Effects Of Adding Arginine And Leucine In Sperm Preservation Solution On Fertilization And Hatching Of Hybrid Yellow Catfish (Pelteobagrus Fulvidraco ♀×P.Vachelli ♂)

Yellow catfish(Pelteobagrus fulvidraco)is one of the top aquaculture fish species with high annual production in China.The birth of the new variety,hybrid yellow catfish“Huangyou No.1”(GS-02-001-2018)has made a great contribution to the sustained and stable growth of yellow catfish industry.Since“Huangyou No.1”is a variety based on the crossbreeding between yellow catfish(♀)and darkbarbel catfish(♂),it is affected by interspecific reproductive isolation,which not only affects fertilization,but also affects hatching due to the high proportion of abnormal embryos produced during embryo development,resulting in a high deformity rate of in the progeny of the hybrid.Previously,we found that the high deformity rate of hybrid yellow catfish may be due to the impaired autophagy and delayed elimination of paternal mt DNA during early embryogenesis,while leucine can regulate cellular autophagy,and sperm viability can be improved by injecting leucine into yellow catfish.Therefore,in this study,L-arginine and L-leucine were added individually or together into the sperm preservation solution to investigate their effects on fertilization and hatching of hybrid yellow catfish,and determine the optimum concentration and explore whether there is a synergistic effect between these two amino acids.What’s more,we invented a new type of hatching barrel which is more suitable for artificial breeding of yellow catfish.Finally,we attemps to establish an efficient and convenient artificial breeding method,and provides scientific basis for guiding the breeding practice of yellow catfish.The main results of the experiment are as follows:1.Effects of different concentrations of L-arginine on fertilization and hatchingThe experiment was designed for the concentration gradient of L-arginine with a single factor double-difference,and the concentration was ranged from 0.25 mmol/L to8.00 mmol/L.The experimental results showed that the fertilization rate increased first and then decreased with the equal fold increase of arginine concentration.The fertilization rate of all concentration groups was significantly higher than that of the control group(p<0.05).Among them,the fertilization rate of the 0.50 mmol/L concentration group was the highest,reaching(69.83±1.52)%,which was significantly higher than that of all other concentration groups except the 1.00 mmol/L concentration group(p<0.05);the highest group added 8.00mmol/L concentration had the lowest fertilization rate(58.08±0.58%),indicating that high concentration had a certain negative effect.Regression analysis showed that the response of fertilization rate to arginine concentration was linearly correlated,and the optimum concentration was 0.449 mmol/L.Unlike fertilization rate,arginine had no significant effect on hatching rate and deformity rate(p>0.05).2.Effects of different concentrations of L-leucine on fertilization and hatchingConsistent with L-arginine,the experiment was designed in the same way for L-leucine,and the concentration was ranged from 0.05 mmol/L to 1.60 mmol/L.The experimental results showed that with the increase of leucine concentration,the fertilization rate and hatching rate increased first and then decreased,while the deformity rate decreased first and then increased.The fertilization rate in the 0.10-0.80 mmol/L concentration groups was significantly higher than that in the control group(p<0.05).Among them,the fertilization rate of the 0.80 mmol/L concentration group was the highest,reaching(72.37±1.92)%,which was significantly higher than the other concentration groups except the 0.20 mmol/L-0.40 mmol/L concentration groups.The hatching rate in the 0.20 mmol/L-1.60 mmol/L concentration group was significantly higher than that in the control group(p<0.05),with the highest hatching rate of(77.94±3.90)%in the 0.80 mmol/L concentration group,significantly higher than that in the 0.05-0.10 mmol/L concentration group and the 1.60 mmol/L concentration group(p<0.05).The deformity rate in the 0.10-1.60 mmol/L concentration groups was significantly lower than that in the control group(p<0.05),with the lowest deformity rate of(6.80±2.17)%in the 0.80 mmol/L concentration group,significantly lower than that in the 0.05 mmol/L-0.10 mmol/L concentration group and the 1.60 mmol/L concentration group(p<0.05).The experiment results also showed that it had a certain negative effect on fertilization rate,hatching rate and deformity rate when the concentrations reached 1.60 mmol/L.Regression analysis showed that the response of fertilization rate,hatching rate