Uses of n-3-hexadecanoic acid in mammalian weaning diets

Abstract

This invention discloses the use of n-3-hexadecanoic acid (HTA) in feed for maintaining intestinal health in weaning mammals. This invention also discloses a nutritional supplement for maintaining intestinal health in weaning mammals, the active ingredient of which includes NTA. The NTA of this invention is derived from seaweed, such as Ulva pertusa. The NTA of this invention can enhance the immunity of mammalian offspring and maintain intestinal health, thereby effectively reducing their morbidity and mortality.

Description

Technical Field

[0001] This invention belongs to the field of mammalian nutrition technology, specifically relating to a nutritional supplement for mammals during weaning. Background Technology

[0002] The weaning period is a critical and vulnerable stage in the life cycle of mammals. Newborn cubs face multiple challenges after leaving the mother's body. Their immune systems are not yet fully developed, and maternal antibody levels gradually decline, leading to relatively low immunity and susceptibility to pathogens. Simultaneously, the digestive system of mammals is not yet fully mature, the intestinal barrier is weak, and the gut microbiota is in its early stages of establishment, making them prone to malabsorption, diarrhea, and other intestinal health problems. These factors significantly increase the morbidity and mortality rates of newborn mammals, becoming a core challenge in pet breeding and care.

[0003] To meet the nutritional needs of mammals, long-chain polyunsaturated fatty acids (LC-PUFAs), especially n-3 series fatty acids eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA), have been widely used in pet nutritional supplements due to their important roles in regulating immunity, anti-inflammation, and promoting nerve and retinal development.

[0004] There are three main types of n-3 fatty acids. DHA and EPA are derived from animals (such as fish) or seaweed, while the third is octadecanoic acid (alpha-linolenic acid, ALA), which is derived from plants. DHA in the human body can be converted from ALA. For example, patent application CN 201110200029 discloses a nutrient composition and health product that promotes the development of the nervous system.

[0005] However, the conversion rate of alpha-linolenic acid to EPA / DHA in mammals is very low, especially during the breeding period and early development, when mammals have a higher demand for EPA / DHA. Directly supplementing with EPA / DHA from marine animals or algae is a more efficient strategy.

[0006] Current research on n-3 fatty acids mainly focuses on EPA / DHA. Research on members of the n-3 fatty acid family with relatively unique structures and shorter carbon chains, such as n-3 hexadecanoic acid, is relatively scarce, and their potential biological value has not yet been fully recognized and utilized in the field of pet nutrition. Summary of the Invention

[0007] To address the core issues of weak immune defense and vulnerable gut health in weaning mammals, this invention provides a specialized nutritional supplementation program based on short-chain n-3 polyunsaturated fatty acids (PUFAs).

[0008] This invention provides the application of n-3 hexadecanoic acid as a nutritional supplement for weaning mammals. The n-3 hexadecanoic acid of this invention is named cis-4,cis-7,cis-10,cis-13-hexadecanoic acid (C16:4n-3, Hexadecatetraenoic acid), and is abbreviated as HTA in this invention. By providing n-3 hexadecanoic acid to weaning mammals, the immune function of mammalian offspring is significantly enhanced, intestinal barrier stability is strengthened, and intestinal flora composition is optimized, thereby systematically improving the health level of weaning mammals.

[0009] n-3 hexadecanoic acid (HCA) is a short-chain n-3 polyunsaturated fatty acid found in certain marine algae. Ulva pertusa, a rapidly growing and easily cultivated macroalga, is a sustainable source of HCA. Commonly known as sea spinach or sea cabbage, Ulva pertusa is a wild economic algae abundant in my country. Studies have found that the HCA content in Ulva pertusa can be as high as 12.67%. Compared to increasingly depleted marine fishery resources, Ulva pertusa, as a large-scale cultivated algae, provides a sustainable and environmentally friendly pathway to obtaining HCA. Furthermore, algal sources are generally considered to have higher purity and a relatively lower risk of heavy metal contamination.

[0010] This invention provides the use of n-3-hexadecanoic acid in feeds for maintaining intestinal health in weaning mammals.

[0011] Furthermore, the present invention can maintain the intestinal health of weaning mammals by providing them with algae containing n-3 hexadecanoic acid.

[0012] Furthermore, the present invention can maintain the intestinal health of weaning mammals by providing them with Ulva pertusa.

[0013] Furthermore, the present invention can maintain the intestinal health of weaning mammals by providing them with total fat derived from algae containing n-3 hexadecanoic acid.

