Acta Herpetologica 14(2): 123-128, 2019 ISSN 1827-9635 (print) © Firenze University Press ISSN 1827-9643 (online) www.fupress.com/ah DOI: 10.13128/a_h-7750 Hematological parameters of the Bolson tortoise Gopherus flavomarginatus in Mexico Cristina García-De la Peña1,*, Roger Iván Rodríguez-Vivas2, Jorge A. Zegbe-Domínguez3, Luis Manuel Valenzuela-Núñez1, César A. Meza Herrera4, Quetzaly Siller-Rodríguez1, Verónica Ávila-Rodríguez1 1 Facultad de Ciencias Biológicas, Universidad Juárez del Estado de Durango, Gómez Palacio, Durango, México. C.P. 35010. *Corre- sponding author. Email: cristina.g.delapena@gmail.com 2 Facultad de Medicina Veterinaria y Zootecnia, Universidad Autónoma de Yucatán, Mérida, Yucatán, México, C.P. 97100 3 Instituto Nacional de Investigaciones Forestales, Agrícolas y Pecuarias, Campo Experimental Calera, Zacatecas, México, C.P. 98500 4 Universidad Autónoma Chapingo-URUZA, Bermejillo, Durango, México, C.P. 35230 Submitted on: 2018, 21th June; Revised on: 2019, 10th May; Accepted on: 2019, 23rd August Editor: Emilio Sperone Abstract. We present findings of our preliminary study to determine biometry and blood chemistry values of healthy wild individuals of the critically endangered Bolson tortoises (Gopherus flavomarginatus) in Mexico. Given the absence of previously published data regarding hematology parameters for this species, these results represent an important base for additional research. Hematocrit determination, stains, and cell counts were performed, as well as 18 parameters of blood chemistry. Values of biometry and blood chemistry for G. flavomarginatus were similar to ref- erence values those already reported for G. agassizii, G. polyphemus, and G. berlandieri. These similarities reflect the phylogenetic relationships among these species. However, slight differences may point to particular adaptations that each has developed to their own habitat, and so point to questions to be addressed with future research. Keywords. Chelonia, lymphocyte, desert, Mapimí Biosphere Reserve. Hematological analysis is a common technique for evaluating the health status of wild animals (Campbell, 2004; Tavares-Dias et al., 2009). Blood biometry and chemistry of terrestrial, semiaquatic and marine tortoises have been described already (Stacy et al. 2011; Camp- bell, 2015). However, there are still species of ecologi- cal importance whose blood information is unavailable. This is the case of Gopherus flavomarginatus, a tortoise endemic to Mexico and considered the largest terrestrial tortoise in North America, with a carapace length of up to 40 cm (Morafka et al., 1989). This species is in dan- ger of extinction according to the Official Mexican Stand- ard 059 (SEMARNAT, 2010) and critically endangered according to the IUCN red list (Kiester et al., 2018). Its geographical distribution is restricted to the Mapimí Bolson in the Mexican Chihuahuan Desert, where it currently has protected status within the Mapimí Bio- sphere Reserve (CONANP, 2006). Tortoises of the genus Gopherus are keystone organisms for the ecosystems in which they live because, due to their feeding habits (her- bivory), they perform the ecological function of seed dispersal (Carlson et al., 2003), and because the burrows that they excavate are deep and provide shelter for at least 300 species of invertebrates and 60 of vertebrates (Lips, 1991). Due to the importance of protecting this tortoise, we conducted this preliminary study to determine biom- etry and blood chemistry values of healthy wild indi- viduals. This