Application of GALANIN family member in shortening animal labor process and method

Abstract

The invention discloses an application of GALANIN family members in shortening the animal delivery process and a method for promoting mouse delivery. By injecting any polypeptide in members of the GALANIN family, especially GALANIN, SPEXIN and GALP in the members of the GALANIN family, into a mouse to be delivered, the delivery process of the mouse can be remarkably shortened, the delivery process can be shortened to 90.20 min from 203.22 min, and the delivery efficiency is greatly improved. The GALANIN family members can be expected to be applied to other mammals, the delivery efficiency of the mammals can also be improved, and a new solution and thought are provided for the delivery problem of the mammals.

Description

Technical Field

[0001] The present invention relates to the field of animal reproduction and breeding, and in particular to an application and method of GALANIN family members in shortening animal labor. Background Art

[0002] According to currently known mechanisms regulating labor, the labor process can be divided into three stages: the first stage, initiation of labor; the second stage, maintenance of labor; and the third stage, completion of labor. The first stage begins with contractions of the laboring uterus and ends with complete dilation and efflux of the cervix. Progesterone and estrogen play a key regulatory role. Progesterone levels, which maintain uterine quiescence during pregnancy, decrease, while estrogen levels rise, releasing the inhibitory effect on uterine contractions. Uterine contractions begin, and labor begins. The second stage begins with complete dilation of the cervix and ends with delivery of the fetus. During this stage, multiple factors, including prostaglandins and oxytocin, coordinate to regulate rhythmic uterine contractions, facilitating smooth delivery of the fetus. The third stage begins after delivery of the fetus and ends with the expulsion of the placenta. Oxytocin plays a key role in this stage. However, the mechanisms regulating labor remain uncertain and require further research.

[0003] GALANIN is a 29-amino acid neuropeptide (30 in humans) widely expressed in the central and peripheral nervous systems, regulating various biological functions, including pain perception, learning, memory, and appetite. GALANIN selectively binds to distinct GAL receptors (GALRs) in various tissues to regulate their function. GALRs consist of GALR1, GALR2, and GALR3. Members of the GALANIN family include GALANIN, SPEXIN (and SPEXIN2 in non-mammalian vertebrates), KISSPEPTIN, GALP, ALARIN, and GAMP. GALANIN, SPEXIN, and GALP bind to GALRs to exert their effects. SPEXIN is a 14-amino acid neuropeptide. The mature sequences of SPEXIN and GALANIN are highly similar across different vertebrate species and share a conserved structure. GALP is a 60-amino acid non-C-terminally modified peptide with residues (9-21) identical to those of GALANIN (1-13), while residues (1-24) and (41-53) are highly conserved across species. However, the role of GALANIN family members in regulating mammalian parturition has not been reported. Summary of the Invention

[0004] The purpose of the present invention is to provide a method for using GALANIN family members in shortening animal labor and promoting mouse labor, so as to solve the problem of long labor and dystocia, and to provide new ideas and methods for solving mammalian labor problems.

[0005] According to the first aspect of the present invention, there is provided the use of galanin family members in shortening the parturition process of animals. By this application, the parturition of animals can be promoted. In particular, the parturition process of mice can be significantly shortened, thereby improving the parturition efficiency and providing new solutions and methods for the parturition problems of mammals.

[0006] According to the second aspect of the present invention, there is provided the use of galanin family members in the preparation of products for shortening the parturition process of animals. By preparing galanin into corresponding products, it can be used to promote the parturition of animals. In particular, the parturition process of mice can be significantly shortened, thereby improving the parturition efficiency and providing new solutions and methods for the parturition problems of mammals.

[0007] According to the third aspect of the present invention, there is provided the use of galanin family members in promoting the parturition of animals or in the preparation of products for promoting the parturition of animals. Thus, by this application, the parturition of animals can be promoted. In particular, the parturition process of animals can be significantly shortened, thereby improving the parturition efficiency of animals.

[0008] In certain embodiments, the galanin family members include galanin, spexin, kisspeptin, galp, alarin, gamp.

[0009] In certain embodiments, the galanin family members include galanin, spexin, galp.

