ABSTRACT

1. Birchen and Blue Leonesa are two endangered chicken breeds mainly raised in Curueño Valley in North Spain. The establishment of a germplasm bank to guarantee the preservation of these breeds is needed. However, cockerels from different breeder flocks can show variance in semen cryoresistance.

2. The following work focused on the sperm characterisation and cryopreservation of Birchen and Blue Leonesa cockerels from four different breeders. A total of 30 semen pools were analysed. Besides conventional sperm analysis, including motility by computer-aided sperm analysis (CASA) and DNA fragmentation by TUNEL, the present study tested a double staining method (MitoTracker^TM Green FM/propidium iodide). This gave simultaneous assessment of plasma and acrosomal and mitochondrial membranes, which were previously validated by SYBR-14/PI, CASA, aniline blue and TUNEL.

3. No significant differences were found among fresh semen variables between breeds and breeders. For post-thawed variables, significant differences (P < 0.05) were found between breeders in sperm viability (58.0 ± 1.90 breeder D vs. 35.2 ± 7.41 breeder A, 37.2 ± 4.09 breeder B and 22.3 ± 5.92 breeder C) and DNA fragmentation (62.4 ± 9.91 breeder C vs. 31.8 ± 7.08 breeder B and 24.5 ± 5.49 breeder D). The lowest DNA fragmentation values for semen from breeder D birds were coincident with higher integrity of the mitochondrial membrane.

4. The results revealed higher sperm cryoresistance in the cockerels from one of the breeders, possibly due to differences in management system (e.g. diet, housing, control of stress elements and pathogens, reproduction practices or maintenance of genetic diversity). These differences may determine the sperm freezability, and thus the effectiveness of developing a germplasm bank.

摘要

1. Birchen 和 Blue Leonesa 是两个濒临灭绝的鸡种，主要饲养在西班牙北部的 Curueño 山谷。需要建立种质资源库来保证这些品种的保存。然而，来自不同种鸡群的公鸡在精液冷冻抗性方面表现出差异。

2. 以下工作侧重于来自四个不同饲养者的 Birchen 和 Blue Leonesa 公鸡的精子表征和冷冻保存。共分析了 30 个精液池。除了传统的精子分析，包括计算机辅助精子分析 (CASA) 的运动性和 TUNEL 的 DNA 片段化，本研究还测试了双染色方法 (MitoTracker ^TM Green FM/碘化丙啶)。这可以同时评估血浆、顶体和线粒体膜，这些膜之前已通过 SYBR-14/PI、CASA、苯胺蓝和 TUNEL 验证。

3.新鲜精液变量在品种和育种者之间没有显着差异。对于解冻后的变量，育种者之间的精子活力（58.0 ± 1.90 育种者 D对35.2 ± 7.41 育种者 A、37.2 ± 4.09 育种者 B 和 22.3 ± 5.92 育种者 C）和 DNA 碎片（ 62.4 ± 9.91 种鸽 C与31.8 ± 7.08 种鸽 B 和 24.5 ± 5.49 种鸽 D)。D 种鸡精液的最低 DNA 片段化值与线粒体膜的更高完整性一致。

4. 结果表明，其中一名饲养员的公鸡精子抗冻性较高，这可能是由于管理系统的差异（例如饮食、住房、压力因素和病原体的控制、繁殖方式或遗传多样性的维持）。这些差异可能决定精子的可冷冻性，从而决定建立种质库的有效性。