information will serve as a basis for future hematological studies carried out on this tortoise. From May 2015 to September 2017, we captured 44 adult individuals of G. flavomarginatus (16 males and 28 females) within the Mapimí Biosphere Reserve in Mex- 124 Cristina García-De la Peña et alii ico (26°00’ and 26°10’N, and 104°10’ and 103°20’W). Blood samples were collected from the subvertebral vein. Three milliliters of blood were collected from each specimen; one milliliter was placed in a Vacutainer® tube with lithium heparin as anticoagulant and the rest in a red Vacutainer® tube. The tubes were stored in a cooler at a temperature of approximately 4 °C. Each tortoise was determined to be healthy following the observa- tion protocols of Jacobson (2014) and USFWS (2016). Tortoises were then released at the site of their capture. Biometric analysis of blood was performed following the protocols suggested by Thrall et al. (2006) and Turgeon (2012). The volume percentage of red cells (Hematocrit, Ht in%) was determined in the blood contained in the green Vacutainer® tube, using the microhematocrit tech- nique. The total number (TR) and percentage (PR) of red cells were obtained for each blood sample. The formula used to obtain hematocrit values was Ht% = (PR/TR) × 100. Hemoglobin concentration was quantified with the Drabkin colorimetric method, using the Spinreact® com- mercial kit, in a VetTest® spectrophotometer. The reaction product was centrifuged (12,000 g × 5 min) to precipitate the nucleus of the cells and keep them from distorting the color to be measured (Thrall et al., 2006). Erythrocyte and leukocyte counts were carried out using a Thoma pipette with red bead and a Neubauer hemocytometer with Natt and Herrick’s stain (Thrall et al., 2006). The erythrocyte count was carried out under 10× magnification, on both sides of the Neubauer chamber, in the four corner squares and the center square within the large center square of the chamber. The following formula was applied (Kemal, 2014): erythrocytes (×106 ul) = mean erythrocytes × 10000. The leukocyte count was carried out under 40x magnification in the nine large squares on both sides of the Neubauer chamber. The following for- mula was applied: leukocytes (×103 ul) = mean leukocytes × 50 (Kemal, 2014). The erythrocyte indices were calcu- lated according to the formulas described by Ball (2014) and Kemal (2014): mean corpuscular volume, MCV (fL) = Ht% × 10/erythrocytes (×106 ul); mean corpuscular hemoglobin, MCH (pg) = (Hg × 10)/erythrocytes (×106 ul); mean corpuscular hemoglobin concentration, MCHC (g/dl) = (Hg × 100)/Ht%. Two smears were prepared from each sample on glass slides using Wright’s dye (Analyty- ka®). One hundred leukocytes were counted in the body of the smear of each of the two slides. The average of both slides was obtained, and the results were expressed as the proportion of each cell type (relative differential count). To obtain the absolute differential count, the total value of leukocytes (×103 cel/ul) was multiplied by the average percentage of each type of leukocyte and the result was divided by 100 (Thrall et al., 2006). The H:L ratio was obtained by dividing the per- centage of heterophiles by the percentage of lympho- cytes in each sample (Davis et al., 2008). Blood from the red Vacutainer® tube was centrifuged at 12,000 g for five minutes; the serum was separated from the red cells and placed in a sterile plastic tube. No hemoly- sis was observed in any sample. The following param- eters were analyzed: glucose, uric