Biochemical reference intervals for captive bred Crocodylus moreletii and Crocodylus acutus in the Alcuzahue Lagoon, Colima, Mexico

[Intervalos de referência bioquímica para Crocodylus moreletii e Crocodylus acutus criados em cativeiro na Lagoa Alcuzahue, Colima México]

ABSTRACT

Crocodylus moreletii and Crocodylus acutus are two endangered crocodile species endemic to Mexico. In this descriptive cross-sectional study, a total of 58 crocodiles (30 females and 28 males) were examined to determine and evaluate 24 blood biochemical indicators associated with energy, lipid, protein, mineral and enzymatic metabolic processes. Most of the serum biochemistry parameters were similar among sexes by species. However, male crocodiles showed higher triacylglycerol concentration and alkaline phosphatase activity, and lower globulin concentration than females. There were some significant differences between species. Total bilirubin, triacylglycerol, carbon dioxide, and hydrogen carbonate were higher in Crocodylus moreletii, and urea nitrogen, creatinine, alanine aminotransferase, and aspartate aminotransferase were higher in Crocodylus acutus. These reference values are very important for the protection of crocodiles. The calculated confidence intervals could be used to detect alert situations when at least 5% of the sampled crocodiles would fall outside of the calculated reference interval for a given parameter.

Keywords:
blood chemistry; metabolic profile; Crocodylus moreletii; Crocodylus acutus

RESUMO

Crocodylus moreletii e Crocodylus acutus são duas espécies de crocodilos ameaçadas de extinção endêmicas do México. Neste estudo transversal descritivo, um total de 58 crocodilos (30 fêmeas e 28 machos) foi examinado para determinar e avaliar 24 indicadores da bioquímica sanguínea associados a processos metabólicos energéticos, lipídicos, proteicos, minerais e enzimáticos. A maioria dos parâmetros bioquímicos séricos foram semelhantes entre os sexos da espécie. No entanto, os crocodilos machos apresentaram maior concentração de triacilgliceróis e atividade de fosfatase alcalina do que as fêmeas. Com uma concentração de globulina mais baixa do que as fêmeas, houve algumas diferenças significativas entre as espécies. A bilirrubina total, os triacilgliceróis, o dióxido de carbono e o bicarbonato de sódio foram maiores em Crocodylus moreletii, e o nitrogênio ureico, a creatinina, a alanina aminotransferase e o aspartato aminotransferase foram maiores em Crocodylus acutus. Esses valores de referência são muito importantes para a proteção dos crocodilos. Os intervalos de confiança calculados podem ser usados para detectar situações de alerta quando pelo menos 5% dos crocodilos amostrados estiverem fora do intervalo de referência calculado para um determinado parâmetro.

Palavras-chave:
bioquímica sanguínea; perfil metabólico; Crocodylus moreletii; Crocodylus acutus

INTRODUCTION

The natural wealth of planet earth is estimated at 1,700,000 species (Zamorano, 2009). Mexico is one of the five countries with the greatest biological diversity (Cántaro and Cántaro, 2020). Worldwide, it ranks 2^nd in terms of diversity in reptiles (n = 804 spp.), with 173 endemic species (Sánchez, 2011). In this group, crocodiles are aquatic reptiles adapted to water-land interfaces such as swamps, ponds, rivers, and lagoons (Padilla and Weber, 2016). In the Mexican territory the crocodile population was greatly reduced in many areas due to unregulated skin hunting, which occurred mainly in the 1970s (Cedillo et al., 2019). Crocodylus moreletii and Crocodylus acutus are protected by law and are included in the Official Mexican Directive (NOM-059-ECOL-2001), under the list of species that are subject to special protection. They are also listed in appendix I of the Convention on International Trade in Endangered Species of Wild Fauna and Flora (Convention…, 2021) and in the vulnerable category by the International Union for Conservation of Nature (Elbers, 2011). Therefore, Mexico established captive breeding programs in the Wildlife Management Unit (UMA-La Colorada), in the Alcuzahue Lagoon, Colima. Biochemical profile is a relatively non-invasive method, its blood parameters such as energy, protein, minerals, and enzymes could provide information on internal organs, nutritional status, and metabolic state (Puppel and Kuczyńska, 2016). Given their rich diversity, they are a strategic tool in animal resource conservation programs (Brakes et al., 2019). However, to date, there is still not a comprehensive and complete reference range on biochemistry parameters for Crocodylus moreletii and Crocodylus acutus. Therefore, the study was planned to generate the reference values in the serum biochemical profile of captive bred Crocodylus moreletii and Crocodylus acutus in the Alcuzahue Lagoon, Colima Mexico.

