Sperm cryopreservation has become an important technique in the conservation of native Ryukyu-Agu pigs, in which signs of inbreeding depression have begun to show. In this breed, sperm from total ejaculated semen has been used for freezing because its sperm rich fraction (SRF) cannot be distinguished visually unlike those of other commercial breeds. In this study, in order to develop an effective cryopreservation technique of Agu sperm, we first examined whether there was a SRF in the first 100 mL after ejaculation (Portion 1);the later fraction was defined as Portion 2. As a result, the sperm concentration of these fractions were 7.6×10⁸ and 0.7×10⁸ spermmL and that of Portion 1 was significantly higher (P<0.05). Moreover, the sperm of Portion 1 showed significantly higher motility index after freezing-thawing when compared with that of Portion 2 (P<0.05). To examine whether the difference of freezability between the fractions depended on seminal plasma, we performed an experiment to replace the seminal plasma of both fractions with each other. However, the seminal plasma did not affect sperm motility index after freezing-thawing in both Portion 1 and Portion 2. We speculated that this may have resulted because Portion 1 contained post-SRF fraction and its seminal plasma was similar to that in Portion 2. Thus, in place of Portion 1, the ejaculated semen was collected in a series of 25-mL fractions (Portion 1-1, Portion 1-2, Portion 1-3and Portion 1-4), and sperm parameters such as sperm concentration or motility index were investigated. Although the significant difference was not observed in the sperm motility index among fractions, sperm concentration of the fractions were 23.6 (Portion 1-1), 14.4 (Portion 1-2), 2.9 (Portion 1-3) and 1.4 (Portion 1-4)×10⁸ spermmL, and it was revealed that Portions 1-1 and 1-2 were SRF. Sperm in SRF (the first 50 mL of ejaculate) showed higher freezability, and addition of seminal plasma of Portions 1-3 and 1-4 reduced sperm quality after freezing-thawing, suggesting that seminal plasma controlled sperm freezability. Based on these results, we devised a technique to use the first 50 mL of semen after ejaculation for sperm freezing. When comparative analysis between the novel method (using the first 50 mL of ejaculate) and conventional method (using total ejaculate, control) was performed, the motility rate after thawing of sperm frozen by the novel method was significantly higher (82% vs. 25%, P<0.05). From these results, it is expected that the technique developed in this study will greatly contribute to the preservation of genetic resources of valuable Agu pigs.

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