Application of gallnut extract as feed additive in sow production

Abstract

The invention belongs to the technical field of animal husbandry production, and particularly relates to application of a gallnut extract as a feed additive in sow production. The invention relates to a production feed additive for pregnant sows. The production feed additive for the pregnant sows is a gallnut extract, wherein the gallnut extract is ellagic acid and tannic acid. The purpose of the invention is to provide the application of ellagic acid and tannic acid as feed additives in sows at the later stage of pregnancy and sows at the early stage of pregnancy, the ellagic acid and tannic acid are added into daily ration to improve the reproductive performance of the sows, and the feed additive has the effects of high safety, no negative influence on the production performance of the sows and improvement of the reproductive performance of the sows.

Description

Technical Field

[0001] The invention belongs to the technical field of animal husbandry production, and in particular relates to application of a gallnut extract as a feed additive in sow production. Background Art

[0002] Reproductive traits are important economic traits for pigs, among which sow reproductive performance is a key parameter for evaluating pig farm breeding models and breeding techniques. The level of this parameter is closely related to the breeding efficiency of the pig farm. In pig production, sow reproductive performance is closely related to the sow's pregnancy process. The sow's pregnancy process is affected by various factors, which in turn affect the sow's pregnancy outcome. In early pregnancy, the sow's reproductive data such as ovulation, fertilization, early embryo implantation, and placenta formation may experience oxidative stress and inflammatory responses, leading to embryo implantation failure and abnormal embryo development, and then early embryonic death. In late pregnancy, inflammation and oxidative stress in the sow's placenta may lead to abnormal placental angiogenesis, energy metabolism disorders, and transport function disorders, which in turn affect the growth and development of the fetus.

[0003] Ellagic acid (EA) and tannins (TA) are natural polyphenolic compounds with anti-inflammatory, antioxidant, antibacterial, and anti-tumor properties. They are widely found in plants such as gallnut, chestnut, and tara. Studies have shown that EA has potent anti-inflammatory and anti-inflammatory effects, achieving these effects by reducing the levels of inflammatory factors in cells or tissues and by modulating inflammatory signaling pathways to reduce their expression. TA has strong antioxidant capacity and is a natural antioxidant that can exert its antioxidant activity by scavenging ROS and alleviating oxidative stress. Reports also indicate that adding a certain amount of EA and TA to animal diets can improve production and reproductive performance. Dietary EA can improve feed digestibility and growth performance in growing and finishing pigs. Supplementing weaned piglets with 0.1% EA improves the intestinal microbiome, promotes growth performance, and reduces intestinal damage and oxidative stress. Furthermore, adding 190 mg of TA to 1 mL of liquid milk replacer significantly increased the average daily weight gain of normal piglets and piglets infected with porcine epidemic diarrhea virus (PEDV), significantly reduced the feed-to-gain ratio of piglets infected with PEDV, and improved piglet growth performance. Adding an appropriate amount of TA to the diet can significantly increase ileal villus height, reduce intestinal permeability, and improve intestinal health in weaned piglets. Summary of the Invention

[0004] Therefore, in order to study the effects of gallnut extract (ellagic acid, tannic acid) on the reproductive performance of sows, this application adds gallnut ellagic acid (GEA) to the diet of sows in late pregnancy, explores the effects of different concentrations of GEA on the reproductive performance of sows, screens out the optimal concentration of GEA for regulating the reproductive performance of sows in late pregnancy, and verifies its effects on the gene expression levels of inflammatory factors in the piglet placenta and placental tissue morphology; adds gallnut tannin acid (GTA) to the diet of sows in early pregnancy, explores the effects of different concentrations of GTA on the reproductive performance of sows, screens out the optimal concentration of GTA for controlling the reproductive performance of sows in early pregnancy, and verifies its effects on the levels of antioxidant and inflammatory factors in sow serum, which has important research significance.

