CN112335556B - Novel captive wild animal management method - Google Patents
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- CN112335556B CN112335556B CN202011117141.3A CN202011117141A CN112335556B CN 112335556 B CN112335556 B CN 112335556B CN 202011117141 A CN202011117141 A CN 202011117141A CN 112335556 B CN112335556 B CN 112335556B
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Images
Classifications
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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
- A01K1/00—Housing animals; Equipment therefor
- A01K1/015—Floor coverings, e.g. bedding-down sheets ; Stable floors
- A01K1/0152—Litter
- A01K1/0155—Litter comprising organic material
-
- 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
- A01K1/00—Housing animals; Equipment therefor
- A01K1/02—Pigsties; Dog-kennels; Rabbit-hutches or the like
-
- 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
- A01K1/00—Housing animals; Equipment therefor
- A01K1/02—Pigsties; Dog-kennels; Rabbit-hutches or the like
- A01K1/035—Devices for use in keeping domestic animals, e.g. fittings in housings or dog beds
-
- 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
- A01K31/00—Housing birds
- A01K31/04—Dropping-boards; Devices for removing excrement
-
- 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
- A01K31/00—Housing birds
- A01K31/06—Cages, e.g. for singing birds
-
- 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
- A01K67/00—Rearing or breeding animals, not otherwise provided for; New or modified breeds of animals
- A01K67/02—Breeding vertebrates
Landscapes
- Life Sciences & Earth Sciences (AREA)
- Environmental Sciences (AREA)
- Zoology (AREA)
- Animal Husbandry (AREA)
- Biodiversity & Conservation Biology (AREA)
- Birds (AREA)
- Animal Behavior & Ethology (AREA)
- Housing For Livestock And Birds (AREA)
Abstract
The invention discloses a novel method for managing captive wild animals, which is characterized by comprising the following steps: (1) laying a fermentation bed and/or a padding layer: laying a bedding layer or a fermentation bed based on pine barks and probiotics on the indoor ground and/or the outdoor ground of the animal cage or a show-window type exhibition house, or combining the bedding layer with the simulated ecological garden landscape to simulate the natural habitat environment of animals; (2) daily management: the cage or the show window type exhibition room does not need to be cleaned every day; (3) and (3) disinfection: regular and routine disinfection of animal cages or show-window type shows is not required. The management method of the invention provides a more natural and comfortable living environment for animals, is beneficial to the expression of natural behaviors of the animals, enriches microbial communities in the living environment of the animals, simplifies the daily operation process, reduces the disinfection frequency of animal cages, simplifies the daily management process, has a certain regulation effect on indoor temperature and humidity environments, and improves the welfare and health level of the animals.
Description
Technical Field
The invention relates to the field of wild animal management, in particular to a novel captive wild animal management method.
Background
The basic structure of the animal cage of domestic zoos and other wild animal breeding units comprises: (1) interior room or indoor exhibition room: basically all concrete floors or other similar hard floors; (2) outdoor playground: mainly comprises a mud land, a concrete ground or other similar hard ground or sand paved on the ground, and more wild animal cages are used for planting lawns or other plants or adding pools, waterfalls and other simulated ecological landscapes in recent years.
According to the national people's republic of China industry Standard animal park management Specification (CJJ/T263-2017), the zoo should establish the specification requirements of animal feeding operation regulations, animal sanitation operation regulations and the like. The daily management comprises the following steps: (1) cleaning the cage and washing the indoor surface with water every day (Specification for zoo rearing management); (2) regular disinfection of the cages and the environment is carried out regularly. The specific requirements of the zoo disinfection management norm (draft of comments) on disinfection of cages are: a) the internal house (inner room) should be used 1 time per week in winter, and 2 times per week in other seasons; b) the outer house (outdoor playground) is preferably disinfected 1-2 times per month, the frequency is preferably increased in high-temperature seasons in the south, and the frequency is reduced in winter in the north.
The existing zoo animal cage and the daily management thereof have the following defects:
1. the zoo animal cages are greatly influenced by climate, the ground surface temperature is low in winter, the humidity is too high in humid weather, and particularly after the ground is cleaned, the zoo animal cages are not easy to dry, and the comfort of animal life is also influenced due to too dry climate.
2. The concrete floor is too hard, the surface is too rough or too smooth, and the foot pads and joints of animals are easy to be injured.
3. In order to ensure the health of animals and the cleanness and sanitation of the feeding environment, animal cages need to be cleaned, the ground needs to be washed by water, and the accumulated water on the ground needs to be dried by a fan every day, so that the labor and water resources are consumed, the accumulated water on the ground is not easy to dry, and the mildew or other pathogenic microorganisms are easy to breed, especially under the warm and humid climatic conditions in the south.
4. Dust is easily generated when cleaning mud, and the ground is easily muddy in rainy season; the sand does not absorb water, and the heat radiation is large in summer; grasslands solve the aforementioned problems to some extent, but are susceptible to microclimate in cages or to damage by animals, and lawns or other plants grown in many animal cages are difficult to survive; and when the green plants are too luxuriant, the ventilation is also blocked.
5. In order to prevent animal epidemic diseases, the traditional method is to frequently disinfect animal cages, so that the harmful microorganisms in the environment of the cages are killed, the beneficial microorganisms in the living environment of the animals are killed, and the microbial communities in the animals are indirectly influenced, so that the immune function of the animal body is adversely affected. Moreover, the disinfection effect is limited to the time when the disinfection medicine has the drug effect, and the environment of the cage cannot be protected from new pollution after the disinfection process is finished.
In livestock and poultry breeding industry, fermentation beds or padding are adopted in part of breeding units. However, the management of wild animal breeding under zoo containment conditions is substantially different from animal husbandry production. The main body is as follows: (1) different purposes of raising animals are specifically required in zoo management regulations (CJJ/T263-plus 2017), the zoo is the first place for the public to recognize the animals visually and understand the protection of the animals, and the relevant information spread by the zoo is required to be accurate and does not violate natural laws and scientific ethics; in order to ensure that animals live healthily in an environment conforming to physiological habits, show natural behaviors, enable the public to visually know the geographical environment of the animals and transmit protection information, the cage design is required to simulate the habitat of the original habitat in the field as much as possible, and suitable auxiliary facilities are provided according to the characteristics of the animals. Emphasis is placed on the natural ecology of the environment and the benefit of demonstrating the natural behavior of animals. The aim of animal husbandry production is to obtain animal products such as meat, eggs, milk and the like, and the emphasis is on production efficiency and the emphasis on input-output ratio; (2) the domestication degree of the animals is different, and the wild animals are not domesticated animals and are more sensitive to various stressors in living environment; the livestock and poultry are domesticated for a long time, and have strong adaptability to artificial environment; (3) the number of the raised animals in unit area is less than that of the wild animal cages in unit area so as to meet the display requirement of natural behaviors of the animals, and the number of the produced excrement is relatively less; modern animal husbandry mostly adopts an intensive method, mainly based on the requirement of improving production efficiency, and the produced animal excrement is relatively more. Therefore, the method for raising and managing the captive wild animals is fundamentally different from the method for animal husbandry production.
The fermentation bed or padding adopted by animal husbandry production units is designed based on the requirements of animal husbandry production, the simulation of animal field habitat is not required, the requirement on the decomposition and fermentation efficiency of animal excrement is high, and intensive production can produce a large amount of animal excrement, so that the material of the fermentation bed or padding in animal husbandry production basically contains crushed plant (such as straw or other plant) components, and an auxiliary manual or mechanical method is used for continuously stirring the fermentation bed to promote the sufficient mixing of the animal excrement and the fermentation material, so that the fermentation reaction process is accelerated. In addition, animal husbandry is generally not carried out for a long period, usually about 3 to 8 months, and the fermentation beds or bedding used are removed with the end of the animal husbandry period.
In the breeding of wild animals in zoos, the fermentation bed or padding is firstly considered to meet the requirements of transmitting natural protection information to the public and the requirements of expressing natural behaviors of animals, and the habitat of animal habitats needs to be simulated as much as possible. Secondly, as the major mission of zoos is the species protection of endangered wild animals, the health care of the breeding subjects is all-round, and animal welfare is put to an important position, not only to prevent the animals from being infected with epidemic diseases. Researches show that the living environment of wild animals, including the environment of microorganisms, is closely related to the health of animals, and not only do pathogenic microorganisms harm the health of animals, but the abundance of the environmental microorganisms can also indirectly influence the health of animals by influencing the intestinal flora of the animals. Therefore, it is necessary to create a microbial environment for wild animals that is beneficial to the health of the animals. In addition, as the wild animals are bred for a long period, the domestication degree of the animals is extremely low, and the natural behaviors are richer, the ground padding is beneficial to protecting the foot pads and joints of the animals, and the ground padding can be preferably provided with a temperature and humidity environment which is as comfortable as possible so as to reduce the occurrence of trauma, infection, stress and the like.