and deformity rate to leucine concentration were exponentially correlated,yielding optimum concentrations of 0.834mmol/L,0.969 mmol/L and 0.968 mmol/L,respectively.3.Effect of different concentrations co-action of L-arginine and L-leucine on fertilization and hatchingA 3~2 orthogonal design with 2 factors and 3 levels was used for L-arginine and L-leucine.The concentration level of arginine was 0.2 mmol/L,and the concentration level of leucine was 0.5 mmol/L.The experimental results showed that the 0.2 mmol/L arginine concentration corresponding to the 1.5 mmol/L leucine level group,the 0.4 mmol/L arginine concentration corresponding to the 3 level groups of leucine and the 0.6 mmol/L arginine concentration corresponding to the 1.0 mmol/L-1.5 mmol/L leucine level groups had significantly higher fertilization rates than the control group(p<0.05),with the fertilization rates increased by 29.19%-44.85%.The hatching rates of the two amino acid added groups were significantly higher(p<0.05)than that of the control group,which increased by 19.44%-38.06%.Contrary to the hatching rate,the deformity rate in each level group was significantly lower(p<0.05)than that in the control group,which decreased by 48.49%-77.11%.It was shown that the optimum concentrations of two amino acid combinations were arginine 0.4 mmol/L and leucine 1.0 mmol/L,compared with the control group,the fertilization rate and hatching rate were increased by 44.85%and 38.06%,respectively,and the deformity rate was reduced up 77.11%.To identify the reasons for changes in reproductive efficiency,sperm motility was further verified using CASA.The three treatment groups were 0.4 mmol/L arginine,1.0mmol/L leucine and 0.4 mmol/L arginine+1.0 mmol/L leucine.The results showed that sperm motility in the treatment group was significantly improved compared to the control group(p<0.05),with the highest parameters being achieved by the addition of both arginine and leucine,such as VAP(120.76±3.76μm/s),VSL(123.19±3.11μm/s),VCL(126.28±5.98μm/s)and the percentage of 30 s motile sperm(53.68±7.48%).At the same time,sperm motility time was also greatly increased,among which the combined treatment of 0.4 mmol/L arginine+1.0 mmol/L leucine had the longest motility time,which was significantly higher than that of the control group about 30 S,both fast and slow-moving sperm increased motility time.The results show that arginine and leucine have a synergistic effect to significantly improve sperm motility.4.Effect of new hatching tank on fertilization and hatchingWe tested the effect of two different hatching buckets on the hybridization efficiency of yellow catfish.The hatching rate of the designed new hatching tub were significantly higher than those of the traditional hatching tub(p<0.05),but the deformity rate was significantly lower(p<0.05).The fertilization rate and hatching rate were respectively increased by 15.13%and 20.76%compared with the traditional hatching tub;the deformity rate was reduced by 20.25%compared with the traditional hatching tub.For yellow catfish fingerlings,with or without amino acid addition,interspecies hybridization was carried out using a new type of hatching tank for further productivity verification.The results showed that the fertilization rate and hatching rate of intraspecific crossbreeding were significantly higher than those of interspecific crossbreeding,while the deformity rate was significantly lower than that of interspecific crossbreeding(p<0.05).With the addition of the optimum concentration of 0.4 mmol/L arginine and 1.0 mmol/L leucine,the fertilization rate of interspecific crossbreeding can be restored to the level of88.48%of intraspecific crossbreeding,and the hatching rate is higher than that of intraspecific crossbreeding,but the hatching rate is higher than that of intraspecific crossbreeding,the deformity rate was lower than that of intraspecific crossbreeding,but there was no significant difference(p>0.05),indicating that the hatching rate and deformity rate could be restored to the level of the intraspecific crossbreeding.Combined with all the results of this study,it is shown that the adding arginine and leucine to sperm preservation solution had a significant promoting and synergistic effects on improving the rates of fertilization and hatching.The fertilization rate,hatching rate and deformity rate of the new hatching bucket can meet the needs of artificial hatching and production practice.This will help to improve the efficiency of artificial propagation and is the key to achieve high efficiency in the promotion of new varieties,which is of great significance for the industrialization of new varieties and the development of the yellow catfish industry.