Gender Determination by PCR Assay for The Sex-Determining Region Y(SRY) Gene Amplification in Linnaeus’s Two-Toed Sloth (Choloepus Didactylus)

Year 2023, , 24 - 28, 01.07.2023

Sena Ardıclı , Berkay Bozkurt Ezgi Vatansever

https://doi.org/10.30782/jrvm.1283245

Abstract

In Linnaeus’s two-toed sloths (Choloepus didactylus), there is no distinct sexual dimorphism. It is an obstacle for gender determination from
the external genitalia, especially in newborns or young sloths. Hence, easy, rapid, and reliable genetics-based methods for gender identification
of the sloths are needed to continue captive breeding more successfully. In this study, a PCR-based technique that allows gender determination
of two-toed sloths by using a sex-determining region Y (SRY) gene marker was described. The hair samples from young (suspect
gender) and adult sloths (known gender) were used in genetic analysis. Initially, genomic DNA was isolated from hair root samples using
Roche high pure PCR template preparation kit. The SRY primers were specifically designed based on the NCBI and Ensembl databases, and
they were verified with the BLAST program concerning the two-toed sloth genome. PCR amplification with the SRY-specific primers was
carried out by a programmable thermal cycler device using FastStart High Fidelity PCR System, Roche dNTPack. The samples were then electrophoresed
on 2% agarose gels and were visualized by a gel documentation and analysis system. A specific band in the electrophoresis pattern
is diagnostic for a male individual with a partial SRY region. Hence, the analysis demonstrated that the samples belonged to a male two-toed
sloth. Two-toed sloth species are commonly preferred animals in zoos. Gender determination is inevitable for these animals in captivity to
be raised successfully and healthily. Molecular genetic techniques allow high efficiency in taxonomic evaluations and gender identification in
species that do not display sexual dimorphism. The PCR assay described here may be helpful for a rapid genetic analysis that can be widely
used in gender determination for two-toed sloths.

Keywords

Two-toed sloth, PCR, Sex identification, SRY gene

Thanks

The authors gratefully thank Faruk Yalçın Zoo for the support.

References

• 1.Murata K, Masuda R. Gender determination of the Linne's two-toed sloth (Choloepus didactylus) using SRY amplified from hair. J Vet Med. 1996;58(12):1157-59.
• 2. Nowak RM, Walker EP. Walker's Mammals of the World: John Hopkins University Press; 1999.
• 3. Steiner CC, Houck ML, Ryder OA. Species identification and chromosome variation of captive two-toed sloths. Zoo Biol. 2011;30(6):623-35.
• 4. Richard-Hansen C, Vié J-C, Vidal N, Kéravec J. Body measurements on 40 species of mammals from French Guiana. Journal of Zool. 1999;247(4):419-28.
• 5. Stewart HD, Tighe E, Griffiths BM. Patterns of visitation of the Linnaeus’s two-toed sloth (Choloepus didactylus) at Amazonian mineral licks. Eur. J Wildl Res 2022;68(2):25.
• 6. Adam PJ. Choloepus didactylus. Mamm Species. 1999(621):1-8.
• 7. Shahidehnia M. Epigenetic effects of endocrine disrupting chemicals. J Environ Anal Toxicol. 2016;6(4):381. DOI: 10.4172/2161-0525.1000381
• 8. Nishino K, Hattori N, Tanaka S, Shiota K. DNA methylation-mediated control of SRY gene expression in mouse gonadal development. J Biol Chem. 2004;279(21):22306-22313.
• 9. Harley VR, Goodfellow PN. The biochemical role of SRY in sex determination. Mol Reprod Dev. 1994;39:184-193.
• 10. Schartl M, Lamatsch DK. How to manage without a Y chromosome. Proc Natl Acad Sci USA. 2023;120(2):e2218839120.
• 11. Charlesworth B. The evolution of sex chromosomes. Science. 1991;251:1030–1033.
• 12. Roy SW. How did a novel X-linked gene become essential for male determination? Cell Res. 2022;32:7–8.
• 13. Hawkins JR. Mutational analysis of SRY in XY females.Human Mutat. 1993;2:347–350.
• 14. Bull JJ, Bulmer MG. The evolution of XY females in mammals. Heredity. 1981;47:347–365.
• 15. Burt A, Trivers R. Genes in Conflict. The Biology of Selfish Genetic Elements. Belknap Press-Harvard University Press, Cambridge, MA, ISBN 9780674027220; 2008.
• 16. Zhan J, Cui P, Yu Z, et al. SDX on the X chromosome is required for male sex determination. Cell Res. 2022;32:99–102.
• 17. National Center for Biotechnology Information (NCBI). SRY: sex determining region Y Choloepus didactylus (southern two-toed sloth). 2023. https://www. ncbi.nlm.nih.gov/gene/?term=XM_037824592.1
• 18. Ensembl Genome Browser. SRY-box transcription factor_Sloth. 2023. https://www.ensembl.org/index.html
• 19. Basic Local Alignment Search Tool (BLAST). Choloepus didactylus sex determining region Y (SRY). 2023. https://blast.ncbi.nlm.nih.gov/Blast.cgi#1934645850
• 20. Varela-Lasheras I, Bakker AJ, van der Mije SD, et al. Breaking evolutionary and pleiotropic constraints in mammals: on sloths, manatees and homeotic mutations. EvoDevoç 2011;2:1-27.
• 21. Jorge W, Orsi-Souza A, Best R. The somatic chromosomes of Xenarthra. The evolution and ecology of armadillos, sloths, and vermilinguas (GG Montgomery, ed.). Smithsonian Institution Press, Washington, DC 1985:121-129.
• 22. Sekido R, Lovell-Badge R. Sex determination involves synergistic action of SRY and SF1 on a specific Sox9 enhancer. Nature. 2008;453(7197):930-34.
• 23. Pamilo P, O'Neill R. Evolution of the SRY genes. Mol Biol Evol. 1997;14(1):49-55.
• 24. Chen Y, Dong Y, Xiang X, Zhang X, Zhu B. Sex determination of Microtus mandarinus mandarinus is independent of SRY gene. Mamm Genome. 2008;19:61- 68.
• 25. Foster JW, Graves J. An SRY-related sequence on the marsupial X chromosome: implications for the evolution of the mammalian testis-determining gene. Proc Natl Acad Sci USA. 1994;91(5):1927-1931.
• 26. Graves JM. Sex chromosomes and sex determination in weird mammals. Cytogenet Genome Res. 2002;96(1-4):161-168.
• 27. Yu H-T, Ma G-C, Lee D-J, et al. Molecular delineation of the Y-borne SRY gene in the Formosan pangolin (Manis pentadactyla pentadactyla) and its phylogenetic implications for Pholidota in extant mammals. Theriogenology. 2011;75(1):55-64.