Figueroa E, Valdebenito I, Merino O, Ubilla A, Risopatrón J, Farias J G
School of Aquaculture, Catholic University of Temuco, Temuco, Chile.
Departamento de Ingeniería Química, Facultad de Ingeniería y Ciencias, Universidad de La Frontera, Temuco, Chile.
J Fish Biol. 2016 Sep;89(3):1537-50. doi: 10.1111/jfb.13052. Epub 2016 Jul 12.
The objective of this study was to determine the effect of freezing on the function in Atlantic salmon Salmo salar spermatozoa. The semen was frozen in Cortland's medium + 1.3M dimethyl sulphoxide + 0.3M glucose + 2% bovine serum albumin (final concentration) in a ratio of 1:3 (semen:cryoprotectant) as the treatment (T) and fresh semen as the control (F). Straws of 0·5 ml of sperm suspension were frozen in 4 cm of N2 L. They were thawed in a thermoregulated bath (40° C). After thawing, the percentage of spermatozoa with fragmented DNA [transferase dUTP (deoxyuridine triphosphate) nick-end labelling (TUNEL)], plasma membrane integrity (SYBR-14/PI) and mitochondrial membrane potential (ΔΨMMit, JC-1) were evaluated by flow cytometry and motility was evaluated by optical microscope under stroboscopic light. The fertilization rates of the control and treatment semen were tested at a sperm density of 1·5 × 10(7) spermatozoa oocyte(-1) , by observation of the first cleavages after 16 h incubation at 10° C. In the cryopreserved semen (T), the mean ± s.d. DNA fragmentation was 4·8 ± 2·5%; plasma membrane integrity 75·2 ± 6·3%; mitochondrial membrane potential 51·7 ± 3·6%; motility 58·5 ± 5·3%; curved line velocity (VCL ) 61·2 ± 17·4 µm s(-1) ; average-path velocity (VAP ) 50·1 ± 17·3 µm s(-1) ; straight-line velocity (VSL ) 59·1 ± 18·4 µm s(-1) ; fertilization rate 81·6 ± 1·9%. There were significant differences in the plasma membrane integrity, mitochondrial membrane potential, motility, fertilization rate, VCL , VAP and VSL compared with the controls (P < 0·05). Also the mitochondrial membrane potential correlated with motility, fertilization rate, VCL and VSL (r = 0·75; r = 0·59; r = 0·77 and r = 0·79, respectively; P < 0·05); and the fertilization rate correlated with VCL and VSL (r = 0·59 and r = 0·55, respectively).
本研究的目的是确定冷冻对大西洋鲑（Salmo salar）精子功能的影响。精液在科特兰培养基+1.3M二甲基亚砜+0.3M葡萄糖+2%牛血清白蛋白（终浓度）中按1:3（精液:冷冻保护剂）的比例冷冻作为处理组（T），新鲜精液作为对照组（F）。将0.5毫升精子悬液吸管在4厘米液氮中冷冻。在温度调节水浴（40°C）中解冻。解冻后，通过流式细胞术评估DNA片段化精子（转脱氧尿苷三磷酸末端转移酶缺口末端标记法，即TUNEL法）的百分比、质膜完整性（SYBR-14/PI法）和线粒体膜电位（ΔΨM，JC-1法），并在频闪灯下通过光学显微镜评估活力。在精子密度为1.5×10⁷精子/卵母细胞时，通过观察在10°C下孵育16小时后的首次卵裂来检测对照精液和处理精液的受精率。在冷冻保存的精液（T）中，DNA片段化的平均值±标准差为4.8±2.5%；质膜完整性为75.2±6.3%；线粒体膜电位为51.7±3.6%；活力为58.5±5.3%；曲线速度（VCL）为61.2±17.4微米/秒；平均路径速度（VAP）为50.1±17.3微米/秒；直线速度（VSL）为59.1±18.4微米/秒；受精率为81.6±1.9%。与对照组相比，质膜完整性、线粒体膜电位、活力、受精率、VCL、VAP和VSL存在显著差异（P<0.05）。此外，线粒体膜电位与活力、受精率、VCL和VSL相关（r分别为0.75、0.59、0.77和0.79；P<0.05）；受精率与VCL和VSL相关（r分别为0.59和0.55）。
