History
An approximately 5-year-old intact miniature equid was presented for evaluation of ambiguous external genitalia and gonadectomy. Additionally, the equid had features consistent with a donkey-horse hybrid, but it was unknown whether the equid was a Miniature Donkey, hinny, or mule. The equid was purchased from an auction by a rescue organization. Members of the rescue organization noticed that the animal had external gonads, but postured to urinate in a manner typical of a mare, such that urine was expelled caudally from under the tail, rather than ventrally. Visual examination of the perineum revealed a vulva-like structure that extended ventrally beginning approximately 3 cm ventral to the anus. The animal was semiferal and could not be handled sufficiently to evaluate the condition any further. Estrous behavior was not noted, and although the animal could be aggressive when it was approached, male-specific behaviors (eg, mounting females, penile erection) were not noted.
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Diagnosis
Differential diagnoses included male pseudohermaphroditism, true hermaphroditism, hypospadias, mosaicism, and chimerism.
Due to the animal’s temperament, general anesthesia was required to facilitate a complete physical examination (premedication: acepromazine, 0.02 mg/kg, IM; detomidine, 0.02 mg/kg, IM; xylazine, 0.2 mg/kg, IV; and butorphanol, 0.02 mg/kg, IV; induction: midazolam, 0.06 mg/kg, IV; and ketamine, 1.3 mg/kg, IV). On physical examination, the animal had a scrotum with 2 external gonads palpably consistent with testes and associated epididymides. The gonads were apposed to the body wall and therefore were considered incompletely descended. The external genitalia most closely resembled an abnormal penis. A rudimentary penile shaft was present and the glans penis was directed caudally. Incomplete fusion of the glans penis was present ventrally. Incomplete ventral fusion extended the length of the urethra along the rudimentary penile shaft and perineum to approximately 3 cm ventral to the anus. From that point, a fully closed pelvic urethra extended cranially to the bladder (Figure 1).
Severe hypospadias in an approximately 5-year-old miniature equid. A—Scrotum with gonads closely apposed to body wall (white outlined arrows) and lack of fusion of ventral glans penis and urethra. The glans penis-like structure can be seen pointing caudally, at the termination of the urethra (solid white arrow). The gloved hand is holding a portion of what appeared to be a penile sheath. B—Lack of fusion of urethra continues dorsally to approximately 3 cm ventral to the anus. The glans penis-like structure is indicated by the solid white arrow.
Citation: Journal of the American Veterinary Medical Association 261, 9; 10.2460/javma.23.03.0175
Ultrasonographic examination per rectum revealed well-developed male accessory sex glands (paired ampullae, paired seminal vesicles, a bilobed prostate and paired bulbourethral glands; Figure 2). The size and character of the accessory glands were consistent with exposure to androgens.
Transrectal ultrasonographic image of the animal described in Figure 1. Image shows presence of a well-developed ampulla (solid white outline) and prostate (dashed white outline) as well as a seminal vesicle filled with anechoic fluid (white arrow), all consistent with male accessory sex glands that have been exposed to androgens.
Citation: Journal of the American Veterinary Medical Association 261, 9; 10.2460/javma.23.03.0175
Blood was obtained to perform short-term lymphocyte cultures for karyotyping to establish chromosomal sex and to determine whether the animal was a Miniature Donkey, hinny, or mule. PCR analysis was performed on isolated genomic DNA (QIAmp DNA Blood Mini Kit; Qiagen) to screen for the presence of the Y-linked SRY gene. PCR analysis included normal male and female genomic DNA as controls. PCR analysis with primers specific for a 294-bp portion of the X-linked androgen receptor gene served as a positive technical control for PCR amplification. Chromosome analysis confirmed that the animal was a horse-donkey hybrid with 63, XY male karyotype. Fluorescence in situ hybridization analysis using equid X- and Y-chromosome–specific fluorescent probes confirmed the presence of 1 X and 1 Y chromosome (male genetic sex). Chromosomal banding identified the X chromosome as originating from a horse dam and the Y chromosome as originating from a donkey sire, confirming that the animal was a mule (Figure 3). We observed a relatively high frequency (approx 10%) of polyploid cells in cultured lymphocytes. PCR analysis confirmed the presence of the Y-linked SRY gene and a 294-bp portion of the X-linked androgen receptor gene, both normal for a male.
Fluorescence in situ hybridization analysis of a metaphase chromosome spread from the animal described in Figure 1. A—Fluorescent probes are specific to equid X (green) and Y (red) chromosomes. This image shows a 2n=63 hybrid karyotype and confirms the presence of equid X and Y chromosomes (arrows). Chromosomes are counterstained with DAPI (blue). B—The same metaphase spread with inverted DAPI banding for chromosome identification. Banding patterns confirm that the X is of equine origin and the Y is of donkey origin.
Citation: Journal of the American Veterinary Medical Association 261, 9; 10.2460/javma.23.03.0175
Treatment and Outcome
Surgical closure of the urethra was considered but ultimately not performed because no adverse effects of the defect on the animal’s well-being were apparent and because of the risk of stricture formation. A routine closed castration was performed, and the tissue was submitted for histologic evaluation. Based on the presence of seminiferous tubules with Sertoli cells, spermatogonia, and, infrequently, cells consistent with primary spermatocytes, and based also on the absence of complete spermatogenesis, the gonads were considered consistent with either mule testes or partially retained/cryptorchid stallion testes1 (male gonadal sex; Figure 4). Based on these findings, the animal was diagnosed as a male mule with hypospadias; however, a male mule pseudohermaphrodite could not be ruled out.
