History
A 3-year-old Holstein Friesian cow from the Rayner Dairy Research and Teaching Facility, University of Saskatchewan, was bred successfully by artificial insemination at 135 days in milk (DIM) during the first lactation. Pregnancy was diagnosed at 168 DIM and confirmed at 196 and 238 DIM via transrectal ultrasonography. At 278 days of gestation, the cow delivered a healthy heifer calf that weighed 40 kg followed by the expulsion of an abnormal mass. The fetal membranes were expelled 2 hours after delivery of the calf and mass.
Clinically, at 1 day of age, the heifer calf had a prominent tuft of hair below the vulva and a small and slit-like vulvar opening, which did not allow passage of a lubricated 5-mm-thick round-ended plastic probe into the vestibule. The heifer had a 5.8-cm anogenital distance (from the center of the anus to the base of the clitoris), which was smaller than the mean anogenital distance (7.0 cm; D Ambrose, PhD, University of Alberta, unpublished data, 2019) for a typical 1-day-old heifer. The abnormal mass weighed 0.5 kg, had a length of 20 cm and width of 15 cm, and had a mottled pink to red smooth outer surface (Figure 1). The abnormal mass was submitted to the Prairie Diagnostic Services, Saskatoon, for gross and histologic evaluations. The cow underwent a routine reproductive examination at 7 DIM of the second lactation. Two regressing corpora lutea from the previous pregnancy, 1 on each ovary, were confirmed by transrectal ultrasonography.
Photographs of the perineal region of a live 1-day-old heifer calf (A) that was spontaneously delivered followed immediately by the expulsion of an abnormal mass (B) by a 3-year-old Holstein Friesian cow. A—The heifer calf was delivered at full term and weighed 40 kg. B—The mass weighed 0.5 kg, had a length of 20 cm and width of 15 cm, and had a mottled pink to red smooth outer surface.
Citation: Journal of the American Veterinary Medical Association 260, 2; 10.2460/javma.21.06.0285
An initial histologic report confirmed the presence of testicular tissue in the abnormal mass. Initially, the owner intended to keep the heifer calf for feedlot purposes, which allowed follow-up observations on the calf. When the heifer was 13 days old, a blood sample (approx 18 mL) was obtained by jugular venipuncture into three blood collection tubes (6ml) containing sodium heparin as an anticoagulant. The blood sample was submitted for karyotyping to the University of Guelph. At the time of disbudding at 21 days old, the calf was reevaluated, and we were again unable to introduce the 5-mm-thick round-ended plastic probe through the vulvar opening to assess the length of the vagina. With the owner’s consent, the calf was euthanized at 33 days old (68 kg) with a penetrating captive bolt in accordance with the guidelines of the Canadian Council on Animal Care and submitted to Prairie Diagnostic Services for a necropsy and detailed examination of the reproductive tract and associated structures.
Question
What is the most likely diagnosis or description for the abnormal mass and heifer calf?
Answer
Amorphus globosus and a freemartin heifer calf.
Results
The round mass was identified as an amorphus globosus because no organ system was discernible. Inside the mass, there were multiple empty chambers with gross features resembling the ruminant stomach. Other identified structures included a round structure that resembled a lymph node on cut section, a fibrous tissue wall with hair on the inner surface, and a third smooth fluid-filled nodule suggestive of a primitive eye. Additionally, there were focal areas indicative of skeletal muscle, fat, and bony tissues.
A key histologic finding in the amorphous globosus was the presence of testicular tissue with a characteristic fibrous capsule surrounding a mass of tortuous seminiferous tubules (Supplementary Figure S1). The tubules were lined by cuboidal to columnar epithelial cells with round dark nuclei (Sertoli cells) and surrounded by polygonal Leydig cells. Other histologic sections revealed components of the integumentary system (skin, hair follicles, and sebaceous glands), musculoskeletal system (long bone, cartilage, and skeletal and smooth muscles), gastrointestinal system (primitive esophagus, rumen, reticulum, omasum, abomasum, and intestines), and structures of the lymphoid and nervous systems as well as adipose tissue.
Morphological examination of the heifer calf during necropsy confirmed abnormalities of external genitalia. Detailed evaluation of the reproductive tract revealed a ventrally placed slit-like vulvar opening, which progressed to an 8.5-cm-long vestibule with a narrow lumen (2 to 3 mm in diameter) that ended in a transverse septum cranial to the urethral opening. Also, paired white elongated structures (length, 3 mm; width, 2 mm) suggestive of poorly developed seminal vesicular glands were observed approximately 2 cm cranial to the transverse septum between the vestibule and vagina (Figure 2). The vagina was approximately 11 cm in length and 1.5 cm in diameter and contained slightly cloudy mucoid material that could not be expressed into the vestibule owing to the imperforate transverse septum. The cervix was grossly normal. The paired paramesonephric ducts (uterine horns) did not fuse to form a single uterine body but rather separately joined the single cervical lumen. There were no other pathological abnormalities found during gross necropsy.
