Pharmaceutical composition for preventing or treating subclinical ketosis of dairy cow and application

Abstract

The invention discloses a pharmaceutical composition for preventing or treating subclinical ketosis of dairy cows and application, and belongs to the technical field of animal nutrition and feed science. The invention aims to solve the problem that the prevention and treatment effects are not uniform due to high incidence of ketosis of dairy cows in large-scale pastures. The pharmaceutical composition for preventing or treating the subclinical ketosis of the dairy cow comprises taurine and propylene glycol as active ingredients. The taurine and the propylene glycol are combined, so that the SCK positive rate is effectively reduced, and the energy metabolism, the liver function state, the oxidative stress and the production performance of the SCK dairy cow are improved.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of animal nutrition and feed science and technology, and particularly relates to a pharmaceutical composition for preventing or treating subclinical ketosis of dairy cows and application. BACKGROUND

[0002] In recent years, with the continuous increase of the single output of dairy cows in large-scale farms in China, subclinical ketosis (SCK) of dairy cows, as a common group of metabolic diseases, has brought huge economic losses to the dairy farming industry. SCK of dairy cows is a metabolic disease caused by metabolic disorders of glucose, volatile fatty acid and other substances in the body of dairy cows. The disease is mostly seen in dairy cows 1-3 weeks after delivery, and its harm cannot be underestimated. Ketosis of dairy cows is characterized by elevated serum ketone body levels. It is divided into clinical ketosis (CK) (BHBA concentration ≥ 2.90 mmol / L, with obvious clinical symptoms) and SCK (1.20 mmol / L≤BHBA concentration<2.90 mmol / L and no obvious symptoms) according to the concentration of β-hydroxybutyric acid (BHBA) in blood and the presence or absence of symptoms; according to the pathogenesis, ketosis of dairy cows can be divided into type I ketosis, type II ketosis and type III ketosis. The occurrence of SCK of dairy cows is related to many factors, such as breed, environment, body condition, parity, diet, feeding management, liver function and disease factors, and controlling these risk factors will help to reduce the incidence. At present, the global incidence of SCK of dairy cows is 22.7%, and that in China is 10-30%, and foreign reports are lower than 10%. SCK has become one of the most common metabolic diseases in large-scale dairy farms. Therefore, effectively reducing the incidence of SCK of dairy cows has become an important task for disease prevention and control in large-scale dairy farms.

[0003] In modern dairy farming, SCK of dairy cows has become a common group of metabolic diseases, which can bring great economic losses to dairy farms, so reducing SCK of dairy cows can ensure the health and efficient production of dairy cows. There is no report on the combined use of propylene glycol and taurine for treating SCK of dairy cows. SUMMARY

[0004] The present application provides a pharmaceutical composition for preventing or treating subclinical ketosis of dairy cows and application to solve the problem of high incidence of ketosis of dairy cows in large-scale farms and inconsistent prevention and treatment effects.

[0005] In order to achieve the above technical problems, the present application adopts the following technical solutions: The present application aims to provide a pharmaceutical composition for preventing or treating subclinical ketosis of dairy cows, which comprises taurine and propylene glycol as active ingredients.

[0006] Further limitation, the dosage form of the pharmaceutical composition is oral liquid, sustained-release gel or rumen sustained-release granules.

[0007] Further limitation, the pharmaceutical composition further comprises a pharmaceutically acceptable carrier.

[0008] Further limitation, the carrier comprises at least one of diatomite, sodium carboxymethyl cellulose or glycerol.

[0009] The application also provides the use of taurine and propylene glycol in combination in the preparation of an oral medicine for preventing or treating subclinical ketosis of dairy cows. Further limitation, 100 g of taurine is orally administered to each dairy cow per day, and 500 mL of propylene glycol is orally administered to each dairy cow every 1 day, Further limitation, the administration time is 6-7 days after delivery, and the taurine is administered once a day for 7 consecutive days, and the propylene glycol is administered on the 1st day, the 3rd day, the 5th day and the 7th day.

[0010] Further limitation, the criterion for judging the subclinical ketosis is that the blood β-hydroxybutyric acid (BHBA) concentration is ≥1.2 mmol / L and ≤3.0 mmol / L.