Due to the great differences in the feeding purpose, feeding density and animal domestication degree, the animal husbandry production and the management method of wild animals in zoos also have great differences.
Disclosure of Invention
In order to solve the problems, the invention aims to provide a novel method for managing wild animals in captivity, which is simple and easy to implement, provides a more natural and comfortable living environment for the animals, is beneficial to the expression of natural behaviors of the animals, enriches microbial communities in the living environment of the animals, does not need to clean animal cages every day, reduces the disinfection frequency of the animal cages, simplifies the daily management process, has a certain regulation effect on indoor temperature and humidity environments, and indirectly influences the health level of the animals.
The purpose of the invention is realized as follows: a novel method for managing captive wild animals is characterized by comprising the following steps: (1) laying a fermentation bed and/or a padding layer: laying pine barks mixed with probiotics on the indoor ground of the animal cage as a fermentation bed, laying pine barks mixed with probiotics on the outdoor ground of the animal cage or a show-window type exhibition house as a padding layer, or combining the padding layer with the quasi-ecological landscape to simulate the natural animal habitat; (2) daily management: the cage or the show house in a show window type does not need to be cleaned every day, but the residual food of the animals needs to be removed; (3) and (3) disinfection: regular and routine disinfection of animal cages or window type show houses is not required.
The replacement of the padding layer or the fermentation bed: when the pine bark becomes brittle in texture, crumbles easily, or is heavily contaminated, or when the animal becomes epidemic or dies, it needs to be replaced in whole or in part.
The pine bark is all tissues of a plant of Pinus of Pinaceae wrapped outside a secondary xylem of a trunk, a branch and a root, and the length of each block is 2 cm to 25 cm; the thickness of the padding layer is 5 cm to 30 cm; the thickness of the bedding of the fermentation bed is 30 cm to 100 cm; the pine bark is from non-epidemic areas, is far away from animal farms by more than 3 kilometers, and is treated by a physical disinfection method of sunlight exposure, ultraviolet irradiation or radiation or formalin fumigation before use.
The bedding thickness of the fermentation bed is as follows: the small animal is 30-40 cm; the size of the medium and small animals is 40-60 cm; the large animal is 60-100 cm; the bedding thickness of the bedding layer is as follows: the small animal is 5-10 cm; the size of the small and medium animals is 10-15 cm; the large animal is 15-30 cm; the small animals are mammals, amphibians or birds with the weight of less than 10 kg, the small animals are mammals, amphibians or birds with the weight of 10 kg to 200 kg, and the large animals are mammals and amphibians with the weight of more than 200 kg.
The animal cage or the show window type exhibition house is provided with a drainage facility, and a metal separation net is arranged at a water outlet of the drainage facility; the fermentation bed and the base of the padding layer have slopes and incline towards the water outlet.
The addition amount of the probiotics is 200-400 g/cubic meter of pine bark; adding frequency: the fermentation bed is laid and added for the first time, and is not needed to be added before the environment is thoroughly disinfected for the next time, and the fermentation bed is added again after the environment is thoroughly disinfected.
The probiotic is Lactobacillus acidophilus with a viable count of not less than 1 × 108cfu/g。
Urine excreted by animals is directly absorbed and decomposed by the padding or the fermentation bed; the excrement of animals is treated by respectively embedding the animal excrement into the fermentation bed/bedding layer by turning the fermentation bed/bedding layer according to different animal species or locally flushing the animal excrement into the fermentation bed/bedding layer by water.
A disinfection pond or a disinfection pad is arranged at an entrance and an exit of workers of the animal cage.
Except for severe pollution or epidemic or death of animals, the bedding layer or fermentation bed of cages for primates, carnivores, birds or amphibians does not need to be replaced for more than 2 years, and the bedding layer or fermentation bed of cages for herbivores and omnivores except primates does not need to be replaced for more than 1 year.
The management method of the invention provides a more natural and comfortable living environment for animals, is beneficial to the expression of natural behaviors of the animals, enriches microbial communities in the living environment of the animals, does not need to wash the animal cages every day, reduces the disinfection frequency of the animal cages, simplifies the daily management process, has a certain regulation effect on indoor temperature and humidity environments, and improves the welfare and health level of the animals.
Drawings
FIG. 1 is a cross-sectional view of a layer of fermentation bed or bedding of the present invention.
FIG. 2 is a schematic view of an inner chamber of a fermentation bed before being laid in the embodiment 1 of the present invention; FIG. 3 is an effect diagram of the inner chamber fermentation bed of example 1 after being laid; fig. 4 is a diagram showing effects of the bedding layer for outdoor sports fields according to example 1.
FIG. 5 shows the winter environmental temperature and the internal temperature of the fermentation bed in the experimental study 1 of the present invention, and FIG. 6 shows the summer environmental temperature and the internal temperature of the fermentation bed in the experimental study 1 of the present invention; FIG. 7 shows the environmental humidity in winter and the internal humidity of the fermentation bed in the experimental study 1 of the present invention, and FIG. 8 shows the environmental humidity in summer and the internal humidity of the fermentation bed in the experimental study 1 of the present invention; FIG. 9 shows the monitoring of ammonia concentration in air after indoor bedding of a fermentation bed according to the experimental study 1 of the present invention.
FIG. 10 is a graph of the AWCD values versus incubation time for 6 pine bark samples from study 2 of the present invention; FIG. 11 is a graph of the AWCD values versus incubation time for 4 samples of wood shavings from Experimental study 2 of the present invention; FIG. 12 is a graph of the AWCD values versus culture time for 3 moss specimens from study 2 of the present invention; fig. 13 is a plot of AWCD values versus incubation time for different types of dunnage samples from experimental study 2 of the present invention.
FIG. 14 is a graph of the analysis of the species composition in Experimental study 3 of the present invention, in which A is the relative abundance of bacteria at phylum level and B is the relative abundance of bacteria at genus level; FIG. 15 is a Beta diversity analysis of the experimental study 3 of the present invention, wherein A:4 group samples are analyzed by PCA at OUT level (16S), B: exhibition 4 and exhibition 2 samples principal component analysis comparison, C: exhibition 4 and exhibition 1 samples principal component analysis comparison, and D: exhibition 4 and exhibition 3 samples principal component analysis comparison; FIG. 16 is a volcanic plot of the various groups of differential genes in Experimental study 3 of the present invention.
FIG. 17 shows the results of the present invention in experimental study 4 in which the cells were cultured on a Sabouraud's medium at 37 ℃ for 48 hours; FIG. 18 shows the results of the wort culture at 26 ℃ for 168 hours in the experimental study 4 of the present invention; FIG. 19 shows the results of the potato dextrose culture at 26 ℃ for 168 hours in Experimental study 4 of the present invention; FIG. 20 shows the microscopic fungus and conidia morphology of 48 h.times.10 in the experimental study 4 of the present invention.
Detailed Description
The invention relates to a novel captive wild animal management method, which comprises the following steps:
(1) the method is characterized in that simple transformation is carried out on the basis of the existing animal cage and show window type exhibition houses, and a fermentation bed and/or a padding layer are laid: the method comprises the steps of laying pine barks mixed with probiotics on the indoor ground of an animal cage as a fermentation bed, laying pine barks mixed with the probiotics on the outdoor ground of the animal cage or a show-window type exhibition house as a padding layer, or organically combining a quasi-ecological landscape with the padding layer, and laying the padding layer on the ground where green plants are not easy to grow or the ground where animals frequently lie or trample.
