Adult Chinese pangolin feed and application thereof

Abstract

The adult Chinese pangolin feed is prepared from the following raw materials in percentage by mass: 11.00 percent to 15.20 percent of pears, 4.40 percent to 6.00 percent of bee pupae, 5.80 percent to 8.60 percent of dried silkworm pupae, 2.00 percent to 8.10 percent of dried earthworms, 23.00 percent to 38.80 percent of black ants, 3.00 percent to 13.80 percent of dried hermetia illucens larvae, 6.50 percent to 10.20 percent of dried tenebrio molitor, 2.00 percent to 9.80 percent of coconut powder, 2.00 percent to 4.00 percent of dried haws and 12.50 percent to 15.50 percent of soil. The adult Chinese pangolin feed and water are crushed and uniformly mixed according to the mass ratio of 1: 1.2, and the feed is fed once a day, so that the daily requirements of the adult Chinese pangolin can be met, a good nutritional condition is maintained, overnutrition does not exist, and digestive absorption and immune defense of the Chinese pangolin can be guaranteed.

Description

Technical Field

[0001] The invention belongs to the technical field of animal feed, and in particular relates to a feed for adult Chinese pangolins and application thereof. Background Art

[0002] The Chinese pangolin (Manis pentadactyla) belongs to the genus Manis, family Manidae, order Pholidota. It is a national first-class protected animal, a critically endangered species on the International Union for Conservation of Nature (ICUN) Red List of Threatened Species, and is listed in Appendix I of CITES by the Convention on International Trade in Endangered Species of Wild Fauna and Flora. With the continuous decline in the number of wild populations, artificial rescue and breeding of pangolins have become an effective measure to protect this critically endangered species. However, in captivity, it is unclear whether the main nutrients such as crude protein in artificial feed can meet the needs of adult Chinese pangolins.

[0003] Protein is an important component of the animal body and the basis for animal metabolism and production. Crude protein in feed is the main source of protein required for animal growth and is an important nutritional indicator that determines the quality of feed. An appropriate level of protein in the feed can maintain the intestinal health of animals and ensure the digestion, absorption and immune defense of animals. In the wild, pangolins mainly feed on a variety of ants and termites, and occasionally eat some insect larvae and arthropods. In the relevant research on pangolins' wild food, Ke Yayong et al. (1999) recorded that the protein content of three ants exceeded 50% of the dry weight; in the study of Li Xu et al. (2010), the crude protein content of pangolins eating ants accounted for 30% to 70% of the air-dried weight; in the analysis of the colony of the double-toothed polyspinous ant by Xu et al. (2022), its crude protein content exceeded 60%. In the captive environment of pangolins, Wang et al. (2022) reported that there were large differences in the feed formulas and nutritional components of 10 different pangolin protection institutions, with the crude protein content ranging from 32.41% to 55.11%.

[0004] Therefore, it remains unclear what protein level can meet the daily nutritional needs of Chinese pangolins, hindering the development of diets with appropriate protein levels that can meet the daily nutritional needs of Chinese pangolins. Summary of the invention

[0005] In order to overcome the deficiencies of the prior art, a feed for an adult Chinese pangolin and application thereof are provided.

[0006] The invention aims to provide a feed for an adult Chinese pangolin, which is composed of the following components, calculated by mass percentage of feed raw materials: 11.00%-15.20% of pears, 4.40%-6.00% of bee pupae, 5.80%-8.60% of dried silkworm pupae, 2.00%-8.10% of dried earthworms, 23.00%-38.80% of black ants, 3.00%-13.80% of dried black soldier fly larvae, 6.50%-10.20% of dried mealworms, 2.00%-9.80% of coconut powder, 2.00%-4.00% of dried hawthorn and 12.50%-15.50% of soil.

[0007] Preferably, the adult Chinese pangolin feed, calculated by mass percentage of feed raw materials, is composed of: 15.00% pears, 6.00% bee pupae, 6.80% dried silkworm pupae, 3.00% dried earthworms, 30.00% black ants, 9.40% dried black soldier fly larvae, 10.20% dried mealworms, 2.60% coconut powder, 2.00% dried hawthorn, and 15.00% soil.

[0008] The present invention also provides use of the adult Chinese pangolin feed in feeding adult Chinese pangolins.

