏进行固定，制备组织切片，
(2)使用预冷的PBS清洗3次；
(3)用4％多聚甲醛溶液组织固定液固定24h；
(4)以80％-95％-100％梯度乙醇脱水；
(5)石蜡包埋切片：用二甲苯透明后，石蜡中包埋，冷却凝固后切片机切5μm的切片；
(6)将切片贴于载玻片，45℃恒温箱中烘干，然后二甲苯脱蜡两次每次15min；
(7)先以100％乙醇复水5min，进行2次重复，再以80％乙醇复水5min；
(8)苏木精和曙红(HE)染色；
(9)常规方法制片后，显微镜拍照并保存，结果见图1和图2。
由图1可以看出，尼罗罗非鱼饱食会导致肝细胞脂质增加，甚至出现细胞核偏移，细胞空泡化，饥饿7～14天，可以显著改善这种状况，使肝细胞形态恢复，但饥饿时间超过14天，可能会导致炎症反应。
由图2可以看出，尼罗罗非鱼经过饥饿后，立即进行饱食投喂(按鱼体重的5％的投喂量投喂)，会加重肝细胞脂质化现象，伤及尼罗罗非鱼的肝脏，而进行适当的饥饿(7-14天为宜)后，以递增式投喂量投喂(1％-2.5％-5％)可以改善因饱食投喂造成的伤害。
2、血清中炎症因子IL-6与IL-1β的表达变化
通过荧光定量PCR对炎症调控因子Hsp70的表达量进行检测。具体 步骤包括：根据尼罗罗非鱼序列的保守区设计特异性引物，以β-actin基因作为内参，经qRT-PCR分析饥饿状态下尼罗罗非鱼血液中炎症因子IL-6与IL-1β的相对表达量，结果见图3和图4。
由图3和图4结果表明，断食处理的尼罗罗非鱼血液中的炎症因子显著低于饱食后的尼罗罗非鱼，但断食14天后，炎症因子表达量明显增加。因此，适当时间断食处理并不影响尼罗罗非鱼的健康状况，但断食时间过长会导致尼罗罗非鱼的炎症反应。
3、采用检测GPT、GOT、LDH、AKP的试剂盒检测尼罗罗非鱼血清中GPT、GOT、LDH、AKP的活性变化情况，试剂盒均购于南京建成生物工程研究所。
(1)GPT检测
S1.按照以下比例配置反应体系
表2 GPT检测反应体系
Figure PCTCN2022097273-appb-000003
S2.小心振荡酶标板混匀，室温静置15min，酶标仪波长510nm测定各孔吸光度OD值(n＝4)；
S3.将测得的OD值代入试剂盒标准曲线直接计算获得血清中GPT活性。结果见表6。
(2)GOT检测
S1.按照以下比例配置反应体系
表3 GOT检测反应体系
Figure PCTCN2022097273-appb-000004
Figure PCTCN2022097273-appb-000005
S2.小心振荡酶标板混匀，室温静置15min，酶标仪波长510nm测定各孔吸光度OD值(n＝4)；
S3.将测得的OD值代入试剂盒标准曲线直接计算获得血清中GOT活性。结果见表6。
(3)LDH检测
S1.按照以下比例配置反应体系
表4 LDH检测反应体系
Figure PCTCN2022097273-appb-000006
S2.小心振荡酶标板混匀，酶标仪波长520nm测定各孔吸光度OD值(n＝4)；
S3.将测得的OD值代入试剂盒标准曲线直接计算获得血清中LDH活性。结果见表6。
(4)AKP检测
S1.按照以下比例配置反应体系
表5 AKP检测反应体系
Figure PCTCN2022097273-appb-000007
S2.小心振荡酶标板混匀，酶标仪波长520nm测定各孔吸光度OD值。
S3.根据如下计算公式计算：血清中AKP活力＝(测定OD值-空白OD值)/(标准OD值-空白OD值)×标准品浓度(0.1mg/mL)×样品稀释倍数。结果见表6。
表6各阶段血清中GPT、AKP、GOT、LDH的活性
Figure PCTCN2022097273-appb-000008
Figure PCTCN2022097273-appb-000009
注：ns表示没有显著性差异(P＞0.05)，s表示有显著性差异(P＜0.05)
由表6结果表明，饥饿期间尼罗罗非鱼血清中谷丙转氨酶(GPT)、谷草转氨酶(GOT)、乳酸脱氢酶(LDH)、碱性磷酸酶(AKP)的活性显著降，表明经断食处理的尼罗罗非鱼脂肪肝病风险有所改善。
以上所述仅是本发明的优选实施方式，应当指出，对于本技术领域的普通技术人员来说，在不脱离本发明原理的前提下，还可以做出若干改进和润饰，这些改进和润饰也应视为本发明的保护范围

Claims (10)

  1. 一种预防罗非鱼脂肪肝病的养殖方法，其特征在于，包括以下步骤：将罗非鱼苗投放于养殖池塘中投喂养殖，28～31天后进行断食处理，然后再投喂养殖至收获。
  2. 根据权利要求1所述的养殖方法，其特征在于，所述罗非鱼苗质量规格为28～32g。
  3. 根据权利要求1所述的养殖方法，其特征在于，所述罗非鱼苗投放密度为1800～2200尾/亩。
  4. 根据权利要求1所述的养殖方法，其特征在于，所述投喂养殖为每天按鱼体重的3～8％投饵料，每天于6:00点和17:00点投饵料两次。
  5. 根据权利要求4所述的养殖方法，其特征在于，所述投饵料两次的量相同。
  6. 根据权利要求1所述的养殖方法，其特征在于，所述断食处理的时间小于21天。
  7. 根据权利要求6所述的养殖方法，其特征在于，所述断食处理的时间为7～14天。
  8. 根据权利要求1所述的养殖方法，其特征在于，所述再投喂方式为递增式投喂。
  9. 根据权利要求8所述的养殖方法，其特征在于，所述递增式投喂的日投饵量每个阶段按鱼体重的1～4％递增量投喂，直到最终投喂量为3～8％。
  10. 根据权利要求9所述的养殖方法，其特征在于，所述递增式投喂每个阶段的养殖时间为4～11天。
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