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Introduction
Crocodile farming is commonly conducted under improper management practices, causing stress in the animals and a concurrent increase in corticosterone (CORT) plasma levels (Isberg and Shilton, 2013; Finger et al., 2015), which is the main glucocorticoid (GCs) in reptiles (Cockrem, 2013). While acute increases in GCs after short-term stress are beneficial for the survival of the individual (Dantzer et al., 2014), long-term increases in GCs after chronic stress have deleterious effects on reproduction, immunity, and growth (Sapolsky et al., 2000). In farmed individuals, including crocodilians, chronic stress affects their health, reproduction, and productivity (Morici et al., 1997). High CORT correlates negatively with concentrations of sex hormones and reproductive success (Lance and Elsey, 1986; Elsey et al., 1991; 1994; Guillette et al., 1995).
Data on the relationship between plasma CORT levels and sex hormones are available on the American alligator (Alligator mississippiensis; Lance and Elsey, 1986; Elsey et al., 1990; 1991; Guillette et al., 1997) and the Cuban crocodile (Crocodylus rhombifer; Augustine et al., 2020). Nevertheless, there is no information in this regard in Morelet’s crocodile (Crocodylus moreletii). Morelet’s crocodile is native to Mexico and is included in Appendix II of the Convention on International Trade in Endangered Species of Wild Fauna and Flora (CITES, 2022). Hence, its legal use for conservation, research, or commercial purposes occurs in farms (Sánchez-Herrera et al., 2011; Cedeño-Vázquez et al., 2012) where crocodiles are usually housed in groups according to their size. However, confinement of crocodiles increases their aggressive behavior, particularly in the breeding season, and leads to high levels of GCs (Elsey et al., 1990; 1994). Evaluation of CORT and sex hormones patterns in crocodilians can help monitor animal welfare and reproductive status (Augustine et al., 2020).
The present study hypothesized that CORT correlates negatively with sex hormones levels in intensively managed Morelet’s crocodiles. Therefore, we assessed serum concentrations of CORT and sex hormones and their relationship in farmed C. moreletii female and male adults during non-breeding (NBS) and breeding (BS) seasons.
Materials and Methods
Ethical considerations
The experiment complied with guidelines by the Bioethics and Animal Welfare Commission of the School of Veterinary Medicine and Zootechnics of Universidad Veracruzana (Act 07/22, November 24, 2022).
Study characteristics
The study was conducted in Veracruz, Mexico (Lat. 19°22' N, Long. 96°22' W) at El Colibrí de la Antigua crocodile farm during two consecutive years (2017-2019) to evaluate hormone concentrations in NBS (November) and BS (March). The breeding season in C. moreletii starts with courtships in February or March and ends with egg laying in July (Pérez-Higareda, 1980; Lazcano, 1982). During the study, mean annual temperature and precipitation were 23.5°C and 34 mm during NBS, and 26.7°C and 15 mm in BS.
Experimental animals
The study included 59 apparently healthy adult Morelet’s crocodiles (≥1.51 m length, 29.2 ± 9.1 kg live weight; 29 females and 30 males). Crocodiles were randomly selected from the general population of adult individuals that had reached commercial size. In NBS, blood samples were collected from 31 individuals (15 females, 16 males), and in BS from 28 (14 females, 14 males). Different crocodiles were included in each evaluation to determine hormone concentrations that could be representative of the population and not concentrations in specific individuals. In addition, since the crocodiles had reached commercial size, they were slaughtered a few days after sampling.
Crocodiles were housed in outdoor concrete enclosures with both females and males. Each enclosure was 18 m length×8 m breadth×1.4 m height and had a pool of 17×6×1.4 m filled with water at a depth of 80 cm, a basking area at each side, and a shaded area of 12×1.5 m. The stocking density was similar in both seasons of the year, and it was 0.1 to 0.2 individuals/m2.