MATERIALS AND METHODS

This work was conducted as part of a population health assessment approved and supported by the Network Advances in Agricultural Research in Mexico. All the handling and sampling were performed in compliance with standard vertebrate protocols and veterinary practices, and in accordance with Bioethics and Animal Welfare Commission of the Faculty of Veterinary Medicine and Zootechnics of the University of Colima. Evaluation act: No. 1, February 3, 2021.

Fifty-eight crocodiles: 29 Crocodylus moreletii and 29 Crocodylus acutus (15 females and 14 males per species), were captured in the Alcuzahue Lagoon of the Wildlife Management Unit (UMA-La Colorada), located on 18km east of Tecomán (Environmental…, 2021), 47m snm altitude (Figure 1), with a warm sub-humid climate (Köppen Cfb) (Peel et al., 2007), temperature between 12.1°C in Jan and 37.1°C in May with rainfall of 484mm/year (Sedesol, 2021). Capture was achieved using aluminum poles with a stainless-steel cable, none of the crocodiles were sedated to avoid the interference of anesthetic drugs on the blood parameters.

Figure 1
Location of Alcuzahue Lagoon of the Wildlife Management Unit; UMA-La Colorada., Colima, Mexico.

The crocodiles were examined for clinical signs of trauma and illness, and the gender was determined by digital palpation of genital organs in the cloacae (Ziegler and Olbort, 2007). The average age was 3 years, weight 70.60±17.50kg (males), 60.10±7.50kg (females), and length 250.20±30.15cm for both sexes. Crocodiles were fed once a week, primarily on locally available fresh fish, chicken, and bovine viscera (lung and heart).

Immediately after capture, blood was taken from the post-occipital venous sinus located at the dorsal midline and caudal to the base of the head (Ragaza et al., 2021) using sterile 10mL syringes with 20g needles (Figure 2).

Figure 2
Post-sampling isolated crocodiles. Alcuzahue Lagoon of the Wildlife Management Unit; UMA-La Colorada., Colima, Mexico.

Blood was aliquoted in 8.5mL vacuum tubes with clot activator and serum separator gel (BD Vacutainer 367988; Becton-Dickinson Co., Franklin Lakes, United States). Prior to blood collection, all individuals were fasted for 24h to reduce the possibility of obtaining highly lipemic samples. To obtain serum, the blood samples were centrifuged directly at the UMA-La Colorada, at 1 500 x g for 10min using a portable centrifuge (Porta-Spin C828; UNICO., Dayton, United States). Subsequently, the serum samples were separated using 1.5mL tubes with lid (Tubes Safe-Lock 3810X; Eppendorf., Madrid, Spain) and transported at 4°C in a portable refrigerator (Thermoelectric Cooler Car/Home M5644-710; Coleman Company., Kansas, United States) to the clinical laboratories at the Autonomous Metropolitan University and the University of Colima where they were frozen at -20°C until analysis.

The concentration of each analyte was determined with an UV-Vis double beam spectrophotometer (Biochemistry Analyzer ES-218; KONTROLab., Guidonia, Italy). The biochemical analytes, the analytical method for each parameter, the units in which the results were expressed, and the corresponding commercial reagents, are described in Table 1.

