Determination of a cooling-rate frame for antibiotic-free preservation of boar semen at 5°C
Determination of a cooling-rate frame for antibiotic-free preservation of boar semen at 5°C
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DOI：
10.1371/journal.pone.0234339
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发表时间：
2020-06-09
期刊：
影响因子：
3.7
通讯作者：
Waberski, Dagmar
中科院分区：
文献类型：
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作者：
Paschoal, Aline F. L.;Luther, Anne-Marie;Waberski, Dagmar
只要保持精子质量并防止细菌生长，猪精液的低温保存就有可能省去精液稀释液中的抗生素。本研究的目的是确定猪精液在不含抗生素的稀释液中5℃保存的最佳降温速率范围。用7种不同的冷却速度将8头公猪的精液从30摄氏度冷却到5摄氏度，最初的冷却速度为0.01至0.36摄氏度分钟(-1)，稀释后2小时至24小时达到5摄氏度。精子活力、膜完整性、膜流动性、线粒体膜电位和对获能刺激碳酸氢盐的反应在5℃下保持较高水平144小时，此时精液在30~25℃的降温范围内冷却0.01~0.09℃min(-1)，然后在0.02~0.06℃min(-1)~10℃和0.01~0.02℃min(-1)的最终保存温度下冷却。在30到10摄氏度的温度范围内，0.07摄氏度的降温速度分钟(-1)导致对碳酸氢盐的反应降低(P<；0.01)，在1小时内快速降温至5摄氏度，降温速度为0.31摄氏度分钟(-1)，所有精子参数的值都较低(P>；0.05)。在进一步的实验中，在22℃下缓慢冷却6h后，在无精子接种的扩展器中，大肠杆菌的数量暂时增加了0.5×10(3)到2.4×10(3)cfu mL(-1)。总体而言，储存精液中的嗜中性菌载量低于6×10(3)cfu mL(-1)，这一水平被认为不是精子质量的关键。总之，为猪精液在5摄氏度下的无抗生素保存建立了合适的冷却方案，从而允许在研究和人工授精中应用低温保存。
Hypothermic storage of boar semen provides the possibility to omit antibiotics from semen extenders so long as sperm quality is maintained and bacterial growth prevented. The objective of this study was to determine an optimal cooling-rate frame for boar semen preserved at 5 degrees C in an antibiotic-free extender. Semen from eight boars extended in AndroStar(R) Premium was cooled from 30 degrees C to 5 degrees C using seven different cooling rates, ranging initially from 0.01 to 0.36 degrees C min(-1) and reaching 5 degrees C between 2 h and 24 h after dilution. Sperm motility, membrane integrity, membrane fluidity, mitochondrial membrane potential and the response to the capacitation stimulus bicarbonate remained at a high level for 144 h at 5 degrees C when the semen was initially cooled in a cooling-rate frame ranging from 0.01 to 0.09 degrees C min(-1) in the temperature zone from 30 to 25 degrees C, followed by 0.02 to 0.06 degrees C min(-1) to 10 degrees C and 0.01 to 0.02 degrees C min(-1) to the final storage temperature. A cooling rate of 0.07 degrees C min(-1) in the temperature zone from 30 to 10 degrees C led to a reduced response to bicarbonate (P < 0.01) and fast cooling to 5 degrees C within 1 h with a cooling rate of 0.31 degrees C min(-1) resulted in lower values (P > 0.05) of all sperm parameters. In a further experiment, slow cooling with a holding time of 6 h at 22 degrees C induced after 6 h storage a temporary increase in Escherichia coli of 0.5 x 10(3) to 2.4 x 10(3) CFU mL(-1) in the sperm-free inoculated extender. Overall, the load of mesophilic bacteria in the stored semen was below 6 x 10(3) CFU mL(-1), a level that is not regarded as critical for sperm quality. In conclusion, appropriate cooling protocols were established for the antibiotic-free storage of boar semen at 5 degrees C, allowing the application of hypothermic preservation in research and in artificial insemination.