阅读说明：本技术 一种利用粪菌移植提高昆明犬幼犬工作性能的方法 (Method for improving working performance of Kunming dog puppy by using fecal strain transplantation ) 是由 曹振辉 杜津京 李�杰 徐虎 林秋叶 王孝义 殷桦娟 茶光辉 胡天念 李居东 王 于 2021-07-06 设计创作，主要内容包括：本发明公开了一种利用粪菌移植提高昆明犬幼犬工作性能的方法,属于生物技术领域。包括以下步骤：粪便供体筛选；选择养殖条件相同、工作性能优良幼公犬的外祖母作为菌源供体；制备粪菌原液,低温保存；移植时,制备粪菌移植液；灌胃至来源于同一健康外祖母、45～55日龄的不同健康幼公犬,连续灌胃≥14天；饲喂无抗饲料。本发明可以明显提升幼犬工作素质行为即幼犬工作性能。(The invention discloses a method for improving the working performance of a Kunming dog puppy by using fecal strain transplantation, belonging to the technical field of biology. The method comprises the following steps: screening a feces donor; selecting a grandmother of a puppy with the same culture conditions and excellent working performance as a bacteria source donor; preparing a stock solution of the fecal strain, and storing at low temperature; preparing a fecal strain transplanting solution during transplanting; performing intragastric lavage to different healthy young male dogs of 45-55 days old from the same healthy grandmother, wherein the continuous intragastric lavage is not less than 14 days; feeding antibiotic-free feed. The invention can obviously improve the working quality behavior of the puppy, namely the working performance of the puppy.)

1. A method for improving the working performance of a Kunming dog puppy by using fecal strain transplantation is characterized by comprising the following steps:

(1) screening of fecal donors: selecting a grandmother of a puppy with the same culture conditions and excellent working performance as a bacteria source donor;

(2) collecting fresh excrement of the donor for pretreatment, preparing a fecal bacteria stock solution by dissolving, filtering, centrifuging and suspending under an aseptic condition, adding aseptic glycerol to obtain the fecal bacteria stock solution, and freezing for later use;

(3) during transplantation, dissolving the frozen stock solution of the fecal bacteria, centrifuging to remove glycerol and supernate, adding 0.9% sterile normal saline, and carrying out heavy suspension precipitation to prepare a fecal bacteria transplantation solution;

(4) perfusing the fecal strain transplanting liquid into different healthy young male dogs of 45-55 days old from the same healthy grandmother, and continuously perfusing for more than or equal to 14 days; feeding antibiotic-free feed, and performing working performance training every day.

2. The method of claim 1, wherein the donor of step (1) is Kunming dog of the pennisetum purpureum strain, and the selection criteria are as follows:

(1) the system information is clear, accurate and traceable;

(2) normally injecting vaccine and expelling parasites, having no history for three months and not using antibiotics;

(3) no rabies, canine distemper, parvovirus and canine infectious hepatitis;

(4) the mental state is good, the people can eat the antibiotic-free granulated feed actively, and the body temperature is not abnormal;

(5) defecation is performed for 1-2 times every day, the water content of the excrement is 60% -70%, the color of the excrement is golden yellow or brown, the shape of the excrement is cylindrical, the size of the excrement is moderate, and the excrement has luster and normal smell.

3. The method of claim 1, wherein the pretreatment of step (2) comprises: sterile glycerol with the same mass and the same concentration of 15 percent of the fresh excrement is added and placed on ice.

4. The method of claim 1, wherein the solubilizing of step (2) comprises: adding 0.9% sterile normal saline to dissolve until the concentration of feces is 0.2g/mL, and stirring well.

5. The method of claim 1, wherein the filtering of step (2) comprises: sequentially sieving with size of 2.0mm, 1.0mm, 0.5mm, and 0.25mm to remove residue and particulate matter, filtering, and stirring.

6. The method of claim 1, wherein the centrifugation of step (2) is performed by: 6000 r/min; the time is 15 min.

7. The method of claim 1, wherein the suspending of step (2) comprises: and removing the supernatant of the centrifuged liquid, adding sterile physiological saline with the volume of 0.9 percent which is the same as that in the dissolving step, and carrying out heavy suspension precipitation to obtain the fecal strain stock solution.