[0009] Preferably, the application is to crush and evenly mix the adult Chinese pangolin feed and water in a mass ratio of 1:1.2, feed the adult Chinese pangolin once a day, and allow the adult pangolin to feed freely.

[0010] Preferably, the feeding is feeding at a fixed time every day.

[0011] Beneficial effects of the present invention:

[0012] The present invention designs feeds for adult Chinese pangolins with different crude protein levels, and explores that the crude protein level of the feed suitable for adult Chinese pangolins is 38.23%. The feed for adult Chinese pangolins provided by the present invention can meet the daily needs of pangolins, maintain a good nutritional status without excess nutrition, and can also reduce the MDA content of the adult Chinese pangolin body, improve the apparent digestibility of nutrients, be easy to digest, help improve the body's antioxidant capacity, maintain the intestinal health of the Chinese pangolin, ensure the digestion, absorption and immune defense of the Chinese pangolin, and provide a suitable feed for artificial breeding of Chinese pangolins. BRIEF DESCRIPTION OF THE DRAWINGS

[0013] Figure 1 This is a graph showing the differences in ALT, CF, GSSG, and MDA in adult pangolins after being fed 6 groups of diets with different crude protein levels. DETAILED DESCRIPTION

[0014] The following examples are provided to further illustrate the present invention, rather than to limit the present invention.

[0015] The following examples were conducted at the Pangolin Conservation and Research Center of the State Forestry and Grassland Administration (hereinafter referred to as the "Pangolin Conservation and Research Center"), which has been carrying out pangolin rescue and breeding work since 2020, with a rescue success rate of 80%, and many Chinese pangolins have successfully become pregnant and given birth to cubs. The following examples set 6 different dietary protein levels to observe the effects of different crude protein levels in the diet on the weight, feed intake, fecal scores, blood biochemical indicators and antioxidant indicators, and apparent digestibility of nutrients of adult Chinese pangolins in captivity, and to provide a diet suitable for artificially rearing Chinese pangolins.

[0016] Example 1

[0017] 1. Materials and Methods

[0018] 1.1 Experimental subjects and experimental design

[0019] As an extremely endangered species, the Chinese pangolin has a small number of artificially bred populations. This embodiment relies on the Pangolin Conservation and Research Center of the State Forestry and Grassland Administration as a test base. When the experiment was carried out (August to November 2022), the center had a total of 24 Chinese pangolins, which is one of the largest artificially bred populations of Chinese pangolins in China. The population includes lactating mothers, cubs, subadults, and adults, of which there are 21 adult Chinese pangolins. Therefore, this embodiment can only select 6 healthy individuals of similar weight (3 females and 3 males) from adult pangolins as test subjects, using a 6×6 Latin square design, and setting 6 feed crude protein levels of 34% (Group I), 36% (Group II), 38% (Group III), 40% (Group IV), 42% (Group V), and 44% (Group VI). The experiment consists of six stages (6 protein level tests, respectively), each stage is 14d, including a pre-test period of 7d and a regular test period of 7d.

[0020] 1.2 Experimental diet and feeding management

[0021] The percentages were calculated by mass. The composition of the experimental diet and the measured nutritional level of the diet in the air-dried state are shown in Table 1. According to the pangolin feeding and management regulations, all experimental animals were kept in single cages during the experiment, cleaned daily, and disinfected regularly; they were fed once a day at 16:00, and each feeding amount was 160g (feed raw materials: water = 1:1.2), and they were allowed to eat and drink freely. The actual feed intake was recorded at 09:00 the next day. During the experiment, the physical condition of the experimental animals was monitored, and the fasting body weight was tested once a day. The sources of the raw materials of each component of the diet are shown in Table 2. Preparation method of the diet: Weigh the prepared feed raw materials according to the mass ratio of the diet composition in Table 1, add the weighed feed raw materials and water in a mass ratio of 1:1.2 to a wall breaker, and crush and mix evenly.

[0022] Table 1 Feed composition and its measured nutritional level (%)

[0023]

[0024]

[0025] Note: All nutritional indicators are measured values.