The precision and reliability of the techniques was controlled using lyophilized control serum (SPINTROL NORMAL 1002100; Spinreact., Girona, Spain) and (Assayed Multi-Sera AL 1027; Randox Laboratories., Northern Ireland, United Kingdom). Duplicate tests were performed for every specimen in each parameter. Hemolysis of serum was recorded on a qualitative scale of 0 (none) to 3 (dark). Samples showing hemolysis scores of 2 and above constituted less than 2% of all samples and did not introduce a significant bias in any of the tested models; thus, the influence of serum hemolysis was ignored.

We processed the data using software SPSS (2013). The statistical procedure used to calculate 95% confidence intervals for the biochemical analytes follow the recommendation of the International Federation of Clinical Chemistry (Solberg, 1987). All variables were tested for normality distribution (Shapiro-Wilk test) and homogeneity of variances (Levene test). The results were compared among sexes within each species and within species. Significant difference between means was determined using an independent sample t-test model (SPSS…, 2013). The linear relationships between the biochemical analytes were identified using a Pearson Correlation Coefficient matrix. Results were considered significant at (P<0.05).

Table 1
Biochemical analytes, units, analytical methods, and corresponding commercial reagents

Analyte*	Unit	Method	Reagent
Energy-lipid profile
Glucose (GLU)	mM	Trinder. GOD-POD1	1001190a
Total cholesterol (COL-T)	mM	CHOD-POD. Liquid2	41020a
Total bilirubin (TB)	μM	DMSO. Colorimetric3	1001044a
Triacylglycerol (TAG)	mM	GPO-POD. Liquid4	41032a
β-hydroxybutyrate (β-HBA)	mM	Enzymatic5	RB1007b
Protein profile
Blood urea nitrogen (BUN)	mM	Urease-GLDH. Kinetic UV6	1001333a
Albumin (ALB)	g/L	Bromcresol green. Colorimetric	1001020a
Globulin (GLOB)	g/L	(PROT-T) - (ALB)	Difference
Creatinine (Cre)	μM	Jaffé. Colorimetric-kinetic7	1001111a
Total protein (PROT-T)	g/L	Biuret. Colorimetric8	1001291a
Mineral-electrolyte profile
Calcium ion (Ca2+)	mM	Arsenazo III. Colorimetric	CA2391b
Inorganic phosphate (Pi )	mM	Phosphomolybdate. UV9	1001155a
Sodium ion (Na+)	mM	Enzymatic. Colorimetric10	1001385a
Potassium ion (K+)	mM	UV Test11	1001395a
Magnesium ion (Mg2+)	mM	Xylidyl Blue. Colorimetric12	1001286a
Chloride ion (Cl-)	mM	Thiocyanate-Hg. Colorimetric13	1001360a
Carbon dioxide (CO2)	mM	Enzymatic14	CD127b
Hydrogen carbonate (HCO3 -)	mM	Enzymatic by CO2 total and gas dissolved	99852c
Anion gap (A-gap)	mM	[(Na+ + K+) - (Cl- + HCO3 -)]	Difference
Enzyme profile
Alanine aminotransferase (ALT)	U/L	NADH. Kinetic UV. IFCC rec. Liquid15	41274a
Alkaline phosphatase (ALP)	U/L	p-Nitrophenylphosphate. kinetic. Liquid16	41233a
Aspartate aminotransferase (AST)	U/L	NADH. Kinetic UV. IFCC rec. Liquid17	41264a
Creatine kinase (CK)	U/L	NAC. Kinetic UV. Liquid18	41250a
γ-glutamyl transferase (γ-GT)	U/L	Carboxy substrate. Kinetic. Liquid19	41288a

