| 1.1 Background and objectiveGermfree animals(GF)refers to animals that are not detectable in all living organisms.Furthermore,they refer to the use of existing detection techniques to see no live microorganisms and parasites in any part of the animal's body animal.Germfree animals have the following major characteristics: First,it can completely eliminate the background of microbial interference.The germfree animals background is clear and does not carry any live microorganisms and parasites.It use for the construction of flora,strain transplant model,and can be determined to exclude background microbial interference.Second,the human body is a "super-organisms" which has constitute the body cells and microbial cells together.Germfree animals are indispensible for the analysis of the function of a bacterial genome.The use of germfree animals technology to "transfer"(Colonized bacteria)or "knockout"(sterilize)flora is essential to resolve the impact of a specific metagenomics for diseaseas,as important as analyzing the contribution of the gene to the disease for using genetic engineering animal technology knock in or knock out a gene.With the development of high-throughput sequencing technology,more and more scientists have found that there is a correlation between intestinal flora and many diseases.The flora-associated transplantation model and gnotobiotes animal model was established by using GF mice,can analysis of the relationship between bacteria and human.Therefore,the demand for GF mice was increasing year by year.However,the existing commercial GF mice can not meet the needs of the market.At present,the breeding of GF mice is difficult to get the first generation mice,and the breeding rate is low.The GF mice were cultured by the method of biological purification,which was obtained by caesarean section.The suckling mice were feed by artificial feeding.The key to the successful feeding of the GF mice is the development of a nutritional formula suitable for the growth and development of the GF mice,including the milk products for lactation and the feed for growth and reproduction stages.However,there are relatively few studies on the nutritional needs of GF mice.There is no standard feed in the world for the lactation and weaning period of the GF mice,which greatly affected the work of the establishment of standardized GF mice.In order to adapt to the sterile environment,the biological characteristics of germfree mice have changed dramatically.The cecum of the germfree mice became enlarged because of its inability to ferment the polysaccharide in the food.In order to absorb more nutrients,the intestinal wall is thinner and villi is longer.The immune system of mice was at low level due to lack of microbial antigen.And the germfree animals basal metabolic rate is higher,not easy to accumulate fat.The stress response and behavioral characteristics of the mice were different from that of the normal animals.Therefore,the nutritional requirements of germfree mice are different from those of normal mice.Normal mouse intestinal flora can synthesize the B vitamins and vitamin K needed for the body's metabolism.But there was no bacteria in the germ free mice,and the content of vitamin B and vitamin K could not meet the needs of normal mice.In addition,after sterilized by high dose irradiation,the feed nutrition were severely damaged.Therefore,according to its biological characteristics and the nutritional needs of the living environment of GF mice is different from ordinary mice.Therefore,in order to solve the above problems,the research combined with the physiological and biochemical indexes and nutritional requirements of normal mice during lactation and growth and reproduction stages,ultimately determine the artificial milk formula suitable for germfree mice during lactation growth,and the fortified feed for growth stage.The IHFA model was established by using GF mice.The results of 16 S rDNA high throughput sequencing analysis further confirmed the relationship between the fortified feed and the flora-associated transplantation model.1.2 methods(1)According to the nutritional composition of mouse milk,the growth characteristics of KM mice,such as open the ears?open the eyes and so on,were compared in the infant milk powder group,the artificial milk group and the normal control group.We estimated the ablactation rate.We collected data on body weight and tail length during lactation,and compared pathological section of small intestine.The GraphPad Prism6.0 analysis software was used to analyze the results of blood routine test and the physical and chemical indexes of blood gas,so as to evaluate the difference between the infant milk powder group,the artificial milk group and the normal control group.(2)According to the standard of AIN-93 G,we compared the degree of loss of nutrients in the irradiated feed.Combined with the characteristics of vitamin B group and vitamin K which can not be synthesized in the GF mice,the invention can be prepared into a fortified feed.We used SPSS19.0 to analyze the changes of body weight in the common diet group(group 1),the fortified feed group(group 2)and the normal control group.The mice were mated 3 times after sexual maturation.We analyzed the pregnancy rate,litter size and number of weaning.The 5 mice of each group were randomly selected,comparing the coagulation four and the pathological section of heart,to evaluate the differences between the common diet group(group 1),the fortified feed group(experimental group 2)and the normal control group.(3)The model mice of normal infant intestinal flora,fed the fortified feed and infant milk powder which the same as the host.Mice feces were sequenced by 16 S rDNA to analyze the similarity of bacteria at the colonization of bacteria for 14 days and 28 days.1.3 Results(1)137 suckling mice were fed with infant milk powder by artificial feeding method,and only 0 were isolated.In the artificial milk group,124 suckling mice were fed with artificial milk,and weaning 72.The results showed that the ablactation rate was about 58%.There was a significant difference in body weight and tail length between infant milk powder group and normal control group(P < 0.01).The growth of body weight and tail length of artificial formula milk was similar to that of normal control group(P > 0.05).There was no significant difference in red blood cell count,white blood cell count,platelet count and blood pH(P > 0.05).There was significant difference in oxygen partial pressure(P < 0.05).It can be seen that the artificial formula can basically meet the needs of the growth of GF mouse lactation.(2)Compared with the normal control group,there was significant difference in the body weight increase of 3-6 weeks(P < 0.05)in the common diet group(group 1).There was no significant difference between the control group and the fortified feed group(P > 0.05).After reaching maturity,there was a significant difference in the pregnancy rate and litter size of the common diet group(group 1)(P < 0.05).Compared with the normal control group,there was no significant difference in the pregnancy rate and litter size of the fortified feed group(group 2)(P > 0.05).There was no significant difference in the coagulation four in t the fortified feed group(group 2)(P > 0.05).There were a few bleeding spots in the pathological sections of mice in the common diet group(group 1).This group of mice showed pathological bleeding.The common feed can not be used for GF mice feeding.And through the improvement of nutrition after the feed can basically meet the needs of GF mice growth and reproduction.(3)Flora-associated mice were fed with different diets which are milk and feed.At the colonization of bacteria for 14 days,we found that the mice fed milk powder,at the "order" level,was similar to that of Clostridiales and Enterobacteriales in infant fecal Clostridiales and Enterobacteriales with the method of 16 S rDNA sequencing.The results suggested that the infant human flora-associated mice with the same diet was closer to the host.At the colonization of bacteria for 28 days,there was no significant difference in intestinal microflora between the fortified feed group and the milk powder group.The fortified feed is suitable for feeding the flora-associated model mice.1.4Conclusions:(1)The artificial formula can basically meet the needs of the growth of GF mouse lactation.(2)The nutritional components of the fortified feed can basically meet the nutritional needs of GF mice.(3)There was little effect on the bacterial flora of the flora-associated model mice with the fortified feed.It is suitable for feeding that mice. |
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