Garde J J, Soler A J, Cassinello J, Crespo C, Malo A F, Espeso G, Gomendio M, Roldan E R S
Departamento de Ciencia y Tecnología Agroforestal, ETSI Agrónomos, Universidad de Castilla-La Mancha, 02071-Albacete, Spain.
Biol Reprod. 2003 Aug;69(2):602-11. doi: 10.1095/biolreprod.102.012914. Epub 2003 Apr 16.
Long-term storage of semen by cryopreservation, with high recovery rates on thawing, is essential for the establishment of genetic resource banks of endangered species. The purpose of the present study was to evaluate various diluents for the cryopreservation of spermatozoa from three species of gazelles (genus Gazella) in a captive breeding program. The diluents compared were Tes (N-tris(hydroxymethyl)methyl-2 aminoethane sulfonic acid)-Tris with 5% egg yolk and 6% glycerol (TEST) and Triladyl, yolk-citrate, Tris-trehalose, and Tris-lactose-all of them with 20% egg yolk and 6% (Triladyl) or 8% glycerol. Semen was obtained by electroejaculation from 12 G. cuvieri, 12 G. dama, and 13 G. dorcas males. Samples with less than 50% motile sperm, positive endosmosis, or acrosome integrity were not used. Diluted samples were loaded into 0.25-ml straws, cooled slowly to 5 degrees C over 1.5 h (-0.16 degrees C/min), equilibrated at that temperature for 2 h, frozen in nitrogen vapors for 10 min, and plunged into liquid nitrogen. Subsamples were assessed fresh, after refrigeration-equilibration, after freezing and thawing, and 2 h after thawing. Differences were seen between diluents, with best overall recovery rates after freezing and thawing found with Triladyl, TEST, and Tris-trehalose in G. cuvieri, TEST in G. dama, and Triladyl and TEST in G. dorcas. Differences were observed between species in the ability to withstand freezing and thawing, with best results seen in G. dorcas, intermediate results in G. dama, and worst results in G. cuvieri. These differences were inversely related to the average values of inbreeding of these populations. The underlying mechanism responsible for these differences may be a differential resistance to osmotic shock.
通过冷冻保存精液并在解冻时保持高回收率,对于建立濒危物种的遗传资源库至关重要。本研究的目的是评估在圈养繁殖计划中用于三种瞪羚(瞪羚属)精子冷冻保存的各种稀释剂。所比较的稀释剂有含5%蛋黄和6%甘油的Tes(N-三(羟甲基)甲基-2-氨基乙烷磺酸)-Tris(TEST)以及Triladyl、蛋黄柠檬酸盐、Tris-海藻糖和Tris-乳糖,它们均含有20%蛋黄和6%(Triladyl)或8%甘油。通过电刺激从12只曲角羚、12只小鹿瞪羚和1只阿拉伯瞪羚雄性获取精液。未使用活动精子少于50%、存在正向内渗或顶体完整性呈阳性的样本。将稀释后的样本装入0.25毫升的细管中,在1.5小时内缓慢冷却至5摄氏度(每分钟-0.16摄氏度),在该温度下平衡2小时,在液氮蒸气中冷冻10分钟,然后投入液氮中。对新鲜的、冷藏平衡后的、冷冻和解冻后的以及解冻后2小时的子样本进行评估。不同稀释剂之间存在差异,在曲角羚中,冷冻和解冻后总体回收率最佳的是Triladyl、TEST和Tris-海藻糖;在小鹿瞪羚中是TEST;在阿拉伯瞪羚中是Triladyl和TEST。在耐受冷冻和解冻的能力方面,不同物种之间存在差异,阿拉伯瞪羚的结果最佳,小鹿瞪羚的结果居中,曲角羚的结果最差。这些差异与这些种群的平均近亲繁殖值呈负相关。造成这些差异的潜在机制可能是对渗透压休克的不同抵抗力。