Successful cryopreservation of Asian elephant (Elephas maximus) spermatozoa.

Reproduction in captive elephants is low and infant mortality is high, collectively leading to possible population extinction. Artificial insemination was developed a decade ago; however, it relies on fresh-chilled semen from just a handful of bulls with inconsistent sperm quality. Artificial insemination with frozen-thawed sperm has never been described, probably, in part, due to low semen quality after cryopreservation. The present study was designed with the aim of finding a reliable semen freezing protocol. Screening tests included freezing semen with varying concentrations of ethylene glycol, propylene glycol, trehalose, dimethyl sulfoxide and glycerol as cryoprotectants and assessing cushioned centrifugation, rapid chilling to suprazero temperatures, freezing extender osmolarity, egg yolk concentration, post-thaw dilution with cryoprotectant-free BC solution and the addition of 10% (v/v) of autologous seminal plasma. The resulting optimal freezing protocol uses cushioned centrifugation, two-step dilution with isothermal 285 m Osm/kg Berliner Cryomedium (BC) with final glycerol concentration of 7% and 16% egg yolk, and freezing in large volume by the directional freezing technique. After thawing, samples are diluted 1:1 with BC solution. Using this protocol, post-thaw evaluations results were: motility upon thawing: 57.2+/-5.4%, motility following 30 min incubation at 37 degrees C: 58.5+/-6.0% and following 3h incubation: 21.7+/-7.6%, intact acrosome: 57.1+/-5.2%, normal morphology: 52.0+/-5.8% and viability: 67.3+/-6.1%. With this protocol, good quality semen can be accumulated for future use in artificial inseminations when and where needed.

Sperm sex-sorting in the Asian elephant (Elephas maximus).

In captive Asian elephants, there is a strong need for production of female offspring to enhance reproduction, counter premature aging processes in female animals and reduce challenging management situations derived from husbandry of several bulls in one institution. Artificial insemination of flow cytometrically sex-sorted spermatozoa offers the possibility to predetermine the sex of offspring with high accuracy. The aims of this study were to determine a suitable semen extender and basic parameters for flow cytometrical sex-sorting of Asian elephant spermatozoa. In total 18 semen samples were collected by manual rectal stimulation from one bull. Sperm quality parameters and sex sortability of spermatozoa were evaluated after dilution in three semen extenders (MES-HEPES-skim milk, MES-HEPES, TRIS-citric acid) and DNA staining. MES-HEPES-skim milk was the only semen extender found suitable to sex Asian elephant spermatozoa. From 18 ejaculates collected, 12 were successfully sorted with a purity of 94.5+/-0.7% at an average sort rate of 1945.5+/-187.5 spermatozoa per second. Sperm integrity, progressive and total motility were 42.6+/-3.9%, 48.1+/-3.3%, 59.4+/-3.8% after DNA labelling, and 64.8+/-3.2%, 58.0+/-5.0%, 70.8+/-4.4% after sorting, respectively. After liquid storage of sorted spermatozoa for 12h at 4 degrees C, sperm integrity, progressive and total motility were 46.4+/-5.2%, 32.2+/-4.2% and 58.2+/-3.9%, respectively. The obtained results provide a promising base to inseminate Asian elephants with sexed semen.