The pine bark is the whole tissue of the Pinaceae Pinus (Pinus) plant, which is wrapped outside the secondary xylem of trunk, branch and root, also called red pine bark, red dragon scale, pine bark, red dragon bark and the like, and is preferably the lower scaly bark of the old trees such as Pinus massoniana, Pinus tabulaeformis, Pinus sylvestris and the like, and the bark of other pine tree species of the same genus can also be used. According to the record of the compendium of materia Medica: pine bark is bitter and astringent in taste and warm in nature, enters lung and large intestine channels, has the effects of astringing and promoting granulation, and is externally used for treating burns, scalds, infantile eczema and the like. The Chinese herbal medicine compilation records: cortex Pini (cortex Pini) has effects in expelling pathogenic wind, removing dampness, promoting blood circulation, stopping bleeding, healing sore, and promoting granulation; can be used for treating rheumatic osteodynia, traumatic injury, incised wound, hematochezia, chronic dysentery, eczema, burn, scald, and carbuncle. In addition, the pine bark has the characteristics of high organic matter content, high void ratio, wide total cut-off surface, good adsorption effect, stable pH value, corrosion resistance and the like, and has good effects of filtering waste gas, retaining water and bearing microorganism groups in a biofilter in industry. The invention adopts pine bark as a base material for wild animal cages, is not only harmless to animals, but also has a plurality of advantages.
Pine trees are widely distributed in the nature, and after the pine barks are used for animal cages, the animal cages look more natural, interesting and ecological, which is beneficial to improving the landscape effect; the pine bark has good compatibility with plants, can be well matched with trees, shrubs and herbs, and can be used as a substrate for planting the plants, so that the pine bark can achieve good effects when being used for animal cages regardless of a simple padding layer or a padding layer with garden landscapes.
The pine bark has the characteristics of elasticity, sound insulation and the like, and can improve the ground texture, protect the foot pad and the joint of an animal and reduce the noise in the animal cage when being paved on a hard ground.
The pine bark is a poor heat conductor, so that the surfaces of the padding and the fermentation bed cannot be cooled even in cold winter, the fermentation bed can generate heat in the fermentation process, the internal temperature of the fermentation bed is always higher than the ambient temperature, a comfortable 'hotbed' is provided for animals, and the heat preservation effect similar to that of a carpet can be achieved; when the temperature is high, as long as the cage keeps ventilation, the influence of the heat generated in the fermentation bed on the environmental temperature can be ignored.
In the humid weather in the south, the humidity in the cage is very high, particularly after the ground is washed by water, the cage is difficult to dry even if a fan blows, and the property of porous, water-absorbing and corrosion-resistant pine bark can always keep the cage dry; when the air humidity is low, part of moisture adsorbed in the pine bark can be supplemented into the air, so that the comfortable humidity in the cage can be kept. Therefore, the invention has certain regulation effect on the temperature and the humidity in the animal cage, particularly solves the problem of long-term dampness of the animal cage under the warm and humid southern climatic condition, and is beneficial to reducing the occurrence of diseases such as animal skin diseases and improving the welfare level of animals.
The natural characteristics of the pine bark can play a role similar to a 'bacteria house', which is beneficial to the growth of microorganisms, and the natural excrement of animals is natural nutrient for the growth of probiotics. Research shows that when the pine bark is used as padding, the pine bark is richer in environmental microorganisms than wood shavings, moss, sand, soil and other materials, and the abundance of the microorganisms in the living environment of the animals is related to the abundance of the intestinal microflora of the animals, so that the pine bark has favorable influence on the health of the animals.
And the pine bark has good adsorption and absorption performance, and a large amount of probiotics have good absorption and decomposition effects on animal excrement. Urine excreted by animals is quickly absorbed by the pine barks and decomposed as nutrition of probiotics, so that the surfaces of the fermentation bed and the padding are kept dry and comfortable; the excrement discharged by the animals can be utilized and decomposed by the probiotics, so that the labor amount for manually removing the excrement of the animals is reduced, and the excrement has a certain purification effect on indoor air. Through the long-term tracking detection of harmful gas in the inner chamber of the cage with the fermentation bed, the results are as follows: hydrogen sulfide and sulfur dioxide are not detected, the concentration of ammonia gas is lower than 1.0ppm under the condition of indoor heat preservation in winter, and the hydrogen sulfide and the sulfur dioxide are not detected under the condition of windowing and ventilation.
The pine bark is from non-epidemic areas, is far away from animal farms by more than 3 kilometers, and is treated by a physical disinfection method of sunlight exposure, ultraviolet irradiation or radiation or formalin fumigation before use. Preferably, the length of each pine bark is from 3 cm to 20 cm according to the species and habit of the animals to be raised, and for small and medium-sized animals such as apes and monkeys, leopards, etc., the length of the pine bark is preferably from 5 cm to 10 cm; for large animals such as rhinoceros and elephants, the pine bark preferably has a length of 5 cm to 15 cm.
Preferably, the thickness of the matting is divided into two categories: firstly, the material is used as a bedding layer, namely, the material is directly paved on a hard ground or a muddy ground, and the thickness of the material is from 5 cm to 30 cm; secondly, the mattress is used as a fermentation bed, and the thickness of the mattress is different from 30 cm to 100 cm. More preferably, the thickness of the fermentation bed for small and medium animals such as apes and leopards is 40-60 cm; for small animals such as squirrel and monkey, the thickness of the mattress of the fermentation bed is 30-40 cm; for large animals such as Rhinoceros, elephant, etc., the thickness of the mattress of the fermentation bed is 60-100 cm. Most preferably, the thickness of the pine bark mat used as a fermentation bed is preferably 50 cm for small and medium animals such as simians and leopards; for small animals such as squirrel and monkey, the thickness of the pine bark cushion layer used in the fermentation bed is 30 cm; for large animals such as rhinoceros, the thickness of the pine bark mat used as the fermentation bed is preferably 100 cm. The fermentation bed and the padding layer can be used independently or can be combined together, and the difference is that the decomposition efficiency of the fermentation bed on the animal excrement is higher. Preferably, a fermentation bed is used in the interior of the cage, and a bedding layer is used in an outdoor playground or a shop-window show. Preferably, the fermentation bed is preferentially used for herbivores producing large quantities of excreta.
The small animals are mammals, amphibians or birds with the weight of less than 10 kg, the small animals are mammals, amphibians or birds with the weight of 10 kg to 200 kg, and the large animals are mammals and amphibians with the weight of more than 200 kg.
Preferably, as shown in fig. 1, the animal cage or the showroom is provided with a drainage facility 3, and a stainless steel metal separation net 2 (for example, a separation net with a pore diameter of 1 cm) is arranged at a water inlet of the drainage facility to prevent barks from entering the drainage facility; the periphery of the fermentation bed 1 (or a bedding layer) is bricked into a pool shape, and the treated pine bark 12 is put into the pool according to the required bedding thickness; the base 11 of the fermentation bed 1 (or bedding layer) has a slope (in the example shown in the figures the slope is about 5 °) which is inclined towards the drainage means 3 to facilitate drainage.
Preferably, the addition amount of the probiotics is 200-400 g/cubic meter of pine bark; adding frequency: usually, the fermentation bed is added once, the fermentation bed is laid and does not need to be added after the fermentation bed is added for the first time until the environment is thoroughly disinfected next time, and the fermentation bed is added again under special conditions, such as after the environment is thoroughly disinfected. The probiotics can be added by uniformly mixing with the pine bark 12 before laying, or in the laying process, namely, laying a layer of pine bark, adding a layer of probiotics, uniformly splashing and stirring.
Preferably, lactobacillus acidophilus is added to the pine bark in order to promote the rapid establishment of beneficial microorganisms in the bedding or/and the fermentation bed, or/and to inhibit the growth of harmful microorganisms. Most preferably, the viable count of Lactobacillus acidophilus is not less than 1 × 108cfu/g。
(2) Daily management and maintenance: due to the filtering effect and the absorption effect of the padding layer or/and the fermentation bed on the animal excrement and the decomposition effect of the probiotics on organic matters, the animal excrement can be effectively treated, so that the cage or the show-window type exhibition house does not need to be cleaned every day, and the residual food of the animals still needs to be removed in time to prevent the animals from eating the deteriorated food. The animal excrement can be used as a nutritional raw material of the fermentation bed, the animal excrement does not need to be removed, when the amount of the animal excrement remained on the surface of the fermentation bed or the padding is large, and the landscape is obviously affected, the padding can be locally turned over to embed the animal excrement into the fermentation bed/padding layer to accelerate the decomposition of the animal excrement, or the animal excrement is locally flushed into the fermentation bed/padding layer to accelerate the decomposition of the animal excrement by using water. Preferably, primates, other omnivores or carnivores, amphireptiles and birds with low excrement discharge are cleaned once a week; for herbivores with large excrement discharge amount, the excrement of the animals can be flushed into the fermentation bed/padding layer by local water without clearing.