• *Official abbreviation of the International Union of Pure and Applied Chemistry; ¹Glucose oxidase-peroxidase; ²Cholesterol oxidase-peroxidase; ³Solubilization with dimethylsulfoxide; ⁴Glycerol-3-oxidasa-peroxidase; ⁵β-hydroxybutyrate dehydrogenase; ⁶Urease-glutamate dehydrogenase; ⁷Sodium picrate; ⁸Copper salts in an alkaline medium; ⁹Ammonium molybdate-phosphomolybdate; ¹⁰Galactosidase-o-nitrophenyl-d-galactopyranose; ¹¹Pyruvate kinase-phosphoenolpyruvate; ¹²Magon sulfonate in alkaline solution; ¹³Mercuric thiocyanate-red ferric thiocyanate; ¹⁴Phosphoenolpyruvate-phosphoenolpyruvate carboxylase-malate dehydrogenase; ¹⁵α-ketoglutarate-glutamate-piruvate-lactate-NADH; ¹⁶Pnitrophenyl phosphate-p-nitrophenol-phosphate; ¹⁷Aspartate-α-ketoglutarate-glutamate-oxalacetate-malate-NADH; ¹⁸Creatine phosphate-ADP-creatine-ATP-D-glucose-ADP-glucose-6-phosphate-NADP-6-phosphogluconate-NADPH; ¹⁹γ-L-glutamyl-3-carboxy-4-nitroanilide-glycylglycine-γ-L-glutamyl-glycylglycine-2-nitro-5-aminobenzoic acid; ^aSpinreact., Girona, Spain; ^bRandox Laboratories., Northern Ireland, United Kingdom; ^cBiolabo Laboratory., Grandcamp-Maisy, France.

RESULTS

The descriptive statistic for glucose (GLU), total cholesterol (COL-T), total bilirubin (TB), triacylglycerol (TAG), β-hydroxybutyrate (β-HBA), blood urea nitrogen (BUN), albumin (ALB), globulin (GLOB), creatinine (Cre), total protein (PROT-T), calcium ion (Ca²⁺), inorganic phosphate (P_i ), sodium ion (Na⁺), potassium ion (K⁺), magnesium ion (Mg²⁺), chloride ion (Cl^-), carbon dioxide (CO₂), hydrogen carbonate (HCO₃ ^-), anion gap (A-gap), alanine aminotransferase (ALT), alkaline phosphatase (ALP), aspartate aminotransferase (AST), creatine kinase (CK), and γ-glutamyl transpeptidase (γ-GT), determined from 58 blood serums of 29 Crocodylus moreletii and 29 Crocodylus acutus, and its respective international range values are shown in Table 2.

Table 2
Serum biochemistry parameters of crocodiles sampled in the Alcuzahue Lagoon of the Wildlife Management Unit; UMA-La Colorada., Colima, Mexico (n = 29 Crocodylus moreletii and 29 Crocodylus acutus, respectively)

