Use of pgd2 in promoting rumen development in young ruminants

Abstract

The application discloses application of PGD2 in promoting rumen development of young ruminants, and application of PGD2 in young ruminants promotes proliferation and differentiation of rumen wall cells, and further promotes rumen development and improves the digestive and absorptive capacity of the rumen. 2+ It is found that PGD2 can promote rumen development by activating Ca signal pathways, promoting expression of CAMK2A protein, promoting cell cycle progression, promoting rumen development, and ensuring animal health. Therefore, PGD2 can be used for preparing a medicine for promoting rumen development of young ruminants.

Description

Technical Field

[0001] This invention relates to the application of PGD2 in promoting rumen development in young ruminants. Background Technology

[0002] With the improvement of living standards in urban and rural areas and population growth, the supply and demand of grain and livestock products will remain unbalanced for a long time. Developing grain-saving animal husbandry can alleviate the contradiction between grain supply and demand and enrich the dietary structure of residents, while the ruminant animal industry has long occupied an important position in animal husbandry.

[0003] As the nutritional value of mutton is widely recognized by consumers, its consumption has been increasing year by year, becoming an important choice for people seeking to improve their quality of life and pursue a healthy diet. However, the current situation in sheep farming is one of supply falling short of demand, leading to the rapid development of the mutton sheep industry driven by consumers. The transition of lambs from the non-ruminant to the ruminant stage involves changes in their diet and the stress of separation from their mothers, which negatively impacts their growth, development, and health, resulting in problems such as weight loss, increased morbidity, and higher mortality rates, causing significant economic losses to the mutton sheep industry.

[0004] The rumen of young ruminants is in a rapid development stage and has strong plasticity. Its developmental level significantly impacts the health and productivity of lambs after weaning and into adulthood. Therefore, the early rumen stage is the optimal window for regulating rumen development. If rumen development is poorly regulated during this critical transition period, it can easily lead to insufficient rumen digestive capacity and motility, resulting in reduced rumination frequency and duration, decreased feed intake, and lower productivity. Currently, how to rationally regulate healthy rumen development has become one of the urgent problems to be solved in modern large-scale ruminant farming.

[0005] Prostaglandin D2 (PGD2), molecular formula: C 20 H 32 O5 is a type of prostaglandin (PG). It is produced by the isomerization of the unstable intermediate PGH2 catalyzed by prostaglandin D synthase (PGDS). PGD2 has certain biological functions, including promoting sleep, inhibiting inflammation, and relaxing smooth muscle. In medicine and pharmaceuticals, PGD2 can promote uterine smooth muscle relaxation and uterine contractions through PGD2 receptor mediation. However, its application in livestock feed has not been reported.

[0006] Purpose of the invention

[0007] Summary of the Invention: Addressing the problems existing in the prior art, this invention provides the application of PGD2 in promoting rumen development in young ruminants. This invention proposes a novel function for PGD2, allowing for oral administration to young ruminants in addition to their milk replacer diet, effectively promoting the proliferation of rumen parietal cells through calcium absorption. 2+ Signaling pathways mediate and regulate rumen development, thereby ensuring lambs' digestion of starter feed, enabling early weaning, reducing weaning stress, and increasing the supply of mutton to the market.

[0008] Technical solution: In order to achieve the above objectives, the present invention describes the application of PGD2 in promoting rumen development in young ruminants.

[0009] The present invention describes the application of PGD2 in promoting the proliferation of rumen parietal cells in young ruminants, mediating and regulating rumen development, digesting starter feed, achieving early weaning, and reducing weaning stress.

[0010] The young ruminants mentioned are lambs or calves.

[0011] Among them, PGD2 is used as a drug additive in animal feed to promote rumen development and rumen motility in young ruminants.

[0012] The animal feed is either a milk replacer or a concentrated starter feed.

[0013] The PGD2 is administered orally to young ruminants in addition to milk replacer or concentrate starter feed.

[0014] The oral administration of PGD2 is 10-30 μg / kg body weight in young ruminants.

[0015] Preferably, the oral administration of PGD2 is 20 μg / kg body weight in young ruminants.

[0016] The lambs or calves referred to are those within six weeks after birth and after weaning.