(3) Disinfection (disinfection) refers to a process of removing or killing pathogens from the surface of animals and their environment by physical, chemical or biological methods, etc. to make them harmless. The disinfection process not only kills harmful microorganisms, but also kills probiotics beneficial to animals. For safety, before the fermentation bed and the padding layer are laid, the environment in the cage or the showcase needs to be thoroughly disinfected once to kill pathogenic bacteria possibly existing in the environment of the cage or the showcase. Pine bark used as bedding is disinfected before being used and mixed with a large amount of probiotics, and the function of the pine bark is to artificially and rapidly establish a dominant flora which is beneficial to animals in the environment so as to competitively inhibit the growth of other bacteria. After that, the characteristics of adsorption, porosity and the like of the pine bark and continuous animal excrement provide proper conditions for the growth of the probiotics, so that the probiotics in the padding layer and the fermentation bed are always in relatively rich states, the growth of pathogenic microorganisms in the environment is inhibited, and a rich microbial ecological environment is provided for animals. Therefore, once the padding layer and/or the fermentation bed are/is paved, regular and conventional disinfection operation is not needed in a cage or a window type exhibition room, and the disinfection operation is not suitable, so that the traditional wild animal daily management process is improved, and the wild animal daily management process is simpler and more efficient. For safety, only when the cages are maintained or the animals are infected with epidemic diseases, die and other events occur, padding and the environment in the cages or the showcase type exhibition houses need to be thoroughly sterilized, and probiotics are timely supplemented after the sterilization operation so as to maintain the dominant bacteria status of the probiotics in the environment; if chemical medicines are adopted for disinfection, probiotics are added after the medicine effect of the disinfection medicines disappears so as to prevent the residual disinfection medicines from reducing the activity of the added probiotics.
No matter the physical or chemical disinfection method, the microorganism killing effect is only exerted in the disinfection process, the microorganism killing effect disappears after the disinfection process is finished, and the environment of the cage or the showcase is still exposed to various microorganisms including pathogenic microorganisms. The padding and the fermentation bed of the invention have a large amount of probiotics, starting from the padding material layer and the fermentation bed, a mechanism for inhibiting pathogenic microorganisms is established in a cage or a show-window type exhibition house, the existence of animal excrement strengthens the dominant bacterial status of the probiotics, so that the pathogenic microorganisms are difficult to grow, and the environment for inhibiting the pathogenic microorganisms for a long time and stably is established. Moreover, researches show that abundant microbial environment can influence the abundance of animal intestinal bacterial colonies, so that the animal intestinal bacterial colonies have beneficial effects on the immune function and the digestion function of animals.
(4) Replacement of bedding or fermentation bed: according to the actual conditions of animal species, quantity, area of a cage, specific environment and the like, when the pine bark becomes fragile in texture and easy to crush, or is seriously polluted or subjected to other special conditions (for example, the animals are in epidemic situation or die and cannot be solved only by disinfection), all or part of the pine bark needs to be replaced in time. In general (except for the special cases of serious pollution, epidemic situation or death of animals, such as normal weathering of pine bark or crushing by trampling by animals), the padding or fermentation bed of animal cages such as primates, carnivores, birds and amphibians can be maintained for more than two years without replacement; bedding or fermentation beds for herbivores, and omnivores other than primates, can last more than one year without replacement.
(5) Set up disinfection pond or disinfection pad at the staff access & exit of cage, the purpose reduces to come in and go out the possibility that brings the pathogenic microorganism outside the cage into the cage because of the staff. Personnel must wear special work clothes and work shoes when getting in and out of the animal cage, and must pass through a disinfection pad or a disinfection pond to prevent harmful microorganisms from being brought into the cage from the outside of the cage.
After the bedding layer or the fermentation bed is paved, the living environment of the animals looks more natural and ecological, the animals can also turn over the barks on the bedding layer or the fermentation bed frequently, the behaviors are more abundant, the occurrence of stereotyped behaviors which are not beneficial to the health of the animals is reduced, the ornamental effect is better, and the animals are more active.
Example 1 (Black monkey cage)
The Guangzhou zoo black-leaf monkey cage is divided into an inner room and an outdoor sports ground, and 1 pair of adult monkeys and 3 immature offspring thereof are raised. Wherein the inner chamber part is about 7 square meters, and has two doors which are respectively communicated with the outdoor playground and the staff passage, the ground of the inner chamber is of a concrete structure (as shown in figure 2), the ground has a certain gradient, and a sewer (namely a drainage facility) is arranged at one corner with lower terrain of the inner chamber; the outdoor playground part is about 100 square meters, and is also provided with two doors which are respectively communicated with the inner chamber and the staff passage, the ground of the playground has a natural gradient, one lower end of the playground is provided with a drainage ditch (namely a drainage facility) with a fence, the original ground is mud ground and sand ground, and a small amount of green plants are planted on the original ground.
1.1 engineering construction and Disinfection
An inner chamber: firstly, bricking the inner sides of two doors of an inner chamber into a retaining wall with the height of 55 cm by bricks, and requiring no influence on normal door opening and closing operation; a stainless steel net with the aperture of 1 cm is covered on the drainage port of the existing sewer.
Outdoor playground: the fence type cover plate on the existing drainage ditch is opened, a layer of stainless steel mesh with the aperture of 1 cm is added below the cover plate, and then the fence type cover plate is covered again.
The purpose of the stainless steel net is to prevent the pine bark from clogging the drainage facility.
And (3) disinfection: after construction, the environment in the cage is disinfected by spraying 1:2000 diluted Guangxiang (glutaraldehyde decamethyl ammonium bromide solution, produced by frontier animal health Co., Ltd., Guangdong province) according to a conventional method, and residual disinfectant is washed away by clear water after disinfection. The cushion material layer and the fermentation bed are not disinfected after being paved.
A disinfection pad is arranged outside an entrance and an exit of cage workers, the disinfection pad is sprayed with Guangxian diluted by 1:2000 every day and is kept moist, and the workers need to step on the disinfection pad when entering and exiting the cage.
1.2 preparation of fermentation beds and bedding Material
The Pinus massoniana Lamb is prepared by using Pinus massoniana Lamb as main trunk and scaly skin with the length diameter of 5-10 cm, which is purchased from a production place directly and used after being exposed to the sun. The raw materials are uniform in size, fresh, clean, dry and free of mildew. It is known that animal epidemic diseases do not occur around the producing area, and animal-free farms are nearby within 3 kilometers. Adding probiotics (the viable count of Lactobacillus acidophilus is more than or equal to 1 × 10) before laying and using8cfu/g) in an amount of 400 g/m pine bark, mixed as uniformly as possible and used immediately.
1.3 preparation of fermentation beds
The pine bark material containing probiotic bacteria is poured into the inner chamber, and the thickness of the mat is 50 cm (as shown in figure 3). Embedding a temperature and humidity probe in the center of the fermentation bed, and recording the temperature and humidity of the fermentation bed every day. And recording the concentrations of ammonia gas, sulfur dioxide, hydrogen sulfide and carbon monoxide in the room by using a gas detector every day. The cross-sectional structure of the fermentation bed obtained by the preparation method is shown in figure 1.
1.4 preparation of bedding layer
And (3) paving pine bark raw materials containing probiotics on the mud land and the sand land of the outdoor playground, wherein the thickness of the paving is about 10 cm, and part of the native and well-grown vegetation is still remained, as shown in figure 4. The original withered trees, roost, wood pavilion and other facilities are reserved on the bedding layer, and the landscape is more natural and ecological.
1.5 daily management
When going on duty, the worker wears the working clothes and the working shoes according to the traditional requirements.
The worker feeds animal feed and changes clean drinking water every day according to the traditional requirement, clears up the remaining feed of animal every day, prevents to putrefy.
Because the number of the animals raised in the cage is only 5, the number of the animal excrements is small, the excrements in an outdoor playground do not need to be cleaned, and the residual animal excrements on the surface of the indoor fermentation bed are washed by tap water 2 times a week and naturally permeate into the fermentation bed. A comparison of the working of breeders before and after using the present invention is shown in Table 1.
TABLE 1 comparison of before and after breeder's job using the present invention
Example 2 (Black monkey house)
The black-leaf monkeys adjacent to example 1 were housed in an interior space of about 7 square meters and an exterior stadium of about 80 square meters, and 2 sub-adult black-leaf monkeys were housed.