8. The method of claim 1, wherein the solubilizing of step (3) comprises: unfreezing in a 37 ℃ water bath for 8-10 min; and (3) centrifuging: 6000r/min, time 15 min.

9. The method for improving the transport stress resistance of the Kunming dog puppy by using fecal strain transplantation as claimed in claim 1, wherein the addition amount of the 0.9% sterile physiological saline in the step (3) is 1/2-2 times of the volume of the fecal strain stock solution.

10. The method for improving the working performance of a Kunming dog puppy by using fecal bacteria transplantation as claimed in claim 1, wherein the gavage amount in step (4) is 1mL 1 time per day.

Technical Field

The invention relates to the technical field of biology, in particular to a method for improving the working performance of a Kunming dog puppy by using fecal strain transplantation.

Background

Puppies are the key period for forming working performance, improvement of the working performance of the puppies is promoted mainly by means of strengthening puppy training at present, after part of puppies are trained, the working performance does not reach the standard and is eliminated, and huge waste of manpower, material resources and financial resources is caused.

Fecal Transplantation (FMT) refers to the Transplantation of functional flora of feces of healthy donor animals into gastrointestinal tracts of recipient animals, and achieves the purposes of treating animal diseases, promoting growth and improving immunity by adjusting, repairing and remodeling intestinal flora of the recipient animals. It has been proved that FMT can treat inflammatory bowel disease and irritable bowel syndrome of human patients, and improve the growth performance and immunity of pigs and chickens. However, no literature report is available on a method for improving the working performance of puppies by utilizing the FMT technology.

Therefore, how to provide a method for improving the working performance of a Kunming dog puppy by using fecal bacteria transplantation is a problem to be solved urgently by the technical personnel in the field.

Disclosure of Invention

In view of the above, the invention provides a method for improving the working performance of a Kunming dog puppy by using fecal strain transplantation, which is simple and easy to implement, strong in operability and wide in application prospect.

In order to achieve the purpose, the invention adopts the following technical scheme:

a method for improving the working performance of a Kunming dog puppy by using fecal strain transplantation comprises the following steps:

(1) screening of fecal donors: selecting a grandmother of a puppy with the same culture conditions and excellent working performance as a bacteria source donor;

(2) collecting fresh feces of a donor, pretreating, preparing a fecal strain stock solution by dissolving, filtering, centrifuging and suspending under an aseptic condition, adding aseptic glycerol to obtain the fecal strain stock solution, and freezing for later use;

(3) during transplantation, dissolving the frozen stock solution of the fecal bacteria, centrifuging to remove glycerol and supernate, adding 0.9% sterile normal saline, and carrying out heavy suspension precipitation to prepare a fecal bacteria transplantation solution;

(4) perfusing the fecal strain transplanting liquid into different healthy young male dogs of 45-55 days old from the same healthy grandmother, and continuously perfusing for more than or equal to 14 days; feeding antibiotic-free feed, and performing working performance training every day.

Further: the breeding conditions of the puppies and the grandparents are the same.

The working performance is excellent: according to the working performance scoring standard of the puppies, the score is over 37 points.

Has the advantages that: the newborn mammal intestinal flora has strong maternal characteristics, the maternal microorganisms play an important role in the formation process of the offspring intestinal flora, and the intestinal flora of the young animal plays an important role in nutrient metabolism, activation of an immune system and construction of an intestinal barrier function, so that the father line of the young dog is not selected as a donor. On the other hand, generally, 7 to 9 puppies can be born per birth of each bitch on average, if the mother of the puppy is selected, the excrement of several or even more puppy mothers must be mixed, the mixing operation can destroy the abundance and diversity of the anaerobic bacteria playing a role in the excrement liquid, thereby affecting the excrement bacteria transplanting effect, and the external grandmother of the puppy is selected, the excrement is collected to prepare the excrement bacteria transplanting liquid, so that the operation can be reduced, the number and abundance of the anaerobic bacteria in the excrement liquid are increased, and the excrement bacteria transplanting effect is ensured.

