Nutritional additive for livestock and application

Abstract

The invention discloses a nutritional additive for livestock, which is prepared from thiamine and nicotinamide in a mass ratio of (1-2): (1-2), and is a brand-new nutritional additive capable of effectively relieving the industrial problems of sheep breast immunity, oxidative damage, lactation performance reduction and the like caused by heat stress. Under the optimized additive amount, by applying the nutritional additive, the functions of mammary gland cells can be remarkably improved, and the milk yield and the content of milk components such as milk fat, milk protein and lactose can be increased; according to the nutritional additive, a safe and stable daily ration formula can be established, and the residual risk of traditional chemical drugs or excessive vitamins is avoided.

Description

Technical Field

[0001] This invention relates to livestock breeding technology, and more particularly to a nutritional additive for livestock and its application. Background Technology

[0002] Heat stress is a significant environmental factor limiting the production performance of ruminants, especially in subtropical and temperate regions during the summer when high temperature and humidity exacerbate its effects. When the ambient temperature exceeds the ruminant's comfort zone, the animal will attempt to maintain body temperature balance through physiological responses such as increasing respiratory rate and reducing feed intake, leading to energy metabolism disorders and damage to multiple physiological functions.

[0003] As an important category of ruminants, sheep experience a significant decrease in feed intake and inhibition of nutrient uptake and utilization by the mammary glands during lactation due to heat stress. This leads to a decline in milk production and a decrease in milk quality indicators such as milk fat percentage and milk protein percentage. Furthermore, heat stress can induce oxidative stress and immunosuppression, increasing the risk of mastitis and further affecting the sustainability of lactation and overall health.

[0004] To mitigate the adverse effects of heat stress, current methods primarily employ physical cooling and feeding management strategies, such as setting up shade nets, implementing spray cooling, and adjusting feeding times. However, these methods are often costly and complex to operate, making them difficult to promote and apply in large-scale farming. Therefore, nutritional intervention has become a research hotspot, including adding antioxidants such as electrolytes, vitamin C, vitamin E, and organic chromium to the diet, as well as vasodilators such as niacin (nicotinic acid) and nicotinamide to promote heat dissipation. However, long-term use of certain additives, such as niacin, may increase the risk of rumen acidosis; while yeast cultures help stabilize rumen pH, their effect on improving the negative energy balance caused by heat stress is limited. Traditional Chinese medicine compound additives exert anti-stress effects through multiple targets and pathways, but they suffer from limitations such as complex composition, high cost, and unclear mechanisms of action. While existing regulatory methods can alleviate heat stress responses to some extent, efficient, stable solutions suitable for large-scale production are still lacking. Summary of the Invention

[0005] Purpose of the invention: The purpose of this invention is to provide a nutritional additive that can effectively improve the function of mammary gland tissue in livestock, especially the lactation performance of heat-stressed ruminants. The second purpose is to provide the application of this nutritional additive for livestock.

[0006] Technical solution: The animal nutritional additive of the present invention contains thiamine and nicotinamide in a mass ratio of 1-2:1-2.

[0007] Preferably, the mass ratio of thiamine to nicotinamide is 1:1-2.

[0008] The application of the animal nutritional additive described in this invention in improving the function of mammary gland tissue in livestock.

[0009] Preferably, the application is used to improve the function of mammary gland tissue in heat-stressed livestock.

[0010] Preferably, the application is for improving the lactation performance of heat-stressed livestock.

[0011] Preferably, the livestock is a ruminant.

[0012] Preferably, the ruminant livestock is a sheep.

[0013] Preferably, the lactation performance includes milk yield and the content of nutrients in the milk.

[0014] Preferably, the application involves adding 200-300 mg of animal nutritional additive per kilogram of dry matter intake.

[0015] Preferably, the animal nutritional additive added per kilogram of dry matter intake contains 70-130 mg of thiamine and 120-180 mg of nicotinamide.

[0016] Beneficial effects: Compared with the prior art, the present invention has the following significant advantages: 1. By using thiamine and nicotinamide in combination, a novel nutritional additive is provided that can effectively alleviate industrial problems such as decreased milk production, oxidative damage, and reduced lactation performance of sheep mammary glands caused by heat stress; 2. By optimizing the dosage, the application of this nutritional additive can significantly improve mammary gland cell function and increase milk yield and the content of milk components such as milk fat, milk protein, and lactose; 3. A safe and stable diet formula can be established based on this nutritional additive, avoiding the risk of residues from traditional chemical drugs or excessive vitamins. Detailed Implementation

[0017] The technical solution of the present invention will be further described below.