[0011] The application has the following beneficial effects: The combination of taurine and propylene glycol effectively reduces the SCK positive rate, and improves the energy metabolism, liver function, oxidative stress and production performance of SCK dairy cows. P <0.01), the mating rate in 21 d ( P <0.01), the pregnancy rate and the conception rate are all increased ( P <0.01), and the proportion of non-pregnant cows in 150 d is reduced ( P<0.01); taurine and propylene glycol combination improved the SCK cow's NEB state (the levels of BHBA and NEFA in blood decreased, the level of GLU increased), liver function state (the levels of AST, TBIL and TC in blood decreased) and OS state (the level of MDA in blood decreased, the levels of SOD, T-AOC and GSH-PX increased), and the effect of TPG was the best. (2) In test 2, there were 257 differential metabolites (100 down-regulated and 157 up-regulated) at 3 d, 207 differential metabolites (113 down-regulated and 94 up-regulated) at 7 d, and 221 differential metabolites (143 down-regulated and 78 up-regulated) at 14 d, and there were 30 differential metabolites common to the three time points; the first nine significant metabolic pathways enriched were central carbon metabolism in cancer, mTOR signaling pathway, cocaine addiction, amphetamine addiction, ethanol addiction, digestion and absorption of proteins, phenylalanine metabolism, glutamatergic synapse and arginine biosynthesis. In addition, the correlation analysis of blood biochemical indicators and differential metabolites showed that at 3 d, NEFA in blood was significantly positively correlated with N-acetyl-L-glutamic acid (P < 0.05), and ALB was extremely significantly negatively correlated with N-acetyl-L-glutamic acid (P < 0.01); at 7 d, NEFA in blood was extremely significantly positively correlated with N-acetyl-L-glutamic acid (P < 0.01), and GLU was extremely significantly negatively correlated with N-acetyl-L-glutamic acid (P < 0.01); at 14 d, BHBA in blood was significantly positively correlated with N-acetyl-L-glutamic acid (P < 0.05), and NEFA, AST and MDA were extremely significantly positively correlated with N-acetyl-L-glutamic acid (P < 0.01), and T-AOC was significantly negatively correlated with N-acetyl-L-glutamic acid (P < 0.05), and GLU and GSH-PX were extremely significantly negatively correlated with N-acetyl-L-glutamic acid (P < 0.01). P <0.05), ALB was extremely significantly negatively correlated with N-acetyl-L-glutamic acid (P < 0.01); at 7 d, NEFA in blood was extremely significantly positively correlated with N-acetyl-L-glutamic acid (P < 0.01), and GLU was extremely significantly negatively correlated with N-acetyl-L-glutamic acid (P < 0.01); at 14 d, BHBA in blood was significantly positively correlated with N-acetyl-L-glutamic acid (P < 0.05), and NEFA, AST and MDA were extremely significantly positively correlated with N-acetyl-L-glutamic acid (P < 0.01), and T-AOC was significantly negatively correlated with N-acetyl-L-glutamic acid (P < 0.05), and GLU and GSH-PX were extremely significantly negatively correlated with N-acetyl-L-glutamic acid (P < 0.01). P <0.01), and GLU and GSH-PX were extremely significantly negatively correlated with N-acetyl-L-glutamic acid (P < 0.01). P <0.05), and GLU and GSH-PX were extremely significantly negatively correlated with N-acetyl-L-glutamic acid (P < 0.01). P <0.01), and GLU and GSH-PX were extremely significantly negatively correlated with N-acetyl-L-glutamic acid (P < 0.01). P <0.01). P <0.01).

[0012] Taurine and propylene glycol combination effectively reduced the SCK positive rate, and improved the energy metabolism, liver function state, oxidative stress and production performance of SCK cows. The metabolomics mechanism of taurine and propylene glycol combination in treating SCK cows involves eight main enriched metabolic pathways. This lays a foundation for future new strategies for treating SCK in cows.

[0013] In order to further understand the features and technical contents of the present application, please refer to the following detailed description of the present application and the accompanying drawings, however, the accompanying drawings are provided for reference and illustration only, and are not used to limit the present application. BRIEF DESCRIPTION OF DRAWINGS

[0014] Figure 1A is the cure rate change of four groups of subclinical ketosis cows with treatment days, PC: positive group, PG: propylene glycol group, TAU: taurine monomer group, TPG: taurine + propylene glycol group; Figure 1B The average cure rate for subclinical ketosis in dairy cows is shown in the figures: PC: positive group, PG: propylene glycol group, TAU: taurine monomer group, and TPG: taurine + propylene glycol group. Figure 2 These are the changes in the concentrations of BHBA, GLU, and NEFA in the blood of four groups of dairy cows in the experiment: A: BHBA, B: GLU, C: NEFA. P <0.05, significantly different from the PC group; P <0.01, which is highly significant compared to the PC group; Figure 3 These are the changes in AST, TP, ALB, TBIL, and TC in the blood of four groups of dairy cows in an experiment: A: AST, B: TP, C: ALB, D: TBIL, E: TC. P <0.05, significantly different from the PC group; P <0.01, which is highly significant compared to the PC group; Figure 4 The changes in SOD, T-AOC, GSH-PX, and MDA in the blood of four groups of dairy cows in the experiment are shown. A: SOD, B: T-AOC, C: GSH-PX, D: MDA. Figure 5 These are the levels of milk yield and milk composition in four groups of experimental dairy cows.* P <0.05, significantly different from the PC group;** P <0.01, extremely significant difference compared with PC group, A: milk fat percentage, B: milk protein percentage, C: milk yield, D: somatic cell count, E: blood urea nitrogen, F: lactose, PC: positive group, PG: propylene glycol group, TAU: taurine monomer group, TPG: taurine + propylene glycol group. Figure 6 The results show the changes in reproductive performance of four groups of dairy cows in the experiment: A: mating rate at 21 days, B: pregnancy rate at 21 days, C: conception rate at 21 days, D: non-pregnant rate at 150 days, PC: positive group, PG: propylene glycol group, TAU: taurine monomer group, and TPG: taurine + propylene glycol group. Detailed Implementation

[0015] The present invention will now be described in detail with reference to specific embodiments. These embodiments will help those skilled in the art to further understand the present invention, while not limiting the invention in any way. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of the present invention. These all fall within the protection scope of the present invention.

[0016] Example 1: Application of taurine and propylene glycol in the preparation of oral medications for the prevention or treatment of subclinical ketosis in dairy cows. Each dairy cow was given 100 g of taurine orally daily, and 500 mL of propylene glycol orally every other day. The medication is administered 6-7 days postpartum, once daily via gavage. Taurine is administered continuously for 7 days, and propylene glycol is administered on days 1, 3, 5, and 7. The following tests were used to verify the effectiveness of the invention. 1. Materials and Methods 1.1 Test Materials 1.1.1 Main Instruments and Equipment The main instruments are shown in Table 1.

[0017] Table 1 Main Instruments for the Experiment

[0018] 1.1.2 Main Reagents and Consumables The main reagents and chemicals used in the experiment are shown in Table 2.

