我们的目标是探索在精液补充剂中添加柚皮素对解冻后1)精子质量的影响,2)生育相关基因表达,3)水牛公牛精子的受精潜力。在实验1中,将来自四个Nili-Ravi水牛公牛的精液样品(n=32)合并(n=8),并用含有不同浓度柚皮素的tris-柠檬酸(TCF-EY)补充剂稀释,即,安慰剂(DMSO),0(控制),50、100、150和200μM柚皮素。稀释后,精液样本装在0.5毫升法国吸管中,冷冻保存并分析解冻后精子质量和基因表达。计算机辅助精液分析,低渗肿胀试验,正常根尖脊测定,罗丹明123,吖啶橙,进行碘化丙啶染色和硫代巴比妥酸反应性物质测定以评估精子运动参数,质膜功能,顶体完整性,线粒体膜电位,DNA完整性,活力和脂质过氧化,分别。精子顶体相关的SPACA3,DNA缩合相关的PRM1,抗凋亡的BCL2,促凋亡的BAX的表达水平,和氧化应激相关的ROMO1基因通过qPCR进行评估。结果表明,总运动和渐进运动,质膜功能,顶体完整性,线粒体膜电位,与200μM柚皮素相比,50、100和150μM柚皮素的DNA完整性和活力更高(P<0.05),安慰剂组和对照组。此外,所有柚皮素治疗组改善过氧化氢酶活性,与安慰剂组和对照组相比,脂质过氧化降低(P<0.05)。150μM柚皮素的SPACA3和PRM1基因的相对表达水平高于100μM(P>0.05)。各组间BCL2基因表达水平无差异(P>0.05)。此外,200μM柚皮素组中BAX基因表达较高(P<0.05),其余各组的表达无差异(P>0.05)。此外,与对照组相比,ROMO1基因在所有柚皮素处理组中表达更低(P<0.05)。在实验2中,将含有最佳浓度的柚皮素(150μM;实验1中描述的体外精子质量更好)的精液剂量(n=400;200/组)的体内生育力与繁殖季节的对照进行了比较。水牛在自然发情期开始后24小时授精,并在授精后至少60天经直肠触诊妊娠。150μM柚皮素组的生育率高于对照组(P=0.0366)[57.00±0.03%(114/200)。46.50±0.04%(93/200),分别]。一起来看,结论是,精液补充剂中补充柚皮素可以改善解冻后的质量,水牛公牛精子的生育相关基因表达和受精潜力,更明显地在150μM浓度。
Our objectives were to explore the effect of
naringenin addition in the semen extender on the post-thaw 1) sperm quality, 2) fertility-associated gene expression, and 3) fertilization potential of buffalo bull sperm. In experiment 1, semen samples (n = 32) from four Nili-Ravi buffalo bulls were pooled (n = 8) and diluted with the tris-citric acid (TCF-EY) extender containing different concentrations of naringenin, i.e., placebo (DMSO), 0 (control), 50, 100, 150 and 200 μM
naringenin. After dilution, semen samples were packed in 0.5 mL French straws, cryopreserved and analyzed for post-thawed sperm quality and gene expression. Computer-assisted Semen Analysis, Hypo-osmotic Swelling test, Normal Apical Ridge assay, Rhodamine 123, Acridine orange, Propidium iodide staining and Thiobarbituric Acid Reactive Substances assay were performed to assess sperm motility parameters, plasma membrane functionality, acrosome integrity, mitochondrial membrane potential, DNA integrity, viability and lipid peroxidation, respectively. Expression levels of sperm acrosome-associated SPACA3, DNA condensation-related PRM1, anti-apoptotic BCL2, pro-apoptotic BAX, and oxidative stress-associated ROMO1 genes were evaluated through qPCR. Results revealed that total and progressive motility, plasma membrane functionality, acrosome integrity, mitochondrial membrane potential, DNA integrity and viability were higher (P < 0.05) with 50, 100 and 150 μM naringenin compared to 200 μM
naringenin, placebo and control groups. Moreover, all naringenin-treated groups improved catalase activity, and reduced lipid peroxidation compared to placebo and control groups (P < 0.05). Relative expression levels of SPACA3 and PRM1 genes were higher (P < 0.05) with 150μM naringenin compared to all groups except 100 μM (P > 0.05). No difference (P > 0.05) in the expression level of BCL2 gene was observed among all groups. Furthermore, BAX gene was expressed higher (P < 0.05) in the 200 μM naringenin group, whereas no difference (P > 0.05) in expression was noticed among the remaining groups. In addition, ROMO1 gene was expressed lower (P < 0.05) in all naringenin-treated groups compared to the control. In experiment 2, the in vivo fertility of semen doses (n = 400; 200/group) containing optimum concentration of naringenin (150 μM; depicted better in vitro sperm quality in experiment 1) was compared with control during the breeding season. Buffaloes were inseminated 24 h after the onset of natural estrus and palpated transrectal for pregnancy at least 60 days post-insemination. The fertility rate of 150 μM
naringenin group was higher (P = 0.0366) compared to the control [57.00 ± 0.03% (114/200) vs. 46.50 ± 0.04% (93/200), respectively]. Taken together, it is concluded that
naringenin supplementation in semen extender improves post-thaw quality, fertility-associated gene expression and fertilization potential of buffalo bull sperm, more apparently at 150 μM concentration.