Crocodiles were fed a mixture of minced chicken and fish prepared at the farm (40% crude protein) at amounts of 10% of their live weight once or twice a week throughout the study. The food was spread throughout the basking area so each crocodile could get some. They received mebendazole, as a dewormer, mixed with food every four months.
Blood sample collection and processing
Blood samples were collected to determine serum concentrations of CORT and sex hormones (estradiol [E2], progesterone [P4], and testosterone [T]). For blood sample collection, the crocodiles were captured from their enclosure one at a time. Once captured, they were physically restrained, and their snout taped shut to assure their own and the handlers’ safety. Sex was determined and animals were examined visually to exclude sick, wounded, or emaciated individuals.
From each animal, a blood sample (3 to 5 mL) was aseptically collected from the post-occipital venous sinus within 3 min of capture (Romero and Reed, 2005), transferred into 6 mL plastic tubes without anticoagulant, and kept in a cooler for 1 to 2 h until the sampling of all the individuals was completed. Then, blood samples were centrifuged at 810 x g for 10 min to obtain the serum, which was stored at -20ºC until hormone analysis. Blood samples were collected once during the NBS and once in BS in each year of the study, totaling four blood samplings (November 2017/2018 and March 2018/2019). After sampling, each crocodile was measured and weighed to determine its total length and weight.
Determination of serum hormone concentrations
Serum concentrations of CORT were measured in both females and males; E2 and P4 in females, and T in males, all by solid phase enzyme-linked immunosorbent assay (ELISA) using commercial kits (DRG International, Inc., Springfield, NJ, USA). The hormone assays were validated for crocodiles using one mammalian known sample as positive control in each run of each hormone to confirm that the assay detected the hormone and measured its levels in serum. Validation values of the standard curves constructed to calculate the results for each hormone were: Corticosterone: R=0.9891, Rsqr=0.9881, Adj Rsqr: 9852, SE of estimate: 0.0279; Estradiol: R=0.9987, Rsqr=0.9974, Adj Rsqr=0.9949, SE of estimate=0.0123; Progesterone: R=0.9984, Rsqr=0.9968, Adj Rsqr=0.9936, SE of estimate=0.0085; Testosterone: R=0.9990, Rsqr=0.9980, Adj Rsqr=0.9961, SE of estimate=0.0512.
Corticosterone. The DRG® Corticosterone ELISA kit was used (DRG International, Inc., Springfield, NJ, USA). The assay indicated cross-reactivity with CORT (100%), P4 (7.4%), deoxycorticosterone (3.4%), 11-dehydrocorticosterone (1.6%) and cortisol (0.3%). The assay range was 0 to 240 nmol/L, with sensitivity <1.6 nmol/L. The intra- and inter-assay coefficients of variation (CV) were 3.1 and 6.0%. The resultant concentrations were transformed from nmol/L into ng/mL by dividing them by 2.89.
Estradiol. The kit used was DRG® Estradiol ELISA (DRG International, Inc., Springfield, NJ, USA). The assay indicated cross-reactivity with estradiol-17β (100%), estrone (0.2%) and estriol (0.05%). The assay range was 9.7 to 2,000 pg/mL, and 9.7 pg/mL sensitivity. Intra- and inter-assay CVs were 4.5 and 7.8%.
Progesterone. The DRG® Progesterone ELISA kit was used (DRG International, Inc., Springfield, NJ, USA). The assay indicated cross-reactivity with P4 (100%), 11-desoxycorticosterone (1.1%), pregnenolone (0.35%), 17α-hydroxyprogesterone (0.3%) and CORT (0.2%). The assay range was 0 to 40 ng/mL, and 0.04 ng/mL sensitivity. Intra- and inter-assay CVs were 6.4 and 6.6%.
Testosterone. The DRG® Testosterone ELISA kit was used (DRG International, Inc., Springfield, NJ, USA). The assay indicated cross-reactivity with T (100%), 11β-hydroxytestosterone (3.3%), 19-nortestosterone (3.3%), androstenedione (0.9%) and 5α-dihydrotestosterone (0.8%). The assay range was 0.08 to 16 ng/mL, with 0.08 ng/mL sensitivity. Intra- and inter-assay CVs were 3.5 and 7.1%.