[0014] Furthermore, the present invention can maintain the intestinal health of weaning mammals by providing them with total fat derived from Ulva pertusa.

[0015] Furthermore, the present invention can maintain the intestinal health of weaning mammals by providing them with unsaturated fatty acids derived from algae containing n-3 hexadecanoic acid.

[0016] Furthermore, the present invention can maintain the intestinal health of weaning mammals by providing them with unsaturated fatty acids derived from Ulva pertusa.

[0017] Furthermore, the present invention can maintain the intestinal health of weaning mammals by directly providing them with n-3-hexadecanoic acid from algae containing n-3-hexadecanoic acid.

[0018] Furthermore, the present invention can maintain the intestinal health of weaning mammals by directly providing them with n-3 hexadecanoic acid derived from Ulva pertusa.

[0019] Furthermore, the n-3-hexadecanoic acid of the present invention can be a high-purity active ingredient obtained by lipid extraction and purification using seaweed containing n-3-hexadecanoic acid as raw material.

[0020] Furthermore, the n-3-hexadecanoic acid of the present invention can be a high-purity active ingredient obtained from Ulva perforata through lipid extraction and purification processes.

[0021] Furthermore, the feed addition amount of the above-mentioned n-3 hexadecanoic acid can be 20-40 mg / kg body weight.

[0022] Furthermore, the aforementioned mammals can be dogs or cats.

[0023] The present invention also provides a nutritional supplement for maintaining intestinal health in weaning mammals, the active ingredient of which includes n-3-hexadecanoic acid.

[0024] Furthermore, the aforementioned n-3 hexadecanoic acid can be derived from seaweed containing n-3 hexadecanoic acid.

[0025] Furthermore, the aforementioned n-3 hexadecanoic acid can be derived from Ulva prostrata.

[0026] Furthermore, the aforementioned n-3 hexadecanoic acid can be the total fat derived from seaweed containing n-3 hexadecanoic acid.

[0027] Furthermore, the aforementioned n-3 hexadecanoic acid can be the total fat derived from Ulva prostrata.

[0028] Furthermore, the aforementioned n-3 hexadecanoic acid can be an unsaturated fatty acid derived from seaweed containing n-3 hexadecanoic acid.

[0029] Furthermore, the aforementioned n-3 hexadecanoic acid can be an unsaturated fatty acid derived from Ulva pertusa.

[0030] Furthermore, the aforementioned n-3 hexadecanoic acid can be purified n-3 hexadecanoic acid derived from seaweed containing n-3 hexadecanoic acid.

[0031] Furthermore, the aforementioned n-3 hexadecanoic acid can be purified n-3 hexadecanoic acid derived from Ulva prostrata.

[0032] Furthermore, the feed addition amount of the above-mentioned n-3 hexadecanoic acid can be 20-40 mg / kg body weight.

[0033] Furthermore, the aforementioned mammals can be dogs or cats.

[0034] Specifically, the present invention is as follows.

[0035] 1. Use of n-3-hexadecanoic acid in feeds for maintaining intestinal health in weaning mammals.

[0036] 2. In the above-mentioned uses, the feed addition amount of the n-3-hexadecanoic acid is 20-40 mg / kg body weight.

[0037] 3. In the above uses, the mammal is a dog or a cat.

[0038] 4. A nutritional supplement for maintaining gut health in weaning mammals, the active ingredient of which includes n-3-hexadecanoic acid.

[0039] 5. In the above-mentioned nutritional supplements, the n-3-hexadecanoic acid is derived from seaweed.

[0040] 6. In the above-mentioned nutritional supplements, the n-3 hexadecanoic acid is derived from Ulva prostrata.

[0041] 7. In the above-mentioned nutritional supplements, the n-3 hexadecanoic acid is the total fat derived from Ulva pertusa.

[0042] 8. In the above-mentioned nutritional supplements, the n-3 hexadecanoic acid is an unsaturated fatty acid derived from Ulva pertusa.

[0043] 9. In the above-mentioned nutritional supplements, the feed addition amount of the n-3 hexadecanoic acid is 20-40 mg / kg body weight.

[0044] 10. In the above-mentioned nutritional supplements, the mammal is a dog or a cat.

[0045] The beneficial effects of this invention are as follows:

[0046] I. This invention is the first to apply n-3 hexadecanoic acid (HCA) to the nutrition of mammalian pups during the weaning period. Utilizing the bioactivity of its short-chain n-3 polyunsaturated fatty acids, it specifically addresses immune and gut health issues in mammalian pups. By providing short-chain n-3 polyunsaturated fatty acids with potentially unique bioactivity, it enhances the immunity of mammalian pups and maintains gut health, thereby effectively reducing morbidity and mortality rates and improving the overall level of mammalian pet breeding and care.

