Characteristics and in vitro fertilizing ability of giant panda (Ailuropoda melanoleuca) frozen‐thawed epididymal spermatozoa obtained 4 hours postmortem: A case report

Abstract

When Chu-Lin, a male giant panda (studbook #249), died at Madrid Zoo, his reproductive tract was removed 4 hr postmortem and the epididymal spermatozoa were collected. Extended sperm were kept at 51C for 4 hr, loaded into straws, and frozen for 7 min in liquid nitrogen vapor before the straws were plunged into liquid nitrogen. Two straws were thawed and evaluated. Sperm motility was assessed in fresh, refrigerated, and thawed spermatozoa (75%, 60%, 35%, respectively). Sperm viability and acrosome status were estimated using a triplestain technique (TST). The results showed 33% live sperm with intact acrosomes after thawing. A hypoosmotic swelling (HOS) test demonstrated the retention of membrane integrity in 72% of thawed sperm. To evaluate the in vitro fertilizing ability of thawed sperm, a sperm penetration assay (SPA) was performed. The values obtained for the percentage of penetration and the penetration index were 62% and 1.78 sperm/oocyte, respectively. The results obtained demonstrate that epididymal sperm recovered from a giant panda postmortem can be successfully cryopreserved. The sperm fertilizing ability demonstrated in vitro after thawing may provide a final opportunity for this male to contribute to the currently small germplasm reserves of this endangered species, and to reproduce in the future through assisted reproductive technology.

Hypoosmotic Swelling Test in Alpaca (Vicugna pacos) Epididymal Spermatozoa

“…1997), elephants, emus and baboons (Matson et al. 2009), giant pandas (Pérez‐Garnelo et al. 2004), hippopotamuses (Saragusty et al.…”

“…The HOS test has been routinely used in several species, applying the original conditions described and standardized for human samples (Jeyendran et al 1984). At present, the use of HOS test has been reported in boars (Vazquez et al 1997), bulls (Correa and Zavos 1994), dogs (Kumi-Diaka 1993), rabbits (Ducci et al 2002), turkeys (Donoghue et al 1996), horses (de la Cueva et al 1997, elephants, emus and baboons (Matson et al 2009), giant pandas (Pe´rez-Garnelo et al 2004), hippopotamuses (Saragusty et al 2010) and red deer (Ferna´ndez-Santos et al 2007), confirming that the test is of great use for spermatozoa assessment. The majority of studies cited above have used semen and should be suited to epididymal spermatozoa as it is well known that there are important differences in the physiological characteristics of epididymal vs ejaculated spermatozoa, especially in their membranes properties (Hammond 1930;Walton 1930;Songsasen and Leibo 1998;Martinez-Pastor et al 2006).…”

Effects of post-mortem storage conditions of bovine epididymides on sperm characteristics: investigating a tool for preservation of sperm from endangered species

“…Using material from endangered species to develop suitable preservation protocols is a challenge because of the typical low number of animals available for experimentation, so using farm animals such as the bovid is a well-suited model to obtain knowledge that can aid the development of protocols for other species. Recovery of epididymal spermatozoa has been described as a possible method for conserving genetic variation in species such as Sumatran rhinoceros (O'Brien and Roth, 2000), sheep (Kaabi et al ., 2003), marmosets, baboons and chimpanzees (O'Brien et al ., 2003), giant pandas (Pérez-Garnelo et al ., 2004), goats (Santiago-Moreno et al ., 2006), rhesus monkeys (Dong et al ., 2008), black and white tegus (Young et al ., 2013) and saltwater crocodiles (Johnston et al ., 2014). In these studies, different practical conditions for handling the testicles (storage time and temperature between death and spermatozoa collection) and different approaches to obtain the spermatozoa (slicing, cutting, opening and flushing) have been used.…”

Optimization of Glycerol Concentration and Freezing Rate in the Cryopreservation of Ejaculate From Brown Bear (Ursus arctos)

“…With regard to Ursidae, Giant panda ( Ailuropoda melanoleuca ) spermatozoa have been cryopreserved using 5% (Spindler et al. 2004) and 6% (Perez‐Garnelo et al. 2004) glycerol.…”