	Crocodylus moreletii			Crocodylus acutus
	x ± SD	CI		x ± SD	CI		Reference
Energy-lipid profile
Glucose (mM)	3.90±0.47	3.72-4.08		4.02±0.45	3.85-4.19		3.24±0.30¥
Total cholesterol (mM)	6.14±0.77	5.84-6.43		5.95±0.71	5.68-6.22		6.21±0.94†
Total bilirubin (μM)	1.24±0.08	1.20-1.27		1.19±0.06	1.16-1.21		1.20±0.47†
Triacylglycerol (mM)	8.92±0.40	8.76-9.07		7.59±0.35	7.45-7.72		9.02±1.07†
β-hydroxybutyrate (mM)	0.85±0.14	0.79-0.91		0.79±0.19	0.72-0.86
Protein profile
Blood urea nitrogen (mM)	0.47±0.03	0.45-0.48		0.52±0.01	0.52-0.53		0.44±0.07†
Albumin (g/L)	26.25±0.32	26.12-26.37		26.31±0.29	26.20-26.43		26.47±3.75†
Globulin (g/L)	46.11±3.00	44.97-47.25		45.04±3.16	43.83-46.24		44.41±5.85†
Creatinine (μM)	38.03±1.66	37.39-38.66		39.19±1.66	38.55-39.82		31.19±14.54†
Total protein (g/L)	72.36±3.22	71.13-73.59		71.44±3.26	70.20-72.68		73.77±7.39†
Mineral-electrolyte profile
Calcium ion (mM)	3.24±0.18	3.17-3.31		3.26±0.15	3.21-3.32		3.26±0.13¥
Inorganic phosphate (Pi )	1.70±0.27	1.60-1.81		1.62±0.20	1.55-1.70		1.61±0.20¥
Sodium ion (mM)	140.19±2.05	139.93-140.46		140.64±3.68	139.24-142.04		141.07±1.95¥
Potassium ion (mM)	4.37±0.53	4.16-4.57		4.34±0.62	4.10-4.57		4.33±0.40†
Magnesium ion (mM)	1.11±0.19	1.04-1.19		1.12±0.19	1.05-1.20
Chloride ion (mM)	85.29±4.50	83.57-87.00		84.45±4.47	82.74-86.15		82.74±7.79†
Carbon dioxide (mM)	26.78±1.02	26.39-27.17		25.85±1.01	25.47-26.24
Hydrogen carbonate (mM)	24.10±0.73	23.82-24.38		23.26±0.74	22.98-23.55
Anion gap (mM)	35.16±6.91	32.53-37.79		37.26±7.80	34.30-40.23
Enzyme profile
Alanine aminotransferase (U/L)	87.81±1.99	87.05-88.57		88.70±1.24	88.22-89.17		89.07±3.72†
Alkaline phosphatase (U/L)	48.15±4.20	46.55-49.75		48.45±3.38	47.16-49.73		42.10±2.03†
Aspartate aminotransferase (U/L)	194.09±2.58	193.11-195.08		196.84±1.53	196.26-197.43		193.32±4.64†
Creatine kinase (U/L)	147.36±8.15	144.26-150.47		148.05±8.08	144.97-151.13		149.22±5.07†
γ-glutamyl transferase (U/L)	11.00±3.15	9.80-12.20		10.00±3.15	8.80-11.20

• Values are expressed as mean (x), standard deviation (SD), and as a confidence interval of 95% (CI); ^† Alligator sinensis, active period (et al.); ^¥ Crocodylus palustris (Stacy and Whitaker, 2000).

Quantified concentrations for serum biochemistry parameters are unprecedented at country level. Therefore, it was only possible to compare it with some parameters reported in Alligator sinensis (Peng et al., 2018) and Crocodylus palustris (Stacy and Whitaker, 2000), but the differences were not statistically significant. Some differences in relation to sex were found (Table 3). The male crocodiles had significantly higher TAG and ALP activity, and significantly lower GLOB concentration than the females. The concentration of the other analytes did not show differences between sexes by species.

Meanwhile, differences in relation to species were found (Table 4). TB, TAG, CO₂ and HCO₃ ^- were higher in Crocodylus moreletii and BUN, Cre, ALT and AST were higher in Crocodylus acutus. The concentration of the other analytes did not show differences between species.

DISCUSSION

The health evaluations are very important for the protection of animal species in danger of extinction. Therefore, it is essential to establish reference ranges for serum biochemical profile, which could aid in the identification of alterations in animal metabolism. TAG and COL-T are often used as indexes in the assessment of nutritional status, and hyperlipidemia in obese animals (Kawasumi et al., 2014). BUN and Cre are biochemical analytes related to each other, and very helpful to diagnose kidney pathologies in animals (Kaneko et al., 2008). These conditions might well be associated, say because of a positive correlation (P<0.05) between BUN and Cre found in our study (Table 5). However, the concentration of TAG, COL-T, BUN, and Cre showed serum levels within the normal range. Our analysis thus suggests a correct protein and lipid metabolism.

