Establishment and Optimization of HPLCMS/MS for Detection of Progesterone Residue in Raw Milk

Zhen ZHANG Jun ZHANG Lei YAN Yuxi ZHAO Aifeng ZHANG Yuefei YAN

AbstractThe losses caused by outbreaks of various diseases in aquaculture intensive farming are becoming more and more serious. Fish diseases have become one of the problems that constrain the healthy development of aquaculture. Among them, fish parasitic diseases have also caused some losses to the aquaculture industry. In this paper, 17 species of fish parasites in Huaihua area, belonging to 15 genera in 13 families of 13 orders, are reviewed. In particular, the 10 kinds of fish parasitic diseases that are more harmful to farmed fishes are described in detail in order to provide a basis for the prevention of fish diseases in the future.

Key wordsFreshwater fishes; Huaihua; Parasitic diseases (fishes); Taxonomy; Investigation; Prevention

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Received: September 3, 2018Accepted: October 26, 2018

Guangzhong HUANG (1967-), male, P. R. China, associate professor, master, devoted to teaching and technical services of aquaculture.

*Corresponding author. Email: hzhgzh@126.com.

Huaihua City is located in the southwestern part of Hunan Province, and the middle and upper reaches of Yuan River, known as the "Land of Five Rivers". The important tributaries in the territory include You River, Chen River, Xu River, Wu River and Qu River. Centered on Huaihua, Huaihua City is a typical mountainous area in South China. The waters have the characteristic of scattered distribution and different sizes. In addition to rivers, other water bodies are mostly stocked with fishes. The most common species are Mylopharyngodon piceus, Ctenopharyngodon idellus, Hypophthalmichthys molitrix, Aristichthys nobilis, Cyprinus carpio Linnaeus, etc., but the yield is low. Pond farming is a major component of the Huaihua aquaculture industry, and its development is of great significance for improving fishery production. Diseases with parasites as pathogens are a major type of fish disease, with a wide variety of pathogens and complex developmental processes, seriously endangering the health of fishes. In the process of occurrence and epidemic of parasitic diseases, the relationship between fishes (hosts), parasites (pathogens), and the external environment is very close, and the contradiction between the three directly affects the progress of diseases. The mechanism of fish diseases can be summarized by the tricyclic relationship. When the pathogen exists, the host body is in poor condition, and the environmental conditions deteriorate, the disease occurs［1］. When a disease occurs, the relationship between pathogens, hosts and the environment is extremely complex. From September 2012 to December 2015, fish parasites in natural waters and artificial fish farms in Huaihua City were surveyed and collected. On the basis of taxonomy, the fauna composition of fish parasites in Huaihua is further understood. This study will provide a basis for the prevention and treatment of endemic fish diseases and the prevention of the spread of pathogens.

Materials and Methods

Species and quantity of fishes for microscopic examination

The waters in the 13 counties, cities and districts of Huaihua City were surveyed. The common economic fishes in three natural waters of each county/city/district were investigated. The common fishes in the waters of Huaihua City were investigated one by one. For each natural water and each fish in each pond, at least 10 fish were subjected to microscopic examination. For each adult fish, at least 3-5 fish were subjected to microscopic examination. A total of 411 fish, belonging to 10 species (Table 1) were investigated.

Table 1Species and quantity of fishes subjected to microscopic examination

Fish speciesQuantity

Cyprinus carpio Linnaeus93

Carassius auratus Linnaeus62

Mylopharyngodon piceus37

Hypophthalmichthys molitrix58

Ctenopharyngodon idellus34

Aristichthys nobilis23

Ergthroculter ilishaeformis31

Megalobrama amblycephala Yih23

Sinilabeo decorus tungting23

Culter erythropterus Basil27

Examination method

Live fishes randomly collected from the ponds and natural waters of Huaihua City were subjected to microscopic examination. First, the fishes were observed by naked eyes to see whether there were visible parasites and lesions on the body surface, mouth, gill and other parts of each of the fishes. Then, the internal organs of each of the fishes were dissected and microscopically examined［2］. The relevant parts, methods and requirements for the specific inspection were carried out in accordance with the methods described in the second edition of the Fish Disease Investigation Manual.

Collection and preservation of specimens

All kinds of pathogens found during the inspection were subjected to living body observation. They were collected as more as possible to make temporary and permanent specimens. Tiny protozoa, flagellates and ciliates were examined by smear test. After being fixed, they were transferred to preservation solution for storage. The large pathogens of Monogenea, small nematodes and other individuals were detected and separated under a dissecting microscope, fixed and stored with different solutions. The large parasites such as crustaceans and aphids were directly separated, fixed and preserved［3］. At last, the detected parasites were fixed, stored and made into paraffin slices. The specific specimen collection, preservation and production methods were in accordance with the operation methods described in the second edition of the Fish Disease Investigation Manual.