Photographs of the reproductive tract of a 33-day-old (68-kg) freemartin heifer calf that was delivered with a male amorphus globosus before (A) and after (B) fixation in formalin. A—The vagina (a) was 11 cm long and 1.5 cm in diameter and contained slightly cloudy mucoid material. Also visible in this photograph are paired structures believed to be rudimentary seminal vesicular glands (b) adjacent to the vagina 2 cm cranial to a transverse septum between the vagina and vestibule (c) and rectum (d). B—In the formalin-fixed specimen, a gonad (e), uterine tubes (f), uterine horns (g), and cervix (h) are identified. Notice that the paired uterine horns did not fuse to form a single uterine body but rather separately joined the single cervix lumen.
Citation: Journal of the American Veterinary Medical Association 260, 2; 10.2460/javma.21.06.0285
Histologic assessment of the reproductive tract of the heifer calf revealed paired hypoplastic seminal vesicular glands adjacent to the junction of vestibule and vagina. Both gonads of the heifer calf were histologically consistent with ovaries characterized by a cortex composed of small numbers of primordial follicles surrounded by a stroma composed of spindled cells and thin strands of collagen fibers. The ovarian medulla was composed of hilus cells and large numbers of blood vessels suggestive of rete ovarii. Sections from the remainder of tissues from the heifer calf revealed no other significant histologic lesions.
Blood leukocytes obtained from the heifer calf were cultured and stained with Giemsa stain for karyotyping. One hundred five mitotic cells were studied, and all examined cells contained 60 XX chromosomes (Figure 3). Additionally, DNA extracted from testicular tissue (1 section) of the amorphus globosus and ovarian tissues (2 sections) of the heifer calf underwent a conventional PCR assay designed to target Y-chromosome–specific gene sequences at the Molecular Microbiology Laboratory, Western College of Veterinary Medicine. The PCR assay successfully amplified the 210-base pair (bp) sequence for the Y-chromosome gene from DNA extracted from sections of the amorphus globosus testicular and heifer ovarian tissues.
Photomicrograph of a metaphase plate of cultured leukocytes from the heifer calf of Figure 1 used for karyotype determination (A) and photograph of results for a Y-chromosome–specific gene PCR assay performed on tissues obtained from the heifer calf and amorphus globosus of Figure 1 (B). A—The photomicrograph depicts 2 submetacentric × chromosomes (arrows), which suggested that the calf had a female karyotype. Giemsa stain. B—Results are provided for a nontemplate control (lane NTC), 100-base pair DNA ladder (lane DNA L), DNA extracted from testicular tissue obtained from the amorphus globosus (lane 1), DNA extracted from 2 different sections of ovarian tissue obtained from the freemartin heifer calf (lanes 2 and 3), DNA obtained from a bull (positive control; lane B), and DNA obtained from a nonpregnant cow (negative control; lane C). Lanes 1, 2, 3, and B are all positive for the 210-base pair sequence specific for the Y-chromosome gene; a similar band was not observed in lane NTC or C. These results definitively identified the amorphus globosus as male and the heifer as a freemartin.
Citation: Journal of the American Veterinary Medical Association 260, 2; 10.2460/javma.21.06.0285
Discussion
The term amorphus globosus describes the presence of undifferentiated organ systems within a spherical mass encapsulated by skin.1 The outer covering (skin) of an amorphus globosus has a varying degree of hair and can range from completely covered with hair to an apparently hairless membrane.1,2 The absence of a heart is a pathognomonic feature of an amorphus globosus, and such an object is often called acardius amorphus. The first amorphus globosus described in cattle was reported in Germany almost 188 years ago,3 and since then amorphus globosus has been reported in other species. It is projected that approximately 1 in 3,500 pregnancies in Holstein cows results in amorphus globosus.4
The present report described a rare occurrence of a dairy cow giving birth to a live freemartin heifer calf along with an amorphus globosus containing male reproductive tissues. Traditionally, fetal twins are classified as monozygotic when the inner cell mass of a single zygote splits into 2 and dizygotic when 2 oocytes are simultaneously fertilized by different spermatozoa. For the cow of this report, a regressing corpus luteum was identified on each ovary during the first postpartum reproductive tract examination at 7 DIM. This was suggestive of dizygotic twins, as was the sex discrepancy between the live heifer calf and male amorphus globosus. The development of an amorphus globosus is commonly described by the twin-reversed arterial perfusion theory, whereby monozygotic twins have extensive placental vascular anastomosis through monochorionic placentation. Consequently, one fetus (ie, the pump twin) pumps hypoxic blood to the other fetus (ie, the acardiac twin) first, then to the mother, resulting in dysmorphic growth of the acardiac twin. Placental vascular anastomosis can potentially lead to monochorionic placentation in the case of dizygotic twins and has been reported in humans.5 The entire fetal membranes were not available for detailed examination of placental vascular anastomoses for the present case.
