Accurate gavage device and method for early life

Abstract

The invention discloses a precise gavage device and method for the early life stage, relates to the technical field of biological experiment equipment, and solves the problems that an existing gavage method and tool are high in damage rate and death rate, strong in stress reaction and inaccurate in administration dosage. The device comprises an injector module and a flexible lavage catheter, wherein the output end of the injector module is detachably connected with the flexible lavage catheter; the output end of the flexible gavage catheter is arc-shaped, and the end part is passivated. The flexible gavage catheter comprises a connecting sleeve and a hose, and the syringe interface module, the connecting sleeve and the hose are connected in sequence; and the hose is arc-shaped. And the output end of the hose is spherical or hemispherical. According to the design of the integrated gavage device, operation damage and animal stress can be remarkably reduced, and the dosage accuracy of dosing or feeding is ensured, so that the success rate of long-term and continuous gavage experiments and the reliability of data are effectively improved.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of biological experiment equipment, in particular to a precise gavage device and gavage method for early life. BACKGROUND

[0002] Early life is recognized as a decisive window for individual growth and development, which has a profound impact on the lifelong health trajectory of individuals. The core feature of this period is the establishment of gut microbiota and the maturation of the immune system, and these two processes are closely intertwined and occur simultaneously (Lin, C.; Lin, Y.; Zhang, H.; Wang, G.; Zhao, J.; Zhang, H.; Chen, W. Intestinal ‘Infant-Type’ Bifidobacteria Mediate Immune System Development in the First 1000 Days of Life. Nutrients 2022, 14, 1498. https: / / doi.org / 10.3390 / nu14071498). Although the precise composition of the prenatal microbial environment is still controversial, it is a consensus that the newborn's gut is rapidly colonized by a large number of microorganisms after delivery. In this process, Bifidobacterium plays a key role as a pioneer and cornerstone species. However, the specific regulatory mechanisms behind it remain to be elucidated. Bifidobacterium

[0003] However, directly exploring these issues in human infants faces many challenges. First, human studies are difficult to exclude the interference of uncontrollable variables such as environmental microbial intake. Second, due to ethical and operational limitations, the collection of infant samples (such as limited to peripheral blood) and the detection window period are strictly limited, making it difficult to comprehensively and dynamically assess systemic effects. In view of this, neonatal mouse models provide an ideal research paradigm for analyzing these complex human biological problems. In the fields of life sciences, pharmacology and nutrition, neonatal rodent models have become an indispensable research tool due to their high similarity in simulating human infant growth and development, disease progression, drug metabolism and nutritional response. This model not only simplifies and focuses on complex interaction relationships, but more importantly, it allows researchers to systematically explore the deep effects of Bifidobacterium colonization on early life immunity and intestinal health and its molecular mechanisms through precise artificial feeding, strain colonization and drug intervention under strictly controlled conditions.

[0004] Currently, gavage operation on neonatal rodents mainly relies on the following ways: ​Simulated nursing / sucking method: This method attempts to mimic natural nursing behavior by using a dropper or micropipette to drop liquid onto the animal's mouth, encouraging it to suck on its own. However, this method has fundamental flaws that cannot be overcome: First, it is impossible to accurately control the amount of liquid administered in a single feeding, and the actual intake of the animal is unknown, which is not conducive to experiments that require strict dosing or nutritional comparison studies; second, animals are prone to swallowing large amounts of air into their stomachs during sucking, causing severe abdominal distension and indigestion, often leading to the failure of the experiment; finally, this method is time-consuming and inefficient, making it difficult to apply to large-scale animal experiments. Therefore, in order to ensure the accuracy of quantitative and operational efficiency, researchers generally turn to forced gavage.

[0005] Metal gavage needle: After determining the use of gavage, researchers will first try to use commercially available standardized metal gavage needles. Such tools are usually designed for adult animals, and their hard material and fixed size can easily cause scratches or even perforations in the mouth, throat, and esophagus of newborn animals, which are small in size, delicate in structure, and have a curved and slender esophagus, leading to infection, refusal to eat, and ultimately death. This not only raises ethical issues, but also causes a huge waste of experimental animals and research costs.

[0006] Temporarily modified catheter: Some researchers will use intravenous catheter or capsule shell for temporary modification. However, this modification lacks standardization, and the hardness, tip treatment, and size selection of the catheter often depend on the personal experience of the operator, making it difficult to ensure consistency and safety. More importantly, newborn animals will experience significant growth in size and internal organ dimensions (such as esophageal length and internal diameter) within a few days after birth. Any single size of gavage tool available cannot dynamically adapt to such rapid physiological changes, resulting in significant fluctuations in operation success rate and safety at different stages of the experimental period.

[0007] In summary, the existing technology presents a dilemma: in terms of method selection, the sucking method is not desirable due to its inability to quantify, while the forced gavage method is the inevitable choice to achieve precise dosing; but in terms of specific implementation, the existing gavage tools (whether metal needles or temporarily modified catheters) cannot safely and effectively adapt to the unique and dynamically changing physiological structure of newborn animals.

[0008] The combined deficiencies of these existing technologies and methods collectively lead to the following serious technical problems: High injury rate and high mortality rate: mismatched tools lead to high failure rate of operation, serious injury and death of animals, making it difficult to conduct experiments that require continuous gavage in the neonatal period.

[0009] Strong stress response: pain and discomfort can trigger a strong stress response, releasing hormones such as cortisol, which can severely interfere with the normal physiological state of the animal, becoming a major experimental confounding factor.

[0010] Inaccurate dosing: the difficulty of operation can cause liquid leakage or animal reflux, so that the actual dose into the stomach cannot be guaranteed, directly affecting the accuracy of the experimental results.

