A natural inducer for promoting the synchronization of estrus in cows, preparation method and application

Abstract

The present application relates to the technical field of livestock breeding regulation, and discloses a natural inducer for promoting the estrus synchronization of cows, a preparation method and application, comprising the following steps: first, collecting natural musk of adult male forest musk deer and uniformly mixing the same to obtain musk raw materials; then, adding the musk raw materials into a solvent to obtain a solid-liquid mixture; then, placing the solid-liquid mixture in an ultrasonic device for ultrasonic extraction, so that the active ingredients in the musk raw materials are dissolved; finally, removing the precipitate and collecting the supernatant to obtain the natural inducer. The chemical signal is transmitted to the hypothalamus through the olfactory pathway, avoiding the pain stress and the risk of inflammation at the injection site caused by muscle injection; at the same time, the reproductive cycle is regulated by activating the neuroendocrine system of the cow itself, rather than directly supplementing exogenous hormones, thereby fundamentally eliminating the hidden danger of exogenous hormone residues in cow's milk or beef, meeting the requirements of modern livestock farming for green and healthy breeding.

Description

Technical Field

[0001] This invention relates to the field of livestock reproductive regulation technology, specifically to a natural inducer for promoting estrus synchronization in cows, its preparation method, and its application. Background Technology

[0002] In modern intensive livestock farming, estrus regulation technology in cattle is a key support for achieving efficient mass production. To shorten calving intervals, optimize genetic progression, and reduce reliance on artificial observation of natural estrus, the industry has established a reproductive management system centered on hormone programming. This system primarily uses a combination of exogenous hormones such as gonadotropin-releasing hormone, prostaglandins, and intravaginal progesterone release devices to artificially control the timing of estrus and ovulation in cows, achieving synchronized estrus or timed insemination. While this management model has improved reproductive efficiency to some extent, it still faces many challenges in practical application.

[0003] Current hormone regulation methods are generally invasive procedures. Frequent intramuscular injections not only cause direct pain stress in cows but may also lead to inflammatory reactions at the injection site. Furthermore, long-term placement of vaginal plugs can easily induce local reproductive tract infections, thus affecting conception rates. More importantly, traditional methods rely on directly supplementing animals with high concentrations of exogenous synthetic hormones. This not only disrupts the cow's own endocrine balance but also inevitably introduces the risk of hormone residues into milk or beef products, failing to meet the stringent requirements of modern society for green and healthy livestock products. Therefore, exploring a non-invasive regulation method that avoids physical damage and eliminates the risk of hormone residues has become an urgent need for the industry's development.

[0004] Biostimulation methods utilize chemical signals to regulate the reproductive cycle through the olfactory pathway, offering the natural advantages of being stress-free and residue-free. Natural musk, a traditional and precious Chinese medicinal material, primarily originates from the secretions of the musk glands of musk deer such as the forest musk deer. It contains various active ingredients, including macrocyclic ketones, steroids, and proteins. Pharmacological studies have shown that musk and its characteristic component, muscone, have significant effects on stimulating the mind and spirit, bidirectionally regulating the central nervous system, and positively impacting the cardiovascular and reproductive systems. However, currently, there are no comprehensive reports or applications of using the volatile odor substances of natural musk as exogenous signals to activate the hypothalamus, pituitary gland, and gonadal axis in cows through the olfactory nerve pathway, thereby initiating an endogenous reproductive program to replace traditional hormone injection-based estrus synchronization techniques. Summary of the Invention

[0005] To address the shortcomings of existing technologies, this invention provides a natural inducing agent for promoting estrus synchronization in cows, its preparation method, and its application, thus solving the problem that existing technologies rely heavily on invasive procedures.

[0006] To address the above problems, the present invention provides the following technical solution: First aspect This invention provides a method for preparing a natural inducing agent to promote estrus synchronization in cows, using the following technical solution: A method for preparing a natural estrus inducer to promote estrus synchronization in cows includes the following steps: First, natural musk from adult male musk deer is collected and mixed evenly to obtain musk raw material; then, the musk raw material is added to a solvent to obtain a solid-liquid mixture; next, the solid-liquid mixture is placed in an ultrasonic device for ultrasonic extraction to dissolve the active ingredients in the musk raw material; finally, the ultrasonically extracted mixture is centrifuged to remove the precipitate and collect the supernatant to obtain the natural inducer.