[0047] Second, using artificially cultivated seaweed to replace traditional fish oil resources avoids the risks of overfishing and marine pollution, ensuring the resource sustainability, stability, and environmental friendliness of n-3 hexadecanoic acid supply. Attached Figure Description

[0048] Figure 1 This is a gas chromatogram of the purified n-3-hexadecanoic acid of this invention. Detailed Implementation

[0049] To better understand this invention, the following embodiments are provided in conjunction with the accompanying drawings. It should be understood that the embodiments of this invention are for illustrative purposes only and not for limiting the invention; the scope of protection of this invention is defined solely by the claims. The embodiments provided are merely preferred embodiments and are not intended to limit the invention in any way. Those skilled in the art can make changes, equivalent substitutions, or modifications based on the content of this invention to form different implementations. However, any changes and modifications, and any equivalent substitutions made to the method of this invention without departing from the inventive concept are within the scope of protection of this invention.

[0050] Example 1: Preparation of n-3-hexadecanoic acid

[0051] 1.1 Extraction of total fat from Ulva procumbens

[0052] Total fat extraction from Ulva procumbens was performed using the Folch method with appropriate modifications. The extraction solvent was dichloromethane-methanol (2:1, volume ratio). The dried Ulva procumbens powder was mixed with 10 times its volume of solvent and stirred thoroughly for 24 hours. After filtration, 1 / 4 volume of ultrapure water was added to the extract, mixed thoroughly, and transferred to a separatory funnel. After standing for 12 hours, the lower organic phase was collected, and the organic solvent was removed by vacuum concentration to obtain the total fat from Ulva procumbens.

[0053] 1.2 Preparation of Free Fatty Acids

[0054] The total fat from *Ulva pertusa* was saponified using a 1 mol / L NaOH-ethanol saponification solution. After adding an appropriate amount of water, the mixture was extracted three times with petroleum ether to remove unsaponifiable matter. The extraction was repeated three times with petroleum ether, and the petroleum ether layer was collected, concentrated, and then the free fatty acids were obtained.

[0055] 1.3. Urea packaging method for separating unsaturated fatty acids

[0056] First, a urea-ethanol solution was prepared by dissolving urea in an ethanol solution at a ratio of 1:10 in a water bath at 70°C. Then, free fatty acids were added at a fat-to-urea ratio of 1:2, and the solution was crystallized at 4°C for 12 hours. The urea crystals were removed by filtration, yielding a filtrate. A chloroform-methanol solution (chloroform:methanol = 2:1) was added and shaken thoroughly. Then, 1 / 4 volume of water was added, and after separation, the chloroform layer was collected and concentrated under reduced pressure to obtain the unsaturated fatty acids.

[0057] 1.4 Separation of n-3-hexadecanoic acid by molecular distillation

[0058] Unsaturated fatty acids were collected, and the molecular distillation apparatus was set to a vacuum of 6.6 Pa, a rectification temperature of 80 °C, and a feed rate of 2 ml / min for molecular distillation to separate n-3 hexadecanoic acid (cis-4, cis-7, cis-10, cis-13-hexadecanoic acid).

[0059] 1.5 Gas Chromatography Analysis

[0060] The separated n-3-hexadecanoic acid sample was analyzed using an Agilent 7860A gas chromatograph (flame ionization detector). The chromatographic column was a quartz capillary column (30m × 0.320mm × 0.25μm) manufactured by Lanzhou Zhongke Antai Analytical Technology Co., Ltd. Nitrogen was used as the carrier gas, and the injection port was in split mode with a split ratio of 20:1. The control mode was constant flow mode, with a nitrogen flow rate of 1 mL / min, a hydrogen flow rate of 30 mL / min, and an air flow rate of 400 mL / min. The column oven initial temperature was 170℃, the programmed temperature ramp rate was 2.8℃ / min, the final temperature was 220℃, the final temperature hold time was 23 min, the detector temperature was 250℃, and the run time was 58 min.

[0061] Fatty acid methyl ester standards were purchased from Sigma-Aldrich and used to determine the peak position of n-3-hexadecanoic acid. Quantification was performed based on the principle that the detector response to the analyte is proportional to the amount of the analyte. The peaks in the sample spectrum were integrated, and the composition of each fatty acid was expressed as a percentage of the total fatty acid content.