[0011] Therefore, there is an urgent need in the art for a technical solution that can systematically solve the problem of precise gavage of newborn mice, which not only provides a safe tool, but also adapts to the rapid growth and physiological characteristics of the animal, thereby ensuring standardization, high success rate and low interference during the operation throughout the experimental period, ensuring the authenticity and reliability of scientific data. SUMMARY

[0012] In order to solve the problems of high damage rate, high mortality rate, strong stress reaction and inaccurate dosing of the existing gavage method and tool mentioned above, a precise gavage device and method for early life are proposed. The present application can be conveniently adapted with a conventional syringe, and the hose is made of polyethylene material with good biocompatibility. The length and thickness of the hose are precisely calculated and designed to accurately match the digestive tract structure of newborn mice which has not yet developed completely, while ensuring that the liquid medicine or nutrient liquid can flow smoothly and unobstructed. The integrated design can significantly reduce the operation damage and animal stress, ensure the accuracy of the dosing or feeding, thereby effectively improving the success rate of long-term and continuous gavage experiments and the reliability of the data.

[0013] The present application proposes a precise gavage device for early life, which specifically comprises a syringe module and a flexible gavage catheter, and the output end of the syringe module is detachably connected with the flexible gavage catheter; the output end of the flexible gavage catheter is arc-shaped, and the end is passivated.

[0014] Further, the flexible gavage catheter comprises a connecting sleeve and a hose, and the syringe interface module, the connecting sleeve and the hose are connected in sequence; the hose is arc-shaped.

[0015] Further, the output end of the hose is spherical or hemispherical.

[0016] Further, the material of the hose is polyethylene.

[0017] A gavage method using the above-mentioned precise gavage device for early life, comprising the following steps: Step one, evaluation and selection: according to the age or weight of the newborn mouse to be operated, select a hose that matches the growth stage of the animal; Step two, assembly and shaping: connect one end of the connecting sleeve with the output end of the syringe module, and connect the other end with the hose to form a nested structure; after assembly, shape the arc of the exposed part of the hose as needed to fit the physiological curvature of the animal.

[0018] Step three, gavage: insert the hose into the esophagus of the newborn mouse; after the hose end reaches the stomach, push the syringe module for gavage.

[0019] The beneficial effects of the precise gavage device and method for early life according to the present application are: (1) The precise gavage device and method for early life according to the present application has extremely high safety and extremely low damage rate: through the design concept of "dynamic adaptation", i.e. selecting a hose with accurate size according to the age, the best matching of the outer diameter of the hose and the inner diameter of the animal esophagus is ensured. Combined with flexible material and smooth head design, mechanical damage to the delicate tissues of the newborn animal is fundamentally avoided, the success rate of gavage operation is increased to nearly 100%, and the animal mortality is significantly reduced.

[0020] (2) The precise gavage device and method for early life according to the present application has high plasticity and adaptability: the hose body is made of PE material and can be conveniently used with a conventional 1 mL syringe. The core technical advantage of this design is that the PE material gives the hose the unique function of manual shaping, allowing the operator to adjust the hose curvature instantly and accurately according to their own gavage habits, angle preferences, and individual differences in the size and physiological curvature of the esophagus of newborn mice. The shaped hose can accurately fit the esophageal wall profile of the animal, ensuring that the best insertion trajectory and angle are maintained under different operating postures (such as vertical or inclined body position), thereby minimizing the mechanical friction and tension stimulation of the esophageal mucosa by the tube, effectively avoiding potential damage such as perforation and scratching caused by mismatch between the instrument and the tissue.

[0021] (3) The precise gavage device and method for early life according to the present application is simple to operate and has high standardization: a clear "selection-use" process is provided, which converts complex empirical operations into standardized technical steps. Even beginners can quickly master it, ensuring consistency between different operators and at different time points within the same experiment, and improving the repeatability of the experiment.

[0022] (4) The precise gavage device and method for early life according to the present application ensures the feasibility of long-term experiments and the reliability of data: as the operation trauma and stress are minimized, it is possible to perform continuous gavage on newborn mice for several weeks. This eliminates the significant physiological disturbance introduced by the operation itself, allowing the experimental results to truly reflect the biological effects of different interventions, greatly improving the reliability and validity of scientific data. BRIEF DESCRIPTION OF DRAWINGS

[0023] The accompanying drawings, which form a part of this application, are included to provide a further understanding of the application and are incorporated in and constitute a part of this specification. The illustrations are shown schematically and are not limiting of the application.

