Zoo Biology 35: 95–103 (2016) RESEARCH ARTICLE Chelonian Perivitelline Membrane-Bound Sperm Detection: A New Breeding Management Tool Kaitlin Croyle,1,2 Paul Gibbons,3 Christine Light,3 Eric Goode,3 Barbara Durrant,1 and Thomas Jensen1* 1San Diego Zoo Institute for Conservation Research, Escondido, California 2Department of Biological Sciences, California State University San Marcos, San Marcos, California 3Turtle Conservancy, New York, New York Perivitelline membrane (PVM)-bound sperm detection has recently been incorporated into avian breeding programs to assess egg fertility, confirm successful copulation, and to evaluate male reproductive status and pair compatibility. Due to the similarities between avian and chelonian egg structure and development, and because fertility determination in chelonian eggs lacking embryonic growth is equally challenging, PVM-bound sperm detection may also be a promising tool for the reproductive management of turtles and tortoises. This study is the first to successfully demonstrate the use of PVM-bound sperm detection in chelonian eggs. Recovered membranes were stained with Hoechst 33342 and examined for sperm presence using fluorescence microscopy. Sperm were positively identified for up to 206 days post-oviposition, following storage, diapause, and/or incubation, in 52 opportunistically collected eggs representing 12 species. However, advanced microbial infection frequently hindered the ability to detect membrane-bound sperm. Fertile Centrochelys sulcata, Manouria emys,andStigmochelys pardalis eggs were used to evaluate the impact of incubation and storage on the ability to detect sperm. Storage at À20°C or in formalin were found to be the best methods for egg preservation prior to sperm detection. Additionally, sperm-derived mtDNA was isolated and PCR amplified from Astrochelys radiata, C. sulcata,andS. pardalis eggs. PVM-bound sperm detection has the potential to substantially improve studies of artificial incubation and sperm storage, and could be used to evaluate the success of artificial insemination in chelonian species. Mitochondrial DNA from PVM-bound sperm has applications for parentage analysis, the study of sperm competition, and potentially species identification. Zoo Biol. 35:95–103, 2016. © 2016 Wiley Periodicals, Inc. Keywords: turtle; tortoise; conservation; fertility assessment; PVM; sperm; chelonian INTRODUCTION artificial incubation, temperature manipulation for sex selection [Girondot et al., 1998], and protection of eggs Turtles and tortoises are important targets for from nest predation [Noel et al., 2012]. Eggs that fail to conservation programs, with many chelonian species facing exhibit embryonic development during incubation can be a the threat of extinction due to habitat destruction and problem for pair/herd management because developmental excessive harvesting [Turtle Conservation Fund, 2002]. failure can be caused by a variety of factors requiring Conservation initiatives frequently include captive breeding programs for reintroductions and to serve as genetic à assurance colonies. Therefore, any technique that can Correspondence to: Thomas Jensen, San Diego Zoo Institute for enhance a program’s success are valuable, as demonstrated Conservation Research, 15600 San Pasqual Valley Road, Escondido, CA 92027. E-mail: [email protected] by the Batagur baska conservation program, where a few successful clutches had a significant impact on the sustainability of the species [Weissenbacher et al., 2015]. Received 17 September 2015; Revised 26 January 2016; Accepted 04 February 2016 Due to limited success with turtle and tortoise semen collection and cryopreservation [Browne et al., 2011], DOI: 10.1002/zoo.21273 chelonian assisted reproductive techniques primarily involve Published online 17 February 2016 in Wiley Online Library pair management for maintenance of genetic diversity, (wileyonlinelibrary.com). © 2016 Wiley Periodicals, Inc. 96 Croyle et al. different solutions. These include egg infertility due to knowledge, this study is the first to demonstrate the use of copulatory organ deformity, lack of copulation, low-quality PVM-bound sperm detection in any reptile species. sperm in ejaculate, genetic incompatibility, inappropriate incubation parameters, or the persistence of embryonic MATERIALS AND METHODS diapause [Peters et al., 1994]. Although eggshell chalk formation and visualization of vasculature by candling This study was reviewed and approved by the San indicate early embryonic development, incubated eggs Diego Zoo Global IACUC (assurance# A3675-01) in lacking outward signs of development require necropsy to compliance with the guidelines for the use of animals in assess fertility. However, distinguishing between a blasto- research [OLAW/ARENA, 