[0005] The purpose of the present invention is to provide the use of ellagic acid and tannic acid as feed additives for sows in late pregnancy and early pregnancy, and to add them to the diet to improve the reproductive performance of sows. The invention has the advantages of high safety, no negative impact on the production performance of sows, and the effect of improving the reproductive performance of sows.

[0006] The present invention provides a production feed additive for pregnant sows, wherein the production feed additive for pregnant sows is a gallnut extract;

[0007] Among them, the gallnut extract is ellagic acid and tannic acid.

[0008] The present invention provides a feed additive containing ellagic acid, wherein the dosage of the ellagic acid is 0.02%-0.1% of the basic feed.

[0009] The invention provides a feed additive containing tannic acid, wherein the dosage of the tannic acid is 0.05%-0.2% of the basic feed.

[0010] The invention provides a production feed for pregnant sows, which contains a feed additive.

[0011] The present invention provides use of a feed additive containing ellagic acid in preparing feed for pregnant sows.

[0012] The present invention provides use of a feed additive containing tannic acid in preparing feed for pregnant sows.

[0013] Compared with the prior art, the present invention has the following beneficial effects:

[0014] (1) The present invention adds different levels of GEA (0, 0.02%, 0.06%, 0.1%) to the diet of dual-purpose sows in late pregnancy to explore the effect of adding different levels of GEA on the reproductive performance of sows and screen out the optimal concentration. Then, qPCR experiments and histological experiments are used to examine the effect of GEA on the relative expression of inflammatory factor-related gene mRNA and the number of capillaries in the placenta. The results showed that adding 0.06% GEA to the diet of sows in late pregnancy could increase the birth weight and litter weight of piglets by 8.15% and 9.72%, respectively (P=0.069, P=0.085), and significantly reduce the rate of weak piglets by 9.83% (P<0.05); adding 0.06% GEA could significantly increase the relative mRNA expression of anti-inflammatory factors TRAF5 and IL-10 in the placenta (P<0.05), and reduce the relative mRNA expression of pro-inflammatory factors TNF and CXCL2 (P<0.05); adding 0.06% GEA significantly increased the number of blood vessels per unit length in the placenta tissue (P<0.05).

[0015] (2) The present invention adds different levels of GTA (0, 0.05%, 0.1%, 0.2%) to the diet of Large White sows in early pregnancy to explore the effect of adding different levels of GTA on the reproductive performance of sows and screen out the optimal concentration. Then, the effects of GTA on serum oxidative stress indicators and inflammatory factor levels are tested by antioxidant capacity test and ELISA test. The results show that adding 0.1% GTA to the diet of sows in early pregnancy can increase the average litter size by 12.17% (P=0.068), and has no significant effect on the average number of live piglets born per litter, stillbirth rate and average litter weight (P>0.05); adding 0.1% GTA can significantly increase the levels of CAT, GSH-Px and T-SOD in serum (P<0.05), and reduce the level of MDA (P>0.05); adding 0.1% GTA can significantly reduce the level of serum IL-6 (P<0.05).

[0016] (3) Adding GEA and GTA to the sow's basic diet has no adverse effects on the sow's growth and production. It is highly safe and can be used as a feed additive for a long time. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 This is a graph showing the effect of GEA on sow reproductive performance in Example 1, wherein A represents the effect of GEA on average litter birth weight; B represents the effect of GEA on average litter weight.

[0018] Figure 2 This is a diagram showing the effect of GEA on the mRNA transcription levels of genes related to piglet placental inflammation in Example 1, where * indicates significant differences between groups (P < 0.05).

[0019] Figure 3 1 is a comparison of the placental tissue morphology of piglets in the NC group and the E2 group in Example 1, wherein A is a comparison of the placental tissue morphology; B is a comparison of the number of placental blood vessels.

[0020] Figure 4 This figure shows the effects of dietary GTA supplementation at different levels on serum antioxidant indices in sows (n = 8) in Example 2. AE represents CAT, GSH-Px, T-SOD concentrations, total antioxidant capacity, and MDA levels, respectively. Values ​​are expressed as mean ± standard error; * indicates a significant difference (0.01 < P < 0.05), and ** indicates an extremely significant difference (P < 0.01).

