| The yolk sac is an important organ in the process of embryonic development.The yolk sac of poμltry embryos stores a large amoμnt of yolk,which provides nμtrition for the development of varioμs organs of the embryo,and is also the place where blood vessels and blood cells are formed first.The yolk sac is mainly composed of extraembryonic ectoderm,extraembryonic mesoderm and extraembryonic endoderm,and the supply of nutrients is mainly completed through the endocytosis of extraembryonic endoderm cells,so the yolk sac is very important for embryonic development.In production practice,gender-differentiated management of poultry for different production purposes is conducive to saving costs and improving economic benefits;some studies have shown that the yolk sacs of different sexes have certain differences in morphology and structure.Morphological and structural changes during the embryonic period and explore its rules,and then explore the method of sex identification of early embryos.In this experiment,Jilin white goose was μ sed as the research object.The sex of the embryo was preliminarily judged by observing the vascular morphology on the surface of the yolk sac by taking eggs.In order to explore the changes in the morphology of the yolk sac tissue,HE staining was used to observe the morphological development process of the yolk sac on the10th-28th(E10-28)day of the embryonic period;to explore the special time nodes of the yolk sac development,and then through ALK,PAS,and IHC staining,Observe the expression and distribution of enzymes,glycogen and Ig Y in the yolk sac of different sexes at the developmental nodes;in order to further verify the relationship between the yolk sac and gender,the total amino acid content in the yolk fluid from E10 to E27 was measured to observe whether the changes in amino acid content between males and females The same;the total protein concentration of E10-E27 was measured to observe the difference between male and female;q PCR technology was used to measure the m RNA expression of amino acid metabolism-related genes LAP3,CBS,and SARDH in the back skin tissue of embryos of different sexes,and to explore the differences between females.The difference between them,and the connection with the yolk sac was initially established.The test results are as follows:(1)Observing the relationship between the distribution of blood vessels in the yolk sac and sex by lighting eggs.246 eggs of Jilin White Goose were randomly selected for hatching,and the eggs were illuminated at E5 of embryonic development.The vascular morphology of the yolk sac was observed,and the sex of the embryo was preliminarily judged.The sex of the embryo was verified by micro-quantity whole blood PCR.The results showed that during E5-E8,the blood vessels on the yolk sac of male embryos were distributed symmetrically,while those of female embryos were asymmetrically distributed;in addition,the allantois would block the blood vessels of the yolk sac at E8,while the blood vessels were most clearly distributed and observed in the largest area at E7,E7 is determined to be the best observation time point,and the accuracy rate of judging the gender of eggs at this time can reach 79%.Subsequently,3female and male eggs with similar egg weight after sex identification were selected for embryo weight weighing.The results showed that there was no significant difference in embryo weight between eggs of different sexes(p>0.05).(2)Observe the development process of the yolk sac and the expression of various substances through histochemical staining,and determine the content of total amino acids and total proteins in the yolk fluid.The results showed that the special change period of the structure of the yolk sac was: around the 10 th day,the blood vessels inside the yolk sac had been covered by tissues and began to extend and grow.On the 13 th day,the hematopoietic tissue grows rapidly,and the folds of the yolk sac penetrate deep into the yolk.The cross-sectional area of the yolk sac blood vessels was the largest at E18,and the blood vessel walls were the thickest.Degeneration was seen in the following days,and we specμlated that the degeneration began after 18 days.At E23,the yolk sac begins to enter the abdominal cavity of the embryo,and there is basically no hematopoietic tissue.At E28,most of the yolk sac enters the abdominal cavity of the embryo,and the yolk fluid in it is basically absorbed;the hematopoietic tissue disappears,and the blood vessels decrease or disappear.The enzymes in the yolk sac were mainly concentrated in the hematopoietic tissue and blood,and the most obvious on the 18 th day,with the largest expression area;the glycogen was mainly concentrated in the extraembryonic endoderm cells,and the glycogen was the most concentrated on the 18 th day;Ig Y was mainly concentrated in the serum and In the cell membrane and cytoplasm of the extraembryonic endoderm,it is stably expressed on the cell membrane at various stages,but the positive reaction is the most in the cytoplasm of E18,and the male is more obvious than the female;the female amino acid content reaches the highest on the 19 th day,and the male is the highest on the 21 st day;verified the yolk sac relationship with gender.And we found that the key time node of hair follicle development is basically the same as the node of yolk sac development,so it is speculated that there may be a certain connection between yolk sac and hair follicle development.(3)Determination of the m RNA expression of LAP3,CBS,and SARDH genes in the feather follicle tissue of the back skin of goose embryos.The results showed that the relative expression of LAP3 gene m RNA: males showed a trend of rising,then falling and then rising,and E18 had the highest expression level,which was significantly different from E10,E13,E23,and E28(p<0.05);females showed a trend of falling first and then rising,reached the highest at E28,and the difference was extremely significant(p<0.01)with E10,E13,E18,and E23;the relative expression of CBS gene m RNA: the male showed a sudden rise and fall,and the highest expression at E18 was the same as that at E10,E13,and E23,E28,the difference was extremely significant(p<0.01);females showed a trend of decreasing first and then increasing,and the expression was highest at E28,which was significantly different from E10,E13,E18,and E23(p<0.05);the relative expression of SARDH gene m RNA: males showed There was a downward trend after an increase,and the highest expression level of E18 was significantly different from that of E10,E13,E23,and E28(p<0.05);females were in a stable state,and the expression levels of E10,E13,E18,E23,and E28 were not significantly different(p>0.05).The expression levels of LAP3,CBS,and SARDH genes were much higher in males than in females on the 18 th day(p<0.05).It is speculated that the sex of embryos can be determined by studying the expression levels of these genes.To sum up,we have preliminarily determined the method of early sex judgment through candling eggs and observing the structure of the yolk sac,which provides a theoretical basis for early sex distinction in production practice,and understands the expression rules of the components of the yolk sac at different stages.The relationship between the yolk sac and skin feather follicle development was preliminarily discussed,which provided some ideas for future research on the yolk sac of poultry. |
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