[0019] Table 2. Main reagents and chemicals used in the experiment

[0020] 1.2 Test Methods 1.2.1 Experimental Animals This invention conducted two experiments at a large-scale ranch in eastern Heilongjiang Province: the therapeutic effects of taurine, propylene glycol, and their combination on subclinical ketosis in dairy cows, and the metabolomics mechanism of the combined use of taurine and propylene glycol in treating subclinical ketosis in dairy cows. The experimental procedures followed the requirements of the Animal Ethics Committee of Heilongjiang Bayi Agricultural Reclamation University (DWKJXY2024017). Holstein dairy cows with similar age, parity, body condition, previous milk yield, and postpartum days were selected as experimental animals. The experimental cows were divided into healthy cows and subclinical ketosis cows based on their serum BHBA concentration 6-7 days postpartum (determination criteria: healthy cows had a serum BHBA level less than 1.20 mmol / L and no clinical symptoms; subclinical ketosis cows had a serum BHBA level between 1.20 and 2.90 mmol / L and no obvious clinical symptoms). All experimental dairy cows were fed using the TMR (Total Mixed Feeding) method, with free access to feed and water. The cows were milked three times a day, at 04:00, 12:00, and 18:00.

[0021] The basal diet for the experimental dairy cows at this ranch consisted of the following: Pre-partum period: soybean meal (2.66%), DDGS (3.27%), alfalfa (2.21%), beet meal (8.86%), corn gluten meal (4.43%), oat hay (11.52%), high-moisture corn (8.86%), whole-plant corn silage (39.86%), limestone (0.89%), probiotic premix (0.71%), 5% premix (3.00%), and water (13.73%); Post-partum period: soybean meal (4.31%), alfalfa (5%), and so on. 0.47%), beet meal (2.87%), corn gluten meal (1.72%), oat straw (2.30%), high-moisture corn (17.51%), whole-plant corn silage (34.45%), probiotic premix (1.44%), 5% premix (1.44%), water (12.55%), baking soda (0.72%), calcium bicarbonate (0.29%), fat meal (0.86%), cottonseed (2.87%), coated urea (0.52%), soybean hulls (7.18%), and molasses (2.87%). The nutritional composition was as follows: pre-partum period: crude protein (15.7%), starch (16.8%), neutral detergent fiber (33.7%), calcium (1%), phosphorus (0.4%), and net energy for lactation (1.52 Mcal / kg); post-partum period: crude protein (16.7%), starch (21.5%), neutral detergent fiber (30.7%), calcium (1%), phosphorus (0.4%), and net energy for lactation (1.76 Mcal / kg). The experimental dairy cows did not receive postpartum care.

[0022] To investigate the therapeutic effects of taurine, propylene glycol, and their combination on subclinical ketosis in dairy cows, Experiment 1, based on the preliminary experimental determination that the taurine dose of 100 g was optimal, selected 48 dairy cows with subclinical ketosis according to the criteria of no other symptoms and clinical manifestations and a blood BHBA concentration ≥1.20 mmol / L when blood was collected from calving to 6 days postpartum. They were randomly divided into 4 groups (12 cows in each group): (1) Positive group (PC), no treatment; (2) Taurine monomer group (TAU), 100 g of taurine monomer orally for 7 consecutive days; (3) Propylene glycol group (PG), 500 mL of propylene glycol orally on the 1st, 3rd, 5th, and 7th days of treatment; (4) Taurine + propylene glycol group (TPG), 100 g of taurine orally for 7 consecutive days, and 500 mL of PG orally on the 1st, 3rd, 5th, and 7th days. The trial lasted for 14 days, with the first 7 days being the treatment period and the last 7 days being the observation period. The trial ended approximately 20 days postpartum.

[0023] To investigate the metabolomics mechanism of taurine combined with propylene glycol in treating subclinical ketosis in dairy cows, Experiment 2 selected two groups of dairy cows: a positive control group (PC) and a TPG group, with eight cows in each group. The background information and physicochemical indicators of the cows in each group were similar. Forty-eight serum samples were collected from the two groups at time points 3, 7, and 14 during the experiment for LC-MS detection. The LC-MS experimental design is shown in Table 3. The two groups of cows were compared at the three time points of 3, 7, and 14. For information on the grouping of the two experimental groups, sample collection, and physicochemical indicators, please refer to section 3.1.

[0024] Table 3 Comparison of LC-MS detection schemes between the two groups of experimental dairy cows

[0025] Note: PCA: positive control group cows at 3 days; PCB: positive control group cows at 7 days; PCC: positive control group cows at 14 days; TPGA: taurine + propylene glycol group cows at 3 days; TPGB: taurine + propylene glycol group cows at 7 days; TPGC: taurine + propylene glycol group cows at 14 days.

[0026] 1.2.2 Background Information on Experimental Animals Information such as age, parity, postpartum days, and milk yield of experimental dairy cows were collected one day before treatment using the farm's Dweb dairy cow production management system (Yimu Technology Beijing Co., Ltd., China). Postpartum production performance data, including 21-day pregnancy rate, 21-day mating rate, conception rate, and 150-day non-pregnant percentage, were collected at 60 and 150 days of lactation. After treatment, lactation performance indicators such as milk fat percentage, milk protein percentage, milk yield, somatic cell count, blood urea nitrogen, and lactose were collected for the month. One day before treatment, the body condition score (out of 5) of the dairy cows was measured on-site using BCS Cowdition software (1.2.2, Bayer Animal Health GmbH).