[0021] Figure 5 This figure shows the effect of adding different levels of GTA to the diet on the levels of inflammatory cytokines in the serum of sows in Example 2 (n=8); wherein A is the IL-6 level; B is the TNF-α level; the values ​​are expressed as mean ± standard error; * indicates a significant difference (0.01 < P < 0.05), and ** indicates an extremely significant difference (P < 0.01). DETAILED DESCRIPTION

[0022] 1. Test materials

[0023] The grown-up dual-purpose sows and Large White pigs used in the experiment of the present invention were respectively sourced from a pig farm under Guizhou Fuzhiyuan Technology Group Co., Ltd. and a pig farm under Guizhou Wenshi Breeding Pig Technology Co., Ltd.

[0024] The gallnut ellagic acid (GEA, Dudukang 201) and gallnut tannin acid (GTA, Dudukang Duobao) used in the experiment were provided by Guiyang Beilong Biotechnology Co., Ltd. (purity ≥40%).

[0025] 2. Feeding design

[0026] GEA feeding for sows in late pregnancy:

[0027] One hundred healthy, late-gestation, two-parity, grown-up sows were randomly divided into four groups: control (NC), experimental group I (E1), experimental group II (E2), and experimental group III (E3), with 25 sows in each group. The NC group was fed a basal diet, while sows in experimental groups I, II, and III were fed a basal diet supplemented with 0.02%, 0.06%, and 0.1% GEA, respectively.

[0028] The pilot experiment lasted for 7 days, and the feeding period was from the 85th day of gestation to farrowing of the sows.

[0029] Fresh placental tissue samples were collected from piglets after natural delivery of sows (15 piglets were randomly collected from each group, covering different birth weights: low birth weight (<0.08 kg), normal birth weight (0.08 kg-1.5 kg), and high birth weight (>1.5 kg)).

[0030] GTA feeding for early gestation sows:

[0031] One hundred healthy early pregnant Large White sows with 2-6 parities were selected and randomly divided into 4 groups: CON, Ⅰ, Ⅱ and Ⅲ, with 25 sows in each group.

[0032] The CON group was fed a basal diet, while groups I, II, and III were fed a basal diet supplemented with 0.05%, 0.1%, and 0.2% GTA, respectively (the amount of GTA added was the mass percentage of the basal diet. For example, if the basal diet was 2.5 kg per day, the amount of GTA added per day in group I was 2500*0.05%=1.25 g).

[0033] The pre-experiment lasted 7 days, and the feeding period was from gestational day 7 to 30. After that, all sows were fed a basal diet. Serum samples were obtained by collecting blood from the jugular vein on gestational day 30 (8 sows were randomly selected from each group);

[0034] Among them, the serum sample was obtained: blood was collected using a coagulant tube, and it was allowed to stand for 2 to 3 hours. After the serum and plasma were separated, the serum in the coagulant tube was separated into a 2 ml centrifuge tube using a Pasteur pipette, and centrifuged at 3000 g for 15 minutes. The supernatant was aspirated and divided into 200 μL PCR tubes and labeled.

[0035] Example 1

[0036] 1. Placental total RNA extraction and cDNA synthesis

[0037] Total RNA from placental tissues of the NC and E2 groups was extracted using Trizol reagent (Novozymes Biotech Co., Ltd., Nanjing, China). The concentration and purity of RNA were determined using an ultramicrospectrophotometer. Qualified RNA (no contamination and OD260 / OD280 = 1.8-2.0) was reverse transcribed and synthesized into cDNA.

[0038] Subsequently, reverse transcription was performed according to the instructions of the reverse transcription kit (Thermo Fisher Scientific, USA) to synthesize the corresponding cDNA, and the reverse transcribed cDNA was stored in a -20°C refrigerator for future use.