[0018] Example 1: Heat stress treatment of lactating Hu sheep ewes

[0019] Forty lactating ewes were selected, with the following inclusion criteria: parity 1-2; lactation days 14±1 days; milk yield 1.25±0.08 liters / day; body weight 52.6±2.7 kg; and body condition score 2.62±0.12. All ewes underwent a 7-day acclimatization period under conditions of 22.8±0.39℃, relative humidity 45.2±0.06%, and a temperature and humidity index (THI) of 72±1.57. The THI calculation formula is as follows:

[0020] THI=(1.8T+32)-[(0.55-0.0055RH)×(1.8T-26.8)],

[0021] Where T is the air temperature (°C) and RH is the relative humidity (%).

[0022] After the acclimatization period, the ewes were randomly assigned to the following five groups for 21 days: heat-neutral diet without supplementation (CON), heat stress diet without supplementation (HS), heat stress diet supplemented with thiamine (THS), heat stress diet supplemented with nicotinamide (NHS), and heat stress diet supplemented with thiamine + nicotinamide (TNHS), with 8 ewes in each group.

[0023] For the thermoneutral group, the daily ambient temperature was maintained at 22.5±0.5℃, and the THI was below 80. For the heat stress group, the temperature rose from 27℃ to 37℃ at 0600 and then dropped from 37℃ to 27℃ at 1800, with the THI exceeding 80. The heat stress supplemented diet thiamine group was given 100mg thiamine per kilogram of dry matter intake, the heat stress supplemented diet nicotinamide group was given 150mg nicotinamide per kilogram of dry matter intake, and the heat stress supplemented diet thiamine + nicotinamide group was given both 100mg thiamine and 150mg nicotinamide per kilogram of dry matter intake.

[0024] The basic diet formulas for all groups of sheep are shown in Table 1 below.

[0025] Table 1. Basal Diet Formula

[0026]

[0027]

[0028] Of these, 4% of the cow and sheep premix was purchased from Xinxiang Huachu Trade Co., Ltd.

[0029] The nutritional composition of the basic diet is shown in Table 2 below.

[0030] Table 2 Nutritional composition of basal diet

[0031] name content Metabolizable energy (ME) 9.12 MJ / kg DM Crude protein (CP) 10.66% of DM Neutral detergent fiber (NDF) 41.28% of DM Acid detergent fiber (AD) 22.19% of DM calcium 0.88% of DM phosphorus 0.51% of DM

[0032] During the 21-day experimental period, each sheep was housed in a single pen within a 2m x 1.8m enclosure. They were fed their daily rations at 07:00 and 17:00 daily, with free access to water and feed. The daily feed intake was managed to maintain a 5% uneaten feed level.

[0033] The statistical results of daily temperature, relative humidity, and temperature and humidity index of the heat-neutral treatment group and the heat stress treatment group during the experimental period are shown in Table 3 below.

[0034] Table 3. Statistical results of ambient temperature, relative humidity, and temperature-humidity index during the experiment.

[0035]

[0036] Throughout the experiment, the environmental THI of the heat stress treatment group consistently exceeded 80, significantly higher than that of the heat neutral treatment group, indicating that the heat stress treatment was successful.

[0037] Example 2: Collection and analysis of milk samples from lactating Hu sheep ewes

[0038] During the experiment in Example 1, manual milking was performed at 08:00 and 18:00 daily. The daily milk production was measured using an electronic balance. Milk samples from each ewe were mixed in a 6:4 ratio between the morning (08:00) and evening (18:00) milk. The statistical results of the daily dry matter intake (DMI) and milk production of each group of Hu sheep are shown in Table 4.

[0039] Table 4. Statistical Results of Dry Matter Intake and Milk Production

[0040] CON HS THS NHS TNHS SEM P-value DMI, kg / d <![CDATA[2.19 a ]]> <![CDATA[1.25 b ]]> <![CDATA[1.28 b ]]> <![CDATA[1.24 b ]]> <![CDATA[1.27 b ]]> 0.17 0.008 Milk production, kg / d <![CDATA[1.36 a ]]> <![CDATA[0.72 c ]]> <![CDATA[1.05 b ]]> <![CDATA[1.01 b ]]> <![CDATA[1.33 a ]]> 0.08 0.009

[0041] Wherein, SEM represents the pooled standard error, a, b, and c indicate significant differences between groups (P<0.05), and the same letter indicates no statistical difference (P>0.05).

[0042] 1. Analysis of milk fat, milk protein and lactose content

[0043] Milk samples were taken from each sheep on day 21 of the experimental period. The contents of milk fat, milk protein, and lactose were analyzed using mid-infrared spectroscopy, and the results are shown in Table 5.