[0027] 1.2.3 Blood Collection and Testing ① Blood sample collection: 10 mL of blood was collected from the tail vein of the four experimental dairy cows 1 day before treatment and on days 1, 3, 5, 7 and 14 after treatment in the early morning before feeding. 1 mL of blood was used to measure the BHBA concentration on-site using a blood ketone meter, and the remaining 9 mL of blood was placed in disposable vacuum blood collection tubes. After centrifugation at 4000 r / min for 10 min, the supernatant was aliquoted into 1.5 mL EP tubes, centrifuged again at 10000 r / min for 5 min, and then aliquoted into serum. The serum was flash-frozen in liquid nitrogen and stored at -80℃ for the detection of blood biochemical indicators and subsequent omics experiments.

[0028] ② Energy metabolism indicators: glucose (GLU, mmol / L, oxidase method), free fatty acids (NEFA, mmol / L, enzymatic method), β-hydroxybutyrate (BHBA, mmol / L, electrochemical method).

[0029] ③ Liver function indicators: Aspartate aminotransferase (AST, U / L, IFCC method), total bilirubin (T-BIL, umoI / L, vanadate oxidation method), albumin (ALB, g / L, bromocresol green method), total protein (TP, g / L, biuret method); total cholesterol (TC, mmol / L, oxidase method).

[0030] ④OS indicators: total antioxidant capacity (T-AOC, U / mL, FRAP method), superoxide dismutase (SOD, U / mL, WST-8 method), malondialdehyde (MDA, nmol / mL, thiobarbituric acid method), glutathione peroxidase (GSH-Px, umoI / L, DTNB method).

[0031] ⑤ Prevalence of subclinical ketosis:

[0032] ⑥ Criteria for determining the cure of subclinical ketosis: The determination is based on the concentration of BHBA in the blood of dairy cows. When the concentration of BHBA in the blood of dairy cows is <1.00 mmol / L, it is considered cured.

[0033] 2.2.4 LC-MS Analysis ① Extraction of serum metabolites: The serum sample was thawed at 4℃, and after vortexing for 60s, 50μL of serum sample was placed in a 2 mL centrifuge tube. 400 µL of methanol solution was added to the tube, and the mixture was vortexed for 60s. Subsequently, the sample was centrifuged at 12000 rpm for 10 min at 4℃. All supernatant was collected and transferred to a 2 mL centrifuge tube, concentrated and dried. Then, 150 µL of 2-chloro-L-phenylalanine (4 ppm) solution prepared with 80% methanol and water was accurately added to reconstitute the sample. The supernatant was filtered through a 0.22 μm membrane, and the filtrate was added to the detection bottle for LC-MS detection.

[0034] ② On-machine testing: This experiment uses the Thermo-Vanquish ultra-high performance liquid chromatography system and ACQUITYUPLC. ®Serum metabolomics analysis was performed using an HSS T3 (2.1 × 100 mm, 1.8 µm) column with the following conditions: flow rate of 0.3 mL / min, column temperature of 40 °C, and injection volume of 2 μL. Positive ion mode was used, with a mobile phase of 0.1% formic acid acetonitrile (B2) and 0.1% formic acid water (A2). The gradient elution program was: 0–1 min, 10% B2; 1–5 min, 10%–98% B2; 5–6.5 min, 98% B2; 6.5–6.6 min, 98%–10% B2; 6.6–8 min, 10% B2. In negative ion mode, the mobile phase consisted of acetonitrile (B3) and 5 mM ammonium formate water (A3), with the gradient elution program as follows: 0–1 min, 10% B3; 1–5 min, 10%–98% B3; 5–6.5 min, 98% B3; 6.5–6.6 min, 98%–10% B3; 6.6–8 min, 10% B3.

[0035] In this experiment, a Thermo Q Exactive mass spectrometer was used for detection, and an electrospray ionization (ESI) source was used simultaneously for both positive and negative ion modes to acquire data. The mass spectrometry conditions were set as follows: positive ion spray voltage of 3.50 kV, negative ion spray voltage of -2.50 kV, sheath gas of 40 arb, and auxiliary gas of 10 arb. The capillary temperature was 325℃. A first-stage full scan was performed at a resolution of 60,000 m / z, with a first-stage ion scan range of 100–1000 m / z. Second-stage fragmentation was performed using an HCD with a collision energy of 30% and a second-stage resolution of 17,500 m / z. The first four ions acquired were fragmented, and unnecessary MS / MS information was removed using dynamic exclusion.

[0036] 1.2.5 Statistical Methods In Experiment 1, Microsoft Excel 2019 was used to organize the data on SCK morbidity, production performance, and biochemical indicators of the experimental dairy cows. IBM SPSS Statistics 26 software was then used for data analysis. Chi-square tests and cohort studies were used to analyze the differences in SCK morbidity, other disease morbidity, and reproductive performance in the experimental dairy cows. One-way ANOVA was used to analyze the hematological indicators and lactation performance of the four groups of dairy cows. For the main effects of factors with significant influence, post-hoc multiple comparisons (Tukey method) were used to analyze the differences between means, and data with significant differences were labeled using letters. Repeated measures ANOVA was performed on the hematological indicators of the four groups of dairy cows, using different treatment methods, experimental days, and different treatment methods × experimental days (Trt × Day) as fixed factors. All data results are expressed as mean ± standard deviation. ±SD) indicates that the graph was drawn using GraphPad Prism 10.1.2 software.