Photomicrograph of sections of the gonads removed from the animal described in Figure 1. The presence of seminiferous tubules containing Sertoli cells and spermatogonia confirms that the gonads are testes. A—Seminiferous tubules are devoid of spermatogenesis, as is typical for testicles that are not fully descended into the scrotum. B—In some sections, there were occasional seminiferous tubules containing meiotic germ cell stages, without progression to haploid cells (arrow). This meiotic block is typical of mule testes. H&E stain; bar = 50 μm.
Citation: Journal of the American Veterinary Medical Association 261, 9; 10.2460/javma.23.03.0175
Discussion
Disorders of sexual development (DSDs) are not common in equids, and possibly because of the many variations in phenotype, the literature describing these disorders can be confusing. This case highlights appropriate terminology and shows that a combination of clinical and genetic analyses is the optimal approach for evaluation and diagnosis of DSDs in practice.
A female should have XX sex chromosomes (female genetic sex), 2 functional ovaries (gonadal sex), and female external and internal genitalia (oviducts, uterus, cervix, vagina, vulva and clitoris; phenotypic sex). A male should have XY sex chromosomes (male genetic sex), 2 scrotal testes, and male external and internal genitalia (vas deferens, ampullae, seminal vesicles, prostate, bulbourethral glands, fully formed penis). Any variation in these outcomes is considered a DSD.2
Intersex is an umbrella term used to cover any incongruity among chromosomal, gonadal, and phenotypic sex. If the nature of the gonads can be confirmed, then more specific terms can be employed. A female pseudohermaphrodite is an XX individual with ovaries but ambiguous genitalia, while a male pseudohermaphrodite is an XY individual with testicles (descended or, more often, cryptorchid) but ambiguous genitalia. A true hermaphrodite has both testicular and ovarian tissue intermingled in 1 gonad or both gonads (ie, an ovotestis). A chimera occurs naturally when 2 littermates or twins share genetic material in utero resulting in a genome that reflects, to some degree, the other individual.3 Bovine freemartins are a common example of an intersex condition associated with blood chimerism. Anastomoses of placental vessels allows for in utero exchange of blood between cotwins and creates chimerism in blood and blood-producing tissues.4 Mosaicism occurs during a mitotic nondisjunction event. Many different combinations are possible, all resulting in an animal with cells that originate from more than 1 genetic line. Mosaic animals have been reported with a wide variety of phenotypes.3 Mosaicism and chimerism were deemed unlikely in this animal based on the results of the karyotype analysis.
Hypospadias occurs when the urethra fails to close on the ventral surface of the penis or in the perineal region.2,5 In the past, hypospadias has been considered a mild form of intersexuality, but that is disputed in more current literature. In some cases, intersex anomalies can occur in conjunction with hypospadias, further complicating the terminology.5 Based on our examination, we concluded that the animal in this report would most correctly be considered a miniature male mule with hypospadias. This was because, although ill-formed, a penis with a direct connection to the pelvic urethra was present. Male accessory sex glands and a penile sheath also were present, and the testicles were partially descended into the scrotum. Endocrinologic assays were not performed, but the size and character of the accessory sex glands strongly suggested that the testicles were producing androgens. Additionally, although the penile shaft was present, it was not fused ventrally, and the glans was directly caudal, thus fitting the definition of hypospadias. Although this animal’s hypospadias was severe, there were no inconsistencies among karyotype (XY), gonadal sex (partially descended testes), and genitalia (male internal genitalia and male external genitalia with a congenital genito-urinary malformation), and therefore the animal probably should not be classified as a male pseudohermaphrodite.
Normal sexual development relies on complex signaling pathways involving numerous genes and their protein products. Even in laboratory animal models, the details of these pathways are not yet fully elucidated. In veterinary medicine, assays are available only for the SRY and the androgen receptor genes. Additionally, PCR analysis allows us only to screen for deletions of these genes. Identification of small deletions or point mutations within each gene would require more detailed analysis. These limitations likely explain why the genetic causes of most DSDs in domestic animals are not identified.
Karyotyping, fluorescence in situ hybridization, and chromosome banding can be used to determine chromosome number, genetic sex, and whether an equid is a hinny (jenny dam and stallion sire) or mule (mare dam and jack sire). We used fluorescent markers to confirm a male genetic sex (1 X and 1 Y chromosome) and karyotyping and chromosomal banding to confirm that the animal was a horse-donkey hybrid (2n = 63) and a mule, rather than a hinny (based on banding patterns of the X and Y chromosomes).
Outcome
The animal was discharged routinely 1 day after surgery and experienced no complications. The intent was to find an adoptive home.
Acknowledgments
The authors have nothing to declare.
References
- 1.↑
Neves ES, Chiarini-Garcia H, Franca LR. Comparative testis morphometry and seminiferous epithelium cycle length in donkeys and mules. Biol Reprod. 2002;67(1):247-255. doi:10.1095/biolreprod67.1.247
- 2.↑
Lear TL, McGee RB. Disorders of sexual development in the domestic horse, Equus caballus. Sex Dev. 2012;6(1-3):61-71. doi:10.1159/000334048
- 3.↑
Christensen BW, Meyers-Wallen VN. Sex determination and differentiation. In: McKinnon AO, Squires EL, eds. Equine Reproduction. 2nd ed. Wiley Blackwell; 2011:2211-2221.
- 4.↑
Bleul U, Theiss F, Rutten M, Kahn W. Clinical, cytogenetic and hormonal findings in a stallion with hypospadias—a case report. Vet J. 2007;173(3):679-682. doi:10.1016/j.tvjl.2006.03.005
- 5.↑
Padula AM. The freemartin syndrome: an update. Anim Reprod Sci. 2005;87(1-2):93-109. doi:10.1016/j.anireprosci.2004.09.008