[0044] Table 5 Results of determination of milk fat, lactose and milk protein content

[0045] Milk composition, % CON HS THS NHS TNHS SEM P-value milk fat <![CDATA[5.89 a ]]> 4.18d <![CDATA[5.13 c ]]> <![CDATA[5.11 c ]]> <![CDATA[5.44 b ]]> 0.15 0.016 lactose <![CDATA[5.34 a ]]> <![CDATA[4.02 c ]]> <![CDATA[4.74 b ]]> <![CDATA[4.41 b ]]> <![CDATA[5.31 a ]]> 0.10 0.013 milk protein <![CDATA[5.51 a ]]> <![CDATA[3.95 c ]]> <![CDATA[4.81 b ]]> <![CDATA[4.74 b ]]> <![CDATA[5.46 a ]]> 0.18 0.007

[0046] Wherein, SEM represents the pooled standard error, a, b, and c indicate significant differences between groups (P<0.05), and the same letter indicates no statistical difference (P>0.05).

[0047] Compared with the CON group, the HS group showed significantly lower DMI, milk yield, milk fat, lactose, and protein content (P<0.05). Except for DMI, the THS and NHS groups showed significantly higher values ​​for all of the above indicators than the HS group (P<0.05). Compared with the THS or NHS groups, except for DMI, the TNHS group showed significantly higher values ​​for all of the above indicators (P<0.05).

[0048] 2. Analysis of the fatty acid composition of milk fat

[0049] One liter of milk sample from each sheep on day 21 of the experimental period was taken as the test sample. Fat cake was prepared by centrifugation at 1643×g and 4℃ for 30 min. Milk fat was extracted using the Rhodes-Gottlieb method. Esterified fatty acids were methyl esterified according to the method described in GB5009.168-2016.

[0050] The detection was performed using an Agilent 7890B gas chromatograph equipped with a flame ionization detector (FID) and a high-polarity capillary column (100m × 0.25mm × 0.20μm) such as HP-88 or DB-Wax;

[0051] The chromatographic conditions were as follows: injection port temperature 250℃, split ratio 50:1; FID detector temperature 260℃; carrier gas was high-purity nitrogen (N2), constant flow mode, flow rate 1.0 mL / min; the column oven used a multi-stage temperature program: initial temperature 140℃ held for 5 min, increased to 190℃ at 4℃ / min held for 15 min, then increased to 230℃ at 3℃ / min held for 10 min; injection volume 1 μL.

[0052] After separation under these conditions, fatty acid methyl esters (FAME) were quantified using the area normalization method to obtain the relative percentage composition of each fatty acid in milk fat. The average percentage of fatty acids in the total milk fat of each sample was multiplied by 0.98885 as a correction factor for other milk fat components. The results of the fatty acid composition analysis of milk fat are shown in Table 6.

[0053] Table 6. Analysis of Fatty Acid Composition of Milk Fat

[0054]

[0055]

[0056] Wherein, SEM represents the pooled standard error, a, b, c, and d indicate significant differences between groups (P<0.05), and the same letter indicates no statistical difference (P>0.05).

[0057] The statistical results based on the fatty acid source are shown in Table 7.

[0058] Table 7 Statistical results categorized by fatty acid source

[0059] CON HS THS NHS TNHS SEM P-value fatty acids synthesized from scratch <![CDATA[12.11 a ]]> <![CDATA[6.08 c ]]> <![CDATA[8.95 b ]]> <![CDATA[9.01 b ]]> <![CDATA[11.98 a ]]> 1.08 0.007 Mixed fatty acids <![CDATA[3.49 a ]]> <![CDATA[1.57 d ]]> <![CDATA[2.51 c ]]> <![CDATA[2.33 c ]]> <![CDATA[2.92 b ]]> 0.18 0.012 Preformed fatty acids <![CDATA[8.56 c ]]> <![CDATA[15.94 a ]]> <![CDATA[11.15 b ]]> <![CDATA[11.46 b ]]> <![CDATA[8.83 c ]]> 0.26 0.009

[0060] Among them, de novo synthesized fatty acids (DNFA) refer to fatty acids (fatty acids with less than 16 Cs) synthesized de novo in the mammary gland, mixed fatty acids (MFA) are the sum of palmitic acid (C16:0) and palmitoleic acid (C16:1), and preformed fatty acids (preformed FA) refer to fatty acids (fatty acids with more than 16 Cs) extracted from the blood. a, b, c, and d indicate significant differences between groups (P<0.05), and the same letter indicates no statistical difference (P>0.05).

