Cationic Antimicrobial Peptides Combine with Antibiotics for Liquid Boar Semen Preservation

Autor: WONG, CI-HONG, 翁齊宏
Rok vydání: 2017
Druh dokumentu: 學位論文 ; thesis
Popis: 105
Antibiotics should be added to boar semen extender to increase semen storage time and reduce pathogen proliferation. Although antibiotics easily cause bacterial resistance, a lack of them in boar semen results in bacterial proliferation, a decline in sperm quality, artificial insemination failure, and even harm to the reproductive systems of artificially inseminated sows. Cationic antimicrobial peptides (cAMPs) have a high affinity to bacteria. They are highly stable and provide little bacterial resistance, so they are potential substitutes for antibiotics. However, few studies have been conducted in which antimicrobial peptide has been added to boar-extended semen. This study evaluated the effects of cAMP for bacterial proliferation and sperm quality in extended semen. Two cAMPs, Pleurocidin (PLE-a) and Q4-15a, as well as the antibiotic gentamicin and four types of bacteria, namely Escherichia coli (E. coli), Pseudomonas aeruginosa, Serratia marcescens, and Klebsiella oxytoca were used in the experiment. The experiment was divided into five parts. Experiment 1 evaluated the effects of simulated contamination of common bacteria in boar-extended semen on sperm quality. Experiment 2 evaluated the minimal inhibitory concentration (MIC) of cAMPs on four common bacteria in boar semen. Experiment 3 evaluated the effects of cAMPs on boar sperm quality. Combining the results of three experiments described, experiment 4 evaluated the effects of cAMPs and gentamicin on sperm quality in boar extended semen–simulated contamination of common bacteria and analyzed whether the antimicrobial substance could improve sperm quality in extended semen. Bacterial concentration, pH value, osmotic pressure, agglutination, normal morphology, acrosome integrity, viability, membrane integrity, and mitochondrial functional were detected in these experiments. Finally, experiment 5 evaluated the effects of cAMPs on sperm fertility through in vitro fertilization. The results of experiment 1 showed that the boar-extended semen simulated bacterial contamination at 5 × 106 CFU/mL and stored at 17°C for 72 hours, increased bacterial concentration, decreased the pH value, changed osmotic pressure, decreased sperm motility, and increased sperm agglutination. The results of experiment 2 indicated that 256 μg/mL of PLE-a or Q4-15a in 5 × 106 CFU/mL of extended semen–simulated bacterial contamination could not inhibit the proliferation of E. coli and K. oxytoca. However, the combination of gentamicin, PLE-a, and Q4-15a could decrease the MIC of cAMPs to 128 μg/mL. A total of 5 × 103 CFU/mL of extended semen–simulated bacterial contamination, PLE-a, and Q4-15a could completely inhibit four types of bacteria, and the MICs of cAMPs were 8 μg/mL and 32 μg/mL, respectively. The combination of gentamicin and the two cAMPs also decreased their MIC. Experiment 3 revealed that the high concentration of cAMPs in extended semen could damage the sperm. Extended semen containing more than 25 μg/mL of PLE-a or Q4-15a could seriously damage sperm motility and acrosome integrity. The concentration of cAMPs below 10 μg/mL did not affect sperm quality. Experiment 4 showed that 8 μg/mL of PLE-a combined with gentamicin inhibited bacterial proliferation in extended semen–simulated bacterial contamination but did not affect sperm quality. In Experiment 5, the extended semen supplemented with 8 μg/mL of PLE-a and 4 μg/mL of gentamicin, 4 μg/mL of PLE-a and 32 μg/mL of gentamicin, 16 μg/mL of Q4-15a and 4 μg/mL of gentamicin, or 4 μg/mL of Q4-15a and 32 μg/mL of gentamicin and stored at 17°C for 72 hours could inhibit bacterial proliferation under 103 CFU/mL and maintained more than 70% sperm motility. The results of the in vitro fertilization experiment confirmed that sperm in extended semen could penetrate the oocytes. In conclusion, the bacteria in boar semen can damage semen and sperm quality. The extended semen supplemented with cAMPs could inhibit bacterial proliferation. Ten μg/mL of cAMPs in the extended semen did not affect sperm quality and fertility.
Databáze: Networked Digital Library of Theses & Dissertations
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