2002]. The taxonomic classifi- derm (fertile) and a germinal vesicle (infertile) during cation, naming, and conservation status of turtles and necropsy is difficult. Likewise, locating an early embryo is tortoises is based on the IUCN Red List of Threatened often challenging in eggs exhibiting extensive degradation or Species [IUCN, 2014]. Unless otherwise noted, all chemicals microbial infection following incubation (K. Croyle, were purchased from Fisher Scientific (Pittsburg, PA). personal observation). Yet, the ability to discern between fertile and infertile eggs is critical in troubleshooting clutch Eggs Collection failures, especially in species with embryonic diapause, as A total of 191 chelonian eggs representing 17 species eggs can remain arrested at the blastoderm stage following were used for this study. Chelonian eggs were opportunisti- oviposition for extended periods of time [Miller and cally collected from breeding facilities at San Diego Zoo Dinkelacker, 2008]. Global (Galapagos giant tortoise [Chelonoidis nigra], There are currently no reliable methods to differenti- Chinese three-striped box turtle [Cuora trifasciata], Euro- ate between infertility and early embryonic death (EED) in pean pond turtle [Emys orbicularis], Burmese starred chelonian eggs. Previous studies of chelonian egg fertility tortoise [Geochelone platynota], black-breasted hill turtle and hatching rates are limited to categorizing eggs as [Geoemyda spengleri], Burmese mountain tortoise [Man- undeveloped, without distinguishing between EED and true ouria emys], spider tortoise [Pyxis arachnoides], leopard infertility [Miller, 1999]. In avian species, detection of tortoise [Stigmochelys pardalis]), the Turtle Conservancy ’ sperm bound to the oocyte s perivitelline membranes (radiated tortoise [Astrochelys radiata], ploughshare tortoise (PVM) has been used to study sperm-egg interaction [Astrochelys yniphora], African spurred tortoise [Cen- [Birkhead et al., 1994; Bramwell and Howarth, 1992; trochelys sulcata], Chinese three-striped box turtle [C. Rabbani et al., 2006; Wishart, 1997], sperm competition trifasciata], Burmese starred tortoise [G. platynota], black [Birkhead et al., 1995], sperm storage [Birkhead et al., spotted turtle [Geoclemys hamiltonii], sunburst turtle 1993; Wishart, 1987], estimate sperm production, and to [Heosemys spinosa], serrated hinge-backed tortoise [Kinixys infer egg fertility [Birkhead et al., 2008]. More recently, erosa], Burmese mountain tortoise [M. emys], spider tortoise PVM-bound sperm detection was shown to be a valuable [P. arachnoides], painted wood turtle [Rhinoclemmys management tool for endangered avian breeding programs pulcherrima]), the Santa Barbara Zoo (radiated tortoise [A. [Croyle et al., 2015; Hemmings et al., 2012]. PVM-bound radiata]), Suzhou Zoo (Yangtze giant softshell turtle sperm detection can be used, in combination with [Rafetus swinhoei]), and Dr. Brian Horne’s private collection behavioral observations and reproductive history, to assist (yellow-margined box turtle [Cuora flavomarginata], leop- in more timely breeding management decisions [Croyle ard tortoise [S. pardalis]). Eggs were either freshly laid et al., 2015]. (retrieved <24 hr post-oviposition), removed following Due to the similarities between avian and chelonian embryonic diapause cooling period at 17À20°C, removed egg structure and pre-ovipositional embryonic development from incubation following developmental failure, retrieved [Deeming and Ferguson, 1991], this study investigated the from the oviduct during necropsy (K. erosa), or stored at À20 potential for PVM-bound sperm detection in turtle and or 4°C prior to examination or experimental treatment. Egg tortoise eggs. The feasibility of using PVM-bound sperm failure was determined by lack of visual embryonic growth staining for chelonian conservation was examined using upon candling and/or absence of eggshell chalking. opportunistically collected eggs. As avian PVM-bound sperm have been shown to degrade during incubation Egg Storage and Incubation [Croyle et al., 2015; Small et al., 2000], we examined potential limitations of PVM-bound sperm detection in Incubation data for failed eggs were provided by controlled conditions to evaluate the impact of incubation each facility. Diapause and incubation lengths, as well length, egg storage, and presence of microbial infection. as temperatures, varied greatly according to species and PVM-bound sperm detection is a promising technique for breeding program (Table 1). Incubation temperatures ranged chelonian conservation programs, future studies of chelonian from 25.5 to 33.3°C. Eggs were stored or incubated for up to reproduction, and is currently being used to manage 206 days post-oviposition prior to examination.