The mineral-electrolyte profile also exhibited strong interionic interactions, which lead to strongly correlated movements of the ions. This implies directional correlations between the movements of distinct cations, between the movements of distinct anions, and between the movements of cations and anions (Iqbal et al., 2019). Correlations can be positive, if distinct ions move preferentially into the same direction, e.g., between Ca²⁺, P_i , Na⁺, K⁺, and Mg²⁺. Or negative, if distinct ions move preferentially into opposite directions, e.g., between Cl^- with Ca²⁺, P_i , K⁺, and Mg²⁺. These negative correlations exert a strong influence on the transport properties of the extracellular fluid e.g., Cl^- with A-gap (Figure 3). Like-ion correlations are generally stronger than cation-anion anticorrelations (Table 5) and are particularly strong for the heavier type of ion (Kim et al., 2016).

CK typically presents in skeletal muscle, smooth muscle, heart, and brain. An increase CK activity is probably associated with skeletal muscle and cardiac muscle inflammation (Yousaf and Powell, 2012). As CK activity increases (Table 5), the level of serum TB also increases (Figure 4). Normally, the high level of TB concentration suggested hepatobiliary pathology (Imamura et al., 2021). However, the level of serum CK and BT was quantified within the normal range, which suggests a correct muscle metabolism.

Table 3
Comparison of serum biochemistry parameters of male and female crocodiles sampled in Alcuzahue Lagoon of the Wildlife Management Unit; UMA-La Colorada., Colima, Mexico (n = 14 males and 15 females, per species)

	Crocodylus moreletii			Crocodylus acutus
	Male	Female		Male	Female
	x ± SD
Energy-lipid profile
Glucose (mM)	3.93±0.52	3.86±0.43		3.92±0.34	4.10±0.51
Total cholesterol (mM)	6.30±0.68	5.97±0.85		5.74±0.71	6.12±0.70
Total bilirubin (μM)	1.23±0.09	1.25±0.09		1.18±0.07	1.19±0.05
Triacylglycerol (mM)*	9.19±0.19a	8.62±0.36b		7.88±0.07a	7.35±0.32b
β-hydroxybutyrate (mM)	0.86±0.14	0.84±0.16		0.81±0.21	0.77±0.18
Protein profile
Blood urea nitrogen (mM)	0.47±0.03	0.46±0.03		0.52±0.01	0.53±0.01
Albumin (g/L)	26.29±0.32	26.20±0.32		26.35±0.24	26.35±0.34
Globulin (g/L)*	43.73±3.31b	48.33±2.66a		42.64±1.87b	47.99±1.36a
Creatinine (μM)	37.69±1.55	38.40±1.76		39.61±1.60	38.84±1.69
Total protein (g/L)	72.96±2.82	71.72±31.72		70.19±3.06	72.46±3.14
Mineral-electrolyte profile
Calcium ion (mM)	3.28±0.16	3.20±0.20		3.21±0.13	3.31±0.15
Inorganic phosphate (Pi )	1.74±0.25	1.66±0.29		1.57±0.20	1.67±0.19
Sodium ion (mM)	140.41±1.74	139.95±2.38		139.31±2.86	141.72±4.00
Potassium ion (mM)	4.44±0.44	4.29±0.62		4.17±0.57	4.48±0.57
Magnesium ion (mM)	1.14±0.13	1.08±0.25		1.06±0.17	1.18±0.19
Chloride ion (mM)	84.51±3.94	86.12±5.06		85.37±4.65	83.70±4.33
Carbon dioxide (mM)	26.76±1.29	26.79±0.66		25.88±1.22	25.83±0.83
Hydrogen carbonate (mM)	24.09±0.92	24.12±0.50		23.30±0.89	23.24±0.63
Anion gap (mM)	36.25±5.89	34.00±7.92		34.81±7.00	39.26±8.00
Enzyme profile
Alanine aminotransferase (U/L)	87.49±1.80	88.15±2.19		88.91±1.51	88.52±0.99
Alkaline phosphatase (U/L)*	51.42±4.47a	44.63±4.00b		51.14±1.53a	46.25±2.83b
Aspartate aminotransferase (U/L)	194.20±3.31	193.98±1.59		196.66±1.83	196.99±1.29
Creatine kinase (U/L)	146.06±6.42	148.76±9.74		146.33±9.71	149.45±6.48
γ-glutamyl transferase (U/L)	11.59±3.04	10.36±3.24		10.30±2.82	9.76±3.47

