Clinical Theriogenology 2022; 14: 22 Congenital hypospadias and vertebral malformation in a Barbados Blackbelly neonate Brittany Diehl,a Federico Grosso,b Marley Iredale,c Michael Dark,d Catalina Cabreraa aDepartment of Large Animal Clinical Sciences, bDepartment of Small Animal Clinical Sciences cDepartment of Comparative, Diagnostic and Population Medicine, College of Veterinary Medicine, University of University of Florida, Gainesville, FL, dVeterinary Diagnostic Laboratories, College of Veterinary Medicine and Emerging Pathogens Institute, University of Florida, Gainesville, FL Abstract A male purebred Barbados Blackbelly, twin to another male, was born with obvious external congenital abnormalities. Male twin was normal. Abnormal lamb had a short torso and spinal column. At 1 week, the lamb was presented with dyspnea and lethargy, and euthanasia was elected. Lamb was submitted for full necropsy and for additional diagnostics. After postmortem, in addition to histopathology, radiographs and computed tomographs were obtained. Lamb had congenital vertebral and occipital malfor- mations, meningitis, hypospadias, and renal malformation. To authors’ knowledge, hypospadias and vertebral malformation in the Barbados Blackbelly breed has not been reported. Apparently, Barbados Blackbelly breed harbors closely related genetics, thus vigilant monitoring for genetic malformations is suggested. Keywords: Barbados blackbelly, twin lamb, congenital, hypospadias, vertebral malformation Case presentation A congenitally abnormal, purebred Barbados Blackbelly male lamb was born as a twin to a normal, healthy male lamb. They were born in central Florida to a dam and sire with a history of healthy lamb offspring in previous years; however, this was the first time that they were bred to each other. Owner observed that the lamb had excessively long limbs, a short torso and ver- tebral column, and external genital malformations (Figure 1). Owner described urination to be occurring from the perineal region. Initially, the lamb was unable to stand and was rejected by the ewe. With some assistance, the lamb was able to stand and nurse from a bottle within the first few hours. Lamb was alert, consuming milk replacer from a bottle, and was able to urinate and defecate. At 1 week, the lamb was presented with lethargy and dyspnea. Due to welfare concerns, euthanasia was recommended and elected. Owner submitted the lamb for a full necropsy and further diagnostics. Radiology and computed tomography Immediately after postmortem, orthogonal radiographs of the skull, vertebral column, thorax and abdomen of the lamb were performed with a digital radiography plate (CXDI-50G digital plate, Canon, Lake Success, NY). Additionally, single medio- lateral radiographs of right thoracic and pelvic limbs were ob- tained. Lamb had normal vertebral formula with 7 cervical, 13 thoracic, 7 lumbar, 4 sacral, and 5 coccygeal vertebrae. Occipital condyles were subjectively thickened and elongated. Remain- der of the skull appeared within normal limits. Cervical ver- tebrae were substantially larger than the thoracic and lumbar vertebrae with various sized and shaped vertebral endplates and vertebral bodies. Figure 1. External visualization of the lamb. Note short torso and long appendages. There was severe narrowing of C2 - C3 and C4 - C5 interverte- bral disc spaces with widening of C3 - C4, and especially, C5 - C6 intervertebral disc spaces (Figure 2). Multiple shortened, misshapen and fused thoracic, lumbar, sacral, and coccygeal vertebrae with associated moderate thoracolumbar kyphosis and scoliosis were observed. Thoracic and lumbar vertebrae were markedly widened. Spinous processes of multiple thorac- ic and lumbar vertebrae were fused and misshapen. Severe mid to caudal thoracic rib crowding secondary to the vertebral mal- formations (Figure 3) was observed. Sternum was composed Clinical Theriogenology 2022; 14: 23 of 7 radiographically normal sternebrae with S5 and S6 fusion. Diffuse increases in pulmonary opacity with decreased visual- ization of pulmonary vessels were noted, likely, a postmortem finding attributed to pulmonary atelectasis and pulmonary congestion. Otherwise, the thorax appeared normal. In the ab- domen, a diffuse loss of peritoneal serosal detail was observed, probably associated with lamb’s young age. Gastrointestinal tract contained a mild to moderate amount of gas. Skin mar- gins were irregular in the penile region. No osseous abnormali- ties were observed in right thoracic and pelvic limbs. To further assess radiographic changes, a whole-body post- mortem computed tomography (CT) was performed with a 160-slice (Aquilion Prime) multidetector row, helical CT unit (Toshiba