[0037] In Experiment 2, the raw mass spectrometry files were converted to mzXML file format using the MSConvert tool in the Proteowizard software package (v3.0.8789). Peak detection, peak filtering, and peak alignment were performed using the R XCMS (v3.12.0) software package to obtain a list of metabolite quantifications. The parameters were set as follows: bw=2, ppm=15, peakwidth=c(5, 30), mzwid=0.015, mzdiff=0.01, and method=centWave. Then, support vector regression correction based on quality control (QC) samples was used to eliminate systematic errors. Substances with an RSD > 30% in the QC samples were filtered out during the quality control and quality assurance processes for subsequent data analysis.

[0038] Substance identification (database search) was performed using public databases such as HMDB, massbank, LipidMaps, mzcloud, and KEGG, as well as a self-built standard library. The parameter was set to ppm < 30 ppm to obtain qualitative results for metabolites. Specifically, the molecular weight of the metabolite was determined based on the mass-to-charge ratio (m / z) of the precursor ion in the primary mass spectrometry. The molecular formula was predicted using mass number deviation (ppm) and information such as adduct ions, and then matched with the database. Simultaneously, in the quantification list, metabolites detected in the secondary spectra were matched with fragment ions and other information for each metabolite in the database, achieving secondary identification of the metabolites.

[0039] Principal Component Analysis (PCA) and Orthogonal Partial Least Squares Discriminant Analysis (OPLS-DA) were used to perform dimensionality reduction analysis on the sample data using the R package Robls. Overfitting was tested using the permutation test. R²X and R²Y represent the explanatory power of the model for the X and Y matrices, respectively, and Q² indicates the predictive power of the model. Values ​​closer to 1 indicate a better fit and more accurate classification of the training set samples into their original groups. P-values ​​were calculated based on statistical tests. The OPLS-DA dimensionality reduction method was used to calculate the variable importance for the projection (VIP) and fold change (FC) to measure the influence and explanatory power of each metabolite's content on sample classification, aiding in the screening of marker metabolites.P Metabolite molecules were considered statistically significant when the VIP was <0.05 and >1. Subsequently, pathway enrichment analysis was performed using hypergeometric distribution-based enrichment analysis. The enriched pathways were visualized using the KEGG Mapper tool to view the differential metabolite and pathway maps.

[0040] The Kaitai Bioinformatics Cloud Platform (https: / / kaitai.cloud / tools) was used for Venn analysis and Venn plot viewing of differential metabolites, while the Omic Share Biotechnology Cloud Platform (https: / / www.omicshare.com / tools / ) was used for correlation analysis and correlation heatmap viewing of differential metabolites and blood biochemical indicators.

[0041] 2 Results and Analysis 2.1 The therapeutic effect of taurine and propylene glycol in combination on subclinical ketosis in dairy cows 2.1.1 Background information on the experimental dairy cows Table 4 shows that there were no significant differences in age, parity, body condition, milk yield, and postpartum days among the four groups of experimental dairy cows before treatment. P >0.05).

[0042] Table 4 Comparison of background information of four groups of experimental dairy cows

[0043] Note: when P A difference of <0.05 is considered statistically significant. P When <0.01, the difference between groups was extremely significant; PC: positive group, PG: propylene glycol group, TAU: taurine monomer group, TPG: taurine + propylene glycol group.

[0044] 2.1.2 Disease incidence in experimental dairy cows 2.1.2.1 Cure rate and therapeutic effect of SCK in experimental dairy cows Depend on Figure 1A As shown, compared with before treatment, the cure rate of SCK in all four groups of dairy cows increased with the number of treatment days. At -1 day, the cure rate of SCK in all four groups was 0%. As the trial period extended to 14 days, the cure rates were 16.67% in the PC group, 58.33% in the PG group, 41.67% in the TAU group, and 83.33% in the TPG group, with the TPG group showing the highest cure rate. After 7 days of continuous treatment, the order of SCK cure rate among the four groups was TAU group = TPG group > PG group > PC group. At day 14 after treatment, the order of SCK cure rate was TPG group > PG group > TAU group > PC group. During the trial, the average cure rate of SCK among the four groups was TPG group > PG group ≈ TAU group > PC group. Figure 1B).

[0045] The results showed that the TPG group had the best therapeutic effect on SCK, followed by the TAU group and the PG group.

[0046] As shown in Table 5, the four treatment methods showed significant differences in their effects on SCK treatment in dairy cows. P <0.05, χ 2 =11.33). After the trial, the cure rate was in the following order: TPG group > PG group > TAU group > PC group.

[0047] Table 5. Treatment effects of SCK in four experimental groups of dairy cows.

[0048] Note:*, P <0.05, the difference is significant; **, P <0.01, the difference is extremely significant.

[0049] As shown in Table 6, compared with PC, the RR of SCK in dairy cows treated with TPG was significantly different, decreasing to 5 times the original level (RR=5.000). P <0.01), there was no significant difference in the RR of SCK occurrence between the PG group and the TAU group ( P >0.05).

[0050] Table 6. Relative risk of subclinical ketosis in dairy cows under four treatments.

[0051] Note:*, P <0.05, the difference is significant; **, P <0.01, extremely significant difference; number of comparisons N=6, adjusted significance level α=0.05 / 6=0.008; RR, relative risk; SE(lnRR), standard error of the logarithmic OR; Z, difference between sample data and population mean divided by population standard deviation; 95% CI, 95% confidence interval.