[0061] Compared with CON, the contents of DN FA, MFA, C4:0, C6:0, C8:0, C10:0, C12:0, C14:0, C14:1 and C16:0 in the HS group milk were significantly lower (P<0.05), while the yields of preformed FA, C18:0, C18:1n9c, C18:2n6c, C18:3 and C20:3 were significantly higher (P<0.05). Compared with the HS group, the contents of DN FA, MFA, C4:0, C6:0, C8:0, C10:0, C12:0, C14:0, C14:1 and C16:0 in the milk of the THS and NHS groups were significantly increased (P<0.05), while the contents of preformed FA, C18:0, C18:1n9c, C18:2n6c, C18:3 and C20:3 were significantly decreased (P<0.05). Compared with the THS or NHS groups, the TNHS group showed significantly higher levels of DN FA, MFA, C4:0, C6:0, C8:0, C10:0, C12:0, C14:0, C14:1, and C16:0 (P<0.05), while the TNHS group showed significantly lower levels of preformed FA, C18:0, C18:1n9c, C18:2n6c, C18:3, and C20:3 (P<0.05).

[0062] 3. Determination of milk amino acid content

[0063] 100 mg of milk from each sheep on day 21 of the experimental period was taken as the test sample and transferred to a hydrolysis bottle. It was mixed with 5 mL of hydrolysis solution containing 6 M HCl, 5% mercaptoacetic acid, and 0.1% phenol. The mixture was sealed and hydrolyzed in a hot air oven at 110 °C for 18 h. Subsequently, it was centrifuged at 10000 × g for 10 min, and 100 μL of the supernatant was added to 1 M sodium carbonate until pH = 7 to obtain the neutralized solution.

[0064] Transfer 25 μL of the neutralization solution to a 2 mL GC glass vial, add 50 μL of 200 nmol / mL ortholeucine as an internal standard, and vacuum dry at 60 °C for 1 hour. Then add 50 μL of dichloromethane and dry again for 30 min to remove residual moisture. Finally, add a mixture of 50 μL acetonitrile, 80 μL of N-tert-butyldimethylsilyl-N-methyltrifluoroacetamide (MTBSTFA), and 50 μL of 1% tert-butyldimethylchlorosilane. Seal the sample vial and incubate in a hot air oven at 100 °C for 4 hours to obtain the test sample.

[0065] A 1 μL sample was analyzed using an Agilent GC 7890B / MSD 7000D gas chromatography-mass spectrometry system. The chromatographic conditions were as follows: helium was used as the carrier gas at a flow rate of 1.4 mL / min; the column oven temperature was increased from 130℃ to 190℃ at a rate of 6℃ / min, then increased to 230℃ at a rate of 0.5℃ / s and held for 5 min, and finally increased to 325℃ at a rate of 1℃ / s and held for 6 min; the injection port, transfer line, electron impact ion source, and quadrupole temperatures were maintained at 280℃, 325℃, 240℃, and 180℃, respectively. The results of the determination of lactic acid content are shown in Table 8.

[0066] Table 8. Results of milk amino acid content determination (mg / L)