[0024] In the drawings: Figure 1 is a structural schematic diagram of a precise gavage device for early life according to the present application; Figure 2 is a schematic diagram of the hose selection process of the gavage method according to the present application; Figure 3 is a comparison chart of the effects of different gavage methods on the survival rate of neonatal rats; Figure 4 is a comparison chart of the effects of different gavage methods on the weight gain of neonatal rats; Figure 5 is a comparison chart of the effects of different gavage methods on the incidence of diarrhea in neonatal rats; Figure 6 is a comparison chart of the effects of different gavage methods on the feces form (diarrhea severity) of neonatal rats; Figure 7 is a comparison chart of the effects of different gavage methods on the righting reflex of neonatal rats; Figure 8 is a comparison chart of the effects of different gavage methods on the tendency to land reflex of neonatal rats; Figure 9 is a comparison chart of the effects of different gavage methods on the tendency to land reflex of neonatal rats; Figure 10 is a comparison chart of the effects of different gavage methods on the tendency to land reflex of neonatal rats; Figure 11 is a comparison chart of the effects of different gavage methods on the tendency to land reflex of neonatal rats; Figure 12 is a comparison chart of the effects of different gavage methods on the tendency to land reflex of neonatal rats; Figure 13 is a comparison chart of the effects of different gavage methods on the tendency to land reflex of neonatal rats; Figure 14 is a comparison chart of the effects of different gavage methods on the tendency to land reflex of neonatal rats; Figure 15 is a comparison chart of the effects of different gavage methods on the tendency to land reflex of neonatal rats; + T cells in the intestinal tract of neonatal rats after long-term feeding experiments using the method of the present application. Figure 16 is a comparison chart of the proportion of Th1 cells in the intestinal tract of newborn rats in different feeding groups after long-term feeding experiments using the method of the present application; + T cell proportion comparison chart; Figure 17 is a comparison chart of the proportion of Th cells in the intestinal tract of newborn rats in different feeding groups after long-term feeding experiments using the method of the present application; Figure 18 is a comparison chart of the proportion of Th17 cells in the intestinal tract of newborn rats in different feeding groups after long-term feeding experiments using the method of the present application; + T cell proportion comparison chart; Figure 19 is a comparison chart of the proportion of Th2 cells in the intestinal tract of newborn rats in different feeding groups after long-term feeding experiments using the method of the present application; + T cell proportion comparison chart Figure 20 is a comparison chart of the proportion of Tc cells in the intestinal tract of newborn rats in different feeding groups after long-term feeding experiments using the method of the present application; Figure 21 is a schematic diagram of the ratio of Treg cells and Th17 cells in the intestinal tract of newborn rats in different feeding groups after long-term feeding experiments using the method of the present application; Figure 22 is a schematic diagram of the ratio of Th1 cells and Th2 cells in the intestinal tract of newborn rats in different feeding groups after long-term feeding experiments using the method of the present application; Figure 23 is a schematic diagram of the ratio of Th cells and Tc cells in the intestinal tract of newborn rats in different feeding groups after long-term feeding experiments using the method of the present application; Figure 24 is a comparison chart of the correlation analysis of the composition of Bifidobacterium species and immune indicators in the intestinal tract of newborn rats in different feeding groups after long-term feeding experiments using the method of the present application; Wherein: 1 - syringe module, 2 - connecting sleeve, 3 - hose. DETAILED DESCRIPTION

[0025] The technical solutions in the embodiments of the present application will be clearly and completely described below with reference to the drawings in the embodiments of the present application. It should be noted that the embodiments in the present application and the features in the embodiments can be combined with each other without conflict, and the described embodiments are only a part of the embodiments of the present application, not all the embodiments. Based on the embodiments in the present application, all other embodiments obtained by those skilled in the art without creative labor fall within the scope of protection of the present application.

[0026] Referring to Figures 1-24 Specifically illustrate the present embodiment. The precise gavage device for early life in the present embodiment specifically comprises a syringe module 1 and a flexible gavage catheter, and the output end of the syringe module 1 is detachably connected with the flexible gavage catheter; the output end of the flexible gavage catheter is arc-shaped, and the end portion is blunted.

[0027] The flexible gavage catheter comprises a connecting sleeve 2 and a hose 3, and the syringe interface module 1, the connecting sleeve 2 and the hose 3 are connected in sequence; the hose 3 is arc-shaped.

[0028] The output end of the hose 3 is spherical or semi-spherical. The material of the hose 3 is polyethylene.

[0029] A gavage method using the precise gavage device for early life described above, comprising the following steps: Step one, evaluation and selection: according to the age or body weight of the neonatal mouse to be operated, select the hose 3 matched with the growth stage; Step two, assembly and shaping: connect one end of the connecting sleeve 2 with the output end of the syringe module 1, and connect the other end with the hose 3 to form a nested structure; after assembly, shape the arc of the exposed part of the hose 3 as needed to fit the physiological curvature of the animal.

[0030] Step three, gavage: insert the hose 3 into the esophagus of the neonatal mouse; after the end of the hose 3 reaches the stomach, push the syringe module 1 to perform gavage.

[0031] For neonatal SD rats, the gavage catheter kit is configured in three stages according to the age: 0-2 days after birth, a hose 3 with an outer diameter of 0.35-0.40 mm, an inner diameter of 0.20-0.25 mm and a length of 1.50-2.00 cm is selected; 3-7 days, a hose 3 with an outer diameter of 0.50-0.55 mm, an inner diameter of 0.25-0.30 mm and a length of 2.00-2.50 cm is used; 8-14 days, a hose 3 with an outer diameter of 0.60-0.65 mm, an inner diameter of 0.30-0.35 mm and a length of 2.50-3.00 cm is upgraded.

[0032] For neonatal C57BL / 6 mice, the gavage catheter kit is configured in three stages according to the age: 0-2 days after birth, a hose 3 with an outer diameter of 0.15-0.20 mm, an inner diameter of 0.05-0.10 mm and a length of 0.50-1.00 cm is selected; 3-7 days, a hose 3 with an outer diameter of 0.35-0.40 mm, an inner diameter of 0.20-0.25 mm and a length of 1.50-2.00 cm is used; 8-14 days, a hose 3 with an outer diameter of 0.50-0.55 mm, an inner diameter of 0.25-0.30 mm and a length of 2.00-2.50 cm is upgraded.

[0033] Example 1: Comparative Verification Study of the Gavage Device in this Example and Existing Technologies This embodiment aims to verify, through direct, multi-dimensional comparative experiments, the advantages of the dynamic adaptation precision gavage system and method described in this invention in terms of safety, effectiveness, and impact on animal welfare compared to existing technologies (such as metal gavage needles and single-size tubing).