• Values are expressed as mean (x) and standard deviation (SD); *Significant differences were obtained between groups indicated with different letters (P<0.05).

Table 4
Comparison of serum biochemistry parameters between species of crocodiles sampled in Alcuzahue Lagoon of the Wildlife Management Unit; UMA-La Colorada., Colima, Mexico (n = 29 Crocodylus moreletii and 29 Crocodylus acutus, respectively)

	Crocodylus moreletii	Crocodylus acutus
	x ± SD
Energy-lipid profile
Glucose (mM)	3.90±0.47	4.02±0.45
Total cholesterol (mM)	6.14±0.77	5.95±0.7
Total bilirubin (μM)*	1.24±0.08a	1.19±0.06b
Triacylglycerol (mM)*	8.92±0.40a	7.59±0.35b
β-hydroxybutyrate (mM)	0.85±0.14	0.79±0.19
Protein profile
Blood urea nitrogen (mM)*	0.47±0.03b	0.52±0.01a
Albumin (g/L)	26.25±0.32	26.31±0.29
Globulin (g/L)	46.11±3.00	45.04±3.16
Creatinine (μM)*	38.03±1.66b	39.19±1.66a
Total protein (g/L)	72.36±3.22	71.44±3.26
Mineral-electrolyte profile
Calcium ion (mM)	3.24±0.18	3.26±0.15
Inorganic phosphate (Pi )	1.70±0.27	1.62±0.20
Sodium ion (mM)	140.19±2.05	140.64±3.68
Potassium ion (mM)	4.37±0.53	4.34±0.62
Magnesium ion (mM)	1.11±0.19	1.12±0.19
Chloride ion (mM)	85.29±4.50	84.45±4.47
Carbon dioxide (mM)*	26.78±1.02a	25.85±1.01b
Hydrogen carbonate (mM)*	24.10±0.73a	23.26±0.74b
Anion gap (mM)	35.16±6.91	37.26±7.80
Enzyme profile
Alanine aminotransferase (U/L)*	87.81±1.99b	88.70±1.24a
Alkaline phosphatase (U/L)	48.15±4.20	48.45±3.38
Aspartate aminotransferase (U/L)*	194.09±2.58b	196.84±1.53a
Creatine kinase (U/L)	147.36±8.15	148.05±8.08
γ-glutamyl transferase (U/L)	11.00±3.15	10.00±3.15

Table 5
Pearson correlation coefficients for serum biochemistry parameters of crocodiles sampled in Alcuzahue Lagoon of the Wildlife Management Unit; UMA-La Colorada., Colima, Mexico (n = 58 crocodiles)