acid, urea, blood urea nitrogen (BUN), creatinine, total proteins, albumin, globulins, cholesterol, triglycerides, alanine aminotrans- ferase (ALT), aspartate aminotransferase (AST), alkaline phosphatase (AP), chlorine, sodium, calcium, phospho- rus and osmolality (mOsm/kg=1.86 (Na [mmol/L]) + glucose [mg/dL]/18 + BUN [mg/dL]/2.8 + 9). Human serum Spintrol H Spinreact® was used as control. These analyzes were performed in a VetTest® spectrophotome- ter with Spinreact® reagents. Descriptive statistics (mean, standard error, median, standard deviation, minimum, and maximum) were obtained in PAST 3.14 (Hammer et al., 2001). Blood biometry results for G. flavomarginatus are shown in Table 1 and for blood chemistry in Table 2. A comparison among biometry and chemistry variables of G. flavomarginatus and other Gopherus species are shown in Tables 3 and 4, respectively. Blood biometric and chemistry values obtained for G. flavomarginatus were similar to those reported for G. agassizii (Christopher et al., 1999; Dickinson et al., 2000), G. polyphemus (Taylor and Jacobson, 1982), and G. berlandieri (Teare, 2013a) (Tables 3 and 4). Christopher et al. (1999) reported that the most abundant leukocytes in G. agassizii are het- erophils, followed by lymphocytes, basophils, and finally eosinophils and monocytes. In our findings for G. fla- vomarginatus, the order of abundance of the leukocyte cells was similar to the one mentioned by Duguy (1970), with lymphocytes as the most abundant leukocytes (males = 53.43%, 4.79 × 103 cel/ul; females = 44.03%, 4.20 × 103 cel/ul). This agrees with the findings of Diaz- Figueroa (2005) for G. polyphemus, and other authors who indicate that lymphocytes are predominant in the peripheral blood of most reptile species (Davis et al.2008; Stacy et al., 2011). Heterophils were the second most abundant leu- kocyte cells in G. flavomarginatus. In general, the abun- dance of this type of leukocyte in reptiles ranges from 30 to 45% (Duguy, 1970; Frye, 1991); however, in tortoises it can reach up to 50% (Alleman et al., 1992; Christo- pher et al. 1999). Basophils were the third most abundant leukocytes in the blood of the Bolson tortoise. Alleman et al., (1992), and Duguy, (1970) estimated that the per- centage of basophils in healthy terrestrial and aquatic tortoises can be higher than 40%. In G. flavomargi- 125Hematological parameters of Gopherus flavomarginatus natus, the average was 17.68% for males and 21.42% for females, with wide variation between individuals (min = 6%, max = 46%). Eosinophilic leukocytes were the fourth most abundant leukocytes. The seasons and type of diet can influence the amount of eosinophilic leuko- cytes in some species (Duguy, 1970; Deem et al., 2006). According to Frye (1991), the percentage of eosinophils in healthy reptiles ranges from 7 to 20%; in the case of Table 1. Descriptive statistics of blood biometric variables from male/female Gopherus flavomarginatus. SE = standard error, SD = standard deviation, Min = minimum, Max = maximum. Variable Mean SE Median SD Min Max Hematocit (%) 25.35/23.48 1.41/0.77 25.30/23.61 5.64/4.09 16.47/17.89 35.62/33.33 Hemoglobin (g/dl) 6.53/6.59 0.40/0.26 6.56/7.15 1.62/1.41 4.05/3.89 9.67/8.95 Erythrocytes (×106 cel/ul) 0.54/0.57 0.06/0.03 0.47/0.58 0.25/0.19 0.22/0.32 0.97/0.98 MCV (fl) 539.02/448.98 54.81/27.17 458.56/401.53 219.26/143.78 264.56/196.17 921.72/717.91 MCH (pg) 144.12/122.92 18.96/5.79 124.11/122.53 75.86/30.67 64.66/45.95 292.86/197.78 MCHC (g/dl) 26.37/28.49 1.64/1.23 25.62/28.84 6.56/6.52 