Viability Tests, Active Caspase‐3 and −7, and Chromatin Structure in Ram Sperm Selected Using the Swim‐Up Procedure

Abstract

Swim-up (SU) is a technique that permits the obtaining of motile sperm. Based on the sperm motility of neat ejaculates, we analyzed functional parameters, cytoplasmic esterases, and mitochondrial dehydrogenases of ram sperm using calcein acetomethylester and resazurin, respectively. Active caspase-3 and -7 and chromatin structure were evaluated in ram sperm before and after the SU process. There were no changes in any of the viability parameters after SU in neat semen samples with less or more than 25% motility. The percentage of active caspase-3 and -7 decreased after SU (68.8 6 4.6 vs 54.2 6 6.0), whereas a small but significant increase of chromatin structural abnormalities was observed (DNA fragmentation index [DFI], 287.3 6 3.1 vs 297.2 6 2.4). For the first time, the location of active caspase-3 and -7 was described for ram sperm. Notably, we found active caspases in the implantation fossa region. The presence of active caspases in neat ejaculates and the diminished presence of active caspases in SU-processed ejaculates suggest a role for caspases in motility and possibly in male fertility. The results of this study indicate that the evaluation of more than one cell-function marker is necessary to appropriately evaluate sperm quality. Furthermore, in semen samples with low motility, a lower percentage of sperm with active caspases is obtained after SU, although these sperm present increased values of DFI.

Confocal Microscopy and Image Analysis Indicates a Region-Specific Relation between Active Caspases and Cytoplasm in Ejaculated and Epididymal Sperm

“…However, the ejaculates of ovine normally present a high percentage of sperm with active caspases [8]. On other hand, presence of active caspases could correlate negatively [25] or not correlate with motility [8] but not correlate with viability [8]. Recently, using FLICA to detect active caspases, we observed intense staining in cytoplasmic droplets of boar [15] and ram sperm (observations not published), a similar observation was reported early for human sperm [14].…”

“…The site where cytoplasmic constituents leave the maturing sperm is precisely the middle piece [26], and in this site an intense FLICA-staining has been observed for human [14] and boar sperm [15]. In this work we demonstrated that a strong relationship exists between the presence of active caspases and cytoplasmic esterases for the middle region of ovine sperm; however, intriguingly, active caspases could be present in other regions of sperm apparently not associated with the cytoplasmic droplet, such as equatorial and apical sections in head or principal section in tail [8]. In head and principal section we do not observed correlation between presence of active caspases and esterases.…”

“…The presence of active caspases has been demonstrated in the sperm of ovine [22], [8] and other species [23], [9], including humans [5], [6]. For humans, a high percentage of sperm with active caspases has been related with infertility [24].…”

“…For humans, a high percentage of sperm with active caspases has been related with infertility [24]. However, the ejaculates of ovine normally present a high percentage of sperm with active caspases [8]. On other hand, presence of active caspases could correlate negatively [25] or not correlate with motility [8] but not correlate with viability [8].…”

“…Activation of caspase-3 and -7 is indicative that the cell is committed to die [2]. In this sense, it is intriguing that active caspases are detected in sperm of different species, including man [6], pig [7], and ram [8], and stallion [9]. The physiological role of active caspases in sperm is not known.…”