[0007] By adopting the above technical solution, the mixing of multi-headed musk deer raw materials can eliminate physiological differences between individuals, ensuring that the prepared inducer has stable biological activity between batches; the cavitation effect generated by ultrasound propagation in liquid can destroy the microstructure of musk particles, accelerate the mass transfer process at the solid-liquid interface, and promote the rapid release of macrocyclic ketones and steroids in musk from the glandular secretion matrix into the solvent; at the same time, the centrifugation separation step effectively removes undissolved tissue residues and impurities, obtaining a clear and transparent supernatant, avoiding mechanical obstruction or foreign body irritation to the animal's respiratory tract during subsequent nasal administration.

[0008] Preferably, the solvent used in the step is a 5% (w / v) aqueous solution of dimethyl sulfoxide.

[0009] By adopting the above technical solution, the extraction efficiency of key active substances such as muscone in musk is significantly improved by utilizing the excellent permeability of dimethyl sulfoxide and its ability to dissolve fat-soluble substances. At the same time, the low concentration ratio of 5% takes into account biosafety and avoids chemical burns or irritation to the nasal mucosa of animals caused by high concentrations of organic solvents.

[0010] Preferably, the ratio of musk raw material to solvent in the step is: 1g to 4g of musk raw material is added to every 100mL of solvent.

[0011] By adopting the above technical solution, the effective concentration range of the inducer was established. Extracts within this concentration range, when subsequently diluted, can provide sufficient odor molecule stimulation to meet the threshold required for estrus induction, thus ensuring the stability of the technical effect.

[0012] Preferably, the parameters for ultrasonic extraction in the steps are set as follows: ultrasonic power 200W, ultrasonic frequency 40KHz, and processing time 30min; the parameters for centrifugation are set as follows: rotation speed 5000r / min and centrifugation time 10min.

[0013] By adopting the above technical solution, specific ultrasonic power and frequency can generate appropriate cavitation intensity, which can not only fully break the matrix, but also avoid the degradation of heat-sensitive active ingredients due to local overheating; combined with specific centrifugation parameters, it ensures the complete sedimentation of suspended particles and improves the purity of the formulation.

[0014] Second aspect This invention provides a natural inducing agent to promote estrus synchronization in cows, employing the following technical solution: A natural estrus inducer that promotes estrus synchronization in cows, the natural inducer being prepared by the preparation method described in the first aspect above.

[0015] By adopting the above technical solution, a liquid formulation containing natural musk active ingredients was obtained. This natural inducer fully retains the complex chemical composition of musk, can more comprehensively simulate biochemical signals under natural conditions, and has the characteristics of stable components, high bioavailability, and convenient use, providing a material basis for subsequent non-injection-based regulation of animal reproductive cycles.

[0016] Third aspect This invention provides a method for promoting estrus synchronization in cows using the aforementioned natural inducers, employing the following technical solution: An application method for promoting estrus synchronization in cows using the aforementioned natural inducer, for non-disease treatment purposes, includes the following steps: first, selecting multiparous Holstein cows that are not in estrus as target cows; then, preparing the natural inducer into a working solution; and finally, applying the working solution into the nostrils of the target cows via nasal spray.

[0017] By employing the above technical solution, this invention establishes a non-invasive estrus synchronization regulation system based on the olfactory pathway. Its mechanism of action is as follows: volatile chemical signal molecules in natural musk, after being sprayed into the nasal cavity, bind to olfactory receptors in the vomeronasal organ or the main olfactory system, generating nerve impulses. These nerve signals are transmitted via the olfactory nerve to the hypothalamic-limbic system of the central nervous system, activating the gonadotropin-releasing hormone (GnRH) pulse generator in the hypothalamus. Changes in the frequency and amplitude of the GnRH pulses further act on the anterior pituitary gland, stimulating the secretion and release of follicle-stimulating hormone (FSH) and luteinizing hormone (LH). The elevated FSH and LH levels in the blood synergistically act on the ovaries, breaking the ovarian quiescent state, promoting the recruitment, selection, and maturation of dominant follicles, and ultimately inducing ovulation and corpus luteum formation, thus macroscopically manifesting as the synchronous occurrence of estrus behavior in cows. This method avoids traditional intramuscular injection, reduces stress on cows, meets animal welfare requirements, and utilizes the organism's own neuroendocrine regulatory mechanisms, reducing the risk of exogenous hormone residues.