17.69/16.20 39.36/44.66 Leukocytes (×103 cel/ul) 8.88/9.50 0.82/0.70 8.55/8.55 3.28/3.73 5.00/2.44 17.67/21.33 Heterophils (%) 25.43/29.32 1.99/1.68 26.00/29.00 7.99/8.89 14.00/13.00 39.00/45.00 Eosinophils (%) 2.75/3.17 0.57/0.51 2.00/2.50 2.29/2.74 0.00/0.00 7.00/11.00 Basophils (%) 17.68/21.42 2.11/1.67 15.50/18.50 8.47/8.86 6.00/8.00 29.00/46.00 Lymphocytes (%) 53.43/44.03 2.99/2.06 54.00/42.50 11.99/10.92 34.00/27.00 73.00/70.00 Monocytes (%) 0.68/2.03 0.17/0.40 1.00/1.00 0.70/2.16 0.00/0.00 2.00/9.00 H:L ratio 0.52/0.73 0.06/0.06 0.45/0.71 0.27/0.36 0.22/0.24 1.11/1.59 Heterophils (×103 cel/ul) 2.17/2.81 0.19/0.29 2.09/2.56 0.77/1.55 0.93/0.66 3.45/7.25 Eosinophils (×103 cel/ul) 0.21/0.34 0.04/0.08 0.18/0.21 0.17/0.45 0.00/0.00 0.66/2.35 Basophils (×103 cel/ul) 1.61/1.95 0.28/0.16 1.45/1.80 1.14/0.85 0.30/0.39 5.12/4.16 Lymphocytes (×103 cel/ul) 4.79/4.20 0.57/0.36 3.96/4.04 2.31/1.95 2.57/0.78 10.32/8.96 Monocytes (×103 cel/ul) 0.07/0.18 0.02/0.03 0.06/0.09 0.09/0.20 0.00/0.00 0.35/0.85 Table 2. Descriptive statistics of blood chemistry variables from male/female Gopherus flavomarginatus. SE = standard error, SD = standard deviation, Min = minimum, Max = maximum. Variable Mean SE Median SD Min Max Glucose (mg/dl) 79.19/58.34 7.97/8.28 68.33/40.47 31.90/43.82 43.32/13.34 134.67/165.30 Uric acid (mg/dl) 5.63/5.58 1.07/0.71 5.66/5.96 4.28/3.76 0.27/0.24 12.45/13.48 Urea (mg/dl) 25.60/31.22 0.82/0.76 25.39/31.75 3.30/7.86 20.64/14.67 32.86/51.34 BUN 11.96/14.37 0.38/0.82 11.86/14.22 1.54/4.35 9.64/6.86 15.36/23.99 Creatinine (mg/dl) 0.48/0.43 0.06/0.05 0.47/0.40 0.24/0.26 0.01/0.01 0.95/0.89 Total protein (g/dl) 4.12/3.95 0.53/0.24 3.76/3.78 2.13/1.27 1.29/2.00 8.96/7.13 Albumin (g/dl) 0.87/0.68 0.18/0.10 0.56/0.47 0.74/0.56 0.09/0.04 2.50/2.55 Globulins (g/dl) 3.24/3.27 0.42/0.24 3.23/3.09 1.70/1.30 0.99/1.37 6.46/6.50 Cholesterol (mg/dl) 280.55/214.94 45.28/23.50 269.50/167.43 181.13/124.36 41.46/78.32 692.27/508.20 Triglycerides (mg/dl) 207.85/177.57 13.79/16.18 213.80/164.06 55.18/85.62 114.08/42.35 283.63/402.30 ALT (UI/I) 7.74/8.15 1.06/1.06 7.53/6.48 4.25/5.65 2.04/1.74 15.93/22.43 AST (UI/I) 41.79/45.23 6.30/3.53 32.27/47.92 25.21/18.69 14.95/15.63 105.60/80.42 AP (UI/I) 60.30/66.53 8.08/4.58 56.91/70.05 32.32/24.24 14.11/24.86 102.60/99.32 Chlorine (mmol/l) 119.90/121.64 4.24/2.98 118.90/122.31 16.97/15.81 95.68/94.53 139.62/149.14 Sodium (mmol/l) 137.53/138.24 3.20/2.47 136.24/136.42 12.80/13.11 112.36/117.90 160.90/177.60 Calcium (mg/dl) 11.61/13.44 0.65/0.41 12.64/13.29 2.63/2.19 7.46/10.27 15.67/18.30 Phosphorus (mg/dl) 3.95/4.08 0.56/0.36 3.59/4.29 2.24/1.93 1.02/1.31 7.18/7.16 Osmolality (mOsm/kg) 270.50/273.87 5.92/4.73 268.60/272.56 23.70/23.85 225.43/240.81 316.39/345.66 126 Cristina García-De la Peña et alii Table 3. Comparison among minimum-maximum blood biometry values reported for Gopherus flavomarginatus and reference values of other three species of the genus. Variable G. flavomarginatus1 G.agassizii2 G. polyphemus3,4 G. berlandieri5 Hematocit (%) 16.47-35.62 19.50-37.10 15.00-30.00 12.00-44.80 Hemoglobin (g/dl) 3.89-9.67 4.10-9.90 4.20-8.60 - Erythrocytes (×106 cel/ul) 0.22-0.98 0.36-1.08 0.24-0.91 - MCV (fl) 196.17-921.72 254.00-638.00 200.10-838.60 - MCH (pg) 45.95-292.86 74.00-186.00 - - MCHC (g/dl) 16.20-44.66 20.00-33.00 - - Leukocytes (×103 cel/ul) 2.44-21.33 1.49-10.92 10.00-22.00 0.00-80.650 Heterophils (%) 13.00-45.00 - 10.00-57.00 - Eosinophils (%) 0.00-11.00 - - - Basophils (%) 6.00-46.00 - 2.00-11.00 - Lymphocytes (%) 27.00-73.00 - 32.00-79.00 - Monocytes (%) 0.00-9.00 - 3.00-13.00 - H:L ratio males 0.22-1.59 - - - Heterophils (×103 cel/ul) 0.66-7.25 0.71-7.15 0.00-6.59 0.00-5.21 Eosinophils (×103 cel/ul) 0.00-2.35 0.00-0.95 - - Basophils (×103 cel/ul) 0.30-5.12 0.06-3.57 0.02-0.92 0.00-1.31 Lymphocytes (×103 cel/ul) 0.78-10.32 0.63-2.74 0-4.15 0-3.12 Monocytes (×103 cel/ul) 0.00-0.85 0.00-0.32 - 0.00-0.44 1 Present study. 