[0034] 1. Experimental Design and Methods 1.1 Breeding and feeding of newborn rats Animal experiments were conducted at the Experimental Animal Center of Shenzhen University, and the experimental methods used complied with the EU Guidelines for the Protection of Laboratory Animals (Directive 2010 / 63 / EU). The Experimental Animal Ethics Committee of Shenzhen University reviewed and approved the animal experimental protocol, approval number IACUC-202500072. Eight-week-old SD rats were purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd., and housed in an SPF-grade barrier facility. After entering the barrier, they were randomly assigned to cages and underwent a 7-day acclimatization period. They had free access to food and water daily, with a 12-hour light / dark cycle. The temperature inside the barrier was 25 ± 2℃, and the relative humidity was 60% ± 20%. The growth and reproduction feed was purchased from Jiangsu Xietong Pharmaceutical Biotechnology Co., Ltd. After the acclimatization period, one male and multiple females were housed together for timed mating (1:2 mating). After confirming pregnancy, the female rats were placed in separate cages. The birth date of newborn rats was designated as day 0 (d0), and the ambient temperature was controlled at 30-32℃.

[0035] 1.2 Gavage administration procedure for newborn rats The operation steps of gavage for newborn rats are according to the previous literature reports and make appropriate modifications (Zhu X, Liu J, Zhou S, et al. Technological innovation and breeding of germ-free rats [J]. Experimental Animal Science, 2017, 34(01): 57-61.). First, pinch the neck skin of the newborn rat, fix its body, avoid the head and body struggling and shaking, and make its body keep a straight line. Then, the air in the syringe module 1 and the hose 3 which have sucked the milk is discharged, and then the end of the hose 3 is placed near the mouth of the newborn rat. Push the syringe module 1, and a small amount of milk is dropped into the mouth of the newborn rat, and the swallowing action is observed. Then, the hose 3 is slowly inserted along the upper jaw of the newborn rat. When the gavage hose is in place, slowly and smoothly push the syringe module 1, and the milk is slowly injected, and the change of the milk block in the stomach is observed. When encountering resistance or strong struggling of the newborn rat, the hose 3 should be immediately pulled out and reinserted to prevent injury to the esophagus or misinsertion into the trachea. At the same time, attention should be paid to the angle of insertion to avoid inserting into the contralateral axillary part of the newborn rat to avoid causing abnormal symptoms. After the stomach is completely filled with milk, stop pushing the syringe module 1, and slowly pull out the hose 3. During the whole artificial lactation process, the amount of milk in the stomach should be observed at any time to ensure that the milk is not poured into the intestinal tract. Finally, after wiping the residual milk in the mouth and nose with a cotton swab, gently put the newborn rat into the cage box.

[0036] 1.3 Experimental grouping Take 100 healthy newborn SD rats with a weight difference of ≤1 g, and randomly divide them into 4 groups (25 rats / group) within 6 hours after birth. Each mother mouse only feeds 5 pups to ensure feeding quality. From d0 to d21 (weaning day), artificial gavage infant formula milk is given once every 12 hours, and the dose is calculated as 8 μL / g body weight. The pups are separated from the mother mouse for a short time 20 minutes before gavage to promote gastric emptying and reduce interference. The blank control group does not receive gavage and is naturally breastfed by the mother mouse, but is taken out daily at the same time as the treatment group and is given gentle touch to control stress. The remaining three groups use: ① the precise gavage device of the application; ② a commercially available 6# 5 cm metal gavage needle; ③ a clinical 22G intravenous indwelling needle outer catheter (cut off the needle holder to make a single specification hose).

[0037] 1.4 Survival rate Each group is checked once a day at 8:00 before weaning to accurately count the number of surviving pups.

[0038] 1.5 Growth indicators The body weight of each pup is quickly measured with a 0.01 g precision balance at 8:00 in the morning on d1, d7, d14, and d21.

[0039] 1.6 Fecal evaluation Diarrhea incidence and severity were monitored dynamically according to the color and texture scores of the fecal samples (5: normal feces; 4: slightly soft feces; 3: soft to loose feces; 2: semi-liquid feces; 1: water-like feces) every morning at 8:00 before weaning.

[0040] 1.7 Righting reflex The ability of neonatal rats to turn onto their stomachs was observed at 8:00 in the morning on d7, d14, and d21, with the rats placed on their backs. The performance was scored on a scale of 0 (complete failure) to 5 (quickly turned over and began exploring the surroundings), with three technical repeats (parallel experiments).

[0041] 1.8 Locomotor activity The time taken by neonatal rats to complete a 180° righting turn was recorded at 8:00 in the morning on d7, d14, and d21, with the rats placed head down on a 30° inclined surface. If the time taken was more than 60 s, the test was stopped and recorded as 60 s, with three technical repeats (parallel experiments).

[0042] 1.9 Statistical analysis The experimental data were statistically analyzed and plotted using GraphPad Prism 9.0 and SPSS 26.0, and were expressed as “mean ± standard deviation” or “median-quartile range”. First, the data were subjected to normality test using the Kolmogorov-Smirnov method. For data conforming to normal distribution, one-way analysis of variance (ANOVA) was used for difference analysis. If the data meet the homogeneity of variance requirement, Duncan or Dunnett's (two-tailed) test was used for post-hoc multiple comparisons. If the variance is not equal, Dunnett T3 test is used for multiple comparisons. For non-normally distributed data, Kruskal-Wallis non-parametric test and Dunnett's post-hoc test were used for difference analysis p <0.05 was considered to be significantly different.