Medical Systems, Tustin, CA). Scan was performed in helical acquisition mode with a scan thickness of 0.5 mm, a pitch of 0.8, 100 mA, 120 kVp, tube rotation time of 0.5 sec- onds, and a 512 x 512 matrix. Radiographic and CT findings were similar: multiple vertebral malformations (Figure 4), abnormally elongated occipital condyles (Figure 5), a normal sternum and a normal appendicular skeleton. Necropsy and histopathology There was ~ an 8 cm, shallow, linear defect in the integument and urethra between rectum and prepuce base (Figure 6) that divided the scrotum with 1 testis on either side of the defect (hypospadias). Testes, pelvic urethra, bladder and ureters were grossly and microscopically unremarkable. Right kidney was ~ twice the size of the left kidney and was irregularly shaped. Histopathology revealed no prominent lesions within either kidney. Following removal of head and dissection of masticatory mus- cles, substantial malformation of the occipital condyles was noticed that caused deformation of foramen deformation and magnum and cerebellum herniation. On dissection of the ep- axial and hypaxial muscles, severe irregularity of lumbar verte- brae shape was observed that caused moderate scoliosis and kyphosis. Figure 2. Postmortem lateral radiograph of skull and cervical vertebral column (A), ventrodorsal radiograph of cervical verte- bral column (B) and sagittal multiplanar CT reconstruction in a bone window of lamb (C). Note severe narrowing of C2 - C3 and C4 -C5 intervertebral disc spaces (arrows) with widening of C3 - C4 and C5 - C6 intervertebral disc spaces (arrowheads). Figure 3. Postmortem lateral radiograph (A), and lateral three-dimensional CT reconstruction (B) of the thoracolumbar vertebral column of the lamb. Note multiple shortened, mis- shapen and fused thoracic and lumbar vertebrae (braces) with mid to caudal thoracic rib crowding (wide arrow) and kyphosis (asterisks) secondary to the vertebral malformations, and fused and misshapen spinous processes of multiple thoracic and lumbar vertebrae (braces). Figure 4. Postmortem ventrodorsal radiograph (A), and dor- sal (B) and ventral (C) three-dimensional CT reconstructions of thoracolumbar vertebral column of the lamb. Note multiple shortened, misshapen and fused vertebrae (braces) with mid to caudal thoracic rib crowding and throacolumbar scoliosis (asterisks) secondary to vertebral malformations, and markedly widened multiple vertebrae (wide arrows). Clinical Theriogenology 2022; 14: 24 Figure 5. Caudal three-dimensional CT reconstructions of lamb’s skull. Note thickened occipital and elongated condyles (arrows). Figure 6. Shallow, linear defect in the integument between rec- tum and prepuce base, consistent with hypospadias Sepsis was evident, characterized by meningitis and valvular endocarditis. Definitive cause of sepsis was not histologically identifiable. Cerebellum herniation and sepsis were likely the cause of lethargy. Genetic analysis Lamb’s hair roots were submitted to the University of Califor- nia-Davis Veterinary Genetics Laboratory for DNA analysis to test for ovine hereditary chondrodysplasia (spider lamb syn- drome). Assay used is an allele specific PCR of the c.1719T>A variant. Results revealed N/N, meaning that the lamb was ho- mozygous for tyrosine in the FGFR3 gene, indicating 2 normal copies. No sequencing was performed and thus flanking se- quence was not evaluated. Discussion Ovine hereditary chondrodysplasia (spider lamb syndrome) is a recessive, semilethal inherited disorder in young lambs re- sulting in skeletal deformities.1 This syndrome is a result of a mutation within the gene for fibroblast growth factor receptor 3 (FGFR3). Frequently, affected lambs have spinal deformities, long bone defects, and underdeveloped musculature.1 Although the findings in this lamb were compatible with multiple con- genital vertebral malformations and caudal occipital malfor- mation, hereditary chondrodysplasia was considered a less likely differential diagnosis in the absence of proximal ulnar, glenoid, sternebrae and facial abnormalities.10 Negative genetic testing suggested that this lamb did not have the well-character- ized single-nucleotide polymorphism that causes spider lamb syndrome; however, this does not rule out other, spontaneous mutations in FGFR3. Hypospadias can be defined as an imperfect closure of the exter- nal urethra in a male9 and is commonly classified into 4 types: palanetic, penile, scrotal, and perineal.8 This lamb demonstrat- ed a case of penile, scrotal, and perineal hypospadias. Hypo- spadias has been described in multiple livestock species, and in humans. It is among the most common congenital malfor- mation in human male neonates, whereas