[0052] 2.1.2.2 Prevalence of other diseases in the experimental dairy cows As shown in Table 7, the prevalence of metritis was 16.67% in the PC, PG, and TAU groups, while no cases were observed in the TPG group. The prevalence of diarrhea was 8.33% in the TAU group, while no cases were observed in the PC, PG, and TPG groups. The prevalence of abomasal displacement was 16.67% in the PG group and 8.33% in the TPG group, while no cases were observed in the PC and TAU groups. The prevalence of forestomach atony was 8.33% in both the TAU and TPG groups, while no cases were observed in the PC and PG groups. The prevalence of vaginitis was 16.67% in the PC group, while no cases were observed in the PG, TAU, and TPG groups. The prevalence of mastitis was 8.33% in the PC group and 16.67% in the PG group, while no cases were observed in the TAU and TPG groups. The prevalence of hoof diseases was 8.33% in the PC group, while no cases were observed in the PG, TAU, and TPG groups. The results indicate that at 14 days, the order of prevalence of other diseases in the four groups of dairy cows was PC = PG > TAU > TPG.

[0053] The highest prevalence of metritis (50.01%) was observed in the experimental dairy cows, followed by mastitis and abomasal displacement (25.00%), while hoof diseases and diarrhea had the lowest incidence (8.33%). The prevalence of different diseases was unevenly distributed; some diseases only occurred in specific treatment groups. Cows with ≥2 diseases had a relatively high prevalence across all categories (41.67%), with the highest prevalence in the PC and PG groups (16.67%), followed by the TPG group (8.33%). No cows in the TAU group had more than two diseases.

[0054] Table 7. Prevalence of other diseases in dairy cows under four treatments

[0055] Note: The values ​​in parentheses in the table represent the prevalence of this disease in the dairy cows of that group.

[0056] As shown in Table 8, compared with the PC group, the other three treatments had no significant effect on other production diseases in the experimental dairy cows. P >0.05). The TPG group had the lowest morbidity rate and did not reduce the morbidity of other diseases after treatment.

[0057] Table 8. Effects of the four treatment groups on other production diseases in dairy cows.

[0058] Note: The values ​​in parentheses in the table represent the percentage of cows in that group that have this disease out of the total number of sick cows.

[0059] 2.1.3 Levels of blood biochemical indicators in experimental dairy cows 2.1.3.1 Levels of key energy metabolism indicators in experimental dairy cows Depend on Figure 2 As shown, the four treatments had a significant impact on BHBA, GLU, and NEFA levels in dairy cow blood.P <0.01, the number of experimental days, the interaction between different treatments and the number of experimental days had a significant effect on GLU in dairy cow blood ( P <0.01), but had no significant effect on blood BHBA and NEFA levels ( P >0.05). Before treatment, there were no significant differences in BHBA, GLU, and NEFA levels in the blood of the four groups of dairy cows. P >0.05). On day 7 after the end of treatment, the order of BHBA concentration in the blood of the four groups of dairy cows was PC>PG>TAU>TPG, the order of GLU concentration was TPG>PG>TAU>PC, and the order of NEFA concentration was PC>TAU>TPG>PG. On day 14 after drug withdrawal, the order of BHBA concentration in the blood of dairy cows was PC>TAU>PG>TPG, the order of GLU concentration was TPG>PG>TAU>PC, and the order of NEFA concentration was PC>TAU>PG>TPG.

[0060] As the experimental days increased, the concentrations of NEFA and BHBA in the blood of dairy cows in the PC group were consistently higher than those in the other three groups. NEFA showed a trend of first increasing, then stabilizing, and then increasing again, while BHBA showed a trend of first increasing, then slightly decreasing, and then increasing again, both reaching their highest levels at 14 days. However, the concentration of GLU in the blood was consistently lower than that in the other three groups, showing a trend of first decreasing, then increasing, and then slowly decreasing, reaching its lowest level at 14 days. The concentrations of NEFA, BHBA, and GLU in the blood of dairy cows in the TPG and PG groups showed similar trends. Both NEFA and BHBA showed a trend of first decreasing, then increasing, and then decreasing again, but the concentration in the TPG group was slightly lower than that in the PG group, reaching its lowest level at 14 days. The concentration of GLU in the blood showed a trend of first increasing, then slightly decreasing, then increasing, and then slightly decreasing again, reaching its highest level at 14 days, but the concentration in the TPG group was higher than that in the PG group. Interestingly, the concentrations of NEFA and BHBA in the blood of cows in the TAU group were lower than those in the PC group, and varied between the other two groups, but were higher than the other two groups at 14 days; the concentration of GLU was higher than that in the PC group, and was higher than that in the TPG group in the first three days of treatment, then decreased, and was lower than that in the TPG and PG groups at 14 days.

[0061] The results showed that all three treatment methods could improve the NEB status of SCK dairy cows, with the TPG group showing the best effect. It could more effectively reduce the concentrations of BHBA and NEFA in the blood of dairy cows and increase GLU.

[0062] 2.1.3.2 Levels of liver function indicators in experimental dairy cows Depend on Figure 3 As shown, different treatment methods and the number of experimental days have a significant effect on AST, ALB, and TC in the blood of dairy cows. P <0.01, but had no significant effect on serum TP and TBIL ( P>0.05). The interaction between different treatment methods and the number of experimental days had a significant effect on TC in the blood of dairy cows ( P <0.01, but had no significant effect on serum AST, TP, ALB, and TBIL ( P >0.05). Before treatment, there were no significant differences in AST, TP, ALB, TBIL, and TC levels in the blood of the four groups of dairy cows. P >0.05). On day 7 after the end of treatment and day 14 after drug withdrawal, the order of serum AST and TBIL levels in the four groups of dairy cows was: PC>PG>TAU>TPG; the order of serum TP and ALB concentrations was: TPG>PG>TAU>PC; and the order of serum TC concentrations was: PC>TAU>PG>TPG.