[0039] 2. Primer information and real-time fluorescence quantitative PCR detection

[0040] The sequence information of the target genes was searched in the NCBI database, and primers for TRAF5, MMP3, TNF, IL-1β, IL-6, IL-10, and CXCL2 genes were designed using Primer 6.0 (see Table 1). β-actin was used as the internal reference gene. After specificity verification by BLAST, the primers were commissioned to Beijing Qingke Biotechnology Co., Ltd. for synthesis.

[0041] Table 1 Real-time fluorescence quantitative PCR primer sequence information

[0042]

[0043] Quantitative PCR reaction system (10 μL): SYBR Select Master Mix (5.0 μL), Forward primer (0.3 μL), Reverse primer (0.3 μL), cDNA (1.0 μL), ddH2O (3.4 μL).

[0044] The PCR reaction program was as follows: UDG enzyme activation at 50°C for 2 min, pre-denaturation at 95°C for 2 min, 40 PCR cycles (denaturation at 95°C for 15 s, annealing at 60°C for 15 s, and extension at 72°C for 1 min), and melting curve analysis (95°C for 15 s, 60°C for 1 min, and 95°C for 15 s).

[0045] There were 5 samples in each group and 4 biological replicates for each sample. -ΔΔCt The relative expression of genes was calculated.

[0046] 3. Observation of placental tissue morphology

[0047] The placenta tissues of 8 piglets in the NC group and the E2 group were randomly selected for preparation of paraffin sections, and the tissue morphology was observed after HE staining.

[0048] The paraffin embedding, sectioning and HE staining of placental tissue were commissioned to Wuhan Sevier Biotechnology Co., Ltd.

[0049] Using an upright microscope, two different fields of view were randomly selected for each placental tissue, and the overall tissue morphology and number of blood vessels of the placenta were observed at 40 times the field of view.

[0050] 4. Data statistics and analysis

[0051] All data were preliminarily organized using Excel and analyzed for significance using SPSS 27.0. The average litter weight of sows in late pregnancy, the average birth weight of piglets, and piglet uniformity were analyzed using one-way ANOVA, with Duncan's method used for multiple comparisons. The chi-square test was used for weak litter rate. The relative mRNA expression levels and placental vascular number were analyzed using the T-test.

[0052] The values ​​are expressed as mean ± SD, and P < 0.05 indicated significant differences. The graphing software was GraphPad Prism 9.5.

[0053] The results are as follows Figure 1-3 As shown in Table 2-3:

[0054] Effects of different levels of GEA on sow reproductive performance: The results showed that (Table 2, Table 3, Figure 1 Compared with the NC group, the addition of 0.06% GEA in late gestation showed a marginally significant trend toward increased piglet birth weight and litter weight (P=0.069, P=0.085), with piglet birth weight and litter weight increasing by 8.15% and 9.72%, respectively. Piglet uniformity did not differ significantly among the groups (P>0.05). The E2 group had a significantly lower weak piglet rate than the NC group (P<0.05). The E2 group had the highest weak piglet rate, IUGR rate, average litter weight, and average piglet birth weight among all groups.

[0055] Table 2 Effects of dietary GEA on reproductive performance of sows

[0056]

[0057] Note: *GEA stands for gallic acid. Data in the same row marked with the same lowercase letters indicate no significant difference (P>0.05); data marked with different lowercase letters indicate significant difference (P<0.05).

[0058] Table 3 Effect of adding GEA to the diet on the rate of weak piglets

[0059]

[0060] Effects of feeding GEA in late pregnancy on the level of placental inflammation in piglets: qPCR results of placental tissue showed that ( Figure 2 The addition of 0.06% GEA significantly increased the relative mRNA expression of anti-inflammatory factors TRAF5 and IL-10 in the placenta (P < 0.05), and decreased the relative mRNA expression of pro-inflammatory factors TNF and CXCL2 (P < 0.05). There were no significant differences in the other inflammatory factor-related genes between the NC group and the E2 group (P > 0.05).