The difference from the embodiment 1 is that: the inner chamber is not laid with a fermentation bed, and the inner chamber is cleaned, washed and disinfected as usual every day in management, and the rest is the same as the embodiment 1.
Example 3 (squirrel monkey cage)
The area of the inner chamber is about 6 square meters, the area of the outdoor playground is about 80 square meters, and 11 squirrel monkeys (2 males, 7 females and 2 subexponents) are bred.
The difference from the embodiment 1 is that: the inner chamber is not laid with a fermentation bed, and the inner chamber is cleaned, washed and disinfected as usual every day in management, and the rest is the same as the embodiment 1.
Example 4 (Boa house)
And (5) breeding 2 boa in a show window type show house with the area of about 5 square meters. After the exhibition room is thoroughly disinfected, the treated pine bark containing the probiotics is paved, and the thickness is 10 cm. The probiotic is Lactobacillus acidophilusThe number of viable bacteria is more than or equal to 1 × 108cfu/g, the addition amount is 400 g/cubic meter of pine bark. The daily management does not need other treatment except timely removing residual food, and does not need conventional disinfection.
Example 5 (Laiguana cage)
And (3) culturing 20 pieces of iguana glauca in a show-window exhibition room with the area of about 5 square meters. After the exhibition is thoroughly disinfected, the pine bark which is treated and contains probiotics is paved, and the thickness is 5 cm. The probiotic is Lactobacillus acidophilus with viable count of more than or equal to 1 × 108cfu/g, the addition amount is 400 g/cubic meter of pine bark. The daily management does not need other treatment except timely removing residual food, and does not need conventional disinfection.
Example 6 (giant Exendin cage)
And (3) culturing 3 pieces of iguana glauca in a show-window exhibition room with the area of about 5 square meters. After the exhibition was thoroughly disinfected, the treated pine bark containing probiotic was padded with a thickness of 5 cm. The probiotic is Lactobacillus acidophilus with viable count of more than or equal to 1 × 108cfu/g, the addition amount is 400 g/cubic meter of pine bark. The daily management does not need other treatment except timely removing residual food, and does not need conventional disinfection.
Comparative experiment 1 (chimpanzee cage)
The method comprises the following steps of (1) feeding 8 chimpanzees together in a chimpanzee hall, wherein 4 inner chambers are shared, the area of each inner chamber is about 30 square meters, and filiform wood shavings are used as padding; various vegetation such as arbors, shrubs, herbs and the like are planted in the outdoor sports ground. Daily management is cleaning cages every day, and the playground is disinfected regularly.
Comparative experiment 2 (Boa house)
And (5) breeding 2 boa in a show window type show house with the area of about 5 square meters. The treated moss was spread to a thickness of 10 cm after the exhibition was thoroughly sterilized. The daily management does not need other treatment except timely removing residual food, and does not need conventional disinfection.
Comparative experiment 3 (Laiguana cage)
And (3) culturing 20 pieces of iguana glauca in a show-window exhibition room with the area of about 5 square meters. The treated moss was spread to a thickness of about 5 cm after thorough disinfection of the booth. The daily management does not need other treatment except timely removing residual food, and does not need conventional disinfection.
Contrast experiment 4 (huge lizard house)
And (3) culturing 3 pieces of iguana glauca in a show-window exhibition room with the area of about 5 square meters. After the booth was thoroughly disinfected, the treated moss was layered to a thickness of about 5 cm. The daily management does not need other treatment except timely removing residual food, and does not need conventional disinfection.
1. Purpose of experiment
1.1, monitoring the temperature and humidity changes inside the fermentation bed and the relation between the temperature and humidity changes and the environmental temperature and humidity in the fermentation bed in the example 1;
1.2 the concentration of harmful gases in the indoor air after the application of the fermentation bed, which are related to the decomposition of animal excrement.
2. Apparatus and method
2.1 monitoring temperature and humidity by adopting an LX8013 industrial hygrothermograph produced by Guangzhou Lexun electronics Limited, embedding a temperature probe and a humidity probe in the center of the fermentation bed to read the temperature and the humidity in the fermentation bed every day and simultaneously reading the ambient temperature and the ambient humidity;
2.2 gas detection adopts a Hima AR8500 ammonia gas detector and a Hima AS8900 four-in-one gas detector produced by Wan Dongguan electronic products Co.
3. Results and discussion
3.1 when the temperature is just laid for fermentation, the internal temperature of the fermentation bed is consistent with the ambient temperature, and then the temperature of the fermentation bed is slightly higher than the ambient temperature and fluctuates along with the fluctuation of the ambient temperature, but the fluctuation range of the internal temperature of the fermentation bed is obviously less than the change of the ambient temperature, and particularly when the ambient temperature is higher in summer, the internal temperature of the fermentation bed is relatively stable (fig. 5 and 6). The good heat preservation performance of the pine bark enables the temperature inside the fermentation bed to be more stable than the ambient temperature, and is beneficial to the growth of probiotics.
3.2 humidity in summer and winter, the humidity inside the fermentation bed is almost constant at a high level (fig. 7, fig. 8), but the surface of the fermentation bed is always kept in a drier state, and the ground of the cage without the fermentation bed or the padding is accumulated with water for a long time. The water inside the fermentation bed comes mainly from: guangzhou has high air humidity, animal excrement and residual moisture after workers wash the cage. The higher humidity environment inside the fermentation bed provides favorable conditions for the growth of probiotics.
3.3 detection of harmful gas concentration under the indoor heat preservation condition in winter, the ammonia concentration measured at a position about 30 cm away from the middle surface of the fermentation bed in the inner chamber is up to 1.0ppm, and the ammonia concentration is not detected for most of time (figure 9); gases such as carbon monoxide, sulfur dioxide, hydrogen sulfide, etc. were not detected. In an animal inner chamber paved with a fermentation bed, the air quality is far higher than the relevant regulations of experimental animal specification GB14925-2001 and Industrial Enterprise design sanitary Standard TJ36-79, and the like, and is close to the human indoor air quality Standard GB/T18883-2002 (the indoor air quality Standard GB/T18883-2002 specifies that the maximum allowable concentration of ammonia gas in indoor air is 0.2mg/m3(0.2635 ppm); the Standard of design and hygiene of Industrial enterprises, TJ36-79, states that the maximum permissible concentration of ammonia in the plant air is 30mg/m3(39.53 ppm); the Experimental animal Specification GB14925-2001 stipulates that the ammonia gas concentration in the experimental animal feeding environment should be less than or equal to 14mg/m3(18.452ppm)。
Experimental study 2 (study of microbial Activity in animal cage litter)
To compare the activity of microorganisms in different litters in animal cages, a total of 13 litters samples from 3 primates, 3 reptile cages were collected and analyzed using the Biolog-ECO method, with the results shown: the type of litter in the cages had a greater impact on environmental microbes than the animal species, and the types of litter with microbial activity ranging from high to low in this study were pine bark, wood shavings, and moss in the order.
1 materials and methods
1.1 samples and treatments
Sample collection and processing: about 10g of the litter samples at 5 positions were collected aseptically from the peripheral and central parts of the animal cages according to the examples, sterilized distilled water was added to the samples in a ratio of 1g to 9ml, the samples were placed in a 4 ℃ constant temperature shaking chamber and shaken for 1 hour, the supernatant was diluted 10 times with sterilized distilled water and inoculated onto ECO plates at 150. mu.l/well and incubated at 37 ℃, and OD590 and OD750 values were read every 24 hours, respectively. The sample sources are detailed in table 2.
TABLE 2 sample sources
Adding probiotic bacteria (viable count of Lactobacillus acidophilus is not less than 1 × 10) into cortex Pini8cfu/g) in an amount of 400 g/cubic meter of pine bark
1.2 data processing
The microbial metabolic activity is expressed by an Average color change (AWCD) and is calculated by the following method: AWCD ═ Σ (C-R)/n. In the formula, C is the absorbance value of each carbon source hole at 590nm minus the absorbance value at 750 nm; r is the absorbance value of the control well; n is the number of the carbon source types in the culture medium. The correction is 0 treatment when the value of C-R is less than 0.06.
The obtained AWCD data were processed with SPSS17.0, vegan v2.5-6 and flashClost v1.01-2, respectively.
