Gender Determination of Two-toed Sloths ( Choloepus hoffmanni ) with the SRY Gene

1. Gender Determination of Two-toed Sloths (Choloepushoffmanni) with the SRY Gene Cristina Vaughan, Rebecca Kirby, Jonathan N. Pauli Abstract Identifying the sex of juvenile two-toed sloths (Choloepushoffmanni) is difficult due to incompletely developed genitalia. We utilized a modified polymerase chain reaction (PCR) amplification of a Y chromosome-linked gene, the sex-determining region Y (SRY), to determine the sex of individual sloths. Mature sloths of known sexes were used to test a SRY amplification routine and optimize the technique. Testing was performed on 47 adult sloths of known sex (22 male and 25 female), which resulted in a 6.9% error rate. Of the 35 unknown juveniles tested, 20 were male and 15 were female. The ability to identify sex of juvenile sloths will aid researchers in answering questions of population ecology that require an understanding of sex ratios. Introduction Two-toed sloths (Choloepushoffmanni) are mid-sized arboreal mammals that range throughout South America and southern Central America. They primarily feed on leaves and are solitary, with females bearing one young at a time (Taube et al. 2011). As part of a study on the population ecology of sloths in and around a 120-hectare organic cacao farm near Pueblo Nuevo do Guácimo in Limón, Costa Rica, adult and juvenile sloths were caught by hand while they rested in trees. Researchers sexed, weighed, measured, and pit-tagged each sloth (Vaughan et al., 2007). For genetic analysis, we also collected a small tissue punch from the ear. Most juvenile sloths, however, did not have distinguishable genitalia due to testicles not having descended in the males. An SRY molecular sexing technique for Choloepusdidactylushad been previously used for breeding captive sloths (Murata et al., 1996). This technique involves amplifying the sex-determining region Y, which is only found in males. Determining sex in individuals is important to this research to ascertain sex ratios at birth, sex-biased dispersal, and sex-related survival in this population of sloths. We applied and validated the technique here in UW-Madison. Methods DNA was extracted from collected tissues using Qiagen’sDNeasy Blood and Tissue Extraction kit (Qiagen, Valencia, CA, USA). A polymerase chain reaction (PCR) was performed on the extracted DNA to amplify the sex-determining Y (SRY) gene (Murata et al. 1996) with slight modifications. When electrophoresed on an agarose gel, the amplified SRY region was visualized at 466bp for male individuals. Forty-seven adult sloths of known sex were used to validate the reaction. Subsequently we applied the technique to identify sex of 35 unknown juveniles. Discussion The SRY amplification method proved to be accurate and replicable with an average standard error rate of 6.9% for the SRY optimization runs, which allowed for a 93.1% accuracy per run for each of the three juvenile runs. To ensure accuracy, each of the juveniles was tested three times. Conflicting results arose when a sample resulted in a faint band, or no band at all. From the preliminary tests with adult sloths, we suggest two possible reasons for this: 1) PCR failure due to incomplete mixing during the set up; 2) gel loading error when too much PCR product from a male sloth sample spilled over into a neighboring well with a female sample. Due to the low error rate (6.9%) determined from the initial test runs of 47 adults—in which two consistently showed faint bands while five other individuals had only one run that did not correspond with its others—the tests were considered to be a successful way of sexing otherwise unknown juveniles. This technique can be applied in future work to examine sex ratios at birth, sex survival ratios, and sex dispersal. Finmac, Limón, Costa Rica Male Choloepus Acknowledgements I would like to thank Dr. M Zachariah Peery for the use of his lab and resources, Dr. Jonathan Pauli for his time and mentorship, Rebecca Kirby for teaching me everything about genetic molecular work and working with me so patiently, Wynne Moss for helping me whenever I had questions, Geovanny Herrera for  his dedication and hard work to this project in Costa Rica, and Jorge Mendoza for providing me with his wonderful images of the sloths whom I cannot visit personally. Grassland covertype sloth habitat Results Four SRY optimization runs of the 47 adult sloths of known sexes resulted in an average error rate of 6.9%, with a lowest run error rate of 4.8%. From this, we determined that juveniles could be accurately assigned sex with 93.1% confidence, and increasing the number of PCR runs with consistent results, increased accuracy. Of 35 unknown juveniles, 16 were consistently identified as male and 15 as female across 3 runs. Four individuals were identified as males in only 2 of 3 runs. References Murata, Koichi and Masuda, Ryuichi. 1996. Gender Determination of the Linne’s Two-Toed Sloth (Choloepusdidactylus) Using SRY Amplified from Hair. Journal of Veterinary Medicine Science. 58(12): 1157-1159. Taube, E., J. Keravec, J. Vie, and J. Duplantier. 2001. Reproductive biology and postnatal development in sloths, Bradypus and Choloepus: review with original data from the field (French Guiana) and from captivity. Mamm. Rev. 31:173-188. doi: 10.1046/j.1365-2907.2001.00085.x. Vaughan, Christopher, Ramírez, Herrera, Geovanny, Guries, Raymond. 2007. Spatial ecology and conservation of two sloth species in a cacao landscape in limón, Costa Rica. Springer Science+Business Media B.V. 16:2293-2310 Juvenile PCR performed 11-7-11 with ethidium bromide stained bands appearing for male individuals. Mother Choloepus with her offspring Release of mother and offspring