Effect Of Recombinant Pasmid Of CA? Gene On Egg Fertilization Rate In Hens

The poultry sperm storage tubules?SSTs?are special tubular glands which locate at the uterine and vaginal junctions?UVJ?.This environment allows the sperm to remain stationary and maintain vitality in the hen for a long time.Carbonic anhydrase ??CA??is an important enzyme secreted by SSTs in UVJ.It is speculated that CA? may be involved in avian sperm storage.Therefore,studying the function of CA? in the long-term storage of sperm in the SSTs is of great significance for exploring the physiological mechanism of storing activity in the poultry.This study analyzed the biological information of chicken CA? sequence and found the antigenic epitopes of CA? protein,then constructed the pVAX-CA?-asd recombinant plasmid to immunize hens.Divided 105,38-week-old laying hens into 7groups with 15 hens in each group.The hens of 1^st,2^nd,3^rd,4^th and 5^th group were immunized with 10?50?100?150?200?g of pVAX-CA?-asd recombinant plasmid,respectively.The hens of 6^th group were immunized with 100?g of pVAX-asd empty plasmid and the hens of 7^th group was injected with normal saline.Each group was immunized twice with an interval of 21 days.During the experiment,eggs were collected at each day,body weight was measured every week,and blood was collected every week to detect the CA? antibody and L-lactic acid.The number of sperm penetration of egg yolk membrane were recorded and the fertilization rate was calculated for each hens.The hens were killed at 6 weeks after immunization to measure the expression of CA? mRNA and MCT4 mRNA in hens'genitals.The results showed that:1.The chicken CA? has 316 amino acids which is a membrane-bound protein with strong hydrophilicity and signal peptide.There are abundant?-helix and?-fold in the secondary structure and many antigenic epitopes.The CA? protein has multiple serine and threonine phosphorylation sites and three tyrosine phosphorylation sites,and the asparagine at position 165 may be a glycosylation site.Evolutionary analysis indicated that chicken and turkey CA? were most likely to be derived from the same ancestor because their CA? amino acid sequence has the highest similarity which is94.01%.The lower similarity of chichen CA? with guinea fowl,pheasant,geese and mallard is 92.74%?91.38%?85.55%?84.35%,indicating that the CA? sequence is highly conserved in avian species.2.The anti-CA? antibody levels in the experimental group were significantly higher than the two control groups from the 7^th day to the end of the experiment and reached the peak on the 28^th day after the first immunization,then the antibody level gradually decreased?p<0.05?.3.The CA? mRNA of UVJ in experimental groups was significantly lower than the two control groups.The mRNA of CA? gene in infundibulum funnel showed the valure of experimental groups was significantly lower than the saline control group.The expression of MCT4 mRNA in the UVJ of 4^th and 5^th groups was significantly decreased compared to the saline control group.Likewise,the relative expression of MCT4 mRNA in the infundibulum funnel of the five experimental groups was significantly lower than the saline control group?p<0.05?.4.There was no significant difference in body weight and egg production between the immunized groups and the control groups?p>0.05?.However,fertilization rate of 5^thh group was significantly decreased?p<0.05?.The number of sperm penitration at yolk membrane and L-lactic acid concertation in serum were decreased significantly in all experimental groups?p<0.05?after immunization.In conclusion,our data revealed that the CA? recombinant plasmid had no effects on body weight and egg production.Reduction in the expression of CA? and MCT4genes reduced the fertilization rate,concentration of L-lactic acid in serum and sperm penitration of yolk membrane.Therefore,our results suggested that the MCT4 and CA? genes may regulate the balance of microenvironment in storage sperm tubules together to provide the long-term storage of sperm in SSTs.