[0017] To address the aforementioned problems faced by the lamb farming industry, this invention initially measured and conducted metabolomics studies on the major rumen metabolites of lambs fed goat milk (M) and lambs fed goat milk + alfalfa hay (MH) (e.g. Figure 1 As shown in the figure, in the rumen metabolites of lambs fed with goat milk and alfalfa hay, in addition to significant changes in carbohydrate metabolism, PGD2 in lipid metabolism also showed significant changes. Subsequent random forest analysis identified PGD2 as the highest-ranking characteristic metabolite in the rumen fluid of lambs supplemented with alfalfa hay, paving the way for further research. Based on the preliminary experimental findings, Edu assays were used to detect the growth-promoting effect of different PGD2 concentrations on rumen parietal cells, confirming that PGD2 at 0.1 μM had a good proliferative effect (e.g., ...). Figure 2 As shown in the figure, in order to further verify the promoting effect of PGD2 on rumen development in lambs in vivo, PGD2 was administered orally to Hu sheep lambs. The experiment found that PGD2 can effectively increase the length, width and total epithelial thickness of rumen papillae in lambs, and promote the morphological and functional development of rumen.

[0018] Currently, the change in feeding regimes and separation from ewes during the transition from non-ruminant to ruminant lambs causes significant stress, adversely affecting their growth, development, and health. This results in problems such as weight loss, increased morbidity, and mortality, leading to substantial economic losses. This invention effectively addresses the current situation where supply cannot meet demand and rumen health cannot be guaranteed under intensive farming conditions.

[0019] This invention is the first to propose the application of PGD2 in promoting rumen development and growth performance in young ruminants. Generally, for lambs eating starter feed, since nutrition is mainly digested and absorbed in the rumen, promoting rumen development can effectively promote growth. For lambs that are initially fed milk replacer, PGD2 mainly promotes rumen development, preparing the animals for starting feed and facilitating early weaning in young ruminants.

[0020] Beneficial effects: Compared with the prior art, the present invention has the following advantages:

[0021] This invention proposes a novel function for PGD2: it can be administered orally to young ruminants in addition to their starter feed to promote rumen development. In in vivo experiments, PGD2 significantly increased the length, width, and total epithelial thickness of rumen papillae in lambs, indicating that PGD2 can be added to the starter feed of young ruminants (milk replacer / concentrate) to promote rumen development and the establishment of rumen digestive and absorptive functions. Therefore, PGD2 can be used to prepare drugs that promote rumen development in young ruminants. Attached Figure Description

[0022] Figure 1 Results of rumen fluid metabolomics for groups M and MH;

[0023] Figure 2 The effect of PGD2 on the proliferation of primary rumen parietal cells;

[0024] Figure 3 Comparison of HE slices of rumen from lambs in the control group and PGD2 group;

[0025] Figure 4 The effect of PGD2 on the expression of cell cycle-related genes in rumen tissue;

[0026] Figure 5 The effect of PGD2 treatment on the relative expression levels of key genes in the calcium signaling pathway in rumen tissue. Detailed Implementation

[0027] The present invention will be further described below with reference to the accompanying drawings and embodiments.

[0028] The raw materials and instruments used in the examples are all commercially available.

[0029] PGD2 was purchased from Sigma Aldrich and has a purity of 98%.

[0030] BeyoClick™ Edu-594 Cell Proliferation Detection Kit was purchased from Beyotime.

[0031] Example 1

[0032] To determine the proliferative effect of PGD2 on primary rumen parietal cells and the optimal concentration, three 42-day-old male Hu sheep of the same sex and similar weight were selected for the cell experiment.

[0033] 1. Immediately after slaughter, separate the rumen tissue from the lambs. Rinse repeatedly with D-Hanks solution to remove rumen contents, then rinse 2-3 times until the supernatant is clear. This effectively removes residual bacteria and fungi, yielding approximately 1g of rumen wall sample. Cut the sample into small pieces and add 50mL of 0.25% trypsin solution containing 1× antibiotic-antifungal agent. Digest at 37℃ for 20min. Wash three times with sterile D-Hanks solution, then add 30mL of 0.25% trypsin solution containing 1× antibiotic-antifungal agent again. Digest at 37℃ for 20min, then collect rumen wall cells. Filter through a 70μm pore size cell sieve, centrifuge, resuspend, and seed the cells into culture flasks. Incubate in a 5% CO2, 37℃ incubator. When the cells reach 70-80% confluence, passage the cells. Seed 5000 cells per well in a 96-well plate and culture in complete medium for 24h to obtain primary rumen wall cells.