[0018] Preferably, the steps for screening multiparous Holstein dairy cows that are not in estrus include: selecting multiparous Holstein dairy cows that are 60 to 90 days postpartum, and confirming by rectal examination or B-ultrasound examination that the cow's ovaries have no active corpus luteum and no dominant follicles.

[0019] By adopting the above technical solution, the applicable subjects were strictly defined, excluding individuals who are pregnant, in estrus, or suffering from reproductive system diseases, thus ensuring the accuracy of the induction effect and eliminating the interference of pathological factors on the experimental results.

[0020] Preferably, the step of preparing the working solution from the natural inducer is as follows: select 4g of natural inducer as the stock solution, dilute the stock solution with pure water by 10 times the volume to obtain the working solution.

[0021] By employing the above technical solution, the optimal drug concentration for Holstein dairy cows was determined. At this specific concentration, odor molecules can effectively cover the receptor areas on the nasal mucosa, generating a sufficiently strong stimulation signal, while avoiding olfactory fatigue or reduced adaptability caused by excessive concentration, thus maximizing induction efficiency.

[0022] Preferably, the step of applying the working solution into the nostrils of the target cow by nasal spray specifically includes: starting the application on the 10th day after the end of the previous estrus cycle of the target cow, spraying the target cow twice a day at 8:00 and 20:00, for 10 consecutive days.

[0023] By employing the above-mentioned technical solutions, the timing and window of action for intervention were precisely identified. Day 10 typically falls in the mid-to-late luteal phase, at which point intervention can effectively intervene in the recruitment process of a new wave of follicles. The twice-daily stimulation frequency simulates continuous exposure to biochemical signals in nature, maintaining the sensitivity of the central nervous system to stimulation signals. Furthermore, the continuous treatment for 10 days covers the critical period of follicle development, causing endocrine levels to fluctuate regularly, enabling the treated cows to exhibit estrus behavior within a specific timeframe, significantly improving the estrus synchronization rate.

[0024] This invention provides a natural estrus inducer for promoting estrus synchronization in cows, its preparation method, and its application. It has the following beneficial effects: 1. This invention proposes a novel non-invasive approach to regulating estrus in cows, significantly improving animal welfare and ensuring product safety. Compared to traditional cloprostenol or progesterone suppositories, this invention utilizes the olfactory pathway to transmit chemical signals to the hypothalamus, avoiding the pain stress and injection site inflammation risks associated with intramuscular injections. Furthermore, this method regulates the reproductive cycle by activating the cow's own neuroendocrine system, rather than directly supplementing with exogenous hormones, thus fundamentally eliminating the potential for exogenous hormone residues in milk or beef, meeting the requirements of modern livestock farming for green and healthy breeding.

[0025] 2. This invention employs an ultrasound-assisted extraction process with a specific concentration of dimethyl sulfoxide aqueous solution as a solvent. By utilizing the cavitation effect of ultrasound to disrupt the microstructure of musk gland secretions, the solubility and extraction rate of lipid-soluble macrocyclic ketone active substances such as muscone are significantly improved. This preparation method not only solves the problem of the poor water solubility of the effective components of natural musk, but also removes impurities through centrifugation, obtaining a clear and homogeneous liquid preparation, ensuring the absorption efficiency and safety of the nasal mucosa during subsequent nasal administration.

[0026] 3. This invention significantly improves the synchronization of estrus and the level of reproductive hormones in cows. Experiments show that intervention during the luteal phase and continuous administration of a specific concentration of musk inducer can effectively induce pulsatile secretion of luteinizing hormone and estradiol, accelerating the recruitment and maturation of dominant follicles in the ovary. This application scheme makes the estrus behavior of cows in the treatment group more concentrated in time and the ovulation signs more obvious, which can effectively assist farms in realizing batch estrus detection and artificial insemination, improving the reproductive management efficiency of large-scale farming. Attached Figure Description

[0027] Figure 1 This is a schematic diagram illustrating the natural musk collection process of the present invention. Figure 2 This is a graph showing the estrus hormone levels and ovulation count in mice according to the present invention. Figure 3 This is a diagram illustrating the effects of bovine estrus behavior, follicular ultrasound, and musk on bovine hormone changes, as presented in this invention. Detailed Implementation