[0043] 2. Results and analysis Effect on survival rate (refer to Figure 3 ): As shown in Figure 3 , the survival rate of the blank control group was close to 100%. The survival curve of the inventive group was almost coincident with that of the blank control group, and there was no statistical difference between them, which strongly proved that the inventive method was minimally invasive in physiology and almost did not cause fatal damage. As shown in Figure 3As shown, the group using this invention exhibited an extremely high survival rate of over 95% throughout the entire 21-day experimental period, demonstrating the safety of the method. In contrast, the metal needle group experienced rapid mortality within the first week due to esophageal perforation, infection, and anorexia caused by the procedure, with a 21-day survival rate of less than 30%. Although the single-tube group initially performed better than the metal needle group, as the rats grew, excessively thin tubes caused difficulty in insertion and fluid reflux, or excessively short tubes caused fluid to enter the trachea, resulting in a significantly lower survival rate compared to the group using this invention.

[0044] Effects on growth and development (refer to) Figure 4 ):like Figure 4 As shown, the weight gain curve of the blank control group represents the standard growth pattern of newborn rats of this strain under ideal conditions. The growth curve of the present invention group was highly consistent with that of the blank control group, with no statistically significant difference throughout, indicating that the gavage procedure of the present invention did not have an observable negative impact on the animals' normal nutrient absorption and growth and development. In contrast, the metal needle group and the single-tube group showed significant growth retardation due to continuous gavage trauma, stress response, and possible insufficient nutrient intake, and their average weight was much lower than that of the present invention group in the later stages of the experiment.

[0045] Impact on health (see reference) Figures 5-8 ):like Figures 5-8 As shown, the blank control group performed best in comprehensive health assessments, including diarrhea incidence, diarrhea severity, and mental state (rolling reflex score and geotropism test). The health indicators of the present invention group showed no significant differences compared to the blank control group, and were significantly better than both the metal needle group and the single-tube group. This indicates that the method of the present invention successfully avoids gastrointestinal disturbances and nervous system stress responses caused by improper operation, thus maximizing the maintenance of physiological homeostasis in the animals.

[0046] Conclusion of this embodiment: Through direct comparison with existing mainstream gavage techniques, this embodiment demonstrates, from multiple key dimensions such as survival rate, growth and development, and overall health status, that the dynamic adaptation gavage system and method of this invention can fundamentally solve the problems of high injury and high mortality rates in gavage procedures for newborn rodents, and its technical effects far exceed those of existing technologies. It enables newborn rats under artificial intervention to achieve survival rates, growth and development, and overall health status similar to those in natural lactation. This means that this invention can minimize the "noise" and "errors" introduced by the experimental operation itself, providing an ideal technical guarantee for obtaining real and reliable scientific research data.

[0047] Example 2: Innovative research on the efficacy of infant formula milk powder using the system of the present invention. This embodiment aims to demonstrate the significant effectiveness of the present invention, as verified in Example 1, enabling the successful conduct of long-term, sophisticated scientific research that was previously difficult to accomplish due to technological limitations, thereby obtaining high-value biological data. The study investigates the effects of infant formula milk products from different sources (breast milk, cow's milk without HMOs, cow's milk with HMOs, sheep's milk, and goat's milk) on gut microbiota colonization and immune system development in neonatal rats.

[0048] 1. Experimental Design and Methods 1.1 Breeding and feeding of newborn rats Animal experiments were conducted at the Experimental Animal Center of Shenzhen University, and the experimental methods used complied with the EU Guidelines for the Protection of Laboratory Animals (Directive 2010 / 63 / EU). The Experimental Animal Ethics Committee of Shenzhen University reviewed and approved the animal experimental protocol, approval number IACUC-202500072. Eight-week-old SD rats were purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd., and housed in an SPF-grade barrier facility. After entering the barrier, they were randomly assigned to cages and underwent a 7-day acclimatization period. They had free access to food and water daily, with a 12-hour light / dark cycle. The temperature inside the barrier was 25 ± 2℃, and the relative humidity was 60% ± 20%. The growth and reproduction feed was purchased from Jiangsu Xietong Pharmaceutical Biotechnology Co., Ltd. After the acclimatization period, one male and multiple females were housed together for timed mating (1:2 mating). After confirming pregnancy, the female rats were placed in separate cages. The birth date of newborn rats was designated as day 0 (d0), and the ambient temperature was controlled at 30-32℃.

[0049] 1.2 Gavage administration procedure for newborn rats The procedure for gavage of neonatal rats was based on previous literature reports with appropriate modifications (Zhu X, Liu JF, Zhou SP, et al. Technological innovation and breeding of germ-free rats [J]. Experimental Animal Science, 2017, 34(01): 57-61.). First, the neck skin of the neonatal rat was pinched to fix its body, avoiding the head and body from struggling and shaking, so that its body remained in a straight line. Then, the air in the syringe module 1 and the hose 3 that had sucked the milk was discharged, and then the end of the hose 3 was placed near the mouth of the neonatal rat. The syringe module 1 was pushed gently, and a small amount of milk was dropped into the mouth of the neonatal rat, and the swallowing action was observed. Then, the hose 3 was slowly inserted along the upper jaw of the neonatal rat. When the gavage hose was in place, the syringe module 1 was slowly and smoothly pushed to slowly inject the milk, and the change of the milk block in the stomach was observed at the same time. When resistance or strong struggling of the neonatal rat was encountered, the hose 3 should be immediately withdrawn and reinserted to prevent injury to the esophagus or misinsertion into the trachea. At the same time, attention should be paid to the angle of insertion to avoid inserting into the contralateral axillary part of the neonatal rat to avoid causing abnormal symptoms. After the stomach was completely filled with milk, the syringe module 1 was stopped and the hose 3 was slowly pulled out. During the entire artificial lactation process, the amount of milk in the stomach should be observed at any time to ensure that the milk is not poured into the intestinal tract. Finally, after using a cotton swab to wipe off the residual milk from the mouth and nose, the neonatal rat was gently placed into the cage box.