[0026] Table 2 Sources of feed raw materials

[0027]

[0028] 1.3 Sample collection and indicator testing

[0029] 1.3.1 Stool score and water content

[0030] Observe and record pangolin feces every day, and score pangolin feces. The feces scoring standard was formulated with reference to the methods of Grellet (2012) and Clark (2016), etc. See Table 3. The feces were placed in a constant temperature of 60°C for drying and weighed to calculate the water content of the feces.

[0031] Table 3 Fecal scoring criteria

[0032]

[0033]

[0034] 1.3.2 Biochemical indicators

[0035] On the morning of the second day after the end of each phase of the trial, 2-3 mL of pangolin blood was collected on an empty stomach and placed in a 1.5 mL centrifuge tube and a heparin-treated tube, respectively. Serum and plasma were collected and centrifuged at 3000 × g for 10 min. The supernatant was placed in another clean 1.5 mL centrifuge tube and stored at -80°C for blood biochemical analysis and antioxidant index detection.

[0036] The prepared plasma was sent to the Institute of Subtropical Agriculture and Ecology, Chinese Academy of Sciences for the determination of plasma biochemical indicators. The test was performed using a fully automatic biochemical analyzer (Roche cobas c311, Switzerland). The reagents were from Roche. The test indicators and reagents used included plasma glucose (GLU, catalog number 04404483190), triglycerides (TG, catalog number 20767107322), total protein (TP, catalog number 03183734190), albumin (ALB, catalog number 03183688122), alanine aminotransferase (ALT, catalog number 20764957322), alkaline phosphatase (ALP, catalog number 03333701190), lactate dehydrogenase (LDH, catalog number is 03004732122), blood urea nitrogen (BUN, catalog number is 04460715190), cholesterol (CHOL, catalog number is 03039773190), low-density lipoprotein (LDL-C, catalog number is 03038866322), high-density lipoprotein (HDL-C, catalog number is 04399803190), and lipase (LIP, catalog number is 03029590322).

[0037] 1.3.3 Antioxidant index

[0038] The prepared serum was sent to Nanjing Boyan Biological Co., Ltd. for the determination of serum antioxidant indexes. The kit from Nanjing Boyan Biological Co., Ltd. was used, and the reagents used included reduced glutathione (GSH, item number BYSH-0206W), oxidized glutathione (GSSG, item number BYSH-0207W), malondialdehyde (MDA, item number BYSH-0109W), glutathione peroxidase (GPx, item number BYSH-0204W), catalase (CAT, item number BYSH-0105W), and superoxide dismutase (SOD, item number BYSH-0103W) for detecting the content of antioxidant indexes.

[0039] 1.3.4 Apparent digestibility of nutrients

[0040] During the trial period, the feces of each pangolin before feeding were collected by the full feces collection method, which was used as the complete feces of the individual on the previous day. The feces were collected for 7 consecutive days, weighed and recorded, and placed in a 60℃ oven for drying and stored for later use. During the trial period, 30g of feed samples were collected every day, placed in a 60℃ oven for drying and stored for later use.

[0041] The dry matter (DM) in the feed and feces was determined according to the "Determination of Water in Feed" (GB / T 6435-2006), the crude ash (Ash) was determined according to the "Determination of Crude Ash in Feed" (GB / T 6438-2007), the crude protein (CP) was determined according to the "Determination of Crude Protein in Feed by Kjeldahl Method" (GB / T 6432-2018), the crude fat (EE) was determined according to the "Determination of Crude Fat in Feed" (GB / T 6433-2006), the calcium (Ca) was determined according to the "Determination of Calcium in Feed" (GB / T 6436-2018), the total phosphorus (TP) was determined according to the "Determination of Total Phosphorus in Feed by Spectrophotometry" (GB / T 6437-2018), and the gross energy (GE) was analyzed by the HXR-6000 fully automatic oxygen bomb calorimeter (purchased from Hunan Huaxing Energy Technology Co., Ltd.). The calculation formulas for the relevant indicators are as follows:

[0042] Apparent digestibility of dry matter (%) = (dry matter intake - dry matter excretion in feces) / dry matter intake × 100%;

[0043] Apparent digestibility of energy (%) = (intake energy - fecal energy) / intake energy × 100%;

[0044] Apparent digestibility of each nutrient (%) = (nutrient intake - nutrient excretion in feces) / nutrient intake × 100%.