	BUNb	GLOBc	Cred	Ca2+e	Pi f	Na+g	K+h	Mg2+i	Cl-j	A-gapk	ALTl	ALPm	ASTn	CKo
BTa	-0.05	0.05	0.09	-0.03	0.05	-0.04	0.03	0.01	0.09	0.04	0.26	0.25	0.26	0.71*
	BUN	0.22	0.46*	0.29	0.24	0.24	0.22	0.23	0.25	0.25	-0.01	-0.02	-0.01	0.20
		GLOB	0.26	0.17	0.20	0.22	0.25	0.22	-0.15	0.27	-0.02	0.67*	0.10	0.26
			Cre	-0.20	-0.24	0.25	0.20	0.23	0.23	0.27	-0.01	0.08	0.17	-0.03
				Ca2+	0.81*	0.59*	0.80*	0.61*	-0.50*	0.79*	-0.03	-0.01	0.01	-0.01
					Pi	0.40*	0.87*	0.68*	-0.56*	0.89*	0.01	0.04	0.15	-0.01
						Na+	0.24	0.15	0.16	0.64*	-0.03	-0.02	-0.01	0.14
							K+	0.65*	-0.54*	0.82*	-0.01	0.01	0.06	-0.02
								Mg2+	-0.41*	0.51*	-0.11	-0.06	-0.11	-0.02
									Cl-	-0.75*	0.01	0.03	0.10	-0.02
										A-gap	-0.01	0.03	0.07	-0.01
											ALT	0.25	0.67*	0.48*
												ALP	0.10	0.10
													AST	0.45*
														CK

• ^a
  Total bilirubin; ^bBlood urea nitrogen; ^cGlobulin; ^dCreatinine; ^eCalcium ion; ^fInorganic phosphate; ^gSodium ion; ^hPotassium ion; ⁱMagnesium ion; ^jChloride ion; ^kAnion gap; ^lAlanine aminotransferase; ^mAlkaline phosphatase; ⁿAspartate aminotransferase; and ^oCreatine kinase; *P<0.05.

Figure 3
Relationship between chloride ion and anion gap. Chloride ion (●); predicted response (-).

Figure 4
Relationship between total bilirubin and creatine kinase. Total bilirubin (●); predicted response (-).

ALP is a membrane-bound enzyme found in a wide variety of tissues, including liver and bone (Zender et al., 2016). The level of serum ALP is used widely in the diagnosis of hepatobiliary pathology and various bone disorders (Ali et al., 2006). ALP works at an alkaline pH and the difference in adipose tissue between sexes may influence its release from the liver into the circulation (Ali et al., 2005). Therefore, the level of serum ALP of crocodiles could be influenced by the greater mass of total and abdominal adipose tissue of males compared to females (Table 3). Finally, AST is a liver-specific enzyme in humans and animals. The level of AST concentration in crocodiles is probably related to the enhancement of hepatic metabolism. Additionally, we found that the AST activity of Crocodylus moreletii and Crocodylus acutus was similar to Alligator sinensis in active period (Peng et al., 2018), but greater than that of Caiman crocodilus crocodilus (Rossini et al., 2011), and less than that of Alligator mississippiensis (Hamilton et al., 2016). However, the concentration of ALT and ALP showed serum levels within the normal range. Our analysis thus suggests that liver function within this population has not been affected adversely by any current level of hepatic steatosis.

CONCLUSIONS

This report provided the first comprehensive reference ranges of serum biochemistry for captive bred Crocodylus moreletii and Crocodylus acutus. Most of the serum biochemistry parameters were similar among the sexes by species, only three significant differences existed in triacylglycerol, alkaline phosphatase, and globulin values. Some significant differences were found between species. Total bilirubin, triacylglycerol, carbon dioxide, and hydrogen carbonate were higher in Crocodylus moreletii, and urea nitrogen, creatinine, alanine aminotransferase, and aspartate aminotransferase were higher in Crocodylus acutus. These reference values are very important for the protection of crocodiles in danger of extinction. The calculated confidence intervals could be used to detect alert situations when at least 5% of the sampled crocodiles would fall outside of the calculated reference interval for a given parameter.

ACKNOWLEDGMENTS

This project was supported by the National Council of Science and Technology-México (CONACyT-México) and the Network Advances in Agricultural Research in Mexico. The authors wish to thank the colleagues Jesús Pérez de la Mora and Armando Rodríguez Vásquez for their invaluable assistance.

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Publication Dates

• Publication in this collection
  06 Jan 2023
• Date of issue
  Nov-Dec 2022

History

• Received
  07 Feb 2022
• Accepted
  21 July 2022