Association of the polymorphism of the Pon-1 locus with the concentration of Paraoxonase-1 and post-thaw sperm viability in pig

Sandra Elena Montaño Garcia, Patricia Cervantes Acosta, María de Lourdez Juarez Mosqueda, A. H. Beltran, B. Mancera, Manuel Barrientos Villeda, Dora Romero Salas, Manuel Barrientos Morales
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Abstract

The objective of the present work was to correlate the expression of the PON-1 locus polymorphism, the concentration of the enzyme paraoxonase 1 in seminal plasma and the effect on post-thawing sperm viability in the pig. Nine ejaculates from 3 boars were used, to obtain seminal plasma; part of the ejaculates was centrifuged at 800 g/10 min; the material obtained was stored at -20°C until use. To freeze the semen, the samples were diluted 1:1 in commercial diluent (Magapor®) at 37°C and the temperature was gradually lowered to 16°C. They were transported to the laboratory protected from light, and kept at the same temperature in the laboratory for 24 h. After this, they were frozen using the glycerolation technique and a portion was cryopreserved by adding 20% of seminal plasma to the conservation medium and another portion was made without seminal plasma. The concentration of PON-1 was determined by an enzyme-linked immunosorbent assay (ELISA). The functional status of the plasma membrane was evaluated by chlortetracycline staining. The amplification of a fragment of the PON-1 gene was carried out by PCR and the identification of allelic variability by the analysis of Restriction Fragment Length Polymorphism (RFLP), with the enzymes HinfI and FokI, from a DNA sample of each individual. The results were analyzed by analysis of variance (ANOVA) and Spearman correlation, with the use of statistical software (Statistica V10 for Windows). In electrophoresis of enzymatic digestion with the enzyme HinfI All the expected fragments were obtained, with the FokI enzyme a mutation was observed in one individual. No correlation was observed (R=-0.1, P>0.05) between the concentration of the paraoxonase-1 enzyme in seminal plasma and the observed mutations. As well as the concentration of the enzyme paraoxonase-1 in the ejaculates was 1.8 to 2.7 ng/mL and this had no statistically significant difference in the seminal plasma of the ejaculates analyzed (P>0.05). As well as with sperm viability in thawed semen (P>0.05).
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猪 Pon-1 基因位点的多态性与副氧合酶-1 的浓度和解冻后精子活力的关系
本研究的目的是将 PON-1 基因座多态性的表达、精浆中副氧自由基酶 1 的浓度及其对猪解冻后精子活力的影响联系起来。用 3 头公猪的 9 次射精获得精浆;部分射精以 800 克/10 分钟的速度离心;获得的材料在使用前保存在 -20°C 下。为了冷冻精液,样品在 37°C 下用商用稀释剂(Magapor®)以 1:1 的比例稀释,然后逐渐降温至 16°C。之后,使用甘油隔离技术将精液冷冻,并在保存介质中加入 20% 的精浆对其中一部分进行冷冻保存,而另一部分则不加精浆。PON-1的浓度是通过酶联免疫吸附试验(ELISA)测定的。质膜的功能状态通过金霉素染色法进行评估。通过 PCR 扩增 PON-1 基因片段,并用 HinfI 和 FokI 酶对每个个体的 DNA 样本进行限制性片段长度多态性(RFLP)分析,以确定等位基因的变异性。使用统计软件(Statistica V10 for Windows)对结果进行方差分析(ANOVA)和斯皮尔曼相关分析。在用 HinfI 酶进行酶解的电泳中,得到了所有预期的片段,而用 FokI 酶则在一个个体中发现了突变。精浆中副氧合酶-1 的浓度与观察到的突变之间没有相关性(R=-0.1,P>0.05)。此外,射精中副氧合酶-1 的浓度为 1.8 至 2.7 纳克/毫升,这与所分析的射精精浆中副氧合酶-1 的浓度没有显著的统计学差异(P>0.05)。解冻精液中的精子存活率也是如此(P>0.05)。
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