[0062] Gas chromatographic analysis results of n-3-hexadecanoic acid separated by molecular distillation are as follows: Figure 1 As shown, calculations revealed that the relative content of n-3 hexadecanoic acid (HTA) in the sample obtained by molecular distillation was 78%.

[0063] Example 2: Fecal scoring method for experimental animals

[0064] Weaning puppies / kittens were selected as the experimental subjects.

[0065] Sampling was conducted at regular intervals daily. Within 2 hours after morning feeding, fresh fecal samples were observed and collected from the first excretion of each animal to avoid the influence of environmental factors on the samples.

[0066] Continuous monitoring. Stool samples were collected daily throughout the experiment, and fecal characteristics were recorded.

[0067] The sample collection guidelines are as follows.

[0068] Use a sterile sampling spoon to collect approximately 2g of feces from the middle, avoiding contact with ground contaminants.

[0069] The samples were placed in pre-labeled sterile sealed tubes and their characteristics were immediately assessed.

[0070] Samples requiring microbial community analysis should be temporarily stored in an ultra-low temperature freezer at −80℃ until analysis.

[0071] The criteria for scoring the feces of the experimental animals are shown in Table 1.

[0072] Table 1. Basis for Stool Scoring

[0073] Classification Scoring criteria 1 point (diarrhea) Completely liquid, unable to take shape, often contains mucus or blood, leaving obvious fecal stains on the ground when picked up. 2 points (soft stool) High humidity, misshapen, resembling a "cowhide cake," easily deformed when picked up, and may adhere around the anus. 3 points (slightly soft but well-formed) Damp cylindrical shape, some residue remains on the ground when picked up, easily broken. 4 points (ideal situation) Hard cylindrical shape, textured surface, leaves little or no residue when picked up. 5 points (constipation) Extremely dry and hard, in lumps or spheres, leaving no residue when picked up, causing difficulty in defecation.

[0074] Example 3: Determination of serum immune and inflammatory markers

[0075] Five mL of venous blood was collected from the experimental animals in the early morning under quiet conditions and on an empty stomach. The blood was injected into a vacuum blood collection tube without anticoagulant. Serum without hemolysis was separated within 2 hours after blood collection, and the test was completed within 8 hours after blood collection.

[0076] Immunoglobulins in the serum of experimental animals were determined. The levels of immunoglobulin A (IgA), immunoglobulin G (IgG), and immunoglobulin M (IgM) in the serum of experimental animals were measured using kits from Jingmei Biotechnology Co., Ltd., following the instructions in the kit's manual.

[0077] Immunoglobulin A (IgA) primarily functions on mucosal surfaces, protecting immune barriers such as the respiratory and digestive tracts. Immunoglobulin G (IgG) mainly functions in the blood and is the most common immunoglobulin in the body, providing long-term immune protection. Immunoglobulin M (IgM) is an important member of the rapid immune response to primary infection, capable of neutralizing pathogens in the early stages of infection.

[0078] Simultaneously, inflammatory markers in the serum of the experimental animals were measured. Interleukin-6 (IL-6), interleukin-10 (IL-10), and tumor necrosis factor-α (TNF-α) in the serum of the experimental animals were measured using kits from Jingmei Biotechnology Co., Ltd., following the instructions for use.

[0079] Interleukin-6 (IL-6) and tumor necrosis factor-α (TNF-α) are pro-inflammatory cytokines that promote inflammation in the immune response and help clear pathogens, but overactivity can lead to chronic inflammation or tissue damage. Interleukin-10 (IL-10) is an anti-inflammatory cytokine that can suppress excessive immune responses, help maintain the balance of the immune system, and prevent the occurrence of autoimmune diseases.

[0080] Example 4: Dietary intervention for puppies

[0081] All puppies were fed commercially available weaning supplements that meet the nutritional standards for puppies of the American Association of Feed Control Officials (AAFCO). The basic nutritional composition (dry matter basis) is shown in Table 2.

[0082] Table 2. Nutritional composition of basic supplementary food for puppies (dry matter basis)

[0083] Element content crude protein ≥28% Crude fat ≥20% crude fiber ≤3% Moisture ≤10% Coarse ash ≤10%

[0084] Thirty healthy 1-month-old Beagle puppies (15 males and 15 females) were randomly divided into three groups of 10 each. The control group was fed a basic complementary diet; the low-dose HTA group was fed a basic complementary diet plus hexadecanoic acid supplement (20 mg / kg body weight); and the high-dose HTA group was fed a basic complementary diet plus hexadecanoic acid supplement (40 mg / kg body weight). n-3-hexadecanoic acid (HTA) was evenly mixed into the basic complementary diet. The dietary intervention lasted for 30 days, and all puppies had free access to water.