Preferably: the donor in the step (1) is a chamaemelum wolfsbane strain Kunming dog, and the selection standard is as follows:

(1) the system information is clear, accurate and traceable;

(2) normally injecting vaccine and expelling parasites, having no history for three months and not using antibiotics;

(3) no rabies, canine distemper, parvovirus and canine infectious hepatitis;

(4) the mental state is good, the people can eat the antibiotic-free granulated feed actively, and the body temperature is not abnormal;

(5) defecation is performed for 1-2 times every day, the water content of the excrement is 60% -70%, the color of the excrement is golden yellow or brown, the shape of the excrement is cylindrical, the size of the excrement is moderate, and the excrement has luster and normal smell.

Preferably: step (2) pretreatment: sterile glycerol with the same mass and 15% concentration as fresh excrement is added and placed on ice.

Preferably: dissolving in step (2): adding 0.9% sterile normal saline to dissolve until the concentration of feces is 0.2g/mL, and stirring well.

Preferably: step (2), filtering: sequentially sieving with size of 2.0mm, 1.0mm, 0.5mm, and 0.25mm to remove residue and particulate matter, filtering, and stirring.

Preferably: centrifuging: 6000 r/min; the time is 15 min.

Preferably: suspending in step (2): removing supernatant from the centrifuged liquid, adding sterile physiological saline with the volume of 0.9 percent of that in the dissolving step, and re-suspending the precipitate, namely the fecal strain stock solution.

Preferably: and (3) dissolving: unfreezing in a 37 ℃ water bath for 8-10 min; centrifuging: 6000r/min, time 15 min.

Preferably: and (3) adding 0.9% of sterile normal saline into the liquid manure in an amount which is 1/2-2 times of the volume of the liquid manure.

More preferably, the volume of the liquid is 2 times of the volume of the stock solution of the coprinus.

Preferably: the stomach filling amount in the step (4) is 1 time and 1mL per day for 1 time.

According to the technical scheme, compared with the prior art, the method for improving the working performance of the Kunming dog puppy by using fecal strain transplantation can obviously improve the working quality behavior of the puppy, namely the working performance of the puppy.

Detailed Description

The following will clearly and completely describe the technical solutions in the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.

The embodiment of the invention discloses a method for improving the working performance of a Kunming dog puppy by using fecal strain transplantation.

Example 1

(1) The method selects the foreign grandmother of FMT receptor of the young beagle dog of the healthy wolfsbane strain with the same breeding conditions (the young granddog and the foreign grandmother) and excellent working performance as a bacteria source donor.

The selection criteria were as follows: 1) the system information is clear, accurate and traceable; 2) normally injecting vaccine and expelling parasites, having no history for three months and not using antibiotics; 3) the serum Elisa antigen kit detects that rabies, canine distemper, parvovirus and canine infectious hepatitis are negative; 4) the mental state is good, the people can eat the antibiotic-free granulated feed actively, and the body temperature is not abnormal; 5) defecation is performed for about 2 times every day, the water content of the excrement is 60% -70%, the color of the excrement is golden yellow or brown, the shape of the excrement is cylindrical, the size of the excrement is moderate, and the excrement has luster and normal smell.

(2) Collecting fresh feces of donor dogs, filling the feces into a sterile wide-mouth bottle, adding 15% sterile glycerol with the same mass and concentration, placing on ice, and transporting to a laboratory within 1 hour; performing low-temperature operation in an aseptic operation table, putting all collected feces into an aseptic container, adding 0.9% of aseptic normal saline according to the ratio of 0.2g/mL of the feces for dissolving, and uniformly stirring; sequentially passing through sterile stainless steel laboratory screens of 2.0mm, 1.0mm, 0.5mm and 0.25mm, filtering out residues and particulate matters, and stirring uniformly again; subpackaging into 50mL sterile centrifuge tubes, centrifuging at 6000r/min for 15min in a centrifuge, pouring out supernatant after centrifugation, adding 0.9% sterile physiological saline with the same volume as that in the dissolving step, and uniformly suspending the precipitate to obtain a stock solution of fecal bacteria; subpackaging the prepared stock solution of the coprinus comatus into 50mL of a bacteria freezing tube, adding sterile glycerol with the same volume and the concentration of 30%, and freezing and storing in a refrigerator at the temperature of-80 ℃.