[0067] CON HS THS NHS TNHS SEM P-value Arginine <![CDATA[12.16 a ]]> <![CDATA[7.01 c ]]> <![CDATA[9.87 b ]]> <![CDATA[9.17 b ]]> <![CDATA[11.98 a ]]> 1.27 0.008 Histidine <![CDATA[9.13 a ]]> <![CDATA[6.56 b ]]> <![CDATA[6.69 b ]]> <![CDATA[6.52 b ]]> <![CDATA[9.01 a ]]> 0.93 0.025 Isoleucine <![CDATA[14.57 a ]]> <![CDATA[8.94 b ]]> <![CDATA[9.02 b ]]> <![CDATA[8.97 b ]]> <![CDATA[9.08 b ]]> 1.13 0.011 Leucine <![CDATA[31.25 a ]]> <![CDATA[15.27 c ]]> <![CDATA[22.97 b ]]> <![CDATA[20.87 b ]]> <![CDATA[29.37 a ]]> 1.76 0.007 Lysine 8.19 8.21 8.27 8.18 8.26 0.97 0.105 Methionine <![CDATA[27.84 a ]]> <![CDATA[19.36 c ]]> <![CDATA[19.27 c ]]> <![CDATA[19.52 c ]]> <![CDATA[23.17 b ]]> 1.18 0.013 Phenylalanine 21.25 22.17 21.76 22.01 21.62 1.47 0.223 threonine 17.27 16.93 17.08 17.18 16.97 1.86 0.119 Tryptophan <![CDATA[14.26 a ]]> <![CDATA[9.89 b ]]> <![CDATA[9.97 b ]]> <![CDATA[10.01 b ]]> <![CDATA[14.17 a ]]> 1.03 0.008 Valine <![CDATA[23.23 a ]]> <![CDATA[12.26 d ]]> <![CDATA[16.27 c ]]> <![CDATA[15.97 c ]]> <![CDATA[19.28 b ]]> 1.56 0.017 Cysteine 3.15 3.11 3.23 3.18 3.27 0.16 0.125 alanine <![CDATA[12.36 a ]]> <![CDATA[6.48 c ]]> <![CDATA[9.27 b ]]> <![CDATA[9.31 b ]]> <![CDATA[12.27 a ]]> 1.08 0.007 Aspartic acid <![CDATA[29.16 a ]]> <![CDATA[16.25 c ]]> <![CDATA[21.67 b ]]> <![CDATA[20.89 b ]]> <![CDATA[28.78 a ]]> 2.13 0.009 glutamic acid <![CDATA[76.26 a ]]> <![CDATA[47.27 c ]]> <![CDATA[62.16 b ]]> <![CDATA[63.17 b ]]> <![CDATA[76.12 a ]]> 1.62 0.011 glutamine <![CDATA[52.13 a ]]> <![CDATA[30.27 d ]]> <![CDATA[41.26 c ]]> <![CDATA[40.12 c ]]> <![CDATA[45.28 b ]]> 1.18 0.010 ornithine <![CDATA[8.89 a ]]> <![CDATA[4.15 d ]]> <![CDATA[6.13 c ]]> <![CDATA[6.01 c ]]> <![CDATA[7.27 b ]]> 0.19 0.008 proline <![CDATA[31.27 a ]]> <![CDATA[17.18 c ]]> <![CDATA[25.83 b ]]> <![CDATA[24.99 b ]]> <![CDATA[31.18 a ]]> 1.86 0.007

[0068] Wherein, SEM represents the pooled standard error, a, b, c, and d indicate significant differences between groups (P<0.05), and the same letter indicates no statistical difference (P>0.05).

[0069] Compared with the CON group, the HS group showed significantly lower levels of arginine, histidine, isoleucine, leucine, methionine, tryptophan, valine, alanine, aspartic acid, glutamic acid, glutamine, ornithine, and proline in milk (P<0.05). Compared with the HS group, these indicators were significantly increased in the THS and NHS groups (P<0.05), while the levels of lysine, phenylalanine, threonine, and cysteine ​​in milk showed no significant differences among the three groups (P>0.05). Compared with the THS or NHS groups, the TNHS group showed significantly increased levels of arginine, histidine, isoleucine, leucine, methionine, tryptophan, valine, alanine, aspartic acid, glutamic acid, glutamine, ornithine, and proline in milk (P<0.05).

[0070] Example 3: Analysis of mammary gland tissue in lactating Hu sheep ewes

[0071] At 08:00 on day 21 of the experiment in Example 1, blood samples were collected from the mammary vein using a 5 ml vacuum blood collection tube containing sodium heparin. The blood samples were centrifuged at 3000×g for 15 min at 4°C to obtain plasma for subsequent analysis and detection.

[0072] Subsequently, all sheep were euthanized and mammary gland tissue was collected, which was then cut into 1cm pieces. 3 Small pieces were rinsed with physiological saline to remove surface milk and blood, then flash-frozen in liquid nitrogen and collected in 5 ml cryovials for subsequent analysis and detection.

[0073] 1. Antioxidant activity assay

[0074] 0.1 g of breast tissue was homogenized and then centrifuged at 15000 × g for 15 minutes at 4 °C in 1 mL of lysis buffer containing 50 mM Tris-HCl (pH 7.4), 150 mM NaCl, 1 mM EDTA, 10% glycerol (v / v), and 0.5% Triton X-100. The supernatant was collected for the detection of oxidative stress-related markers, antioxidant enzyme activity, and triglycerides.

[0075] Using Pierce TM The BCA protein assay kit is used to detect the protein concentration in homogenized breast tissue.

[0076] The levels of triglycerides, malondialdehyde (MDA), total antioxidant capacity (T-AOC), and the activities of superoxide dismutase (SOD) and glutathione peroxidase (GSH-PX) in mammary venous blood and homogenized mammary tissue were measured using commercial ELISA kits (purchased from Nanjing Jiancheng Biotechnology Institute or Shanghai Chutai Biotechnology Co., Ltd.).

[0077] The results of the heat stress-related indicators are shown in Table 9-10 below.

[0078] Table 9. Results of measurements of heat stress-related indicators in mammary venous blood.

