Optimization Of Antibiotics In Sheep Semen Diluent

With the long-term use of penicillin leading to increasing bacterial resistance,coupled with the fact that antibiotics in diluent are mainly added to penicillin,the preservation effect of sheep semen diluent is affected,to address this outstanding problem.In this study,we screened the optimal concentration of enrofloxacin in Kunlun I diluent,examined the diversity of microorganisms in sheep semen and their drug sensitivity;and investigated the effect of adding different antibiotics to the diluent on the expression of sperm apoptosis genes,in order to achieve the effect of low temperature preservation of sheep semen.1.To examine the microbial diversity in sheep semen and its sensitivity to antibiotics.In this study,the bacteria were isolated and purified by selecting diluted concentrations of 5×,10×and 100×sperm suspensions applied to Mc Conkey agar medium,Erythromax blue agar medium,SS agar medium,brain heart leaching agar medium and Baird-Parker agar medium using the dilution coating plate method,then the different morphological strains were isolated and purified by the plate scribing method,and the The strains were identified by 16s r RNA sequencing,and the drug sensitivity test was performed by paper diffusion method,and the selected antibiotics were penicillin,streptomycin and enrofloxacin.The results showed that 39 strains were isolated,and 16 different strains were isolated by removing the same results of different media,among which Escherichia,Shigella and Staphylococcus had a greater impact on semen quality.Among the 16 strains,3 were sensitive to penicillin,13 were resistant to penicillin,16 were resistant to streptomycin,4 were sensitive to enrofloxacin,4 were moderately sensitive and 8 were resistant to enrofloxacin,4 of which had no inhibition circle.The results showed that sheep semen is rich in microorganisms,and enrofloxacin has inhibitory effect on most bacteria.2.To investigate the effect of adding different concentrations of enrofloxacin to Kunlun I dilution solution on the effect of low temperature preservation of sheep semen.In this experiment,sheep semen was low temperature preservation at different concentrations(E₀ group:0μg/m L;E₂₅ group:25μg/m L;E₅₀group:50μg/m L;E₇₅ group:75μg/m L;E₁₀₀ group:100μg/m L)by adding 5 groups of enrofloxacin at different concentrations(E₀ group:0μg/m L;E₂₅ group:25μg/m L;E₅₀ group:50μg/m L;E₇₅ group:75μg/m L;E₁₀₀ group:100μg/m L)to Kunlun I dilution solution,and using Kunlun I dilution solution（C group）as the control group,and the sheep semen was low temperature preservation for an extra long time at 0 d,2 d,4 d,6 d,8 d,10 d and 12 d,the sperm viability,acrosome integrity rate and bacterial count were examined and analyzed.The results showed that the sperm viability of the diluted sheep semen low temperature preservation in the E₇₅ group was significantly higher than that of the remaining five groups from the 8 d onwards（P<0.01）and was the highest among the five groups at the 12 d of preservation（50.67±1.16）%;the sperm viability of sheep low temperature preservation in the E₀ group with diluted fluid was lower than that of the remaining five groups from the 2 d onwards and was significantly lower than that of the E₅₀,E₇₅ and C groups from the 6 d onwards（P<0.01）.The sperm acrosome integrity rate in the E₇₅ group was significantly higher than that in the remaining 5 groups only on the 10 d（P<0.05）,while the other days were not significantly different from those in the E₂₅,E₅₀,E₁₀₀ and C groups（P>0.05）,while the sperm acrosome integrity rate in the E₀group was significantly lower than that in the remaining 5 groups from the 6 d（P<0.05）;the semen bacterial count in the E₀ group was significantly higher than that in the remaining 5 groups from the 4 d（P<0.05）.The bacterial count of semen in the E₀ group was significantly higher than that in the remaining 5 groups from the 4 d（P<0.01）,but the bacterial counts of semen in all 6groups were within the national standard.It can be seen that the addition of 75μg/m L enrofloxacin to KunlunⅠdilution solution was more effective in preserving sheep semen at low temperature,while the addition of 50μg/m L enrofloxacin had the next best effect with KunlunⅠdilution solution,and all three groups could achieve the effect of long-lasting preservation of sheep semen.3.To test the effect of adding different antibiotics on the expression of apoptotic genes in sheep spermatozoa.In this experiment,we extracted the sperm RNA of sheep sperm without the addition of antibiotics Kunlun I dilution,Kunlun I dilution with 75μg/m L enrofloxacin and Kunlun I dilution with low temperature preservation at the 6 d.The pro-apoptotic genes Bax,Caspase-3,Caspase-8,Caspase-9,TNF-α,P53 and the expression of the apoptosis inhibitory genes Bcl-2 was measured by q RT-PCR.The results showed that the addition of 75μg/m L enrofloxacin Kunlun I diluent group and Kunlun I diluent group could reduce the expression of pro-apoptotic genes Bax,Caspase-3,Caspase-8,Caspase-9,TNF-α,P53,and increase the expression of apoptosis inhibitor gene Bcl-2,but the difference between them was not significant（P>0.05）.The results suggest that the addition of antibiotics in diluent affects the expression of sperm apoptosis-related genes.In conclusion,through three aspects of semen quality testing,microbial diversity identification and molecular testing,it was concluded that the addition of 75μg/m L enrofloxacin to Kunlun I diluent has the best effect and is better than Kunlun I diluent preservation effect and can provide long-lasting low temperature preservation of sheep semen.This study provides a certain experimental basis for the optimization of sheep semen dilutions and semen preservation,and helps to promote the healthy development of sheep breeding industry.