these defects appar- ently occur as a result of a multifactorial disorder that has been associated with genetic and environmental factors.6 However, broadly this process of morphogenesis appears to be incredibly complex and is largely not understood. In a survey of the occur- rence of congenital defects in sheep, the urogenital system was involved in 22.9% of the cases, and the most common male urinary defects were hypospadias and renal agenesis.3 Reported concurrent developmental abnormalities in other an- imals with hypospadias include other urogenital abnormalities, such as penile aplasia, an incomplete ventral sheath, cryptor- chidism, and atresia ani.5,7 None of these concurrent genitoanal abnormalities were present in this lamb. However, this lamb did present with marked, lumbar vertebral and occipital mal- formation, and moderate cerebellar herniation. Limbs and the external genitalia may originate from nearby cell lines during embryogenesis.4 Together with this information, these findings suggest that these congenital malformations may have occurred early in the development (differentiation of the embryo meso- derm). Although hypospadias and associated congenital abnor- malities etiology remain unknown, lack of abnormalities in the twin suggests that an infectious or environmental process had no effect; however, it cannot be ruled out. Therefore, it is more likely that this abnormality was initiated within the develop- mental process of that individual embryo, although it remains unclear if it is product of inheritable genetic mutations. Clinical Theriogenology 2022; 14: 25 Barbados Blackbelly sheep breed originated in Barbados, east- ernmost island of Caribbean chain and West Africa. They are known for their ability to withstand harsh tropical climates and heavy parasite burdens.2 Currently, to our present knowl- edge, there are no specific perpetuating congenital abnormal- ities harboring within the purebred of Barbados Blackbelly lineage. However, the closeness of genetics within the breed makes a case for close monitoring of congenital defects that may be the result of recessive congenital genes. Likewise, due to increasing implications of viruses, plants, and medicinal or chemical products, it is important to continue to be vigilant in our efforts to identify environmental teratogens that may be responsible for congenital defects.3 Producers of sire and dam of this lamb, were informed, to follow up for potential future congenital defects associated with the lines of sire and dam to avoid perpetuating a flaw within the breed. This was the first occurrence in either of their flocks. To our knowledge, a case of hypospadias and vertebral malformation in the Barbados Blackbelly breed has not been reported. Learning points • Hypospadias and concurrent congenital malformations can occur in several mammalian species, often without a defin- itive explanation. • Occurrence of congenital malformations within specific breeds is important to monitor, specifically in those with closely related genetics, • Complications in embryogenesis should be considered when malformations of the external genitalia and append- ages are present, Conflict of Interest Authors disclose no conflicts of interest. Acknowledgement Authors thank the producer for willingness to facilitate further evaluation of the lamb. References 1. Beever JE, Smit MA, Meyers SN, et al: A single-base change in the tyrosine kinase II domain of ovine FGFR3 causes hereditary chondrodysplasia in sheep. Anim Genet 2006;37:66-71. 2. de Almeida AM: Barbados Blackbelly: the Caribbean ovine genetic resource. Trop Amin Health Prod 2018;50:239-250. 3. Dennis SM: Congenital defects of sheep. Vet Clin North Am Food Anim Pract 1993;9:203 217. 4. Herrera AM, Cohn, MJ: Embryonic origin and compartmental organization of the external genitalia. Sci Ref 2014;4:6896. doi: 10.1038/srep06896. 5. Jeong W, Seong Y, Kim I, et al: Radiographic diagnosis of hypospadias in three Korean native calves. J Vet Clin 2008;25:557-559. 6. Joodi M, Amerizadeh F, Hassanian SM, et al: The genetic factors contributing to hypospadias and their clinical utility in its diagnosis. J Cell Physiol 2019;234:5519-5523. 7. Kumi-Diaka J, Osori DI: Perineal hypospadias in two related bull calves, a case report. Theriogenology 1979;11:63-164. 8. Ladds PW: Congenital abnormalities of the genetalia of cattle, sheep, goats, and pigs. Vet Clin North Am Food Anim Pract 1993;9:127-144. 9. Radostitis, OM, Gay CC, Hinchcliff KW, et al: Veterinary Medicine. 10th edition, London; Saunders Co: 2007. p. 573. 10. Vanek JA, Walter PA, Alstad AD: Radiographic diagnosis of hereditary chondrodysplasia in newborn lambs. J Am Vet Med Assoc 1989;194:244-248.