| The test objective was to study estrus synchronization technique of dairy goat,and establish a culture system of dairy goat fetus fibroblast cell in vitro in order toobtain the foundation of setting up the mammary gland bioreactor,and determinationon sex of dairy goat fibroblast cell. The results was as follows:Using three different method to process ewe, the test prove that use CIDR plug,PG and FSH can get the highest level of synchronization of estrus. At the level ofestrus synchronization we can not only use CIDR plug, PG, FSH but also CIDR, PGand PMSG, both these two ways can get a good effect. When we use natural breedinggroup I got the highest rate.Through out surgery to get about the40-45days’ dairy goat fetus. The primarycells of dairy goat fetus were obtained by methods of tissue culture and collagenasedissociation. Using subculture and purified through trypsin digestion and differentialanchoring velocity, at last set up the system of dairy goat fetus fibroblast cell. Theresults showed that the fibroblast cell was cultured with explants need more time, andcultured with trypsinization has more dead cell. Fibroblast cells could long-termmaintain in DMEM medium supplemented with10%fetal bovine serum, passage cellhave a good growth. Fibroblast cell is fusiform shape or oblate star, have apophysis.Observations on determination of viability, dynamic growth, population doublingtime(PDT), and the analysis of karyotype. The results showed that after anabiosis thecell viability was92.3%and has the same growth condition; cell growth tendencypresentation was―S‖and the PDT of cells was approximately40.4h; maintaining anormal diploid chromosome modal, the number was2n=60and which contained29pairs of telocentric chromosomes and a pair of sex; microbial contamination wasnegative. Every index in the cell line met all the standard quality controls of ATCC.According to the sequences of SRY gene and β-lactoglobulin gene of goatpublished in GenBank,2pairs of PCR primers were designed, and the genome DNAfrom fibroblast cell were amplified by PCR. With genome DNA from male andfemale dairy goats as the positive and negative control. The sequence of PCR productwas determined and its homology was analyzed. The results showed that positive andfibroblast cell line had SRY gene272bp part and β-lactoglobulin349bp part both2 amplified bands, negative had β-lactoglobulin349bp part only1amplified band. Theresults of sequence determination showed that the272bp part PCR product was SRYgene, and the cell was male cell line. |
