Differences among studies on sex identification of hatchling turtles

In their recent report, Hernandez-Divers et al¹ described an endoscopic method for visualizing the gonads and identifying the sex of hatchling Chinese box turtles (Cuora flavomarginata; family Geoemydidae) and provided endoscopic pictures of reproductive tracts. Because turtle conservation programs may use these images as a guide during endoscopic sexing, it is imperative that published endoscopic images of juvenile turtle testes and ovaries be correctly identified and labeled.

Illustrations of the ovaries and oviducts in C flavomarginata in this report correspond to those described for other turtles,^2–4 and the correctness of their identification is not questioned. However, illustrations of the “testis” in C flavomarginata show radically different structures from those previously described and illustrated as juvenile turtle testes.^2–4 The close-up of the structure labeled “testis” (Figure 2b) shows a robust, whitish external membrane with thick blood vessels and spots of melanocytes, with no tubular structures visible through it. In contrast, although the endoscopic appearance of hatchling, juvenile, and mature testes varies among turtle families, species, and age groups, the theca testis is always thin and translucent and never contains melanocytes, and tubular structures of various sizes and colors (transparent, white, pinkish, or yellow) or a fine net of surface vasculature is visible in close-up images of the testis in juvenile Astrochelys yniphora⁵ (Testudinidae), Erymnochelys madagascariensis² (Podocnemididae), Caretta caretta³ (Cheloniidae), and Amyda cartilaginea⁴ (Trionychidae) as well as in Batagur affinis and Malayemys macrocephala (Geoemydidae; unpublished data). In hatchling and small juvenile turtles, the testes can be transparent structures, are usually smaller than the ovaries and sometimes smaller than the adjacent adrenal glands, and can easily be overlooked. The vas deferens in small juvenile turtles also is thin and translucent and very different from the robust structure illustrated as the vas deferens by Hernandez-Divers et al (Figure 2c).

I believe that the structure labeled “testis” in illustrations from Hernandez-Divers et al does not represent a testis or any other part of the genital tract. Because the appearance of the male genital tract and testis illustrated in Hernandez-Divers et al is radically different from that of other published studies,^2–5 extreme caution is warranted when considering the use of this publication as guidance to assess sex ratios in conservation programs of endangered turtles.

Gerald Kuchling, PhD

School of Animal Biology, The University of Western Australia, Nedlands, WA, Australia

The authors respond:

We are grateful to Dr. Kuchling for taking the time to write concerning our recent report¹ entitled “An endoscopic method for identifying sex of hatchling Chinese box turtles and comparison of general versus local anesthesia for coelioscopy.” The purpose of the paper was not to describe the general endoscopic appearance of the chelonian gonads for widespread application, as he suggests, but rather to use the Chinese box turtle as a model for demonstrating the value of endoscopy for identifying sex and the need for adequate anesthesia for such procedures. The paper title, abstract, and text clearly restricted our visual interpretations to Cuora flavomarginata alone. The gonads of various chelonian species can vary dramatically in size, shape, and color, and their precise nature must therefore be determined for each species. Nevertheless, our research demonstrates the value of endoscopy for providing unparalleled visualization of the gonads for sex identification purposes.

Dr. Kuchling is correct that the appearance of the testis in C flavomarginata is different from the appearance of the testis in some other species. However, the testicular nature of the tissue was confirmed histologically in several cadaver animals during a pilot phase before the described study was undertaken. We believe that this histologic confirmation of the testis was sufficient to refute the claims of Dr. Kuchling, who by his own admission never actually examined the gonads of Chinese box turtles. In addition, we have spoken to other veterinarians with extensive endoscopy experience in chelonians, and Dr. Sam Rivera of Zoo Atlanta, Dr. Charles Innis of the New England Aquarium, and Dr. Eric Baitchman of Zoo New England have independently confirmed that our identification of the testis is accurate. The interspecies variation that is confusing to Dr. Kuchling testifies to the dangers of extrapolating from one species to the next, particularly when there is no direct experience of the specific species in question.

We suspect that another reason why the gonads and their associated structures look so different to Dr. Kuchling is in part related to the superior quality of the images we obtained. Previous researchers have often failed to abide by established principles of veterinary anesthesia and endoscopy, specifically the need for a dedicated endovideo camera, appropriate insufflation, and adequate general anesthesia.^2–6 We would encourage readers to review and compare the image quality in the cited papers^1–3 to determine which appear clearer and therefore more reliable for the purposes of sex identification.

It is important for all endoscopists to receive formal instruction in appropriate equipment and methods, and the inclusion of a veterinarian on a research team can prove invaluable.^4–6 In addition, endoscopy training courses are regularly offered to veterinarians (and potentially licensed researchers) at several conferences (eg, annual meeting of the Association of Reptile and Amphibian Veterinarians) and universities (eg, the University of Georgia) every year.

We hope these remarks have clarified our position and will encourage others involved in chelonian conservation to realize the true value of endoscopy. In addition, we hope that the real benefits of improved anesthesia and surgery will encourage the inclusion of veterinarians in a multidisciplinary approach to chelonian conservation.