2 Christopher et al. (1999) in summer season. 3 Taylor and Jacobson (1982). 4 Teare (2013b). 5 Teare (2013a). Table 4. Comparison among minimum-maximum blood chemistry values reported for Gopherus flavomarginatus and reference values of other three species of the genus. Variable G. flavomarginatus1 G.agassizii2 G. polyphemus3,4 G. berlandieri5 Glucose (mg/dl) 13.34-165.30 65.00-186.00 55.00-128.00 9.00-157.00 Uric acid (mg/dl) 0.24-13.48 1.70-9.20 0.90-8.50 0.00-8.60 Urea (mg/dl) 14.67-51.34 - 1.00-130.00 - BUN 6.86-23.99 1.00-37.00 2.00-29.00 0.00-13.00 Creatinine (mg/dl) 0.01-0.95 0.20-0.40 0.10-0.40 - Total protein (g/dl) 1.29-8.96 2.30-5.30 1.30-4.60 1.20-7.70 Albumin (g/dl) 0.04-2.55 0.80-1.90 0.50-2.60 0.50-2.70 Globulins (g/dl) 0.99-6.50 1.30-3.90 0.50-4.60 0.60-5.10 Cholesterol (mg/dl) 41.46-692.27 33.00-381.00 19.00-150.00 Triglycerides (mg/dl) 42.35-402.30 7.00-603.00 - - ALT (UI/I) 1.74-22.43 1.00-5.00 2.00-57.00 - AST (UI/I) 14.95-105.60 15.00-123.00 57.00-392.00 0.00-265.00 AP (UI/I) 14.11-102.60 25.00-114.00 11.00-71.00 - Chlorine (mmol/l) 94.53-149.14 101.00-138.00 35.00-128.00 89.00-122.00 Sodium (mmol/l) 112.36-177.60 127.00-176.00 127.00-148.00 123.00-153.00 Calcium (mg/dl) 7.46-18.30 8.60-23.90 10.00-14.00 5.00-17.40 Phosphorus (mg/dl) 1.02-7.18 1.10-6.50 1.00-3.10 0.70-4.90 Osmolality (mOsm/kg) 225.43-345.66 252.00-352.00 - - 1Present study. 2Christopher et al. (1999) in summer season. 3Taylor and Jacobson (1982). 4Teare (2013b). 5Teare (2013a). 127Hematological parameters of Gopherus flavomarginatus G. flavomarginatus, the average percentage found in the present study was 2.75% for males and 3.17% for females, with a total variability of 0-11%. The average percentage of monocytes in the Bolson tortoise was 0.68% for males and 2.03% for females (min = 0, max = 9); these values are within the range of abundance reported for reptiles (Duguy, 1970). The H:L ratio (heterophils/leukocytes) has been used as a reliable method to evaluate the exposure of verte- brates to chronic stress due to the relationship between the leukocyte profile and the adrenal response (produc- tion of cortisol). When the level of cortisol rises, the number of circulating heterophils increases, while the number of lymphocytes decreases (Davis et al., 2011). Davis (2009) indicated that blood biometry studies conducted with terrestrial tortoises in which the pos- sibility of individuals being stressed was not considered (including studies with G. agassizii, G. berlandieri, and G. polyphemus), the H:L ratio was less than 2.0 (mean of 0.65, SD=0.34), which coincides with the values obtained in the present study for G. flavomarginatus. Blood chemistry variables provide important infor- mation for establishing levels of hydration (BUN, uric acid, osmolality), nutrition (glucose, total protein, albu- min, colesterol, phosphorus), and metabolic activity (ala- nine aminotransferas, aspartate aminotransferase, and alkaline phosphatase activities) of desert tortoises (Chris- topher et al., 1999). Christopher et al. (1999) showed that blood chemistry values in G. agassizii change due to the effect of physiological (reproductive cycle, hibernation) and