[0010] According to the fourth aspect of the present invention, there is provided a method for promoting the parturition of mice, the method comprising the following steps: S1: Perfusion osmotic pump: Fill the capsule osmotic pump with any one of the polypeptides in the galanin family members at a concentration of 41 mM; S2: Implant the osmotic pump: Immerse the osmotic pump in 0.9% physiological saline for 4 - 6 hours, and then implant the osmotic pump subcutaneously into the back of the pregnant mouse to be parturited, which can promote the parturition of mice.

[0011] Thus, by this method, the parturition of mice can be promoted. In particular, the parturition process of mice can be significantly shortened, thereby improving the parturition efficiency and providing new solutions and methods for the parturition problems of mammals.

[0012] In certain embodiments, the galanin family members include galanin, spexin, kisspeptin, galp, alarin, gamp.

[0013] In certain embodiments, the GALANIN family members include GALANIN, SPEXIN, and GALP.

[0014] In certain embodiments, the pregnant mouse is a 17.5 dpc mouse.

[0015] According to a fifth aspect of the present invention, the use of the method in promoting delivery in mice is provided. Thus, the method can promote delivery in mice, especially shorten the delivery process and improve delivery efficiency.

[0016] According to a sixth aspect of the present invention, there is provided an application of the method in shortening the labor process of mice, thereby shortening the labor process and improving the labor efficiency.

[0017] The present invention has the beneficial effects of significantly shortening the duration of labor in mice, from 203.22 minutes to 90.20 minutes, by injecting any polypeptide from the GALANIN family, particularly GALANIN, SPEXIN, and GALP, into laboring mice. It is anticipated that the GALANIN family members could be applied to other mammals to improve labor efficiency, providing new solutions and ideas for mammalian labor. DETAILED DESCRIPTION

[0018] The invention is described in further detail below.

[0019] Example 1: The drug concentration was explored by oral administration.

[0020] (1) Animal preparation: The breeding female mice were put together in the afternoon and the plugs were checked the next morning. If a plug was found after the plug check, 12 noon on the day of the plug was found was recorded as 0.5 days. They usually gave birth at 18.5 days after mating (days post coitum, dpc). At 13.5 dpc, each female mouse was placed in a cage alone for adaptation and transition feeding.

[0021] (2) Drug configuration: a. Control group: 10% DMSO, 40% PEG300, 5% Tween-80, and 45% saline.

[0022] b. Receptor inhibitor treatment group: The control group was used as solvent and GALANIN receptor inhibitors (M40 and HT2157, the concentrations of the two inhibitors were the same) were added at concentrations of 10 -4 mM, 10 -3 mM, 10 -2 mM, 10 -1mM, 4×10 -1 mM.

[0023] M40 is an effective non-selective GALANIN receptor inhibitor that competitively inhibits GALR1 and GALR2 and is an inhibitor of GALR1 and GALR2.

[0024] HT2157, also known as SNAP 37889, is a selective high-affinity competitive antagonist of GALR3 and is an inhibitor of GALR3.

[0025] (3) Gavage treatment: After observing the birth of the first pup by the female mouse, immediately gavge 100 μL of the drug. Then, at intervals of 20 minutes, 25 minutes, 30 minutes, 35 minutes, 40 minutes, 45 minutes, and 50 minutes, gavge the 2nd, 3rd, 4th, 5th, 6th, 7th, and 8th times, respectively, with a total of 8 gavages, each time with 100 μL of the drug.

[0026] Record the gavage drug, the female mouse number, and the time of each gavage, and mark the female mouse number on the cage. Immediately move away from the cage after the gavage and avoid talking loudly. During the parturition of the female mouse, record the birth time of each pup. 55 minutes after the 8th gavage (300 minutes after the 1st gavage at this time), record the number of live and dead pups in each cage. Then immediately sacrifice the female mouse to check if there are any unborn pups in its uterus, and record the number of unborn pups for each female mouse.

[0027] The results are shown in Table 1. The results indicate that the completion ratio of parturition in the receptor inhibitor treatment groups is lower than that in the control group. The total parturition duration, the time required for each pup to be born on average, and the number of remaining fetuses in the uterus in the 10 -1 mM and 4×10 -1 mM concentration groups are significantly higher than those in the control group ( P <0.05), the number of live pups in the cage is significantly lower than that in the control group ( P <0.05), and there is no significant difference in the number of dead pups in the cage ( P >0.05). This shows that 10 -1 mM and 4×10 -1 mM are the optimal treatment concentrations, and the combined use of inhibitors of the three GALANIN receptors (GALR1, GALR2, GALR3) during the parturition of pregnant mice will cause dystocia in the female mice and prolong the parturition time. It shows that GALANIN can significantly affect the parturition of mice, such as affecting the labor process and the number of live pups born.