[0085] Fecal samples were collected and assessed in accordance with the method described in Example 2.

[0086] Table 3 shows the fecal scores of puppies during the dietary intervention.

[0087] Table 3. Fecal scores of puppies during the intervention period

[0088] Group control group Low-dose HTA group High-dose HTA group Day 0 <![CDATA[3.25±0.15 a ]]> <![CDATA[3.24±0.23 a ]]> <![CDATA[3.26±0.24 a ]]> Day 10 <![CDATA[3.37±0.22 b ]]> <![CDATA[3.48±0.18 ab ]]> <![CDATA[3.60±0.17 a ]]> Day 20 <![CDATA[3.52±0.13 c ]]> <![CDATA[3.76±0.16 b ]]> <![CDATA[3.91±0.15 a <!-- 5 -->]]> Day 30 <![CDATA[3.68±0.16 c ]]> <![CDATA[3.85±0.12 b ]]> <![CDATA[4.02±0.11 a ]]>

[0089] In Table 3, the superscript letters a, b, and c on the score values ​​indicate that there are significant differences between the experimental groups (P < 0.05).

[0090] As shown in Table 3, HTA supplementation significantly improved fecal formation in puppies. Starting from day 10, the fecal score of the high-dose HTA group (HTA 40 mg / kg body weight) was significantly higher than that of the control group. On day 30 after dietary intervention, the fecal score of the high-dose HTA group reached 4.02 (close to the ideal score of 4), with a 9.2% improvement in fecal formation compared to the control group. Furthermore, the high-dose HTA group was significantly superior to the low-dose HTA group (HTA 20 mg / kg body weight), indicating that HTA supplementation can rapidly, continuously, and dose-dependently optimize the physical form of puppies' feces, with a dose of 40 mg / kg achieving the ideal fecal state.

[0091] The diarrhea rate in puppies during the dietary intervention is shown in Table 4.

[0092] Table 4. Diarrhea rate (including soft stool) in puppies during the trial period

[0093] Group control group Low-dose HTA group High-dose HTA group Days 1-10 15% 12% 9% Days 11-20 10% 7% 5% Days 21-30 8% 5% 3%

[0094] As shown in Table 4, HTA supplementation significantly reduced the incidence of diarrhea (including soft stools) in puppies. During the first 10 days of the experiment, all groups had a certain rate of diarrhea, but the HTA-supplemented group had a lower rate than the control group. The advantage of the HTA-supplemented group became more pronounced as the intervention period lengthened. From days 11 to 20 and from days 21 to 30, the diarrhea rates in both the low-dose and high-dose HTA groups remained consistently lower than those in the control group. In particular, the high-dose HTA group saw its diarrhea rate drop to only 3% in the later stages of the experiment (days 21 to 30), far lower than the 8% in the control group, indicating that HTA supplementation effectively reduces the rate of diarrhea caused by intestinal dysfunction in weaning puppies, with higher doses showing better results.

[0095] Take 1g of fresh fecal sample, dilute it 10-fold serially, spread it on MacConkey agar plates, incubate at 37°C for 24h, count typical Escherichia coli colonies, and express the results as colony forming units per gram of wet weight (CFU / g).

[0096] Table 5 shows the E. coli content in the feces of puppies during dietary intervention.

[0097] Table 5. Escherichia coli content in puppy feces (×10) 7 CFU / g)

[0098] Group control group Low-dose HTA group High-dose HTA group Day 0 <![CDATA[7.68±0.34 a ]]> <![CDATA[7.62±0.25 a ]]> <![CDATA[7.71±0.36 a ]]> Day 10 <![CDATA[7.25±0.26 a ]]> <![CDATA[5.53±0.16 b ]]> <![CDATA[3.68±0.34 c ]]> Day 20 <![CDATA[6.95±0.18 a ]]> <![CDATA[5.11±0.27 b ]]> <![CDATA[3.02±0.19 c ]]> Day 30 <![CDATA[6.12±0.26 a ]]> <![CDATA[4.56±0.32 b ]]> <![CDATA[2.53±0.28 c ]]>

[0099] In Table 5, the superscript letters a, b, and c of the content values ​​indicate that there are significant differences between the experimental groups (P<0.05).