[0028] The technical solution of the present invention will now be clearly and completely described with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0029] The main raw materials and reagents used in the following examples and comparative examples have the following sources and specifications. Reagents not specifically mentioned are all commercially available analytical grade or higher products. Natural musk was collected from the musk sac secretions of adult male musk deer. Dimethyl sulfoxide (DMSO) CAS number: 67-68-5. Pregnant mare serum gonadotropin (PMSG) CAS number: 9002-70-4. Human chorionic gonadotropin (hCG) CAS number: 9002-61-3. Hyaluronidase CAS number: 9001-54-1. All hormone detection kits were commercially available, universal enzyme-linked immunosorbent assay (ELISA) kits.

[0030] Preparation Example 1 This preparation example provides a method for preparing a natural inducing agent that promotes estrus synchronization in cows.

[0031] Healthy adult male musk deer were selected, and natural musk was collected from them in March using a live musk extraction method. The collected musk was then cleaned of impurities and mixed thoroughly as raw material. (See attached document for reference.) Figure 1 100 mL of a 5% (v / v) dimethyl sulfoxide (DMSO) aqueous solution was used as the extraction solvent. 4 g of the above musk raw material was weighed and added to the solvent to obtain a solid-liquid mixture. This mixture was placed in an ultrasonic cleaner and subjected to ultrasonic extraction for 30 min at an ultrasonic power of 200 W and an ultrasonic frequency of 40 kHz, utilizing the cavitation effect to promote the complete dissolution of the active ingredients. After the treatment, the mixture was placed in a centrifuge and centrifuged at 5000 r / min for 10 min. The bottom precipitate was discarded, and the supernatant was collected to obtain the natural inducer.

[0032] Preparation Example 2 This preparation example provides a method for preparing a natural inducing agent that promotes estrus synchronization in cows.

[0033] Except for the weighing of 2g of natural musk raw material, the source of other raw materials, solvent type and amount (100mL 5% DMSO), ultrasonic extraction parameters (200W, 40KHz, 30min), and centrifugation parameters (5000r / min, 10min) were exactly the same as in Preparation Example 1. The supernatant was finally collected to obtain the natural inducer.

[0034] Preparation Example 3 This preparation example provides a method for preparing a natural inducing agent that promotes estrus synchronization in cows.

[0035] Except for the weighing of 1g of natural musk raw material, the sources of other raw materials, solvent types and amounts were: 100mL, 5%, DMSO; the ultrasonic extraction parameters were 200W, 40KHz, 30min, and the centrifugation parameters were 5000r / min, 10min; all were exactly the same as in Preparation Example 1. Finally, the supernatant was collected to obtain the natural inducer.

[0036] Comparative Preparation Example 1 This comparative preparation example provides a method for preparing a blank solvent for a control experiment.

[0037] Take 100 mL of a 5% (v / v) dimethyl sulfoxide (DMSO) aqueous solution without adding any musk. Place the solvent in an ultrasonic cleaner and treat it for 30 min at an ultrasonic power of 200 W and an ultrasonic frequency of 40 kHz. Then, centrifuge it at 5000 r / min for 10 min and collect the liquid as a blank control solution.

[0038] Example

[0039] A method for preparing a natural estrus inducer to promote estrus synchronization in cows, characterized by comprising the following steps: Step S1: Collect natural musk from adult male forest musk deer and mix them evenly to obtain musk raw materials; Step S2: Add the musk raw material to a solvent to obtain a solid-liquid mixture; the solvent is a 5% (w / v) dimethyl sulfoxide aqueous solution; the ratio of the musk raw material to the solvent is: 1g to 4g of the musk raw material is added to every 100mL of the solvent.

[0040] Step S3: Place the solid-liquid mixture in an ultrasonic device for ultrasonic extraction to dissolve the active ingredients in the musk raw material; the parameters for ultrasonic extraction are set as follows: ultrasonic power 200W, ultrasonic frequency 40KHz, and processing time 30min.

[0041] Step S4: Centrifuge the ultrasonically extracted mixture to remove the precipitate and collect the supernatant to obtain the natural inducer; the centrifugation parameters are set as follows: rotation speed 5000 r / min, centrifugation time 10 min.