[0028] Table 1 The parturition situation of pregnant mice after combined gavage of inhibitors of three GALANIN receptors Concentration (mM) Number of female mice Completion rate of parturition (%) Total parturition duration (min) Average time required for each pup to be born (min) Number of remaining fetuses in the uterus Number of live pups in the cage Number of dead pups in the cage 0 14 100 <![CDATA[142.29±17.43 a > <![CDATA[10.26±1.35 a > <![CDATA[0 a > <![CDATA[13.36±0.77 a > <![CDATA[0.71±0.27 a > <![CDATA[10 -4 > 11 82 <![CDATA[212.82±20.22 ab > <![CDATA[13.75±1.12 a > <![CDATA[1.82±1.39 ab > <![CDATA[13.36±1.25 ab > <![CDATA[0.27±0.20 a > <![CDATA[10 -3 > 11 73 <![CDATA[216.36±24.86 ab > <![CDATA[16.44±1.95 a > <![CDATA[3.73±1.58 ab > <![CDATA[8.64±1.74 ab > <![CDATA[1.18±0.71 a > <![CDATA[10 -2 > 12 75 <![CDATA[213.58±19.14 ab > <![CDATA[14.60±1.19 a > <![CDATA[2.67±1.74 ab > <![CDATA[10.83±1.45 ab > <![CDATA[1.08±0.36 a > <![CDATA[10 -1 > 12 42 <![CDATA[252.25±18.73 bc > <![CDATA[120.95±38.85 b > <![CDATA[6.92±1.93 bc > <![CDATA[6.83±1.95 bc > <![CDATA[1.08±0.48 a > <![CDATA[4×10 -1 > 13 46 <![CDATA[254.31±18.53 bc > <![CDATA[124.25±35.72 b > <![CDATA[7.31±2.03 bc > <![CDATA[7.00±1.85 bc > <![CDATA[0.46±0.18 a > Note: Data represent mean ± standard error, and different lowercase letters in the same column indicate significant differences ( P <0.05), the same lowercase letters in the same column indicate no significant difference (P>0.05).

[0029] Example 2: Sustained-release drug administration via implanted osmotic pump.

[0030] (1) Animal preparation: The temperature of the mice is controlled at 20-26℃ by the air-conditioning system. The relative humidity is maintained at 50%-60% and adjusted by a humidifier. Feed and water are added regularly every week, and at other times, feed is added according to the feed stock in the hopper, 3-4 times a week. Breeding females are provided with sufficient breeding feed and fed 0.5-1 gram of sunflower seeds at 9 am every day to supplement vitamin E and energy. Breeding females are put into the cage in the afternoon and the plug is checked the next morning. If a plug is found after the plug is checked, it is recorded as 0.5 days at 12 noon on the day of the plug. The fetus usually gives birth at 18.5 days after mating (days post coitum, dpc).

[0031] (2) Drug configuration: The optimal treatment concentration determined in Example 1 is 10 -1 mM and 4×10 -1 mM, evaluated from the aspects of safety and cost, with 10 -1 The drug concentration perfused into the osmotic pump was calculated based on mM to ensure that there was sufficient drug dose when the osmotic pump was permeated in the animal body, thereby confirming that the drug concentration in the osmotic pump was 41 mM.

[0032] a. Control group (NC): 100% DMSO.

[0033] b. Receptor inhibitor treatment group (RI): M40 (GALR1, GALR2 inhibitor) and HT2157 (GALR3 inhibitor) were dissolved in DMSO to a final concentration of 41 mM.

[0034] c. GALANIN treatment group: GALANIN was dissolved in DMSO to a final concentration of 41 mM.

[0035] d. SPEXIN treatment group: SPEXIN was dissolved in DMSO to a final concentration of 41 mM.

[0036] e. GALP treatment group: GALP was dissolved in DMSO to a final concentration of 41 mM.

