Cost-Effective Method to Transport Manually Collected Gorilla Semen for Long-Term Storage
Since 1994, semen samples have been collected from two western lowland gorillas (Gorilla gorilla gorilla) housed at the Henry Doorly Zoo (HDZ) using a positive reinforcement training program.1 Semen quality collected from gorillas by manual palpation is superior to electroejaculation for obtaining ejaculates with higher percent (up to 80%) motile sperm.2 A protocol for the cryopreservation of gorilla sperm was developed (Loskutoff, unpublished data) and to date, over 500 samples have been stored at the HDZ genome resource bank. The storage of gorilla semen is important not only for use in assisted reproductive techniques, but to maintain genetic diversity in the captive population. Using cryopreserved gorilla sperm collected from a trained animal, embryos have been produced4 and a gorilla baby was born in 1995 by in vitro fertilization.5 Furthermore, gorilla embryos have been produced by intracytoplasmic sperm injection from oocytes collected postmortem.3 Currently, few institutions have the capability to house cryopreserved samples due to the expense and labor involved in maintaining liquid nitrogen (LN2) storage tanks. With an increasing interest by zoos to develop their own training programs for semen collections from gorillas, the goals of this study were to develop a cost-effective method for transporting processed gorilla semen to cryogenic storage facilities and to develop a short-term storage alternative for LN2.
Three ejaculates collected by manual palpation were processed using the standard protocol: 1) dilution (1:1) with TEST-egg yolk buffer + 17.5% egg yolk at room temperature; 2) refrigeration (4–7°C) for 2 hours before dilution with cooled cryodiluent (same as above but with added glycerol for final concentration of 4%), loaded into 0.25 ml straws, and equilibrated for another hour. Straws were frozen using two methods: 1) placed on a rack in an ultralow upright freezer; 2) placed between two 10-lb blocks of dry ice in a Styrofoam container. As a control, some straws were frozen using the standard procedure of placing on a block of dry ice for 10 minutes and then plunged into LN2. Pre-freeze motilities for individual ejaculates were 74%, 68%, and 81% respectively. Post-thaw motilities, expressed as mean±SEM, were determined as percent motile over time from pre-freeze motility estimates (Table 1). Straws were thawed (37°C water bath for 10 sec) at approximate times corresponding to standard express mail delivery courier services: 16 hours, 28 hours, 45 hours, and 75 hours. Straws remaining at the end of 75 hours were plunged into LN2 (Table 2). (VIN editor: Tables 1 and 2 are not available.)
As shown in Table 1, the post-thaw motility of gorilla sperm held in the ultra-low freezer stayed fairly constant over time, while a decrease in motility occurred when straws were stored between dry ice. Overall post-thaw motility rates after plunging into LN2 after 3 days storage were very good, with the ultralow being slightly higher than dry ice, although not statistically significant. This study shows that [VIN editor: The remainder of this paper is not available.]