[0061] Effects of feeding GEA in late pregnancy on the placental tissue morphology of piglets: Figure 3 It can be seen that the villi of the E2 group placenta were better developed than those of the NC group, and the number of blood vessels was significantly higher than that of the large NC group (P < 0.05).

[0062] 0, 0.02%, 0.06% and 0.1% GEA were added to the basal diet of growing dual-purpose sows respectively, and the feeding period was from the 85th day of gestation to the sows' farrowing.

[0063] The results showed that the addition of 0.06% GEA to the diet of sows in late pregnancy could increase the birth weight and litter weight of piglets by 8.15% and 9.72%, respectively (P=0.069, P=0.085), and significantly reduce the rate of weak piglets by 9.83% (P<0.05). (2) The results of qPCR analysis of placental tissue showed that the addition of 0.06% GEA significantly increased the relative mRNA expression of TRAF5 and IL-10 in the placenta (P<0.05), and decreased the relative mRNA expression of TNF and CXCL2 (P<0.05). (3) The results of tissue morphology comparison showed that the addition of 0.06% GEA significantly increased the number of blood vessels per unit length in the placenta (P<0.05).

[0064] Example 2

[0065] Effects of feeding GTA in early pregnancy on serum antioxidant indices and serum cytokine levels in sows

[0066] 1. Detection of serum antioxidant and inflammatory factor indicators

[0067] According to the instructions of the detection kits (Nanjing Jiancheng Bioengineering Institute, Nanjing, China), the ammonium molybdate method, colorimetric method, hydroxylamine method, iron ion reduction / antioxidant capacity method, and thiobarbituric acid method were used to detect catalase (CAT), glutathione peroxidase (GSH-Px), total superoxide dismutase (T-SOD) activities, total antioxidant capacity (T-AOC), and malondialdehyde (MDA) levels in sow serum.

[0068] In addition, the concentrations of interleukin-6 (IL6) and porcine tumor necrosis factor α (TNF-α) in sow serum were determined according to the instructions of the ELISA kit (Jiangsu Jingmei Biotechnology Co., Ltd., Jiangsu, China).

[0069] 2. Data statistics and analysis

[0070] The significance of differences in oxidative stress indices and inflammatory factor levels was analyzed using the one-way ANOVA method of SPSS 27.0; the average litter size, average number of live born per litter, fetal death rate, average litter weight, and body mass index were statistically analyzed using the two-way ANOVA method for differences, and multiple comparisons were performed using the Duncan method. The numerical values were expressed as mean ± standard deviation (mean ± SD). A difference was considered significant when 0.01 < P < 0.05, and extremely significant when P < 0.01.

[0071] Effect of GTA on the reproductive performance of sows in early pregnancy

[0072] Effect of different levels of GTA on the reproductive performance of sows: In this experiment, due to reasons such as estrus return, non-pregnancy, abortion, and death, only 20 litters, 19 litters, 19 litters, and 18 litters of sows in the CON group, Group I, Group II, and Group III gave birth, respectively.

[0073] The comparative analysis results of the reproductive performance indicators of these four groups of sows showed (see Table 4) that compared with the CON group, adding GTA in early pregnancy had a tendency to increase the average litter size of sows, reaching a marginal significance level (P = 0.068), and the highest average litter size (17.05 piglets) was obtained with a 0.1% GTA addition; there were no significant differences in the average number of live born per litter, fetal death rate, and average litter weight among the groups (P > 0.05). The BMI of piglets in Group I was not significantly different from that in the CON group (P > 0.05), but was significantly higher than that in Group II and Group III (P < 0.05).

[0074] In addition, two-way ANOVA showed that there was no significant interaction between GTA and parity on the average litter size, average number of live born per litter, fetal death rate, and average litter weight (P > 0.05), but there was a significant interaction effect on BMI (P < 0.05).