Data analysis
Differences in hormone concentrations between NBS and BS in females and males were analyzed with the Student’s t-test for independent variables. A difference of p<0.05 was considered significant. The Spearman’s correlation coefficient was used to determine the relationship between concentrations of CORT and E2, P4 and T. The statistical program used was Statistica 10® (StatSoft®, Inc., OK, USA).
Results
In females, CORT and P4 concentrations were higher (p<0.05) in NBS, while E2 levels were similar (p>0.05) in NBS and BS (Table 1). No correlation (p>0.05) was observed between CORT and E2 or P4 concentrations in any season (Table 2).
In males, CORT concentrations were similar (p>0.05) in NBS and BS, while T levels were higher (p<0.05) in BS (Table 1). There was no correlation (p>0.05) between CORT and T concentrations in any season (Table 2).
Table 1 Serum concentrations (mean ± SEM) of corticosterone and sex hormones in adult females and males of Morelet’s crocodile (Crocodylus moreletii) during the non-breeding (NBS) and breeding (BS) seasons.
| Non-breeding season | Breeding season | |
|---|---|---|
| Females | ||
| Corticosterone (ng/mL) | 47.8 ± 24.8a | 15.5 ± 9.1b |
| Estradiol (pg/mL) | 203.0 ± 110.2a | 251.6 ± 191.4a |
| Progesterone (ng/mL) | 2.0 ± 1.7a | 0.4 ± 0.5b |
| Males | ||
| Corticosterone (ng/mL) | 27.1 ± 17.5a | 23.4 ± 14.1a |
| Testosterone (ng/mL) | 2.3 ± 1.8a | 8.9 ± 5.1b |
Different superscript letters (a, b) within rows indicate statistical difference by season (p<0.05).
Table 2 Correlation between serum concentrations of corticosterone and sex hormones in adult females and males of Morelet’s crocodile (Crocodylus moreletii) during the non-breeding (NBS) and breeding (BS) seasons.
| Non-breeding season | Breeding season | |
|---|---|---|
| Females | ||
| Corticosterone and estradiol | r=0.02 | r=-0.47 |
| p=0.91 | p=0.08 | |
| Corticosterone and progesterone | r=0.35 | r=0.65 |
| p=0.23 | p=0.058 | |
| Males | ||
| Corticosterone and testosterone | r=0.24 | r=0.16 |
| p=0.36 | p=0.57 |
Discussion
Captive crocodilians will commonly experience some level of stress (Elsey et al., 1994); in consequence, they will have increased plasma CORT (Guillette et al., 1995; Cockrem, 2013). In reptiles, CORT levels are usually higher in BS when they might be necessary for reproduction (Tokarz and Summers, 2011). This was assumed in the present study due to farming conditions where crocodiles would be exposed to several stressors because of increased energy demands (Tokarz and Summers 2011), and high CORT would decrease sex hormones levels. However, none of this occurred. This could be explained as CORT was not high because the animals did not require a CORT release to mobilize energy during BS, like a report in male rattlesnake (Crotalus atrox; Taylor et al., 2004), or that animal management was adequate and thus they had low stress levels, particularly males, which showed similar CORT in NBS and BS. It is known that C. moreletii does well in captivity and shows high tolerance towards conspecifics (Lang, 1987; Ojeda et al., 1998), which could contribute to low stress and CORT levels. However, since there are no reference values for CORT in C. moreletii, it is not possible to accurately know if the levels found in the present study could be considered as normal or if they were elevated, considering that these are captive individuals.
Females had higher CORT in NBS, which could be a female response. Differences in CORT levels are common in reptiles and result from variations, at individual or population level, of the adrenocortical response to the same stressor caused by age, reproductive status, and season of the year (Dunlap and Wingfield, 1995; Moore et al., 2001; Moore and Jessop, 2003). However, as mentioned before, there are no reference values for CORT in C. moreletii that allow to know if lower levels found in the present study could be considered normal or if in both seasons CORT was, indeed, elevated. Therefore, it is necessary to conduct more studies on captive C. moreletii to establish normal values of CORT in females and males to help monitor the stress status throughout the year.