[0100] As shown in Table 5, HTA supplementation significantly inhibited the number of Escherichia coli in puppy feces. After 10 days of intervention, the number of fecal Escherichia coli in the low-dose HTA group and the high-dose HTA group decreased by 24% and 49%, respectively; the antibacterial effect continued to increase over time, with the high-dose HTA group showing a 59% reduction on day 30; at the same time, there were significant differences in Escherichia coli content between the high-dose HTA group and the low-dose HTA group, indicating that its antibacterial effect was dose-dependent.

[0101] The above-mentioned effects of gut microbiota optimization (Table 5), improved fecal formation (Table 3), and decreased diarrhea rate (Table 4) all indicate that HTA effectively inhibits the proliferation of pathogenic bacteria and maintains the intestinal health of weaning puppies.

[0102] Thirty days after the dietary intervention, immune and inflammatory markers were tested according to the method in Example 3.

[0103] The results of serum immune and inflammatory marker measurements in puppies are shown in Table 6.

[0104] Table 6. Results of serum immune and inflammatory marker measurements in puppies

[0105] Group control group Low-dose HTA group High-dose HTA group lgA (g / L) <![CDATA[1.01±0.23 a ]]> <![CDATA[0.96±0.18 a ]]> <![CDATA[1.03±0.25 a ]]> lgG (g / L) <![CDATA[5.23±0.12 a ]]> <![CDATA[5.34±0.24 a ]]> <![CDATA[5.25±0.18 a ]]> lgM (g / L) <![CDATA[0.86±0.11 a ]]> <![CDATA[0.89±0.15 a ]]> <![CDATA[0.85±0.09 a ]]> IL-6 (pg / mL) <![CDATA[128.56±0.67 a ]]> <![CDATA[127.45±0.45 a ]]> <![CDATA[127.89±0.58 a ]]> IL-10 (pg / mL) <![CDATA[15.45±0.24 c ]]> <![CDATA[18.67±0.27 b ]]> <![CDATA[20.34±0.19 a ]]> TNF-α (pg / mL) <![CDATA[39.23±0.42 a ]]> <![CDATA[32.03±0.38 b ]]> <![CDATA[28.01±0.32 c ]]>

[0106] In Table 6, the superscript letters a, b, and c above the measured values ​​indicate that there are significant differences between the experimental groups (P < 0.05).

[0107] As shown in Table 6, HTA supplementation significantly regulated the levels of inflammatory factors in puppies. The anti-inflammatory factor IL-10 in the low-dose and high-dose HTA groups was significantly increased by 21% and 32% compared to the control group, respectively, with significant differences between the two groups. The pro-inflammatory factor TNF-α in the low-dose and high-dose HTA groups was significantly decreased by 18% and 29% compared to the control group, respectively, with significant differences between the two groups. Immunoglobulin (IgA / IgG / IgM) and IL-6 levels remained stable across all groups. This indicates that HTA exerts its immunomodulatory effect by selectively regulating inflammatory pathways rather than basic immune proteins.

[0108] Example 5: Dietary intervention for kittens

[0109] All experimental animals were fed commercially available weaning supplementary food that meets the nutritional standards for kittens of the American Association of Feed Control and Management (AAFCO). The basic composition of the food is shown in Table 7.

[0110] Table 7. Nutritional composition of basic complementary food for kittens (dry matter basis)

[0111] Element content crude protein ≥34% Crude fat ≥16% crude fiber ≤4% Moisture ≤11% Coarse ash ≤10%

[0112] Thirty healthy one-month-old American Shorthair kittens (15 males and 15 females) were randomly divided into three groups of 10 kittens each. The control group was fed a basic complementary diet; the low-dose HTA group was fed a basic complementary diet plus a hexadecanoic acid supplement of 20 mg / kg body weight; and the high-dose HTA group was fed a basic complementary diet plus a hexadecanoic acid supplement of 40 mg / kg body weight. n-3 hexadecanoic acid was evenly mixed into the basic complementary diet. The dietary intervention lasted for 30 days, and all kittens had free access to water.

[0113] Fecal samples were collected and assessed as described in Example 2. Fecal scores of kittens during the dietary intervention are shown in Table 8.

[0114] Table 8. Fecal scores of kittens during the trial period

[0115] Group control group Low-dose HTA group High-dose HTA group Day 0 <![CDATA[3.13±0.19 a ]]> <![CDATA[3.15±0.20 a ]]> <![CDATA[3.11±0.22 a ]]> Day 10 <![CDATA[3.25±0.22 b ]]> <![CDATA[3.39±0.18 a ]]> <![CDATA[3.58±0.16 a ]]> Day 20 <![CDATA[3.42±0.17 c ]]> <![CDATA[3.58±0.16 b ]]> <![CDATA[3.82±0.14 a ]]> Day 30 <![CDATA[3.59±0.16 c ]]> <![CDATA[3.76±0.12 b ]]> <![CDATA[4.00±0.15 a ]]>

[0116] In Table 8, the superscript letters a, b, and c on the score values ​​indicate that there are significant differences between the experimental groups (P < 0.05).