[0050] 1.3 Experimental grouping Each group used the dynamic adaptive gavage system and method verified successfully in Example 1 of the present application for precise daily feeding for 14 days (from d0 to d14). Specifically, 30 healthy neonatal SD rats with a body weight difference of ≤1 g were randomly divided into 5 groups (6 rats / group) within 6 h after birth. From d0 to d14, different sources of infant formula milk (mother's milk, non-HMOs added cow's milk, HMOs added cow's milk, sheep's milk, goat's milk) were artificially gavaged once every 12 h, with a dose of 8 µL / g body weight. The pups were separated from the mother rats for a short time 20 min before gavage to promote gastric emptying and reduce interference.

[0051] 1.4 Sample collection The feces and colon tissues of the animals in each group were collected. Among them, 1 / 2 of the fresh colon tissues were immediately placed in pre-cooled phosphate buffered saline solution to maintain the viability of intestinal cells for flow cytometry analysis. The remaining tissue samples and aliquoted fecal samples were frozen in liquid nitrogen and stored at -80°C at the end of the experiment.

[0052] 1.5 Fecal 16S rDNA and groEL amplicon sequencing Fecal samples of 20 mg were collected and genomic DNA was extracted according to the instruction manual of the kit. The extracted genomic DNA was used as a template for PCR amplification of the V3-V4 region of 16S rDNA and Bifidobacterium groEL gene, respectively. The barcode with 7 bases was used to label the upstream primer to distinguish different samples in the same library. The PCR amplification products were detected by 1.5% agarose gel electrophoresis, and then the target bands were recovered by gel cutting and purification according to the instructions of the kit. The samples were mixed into a mixed system with equal mass concentration at different volumes, the total volume of the library was 50 μL, and sequencing was performed on the Illumina Miseq PE300 platform. Finally, the data were processed by quality detection, filtering, cutting, deduplication, splicing, etc. using the dada2 plugin of the QIIME2 platform. The operational taxonomic unit (OUT) of the V3-V4 region was annotated by the Silva database (version: silva-132-99), and the OTU of Bif-groEL sequence was taxonomically annotated by the self-built local nucleic acid database.

[0053] 1.6 Detection of cytokine and inflammatory marker levels in fecal supernatant The frozen feces were thawed on ice, pre-cooled PBS was added at 1:9 (w / v), and the sample was homogenized at low temperature using a high-throughput tissue grinder, then centrifuged at 8000 × g, 4°C for 20 min, and the supernatant was collected. Subsequently, according to the method described in the ELISA kit (Wuhan Elabscience Biotechnology Co., Ltd.), cytokines (TNF-α, IL-6 and IL-10) and inflammatory markers (calprotectin) in the fecal supernatant were detected, respectively. As a standardization method, the protein content of the fecal supernatant was determined.

[0054] 1.7 Detection of intestinal lamina propria T lymphocyte subpopulation composition Intestinal lamina propria lymphocytes were extracted, and the distribution and balance of T lymphocyte subpopulations were analyzed by flow cytometry, covering CD4 + helper / regulatory populations Th (Th1, Th2 and Th17) and CD4 + , CD25 + , Foxp3 + regulatory T cells (Treg), and CD8 + cytotoxic T cells (Tc) were determined synchronously.

[0055] The extraction method of intestinal lamina propria cells refers to previous studies (Lin C, Wang S, Guo M, et al. Exploiting neonatal host-bifidobacteria interactions to promote intestinal pathogen tolerance and barrier function: Bifidobacterium longum subsp. infantis outperforms Bifidobacterium adolescentis in anti- Salmonella activity andmaintenance of intestinal homeostasis. Food Science and Human Wellness , 2025, 14(4): 9250082. https: / / doi.org / 10.26599 / FSHW.2024.9250082). Briefly, after removing the fat and Peyer's patches of the intestine in pre-cooled D-Hanks medium, the intestine was opened with scissors and its contents were removed, washed thoroughly and cut into 1 cm 2pieces. Next, the intestinal pieces were transferred to a 50 mL conical tube containing 10 mL of Digest 1 (D-Hanks medium containing 5% FBS, 4 mM EDTA, and 1 mM DTT) and incubated on a 37 °C shaker for 15 min (200 rpm) and filtered with a 200 mesh filter after vortexing for 10 s, and the intestinal piece fragments were washed with Digest 1. Subsequently, the washed intestinal piece fragments were transferred to a 50 mL conical tube containing 10 mL of Digest 1 and incubated on a 37 °C shaker for 15 min (200 rpm) and filtered with a 200 mesh filter after vortexing for 10 s, and the intestinal piece fragments were washed with D-Hanks medium. Next, the washed intestinal piece fragments were placed in a 50 mL conical tube containing 10 mL of D-Hanks medium and incubated on a 37 °C shaker for 15 min (200 rpm) and filtered with a 200 mesh filter after vortexing for 10 s, and the intestinal piece fragments were washed with RPMI 1640 medium. Meanwhile, Digest 2 (RPMI 1640 medium containing 5% FBS, 2 mg / ml collagenase 4, and 1 U / mL of Dnase I) was heated at 37 °C for 15 min. Subsequently, the intestinal piece fragments were recovered and placed in a pre-heated 2 mL conical tube containing 1 mL of Digest 2 and incubated on a 37 °C shaker for 20 min (200 rpm) and filtered with a 100 mesh filter after vortexing for 10 s to collect the intestinal lamina propria lymphocytes in the filtrate. Next, the incompletely digested intestinal tissue was continuously collected and re-placed in fresh Digest 2, and the above steps were repeated. Finally, the filtrate was immediately centrifuged at 300 x g for 5 min, and the supernatant was discarded, and the cell pellet was re-suspended in 1 mL of flow cytometry buffer, repeatedly washed by centrifugation twice, and re-suspended in flow cytometry buffer, and the cell concentration was adjusted to 1 x 10 7 cells / mL.