2 results and analysis of carbon utilization of ECO plates by samples
The AWCD value reflects the metabolic activity of the microorganism in the sample, i.e. the ability to utilize a carbon source. The AWCD curves of the 13 samples showed respective regularity depending on the kind of litter, but had no clear correlation with the kind of animal, for example, the pine bark samples were from 5 different animals but the change regularity of the AWCD curves was similar (fig. 10, 11, 12). It is clear from the AWCD mean curves of the 3 padding samples: the slope of the AWCD curve from the moss specimen was greatest within 24h, then flattened and peaked at 96 h; the maximum value of the AWCD curve slope for the wood wool sample appeared in the first 48h and became flat thereafter; the maximum value of the AWCD curve slope of the pine bark sample appears between 24h and 48h, and the curve slope before and after the curve slope is lower. From trend analysis of AWCD curves, the activity of the microbial community of the moss sample using all 31 carbon sources of ECO plates was highest within 24h, but then lagged behind other litter samples; the activity of the microorganisms in the wood shaving sample on all 31 carbon sources of the ECO plate is higher within 48 hours and is lower than that of the pine bark sample; microbial activity in the pine bark samples, although lower during the first 24h, rose rapidly thereafter and continued to be higher than the other two litter samples after 48h chasing the wood wool sample (fig. 13). It can be seen that there is a certain relationship between the type of litter and the change in the AWCD curve, that is, the microbial activity in the environment of the animal cage is mainly related to the type of litter, but is less related to the type of animal.
The AWCD values of the samples cultured for 96h were selected for anova, and as a result, there was a very significant difference in carbon source utilization (P <0.01) for each sample. In order to further analyze the utilization of different types of carbon sources in the ECO plate by each sample microorganism, 31 types of carbon sources in the ECO plate are divided into 6 types (see the application principle of the Biolog ecological plate and the carbon source composition [ J ]. Apron, Yangxue, Green technology, 2011,7: 231-. As a result, the samples showed very significant differences in the utilization of three types of carbon sources such as carbohydrates, amino acids, and amines (P <0.01), but did not show significant differences in the utilization of carbon sources such as carboxylic acids, polymers, and phenolic acids (P > 0.05).
The samples were divided into 3 groups by pad type: the variance analysis is carried out on the AWCD values of the pine bark group, the wood shavings group and the moss group at different culture times respectively, and the AWCD values of 3 sample groups reach a very significant level (P <0.01) in 4 time groups such as 24h, 72h, 96h, 120h and the like except that the AWCD values are significantly different (P <0.05) in 48 h. Wherein, the moss group is significantly higher than the pine bark group (P <0.01) at 24h, the wood shaving group is significantly higher than the pine bark group (P <0.05), but the wood shaving group is not significantly different from the moss group (P > 0.05); the pine bark group and the wood wool group were significantly higher than the moss group at 48h (P <0.05), while the pine bark group and the wood wool group were not significantly different (P > 0.05). At 3 time periods of 72h, 96h, 120h and the like, the wood shaving group is significantly higher than the moss group (P <0.05), and the pine bark group is significantly higher than the moss group (P < 0.01); although statistically the pine bark group and the wood wool group were not significantly different (P >0.05), the pine bark group was consistently higher than the wood wool group since 72 h.
3. Discussion of the related Art
The Biolog method reflects the functional diversity of the microbial community through different utilization types of microorganisms on various carbon substrates, can directly reflect the overall activity of the microbial community, has obvious advantages in representing the space-time scale of the dynamic change of the bacterial community, has the advantages of large data volume, convenience, quickness and the like, and is widely applied to the fields of soil, water bodies, environment and the like in recent years. Among them, ECO plates (ECO plates) are widely used in research on functional diversity of soil microbial communities and also in research on intestinal microorganisms of animals.
Samples collected in this study were from 3 different types of bedding in 3 primate and 3 reptile cages, respectively, and the samples were analyzed for microbial activity using Biolog-ECO plates. As a result, the microbial activities of the samples were very significantly different (P <0.01), and the differences were mainly manifested by the utilization of three types of carbon sources such as carbohydrates, amino acids, and amines (P <0.01), and the utilization of carboxylic acids, polymers, and phenolic acids was not significantly different (P >0.05), indicating that there was some similarity in the environmental microbial communities in these animal cages.
The samples are grouped according to different padding types, and the AWCD curves of the samples from 3 sources such as pine bark, wood shavings, moss and the like show similar regularity, namely, each padding group has respective regularity. Thus, the type of litter within an animal cage has a greater impact on the activity of environmental microorganisms than does the type of animal. Wherein the microbial activity of the moss group samples is higher in the early culture period, while the microbial activity of the pine bark samples is lower in the early culture period, but then rapidly rises and exceeds the other two groups, and after the AWCD value reaches basic stability, the microbial activity of the 3 different padding sample samples is sequentially pine bark planer moss from high to low.
Experimental study 3 (study of relationship between intestinal microorganisms of black-leaf monkey and environment)
The mammalian digestive system contains a complex community of microorganisms that are an integral part of the host digestive system. In recent years, the application and development of high throughput sequencing technologies further reveal that gut microbiota plays an important role in animal health and metabolism. The results of experimental study 2 show that the type of the bedding in the cage is related to the microbial activity contained in the bedding, and the bedding with the microbial activity from high to low is pine bark, wood shavings and moss in turn. In order to further study the influence of the living environment of the animals on the abundance of the microorganisms in the intestinal tracts of the animals, fresh black-leaf monkey feces from different living environments are collected and analyzed by adopting metagenome and a 16S sequencing method.
As an emerging molecular biology technology, metagenomics is widely applied to the development of related researches such as microbial diversity, population structure, evolutionary relationship, functional activity, interrelation and the like, and can reflect the real state of microbial survival more accurately.
Test materials and methods
1.1 sample Collection
Fresh manures of the black-leaf monkeys are respectively collected from 4 animal cages with different management modes and environments, stored at-80 ℃, transported by dry ice, and subjected to metagenome and 16S sequencing analysis by Huada genes. The sample sources are detailed in table 3:
TABLE 3 sampling and grouping case
Injecting: exhibition area 2 is example 1
1.2 test methods
1.2.1 Total DNA extraction microbial genomic DNA from each group of Macaca mulatta stool samples was extracted using a stool genomic DNA extraction kit. After completion of the extraction of the genomic DNA, the extracted genomic DNA was examined by electrophoresis on 1% agarose gel. And finally, the sequencing on the computer and the analysis of partial data are finished by Shenzhen Huada Gen Yongquan GmbH.
1.2.216 amplification, sequencing and analysis of S rRNA A30 ng sample of genomic DNA of acceptable quality was taken for PCR amplification (region V3-V4 of 16S rRNA) and purification. And after the detection is qualified, sequencing by using a HiSeq platform, comparing a sequencing result with a 16S bacteria and archaea ribosome database (silva119) for annotation, then carrying out intestinal microbial diversity analysis on black-leaf monkeys, and carrying out differential analysis on the flora change of 4 groups of black-leaf monkeys.
1.2.3 amplification, sequencing and analysis of metagenome, taking qualified genomic DNA samples, and fragmenting the DNA by using a Covaris ultrasonic instrument to obtain short DNA fragments meeting the length requirement. Libraries were constructed and sequenced by a combination of probe-anchored polymerization techniques (cPAS). And carrying out splitting, quality shearing, pollution removal and other optimization processing on the original sequence from the original sequence, carrying out splicing assembly and gene prediction by using the optimized sequence, and carrying out species and functional annotation and classification on the obtained gene.
2 results
2.1 analysis of species and functional composition of the enterobacteria of black-leaf monkeys shows that Bacteroidetes (bacteroides) and Firmicutes (Firmicutes) account for a higher proportion of all non-human primate enterobacteria. Both 16S rRNA and metagenomic results in this study show: on the phylum level, firmicutes and bacteroidetes belong to the dominant group in the composition of intestinal microorganisms, indicating that the composition of intestinal flora of black-leaf monkeys and the trend among the phyla are similar to that of most mammals. In the research, the environmental abundance of the cages is respectively an exhibition area 2, an exhibition area 1, an exhibition area 3 and an exhibition area 4 from high to low, the abundance of the thick-wall phylum in each exhibition area is increased in sequence, and the abundance of the bacteroidetes in each exhibition area is decreased in sequence. In addition, the Spirochaets occupancy ratio in both the two zones, namely, bay 1 and bay 2, was also slightly higher (similar to the 16S results) than in the other 2 groups; the relative abundance of Proteobacteria in panel 3 was higher than in panel 4, panel 1 and panel 2 (see FIG. 14A).