[0117] As shown in Table 8, HTA supplementation significantly improved fecal formation in kittens in a dose-dependent manner. From day 10, the fecal scores in the HTA-supplemented group were significantly higher than those in the control group. On day 10 of dietary intervention, the fecal formation in the low-dose HTA group and the high-dose HTA group improved by 4.3% and 10.2% compared to the control, respectively. On day 30 of dietary intervention, the fecal score in the high-dose HTA group reached 4.00±0.15a (close to the ideal score of 4), a significant improvement of 11.4% compared to the control group, and significantly better than the low-dose HTA group. This indicates that HTA supplementation can rapidly, continuously, and dose-dependently optimize the physical form of kitten feces, with the high-dose HTA group (40 mg / kg) achieving an ideal intestinal health state.

[0118] The diarrhea rate in kittens during the dietary intervention is shown in Table 9.

[0119] Table 9. Diarrhea rate (including soft stool) in kittens during the trial period

[0120] Group control group Low-dose HTA group High-dose HTA group Days 1-10 17% 14% 10% Days 11-20 12% 8% 6% Days 21-30 9% 6% 4%

[0121] As shown in Table 9, HTA supplementation significantly reduced the incidence of diarrhea (including soft stools) in kittens. During the first 10 days of intervention, all groups had a certain rate of diarrhea, but the HTA-supplemented group had a lower rate than the control group. The advantage of the HTA group became more pronounced as the intervention duration increased. From days 11 to 20 and from days 21 to 30, the diarrhea rates in both the low-dose and high-dose HTA groups remained consistently lower than the control group. In particular, the high-dose HTA group saw its diarrhea rate drop to only 4% in the later stages of the experiment (days 21 to 30), far lower than the 9% in the control group, indicating that HTA supplementation effectively reduces the risk of diarrhea caused by intestinal dysfunction in weaning kittens, with higher doses showing better results.

[0122] Take 1g of fresh fecal sample, dilute it 10-fold serially, spread it on MacConkey agar plates, incubate at 37°C for 24h, count typical Escherichia coli colonies, and express the results as colony forming units per gram of wet weight (CFU / g).

[0123] Table 10 shows the E. coli content in the feces of kittens during dietary intervention.

[0124] Table 10. Escherichia coli content in kitten feces (×10) 7 CFU / g)

[0125] Group control group Low-dose HTA group High-dose HTA group Day 0 <![CDATA[7.82±0.24 a ]]> <![CDATA[7.83±0.15 a ]]> <![CDATA[7.76±0.28 a ]]> Day 10 <![CDATA[7.52±0.19 a ]]> <![CDATA[5.73±0.25 b ]]> <![CDATA[4.16±0.26 c ]]> Day 20 <![CDATA[6.98±0.25 a ]]> <![CDATA[5.24±0.32 b ]]> <![CDATA[3.54±0.18 c ]]> Day 30 <![CDATA[6.37±0.16 a ]]> <![CDATA[4.73±0.21 b ]]> <![CDATA[2.82±0.14 c ]]>

[0126] In Table 10, the superscript letters a, b, and c of the content values ​​indicate that there are significant differences between the experimental groups (P < 0.05).

[0127] As shown in Table 10, HTA supplementation significantly inhibited the number of Escherichia coli in kitten feces. After 10 days of dietary intervention, the number of E. coli in feces decreased by 24% and 45% in the low-dose HTA group and the high-dose HTA group, respectively. The antibacterial effect continued to increase over time, with the high-dose HTA group showing a 56% reduction in E. coli count on day 30 of dietary intervention. Furthermore, there were significant differences in E. coli levels between the high-dose and low-dose HTA groups, indicating that its antibacterial effect was dose-dependent.

[0128] The gut microbiota optimization effect, including the inhibition of Escherichia coli (Table 10), improved fecal formation (Table 8), and decreased diarrhea rate (Table 9), all indicate that HTA effectively inhibits the proliferation of pathogenic bacteria and maintains the intestinal health of weaning kittens.

[0129] Thirty days after the dietary intervention, immune and inflammatory markers were tested according to the method in Example 3.

[0130] The results of serum immune and inflammatory marker measurements in kittens are shown in Table 11.