In females, the E2 similarity observed in NBS and BS was contrary to the expected (higher E2 in BS). In tropical crocodilians, circulating E2 increases in breeding females four to five months before oviposition, as it stimulates vitellogenin production and growth of preovulatory follicles (Uribe and Guillette, 2000; Calderón et al., 2004; Milnes, 2011) and because the oviduct grows during such time (Guillette et al., 1997; Milnes, 2011). Thus, plasma E2 is elevated when vitellogenic follicles are present, namely, right before ovulation (Coutinho et al., 2000). The reason for not obtaining higher E2 in BS could be that the timing and number of samplings did not allow to detect the expected differences that should occur in BS. The samples collected in March corresponded to the start of BS; hence, maybe March was too early into BS to show elevated E2. Thus, it is necessary to evaluate E2 in adult females several times throughout the year to establish normal values for different moments within each season to determine the stage of the breeding cycle in which females are.
Serum P4 levels were higher in NBS, contrary to what was expected and to findings in American alligator females indicating that plasma P4 was barely detectable throughout the year and only increased in the periovulatory period (Lance, 1989) and then declined after oviposition (Guillette et al., 1997). In egg-laying Cubancrocodile (Crocodylus rhombifer) fecal P4 metabolites were higher in the nesting season than in BS and NBS (Augustine et al., 2020), likely because P4 promotes the formation and development of eggs in reptiles (Custodia‐Lora and Collard, 2002). In this study, P4 was not evaluated in the nesting season but at the start of BS, when females were not close yet to ovulating. That might be the reason for not observing differences in P4 between NBS and BS. Hence, it is necessary to evaluate P4 at different moments within BS in adult females to establish normal values for its different stages, including oviposition and nesting, to improve the management of farmed females.
In the present study, CORT did not correlate with E2 and P4. The lack of correlation between CORT and E2 was contrary to the negative correlation observed in female American alligators suggesting that acute stress decreases E2 levels (Elsey et al., 1991). Exposure of females to stressors can decrease E2 levels in some reptiles (Elsey et al., 1991; Ganesh and Yajurvedi, 2002) but the relationship between plasma CORT and E2 or P4 varies greatly with species and reproductive stage (Tokarz and Summers, 2011).
In males, higher levels of T in BS were expected and agreed with the results obtained in American alligators (Lance, 1989; Guillette et al., 1997) and Cuban crocodiles (Augustine et al., 2020), and with reports indicating that plasma T increases concurrently with courtship and copulation in male crocodilians, corresponding with the restart of gonadal activity that begins four months before nesting, and that T declines abruptly at the end of BS (Milnes, 2011). On the other hand, CORT had no correlation with T in NBS and BS, which is contrary to a report where T levels in male reptiles correlate positively with baseline CORT (Eikenaar et al., 2012). In male American alligators, CORT and T were negatively correlated, indicating that CORT inhibits T secretion in males (Lance and Elsey, 1986).
In general, one explanation for the lack of correlation between CORT and E2, P4, or T in NBS and BS could be that CORT was not high enough to influence sex hormones levels, suggesting that crocodiles were not experiencing significant or chronic stress in either season in the present study.
In conclusion, CORT levels did not correlate to those of sex hormones in any sex or season suggesting that crocodile management was adequate for their well-being and optimal reproduction. Only two samplings were conducted throughout each year, which might be insufficient to determine the effect of husbandry on reproduction and welfare. Therefore, it is necessary to conduct more studies on farmed C. moreletii individuals with multiple samplings at different times during NBS and BS to determine basal concentrations of CORT and sex hormones. This would help to better understand the effects of management on reproduction and welfare and to determine optimal husbandry conditions for captive Morelet’s crocodiles.