[0042] A natural inducer to promote estrus synchronization in cows, wherein the natural inducer is prepared by the above-described preparation method.

[0043] An application method for promoting estrus synchronization in cows using natural inducers includes the following steps: Step A: Select multiparous Holstein dairy cows that are not in estrus as target cows; the specific implementation of Step A is as follows: select multiparous Holstein dairy cows that are 60 to 90 days postpartum, and confirm by rectal examination or B-ultrasound examination that the cows' ovaries have no active corpus luteum and no dominant follicles.

[0044] Step B: Prepare the working solution from the natural inducer; the specific implementation of step B is as follows: select 4g of the natural inducer added in step S2 as the stock solution, and dilute the stock solution with pure water by 10 times the volume to obtain the working solution.

[0045] Step C: Apply the working solution into the nostrils of the target cow via nasal spray. Specifically, Step C is implemented by starting application on the 10th day after the end of the target cow's previous estrous cycle, administering the nasal spray twice daily at 8:00 AM and 8:00 PM for 10 consecutive days.

[0046] Application Example 1 This application example verifies the effects of different concentrations of natural inducers on estrus and ovulation in mice.

[0047] Six-week-old female Kunming mice in good condition were randomly divided into four groups of six mice each. The groups were: a high-concentration group (using the natural inducer prepared in Preparation Example 1), a medium-concentration group (using the natural inducer prepared in Preparation Example 2), a low-concentration group (using the natural inducer prepared in Preparation Example 3), and a blank control group (using the blank solvent prepared in Comparative Preparation Example 1).

[0048] Before use, the corresponding natural inducers or blank solvents for each group were diluted 100-fold by volume with ultrapure water to prepare working solutions. Mice were treated twice daily, at 8:00 AM and 8:00 PM, with 1 μL instilled into each nostril (total 2 μL) for 15 consecutive days. Estrus status was monitored daily using vaginal smears during the treatment period. After the experiment, blood was collected from the eyes of mice in estrus. After coagulation at 37°C, serum was separated by centrifugation, and the levels of follicle-stimulating hormone (FSH), luteinizing hormone (LH), estradiol (E2), progesterone (P), and anti-Müllerian hormone (AMH) were detected using an ELISA kit.

[0049] Another batch of age-matched female Kunming mice were treated according to the above grouping and method. On day 13, they were injected with 10 IU of MSG, and 48 hours later, they were injected with 10 IU of hCG. They were sacrificed 14 hours later. The oviducts were collected, the cumulus-oocyte mass was released, and the number of ovulations was counted after digestion with hyaluronidase.

[0050] Experimental results are as follows Figure 2As shown, compared with the blank control group, the levels of FSH, LH, E2, and P in the blood of mice in each experimental group treated with the nasal drop natural inducer were significantly increased during estrus, and this increase was dose-dependent. Among them, the high-concentration group (derived from Preparation Example 1) showed the most significant increase in hormone levels, and the number of ovulations was significantly higher than that of other groups. Therefore, the high-concentration natural inducer prepared in Preparation Example 1 has the best biological activity, and this concentration was selected for subsequent application experiments on estrus synchronization in Holstein dairy cows.

[0051] Application Example 2 This application example verifies the effectiveness of natural inducers in estrus synchronization in Holstein dairy cows.

[0052] Multiparous Holstein dairy cows aged 60-90 days postpartum, in good condition, with normal reproductive records, and confirmed to be in non-estrus (without active corpus luteum and dominant follicles) were randomly divided into a treatment group and a control group, with 10 cows in each group. They were fed under the same feeding and management conditions for 30 days. The treatment group used the natural inducing agent prepared in Example 1 (raw material concentration 4g / 100mL), while the control group used the blank solvent prepared in Comparative Example 1.

[0053] The natural inducer from Preparation Example 1 and the blank solvent from Comparative Preparation Example 1 were diluted 10 times by volume with pure water and placed in clean spray bottles. Starting on the 10th day after the end of the cow's previous estrus cycle, the solution was sprayed into the cow's nasal cavity twice a day at 8:00 and 20:00, with about 1-2 mL per nostril each time, for 10 consecutive days.