[0056] For Th subset analysis, cells were cultured at 37 °C in a 5% CO2 cell incubator and stimulated with cell stimulants and transport inhibitor PMA (50 ng / mL), ionomycin (1 pg / mL), and BFA (5 pg / mL) for 6 h. For surface antibody staining, optimal concentrations of fluorescently labeled antibodies were added to 100 pL of cell suspension, mixed, and incubated on ice for 30 min. Then 1 mL of flow cytometry buffer was added, and a single wash was performed by centrifugation at 350 x g for 5 min. Cells were resuspended in 300 pL of flow cytometry buffer and filtered through a 70 pm cell strainer before acquisition. For antibody staining of intracellular components, cell pellets were resuspended in 200 pL of IC Fixation Buffer A, and after incubation at 4 °C in the dark for 30 min, 500 pL of lx Permeabilization Buffer was added and centrifuged at 450 x g for 5 min. Subsequently, cells were resuspended with 1 mL of lx Permeabilization Buffer and incubated for 15 min, followed by centrifugation at 450 x g for 5 min at 4 °C, and the supernatant was discarded. The fixed / permeabilized cells were resuspended in 100 pL of lx Permeabilization Buffer, and optimal concentrations of fluorescently labeled antibodies were added and vortexed before incubation at 4 °C for 45 min. Then 1 mL of flow cytometry buffer was added, and a single wash was performed by centrifugation at 350 x g for 5 min, and cells were resuspended in 300 pL of flow cytometry buffer and filtered through a 70 pm cell strainer before acquisition. For nuclear antibody staining, cell pellets were resuspended in 500 pL of Fixation / Permeabilization Solution, and after incubation at 4 °C in the dark for 45 min, they were centrifuged at 450 x g for 5 min. Cells were incubated with 1 mL of lx Permeabilization Buffer for 10 min, followed by centrifugation at 450 x g for 5 min at 4 °C, and the fixed / permeabilized cells were resuspended in 100 pL of lx Permeabilization Buffer. Optimal concentrations of fluorescently labeled antibodies were added, vortexed, and incubated at 4 °C for 45 min. Finally, 1 mL of flow cytometry buffer was added, and a single wash was performed by centrifugation at 350 x g for 5 min, and cells were resuspended in 300 pL of flow cytometry buffer and filtered through a 70 pm cell strainer before acquisition. All staining steps were performed in the dark.

[0057] T lymphocyte flow cytometry antibody labeling schemes are shown in Table 1. Flow cytometry sampling was performed using an Attune NxT flow cytometer, and data analysis was completed using FlowJo (10.6.2) software.

[0058] Table 1 Flow cytometry antibody labeling protocol for T lymphocyte subsets

[0059] 1.8 Statistical analysis Statistical analysis and plotting were performed using GraphPad Prism 9.0 and SPSS 22.0. The data were expressed as “mean ± standard deviation” or “median-interquartile range”. First, the data were tested for normality using the Kolmogorov-Smirnov method. For data that met the normal distribution, one-way analysis of variance (ANOVA) was used for difference analysis. If the data met the homogeneity of variance requirement, Duncan or Dunnett's (two-tailed) test was used for post-hoc multiple comparisons. If the variance was not equal, Dunnett T3 test was used for multiple comparisons. For non-normally distributed data, Kruskal-Wallis non-parametric test and Dunnett's post-hoc test were used for difference analysis. To explore the correlation between different types of data, Spearman correlation coefficient was used for analysis. When performing statistical tests on high-dimensional data, the Benjamini-Hochberg method was used to adjust the p-value, with a threshold of 0.05. The analysis and visualization of microbiome data were completed with the help of the online research platform (https: / / www.microbiomeanalyst.ca / ). p

[0060] 2. Results and analysis Effects on intestinal flora (refer to Figures 9-10 ): As shown in Figures 9-10 , the precise feeding technology implemented by the present application clearly presents the differences in intestinal flora colonization patterns of the newborn host under different feeding modes and formula feeding. The results clearly show that the intestinal flora of the breastfed group is dominated by colonization of B. longum subsp. infantis, which is a marker of beneficial bacteria that efficiently utilizes breast milk oligosaccharides. In contrast, formula-fed, especially non-HMO-added cow milk group, is significantly enriched in B. adolescentis, which is associated with adult intestinal flora and in some cases with inflammation. It is strongly demonstrated that the present application technology successfully eliminates the confounding variables introduced by operation due to its low stress and high standardization characteristics, thus accurately revealing the real biological differences determined by feeding decisions.

[0061] Effects on intestinal immune inflammatory state (refer to Figures 11-14 ): As shown in Figures 11-14 ​As shown, the differences in gut microbiota are directly related to changes in immune status. Compared with the formula milk group enriched with Bifidobacterium adolescentis, the breast milk group had significantly lower levels of pro-inflammatory cytokines (TNF-α and IL-6) and inflammatory markers (calprotectin) in their feces, while having higher levels of anti-inflammatory factor (IL-10), indicating that breastfeeding helps maintain intestinal immune homeostasis. This clear difference in the immune profile fully demonstrates that the non-invasive, low-stress gavage technique of this invention avoids interference with intestinal inflammation caused by the procedure itself, and that the observed immunomodulatory effects are attributable to the feeding protocol.

