Abstract：
Poor sperm quality in cooled-shipped semen has been related to subpar fertility in horses. Therefore, this study aimed to evaluate the ability of post-cooling sperm processing to improve sperm parameters of cooled-stored stallion semen for artificial insemination. For all experiments, ejaculates were collected, processed, and diluted in skimmed milk-based (SM) medium and stored at 5 °C/24h. In all experiments an aliquot of unprocessed cooled semen was used as a control. In the first experiment (Exp 1.), cooled-stored semen from 16 stallions (n = 32) was processed by SpermFilter or centrifugation (600×g/10min) and resuspended in an egg yolk-based freezing medium containing permeating cryoprotectants (EY-C) for cryopreservation. Sperm recovery and motility parameters were immediately assessed after sperm resuspension in both groups and compared with unprocessed (Unp) samples. In Exp 2., cooled semen samples from six stallions (n = 18) were processed using SpermFilter and resuspended in SM or EY-C. Motility parameters and plasma membrane integrity were assessed in all groups (Unp, SM, and EY-C). In Exp 3, cooled semen from four stallions (n = 20) was processed by SpermFilter, resuspended in SM, EY-C, or egg yolk-based medium without cryoprotectants (EY-nC); and submitted to a thermoresistance test (37 °C/3h). Motility parameters, plasma membrane integrity and stability, mitochondrial membrane potential, mitochondrial superoxide generation, and DNA fragmentation index were evaluated in all groups. Finally, in Exp 4, 39 estrous cycles of 11 mares were inseminated with unprocessed (n = 6) cooled-stored semen or semen cooled at 5 °C/24h and then processed by SpermFilter and resuspended in SM (n = 5), EY-C (n = 11), EY-nC (n = 11), or centrifuged and resuspended in EY-C (n = 6). Overall, semen processing and resuspension in EY mediums (EY-C and EY-nC) improved sperm parameters compared with those of unprocessed semen (P < 0.05). Centrifugation (91 ± 5 %) recovered more sperm than SpermFilter (84 ± 9 %; P < 0.05). Sperm resuspended in EY-nC maintained better sperm parameters throughout the thermoresistance test than those in the other groups (P < 0.05). The fertility rates were similar between all groups (P > 0.05). In conclusion, processing and resuspension in EY medium can improve sperm parameters in post-cooled-stored stallion semen.
摘要：
冷却精液中精子质量差与马的生育能力不足有关。因此,本研究旨在评估冷却后精子处理改善冷却储存的种马精液人工授精精子参数的能力。对于所有的实验,收集了射精，已处理,并在脱脂乳基(SM)培养基中稀释并在5°C/24h下储存。在所有实验中,将未加工的冷却精液的等分试样用作对照。在第一个实验中(Exp一1。),通过SpermFilter或离心（600×g/10min）处理来自16颗种马（n=32）的冷却储存的精液，并重悬于含有渗透性冷冻保护剂（EY-C）的基于蛋黄的冷冻培养基中进行冷冻保存。两组精子重悬后立即评估精子恢复和运动参数，并与未加工（Unp）样品进行比较。在Exp2中。,使用SpermFilter处理来自六个种马（n=18）的冷却精液样品，并重悬于SM或EY-C中。在所有组中评估了运动参数和质膜完整性（Unp，SM,和EY-C）。在实验3中，通过SpermFilter处理来自四个种马（n=20）的冷却精液，在SM重新暂停，EY-C,或不含冷冻保护剂的基于蛋黄的培养基（EY-nC）；并进行耐热性测试（37°C/3h）。运动性参数,质膜的完整性和稳定性，线粒体膜电位，线粒体超氧化物的产生，并对各组进行DNA片段化指数评价。最后,在实验4中，用未加工（n=6）冷却储存的精液或在5°C/24h冷却的精液对11只母马的39个发情周期进行授精，然后通过SpermFilter进行处理并重新悬浮在SM中（n=5），EY-C（n=11），EY-nC（n=11），或离心并重悬于EY-C(n=6)。总的来说,与未加工精液相比，在EY培养基（EY-C和EY-nC）中的精液加工和重悬可改善精子参数（P<0.05）。离心（91±5％）比SpermFilter（84±9％；P<0.05）回收更多的精子。重悬于EY-nC的精子在整个耐热性测试中保持了比其他组更好的精子参数(P<0.05)。各组生育率差异无统计学意义(P>0.05)。总之,在EY培养基中处理和再悬浮可以改善冷却后储存的种马精液中的精子参数。
