Longobardi Valentina, Salzano Angela, Campanile Giuseppe, Marrone Raffaele, Palumbo Francesco, Vitiello Milena, Zullo Gianluigi, Gasparrini Bianca
Department of Veterinary Medicine and Animal Production, Federico II University, Naples, Italy.
Department of Veterinary Medicine and Animal Production, Federico II University, Naples, Italy.
Theriogenology. 2017 Jan 15;88:236-243. doi: 10.1016/j.theriogenology.2016.09.031. Epub 2016 Sep 20.
The aim of this study was to evaluate the effect of carnitine supplementation of semen extender on fertility parameters of frozen-thawed buffalo sperm. Buffalo semen was cryopreserved in BioXcell containing 0 (control group), 2.5 and 7.5-mM carnitine. After thawing, viability, motility, membrane integrity and capacitation status (assessed by localization of phosphotyrosine-containing proteins and chlortetracycline, chlortetracycline assay) were evaluated. Furthermore, total antioxidant capacity, reactive oxygen species (ROS) and lipid peroxidation levels, as well as adenosine triphosphate (ATP) content and phospholipids concentration were assessed. Finally, in vitro-fertilizing ability was evaluated after heterologous IVF. An increased post-thawing sperm motility and membrane integrity were recorded in both treated groups compared with the control (44.4 ± 3.5, 53.1 ± 3.9, and 52.5 ± 3.6%; P < 0.05 and 48.44 ± 0.69, 55.19 ± 0.54, 59.63 ± 0.30%; P < 0.01 with 0, 2.5-mM, and 7.5-mM carnitine, respectively). Supplementation of carnitine to the freezing extender decreased (P < 0.01) the percentage of sperm displaying fluorescence at both equatorial and anterior acrosomal regions (pattern EA), corresponding to high capacitation level, compared with the control (30.3 ± 3.8, 18.8 ± 2.8, and 7.2 ± 2.9%, respectively, with 0, 2.5-mM, and 7.5-mM carnitine). In agreement with this, carnitine also decreased (P < 0.01) the percentage of sperm displaying chlortetracycline pattern B (capacitated sperm) (63.8 ± 1.8, 46.8 ± 2.2, and 37.2 ± 1.8%, respectively with 0, 2.5-, and 7.5-mM carnitine). Interestingly, carnitine increased total antioxidant capacity and ATP content of buffalo frozen-thawed sperm (1.32 ± 0.02, 1.34 ± 0.01, 1.37 ± 0.01 mM/L and 4.1 ± 0.1, 5.3 ± 0.1 and 8.2 ± 0.4 nM × 10 sperm; P < 0.01, respectively, with 0, 2.5- and 7.5-mM carnitine). Intracellular ROS decreased in carnitine-treated sperm compared with the control, as indicated by dihydroethidium (DHE) values (0.22 ± 0.01, 0.18 ± 0.01, and 0.14 ± 0.0 μM/100 μL dihydroethidium, respectively, with 0, 2.5-, and 7.5-mM carnitine; P < 0.01), whereas lipid peroxidation levels (on average 30.5 ± 0.3 nmol/mL MDA) and phospholipids concentration (on average 0.14 ± 0.00 μg/120 × 10 sperm) were unaffected. Despite the improved sperm quality, the percentage of normospermic penetration after IVF was not influenced (on average 53.5 ± 1.8). In conclusion, enrichment of extender with carnitine improved buffalo sperm quality by increasing ATP generation and modulating ROS production, without affecting in vitro fertilizing ability.
本研究旨在评估在精液稀释液中添加肉碱对冷冻解冻后水牛精子生育参数的影响。将水牛精液冷冻保存在含有0（对照组）、2.5和7.5 mM肉碱的BioXcell中。解冻后，评估精子的活力、运动能力、膜完整性和获能状态（通过含磷酸酪氨酸蛋白的定位以及金霉素、金霉素检测进行评估）。此外，还评估了总抗氧化能力、活性氧（ROS）和脂质过氧化水平，以及三磷酸腺苷（ATP）含量和磷脂浓度。最后，在体外受精后评估体外受精能力。与对照组相比，两个处理组解冻后精子的运动能力和膜完整性均有所提高（分别为44.4±3.5%、53.1±3.9%和52.5±3.6%；P<0.05；以及48.44±0.69%、55.及59.63±0.30%；分别与0、2.5 mM和7.5 mM肉碱组相比，P<0.01）。与对照组相比，在冷冻稀释液中添加肉碱可降低（P<0.01）在赤道和顶体前部区域均显示荧光的精子百分比（模式EA），这对应于高获能水平（分别为30.3±3.8%、18.8±2.8%和7.2±2.9%，0、2.5 mM和7.5 mM肉碱组）。与此一致，肉碱还降低了（P<0.01）显示金霉素模式B（获能精子）的精子百分比（分别为63.8±1.8%、46.8±2.2%和37.2±1.精子，0、2.5 mM和7.5 mM肉碱组）。有趣的是，肉碱提高了水牛冷冻解冻后精子的总抗氧化能力和ATP含量（分别为1.32±0.02、1.34±0.01、1.37±0.01 mM/L和4.1±0.1、5.3±0.1和8.2±0.4 nM×10个精子；分别与0、2.5 mM和7.5 mM肉碱组相比，P<0.01）。与对照组相比，用二氢乙锭（DHE）值表明，肉碱处理的精子细胞内ROS减少（分别为0.22±0.01、0.18±0.01和0.14±0.0μM/100μL二氢乙锭，0、2.5 mM和7.5 mM肉碱组；P<0.01），而脂质过氧化水平（平均30.5±0.3 nmol/mL丙二醛）和磷脂浓度（平均0.14±0.00μg/120×10个精子）未受影响。尽管精子质量有所改善，但体外受精后正常精子穿透的百分比未受影响（平均53.5±1.8）。总之，在稀释液中添加肉碱可通过增加ATP生成和调节ROS产生来提高水牛精子质量，而不影响体外受精能力。