Stephen J. Divers, BVetMed, DZooMed, DACZM

Department of Small Animal Medicine and Surgery, College of Veterinary Medicine, University of Georgia, Athens, Ga

Scott J. Stahl, DVM, DABVP

Stahl Exotic Animal Veterinary Services, Vienna, Va

More on animal models for human disease and treatment

We enjoyed the August 15, 2009, JAVMA Letter to the Editor “Thoughts on animal models for human disease and treatment.”¹ The use of the term “animal model” creates a broad array of connotations, including those related to their ability or inability to predict human disease, as discussed by Drs. Greek and Shank. This complexity can be simplified by classifying animal models on the basis of whether they represent experimentally induced (such as by chemical exposure, implantation of cells, or manipulation of genetics) versus naturally occurring, spontaneous conditions. Experimental models allow parameters to be manipulated in ways that are not possible with spontaneous disease. Conversely, spontaneous models can allow for testing hypotheses (such as the natural history of a disease) that could not be investigated in an experimental model. Anatomic, physiologic, biological, and molecular similarities exist between naturally occurring diseases of humans and companion animals.^2–6 Diseases in these species provide spontaneous models that more closely mimic the human counterpart than most currently used experimental models. In the field of cancer, classic examples include osteosarcoma and lymphoma in dogs.

However, conceptual barriers to the understanding of this class of animal models exist. For example, terms currently used to describe animal models may be perceived differently by different groups. The term “small animal” may imply a mouse to a physician or basic scientist, whereas to a veterinarian, this term implies a dog or cat. Many researchers are unfamiliar with currently existing spontaneous animal models in their research area. Furthermore, the concept of treating companion animals within a clinical trial setting is often so foreign that it constitutes a substantial conceptual gap. This lack of clarity may in part be due to the absence of a descriptive term separate from animal model that accurately reflects the concept.

We propose that a new term be used: SPAREDEL, which stands for Spontaneous PArallel REciprocal moDEL. Cancer and other diseases occur spontaneously in companion animals, as in humans. Diseases in pets parallel the pathophysiology as well as the course, treatment, and outcome in humans, although often on a shorter timescale. Because pets and people share common living environments and lifestyle choices, predisposing factors for diseases (eg, exposure to environmental toxins such as tobacco smoke, overeating, and underexercising) coexist. A reciprocal benefit exists for exchange of relevant findings between the human and veterinary medical professions. Knowledge obtained in one species can be applied to the others and used to emphasize relevant similarities and differences. When diseases occur spontaneously in pets, a unique opportunity is presented for insight into the comparable human disease.

This year marks the 125th anniversary of the University of Pennsylvania School of Veterinary Medicine, for which the motto “Many Species, One Medicine” was coined. Comparison of spontaneous diseases between species can elucidate new questions and foster novel areas of investigation. There is power in a word and in accurately describing a concept. We hope that the term SPAREDEL will serve to facilitate communication and collaboration toward a new frontier in translational research.

Lili Duda, VMD, MBE, DACVR

John R. Lewis, VMD, DAVDC

Sarah H. Kagan, PhD, RN

Sydney M. Evans, VMD, MS

Comparative Oncology Working Group, University of Pennsylvania, Philadelphia, Pa

Questions common vaccination practices in beef calves

I am writing in regard to the study¹ by Dr. Step et al in the September 1, 2009, issue of the JAVMA. Although it did answer the question concerning the efficacy of single versus double vaccination with a modified-live virus (MLV) vaccine, I find no evidence to support the widespread assumption that vaccinating beef calves at the time of purchase with MLV vaccines is effective in preventing bovine respiratory disease. In this study, the two preconditioning vaccination protocol groups had morbidity rates of 36.3% and 44.5%. Most importantly, there were no unvaccinated controls, so the efficacy of the vaccine is open to question. It certainly does not appear to be very good. Had controls been included in the design, their spontaneous changes in titers would have been interesting, especially in comparison to the responses of the vaccinates.

The calves were vaccinated between 16 and 48 hours after arrival, and mean times to first treatment were 7.62 and 7.21 days. Even though the distribution of days to first treatment was not given, this common scenario always puts the vaccine response behind the incubation curve.

The conclusions of phase two of the study would have been more convincing if the performance of properly preconditioned cattle that had not received MLV vaccines and that were also maintained in individual groups during the finishing phase (perhaps with and without vaccination at that time) had been reported. Their response to exposure to cattle persistently infected with bovine viral diarrhea virus would also have been a valuable addition to the work.

I realize the study put more stress on these cattle than normal, as they were put through the chute twice on arrival and incoming calves may have been added to established pens during the arrival period (this was not made clear). In addition, morbidity rates for steers, compared with those for recently castrated bulls, were not included. However, no matter how I look at it, I have to wonder if the current recommendations for vaccination of incoming calves with MLV vaccines are supported by any solid evidence of efficacy. I have read many comparable reports with similar results over the years, so my concerns are not new. I have great respect for the authors, and I may well be missing something, but for now, I remain skeptical about the basis for some of their assumptions.

Walter Hylton, VMD

Staunton, Va