Reproductive morphophysiology of the male scorpion mud turtle (Kinosternon scorpioides Linnaeus, 1766) in captivity

Abstract Kinosternon scorpioides, popularly known as scorpion mud turtle (jurará in Brazil), is a fresh water species. There is little information about its reproduction and the present study aims to morphologically characterize the reproductive organs of male K.scorpioides bred in captivity in two seasons of the year. The reproductive tracts of adult animals under went macroscopic ultrastructural analysis of the lumen, as well as scanning electron and transmission microscopy. Macroscopically, the male genital organs consist of a pair of testicles, epididymis, the vas deferens and a penis. Testicles, epididymis and deferents ducts were characterized by reproductive activity during the rainy season and reproductive inactivity in the dry period. The morphometry regarding the tubular and luminal diameter and epithelial height of the testicles, epididymis and deferents ducts showed changes along the studied periods. The rainy season presented higher averages than the dry period. The penis did not show any changes during both periods. It was concluded that K. scorpioides exhibits reproductive seasonality.

cycle of several animals depends on the hormonal control that directly produces the physiological changes in the reproductive system during the mating season (Reddy & Prasad, 11970).
Understanding the reproduction of the species can enhance breeding and conservation programmes in natural and artificial environments. The present study aims to describe the morphology of male Kinosternon scorpioides in captivity during different periods.

| MATERIAL S AND ME THODS
The study was carried out over 12 months, during the dry (July to December) and rainy season (January to June) in Maranhão state, Brazil. In all, 14 adult K. scorpioides, from the Scientific Breeding Center for Research in K. scorpioides, over 3 years of age, with average carapace and plastron length of 13.29-11.23 cm, respectively, and body weight of 301.50g were studied.
The animals were divided into two experimental groups-the dry and rainy season groups, captured and collected in November 2010 and April 2011, respectively. The 14 animals were anaesthetized with 2% xylazine (40 mg/kg/IM) and 1% ketamine hydrochloride (60 mg/ kg/IM) and euthanized with 2.5% thiopental sodium (60 mg/kg/EV) by catheterization of the cervical venous sinus, according to Schumacher (1996). The coelomic cavity was then opened with a steel handsaw to detach the bone bridge that joins the carapace to the plastron. The gonads were removed and the testes isolated for subsequent microscopy.
Testes were fixed in 4% buffered formaldehyde for 12 hr, processed using routine paraffin embedding techniques and 5-μm thick histological cross-sections were stained with haematoxylin-eosin and Masson trichrome. Images for morphometric studies were obtained with a binocular microscope (Olympus BH-41, São Paulo, Brazil) equipped with a digital camera.
Testis fragments were fixed in 2.5% gluteraldehyde and frozen for 72 hr, then cryofractured in liquid nitrogen, washed in 0.1 M phosphate buffer, postfixed with 1% osmium tetroxide and dehydrated in a series of increasing alcohol concentrations (50%-100%).
Samples were dried in a Balzers CPD 020 critical-point dryer (Balzers Union Ltd, Liechtenstein) with liquid CO 2 , and mounted on aluminium stubs using carbon paste. The samples were then sputtercoated with gold (Emitech K550, Emitech Ltd. Ashford, Kent, UK), analysed and photographed under a scanning electron microscope (Zeiss LEO 435VP, Cambridge, UK).

Analysis of variance was performed with the GraphPad
InSat program to obtain mean and standard deviation and the Cramer-von Mises to test for homoscedastic distribution between variables.