environmental factors (precipitation patterns, avail- ability of water and food). However, we observed similar blood values between G. agassizii and G. flavomarginatus at the same times of year (summer). In general, the phylo- genetic relation among G. flavomarginatus, G. agassizii, G. polyphemus, and G. berlandieri (Reynoso and Montellano- Ballesteros, 2004) may be reflecting the similarity between reported hematology values for these species, and the slight differences may point to particular adaptation con- ditions that each one has developed in their own habitat. Our small sample size (n = 44) precluded the cal- culation of formal reference intervals, a process that would require a minimum of 120 individuals (Geffre, et al., 2009). However, given the limited sample size avail- able, and the lack of previously published data regard- ing hematology parameters in G. flavomarginatus, these results provide a useful starting point for researchers. ACKNOWLEDGMENTS To Fondo Sectorial de Investigación para la Edu- cación SEP-CONACYT Ciencia Básica (220658) for funding this study. To Magdalena Rivas-García for her help in the field work. Cristino Villarreal-Wislar and the Mapimí Biosphere Reserve personnel for logistical sup- port during the realization of this study. To Cameron W. Barrows (University of California, Riverside) for the review of this manuscript. Tortoise samples were collect- ed under the DGVS 07249/15-16-17 permit granted by SEMARNAT, Mexico. REFERENCES Alleman, A.R., Jacobs, E.R., Raskin, R.E. (1992): Morpho- logic and cytochemical characteristics of blood cells from the desert tortoise (Gopherus agassizii). Am. J. Vet. Res. 53: 1645-1651. Campbell, T.W. (2004): Hematology of lower vertebrates. In: 55th Annual Meeting of the American College of Veterinary Pathologists (ACVP) & 39th Annual Meet- ing of the American Society of Clinical Pathology, pp. 1103-1214. ASVCP, ACVP, ASVCP, Eds, Middleton WI, USA. Campbell, T.W. (2015): Exotic Animal Hematology and Cytology. 4th ed. Wiley Blackwell, Oxford, UK. Carlson, J.E., Menges, E.S., Marks, P.L. (2003): Seed dis- persal by Gopherus polyphemus at Archbold Biological Station, Florida. Fla. Sci. 66: 147-154. Christopher, M.M., Berry, K.H., Wallis, I.R., Agy, K.A., Enen, B.T., Peterson, C.C., (1999): Reference intervals and physiologic alterations in hematologic and bio- chemical values of free-ranging desert tortoises in the Mojave Desert. J. Wildl. Dis. 35: 212-238. CONANP. (2006): Programa de Conservación y Manejo Reserva de la Biosfera Mapimí México. CONANP- SEMARNAT, Mexico. Davis, A.K. (2009): The wildlife leukocytes webpage; the ecologists’ source for information about leukocytes of wildlife species. Available at: http://wildlifehematol- ogy.uga.edu. August 17, 2017. Davis, A.K., Maney, D.L., Maerz J.C. (2008): The use of leukocyte profiles to measure stress in vertebrates: a review for ecologists. Funct. Ecol. 22: 760-772. Davis, A.K., Ruyle, L.E., Maerz, J.C. (2011): Effect of trap- ping method on leukocyte profiles of Black-Chested Spiny-Tailed Iguanas (Ctenosaura melanosterna): implications for zoologists in the field. ISRN Zool. 2011: 1-8. Deem, S.L., Dierenfeld, E.S., Sounguet, G.P., Alleman, A.R., Cray, C., Poppenga, R.H., Norton, T.M., Karesh, W.B. (2006): Blood values in free-ranging nesting leatherback sea turtles (Dermochelys coriacea) on the coast of the Republic of Gabon. J. Zoo. Wildl. Med. 37: 464-471. 