In order to further explore the intestinal microbial diversity of the black-leaf monkeys in different feeding environments, dominant species and relative abundance of different samples based on genus level are analyzed by using a community histogram. From the analysis results, unannotated microorganisms (unanswered) were removed from 4 groups, among which Clostridium (Clostridium), Prevotella (Prevotella), Bacteroides (Bacteroides), Alistipes (Alistipes), Ruminococcus (Ruminococcus), Treponema (Treponema), and oscillabacterium (Oscillibacter) belong to the dominant group among the 4 groups (see fig. 14B). The relative abundance of the flora with slightly different abundances, such as Clostridium (Clostridium), in the exhibiting area 4 is higher than that of the other 3 groups; prevotella (Prevotella) and Bacteroides (Bacteroides) were less abundant in span 4 than in the other 3 groups; the relative abundance of Ruminococcus (Ruminococcus) in expanded region 3 was higher than that of the other 3 groups.
In addition, according to the analysis of the colony composition at the microbial species level, it was found that the unannotated microbes (unanswered) were removed from the 4 groups, wherein the relative abundance of Treponema _ succinifiaces in the exhibition area 2 was higher than that of the other 3 groups, which may be related to the exhibition area 2 (example 1) being not sterilized and the environmental microbes being more abundant.
2.2Beta diversity analysis Beta diversity was used to specifically assess differences between microbial communities. It was found by PCA analysis that at the OTU level, apart from some overlap between the span 1 and span 2, the dispersion of the flora composition among the other groups was more evident, with greater differences (see FIG. 15A). PCA analysis by using metagenomic sequencing shows that colonies in the expansion region 4 and the expansion region 3 at the species level have more overlap and smaller difference (see figure 15D); and the population composition of the developed region 4 and the developed region 2 is more discrete than the population composition of the developed region 4 and the developed region 1 (see FIG. 15B, C).
2.3 comparison and analysis of intestinal flora species and functional differences of black-leaf monkeys according to the analysis of 16S rRNA sequencing results, significance test of differences among groups in genus level is carried out, and 7 populations with abundance in Top 15 have significance differences, which are respectively: prevotella _9, Treponema _2, unclassified _ f _ Lachnospiraceae, [ Eubacterium ] _ coprostanogens _ group, Christenseella _ R-7_ group, Ruminococcaceae _ NK4A214_ group. And (3) analyzing the difference of the enterobacteria of the black-leaf monkeys by using metagenome sequencing. The main categories of the samples with the lowest environmental abundance in the two groups of the exhibition region 4 and the exhibition region 3 are that the abundances are significantly different: thermosinus, Thermophagus, and the like. The 2 groups of samples with significant differences between the extended area 4 and the extended area 1 mainly belong to the following categories: unclasfied, Tyzzerella, Tannerella, and the like. The two groups of samples with significant differences in the exhibition area 4 and the exhibition area 2 mainly belong to the following categories: tyzzerella, Tannerella, Ruminococcus, etc., which are more similar to the comparison of panel 4 and panel 1, also indirectly indicate that the abundance of the environment in panel 1 is more similar to that in panel 2.
2.5 analysis of Functional genes of intestinal flora of Macaca mulatta
2.5.1 differential Gene analysis results based on metagenomic sequencing results, each set of differential genes was analyzed by alignment and expressed as a volcanic chart (see FIG. 16). Compared with the other 3 groups, the different genes in the region 4 are more than 4000, and most of the different genes are lower in abundance than other groups, which is probably related to lower environment abundance. The environmental abundance in this study was, in order from high to low: exhibition region 2, exhibition region 1, exhibition region 3 and exhibition region 4, and the results show that the abundance of some differential genes also show similar regularity.
2.5.2 functional analysis of the monkey melanophore intestinal flora differential gene KEGG based on the 16S rRNA sequencing result, performing COG and KEGG functional annotation respectively to obtain annotation information of OTU at each functional level of COG and KEGG and abundance information of each function in different samples. The results show that the COG functional composition of all samples is relatively similar, and no major change is found in different feeding areas. Statistical analysis on the level abundance of the KEGG metabolic pathway level 1 shows that the gene abundances of the 3 pathways, namely Environmental Information Processing, Genetic Information Processing and Metabolism, are the highest. Therefore, the KEGG annotation results were analyzed in combination with the metagenomic sequencing results. The metagenome result shows that the 4 groups have the most genes in Level 1 classification, wherein Level 2 with more genes comprises Global and overview maps, Carbohydrate methyl acetate and Amino acid methyl acetate. While the Environmental Information Processing has only 3 level 2 classification levels, with the maximum number of Membrane and Signal transmissions.
And (4) analyzing functional annotations obtained by comparing the extended region 4 with relatively low environmental abundance with other 3 groups of differential genes by using a KEGG database in combination with metagenome data (the functional annotations in the database are not included). When the spread region 4 was compared with the other 3 groups, the number of genes contained in Metabolim was the largest in the Level 1 classification, followed by Genetic information processing; the level 2 classes containing a larger number of genes all contain Global and overview maps, Replication and repair and Membrane transport. In addition, the differential genes of the expansion region 4 and the expansion region 1 are more overall; but the Level 1 classification Level is the smallest organic system and the least in the other two comparisons is the human discasess.
Discussion of 3
While intestinal microorganisms are closely related to the health of a host, the study of intestinal microorganisms is of great significance as a primate having a recent relationship with humans. In recent years, with the wide application of 16S rRNA and metagenomic sequencing technologies, research on intestinal microbiota has become a hotspot in the field of modern biology at present. The research analyzes the composition and abundance change of intestinal microflora of the black-leaf monkeys under different feeding environments by integrating 16S rRNA and metagenome sequencing, and predicts the differential gene function of the intestinal microflora.
The dominant flora in the enterobacteria of the black-leaf monkeys in the present study are Firmicutes and Bacteroidetes (Bacteroidetes) which play a major role in the digestion of fibrous substances, and are related to the feeding habits of the black-leaf monkeys such as the feeding of leaves, and are consistent with the literature reports. The dominant bacteria such as Clostridium (Clostridium) and Prevotella (Prevotella) identified based on the genus level can help black-leaf monkeys to decompose cellulose in leaves and degrade structural carbohydrates in the leaves, and the intestinal flora plays an important role in nutrition digestion and absorption of the black-leaf monkeys.
The results of the comprehensive differential gene comparison in this study show that the difference between the expansion region 4 and the other 3 groups is more, and the abundance of most of the difference genes is lower than that of the other groups. The environmental abundance in the present study is presumed to be, in order from high to low: exhibition area 2, exhibition area 1, exhibition area 3, exhibition area 4 are relevant. The comparison analysis of the comprehensive environmental abundance and the PCA shows that the discrete degree of the exhibition area 4 and the other 3 groups of intestinal flora increases along with the increase of the environmental abundance, and the microbial community constitution is related to the feeding environment (the concrete ground is added with the disinfection and the killing of environmental microorganisms).
The flora of rumen coccus (Ruminococcus) and the like has a certain relationship with the feeding habits and the metabolism of animals. In the present study, rumen coccus (Ruminococcus) belongs to one of the dominant bacterial groups in intestinal tracts of 4 groups of black-leaf monkeys, and there was a certain difference among the 4 groups. The results of the intestinal flora differential gene function analysis showed that the number of genes associated with metabolism was the greatest in the 4 groups, which was related to the acclimatization temperature of the primate, the type of food and its intake nutrition. Therefore, the intestinal microbial composition structure is closely related to the animal feeding and living environment in the animal evolution process.
According to the research, the intestinal microorganisms of the black-leaf monkeys in different environments are compared and analyzed through 16S rRNA and metagenome sequencing, and the intestinal flora is found to be closely related to the abundance of the environment.
It has been reported that the ratio of bacteroidetes enterobacteria/firmicutes of people with yang-deficiency constitution is lower than that of people with mild constitution, and the abundance of bacteroides enterobacteria of people with yang-deficiency constitution is significantly lower than that of people with mild constitution (P <0.05), and that a significant decrease in the relative abundance of bacteroides is characteristic of the intestinal flora with yang-deficiency constitution. In this study, the ratio of Bacteroides/firmicutes was, in order from high to low: exhibition area 2> exhibition area 1> exhibition area 3> exhibition area 4, consistent with the environmental abundance of 4 exhibition areas from high to low, wherein exhibition area 2 is the cage environment of the example 1 of the present invention. The traditional Chinese medicine considers that the constitution of yang deficiency refers to the dysfunction of viscera of the body, and the manifestations of yang qi deficiency in the body and yang deficiency leading to internal cold are easy to appear. It follows that environmental abundance affects the intestinal microflora of animals and affects the health of the body.