[0034] 2. After primary rumen parietal cells adhered and grew to 80% confluence, they were starved in cell culture serum containing 0.5% fetal bovine serum for 24 h to synchronize the cells. They were then divided into three groups: control group (Con, n = 5), PGD2 group (PGD2, n = 5), and PGD2 + CAMK2 inhibitor group (PGD2 + Ci, n = 5). The PGD2 group was supplemented with PGD2 (Sigma, USA) at a final concentration of 0.1 μM. The PGD2 + CAMK2 inhibitor group was supplemented with CAMK2 inhibitor (KN93; MCE, USA) at a final concentration of 10 μM 1 h before the addition of PGD2. After the addition of PGD2, the cells were cultured for another 24 h, and then samples were collected.

[0035] 3. Pre-prepared cell slides were treated with PGD2 and then 10 μM EdU (Beyotime, China) was added. After culturing for 24 h, the cell culture medium was discarded, and 4% tissue cell fixation solution (Solepro, China) was added for fixation for 15 min. The slides were then washed three times with PBS containing 3% BSA, and permeabilization buffer (Beyotime, China) was added and incubated at room temperature for 12 min. After washing three times, freshly prepared click reaction solution was added and incubated in the dark for 30 min. After washing three times, an anti-fluorescence quencher (Beyotime, China) was added, and cell proliferation was observed using a laser confocal microscope (Zeiss, Germany). Ten fields of view were randomly selected from each sample for photography, and the EdU labeling ratio was calculated.

[0036] 4. Discard the cell culture medium and add 4% tissue cell fixative for half an hour. Then wash three times with PBS, add 0.3% Triton and incubate at room temperature for 15 min. After washing three times with PBS, add 5% PBS solution and incubate on a shaker at room temperature for 60 min. Discard the blocking solution, add primary antibody dilution buffer, and incubate overnight at 4 ℃ in the dark. Wash three times with PBS, 5 min each time, add secondary antibody, and incubate at 37 ℃ for 1 h. Wash three times with PBS, and stain the cell nuclei with DAPI (Soleb, China) for 10 min. Finally, after washing three times with PBS, add anti-fluorescence quencher and scan with a laser confocal microscope to observe the cell protein fluorescence labeling.

[0037] like Figure 2 As shown, compared with the Con group, the proportion of EdU-labeled cells in the PGD2 group was significantly increased (P < 0.05), while the proportion of EdU-labeled cells in the PGD2 + CAMK2 inhibitor group (PGD2 + Ci) was significantly decreased (P < 0.05); compared with the PGD2 group, the proportion of EdU-labeled cells in the PGD2 + Ci group was significantly decreased (P < 0.05). These results indicate that PGD2 (0.1 μM) has a good proliferative effect on rumen parietal cells.

[0038] Example 2

[0039] Dissolve 1 mg of PGD2 powder (Sigma, USA) in 1 mL of DMSO until fully dissolved, then transfer to a centrifuge tube containing 9 mL of DMSO. Mix well and aliquot into 1.5 mL centrifuge tubes (1 mL per tube). Store at -20°C. Before each use, take 1 mL of the solution, add 79 mL of physiological saline, mix well, and then administer perfusion at a rate of 1.6 mL / kg body weight, which is equivalent to an infused PGD2 concentration of 20 μg / kg body weight.

[0040] Fourteen healthy, well-grown, and similarly sized 11-day-old Hu sheep lambs were selected for the experiment. After separation from their mothers, the lambs were fed prepared sheep milk powder and, after a three-day pre-feeding period, were randomly assigned to two groups. One group received PGD2 orally at 1.6 mL / kg body weight (treatment group, PGD2; n = 7; the in vivo perfusion concentration was calculated based on the optimal PGD2 concentration from cell culture). The other group received the same volume of physiological saline orally (control group, Con; n = 7). Oral administration was performed at 8:00 AM daily, and the volume was adjusted weekly based on body weight (maintaining 1.6 mL / kg body weight).

[0041] During the administration period, lambs had free access to goat milk and were not fed solid feed. The goat milk powder used in the experiment was purchased from Shaanxi Yangyangxiang Dairy Co., Ltd., and was prepared before feeding according to a ratio of goat milk powder to water of 1:10. The prepared water temperature was 60℃, and after naturally cooling to 37-40℃, the lambs were fed using a milk bucket with a nipple. Feeding was done four times a day (07:00, 12:00, 17:00, and 22:00), each feeding lasting half an hour to prevent diarrhea caused by excessively low milk temperature. After feeding, the milk bucket was rinsed with clean water and sterilized by soaking in boiling water every 3 days. The amount of milk consumed by the lambs was recorded daily, and their weight was measured weekly before the morning feed. Before the experiment began, there was no significant difference in weight between the two groups of lambs.