128 Cristina García-De la Peña et alii Dickinson, V.M., Jarchow, J.L., Trueblood, M.H. (2002): Hematology and plasma biochemistry reference range values for free-ranging desert tortoises in Arizona. J. Wildl. Dis. 38: 143-153. Diaz-Figueroa, O. (2005): Characterizing the health status of the Louisiana Gopher tortoise (Gopherus polyphe- mus). Unpublished doctoral dissertation. Louisiana State University and Agricultural and Mechanical College. Duguy, R. 1970. Numbers of blood cells and their varia- tion. In: Biology of Reptilia (vol 3), pp. 93-109. Gans, C., Pough, F.H., Eds, Academic Press, New York. Frye, F.L. (1991): Infectious diseases. In: Biomedical and surgical aspects of captive reptile husbandry, pp. 113- 123. Frye, F.L., Ed, Krieger Publishing Co., Malabar, Florida. Geffré, A., Friedrichs, K., Harr, K., Concordet, D., Trumel, C., Braun, J.P. (2009):  Reference values: a review. Vet. Clin. Path. 38: 288-298.  Hammer, Ř., Harper, D.A.T., Ryan, P.D. (2001): PAST: Paleontological statistics software package for educa- tion and data analysis. Palaeontol. Electron. 4: 9. Jackson, D., Milstrey, E.G. (1989): The fauna of gopher tortoise burrows. In: Proceedings of the Gopher Tor- toise Relocation Symposium, pp. 86-98. Diemer, J., Jackson, D., Landers, L., Layne, J., Wood, D., Eds, Florida Game and Freshwater Fish Comm. Nongame Wildlife Program, Technical Report No. 5. Tallahas- see, Florida. Jacobson, E.R. (2014): Health issues of North Ameri- can tortoises. In: Biology and conservation of North American tortoises, pp. 60-76. Rostal, D.C., McCoy, E.D., Mushinsky, H.R., Eds, Johns Hopkins University Press. Baltimore, Maryland. Kemal, J. (2014): Laboratory Manual and Review on Clinical Pathology. OMICS Group eBooks, California, EUA. Kiester, A.R., Palomo-Ramos. R., Ríos-Arana, J., Goode, E.V. (2018). Gopherus flavomarginatus. The IUCN Red List of Threatened Species; e.T9402A112660985. Available at: http://dx.doi.org/10.2305/IUCN. UK.2018-2.RLTS.T9402A112660985.en. [accessed on 3 December 2018] Lips, K.R. (1991): Vertebrates associated with tortoise (Gopherus polyphemus) burrows in four habitats in south-central Florida. J. Herpetol. 25: 477-481. Morafka, D.J., Aguirre, G., Adest, G.A. (1989): Gopherus flavomarginatus Bolson Tortoise. In: The Conserva- tion Biology of Tortoises, pp. 10-13. Swingland, R., Klemens, M.W., Eds, Occasional Papers of the IUCN Species survival Commission (SSC) No. 5. IUCN, Gland, Switzerland. Reynoso, V.H., Montellano-Ballesteros, M. (2004): A new giant turtle of the genus Gopherus (Chelonia: Testu- dinidae) from the Pleistocene of Tamaulipas, México, and a review of the phylogeny and biogeography of gopher tortoises, J. Vert. Paleontol. 24: 822-837. SEMARNAT. (2010): NORMA Oficial Mexicana NOM- 059-SEMARNAT-2010, Protección ambiental-Especies nativas de México de flora y fauna silvestres-Cate- gorías de riesgo y especificaciones para su inclusión, exclusión o cambio-Lista de especies en riesgo. Diario Oficial, 30 Diciembre 2010, México. Stacy, N.I., Alleman, A.R., Sayler, K.A. (2011): Diagnostic hematology of reptiles. Clin. Lab. Med. 31: 87-108. Tavares-Dias, M., Oliveira-Junior, A.A., Silva, M.G., Mar- con, J.L., Barcellos J.F.M. (2009): Comparative hema- tological and biochemical analysis of giant turtles from the Amazon farmed in poor and normal nutri- tional conditions. Vet. Arh. 79: 601-610. Taylor, R.W., Jacobson, E.R. (1982): Hematology and serum chemistry of the gopher tortoise, Gopherus polyphemus. Comp. Biochem. Physiol. 