Investigation On The Breeding Status Of Captive Crested Ibis And Establishment Of PCR Sex Identification Method From Genome Of Crested Ibis Fecal

Crested ibis is a kind of rare wild protected animal in China.In recent years,the population of crested ibis has been greatly expanded by the combination of artificially population breeding and nature reserve establishment.However,the current population distribution of crested ibis is not wide enough,and the ability to withstand risks is relatively weak.Therefore,the protection work still has a long way to go.At present,due to the lack of effective and convenient methods for sex identification in artificial reproduction,the work of sex identification is seriously hindered.Therefore,it is necessary to establish an economical and effective method for sex identification of crested ibis.The reproduction rate of Louguantai artificial crested ibis population were investigated in this study;And the procedure of crested ibis'sex determination was established through the way of crested ibis feces collection with non-invasive sampling and a method of appropriate genomic DNA extraction,as well as design of PCR amplification system.The results are as following:1.The breeding records of Louguantai artificial crested ibis from 2009 to 2018 were collected,and the breeding status of crested ibis was investigated and analyzed.The results showed that the total population,sex ratio,and age structure of artificial ibis were reasonable,but how to properly handle the large proportion of the old age crested ibis is an urgent problem to be solved;Relevant managers have formed a relatively mature and standardized artificial assisted breeding technology,but have not yet properly solved the behavior of their parents to abandon their children;The survival number of chicks can be kept stable by human intervention,but there are significant differences in the annual changes of reproductive data indicators,and the broken eggs have a higher proportion of reproductive failures than dead chicks and rotten eggs.2.Genome samples of female and male crested ibis from blood samples are extracted by a kit in which as a positive template for PCR amplification to verify the feasibility of the universal bird primer 2467F/2530R.The female crested ibis appeared in a single band at358 bp and 552 bp respectively,while the male crested ibis appeared in a single band at552 bp.The results showed that the primer could be used to identify the sex of crested ibis.The sequencing results of the PCR products were identical to those of the NCBI database sequence,and it was confirmed that the PCR products are located in the CHD-1 gene of the sex chromosome.3.The modified GuSCN-SiO₂ method was improved to obtain genomic templates with similar purity and concentration as professional kits,making the practical application of detection becoming more convenient and more conducive and cheaper.In addition,the research determined the composition of the various reagents and the convenient procedures during the experiment.4.47 stool samples?24 female stool samples and 23 male stool samples?were collected and the 40 fecal genome samples?19 female stool samples and 21 male stool samples?with the purity in the normal range of crested ibis was extracted using modified GuSCN-SiO₂ method.The genotyping fragments of PCR that using the primer2467F/2530R consistent with the expected results can be obtained.The results showed that85.11%of the samples were correct and could be used for gender identification.In order to provide suggestions for improving the breeding rate of crested ibis,we investigated the reproductive status of artificially propagated crested ibis population and analyze the data related to the reproduction rate of crested ibis.At the same time,the sex identification procedure of crested ibis was established by modified GuSCN-SiO₂ method and the PCR technology.The experiment proved that the procedures we designed is effective and economical for sex identification,which can avoid the problem of mismatching and support convenience for the management of Crested Ibis artificial population breeding.