[0042] During the experiment, the milk intake of the control group and the PGD2 group was measured daily, and the weight changes of lambs in the control group and the experimental group were monitored weekly. The effect of PGD2 administration on the average daily weight gain of lambs was also measured. The results are shown in Table 1.

[0043] Table 1. Effect of PGD2 administration on the average daily weight gain of lambs

[0044]

[0045] Note: Data are expressed as mean ± standard error, n = 6; ADG, average daily weight gain.

[0046] As shown in Table 1, compared with the control group, gavage administration of PGD2 promoted the average daily weight gain of lambs aged 28-35 days, but had no significant effect on the average daily weight gain of lambs throughout the entire experimental period, indicating that gavage administration of PGD2 did not affect the normal feed intake of lambs.

[0047] Example 3

[0048] Effects of PGD2 oral administration on rumen organ index and rumen epithelial papilla morphology in lambs:

[0049] Lambs from Example 2 were slaughtered and sampled at 42 days of age. The rumen was immediately separated after slaughter, and the contents of the rumen were removed and used for weighing. Three 1×1 cm pieces from the abdominal sac of the rumen were taken. 2The density of rumen papillae was measured using a 2.5x magnifying glass (MG3B-1A, Shanghai). Fifteen rumen papillae were collected from each piece of rumen epithelium from each sheep to determine the length and width of the papillae. The surface area of ​​the papillae was measured per cm². 2 The calculation is nipple length × width × density (nipple count / cm). 2 ×2; Rumen tissue from the rumen sac of each lamb was selected for morphological analysis. Rumen tissue samples fixed in 4% paraformaldehyde were embedded in paraffin, cut into 6μm thick slices, and stained with hematoxylin and eosin. The rumen epithelium was measured using Image Pro Plus (MediaCybernetics, USA) software. Results are shown in Table 2 and... Figure 3 As shown.

[0050] Table 2. Effects of PGD2 gavage on rumen weight and rumen epithelial papillary morphology in lambs.

[0051]

[0052] Note: Data are presented as mean ± standard error, n = 7

[0053] From Table 2 and Figure 3 It was found that PGD2 administration significantly increased the length, width, and total thickness of rumen epithelial papillae. These results indicate that PGD2 administration can effectively promote rumen morphological development.

[0054] Example 4

[0055] To determine the effect of PGD2 on rumen parietal cell proliferation, the relative expression levels of cyclin-related gene mRNAs in rumen parietal cells were detected. RNA was extracted from rumen tissue samples from Example 2, and the relative expression levels of mRNAs were detected by real-time quantitative PCR. The results are as follows: Figure 4 As shown.

[0056] Depend on Figure 4 It can be seen that, compared with the Con group, the relative expression level of CCNE mRNA in the PGD2 group was significantly increased (P < 0.05).

[0057] Example 5

[0058] To determine the effect of PGD2 on rumen parietal cell Ca 2+ The influence of the signaling pathway was detected in Ca. 2+ Relative expression levels of mRNAs related to signaling pathways. RNA was extracted from rumen tissue samples used in Example 2, and the relative expression levels of mRNAs were detected by real-time quantitative PCR. Results are as follows: Figure 5 As shown.

[0059] Depend on Figure 5It can be seen that, compared with the Con group, the PGD2 group and the Ca group... 2+ The relative expression level of the mRNA of the CAMK2 gene, which is closely related to the signaling pathway, was significantly increased (P < 0.05).

[0060] In summary, PGD2 can be administered orally to young ruminants in addition to milk replacer / concentrate starter feed. It effectively promotes rumen morphological development in young ruminants, and by promoting rumen motility and function, it ensures the digestive and absorptive function of the rumen in lambs and other young ruminants, which is conducive to the intake of starter feed, thereby shortening the weaning time of lambs and increasing the supply of mutton to the market. PGD2 can be used to prepare drugs that promote rumen development in young ruminants.

Claims

1. Application of PGD2 in promoting rumen development in young ruminants.

2. Application of PGD2 in promoting rumen parietal cell proliferation, mediating and regulating rumen development, promoting digestion of starter feed, achieving early weaning, and reducing weaning stress in young ruminants.

3. Application of PGD2 in the preparation of drugs that promote rumen development in young ruminants.

4. The application according to any one of claims 1-3, characterized in that, The young ruminants are lambs or calves.

5. The application according to claim 4, characterized in that, The lambs or calves referred to are those within six weeks after birth and after weaning.

Images