Assessment of different sperm functional tests in golden‐headed lion tamarins (Leontopithecus chrysomelas)

Abstract

The golden‐headed lion tamarin (Leontopithecus chrysomelas) is an endangered species endemic to Brazil's Atlantic Forest, a shrinking biodiversity hotspot. As in other Neotropical primates, its semen characteristics and freezability are poorly studied. Hence, reproductive technologies for callitrichids would greatly benefit from reliable methods of semen analysis. In a bid to promote reproductive research in tamarins, we validated simple and inexpensive sperm function tests that can be used to monitor sperm‐egg binding, plasma membrane and acrosome integrity, mitochondrial activity, and DNA fragmentation. Ejaculates from adult males were individually diluted and divided into control and damage‐induced aliquots, and then samples comprising assorted amounts of damaged spermatozoa were examined by organelle‐specific tests. Our findings showed that sperm‐binding in chicken egg perivitelline membrane (EPM) positively correlated with the number of spermatozoa injured by snap‐freezing. Eosin‐nigrosin (EN) and propidium iodide readings were correlated with each other, and both provided robust measurements of plasma membrane integrity. A high correlation between expected and measured amounts of acrosome‐intact spermatozoa was found using Fast Green‐Rose Bengal (FG‐RB), Coomassie Blue (CB), and FITC‐PSA stains, and all three methods exhibited comparable results. Likewise, different percentages of UV‐irradiated spermatozoa were accurately assessed for DNA integrity by Toluidine Blue (TB) and sperm chromatin dispersion (SCD) tests. Comparisons between 3,3′‐diaminobenzidine (DAB) and JC‐1 stains also indicated the reliability of the former assay to ascertain gradual increases in spermatozoa with greater mitochondrial function. These data confirmed that different parts of the tamarin spermatozoa can be simply and consistently evaluated by EPM, EN, FG‐RB, CB, TB, and DAB protocols.

Genetic diversity in ex situ populations of the endangered Leontopithecus chrysomelas and implications for its conservation

“…It is noteworthy that currently there are smaller GHLT groups under human care in 15 other institutions in Brazil (Galbusera P., personal communication) which continue to be included in the ex situ management. In addition, semen analysis and cryopreservation, as tested in the FPZSP [ 83 ] an alternative tool for contributing to the maintenance of genetic diversity in the future [ 6 , 84 ].…”

Semen cryopreservation in golden‐headed lion tamarin,Leontopithecus chrysomelas

“…The sperm‐binding to EPM is a viable, practical, and inexpensive alternative to sperm‐zona pellucida binding assays, and it is based on the high homology among EPM and mammalian zona pellucida glycoproteins (Arakaki, Salgado et al, ; Ganguly, Sharma, & Gupta, ; Lefièvre et al, ; Smith, Paton, Hughes, & Burt, ). To the best of our knowledge, this is the first time this method was used in Nonhuman primates (except for validation tests; Arakaki, Salgado et al, ), and considering the difficulties in obtaining homologous oocytes for this and other wild mammalian groups we believe that it can possibly contribute in providing additional parameters for several wild species. Evidence of this is found in the variety of species in which semen has already been analyzed using sperm‐binding to EPM such as humans, bulls, stallions, dogs, oncillas, and ocelots (Araujo et al, ; Brito et al, ; Losano et al, ; Santos et al, ).…”

“…After inspection and palpation of the testicles, ejaculates were obtained by penile vibrostimulation using an adapted hand‐held personal vibrator (FertiCare® personal, Multicept ApS, Rungsted, Denmark) as previously described for marmosets (Arakaki, Nichi et al, ). Such methodology has been successfully applied for semen collection in golden‐headed lion tamarins (Arakaki, Salgado et al, ). Semen samples were collected into a dry glass tube kept at 37°C until the moment of semen collection.…”

“…At least 100 spermatozoa were counted using a phase‐contrast optical microscope (1000×), and cells were classified in four categories: DAB I, all mitochondria active (100% of the midpiece was stained); DAB II, most mitochondria active (more than 50% of the midpiece was stained); DAB III, most mitochondria inactive (less than 50% of the midpiece was stained) and DAB IV, all mitochondria inactive (absence of staining in the midpiece). Eosin–Nigrosin, Coomassie Blue staining, and DAB assay were previously validated for golden‐headed lion tamarin (Arakaki, Salgado et al, ).…”

“…Frozen‐thawed samples were additionally submitted to a functional sperm test, the sperm‐binding in the chicken egg perivitelline membrane (EPM) assay (Losano et al, ), also validated for golden‐headed lion tamarin (Arakaki, Salgado et al, ). Briefly, EPM was isolated from fresh unfertilized egg yolks and washed with PBS, cut into 0.5 cm 2 segments and incubated with 25 × 10 3 spermatozoa diluted in 250 μl of modified TALP medium at 37°C for 1 hr.…”