[0054] Estrus behavior was observed and recorded during the predicted estrus period. Blood samples were collected from the tail vein on days 0, 6, and 11 of the experiment to detect serum FSH, LH, and E2 levels. At the same time, ultrasound was used to monitor the development of dominant follicles and corpus luteum formation in the ovary.

[0055] The results showed that the estrus behavior in the treatment group was more concentrated in time and the signs of estrus were more typical, such as... Figure 3 As shown in A and B, there is redness and swelling of the vulva, as well as mucus discharge. Figure 3 As shown in Figure C, ultrasound imaging confirmed that the dominant follicles in the treated group developed rapidly, forming clear functional corpora lutea after ovulation, and their development was superior to that in the control group. Figure 3 As shown in Figure D, hormone detection results indicate that the serum estradiol (E2) level in the treated group of dairy cows increased rapidly in the later stage of treatment and induced the synchronous appearance of the luteinizing hormone (LH) peak, which was significantly higher than that in the control group. This suggests that the natural inducer can effectively activate the hypothalamus, pituitary gland and gonadal axis and promote synchronous estrus.

[0056] Although embodiments of the invention have been shown and described, it will be understood by those skilled in the art that various changes, modifications, substitutions and alterations can be made to these embodiments without departing from the principles and spirit of the invention, the scope of which is defined by the appended claims and their equivalents.

Claims

1. A method for preparing a natural estrus inducer to promote estrus synchronization in cows, characterized in that, Includes the following steps: Step S1: Collect natural musk from adult male forest musk deer and mix them evenly to obtain musk raw materials; Step S2: Add the musk raw material to a solvent to obtain a solid-liquid mixture; Step S3: Place the solid-liquid mixture in an ultrasonic device for ultrasonic extraction to dissolve the active ingredients in the musk raw material; Step S4: Centrifuge the mixture after ultrasonic extraction to remove the precipitate and collect the supernatant to obtain the natural inducer.

2. The method for preparing a natural estrus inducer for promoting estrus synchronization in cows according to claim 1, characterized in that: In step S2, the solvent is a 5% (w / v) aqueous solution of dimethyl sulfoxide.

3. The method for preparing a natural estrus inducer for promoting estrus synchronization in cows according to claim 1, characterized in that: In step S2, the ratio of the musk raw material to the solvent is: 1g to 4g of the musk raw material is added to every 100mL of the solvent.

4. The method for preparing a natural inducing agent to promote estrus synchronization in cows according to claim 1, characterized in that: In step S3, the parameters for ultrasonic extraction are set as follows: ultrasonic power 200W, ultrasonic frequency 40KHz, and processing time 30min.

5. The method for preparing a natural estrus inducer for promoting estrus synchronization in cows according to claim 1, characterized in that: In step S4, the parameters for centrifugation are set as follows: rotation speed 5000 r / min, centrifugation time 10 min.

6. A natural inducing agent to promote estrus synchronization in cows, characterized in that, The natural inducer is prepared by the preparation method according to any one of claims 1 to 5.

7. A method for promoting estrus synchronization in cows using a natural inducer, wherein the natural inducer for promoting estrus synchronization in cows according to claim 6 is characterized in that, Includes the following steps: Step A: Select multiparous Holstein dairy cows that are not in estrus as target cows; Step B: Prepare the working solution from the natural inducer; Step C: Apply the working solution into the nostrils of the target cow via nasal spray.

8. The method for promoting estrus synchronization in cows using natural inducers according to claim 7, characterized in that: The specific implementation method of step A is as follows: Select multiparous Holstein dairy cows that are 60 to 90 days postpartum, and confirm by rectal examination or B-ultrasound examination that the cows' ovaries have no active corpus luteum and no dominant follicles.

9. The method for promoting estrus synchronization in cows using natural inducers according to claim 7, characterized in that: The specific implementation method of step B is as follows: select 4g of natural inducer added in step S2 as the stock solution, and dilute the stock solution with pure water by 10 times the volume to obtain the working solution.

10. The method for promoting estrus synchronization in cows using natural inducers according to claim 7, characterized in that: The specific implementation method of step C is as follows: start applying the medication on the 10th day after the end of the previous estrus cycle of the target cow, and spray the target cow with the nasal cavity twice a day at 8:00 and 20:00, for 10 consecutive days.

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