Longobardi Valentina, Albero Giuseppe, De Canditiis Carolina, Salzano Angela, Natale Antonio, Balestrieri Anna, Neglia Gianluca, Campanile Giuseppe, Gasparrini Bianca
Department of Veterinary Medicine and Animal Production, Federico II University, Naples, Italy.
Istituto Zooprofilattico Sperimentale del Mezzogiorno, Portici, Naples, Italy.
Theriogenology. 2017 Feb;89:359-364. doi: 10.1016/j.theriogenology.2016.09.048. Epub 2016 Oct 3.
The aim of this study was to investigate the effect of cholesterol-loaded cyclodextrins (CLC) on motility, viability, capacitation status, and in vivo fertility of buffalo frozen-thawed sperm. After the initial semen assessment, buffalo sperm were diluted in BULLXcell extender containing 0- (control), 1.5-, and 3-mg/mL CLC and cryopreserved. At thawing, sperm motility was evaluated by phase contrast microscopy, and viability-capacitation status was assessed by Hoechst 33258-chlortetracycline (CTC) assay. Capacitation status was also evaluated by an indirect immunofluorescence assay to localize phosphotyrosine-containing proteins. Moreover, buffaloes were artificial inseminated to assess the in vivo-fertilizing potential of CLC-treated semen. No differences among control, 1.5-, and 3-mg/mL CLC-treated groups were recorded in both sperm motility (66.5 ± 5.6, 68.8 ± 4.8, and 68.8 ± 4.8, respectively) and viability (86.5 ± 1.9, 87.6 ± 1.5, 88.4 ± 2.3, respectively). However, the extender supplementation with CLC significantly reduced sperm cryocapacitation. Indeed, CLC treatment decreased (P < 0.01) the proportion of sperm showing the CTC pattern B (capacitated sperm) compared with the control (69.6 ± 3.4, 37.8 ± 1.5, and 51.3 ± 4.7, respectively, with 0, 1.5-, and 3-mg/mL CLC; P < 0.01). Furthermore, the percentage of sperm displaying tyrosine-phosphorylated pattern EA (i.e. high capacitation level) was reduced (P < 0.01) in both CLC-treated groups (10.8 ± 3.3 and 5.6 ± 1.6, respectively, with 1.5- and 3-mg/mL CLC) compared with the control (37.3 ± 6.9), reaching values similar to those recorded in fresh semen (11.0 ± 3.5). In addition, treating sperm with 3-mg/mL CLC increased (P < 0.01) the percentage of nonfluorescent (pattern NF), i.e., non-capacitated sperm (41.8 ± 3.6) compared with fresh semen (11.0 ± 6.9). No differences were recorded in pregnancy rates at 60 days post-artificial insemination among control, 1.5- and 3-mg/mL CLC groups (59.7%, 65.6%, and 56.9%, respectively). In conclusion, CLC treatment of buffalo sperm strongly decreases sperm cryocapacitation damages, without affecting the in vivo fertilizing capability.
本研究的目的是探讨胆固醇负载环糊精（CLC）对水牛冻融精子活力、生存能力、获能状态及体内受精能力的影响。在对精液进行初步评估后，将水牛精子稀释于含有0（对照）、1.5和3mg/mL CLC的BULLXcell稀释液中，并进行冷冻保存。解冻时，通过相差显微镜评估精子活力，通过Hoechst 33258-金霉素（CTC）试验评估生存能力-获能状态。还通过间接免疫荧光试验定位含磷酸酪氨酸的蛋白质来评估获能状态。此外，对水牛进行人工授精以评估经CLC处理的精液的体内受精潜力。对照、1.5mg/mL和3mg/mL CLC处理组在精子活力（分别为66.5±5.6、68.8±4.8和68.8±4.8）和生存能力（分别为86.5±1.9、87.6±1.5、88.4±2.3）方面均未记录到差异。然而，添加CLC的稀释液显著降低了精子的冷冻获能。实际上，与对照组相比，CLC处理降低了（P<0.01）显示CTC模式B（获能精子）的精子比例（0、1.5和3mg/mL CLC组分别为69.6±3.4、37.8±1.5和51.3±4.7；P<0.01）。此外，与对照组（37.3±6.9）相比，两个CLC处理组（1.5和3mg/mL CLC组分别为10.8±3.3和5.6±1.6）中显示酪氨酸磷酸化模式EA（即高获能水平）的精子百分比均降低（P<0.01），达到与新鲜精液中记录的值（11.0±3.5）相似。另外，与新鲜精液（11.0±6.9）相比，用3mg/mL CLC处理精子增加了（P<0.01）非荧光（模式NF）即未获能精子的百分比（41.8±3.6）。对照、1.5mg/mL和3mg/mL CLC组在人工授精后60天的妊娠率未记录到差异（分别为59.7%、65.6%和56.9%）。总之，CLC处理水牛精子可强烈降低精子冷冻获能损伤，而不影响其体内受精能力。