[0045] 1.4 Data Statistics and Analysis

[0046] After the data were sorted by Excel 2021, the normal distribution of the data was confirmed by the Shapiro-Wilk test, and one-way ANOVA was performed using GraphPad Prism9.0 software, and multiple comparisons were performed using the Tukey method. When the data did not conform to the normal distribution, the Kruskal-Wallis test was used for significance. The results are expressed as "mean ± standard error (mean ± SE)", P < 0.01 is extremely significant, P < 0.05 is significant, and 0.05 ≤ P < 0.10 indicates a trend of difference.

[0047] 2. Results

[0048] 2.1 Effects of dietary crude protein levels on body weight and average daily feed intake of adult Chinese pangolins

[0049] Table 4 Effects of dietary crude protein levels on body condition of adult Chinese pangolins

[0050]

[0051] Different lowercase letters in the same column indicate significant differences (P<0.05), different uppercase letters indicate extremely significant differences (P<0.01), and no letters or the same letters indicate no significant differences (P>0.05). The same applies to Tables 5 to 8 below.

[0052] As shown in Table 4, there were no significant differences in the initial weight, final weight and average daily feed intake of pangolins during each experimental period (P>0.05).

[0053] In the daily feeding and management of adult pangolins, body weight and feed intake are important monitoring indicators. In this experiment, the total energy of the designed diets with different crude protein levels was close (Table 1), and the feeding amount of pangolins during the positive trial period of each stage (160g per day, where feed raw materials: water = 1:1.2) was the same, and the average body weight of pangolins remained between 4.33 and 4.43kg (Table 4), indicating that the diet set in this experiment can meet the daily needs of adult pangolins and there is no nutritional surplus.

[0054] 2.2 Effects of dietary crude protein levels on fecal scores and water content of adult Chinese pangolins

[0055] Table 5 Effects of dietary crude protein levels on fecal scores of adult Chinese pangolins

[0056]

[0057] As shown in Table 5, after being fed with diets with different crude protein levels, the fecal scores of pangolins were between 3.67 and 3.92, and the water content was between 54.63% and 58.42%. After being fed with diets with different crude protein levels, there was no significant difference in the fecal scores and water content of Chinese pangolins (P>0.05), both of which were in an ideal state. It can be preliminarily judged that the different crude protein levels of diets set in this experiment had no adverse effects on the intestinal health of Chinese pangolins.

[0058] 2.3 Effects of dietary crude protein levels on plasma biochemical parameters in adult Chinese pangolins

[0059] Table 6 Effects of dietary crude protein levels on plasma biochemical parameters of adult Chinese pangolins

[0060]

[0061] Changes in blood biochemical indicators are closely related to the animal's absorption and metabolism of dietary nutrients. Changes in plasma TP, ALB, and BUN can reflect the animal's protein metabolism and nutritional status. Plasma TP represents the nutritional level of protein in the diet and the degree of digestion and absorption of protein by animals to a certain extent. GLU reflects the body's intake and utilization of glucose; TG and CHOL are the main forms of lipids in the blood. TG participates in the body's material and energy metabolism, and its content reflects the body's absorption, utilization, and metabolism of lipids; LDL-C and HDL-C participate in the metabolic transport of CHOL in and outside the liver. LDH is an important indicator for measuring kidney function; pancreatic damage is the main reason for the increase in LIP content; ALP is an important indicator for measuring liver damage; ALT is closely related to protein metabolism and liver function, and is often used as an indicator for diagnosing liver function.

[0062] As shown in Table 6, the crude protein level in the diet had no significant effect on the TP, ALB, BUN, GLU, TG, CHOL and HDL-C content and ALP activity in the plasma of adult Chinese pangolins (P>0.05), and there was a trend of difference in the effect on the LIP and LDH activity and LDL-C content (0.05≤P<0.10), and there was a significant effect on the ALT activity (P<0.05). The above results show that the crude protein level of the diet in this experiment was set reasonably, the nutritional status of pangolins was good, and the energy and lipid requirements of Chinese pangolins could be met. There were significant differences in the ALT activity of animals in different dietary CP levels, but there were no significant differences in the ALP activity of different dietary CP levels, and none of them exceeded the normal range of healthy adult Chinese pangolins. This shows that the dietary CP level set in each group of experimental diets is within the tolerance range of Chinese pangolins, and there is no liver damage caused by excessively high dietary CP levels.