Experimental study 4 (animal cage bedding pathogenic microorganism detection)
If pathogenic microorganisms exist in the living environment of animals, the health and the life of the animals are seriously threatened. In the traditional wild animal breeding method in zoos, the animal cages and the surrounding environment thereof must be disinfected regularly, and the method has the defects that the harmful microorganisms are killed, the probiotics in the environment are killed, the abundance of the microorganisms in the intestinal tracts of the animals is influenced, and the health of the animals is influenced (see experimental study 3). In order to understand the presence of pathogenic microorganisms that may be present in the animal's environment without periodic sterilization in the present invention, bedding samples from 18 animal cages were tested and the presence of pathogenic microorganisms was not detected.
1. Materials and methods
1.1 samples and treatments
Sterile collecting about 10g of padding samples at 5 positions around and in the center of an animal cage, adding sterilized distilled water into the samples according to the proportion of 1g of samples to 9ml of samples, fully shaking, and then sticking liquid to inoculate a culture medium. The sample sources are detailed in table 4.
TABLE 4 sample sources
Adding probiotic bacteria (viable count of Lactobacillus acidophilus is not less than 1 × 10) into cortex Pini8cfu/g) in an amount of 400 g/cubic meter of pine bark
1.2 Escherichia coli O157 culture
The sample prepared in 1.1 above was inoculated into E.coli O157 color medium (Kyoto Cyclokay, J2294Y) and cultured at 37 ℃ for 24 hours.
1.3 blood agar plate culture
The sample prepared in 1.1 above was inoculated into blood agar plate medium (K1869Y, Kyoto Arkey. Microbiol. Co., Ltd.) and cultured at 37 ℃ for 24 hours.
1.4 fungal culture
The samples prepared in 1.1 above were inoculated with the following media for culture, respectively: (1) culturing in Sa's medium (Kyoto, J2136Y) at 37 deg.C for 48 hr; (2) culturing in malt extract culture medium (20180105, Qingdao high-tech industrial garden Haibo Biotech Co., Ltd.) at 26 deg.C for 168 hr; (3) potato dextrose medium (Guangdong Huanji Microbiol technologies, Inc., 1070091) was cultured at 26 ℃ for 168 hours.
1.5PCR identification
Well-growing colonies were selected from the above-mentioned 1.3 medium, and analyzed by a whole gene sequence analysis of Dianthuqiong parainfluenza virus type 5 and pathogenicity study on mice [ D ]. Guangzhou: total DNA was extracted by the method of 2017, university of agriculture, south China, and PCR amplification was performed using ITS full-sequence universal primers. The instrument comprises the following steps: U.S. Bio-Rad Berle thermocycling PCR instrument T100.
2 results and analysis
3. Results and analysis
2.1 Escherichia coli O157 culture
All 18 samples were inoculated into E.coli O157 chromogenic medium and incubated at 37 ℃ for 24h, and neither magenta nor purple colonies grew.
2.2 blood agar plate culture
After all 18 samples were inoculated on blood agar plate culture medium and cultured at 37 ℃ for 24h, no colony growth with alpha hemolysis or beta hemolysis was observed.
2.3 fungal culture
In both the 11 pine bark samples and the 4 wood wool samples, the following colonies were formed: culturing on a Sasa medium at 37 ℃ for 48h to form a yellow-white, dense and flat colony with a light green plush edge and a colorless reverse side, wherein the result of a sample S1 is shown in FIG. 17; the results of the slow growth on wort medium and potato dextrose medium, formation of brown-green, thick-velvet colonies on wort medium after 168h incubation at 26 ℃ are shown in FIG. 18 for example in sample S1; the results for the formation of smoke-green, thick, fluffy colonies on potato dextrose medium, e.g., sample S1, are shown in fig. 19.
Under a microscope, the hyphae of the conidiophores are transparent and have multiple branches. Conidia are oval or long, single-celled, transparent, colorless, smooth in wall, and 3-5 μm in diameter (see fig. 20).
2.3PCR identification
Taking a colony on a culture medium, extracting total DNA, and performing PCR amplification, sequencing and comparison to obtain the strain, wherein Trichoderma reesei (Trichoderma reesei) has the highest similarity (99.18%) with the gene sequence of the strain.
4. Discussion of the related Art
Samples collected from the study were from 3 different types of bedding in 5 primates and 4 reptile cages, respectively, and neither E.coli O157 nor colony growth with alpha or beta hemolysis was found.
Samples from 11 pine barks and 4 wood shavings all had fungal growth on 3 commonly used fungal media, and were identified by culture characteristics, morphological structure, and PCR as Trichoderma reesei (Trichoderma reesei), a fungus capable of producing cellulolytic enzymes, and no reports of pathogenicity to humans or animals were found.
Combining laboratory culture and identification results, and animal health status, it can be considered that no common pathogenic microorganism is found in the samples collected in this study.
Claims (8)
1. A method for managing captive wild animals is characterized in that: (1) laying a fermentation bed and a padding layer: laying pine barks mixed with probiotics on the indoor ground of the animal cage as a fermentation bed, laying pine barks mixed with probiotics on the outdoor ground of the animal cage or a show-window type exhibition house as a padding layer, or combining the padding layer with the quasi-ecological landscape to simulate the natural animal habitat; the probiotics are lactobacillus acidophilus; the thickness of the mattress of the fermentation bed is as follows: the small animal is 30-40 cm; the size of the medium and small animals is 40-60 cm; the large animal is 60-100 cm; the bedding thickness of the bedding layer is as follows: the size of the small animal is 5-10 cm; the size of the small and medium animals is 10-15 cm; the large animal is 15-30 cm; the small animals are mammals, amphibians or birds with the weight less than 10 kg, the small animals are mammals, amphibians or birds with the weight between 10 kg and 200 kg, and the large animals are mammals and amphibians with the weight more than 200 kg; (2) daily management: the cage or the show window type exhibition room does not need to be cleaned every day, but the residual food of the animals needs to be removed; (3) and (3) disinfection: regular and conventional disinfection of animal cages or show window type exhibition houses is not required; (4) except for severe pollution or epidemic or death of animals, the bedding layer or fermentation bed of cages for primates, carnivores, birds or amphibians does not need to be replaced for more than 2 years, and the bedding layer or fermentation bed of cages for herbivores and omnivores except primates does not need to be replaced for more than 1 year.
2. The method of claim 1 for managing captive wild animals, wherein: the replacement of the padding layer or the fermentation bed: when the pine bark becomes brittle in texture, crumbles easily, or is heavily contaminated, or when the animal becomes epidemic or dies, it needs to be replaced in whole or in part.
3. The method of claim 1 for managing captive wild animals, wherein: the pine bark is all tissues of a plant of Pinus of Pinaceae wrapped outside a secondary xylem of a trunk, a branch and a root, and the length of each block is 2 cm to 25 cm; the pine bark is from non-epidemic areas, is far away from animal farms by more than 3 kilometers, and is treated by sunlight exposure, ultraviolet irradiation, radiation method or formalin fumigation before use.
4. The method of claim 1 for managing captive wild animals, wherein: the animal cage or the show window type exhibition house is provided with a drainage facility, and a metal separation net is arranged at a water outlet of the drainage facility; the fermentation bed and the base of the padding layer have slopes and incline towards the water outlet.
5. The method of claim 1 for managing captive wild animals, wherein: the addition amount of the probiotics is 200-400 g/cubic meter of pine bark; adding frequency: the fermentation bed is laid and added for the first time, and is not needed to be added before the environment is thoroughly disinfected for the next time, and the fermentation bed is added again after the environment is thoroughly disinfected.
6. The method of claim 1 for managing captive wild animals, wherein: the viable count of the lactobacillus acidophilus is more than or equal to 1 multiplied by 108cfu/g。
7. The method of claim 1 for managing captive wild animals, wherein: urine excreted by animals is directly absorbed and decomposed by the padding or the fermentation bed; the excrement of animals is treated by respectively embedding the animal excrement into the fermentation bed/bedding layer by turning over the fermentation bed/bedding layer according to different animal species or locally flushing the animal excrement into the fermentation bed/bedding layer by water.
8. The method of claim 1 for managing captive wild animals, wherein: a disinfection pond or a disinfection pad is arranged at an entrance and an exit of workers of the animal cage.
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