[0079]

[0080]

[0081] Table 10 Results of Measurement of Heat Stress-Related Indicators in Breast Tissue

[0082]

[0083] Wherein, SEM represents the pooled standard error, a, b, c, and d indicate significant differences between groups (P<0.05), and the same letter indicates no statistical difference (P>0.05).

[0084] Compared with the CON group, the HS group showed significantly decreased T-AOC, SOD, GSH-PX activities and triglyceride levels in breast venous blood and breast tissue (P<0.05), while MDA activity was significantly increased (P<0.05). Compared with the HS group, the THS and NHS groups showed significantly increased T-AOC, SOD, GSH-PX activities and triglyceride levels in breast venous blood and breast tissue (P<0.05), while MDA activity was significantly decreased (P<0.05). Compared with the THS or NHS group, the TNHS group showed significantly increased T-AOC, SOD, GSH-PX activities and triglyceride levels in breast venous blood and breast tissue (P<0.05), while MDA activity was significantly decreased (P<0.05).

[0085] 2. Cytokine assay

[0086] The concentrations of tumor necrosis factor-α (purchased from R&D Systems), interleukin-1 (purchased from BioSource / MED Probe), and interleukin-6 (purchased from R&D Systems) were determined using commercial kits.

[0087] The absorbance was measured at 450 nm using an ELISA reader. The results are expressed as ng / g protein in breast tissue and pg / mL in breast venous blood.

[0088] The results of the cytokine assay are shown in Tables 11-12 below.

[0089] Table 11 Results of cytokine measurement in breast venous blood (pg / mL)

[0090] CON HS THS NHS TNHS SEM P-value TNF-α <![CDATA[114.17 c ]]> <![CDATA[163.28 a ]]> <![CDATA[132.56 b ]]> <![CDATA[136.12 b ]]> <![CDATA[115.39 c ]]> 7.82 0.007 IL-6 <![CDATA[102.21 c ]]> <![CDATA[147.27 a ]]> <![CDATA[126.37 b ]]> <![CDATA[121.27 b ]]> <![CDATA[106.21 c ]]> 6.18 0.014 IL-1β <![CDATA[168.28 c ]]> <![CDATA[212.37 a ]]> <![CDATA[189.23 b ]]> <![CDATA[190.21 b ]]> <![CDATA[170.26 c ]]> 11.21 0.009

[0091] Table 12 Results of cytokine assay in breast tissue (ng / g)

[0092]

[0093]

[0094] Wherein, SEM represents the pooled standard error, a, b, and c indicate significant differences between groups (P<0.05), and the same letter indicates no statistical difference (P>0.05).

[0095] Compared with the CON group, the concentrations of TNF-α, IL-6, and IL-1β cytokines in mammary venous blood and breast tissue were significantly increased in the HS group (P<0.05). Compared with the HS group, the concentrations of TNF-α, IL-6, and IL-1β cytokines in mammary venous blood and breast tissue were significantly decreased in the THS and NHS groups (P<0.05). Compared with the THS or NHS groups, the concentrations of TNF-α, IL-6, and IL-1β cytokines in mammary venous blood and breast tissue were significantly decreased in the TNHS group (P<0.05).

[0096] 3. Analysis of relative mRNA expression levels of related genes

[0097] Breast tissue was ground into powder in liquid nitrogen. 50 mg of breast tissue was used to extract total RNA using RNAiso Plus. 1000 ng of the extracted total RNA was then used to synthesize cDNA using the Takara PrimeScript RT Master Mix (Perfect Real Time Kit).

[0098] Primers were designed for the target gene using Premier 6.0 software and synthesized by Shanghai Sangon Biotech Co., Ltd. The primer sequences are shown in Table 9 below.

[0099] Table 13 Target gene primer sequences

[0100]

[0101]

[0102] Real-time quantitative PCR was performed using the Takara SYBR Premix Ex Taq Kit. The reaction system was prepared according to the kit instructions, and the reaction conditions are shown in Table 14 below.

[0103] Table 14 Real-time Quantitative PCR Reaction Conditions

[0104]

[0105] Three replicate reactions were used to obtain the mean Ct value for each gene, and 2... -ΔΔCT The relative transcriptional levels of genes were calculated, with β-actin used as an internal reference gene for transcriptional abundance normalization. The results are shown in Tables 15-20.

