Andrological and cytogenetic investigations of an infertile Przewalski's stallion.

The case of an 8-year-old, sexually active but infertile Przewalski's stallion (Equus ferus przewalskii) was studied. Besides the infertility, the stallion also showed permanent problems with its body condition, being obviously weaker than all the other group members. The horse was kept in a separate place for two years with 12 mares in its harem group (six mares had foals earlier); however, none of the mares covered got pregnant. Andrological and cytogenetic investigations revealed underdeveloped testes, arrested spermatogenesis, azoospermia, and XY/XXY/X0 mosaicism. We classify the case as a mosaic Klinefelter syndrome, the first reported case in Przewalski's horse.


INTRODUCTION
The world's largest managed population of Przewalski's horses (Equus ferus przewalskii) lives under semi-wild conditions in the core zone of the Hortob agy National Park in Eastern Hungary in a reserve of 2,470 ha size. The population of the horses contained 295 (132 male, 163 female) individuals at the time of the investigation (October 2014), all of which were identifiable and genetically tested for parentage. The population goes back to 25 (8 male, 17 female) founders that had been chosen from different zoos and semi-reserves from all over Europe. The first harem group (1 male, 3 females) had been transported to the area in 1997.
The development of the horse population is continuously monitored with the main focus on behavioural studies, health documentation and genetic patterns (Zimmermann et al., 2009). To avoid inbreeding and to ensure a higher genetic diversity in the population, genetically overrepresented stallions are culled each year. In 2014, eleven horses were culled for different reasons, namely chronic diseases, behavioural abnormalities, or high inbreeding factors. One of those horses, the stallion 'Gagarin', was known to be infertile with no sperm cells in his semen taken by electroejaculator (Christine Gohl, personal communication, 2011). Numerical sex chromosome abnormalities, including X polysomy in stallions, are not so rare in infertile domestic horses (Gluchovschi et al., 1970;Bouters et al., 1972;Fretz and Hare, 1976;Halnan et al., 1982;Kubien et al., 1993;M€ akinen et al., 2000;Iannuzzi et al., 2004); however, they have not yet been described in Przewalski's horse.

CASE DESCRIPTION Animal
The stallion 'Gagarin' was born in Munich as a descendent of the highly inbred A-line. Fertility problems in both sexes are frequent in this line. Besides its infertility, the stallion also showed permanent problems with body condition, being obviously weaker than all the other group members. The horse arrived in Hortob agy in 2012. It was kept in a separate place for two years with 12 mares in his harem group (six mares had foals earlier); however, none of the mares covered got pregnant. To detect the cause of its infertility, andrological and cytogenetic investigations have been conducted.

Methods
Chromosome preparation. Blood samples were taken by syringes from the v. jugularis from all 11 individuals (7 stallions, 1 gelding and 3 mares), including the stallion with impotentia generandi and filled into heparinised tubes (Vacuette ® Tube 6 mL LH Lithium Heparin, cat. no. 456088 (Greiner BioOne, Mosonmagyar ov ar, Hungary) Lymphocyte cultures were established with our method elaborated for domestic horses (Chowdhary and Kov acs, 1987). After 30-min sedimentation of the heparinised blood samples, 0.5 mL plasma containing leukocytes was added to 3.0 mL culture medium. The composition of the culture medium used is shown in Table 1. Lymphocyte culture, chromosome preparation and evaluation were carried out as described by Moorhead et al. (1960). The C-banding technique was carried out according to Sumner (1972).
Morphology and histology. The weight of the testes of 'Gagarin' was compared to that of five adult, healthy stallions. Testicular and epididymal structure was evaluated in histological sections. Sugar-lump-sized samples were collected and placed into sample containers filled with 10% neutral buffered formalin solution (Formaldehyde 4%, cat. no. 9713.5000, VWR International Ltd, Radnor, Pennsylvania, USA). After embedding in paraffin, 3-4 mm thick slices were made from the tissue pieces. Histological sections were stained with haematoxylin and eosin staining technique, mounted by Canada balsam (SIAL-C1795, Sigma-Aldrich, St. Louis, Missouri, USA), then coverslipped and evaluated at 3400 magnification using Leica DMRA light microscope (Leica Microsystems, Wetzlar, Germany).
Both caudae epididymides seemed to be empty; their content was collected by retrograde rinsing (Monteiro et al., 2011;Olaciregui et al., 2014). Suspensions were smeared and evaluated by Chicago sky blue and Giemsa viability and acrosome staining (K utv€ olgyi et al., 2006).

RESULTS
In the preparations made from the lymphocyte cultures of the infertile stallion, three different cell lines containing 67, 66 and 65 chromosomes were found. In 73 out of 86 mitoses (84.88%) 66, in 7 (8.14%) 67, and in 6 (6.98%) 65 chromosomes were found. XY/XXY/X0 mosaicism was diagnosed by the karyotypes of the XY, XXY, and X0 cell lines (Figs 1-3) and by the identification of both sex chromosomes: the X having a fine interstitial C-band on its long arm, and the Y being the smallest and darkest element (Matthews and Delhanty, 1979) (Figs 1 and 2).
The smaller body size of 'Gagarin' reminiscent of XO gonadal dysgenesis (human Turner syndrome) could be the only recognised consequence of the XO cell line.
The testicles of the horse were smaller than those of the other adult Przewalski's stallions examined ( Table 2). The histology of the testes showed seminiferous tubules with arrested spermatogenesis at the spermatocyte/spermatid stage showing elongated spermatids as the most advanced stage in spermatogenesis (Figs 4 and 5).
We noticed the lack of normal spermatogenetic cycle in the seminiferous epithelium; the histological structure appeared to be disintegrated. Figure 6 shows several multinucleated cells in the lumen of the corpus epididymidis. Spermatozoa were not found in the tubules or in the lumen of the ductus epididymidis.    The tail of the epididymis appeared macroscopically empty. Azoospermia with very few malformed spermatozoa such as multi-tailed microcephalic cells and other head abnormalities were found in the smears (Fig. 7). There were no morphologically normal spermatozoa present.

DISCUSSION
We obtained satisfactory chromosome preparations after starting the blood cultures under extreme circumstances in the field. According to our experiences, the whole blood method is less convenient, as the red blood cells of Equidae and Felidae are extremely strongly coagulated by the lectins stimulating the lymphocytes.
Ten animals were successfully evaluated by chromosome analysis, nine having 2N 5 66 chromosomes as described by other authors (Benirschke et al., 1965;Matthews and Delhanty, 1979), while in the preparations made from the lymphocyte cultures of the infertile stallion, three different cell lines containing 66, 67 and 65 chromosomes were found.
The histological sections of the testes with incomplete meiosis and azoospermia/teratospermia (Blom, 1944) were similar to the XXXY and XXY cases, reported in domestic horses (Gluchovschi et al., 1970;Bouters et al., 1972;Fretz and Hare, 1976;Halnan et al., 1982;Kubien et al., 1993;M€ akinen et al., 2000;Iannuzzi et al., 2004) and other species, including the mosaic cases of human Klinefelter syndrome (Filadis et al., 1998;Wistuba 2010;Wikstr€ om and Dunkel, 2011;Samplaski et al., 2014). The XXY and X0 cell lines probably resulted from nondisjunction events in the mitoses of multipotent cells in the preimplantation embryo. We classify the present case as a mosaic Klinefelter syndrome, which is, to the best of our knowledge, the first reported case in Przewalski's horse.