[0037] (3) Perfusion osmotic pump: The Alzet osmotic pumps (Alzet Osmotic Pumps, 1003D) were used. Each pump was filled with approximately 100 μL of the drug and slowly released at a rate of 1 μL per hour. The effective action time of the osmotic pump was 72 hours. First, the weight of the empty pump and the pump cap was measured. A 1-ml syringe with a blunt needle compatible with the capsule pump was used to draw the drug solution, and the air in the needle was expelled. The capsule osmotic pump was held upright, and the blunt needle was inserted into the pump until it reached the bottom. The drug solution was slowly injected into the pump until a little overflowed from the pump opening. The needle was carefully removed, the pump cap was fully inserted into the pump, and the excess solution was wiped off. Finally, the weight of the entire pump was measured, and the volume of the injected solution was calculated. After weighing, the volume of the injected liquid should exceed 90% of the pump capacity stated in the instruction manual. If not, it indicated that there was a large amount of gas in the pump and re-infusion was required.

[0038] (4)Implantation of the osmotic pump: The osmotic pump was filled at room temperature. The osmotic pump was immersed in 0.9% physiological saline for 4 - 6 hours to activate the osmotic pump. At 13.5 dpc, each pregnant mouse was placed individually in a cage for acclimation. At 17.5 dpc, the animal was anesthetized, and a horizontal incision about 0.5 cm long was made subcutaneously on the back of the animal. A tissue forceps was used to create a space the size of a capsule under the skin from the incision to the back of the animal. The capsule osmotic pump filled with the drug solution was surgically implanted, and the wound was sutured. After implanting the osmotic pump, the pregnant mouse was returned to the original transparent cage, and a camera was used to record the delivery situation of the pregnant mouse. The start and end times of delivery of the mouse were closely monitored, and the total number of pups born and the number of live pups in the cage were recorded after each pregnant mouse gave birth. At 20.5 dpc, the osmotic pump stopped releasing the drug, and the pregnant mouse was immediately sacrificed to observe whether there were still unborn pups in its uterus to determine whether the pregnant mouse had dystocia.

[0039] (5)Experimental results The experimental results of the combined slow-release of GALANIN three receptor inhibitors on the delivery of pregnant mice are shown in Table 2: The results showed that when pregnant mice were treated with the combined GALANIN three receptor inhibitors, the proportion of pregnant mice that completed delivery was significantly reduced. 60% of the pregnant mice did not give birth after the average start time of delivery at 18.5 dpc and did not start giving birth before the end of the effective time of the drug (20.5 dpc), and there were remaining fetuses in the uterus. The start time of delivery, the total delivery duration, and the time required for each pup to be born of the 40% of the pregnant mice that gave birth were significantly higher than those of the control group of pregnant mice ( P <0.05). This indicates that the combined use of GAL three receptor (GALR1, GALR2, GALR3) inhibitors during the delivery of pregnant mice will cause most pregnant mice to have dystocia. Although some pregnant mice can still give birth, the start time of delivery and the labor process are prolonged.

[0040] Table 2 The effect of the combined slow-release of GALANIN three receptor inhibitors on the delivery of pregnant mice

[0041] Note: The data represent their mean ± standard error. Different lowercase letters in the same row indicate significant differences ( P < 0.05), and the same lowercase letters in the same row indicate no significant differences ( P > 0.05).

[0042] The experimental results of the parturition of pregnant mice after separately releasing three members of the GALANIN family are shown in Table 3 as follows: Comparison of the total parturition duration: In the GALANIN treatment group, compared with the control group (NC), the total parturition duration decreased from 203.22 min in the control group to 90.20 min, and the difference was significant ( P < 0.05); in the SPEXIN treatment group, compared with the control group (NC), the total parturition duration decreased from 203.22 min in the control group to 125.10 min, and the difference was significant ( P < 0.05); in the GALP treatment group, compared with the control group (NC), the total parturition duration decreased from 203.22 min in the control group to 107.30 min, and the difference was significant ( P < 0.05). Comparison of the time required for each pup to be born: In the GALANIN treatment group, compared with the control group (NC), the time required for each pup to be born decreased from 13.52 min in the control group to 5.61 min, and the difference was significant ( P < 0.05); in the SPEXIN treatment group, compared with the control group (NC), the time required for each pup to be born decreased from 13.52 min in the control group to 8.25 min, and the difference was significant ( P < 0.05); in the GALP treatment group, compared with the control group (NC), the time required for each pup to be born decreased from 13.52 min in the control group to 8.44 min, and the difference was significant ( P < 0.05). However, in terms of the parturition completion rate, the start time of parturition, the total number of pups, and the number of live pups, there were no significant differences between the GALANIN, SPEXIN, GALP treatment groups and the control group (NC) ( P > 0.05).