[0106] Table 15 Relative expression levels of genes related to barrier function

[0107] CON HS THS NHS TNHS SEM P-value ZO-1 <![CDATA[1.00 a ]]> <![CDATA[0.63 b ]]> <![CDATA[0.92 a ]]> <![CDATA[0.89 a ]]> <![CDATA[0.94 a ]]> 0.07 0.006 Occludin <![CDATA[1.00 a ]]> <![CDATA[0.46 c ]]> <![CDATA[0.78 b ]]> <![CDATA[0.74 b ]]> <![CDATA[0.96 a ]]> 0.05 0.009 Claudin-1 <![CDATA[1.00 a ]]> <![CDATA[0.78 b ]]> <![CDATA[0.89 ab ]]> <![CDATA[0.87 ab ]]> <![CDATA[0.98 a ]]> 0.08 0.014

[0108] Table 16 Relative expression levels of antioxidant-related genes

[0109] CON HS THS NHS TNHS SEM P-value CAT <![CDATA[1.00 a ]]> <![CDATA[0.37 c ]]> <![CDATA[0.66 b ]]> <![CDATA[0.68 b ]]> <![CDATA[0.99 a ]]> 0.07 0.008 GPX1 <![CDATA[1.00 a ]]> <![CDATA[0.59 c ]]> <![CDATA[0.78 b ]]> <![CDATA[0.72 b ]]> <![CDATA[0.93 a ]]> 0.06 0.014 SOD2 <![CDATA[1.00 a ]]> <![CDATA[0.77 b ]]> <![CDATA[0.93 a ]]> <![CDATA[0.81 b ]]> <![CDATA[0.96 a ]]> 0.11 0.009 Nrf2 <![CDATA[1.00 a ]]> <![CDATA[0.55 c ]]> <![CDATA[0.81 b ]]> <![CDATA[0.77 b ]]> <![CDATA[0.97 a ]]> 0.08 0.007 HO-1 <![CDATA[1.00 a ]]> <![CDATA[0.47 c ]]> <![CDATA[0.79 b ]]> <![CDATA[0.72 b ]]> <![CDATA[0.93 a ]]> 0.06 0.019 NQO1 <![CDATA[1.00 a ]]> <![CDATA[0.66 b ]]> <![CDATA[0.93 a ]]> <![CDATA[0.89 a ]]> <![CDATA[0.96 a ]]> 0.10 0.005

[0110] Table 17 Relative expression levels of genes related to inflammation response

[0111] CON HS THS NHS TNHS SEM P-value TLR-4 <![CDATA[1.00 c ]]> <![CDATA[2.23 a ]]> <![CDATA[1.57 b ]]> <![CDATA[1.62 b ]]> <![CDATA[1.12 c ]]> 0.28 0.009 NF-κB <![CDATA[1.00 b ]]> <![CDATA[1.43 a ]]> <![CDATA[1.17 ab ]]> <![CDATA[1.21 ab ]]> <![CDATA[1.03 b ]]> 0.15 0.011 TNF-α <![CDATA[1.00 c ]]> <![CDATA[1.65 a ]]> <![CDATA[1.28 b ]]> <![CDATA[1.31 b ]]> <![CDATA[1.01 c ]]> 0.11 0.008 IL-1β 1.00 1.13 1.08 1.11 1.05 0.14 0.098 IL-6 <![CDATA[1.00 c ]]> <![CDATA[2.57 a ]]> <![CDATA[1.61 b ]]> <![CDATA[1.65 b ]]> <![CDATA[1.09 c ]]> 0.21 0.006

[0112] Table 18 Relative expression levels of lactose synthesis-related genes

[0113] CON HS THS NHS TNHS SEM P-value GLUT1 <![CDATA[1.00 a ]]> <![CDATA[0.36 c ]]> <![CDATA[0.79 b ]]> <![CDATA[0.74 b ]]> <![CDATA[0.97 a ]]> 0.07 0.013 GLUT3 <![CDATA[1.00 a ]]> <![CDATA[0.51 c ]]> <![CDATA[0.80 b ]]> <![CDATA[0.74 b ]]> <![CDATA[0.91 a ]]> 0.09 0.007 LALBα <![CDATA[1.00 a ]]> <![CDATA[0.29 d ]]> <![CDATA[0.68 c ]]> <![CDATA[0.61 c ]]> <![CDATA[0.80 b ]]> 0.06 0.011

[0114] Table 19 Relative expression levels of genes related to milk fat synthesis