[0131] Table 11. Measurement of serum immune and inflammatory markers in kittens

[0132] Group control group Low-dose HTA group High-dose HTA group lgA (g / L) <![CDATA[1.28±0.23 a ]]> <![CDATA[1.30±0.18 a ]]> <![CDATA[1.26±0.25 a ]]> lgG (g / L) <![CDATA[6.46±0.12 a ]]> <![CDATA[6.52±0.24 a ]]> <![CDATA[6.51±0.18 a ]]> lgM (g / L) <![CDATA[0.82±0.11 a ]]> <![CDATA[0.80±0.15 a ]]> <![CDATA[0.84±0.09 a ]]> IL-6 (pg / mL) <![CDATA[138.45±0.67 a ]]> <![CDATA[138.19±0.45 a ]]> <![CDATA[139.07±0.58 a ]]> IL-10 (pg / mL) <![CDATA[12.25±0.24 c ]]> <![CDATA[14.78±0.27 b ]]> <![CDATA[16.59±0.19 a ]]> TNF-α (pg / mL) <![CDATA[44.53±0.42 a ]]> <![CDATA[36.17±0.38 b ]]> <![CDATA[32.25±0.32 c ]]>

[0133] In Table 11, the superscript letters a, b, and c above the measured values ​​indicate that there are significant differences between the experimental groups (P < 0.05).

[0134] As shown in Table 11, HTA supplementation significantly regulated the levels of inflammatory factors in kittens. The levels of the anti-inflammatory factor IL-10 in the low-dose HTA group and the high-dose HTA group were significantly increased by 21% and 35% compared to the control group, respectively, with significant differences between the two groups. The levels of the pro-inflammatory factor TNF-α in the high-dose HTA group and the low-dose HTA group were significantly decreased by 19% and 8% compared to the control group, respectively, with significant differences between the two groups. Immunoglobulin (IgA / IgG / IgM) and IL-6 levels remained stable across the groups. This indicates that HTA exerts its immunomodulatory effect by selectively regulating inflammatory pathways rather than basic immune proteins.

Claims

1. Use of n-3 hexadecetraenoic acid for the preparation of a feedstuff for maintaining the intestinal health of a weaned mammal; said mammal being a dog or a cat.

2. Use according to claim 1, characterized in that: The feedstuff is added with n-3 hexadecetraenoic acid in an amount of 20-40 mg / kg body weight.

3. Use of n-3 hexadecetraenoic acid for the preparation of a nutritional supplement for maintaining the intestinal health of a weaned mammal; said mammal being a dog or a cat.

4. Use according to claim 3, characterized in that: The n-3 hexadecetraenoic acid is derived from seaweed.

5. Use according to claim 4, characterized in that: The n-3 hexadecetraenoic acid is derived from genus genus genus genus genus genus genus genus genus genus genus genus genus genus genus genus genus genus genus genus genus genus genus genus genus genus genus genus genus genus genus genus genus genus genus genus genus genus genus genus genus genus genus genus genus genus genus genus genus genus genus genus genus genus genus genus genus genus genus genus genus genus genus genus genus genus genus genus genus genus genus genus genus genus genus genus genus genus genus genus genus genus genus genus genus genus genus genus genus genus genus genus genus genus genus genus genus genus genus genus genus genus genus genus genus genus genus genus genus genus genus genus genus genus genus genus genus genus genus genus genus genus genus genus genus genus genus genus genus genus genus genus genus genus genus genus genus genus genus genus genus genus genus genus genus genus genus genus genus genus genus genus genus genus genus genus genus genus genus genus genus genus genus genus genus genus genus genus genus genus genus genus genus genus genus genus genus genus genus genus genus genus genus genus genus genus genus genus genus genus genus genus genus genus genus genus genus genus genus genus genus genus genus genus genus genus genus genus genus genus genus genus genus genus genus genus genus genus genus genus genus genus genus genus genus genus genus genus genus genus genus genus genus genus genus genus genus genus genus genus genus genus genus genus genus genus genus genus genus genus genus genus genus genus genus genus genus genus genus genus genus genus genus genus genus genus genus genus genus genus genus genus genus genus genus genus genus genus genus genus genus genus genus genus genus genus genus genus genus genus genus genus genus genus genus genus genus genus genus genus genus genus genus genus genus genus genus genus genus genus genus genus genus genus genus genus genus genus genus genus genus genus genus genus genus genus genus genus genus 6. Use according to claim 5, characterized in that: ​ 7. Use according to claim 5, characterized in that: ​ 8. Use according to any one of claims 3 to 7, characterized in that: ​

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