[0062] Effects on intestinal T cell differentiation (refer to) Figures 15-23 At a deeper cellular level, this invention reveals the shaping role of early feeding patterns in the differentiation of the adaptive immune system, such as... Figures 15-23 As shown, breastfeeding establishes a healthy environment of immune tolerance and resistance to infection characterized by high Treg, Th1, and Th cell ratios, and promotes the maturation of the immune system towards high Treg / Th17, Th1 / Th2, and Th / Tc ratios. In contrast, standard formula milk, especially milk without added HMOs, may lead to an imbalanced pro-inflammatory and allergic state dominated by Th17, Th2, and Tc cells. The ability to capture subtle biological changes such as T cell subsets is achieved through long-term, stable, and highly reproducible precision feeding, minimizing individual physiological fluctuations in animals and exposing deep immune mechanisms.

[0063] The relationship between gut microbiota and immunity (see reference) Figure 24 ):like Figure 24 As shown, the correlation heatmap analysis provides crucial evidence for the interaction of the "microbiota-immune axis" in early life. The results clearly show that the abundance of *Bifidobacterium longum* subsp. *infantii* is positively correlated with anti-inflammatory markers (such as IL-10, Treg, Th, Th1, Treg / Th17, Th1 / Th2, and Th / Tc) and negatively correlated with pro-inflammatory markers (Th2, Th17, Tc, IL-6, TNF-α, and calprotectin). The abundance of *Bifidobacterium adolescentis*, however, shows the opposite correlation pattern. These experimental results directly reflect the quality of the data, highlighting the experimental precision and data reliability provided by the gavage technique of this invention.

[0064] In summary of the above implementation cases, the precise gavage device and gavage method for early life has high safety and low damage rate: through the design concept of "dynamic adaptation", i.e. selecting a soft tube with accurate size according to the age, the best matching of the outer diameter of the soft tube and the inner diameter of the animal esophagus is ensured. Combined with flexible material and smooth head design, mechanical damage to the delicate tissue of the newborn animal is fundamentally avoided, and the success rate of gavage operation is increased to nearly 100%, and the animal mortality is significantly reduced.

[0065] The precise gavage device and gavage method for early life has high plasticity and adaptability: the soft tube body is made of PE material and can be conveniently matched with a conventional 1 mL syringe. The core technical advantage of this design is that the PE material gives the soft tube the unique function of manual shaping, allowing the operator to adjust the tube curvature in real time and accurately according to their own gavage habits, angle preferences, and individual differences in the size and physiological curvature of the esophagus of newborn mice. The shaped soft tube can accurately fit the contour of the animal's esophagus, ensuring that the best insertion trajectory and angle are maintained under different operating postures (such as vertical or inclined body position), thereby minimizing mechanical friction and tension stimulation of the esophageal mucosa by the tube, effectively avoiding potential damage such as perforation and scratching caused by mismatch between the instrument and the tissue.

[0066] The precise gavage device and gavage method for early life is simple to operate and has high standardization: a clear "selection-use" process is provided, which converts complex empirical operations into standardized technical steps. Even beginners can quickly master it, ensuring consistency between different operators and at different time points in the same experiment, improving the repeatability of the experiment.

[0067] The precise gavage device and gavage method for early life ensures the feasibility and reliability of long-term experiments: as the operation trauma and stress are minimized, it is possible to perform continuous gavage on newborn mice for several weeks. This eliminates the significant physiological disturbance introduced by the operation itself, allowing the experimental results to truly reflect the biological effects of different interventions, greatly improving the reliability and validity of scientific data.

[0068] The above specific embodiments further illustrate the purpose, technical solutions and advantages of the present application. It should be understood that the above description is only a specific embodiment of the present application and is not intended to limit the application. Any modification, equivalent replacement, improvement, etc. made within the spirit and principles of the present application should be included in the protection scope of the present application.

Claims

1. A precise gavage device for early life, characterized in that: It includes a syringe module (1) and a flexible gavage catheter. The output end of the syringe module (1) is detachably connected to the flexible gavage catheter. The output end of the flexible gavage catheter is arc-shaped and the end is blunted.

2. The precise gavage device for early life as described in claim 1, characterized in that: The flexible gavage catheter includes a connecting sleeve (2) and a flexible tube (3), and the syringe interface module (1), the connecting sleeve (2) and the flexible tube (3) are connected in sequence; the flexible tube (3) is arc-shaped.

3. The precise gavage device for early life as described in claim 2, characterized in that: The output end of the hose (3) is spherical or hemispherical.

4. The precise gavage device for early life as described in claim 3, characterized in that: The hose (3) is made of polyethylene.

5. A method for administering a gavage using the precision gavage device for early life as described in claim 4, characterized in that: Includes the following steps: Step 1, Assessment and Selection: Select a tubing that matches the growth stage of the newborn mice / rodents to be operated on (3). Step 2, Assembly and Shaping: Connect one end of the connecting sleeve (2) to the output end of the syringe module (1) and the other end to the hose (3) to form a nested structure; after assembly, shape the curvature of the exposed part of the hose (3) as needed to conform to the physiological curvature of the animal. Step 3, Gavage: Insert the tubing (3) into the esophagus of the newborn mouse / great mouse; after the end of the tubing (3) reaches the stomach, push the syringe module (1) to perform gavage.

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