[0063] 2.4 Effects of dietary crude protein levels on serum antioxidant indices in Chinese pangolins

[0064] Table 7 Effects of dietary crude protein levels on serum antioxidant capacity of adult Chinese pangolins

[0065]

[0066] Changes in the body's antioxidant capacity reflect the health and physiological status of animals to a certain extent. As shown in Table 7, the crude protein level in the diet had no significant effect on the activities of serum SOD, CAT, GPx and GSH content of adult Chinese pangolins (P>0.05). Figure 1 The serum MDA levels of groups D and V were significantly higher than those of group I, and significantly higher than those of groups III and VI (P<0.05). Figure 1The serum GSSG content of group C and IV was higher than that of groups I, II, and III, and was significantly higher than that of group I (P<0.05). The MDA content of group V was higher, and the GSSG content of group IV was higher, both indicating that excessive CP levels in the diet would lead to disorders in the body's nitrogen metabolism and produce more free radicals, thus causing oxidative stress damage. The GSSG and MDA levels of adult Chinese pangolins fed with group III diet were lower, indicating that the CP level of group III diet was more appropriate.

[0067] However, the MDA and GSSG levels in the serum of Chinese pangolins after being fed the diets with six protein levels were still within the normal range, which further demonstrated that the CP levels of the experimental diets set in each group were within the tolerance range of the Chinese pangolins.

[0068] 2.5 Effect of dietary crude protein level on apparent digestibility of nutrients in adult Chinese pangolins

[0069] Table 8 Effects of dietary crude protein levels on apparent digestibility of nutrients in Chinese pangolins

[0070]

[0071] As shown in Table 8, different dietary crude protein levels had no significant effect on the apparent digestibility of DM, CP, Ash, and GE in adult Chinese pangolins (P>0.05). The apparent digestibility of CF in group III was significantly higher than that in group VI (P<0.05). The digestibility of EE in groups II and III was significantly higher than that in group VI (P<0.05).

[0072] ALT is closely related to protein metabolism and liver function, and is often used as an indicator for diagnosing liver function. The CP levels of the experimental diets in each group of this experiment were within the tolerance range of the Chinese pangolin, and no liver damage was caused by excessively high CP levels in the diet. However, ALT was significantly increased in the VI group (p = 0.013), suggesting that excessive protein addition may cause liver damage ( Figure 1 The apparent digestibility of CF in group III was the highest among the six groups and was significantly higher than that in group VI (p=0.032). Therefore, the apparent digestibility of CF decreased when the protein content exceeded that in group III ( Figure 1 B).

[0073] In summary, diets with crude protein levels of 32%-43.88% can be used to feed adult Chinese pangolins, among which the optimal crude protein level for captive adult Chinese pangolins is 38.23% (Group III).

Claims

1. A feed for adult Chinese pangolins, characterized in that: Calculated by the mass percentage of feed raw materials, the composition is as follows: 11.00%-15.20% pears, 4.40%-6.00% bee pupae, 5.80%-8.60% dried silkworm pupae, 2.00%-8.10% dried earthworms, 23.00%-38.80% black ants, 3.00%-13.80% dried black soldier fly larvae, 6.50%-10.20% dried mealworms, 2.00%-9.80% coconut powder, 2.00%-4.00% dried hawthorn, and 12.50%-15.50% soil.

2. The adult Chinese pangolin diet according to claim 1, characterized in that: Calculated by the mass percentage of feed raw materials, the composition is as follows: 15.00% pears, 6.00% bee pupae, 6.80% dried silkworm pupae, 3.00% dried earthworms, 30.00% black ants, 9.40% dried black soldier fly larvae, 10.20% dried mealworms, 2.60% coconut powder, 2.00% dried hawthorn, and 15.00% soil.

3. Use of the adult Chinese pangolin feed according to any one of claims 1 to 2 in feeding adult Chinese pangolins.

4. The use according to claim 3, characterized in that: The adult Chinese pangolin feed according to claim 1 and water are crushed and mixed evenly in a mass ratio of 1:1.2, and the feed is fed to the adult Chinese pangolin once a day, and the adult pangolin is allowed to eat freely.

5. The use according to claim 4, characterized in that: The feeding is performed at a fixed time every day.

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