Results and Analysis

From the nine species of fishes investigated, a total of 17 species of parasites were found, belonging to 15 genera of 13 families in 13 orders (Table 2).

Table 2Taxonomy of parasites in fishes in Huaihua City

ParasitesHostsParasitic sites

Trichodina nobilisHypophthalmichthys molitrix, Ctenopharyngodon idellus, Aristichthys nobilis, Carassius auratus Linnaeus, Cyprinus carpio Linnaeus, Megalobrama amblycephala Yih, Body surface and gills

Trichodina oviformisHypophthalmichthys molitrix, Ctenopharyngodon idellus, Aristichthys nobilis, Carassius auratus Linnaeus, Cyprinus carpio LinnaeusBody surface and gills

Balantidium ctenopharyngodoniCtenopharyngodon idellusIntestine

Chilodonella cyprinid MoroffCyprinus carpio Linnaeus, Hypophthalmichthys molitrix, Ctenopharyngodon idellus, Aristichthys nobilis, Carassius auratus Linnaeus, Mylopharyngodon piceus Gills and body surface

Ichthyophthirius multifiliis Hypophthalmichthys molitrix, Ctenopharyngodon idellus, Aristichthys nobilis, Cyprinus carpio Linnaeus, Carassius auratus Linnaeus, Megalobrama amblycephala YihBody surface and gills

Cryptobia branchialisHypophthalmichthys molitrix, Aristichthys nobilis, Cyprinus carpio Linnaeus, Carassius auratus Linnaeus, Megalobrama amblycephala Yih, Sinilabeo decorus tungtingGills

Cryptobia agitataCtenopharyngodon idellus, Hypophthalmichthys molitrix, Mylopharyngodon piceus, Aristichthys nobilis, Cyprinus carpio Linnaeus Gills and body surface

Ichthyobodo necatorHypophthalmichthys molitrix, Mylopharyngodon piceus, Ctenopharyngodon idellus, Aristichthys nobilis, Megalobrama amblycephala YihGills and body surface

Dactylogyrus spp.Ctenopharyngodon idellus, Hypophthalmichthys molitrix, Aristichthys nobilis, Cyprinus carpio LinnaeusGills

Lernaea ctenopharyngodontis Yin Ctenopharyngodon idellusBody surface

Preventive Measures

Strengthening the nutrition of fish and selecting better compound feed

Immunomodulatory products should be added to the feed regularly, such as Huangqi Duotang Keli, Chongchongcao and other pure Chinese herbal medicine products. Thus, the physical fitness of fish will be improved, and their resistance to pathogenic organisms such as external parasites will be enhanced, thereby effectively preventing the occurrence of parasitic diseases［4］.

Killing parasites carried by fish

There are more or less parasites on the body surface of fish before wintering. Once the water temperature rises, these parasites are easily multiplied, causing large amounts of infection in the fish. Safe, highefficiency, pollutionfree disinfection drugs, such as Jundu Suxiao, will more thoroughly kill a small number of parasites carried by the overwintering fish［5］.

Specific use of tools

Tools for fish farming often become a medium for spreading fish diseases, and when the conditions permit, the tools should be specified to certain pond［6］. If this can not be achieved, the used tools must be disinfected before another use.

Avoiding injuries to fish

The skin, scales, mucous membranes, mucus layer and secretions of various organs of fish body have the function of preventing the invasion of pathogens and parasites. When the fish body is damaged, the pathogens may be easy to enter the fish body and reproduce massively, leading to outbreaks of fish diseases, such as red skin disease and water mold［7-9］.

Doing a good job of water transfer and bottoming

The ponds of less waterexchanged, and the metabolic wastes of various aquatic organisms accumulate in the water, which will produce a large amount of toxic products. Thus, the resistance of fish will be greatly reduced, leading to an increased risk of infection of various diseases including parasites［10］.

Conclusions

In the natural waters and artificial fisheries in Huaihua City, there are many water birds and water plants, as well as many snails, which provide conditions for breeding such as trematodes. The pond should be thoroughly cleaned when the fish are released in spring to kill the intermediate host of snails［11］.

In Huaihua City, the temperature of water is low, and the climate is dry, so the species and quantity of fish parasites are small. In addition to the wide distribution and high infection intensity of trematodes, other parasitic diseases have little infection intensity in the local. Therefore, only from the perspective of fish parasite distribution, Huaihua has the superior conditions for developing fish farming.

For surface parasites, most effective repellents and their concentrations should be screened out. Certain disease can be treated in a small range with the optimal concentration of the drug, which can be then applied to a wide range after the trial. This method can kill surface parasites in time and is safe［12］.

An quarantine system can be established and improved for fish diseases to control foreign pathogens from entering the local. Various production units can conduct systematic survey on fish diseases and establish archives to strengthen the work of fish disease prevention and control［13］.

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