Semen collection, evaluation, and cryopreservation in the bonobo (Pan paniscus)

Captive breeding of bonobos (Pan paniscus) has proven to be successful, but maintaining genetic diversity remains a challenge. Cryopreservation of semen is an important potential tool to maintain genetic diversity by preserving current genetic material for future use, as well as facilitating the transport and exchange of genetic material. This study aimed to develop a protocol for semen collection and cryopreservation in the bonobo. Semen was collected from four healthy adult bonobos under general anesthesia during management translocation procedures. Semen collection utilizing urethral catheterization was not successful (n = 1), however, all males (n = 4) responded well to rectal probe electro-ejaculation. Immediately after collection, ejaculates were evaluated for color and admixtures, volume, motility, and concentration. Eosin-Nigrosin staining was prepared to evaluate morphology and viability. Ejaculates were split into two equal volumes and cryopreserved in two different extenders, using a one-step and a two-step approach. Ejaculates were gradually cooled to 4 °C in two hours, subsequently stored in liquid nitrogen vapor for twenty minutes (0.25 ml straws), and finally dropped into liquid nitrogen.

Pre-freeze evaluation showed thick, white samples with an average ejaculate volume of 450 µl (100-1000 µl), total motility of 59% (40-80%), viability of 69% (38-85%) and 58% (46-72%) normal spermatozoa. Mainly head (22%) and tail (19%) defects were detected on the Eosin-Nigrosin stain. Ejaculates were highly concentrated, nevertheless, due to the coagulum that caused high viscosity and non-homogenous fractions, only estimations of concentration could be made (1000 million/ml). After 24 h of storage, the post-thaw evaluation showed a loss of quality with an average post-thaw total motility of 15% (5-25%) using the one-step freezing medium, and 19% (5-30%) using the two-step medium. Average post-thaw viability was 15% (4-24%) and 21% (15-29%), respectively.

This report on ejaculates from bonobos obtained by rectal probe electro-ejaculation shows that semen parameters of this species are not completely similar to those of its sibling species, the chimpanzee. Further studies are necessary to develop an optimal protocol for the processing and cryopreservation of bonobo spermatozoa.