[0063] As the number of experimental days increased, the levels of AST, TBIL, and TC in the blood of dairy cows in the PC group were consistently higher than those in the other three groups, showing a trend of first increasing and then decreasing, remaining higher than the other three groups on days 7 and 14. The concentrations of TP and ALB in the blood were consistently lower than those in the other three groups, showing a trend of first decreasing and then increasing, reaching their highest point on day 5. The concentrations of AST and TBIL in the blood of dairy cows in the TPG and PG groups showed similar trends, exhibiting a continuous fluctuating decrease, but the TPG group was lower than the PG group, reaching its lowest point on day 14. The concentrations of ALP and TP in dairy cows in both groups showed similar trends, both showing a fluctuating increase, with the PG group reaching its highest point on day 14, while the TPG group reached its highest point on day 5. The concentrations of TC in the blood of dairy cows in the PG and TAU groups showed similar trends, exhibiting an increasing trend, reaching their highest point on day 14, while the TPG group, except for a decrease in concentration on day 14, showed similar trends to the PG and TAU groups at other times. In the TAU group, the concentrations of AST and TBIL in the blood of dairy cows showed a decreasing trend, reaching their lowest point at 14 days. The concentrations of TP and ALB showed similar changes, fluctuating and reaching their highest point at 3 days. TC showed an increasing trend, reaching its highest point at 14 days.

[0064] The results showed that the three treatment methods could improve liver function in SCK dairy cows, with the TPG group showing the best effect, effectively reducing the levels of AST, TBIL, and TC in the blood of dairy cows and increasing the levels of TP and ALB.

[0065] 2.1.3.3 Levels of oxidative stress indicators in the four experimental groups of dairy cows Depend on Figure 4 As shown, different treatment methods have a significant impact on SOD, T-AOC, GSH-PX, and MDA levels in dairy cow blood. P <0.01). The number of experimental days had a significant effect on SOD and T-AOC levels in dairy cow blood ( P <0.01), but had no significant effect on GSH-PX and MDA in bovine blood ( P>0.05). The interaction between different treatment methods and the number of experimental days had a significant effect on SOD, T-AOC, and GSH-PX levels in bovine serum. P <0.01), but had no significant effect on MDA in bovine blood ( P >0.05). Before treatment, there were no significant differences in SOD, T-AOC, GSH-PX, and MDA levels in bovine blood. P >0.05). On day 7 after the end of treatment, the order of SOD and GSH-PX levels in the blood of the four groups of dairy cows was: PG>TPG>TAU>PC; the order of T-AOC concentration in the blood of dairy cows was: TPG>PG>TAU>PC; and the order of MDA concentration in the blood of dairy cows was: PC>PG>TAU>TPG. On day 14 after drug withdrawal, the order of SOD and GSH-PX levels in the blood of the four groups of dairy cows was: TPG>PG>TAU>PC; the order of T-AOC concentration in the blood of dairy cows was: PG>TPG>TAU>PC; and the order of MDA concentration in the blood of dairy cows was: PC>PG>TAU>TPG.

[0066] As the experimental days increased, the concentrations of SOD, T-AOC, and GSH-PX in the blood of dairy cows in the PC group remained lower than those in the other three groups, showing a decreasing trend. In the PG group, the concentrations of T-AOC and GSH-PX in the blood showed similar trends, fluctuating upwards, both reaching their highest levels at 14 days. The SOD concentration also showed a similar trend, reaching its highest level at 7 days. In the TAU group, the concentrations of SOD, T-AOC, and GSH-PX in the blood showed similar changes: SOD fluctuated upwards, T-AOC gradually increased, and GSH-PX increased before decreasing. In the TPG group, the concentrations of SOD, T-AOC, and GSH-PX in the blood all fluctuated upwards, with SOD and T-AOC reaching their highest levels at 14 days, and GSH-PX reaching its highest level at 5 days. MDA levels in all three groups of dairy cows showed a decreasing trend, with the lowest levels in the TPG group at 7 and 14 days, the lowest in the PG group at 7 days, and the lowest in the TAU group at 5 days.

[0067] The results showed that the three treatment methods could improve the OS status of SCK dairy cows, with the TPG group showing the best effect. It could more effectively reduce the concentration of MDA in the blood of dairy cows and increase the levels of SOD, T-AOC and GSH-PX.

[0068] 2.1.4 Levels of production performance indicators in experimental dairy cows 2.1.4.1 Levels of lactation performance indicators in experimental dairy cows Depend on Figure 5 As shown, compared with the PC group, the milk fat percentage and milk protein percentage were significantly higher in the PG group ( P <0.05, the somatic cell count was significantly reduced in the TAU group and TPG group ( P<0.01), there were no significant differences in milk yield, urea nitrogen, peak milk production, and lactose among the groups ( P >0.05).

[0069] Milk fat percentage, in descending order of the four groups of dairy cows, was PG>TPG>TAU>PC; milk protein percentage, in descending order of the four groups of dairy cows, was PG>TPG>TAU>PC; milk somatic cell count, in descending order of the four groups of dairy cows, was PC>PG>TAU>TPG.

[0070] The results showed that TPG had a significant effect on the lactation performance of SCK dairy cows. The treatment with TPG and TAU groups could effectively reduce SCC in dairy milk, while PG could effectively increase the milk fat and milk protein content in dairy milk.