Before FMT experiment, unfreezing the frozen and preserved bacterium liquid in a water bath kettle at 37 ℃ for 9min, then centrifuging the bacterium liquid in a centrifuge at a speed of 6000r/min for 15min, removing glycerol and supernatant, adding 1/2 sterile physiological saline with the concentration of 0.9 percent of the volume of the stock solution of the fecal bacteria, and carrying out heavy suspension precipitation to obtain high-dose FMT bacterium liquid;

adding sterile normal saline with the same volume of the stock solution of the fecal bacteria and the concentration of 0.9 percent for heavy suspension precipitation to obtain medium-dose FMT bacterial liquid;

adding sterile normal saline with the concentration of 0.9 percent and the volume 2 times of the volume of the stock solution of the fecal bacteria for resuspension and precipitation to obtain the low-dose FMT bacterial liquid.

(3) 20 healthy young male dogs of the Queen wolfsbane strain which are from the same external grandmother and are 45-55 days old are selected as FMT receptors and randomly divided into 4 groups of Control (blank Control group), KML (low dose group), KMM (medium dose group) and KMH (high dose group), wherein 5 dogs are selected.

The experimental period is 28d, wherein the 1 st to 14 th days are 10: 00 gavage No. 16 to puppies, and the specific grouping and gavage dose are shown in Table 1.

TABLE 1 test grouping and drenching dosage

The antibiotic-free pellet feed is separately fed to puppies at 8:00, 14:00 and 20:00 every day in a fixed amount, water is freely drunk, the colony house is cleaned after the feeding and the defecation, and the colony house is disinfected by 0.1 percent of new benzalkonium bromide. Puppies were performance trained at 11:00, 17:00 a day. And scoring the various working performances at 15-28 d, wherein the scoring standards of the working performances are shown in Table 2.

TABLE 2 puppy performance scoring criteria

(4) The effect of FMT on the working performance of the Kunming dog is shown in table 3. Compared with a Control group, the crowd shuttling and environmental adaptation scores of FMT groups for evaluating gall content and sensitivity indexes are improved, wherein the difference between a KML group and a KMM group is obvious (P is less than 0.05), and the difference between the KML group and the KMM group is not obvious (P is more than 0.05); compared with the Control group, the evaluation excitability of each FMT group, the guidance performance of the attention index and the item attention score are remarkably improved (P <0.01), and the difference between each FMT group is not significant (P > 0.05); compared with a Control group, the FMT groups have improved evaluation, extraction and holding bite scores of extraction and desire indexes, wherein the KML group and the KMM group have obvious difference (P <0.05), the KML group has extremely obvious difference higher than the Control group (P <0.01), and the KML group has no obvious difference (P >0.05) compared with the KMM group; compared with the Control group, the evaluation occupancy index of each FMT group has obviously improved parabolic pursuit occupancy desire score (P <0.05), wherein the KML group and the KMM group have extremely obvious difference (P <0.01), and the KML group has no obvious difference (P >0.05) compared with the KMM group; compared with the Control group, the evaluation scent search desire of each FMT group and the search desire score of the olfaction ability index are obviously improved (P <0.05), wherein the KML group and the KMM group have extremely obvious difference (P <0.01), and the KML group has no obvious difference (P >0.05) compared with the KMM group; compared with the Control group, the FMT groups have improved scores of the search ability for evaluating the odor search desire and the olfactory ability index, wherein the KML group and the KMM group have obvious difference (P <0.05), and the KML group is extremely higher than the Control group (P < 0.01).

It was shown that FMT can significantly improve the working performance of puppies, and that the low dose group (KML) was most effective.

TABLE 3 Effect of FMT on Kunming dog working Performance

Note: different lower case letters in the same row represent significant differences (P <0.05) and different upper case letters represent very significant differences (P < 0.01).

The embodiments in the present description are described in a progressive manner, each embodiment focuses on differences from other embodiments, and the same and similar parts among the embodiments are referred to each other.

The previous description of the disclosed embodiments is provided to enable any person skilled in the art to make or use the present invention. Various modifications to these embodiments will be readily apparent to those skilled in the art, and the generic principles defined herein may be applied to other embodiments without departing from the spirit or scope of the invention. Thus, the present invention is not intended to be limited to the embodiments shown herein but is to be accorded the widest scope consistent with the principles and novel features disclosed herein.