214USE OF FLOURESCENT PROBES TO ACCESS EPIDIDYMAL SPERMATOZOA OF THE BLUE
WILDEBEEST CONNOCHAETES TAURINUS AND IMPALA ANTELOPE
AEPYCEROS MELAMPUS MELAMPUS
Authors: Spies T. , Olivier F. , Martinez-Pastor F. , Barry D.M. , Bartels P. 
Published: February 2004

Sperm quality assessment may be a useful tool not only for evaluating the reproductive health of free-ranging populations, but also for selecting individuals for future assisted-reproduction technology programs. The aim of this study was to assess the functionality of epididymal spermatozoa collected from blue wildebeest (Connochaetes taurinus) and impala (Aepyceros melampus melampus) during the non-breeding season, using the fluorescent probes, propidium ioide (PI;; Sigma, South Africa) and JC-1 (Molecular Probes, The Netherlands). Six blue wildebeest and eight impala were harvested as part of a wildlife management program on a game ranch in South Africa. Testes were removed and transported to the laboratory within 6 hours while being maintained at 4°C. The cauda epididymides were removed and flushed with 1mL of Tris-citrate egg yolk extender (fraction A, Biladyl;; Minitüb, Germany). The sperm sample was diluted 1:4 in HEPES washing medium (Sigma;; 20mM HEPES, 355mM sucrose, 10mM glucose, 2.5mM KOH;; 400mOsm/kg, pH 7), and centrifuged for 5min at 600g, followed by re-suspending the pellet in 0.1mL of HEPES saline medium (Sigma;; as for washing medium, except 197mM NaCl instead of sucrose). The percentage of motile (MS) and progressively motile (PS) spermatozoa were determined using phase contrast microscopy (×200, 37°C). Sperm plasma membrane integrity and mitochondrial status were assessed using fluorescence microscopy (×400, 450–490nm excitation filter, 510nm dichroic-beam splitter, 520nm barrier filter) after staining with PI (50ngmL−1; 10min, RT) and JC-1 (7.5μM; 30min, 37°C), respectively. Spermatozoa with damaged plasma membranes showed a red fluorescence and spermatozoa with active and inactive mitochondria (MIT) fluoresced orange and green, respectively. Spearman correlation coefficients were calculated between spermatozoa with intact plasma membranes (IPM) and MIT, and with motility (Statistica™ package). A summary of the results is shown in the table 1. Although samples were not collected during the breeding season, sperm quality appeared to be good for the blue wildebeest, but less so for the impala. In general, impala results were more varied. Significant correlations were found for impala (n=8, P&lt;0.05) MS-IPM: 0.75; IPM-MIT: 0.83, and for blue wildebeest (n=6, P&lt;0.05), MS-IPM: 0.84; IPM-MIT: 0.81, and for pooled data (n=14, P&lt;0.01), MS-IPM: 0.93; MS-MIT: 0.87; PS-IPM: 0.67; PS-MIT: 0.66; IPM-MIT: 0.95. These correlations suggest a relationship of functional parameters to sperm motility. Both membrane integrity and mitochondrial status are important for sperm flagellar activity. The correlation between IPM and MIT indicates a relationship or the effect of common factors. In conclusion, sperm collected from blue wildebeest and impala during the non-breeding season appear functional, a fact that may be useful for future conservation programs based on assisted reproduction technology or for assessing the reproductive health status of free-ranging wildlife populations. The fluorescent probes PI and JC-1 appear useful for assessing sperm quality in these two species and should be considered for further sperm quality assessment studies in other antelope species.

Table 1
Results of the analyses, showing mean±SD (max.–min.)