Monozygotic or dizygotic twin pregnancies resulting in the birth of either a heifer or bull calf along with a female or male amorphus globosus have been reported in cattle.1,2 However, to our knowledge, the present report was the first to describe a live heifer calf born co-twin to a nonviable male amorphus globosus. Freemartinism is a distinctive form of intersexuality that affects approximately 90% of dizygotic pregnancies in cattle. Freemartinism results from the crossover of male gonad determinants, such as anti-Müllerian hormone and androgens, from the male fetus to the female fetus through placental vasculature anastomoses. The vascular anastomoses occur by 30 days of gestation, well before initiation of male sexual differentiation (ie, 38 to 42 days of gestation). Because sexual differentiation of a female fetus is initiated at approximately 48 days of gestation, the transfer of XY cells and hormones and other factors from the male fetus alter differentiation of the Müllerian ducts and external genitalia to varying degrees in the female fetus.6 In freemartin heifers, common findings associated with masculinization include an abnormally small vulvar opening, a prominent tuft of hair below the ventral vulvar commissure, and the presence of small vesicular glands,6 all of which were observed in the heifer calf of the present report. Male gonad determinants from the testicular tissue of the amorphus globosus were likely responsible for the development of freemartinism for the heifer calf of the present report.
Karyotyping of cultured peripheral blood leukocytes of neonatal calves is 95% to 99% accurate when hundreds of mitotic cells are assessed.7 Each calf within a pair of newly born heterosexual twins has a similar distribution of XX and XY leukocytes in the peripheral blood, but the ratio of XX and XY cells in the peripheral blood may vary between different sets of newly born heterosexual twins.8 Karyotyping of peripheral blood cells from the heifer calf of the present report revealed only XX cells, which suggested limited crossover of XY cells from the male amorphus globosus, resulting in a misdiagnosis. Karyotyping of cultured fibroblasts from an amorphus globosus has been used to detect chimerism9 but was not pursued in this case. Given the expected similarity in the ratio of XX and XY cell types between heterosexual twins,8 it is likely that karyotyping of tissue from the amorphus globosus of the present report would have revealed only XY cells owing to the limited transfer of cells between the twins. For suspected freemartin heifers, molecular methods such as conventional PCR, short tandem repeats, real-time quantitative PCR, and digital PCR assays and loop-mediated isothermal amplification might be more sensitive for diagnosis of the condition than cytogenetic analysis. McNiel et al10 reported that PCR assay and karyotyping had an accuracy of 97% and 77%, respectively, for diagnosis of freemartinism.
For the freemartin heifer of the present report, we confirmed the presence of XY cells in sections of ovarian tissue by use of a conventional PCR assay that targeted a 210-bp Y-chromosome–specific gene sequence. A relatively lower proportion of XY cells in the ovarian tissues processed for DNA extraction would explain the lighter-intensity bands for the 210-bp Y-chromosome–specific amplicons. The presence of XY cells in the ovarian tissue supported our theory that there was an exchange of cellular and possibly hormonal materials between the potentially dizygotic heterosexual twins because of placental vascular fusion, which resulted in masculinization of the heifer calf. The conventional Y-chromosome–specific PCR assay was used for a subsequent case from a different herd to demonstrate a nonfreemartin Holstein heifer calf born co-twin to a male amorphus globosus with no identifiable testicular tissue.
The present report highlighted the importance of careful evaluation of any expelled mass and fetal membranes along with the sexual phenotype of a neonatal calf to identify potential abnormalities. Further, it may be prudent to use available molecular methods for early confirmation of freemartinism in suspect calves.
Outcome
The cow of this report was artificially inseminated at 73 DIM during the second lactation; was confirmed pregnant at 105, 133, and 175 DIM; and delivered a viable, clinically normal bull calf on February 8, 2021.
Supplementary Materials
Supplementary materials are posted online at the journal website: avmajournals.avma.org
Acknowledgments
No third-party funding or support was received in connection with this study or the writing or publication of the manuscript.
References
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Baghbadorani MK, Oghadam MF. Histological investigation and karyotyping of amorphous globosus cases occurred in a dairy herd throughout one year observation. Reprod Domest Anim. 2017;52(suppl 3):100. 21st Annual Conference of the European Society for Domestic Animal Reproduction (ESDAR) abstract P 105.
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McNiel EA, Madrill NJ, Treeful AE, Buoen LC, Weber AF. Comparison of cytogenetics and polymerase chain reaction based detection of the amelogenin gene polymorphism for the diagnosis of freemartinism in cattle. J Vet Diagn Invest. 2006;18(5):469–472.