72: 425-428. Teare, J.A. (2013a): Gopherus berlandieri, no selection by gender, all ages combined, Conventional American units 2013 CD.html. In: ISIS Physiological Reference Intervals for Captive Wildlife: A CD-ROM Resource. International Species Information System, Ed, Eagan, MN, USA. Teare, J.A. (2013b): Gopherus polyphemus, no selection by gender, all ages combined, Conventional American units 2013 CD.html. In: ISIS Physiological Reference Intervals for Captive Wildlife: A CD-ROM Resource. International Species Information System, Ed, Eagan, MN, USA. Thrall, M.A., Baker, D.C., Campbell, T.W., DeNicola, D., Fettman, M.J., Lassen, E.D., Rebar, A., Weiser, G. (2006): Veterinary hematology and clinical chemistry. Blackwell Publishing, Iowa, USA. Turgeon, M.L. (2012): Clinical Hematology: Theory & Procedures. 5th ed. Wolters Kluwer, Lippincott Wil- liams & Wilkins, Philadelphia, USA. USFWS (2016): Health assessment procedures for the desert tortoise (Gopherus agassizii): A handbook per- tinent to translocation. U.S. Fish and Wildlife Service, Desert Tortoise Recovery Office, Reno, Nevada, USA. Acta Herpetologica Vol. 14, n. 2 - December 2019 Firenze University Press Podarcis siculus latastei (Bedriaga, 1879) of the Western Pontine Islands (Italy) raised to the species rank, and a brief taxonomic overview of Podarcis lizards Gabriele Senczuk1,2,*, Riccardo Castiglia2, Wolfgang Böhme3, Claudia Corti1 Substrate type has a limited impact on the sprint performance of a Mediterranean lizard Pantelis Savvides1,*, Eleni Georgiou1, Panayiotis Pafilis2,3, Spyros Sfenthourakis1 Coping with aliens: how a native gecko manages to persist on Mediterranean islands despite the Black rat? Michel-Jean Delaugerre1,*, Roberto Sacchi2, Marta Biaggini3, Pietro Lo Cascio4, Ridha Ouni5, Claudia Corti 3 PIT-Tags as a technique for marking fossorial reptiles: insights from a long-term field study of the amphisbaenian Trogonophis wiegmanni Pablo Recio, Gonzalo Rodríguez-Ruiz, Jesús Ortega, José Martín* Occurrence of Batrachochytrium dendrobatidis in the Tensift region, with comments on its spreading in Morocco Ait El Cadi Redouane1, Laghzaoui El-Mustapha1, Angelica Crottini2, Slimani Tahar1, Bosch Jaime3,4, EL Mouden El Hassan1,* Hematological parameters of the Bolson tortoise Gopherus flavomarginatus in Mexico Cristina García-De la Peña1,*, Roger Iván Rodríguez-Vivas2, Jorge A. Zegbe-Domínguez3, Luis Manuel Valenzuela-Núñez1, César A. Meza Herrera4, Quetzaly Siller-Rodríguez1, Verónica Ávila-Rodríguez1 Ontogenetic and interspecific variation in skull morphology of two closely related species of toad, Bufo bufo and B. spinosus (Anura: Bufonidae) Giovanni Sanna Visible Implant Alphanumeric (VIA) as a marking method in the lesser snouted treefrog Scinax nasicus Andrea Caballero-Gini1,2,3,*, Diego Bueno Villafañe2,3, Lía Romero2, Marcela Ferreira2,3, Lucas Cañete4, Rafaela Laino2, Karim Musalem2,5 Morphological variation of the newly confirmed population of the Javelin sand boa, Eryx jaculus (Linnaeus, 1758) (Serpentes, Erycidae) in Sicily, Italy Francesco P. Faraone1,*, Salvatore Russotto2, Salvatore A. Barra3, Roberto Chiara3, Gabriele Giacalone4, Mario Lo Valvo3 Variability in the dorsal pattern of the Sardinian grass snake (Natrix natrix cetti) with notes on its ecology Enrico Lunghi1,2,3,4,*, Simone Giachello5, Manuela Mulargia6, Pier Paolo Dore7, Roberto Cogoni8, Claudia Corti1 Estimating abundance of the Stripeless tree-frog Hyla meridionalis by means of replicated call counts Federico Crovetto, Sebastiano Salvidio, Andrea Costa* AT-rich microsatellite loci development for Fejervarya multistriata by Illumina HiSeq sequencing Yan-Mei Wang, Jing-Yi Chen, Guo-Hua Ding*, Zhi-Hua Lin