[0071] 3.1.4.2 Levels of reproductive performance indicators in experimental dairy cows Depend on Figure 6 As shown, compared with PC, the mating rate at 21 days was significantly higher in the PG group ( P <0.05), the 21-day pregnancy rate and 21-day conception rate were significantly increased ( P <0.01); the pregnancy rate at 21 days was significantly higher in the TAU group ( P <0.05), the conception rate increased significantly at 21 days ( P <0.01), the proportion of non-pregnant individuals after 150 days was significantly reduced ( P <0.05); the 21-day mating rate, 21-day pregnancy rate, and 21-day conception rate were significantly increased in the TPG group ( P <0.01), the proportion of non-pregnant individuals after 150 days was significantly reduced ( P <0.01).

[0072] The 21-day mating rate of the four groups of dairy cows was in the following order: TPG > PG > TAU = PC; the 21-day pregnancy rate of the four groups of dairy cows was in the following order: PG > TPG > TAU > PC; the 21-day conception rate of the four groups of dairy cows was in the following order: PG > TAU > TPG > PC; and the 150-day non-pregnancy rate of the four groups of dairy cows was in the following order: PC > PG > TAU > TPG.

[0073] The results showed that the TPG group had a significant impact on the reproductive performance of SCK dairy cows. The PG group increased the 21-day mating rate, 21-day pregnancy rate, and 21-day conception rate by 8.33%, 34.55%, and 35.39%, respectively. The TPG group increased the 21-day mating rate, 21-day pregnancy rate, and 21-day conception rate by 10.71%, 29.55%, and 26.89%, respectively. The TAU group increased the 21-day pregnancy rate and 21-day conception rate by 21.22% and 28.28%, respectively. The TAU group and TPG group reduced the proportion of cows that were not pregnant at 150 days by 15.15% and 16.66%, respectively.

[0074] The combined use of PG and TAU effectively improved the NEB status of SCK dairy cows through glycemic and antiketotic effects.

[0075] The combined use of PG and TAU effectively improves liver function in SCK dairy cows by protecting the liver and promoting bile secretion.

[0076] The combination of PG and TAU has significant advantages in combating OS and reducing oxidative damage, exerting a synergistic effect and improving treatment efficacy.

[0077] Taurine, propylene glycol, and their combination effectively increased the cure rate of SCK and improved production performance, with the taurine + propylene glycol group showing the best effect (SCK positivity rate decreased to 16.67%), manifested as a reduction in SCC in dairy cows and an increase in milk fat and milk protein percentages. The mating rate, pregnancy rate, and conception rate at 21 days were all increased, while the proportion of non-pregnant cows at 150 days was reduced. Furthermore, taurine, propylene glycol, and their combination improved the NEB status (decreased levels of BHBA and NEFA in the blood, and increased levels of GLU), liver function status (decreased levels of AST, TBIL, and TC in the blood), and OS status (decreased levels of MDA in the blood, and increased levels of SOD, T-AOC, and GSH-PX in the blood) in SCK dairy cows, with the taurine + propylene glycol group showing the best effect.

[0078] The combined use of taurine and propylene glycol altered the serum metabolic profile of SCK dairy cows, identifying a total of 554 differential metabolites. Thirty differential metabolites were common across the three time points. Downregulated metabolites included phloroglucinol, butyrylcarnitine, and N-acetyl-L-glutamate; upregulated metabolites included 5-oxohexanoic acid, 5,6-dihydrothymidine, 5-acetamido-6-formylamino-3-methyluracil, 2-hydroxyglutamate, L-glutamate, L-phenylalanine, and sebacic acid; differential metabolites upregulated at 3 days and downregulated at 7 and 14 days included dibenzothiophene sulfone and indole-3-carboxylic acid. In addition, the combined use of taurine and propylene glycol to treat SCK in dairy cows mainly regulates amino acid metabolism and lipid metabolism through enriched metabolic pathways such as central carbon metabolism in cancer, mTOR signaling pathway, cocaine addiction, amphetamine addiction, ethanol addiction, protein digestion and absorption, phenylalanine metabolism, glutamatergic synapses and arginine biosynthesis. It also exerts glucogenic, anti-ketogenic, antioxidant and hepatoprotective effects through the biosynthetic pathways of L-glutamate, N-acetyl-L-glutamate and arginine, thereby improving NEB, liver function and OS in SCK dairy cows.

[0079] The specific embodiments of the present invention have been described in detail above. It should be noted that the present invention is not limited to the specific embodiments described above. Various modifications or alterations can be made by those skilled in the art without departing from the scope of protection defined by the claims, and all such modifications or alterations fall within the technical solutions of the present invention.

Claims

1. A pharmaceutical composition for the prevention or treatment of subclinical ketosis in dairy cows, characterized in that, It contains taurine and propylene glycol as active ingredients.

2. The pharmaceutical composition according to claim 1, characterized in that, Its dosage forms are oral liquid, sustained-release gel or rumen sustained-release granules.

3. The pharmaceutical composition according to claim 1, characterized in that, It contains pharmaceutically acceptable carriers.

4. The pharmaceutical composition according to claim 3, characterized in that, The carrier includes at least one of diatomaceous earth, sodium carboxymethyl cellulose, or glycerol.

5. The use of the pharmaceutical composition according to any one of claims 1–4 in the preparation of an oral remedy for the prevention or treatment of subclinical ketosis in dairy cows.

6. The application according to claim 1, characterized in that, Each dairy cow is given 100 g of taurine orally daily, and 500 mL of propylene glycol orally every other day.

7. The application according to claim 1, characterized in that, The administration period is from 0 to 3 days postpartum, once daily by gavage. Taurine is administered continuously for 7 days, and propylene glycol is administered on days 1, 3, 5, and 7.

8. The application according to claim 4, characterized in that, The criteria for determining subclinical ketosis are a serum β-hydroxybutyrate (BHBA) concentration ≥1.2 mmol / L and ≤3.0 mmol / L.

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