[0043] The results show that treating the female mice separately with three members of the GALANIN family can significantly reduce the total parturition duration and the time required for each pup to be born ( P < 0.05), and there were no significant differences in the start time of parturition, the total number of pups, and the number of live pups. This indicates that separately using three members of the GALANIN family (GALANIN, SPEXIN, GALP) during the parturition of pregnant mice can significantly shorten the parturition process of pregnant mice and does not affect the start time of parturition, the total number of pups, and the number of live pups.

[0044] Table 3 Delivery situations of three members of the GALANIN family with slow release in pregnant mice Group Number of female mice Completion rate of parturition (%) Onset of parturition time (dpc) Total parturition duration (min) Time required for each pup to be born (min) Total number of pups Number of live pups NC 9 100 <![CDATA[18.58±0.07 a > <![CDATA[203.22±28.55 a > <![CDATA[13.52±1.65 a > <![CDATA[15.11±0.54 a > <![CDATA[13.78±0.60 a > GALANIN 10 100 <![CDATA[18.48±0.07 a > <![CDATA[90.20±7.20 b > <![CDATA[5.61±0.27 b > <![CDATA[15.90±0.82 a > <![CDATA[14.50±0.64 a > SPEXIN 10 100 <![CDATA[18.51±0.03 a > <![CDATA[125.10±9.45 b > <![CDATA[8.25±0.62 b > <![CDATA[15.30±0.90 a > <![CDATA[14.20±0.76 a > GALP 10 100 <![CDATA[18.53±0.04 a > <![CDATA[107.30±12.49 b > <![CDATA[8.44±0.95 b > <![CDATA[13.20±0.89 a > <![CDATA[12.10±1.28 a > Note: The data are expressed as mean ± standard error. Different lowercase letters in the same column indicate significant differences ( P < 0.05), and the same lowercase letters in the same column indicate no significant differences ( P > 0.05).

[0045] In summary, by injecting members of the GALANIN family (especially GALANIN, SPEXIN, GALP) into pregnant mice, the labor process of mice can be effectively shortened, and the onset time of labor, total litter size, and number of live offspring are not affected. It shows that members of the GALANIN family can effectively shorten the labor process of mice and promote mouse delivery, and this method can be used in other mammals such as pigs, cows, and sheep to effectively shorten the labor process and improve the delivery efficiency.

Claims

1. Use of galanin family members in shortening the parturition process of animals.

2. Use of galanin family members in the preparation of a product for shortening the parturition process of animals.

3. Use of galanin family members in promoting animal parturition or in the preparation of a product for promoting animal parturition.

4. The application according to any one of claims 1-3, wherein, The galanin family members include galanin, spexin, kisspeptin, galp, alarin, gamp.

5. The application according to claim 4, wherein, The galanin family members include galanin, spexin, galp.

6. A method for promoting parturition in mice, wherein, The method comprises the following steps: S1: Perfusion osmotic pump: Fill the capsule osmotic pump with any one of the polypeptides in the galanin family members at a concentration of 41 mM. S2: Implant the osmotic pump: Immerse the osmotic pump in step S1 in 0.9% physiological saline for 4 - 6 hours, and then implant the osmotic pump subcutaneously in the back of the pregnant mouse to be parturient, which can promote the parturition of mice.

7. According to the method described in claim 6, wherein The galanin family members include galanin, spexin, kisspeptin, galp, alarin, gamp.

8. According to the method described in claim 7, wherein, The galanin family members include galanin, spexin, galp.

9. According to the method described in claim 6, wherein, The pregnant mouse to be parturient refers to a 17.5 dpc mouse.

10. Use of the method according to any one of claims 6 - 9 in promoting the parturition of mice or in shortening the parturition process of mice.

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