[0115] CON HS THS NHS TNHS SEM P-value FASN <![CDATA[1.00 a ]]> <![CDATA[0.36 c ]]> <![CDATA[0.69 b ]]> <![CDATA[0.62 b ]]> <![CDATA[0.93 a ]]> 0.07 0.013 SCD <![CDATA[1.00 a ]]> <![CDATA[0.67 c ]]> <![CDATA[0.81 b ]]> <![CDATA[0.72 b ]]> <![CDATA[0.96 a ]]> 0.12 0.008 ACACα <![CDATA[1.00 a ]]> <![CDATA[0.56 c ]]> <![CDATA[0.77 b ]]> <![CDATA[0.71 b ]]> <![CDATA[0.98 a ]]> 0.09 0.006 PPARγ <![CDATA[1.00 a ]]> <![CDATA[0.47 c ]]> <![CDATA[0.73 b ]]> <![CDATA[0.68 b ]]> <![CDATA[0.91 a ]]> 0.08 0.009 PPARα <![CDATA[1.00 c ]]> <![CDATA[1.76 a ]]> <![CDATA[1.33 b ]]> <![CDATA[1.29 b ]]> <![CDATA[1.03 c ]]> 0.10 0.007

[0116] Table 20 Relative expression levels of genes related to milk protein synthesis

[0117] CON HS THS NHS TNHS SEM P-value SLC7A1 <![CDATA[1.00 a ]]> <![CDATA[0.58 b ]]> <![CDATA[0.89 a ]]> <![CDATA[0.82 a ]]> <![CDATA[0.96 a ]]> 0.11 0.009 SLC1A1 <![CDATA[1.00 a ]]> <![CDATA[0.39 d ]]> <![CDATA[0.68 c ]]> <![CDATA[0.61 c ]]> <![CDATA[0.83 b ]]> 0.06 0.007 SLC7A7 <![CDATA[1.00 a ]]> <![CDATA[0.79 b ]]> <![CDATA[0.88 ab ]]> <![CDATA[0.83 ab ]]> <![CDATA[0.94 a ]]> 0.08 0.011 SLC1A5 <![CDATA[1.00 a ]]> <![CDATA[0.44 c ]]> <![CDATA[0.69 b ]]> <![CDATA[0.63 b ]]> <![CDATA[0.92 a ]]> 0.06 0.009 SLC15A1 <![CDATA[1.00 a ]]> <![CDATA[0.37 c ]]> <![CDATA[0.72 b ]]> <![CDATA[0.73 b ]]> <![CDATA[0.96 a ]]> 0.07 0.005

[0118] Wherein, SEM represents the pooled standard error, a, b, and c indicate significant differences between groups (P<0.05), and the same letter indicates no statistical difference (P>0.05).

[0119] Compared with the CON group, the relative mRNA expression levels of barrier function-related genes, antioxidant-related genes, lactose synthesis-related genes, milk fat synthesis-related genes (except PPARα), and milk protein synthesis-related genes in the breast tissue of the HS group were significantly decreased (P<0.05), while the relative mRNA expression levels of inflammation response-related genes were significantly increased (P<0.05).

[0120] Compared with the HS group, the relative mRNA expression levels of barrier function-related genes, antioxidant-related genes, lactose synthesis-related genes, milk fat synthesis-related genes (excluding PPARα), and milk protein synthesis-related genes in breast tissue of the THS and NHS groups were significantly increased (P<0.05), while the relative mRNA expression levels of inflammation-related genes were significantly decreased (P<0.05). Compared with the THS or NHS groups, the relative mRNA expression levels of barrier function-related genes Occludin and Claudin-1, antioxidant-related genes, lactose synthesis-related genes, milk fat synthesis-related genes (excluding PPARα), and milk protein synthesis-related genes in breast tissue of the TNHS group were significantly increased (P<0.05), while the relative mRNA expression levels of inflammation-related genes were significantly decreased (P<0.05).

Claims

1. A nutritional additive for livestock, characterized in that, The animal nutritional additive contains thiamine and nicotinamide in a mass ratio of 1-2:1-2.

2. The animal nutritional additive according to claim 1, characterized in that, The mass ratio of thiamine to nicotinamide is 1:1-2.

3. The use of the animal nutritional additive as described in claim 1 or 2 in improving the function of mammary gland tissue in livestock.

4. The application according to claim 3, characterized in that, The application is for improving the function of mammary gland tissue in heat-stressed livestock.

5. The application according to claim 4, characterized in that, The application described is for improving the lactation performance of heat-stressed livestock.

6. The application according to claim 5, characterized in that, The livestock in question are ruminants.

7. The application according to claim 6, characterized in that, The ruminant livestock in question is sheep.

8. The application according to claim 5, characterized in that, The lactation performance includes milk yield and the content of nutrients in the milk.

9. The application according to claim 3, characterized in that, The application procedure involves adding 200-300 mg of animal nutritional additives per kilogram of dry matter intake.

10. The application according to claim 9, characterized in that, The animal nutritional additives added per kilogram of dry matter intake contain 70-130 mg of thiamine and 120-180 mg of nicotinamide.

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