[0075] Table 4 Effect of adding GTA in the diet on the reproductive performance of sows

[0076]

[0077] Note: *GTA represents gallotannic acid, #BMI represents body mass index; if the data in the same row are marked with the same lowercase letter, the difference is not significant (P > 0.05); if marked with different lowercase letters, the difference is significant (P < 0.05).

[0078] Effect of feeding GTA in early pregnancy on the serum antioxidant indices of sows: By Figure 4The results showed that the concentration of CAT in the serum of sows in group I was extremely significantly lower than that in group CON (P < 0.01), while the concentration of CAT in the serum of sows in groups II and III was extremely significantly higher than that in group CON (P < 0.01); the concentration of GSH-Px in the serum of sows in groups II and III was extremely significantly higher than that in group CON (P < 0.01), while there was no significant difference between group I and group CON (P > 0.05); the concentration of T-SOD in the serum of sows in groups I and II was significantly higher than that in group CON (P < 0.05), while the concentration of T-SOD in the serum of sows in group III was extremely significantly higher than that in group CON (P < 0.01); the concentration of T-AOC in the serum of sows in groups I and III was extremely significantly higher than that in group CON (P < 0.01), while there was no significant difference between group II and group CON (P > 0.05); the concentration of MDA in the serum of sows in group I was significantly lower than that in group CON (0.01 < P < 0.05), while there was no significant difference between groups II and III and group CON (P > 0.05).

[0079] Effects of feeding GTA in early pregnancy on serum cytokine levels in sows: Figure 5 The results showed that the serum IL-6 level of sows in Group I was significantly lower than that in Group CON (P < 0.01), while the serum IL-6 level of sows in Groups II and III was significantly lower than that in Group CON (P < 0.05). In addition, the serum TNF-α level of sows in Group I was significantly lower than that in Group CON (P < 0.01), while the serum TNF-α level of sows in Group III was significantly higher than that in Group CON (P < 0.01).

[0080] GTA was added to the basal diet of Large White sows at 0, 0.05%, 0.1% and 0.2% respectively, and the feeding period was from the 7th to the 30th day of gestation.

[0081] The results showed that the addition of 0.1% GTA to the diet of sows in early pregnancy could increase the average litter size by 12.17% (P=0.068), but had no significant effect on the average number of live piglets born per litter, the stillbirth rate, and the average litter weight (P>0.05). (2) The results of serum antioxidant level test showed that the addition of 0.1% GTA could significantly increase the levels of CAT, GSH-Px, and T-SOD (P<0.05) and reduce the level of MDA (P>0.05). (3) The results of serum inflammatory factor level test showed that the addition of 0.1% GTA could significantly reduce the level of IL-6 (P>0.05).

[0082] In the above-mentioned Examples 1 and 2, the application results show that the ellagic acid feed additive and the tannic acid feed additive of the present invention have excellent effects on regulating the level of inflammatory factors in sows, alleviating the body's oxidative stress, improving feed digestion and utilization, and improving reproductive performance.

[0083] The embodiments described above are merely descriptions of preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Without departing from the spirit of the present invention, various modifications and improvements made to the technical solutions of the present invention by persons skilled in the art should fall within the scope of protection defined by the claims of the present invention.

Claims

1. A feed additive for pregnant sows, characterized in that: The feed additive for pregnant sows is a gallnut extract; Among them, the gallnut extract is ellagic acid and tannic acid.

2. A feed additive comprising ellagic acid, characterized in that: The dosage of the ellagic acid is 0.02%-0.1% of the basic feed.

3. A feed additive comprising tannic acid, characterized in that The dosage of the tannic acid is 0.05%-0.2% of the basic feed.

4. A production feed for pregnant sows, characterized in that: The feed contains the feed additive according to claim 2 or 3.

5. Use of the feed additive containing ellagic acid as claimed in claim 2 in preparing feed for pregnant sows.

6. Use of the feed additive containing tannic acid as claimed in claim 3 in preparing feed for pregnant sows.

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