Spanner E A, Rickard J P, de Graaf S P
The University of Sydney, Faculty of Science, School of Life and Environmental Sciences, NSW 2006, Australia.
The University of Sydney, Faculty of Science, School of Life and Environmental Sciences, NSW 2006, Australia.
Anim Reprod Sci. 2025 May;276:107822. doi: 10.1016/j.anireprosci.2025.107822. Epub 2025 Apr 3.
The post-thaw quality of pellet-frozen ram semen was evaluated based on (i) sperm concentration, (ii) thawing diluent, and (iii) the number of pellets thawed simultaneously. Ejaculates from three Merino rams were collected, with four replicates per ram (n = 12 ejaculates) for each experiment. In Experiment 1, ejaculates were frozen at 200, 400, 600 or 800 × 10 sperm/ mL. In Experiment 2, ejaculates were frozen at 600 × 10 sperm/ mL and thawed in a dry test tube (control) or with 1 + 3 tris-citrate-fructose, tris-citrate-fructose+egg yolk, PBS+dye, PBS alone or IVF media (Emcare). In Experiment 3, groups of 1, 2 or 3 pellets were thawed in tris-citrate-fructose or a dry test tube (control). Post-thaw samples were tested for motility, acrosome and membrane integrity (FITC-PNA/PI) at 0, 3, and 6 h (all Experiments) and morphology at 0 h (Experiment 1). At 3 and 6 h post-thaw, motility was reduced at 200 × 10⁶ sperm/mL compared to other concentrations (P < 0.05). Across all time points, samples frozen at 800 × 10⁶ sperm/mL (P < 0.05) showed lower viability, while freezing at 200 × 10⁶ sperm/mL increased morphological abnormalities (P < 0.05). Thawing pellets with tris-citrate-fructose media resulted in higher motility, acrosome and membrane integrity 0, 3 and 6 h post-thaw (P > 0.05). Thawing one-pellet in tris-citrate-fructose improved motility and viability post-thaw (P < 0.05) compared to multiple pellets, while the number of pellets thawed had no significant effect when dry-thawed (P > 0.05). The results suggest that the optimal post-thaw quality is achieved when semen is frozen between 400 and 600 × 10 sperm/mL and thawed in tris-citrate-fructose media, one pellet at a time.
（i）精子浓度，（ii）解冻稀释液，以及（iii）同时解冻的颗粒数量。采集三只美利奴公羊的射精样本，每个实验中每只公羊有四个重复样本（n = 12次射精）。在实验1中，射精样本分别以200、400、600或800×10⁶精子/毫升的浓度冷冻。在实验2中，射精样本以600×10⁶精子/毫升的浓度冷冻，并在干燥试管（对照）或用1+3三羟甲基氨基甲烷 - 柠檬酸盐 - 果糖、三羟甲基氨基甲烷 - 柠檬酸盐 - 果糖 + 蛋黄、磷酸盐缓冲液 + 染料、单独的磷酸盐缓冲液或体外受精培养基（Emcare）中解冻。在实验3中，分别将1、2或3个颗粒在三羟甲基氨基甲烷 - 柠檬酸盐 - 果糖或干燥试管（对照）中解冻。解冻后的样本在0、3和6小时（所有实验）检测活力、顶体和膜完整性（异硫氰酸荧光素 - 花生凝集素/碘化丙啶），在0小时（实验1）检测形态。解冻后3小时和6小时，与其他浓度相比，200×10⁶精子/毫升浓度下的活力降低（P < 0.05）。在所有时间点，以800×10⁶精子/毫升浓度冷冻的样本活力较低（P < 0.05），而以200×10⁶精子/毫升浓度冷冻会增加形态异常（P < 0.05）。用三羟甲基氨基甲烷 - 柠檬酸盐 - 果糖培养基解冻颗粒后，在解冻后0、3和6小时活力、顶体和膜完整性更高（P > 0.05）。与多个颗粒相比，在三羟甲基氨基甲烷 - 柠檬酸盐 - 果糖中解冻一个颗粒可提高解冻后的活力和生存能力（P < 0.05），而在干燥解冻时，解冻的颗粒数量没有显著影响（P > 0.05）。结果表明，当精液以400至600×10⁶精子/毫升的浓度冷冻，并在三羟甲基氨基甲烷 - 柠檬酸盐 - 果糖培养基中一次解冻一个颗粒时，可获得最佳解冻后质量。