| RE SULTS
All the male K. scorpioides observed in the present study exhibited a set of male genital organs consisting of a pair of testicles, epididymis and vas deferens located in the coelom, and a penis located in the cloaca. The whitish epididymis, which displayed a macroscopically convoluted shape, was inserted in the mid dorsal edge of the testicles, secured by folds to the cavity wall ( Figure 1). The vas deferens ducts originate from the caudal extremity of the epididymis, have a convoluted shape, and run parallel to the ureters, close to their insertion in the dorsal lateral wall of the cloaca. As light ampouleshaped dilation can be observed (Figure 1a-c). Cross-sections of the tubule lumen showed an irregular mucosa with small longitudinal folds, surrounded by a cylindrical pseudo-stratified epithelium with secretory cells and spermatozoa mass. The sheet itself contains a layer of dense conjunctive tissue with blood vessels and a layer of smooth muscle arrangements.
The testicles of K. scorpioides are bilateral ovoid structures with two poles (cranial and caudal), and two margins (medial and lateral), arranged on each side of the median line and separated by the colon, a segment of the large intestine. They are positioned asymmetrically in the coelomic cavity, the right testicle more cranial than the left, covered by a light yellow to gold-coloured thin transparent capsule, the tunica albuginea. The epididymis is located dorsally to the kidneys and the adrenal glands in their medial phase. The testicles are covered by a fibrous capsule of dense conjunctive tissue, the tunica albuginea, consisting primarily of collagen fibres. Thin septa radiate outwards from the dorsal portion of the tunica albuginea, and separate the testicular parenchyma into seminiferous tubules. In the scorpion mud turtle, these tubules are The vas deferens, originating at the caudal extremity of the epididymis, has a convoluted shape that runs parallel to the ureters.
Near its insertion into the dorsolateral wall of the cloacais a slight ampoule-shaped dilation, exhibiting irregular mucosa with small longitudinal folds, covered by a cylindrical pseudo-stratified epithelium containing secretory cells and spermatozoid mass.
The vas deferens of the K. scorpioides also underwent morphological changes in the dry season, exhibiting a pseudo-stratified epithelium, containing cuboidal cells, and no spermatozoa in the tubular lumen (Figure 6a-b).In the ultrastructure, the cytoplasm is devoid of cytoplasmic organelles (Figure 7a).These structural aspects are compatible with the repair of components for renewed reproduction. The epithelium in the rainy season is pseudo-stratified with cylindrical cells and a lumen filled with spermatozoa (Figure 6c-d).
In the ultrastructure, we observed cylindrical cells with cytoplasmic organelles and secretory vesicles showing the structural organization for the favourable reproductive period (Figure 7b-c).
The penis of the scorpion mud turtle is a muscular dorsally sulcate organ, located on the cloacal floor, divided into the root, body and glans. The root, the initial part of the organ, displays firm consistency, whitish colour and consists of two cavernous bodies that extend as far as the glans. The body is the continuous segment of the root demarcated by a deep groove, denominated ejaculatory duct, from which semen flows, and the blackish, cauliflower-shaped glans is the terminal portion of the organ. Histological cross-sections of the central portion of the penis show that the ejaculatory duct is covered by a stratified prismatic epithelium with mucoid cells that are supported by the lamina propria, which consists of loose connective tissue filled with diffuse lymphoid cells, with smooth muscle fibres forming the spongy body (Figure 8c,d). Dorsolaterally to the spongy body are two cavernous bodies composed of venous sinuses and thick muscle fibre and dorsally to the cavernous bodies is the retractor penis muscle (Figures 8,9a,b, 8,9a,b). Transmission electronic microscopy showed a large amount of collagen and muscle fibres (Figure 9c,d).

| D ISCUSS I ON
The information contained in the literature on the scorpion mud turtle (Kinosternon scorpioides) with respect to the seasonal morphological aspects of the male reproductive organs is incomplete.
As such, this discussion contains references related to the reptile orders.
These data differ in part from those described by Carvalho et al. object of this study. Further research along these lines should be performed to better explain the epithelial cell cycle of the tissues investigated (Viana, Anunciação, et al., 2014b). Guerrero et al. (2004) report the epididymal morphology of Caiman crocodilus from the city of Zambrano, Colombia, as a thin coiled structure, extending along the dorsal surface of the testicles, ending in the vas deferens, covered with a non-ciliated pseudo-stratified columnar epithelium.
These cells have a round nucleus with one to three nucleoli. The basal cells contain darker cytoplasm and exhibit epithelial secretion during the reproductive phase.
In the green anole lizard (Anolis carolinensis), seasonal variations in vas deferens diameter indicate a larger tubule diameter in the period of highest reproductive activity. In Lacerta rhomboidalis, large diameter and irregular boundary caused no visible variation in the vas deferens (Fox, 1952).
In marine turtles, the penis is retractable and located on the cloacal floor. It consists of a pair of cavernous bodies and outer groove known as the sulcus spermaticus. During copulation, the cavernous bodies fill with blood and the sulcus spermaticus contracts into a tubular shape, facilitating the flow of seminal fluid (Wyneken, 2001).
In the scorpion mud turtle (K. scorpioides),the penis is a dark mass located on the ventral wall of the cloaca, covered by a stratified prismatic epithelium with mucoid cells supported by a tunica albuginea, consisting of thick collagen fibres arranged in different directions, and smooth muscle fibres (Abas, Silva, & Pereira, 1998). The penis morphology is divided into the root, body and glans. During sexual stimulation, the sphincter restricts blood flow, causing it to swell, protrude and the groove formed by the cavernous bodies to close, whereby secretion flows via the urethra at ejaculation (Carvalho et al., 2010).
During the dry season, the seminiferous tubules of the Jurará will be characterized by the presence of Sertoli cells and spermatogonia with cytoplasm globe-like, with predominance of pri- In addition to further improving our knowledge of reptile morphology, these findings may be useful in comparative reproductive biology studies, providing the baseline for other physiological studies of K. scorpioides along with the hormonal dosage to propose conservation strategies. Finally, in captivity, this species exhibited reproductive seasonality and morphophysiological alterations of the genital organs.

ACK N OWLED G EM ENTS
The authors thank the Maranhão Foundation for Support to Chaves.

CO N FLI C T S O F I NTE R E S T
The authors have no conflict of interest to declare.

E TH I C S CO M M IT TEE
The project was approved by the System of Biodiversity