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Introduction
There are 7 species of sea turtles globally, 6 of which nest in Mexico (Márquez 1990, Mazaris et al. 2017). Currently, 6 species are listed within an extinction risk category and 1 species is listed as data deficient in the International Union for Conservation of Nature Red list of Threatened Species (IUCN 2020). The main threats are anthropogenic factors such as illegal fishing, pollution, habitat loss, climate change, poaching, illegal egg consumption (Koch et al. 2006, Mancini and Koch 2009, Mazaris et al. 2009). Therefore, implementing sea turtle conservation programs that include nest relocation and management are needed (Hamann et al. 2010, Brost et al. 2015). However, to fulfill this objective, it is necessary to produce viable, high-quality hatchlings, which have greater possibility of survival during incubation and in the open sea and higher chances for successful reproduction.
In situ, or natural incubation, is considered the best technique for the conservation of sea turtles (Mrosovsky 2006). In this method, the female chooses the nesting site on the beach where it will leave the nest to incubate (for ~45 d in the case of the olive ridley), which minimizes the risk of damage to the eggs from handling, movement, and transfer to another location (Kutzari 2006). However, since there are nest losses due to erosion, animal predation, high temperatures, and in certain regions, including Mexico, turtle egg consumption by humans (Mancini and Koch 2009, Hart et al. 2014), ex situ techniques such as beach fenced-off hatcheries and the incubation in polystyrene boxes are implemented.
A characteristic of the beach hatchery is that the eggs are buried by people in the sand, in holes that are similar in size and shape to natural nests. Some advantages of the hatchery are that they have physical protection (a fence or surveillance) and temperatures can be moderated with shade cloth or other material (e.g., palms, irrigation, etc.) (Esteban et al. 2018). The polystyrene boxes are used as containers to protect the eggs from natural threats, and after covering them with sand they are transferred to a room or tent, which depending on local conditions may or may not have controlled temperature and humidity to increase incubation success (Wood and Wood 2009). Polystyrene boxes are generally used on the Pacific coasts of Mexico where there is no space on the beach for a hatchery (e.g., there is no government concession permit, there is rural or touristic development, there is extreme erosion), and in turn they have the advantages of occupying less space and being able to be moved to another site during hurricanes and high tides, which would destroy a beach hatchery (Fuentes et al. 2011).
If management of both ex situ techniques is inadequate or conditions in the nest environment are unfavorable, there may be undesirable results such as higher mortality rates, reduced sizes in neonates, and more malformations compared to in situ incubation (Mortimer and Bresson 1999). Inadequate conditions can affect the sex ratio of a clutch (Morreale et al. 1982, Mrosovsky 1982, Dutton et al. 1985), have detrimental effects on embryonic development (Blanck and Sawyer 1981, Patino-Martínez et al. 2012), and reduce the locomotor performance of hatchlings (Booth et al. 2004, Booth and Evans 2011, Rusli et al. 2015). Environmental variables such as temperature are extremely important for nest incubation, as they have a direct influence on embryonic development in poikilothermic species (Du and Ji 2003, Booth 2006, Les et al. 2007). Sea turtle embryos are very sensitive to temperature fluctuations, with a thermal tolerance limit of 25.00 to 35.00 ºC (Ackerman 1997). Outside of this temperature range, the embryo mortality rate increases (Ackerman 1997, Broderick et al. 2000) or damage can occur in the physiological processes during embryonic development (Maulany et al. 2012).
Locomotive performance (i.e., ability to move with the support of the flippers) plays an important role in the first hours of life for sea turtles (Ischer et al. 2009) because as soon as they hatch, they have to leave their nests, crawl to the sea, avoid predators, and continue their life cycle (Wyneken and Salmon 1992). In this regard, the morphometry of sea turtle hatchlings at birth may be a factor that directly influences locomotion. This has been reported for Dermochelys coriacea hatchlings with narrow shells, which showed an advantage in terrestrial locomotion (Mickelson and Downie 2010). However, if turtles are born with unfavorable phenotypic characteristics that influence their locomotion such as congenital deformities (e.g., shell hypoplasia, fin hypoplasia, or amelia), they will have little or no chance of surviving and breeding (Kaska and Downie 1999, Bárcenas and Maldonado 2009).
Considering all the above, it is extremely important to determine the effectiveness of nest conservation programs through technique assessments and success indicators. The objective of this study was to compare 2 ex situ incubation methods by analyzing biological variables such as nesting temperatures, hatching success, and physical state (morphometry, weight, locomotor performance, and congenital malformations) for olive ridley hatchlings at a conservation project located at Boca de Tomates Beach, Jalisco, Mexico. The present study analyzes the aforementioned factors in both ex situ techniques for the same species, beach, and nesting season with the goal of controlling differences in species, climate, and sand characteristics (e.g., grain size, moisture, organic content). To this date, the authors are unaware of another hatchling quality study with these characteristics.
Materials and methods
Study site
The Boca de Tomates Turtle Project is located in Puerto Vallarta, Jalisco, Mexico (20º40′06.9″ N, 105º16′17.1″ W), with a beach length of 2 km. It has a tropical climate and is one of the most important nesting beaches for olive ridleys (Lepidochelys olivacea) in the region.
Data collection
Night patrols were carried out during July and August 2019, until a total number of 26 olive ridley nests were collected. All nests were immediately excavated upon being found or collected at the end of the oviposition. The eggs were carefully removed and counted. They were then deposited in a clean plastic bag and transported to the conservation center on the beach for artificial incubation.
Of the collected nests, 13 were buried in the beach hatchery and 13 in polystyrene boxes. Both techniques followed the instructions of the sea turtle protection technique manual used on the Pacific coast of Mexico (Kutzari 2006). For the hatchery, a hole approximately 40-50 cm deep was dug in a shape that mimicked an in situ nest (flask shape), and the eggs were then carefully placed in the hole and covered with sand. The nests were placed approximately 1 m from other nests, in alternating rows. The hatchery had a shade cloth to help control sand temperatures. For the polystyrene boxes (43 × 32 × 25 cm), the base of each box was perforated to make multiple holes of approximately 0.5 cm in diameter in order to release excess moisture. Approximately 7 cm of moist sand (the same sand used in the hatchery) was placed in the bottom of the box, and layers of 9 × 6 eggs from the same nest were placed until the box was full, taking care to avoid contact with box wall. The boxes were located adjacent to the hatchery on shelves inside a nylon tent with no other ambient controls.
Temperature
To record the incubation temperature, a thermal sensor (HOBO UA001-08, Onset; Bourne, MA, USA), previously intercalibrated with the other 26 sensors, was placed in the middle of each nest, and it recorded temperature every hour until the nest hatched. Mean daily temperature, daily minimum temperature, daily maximum temperature, variability every 24 h, and the number of hours in which the temperature exceeded 35.00 ºC (upper thermal tolerance limit, Ackerman 1997) were determined. For each nest, the mean and standard deviation of these temperature categories was calculated for the incubation period.
Neonate quality
After day 40 of incubation, the nests were checked daily until hatching. We waited for the most hatchlings to emerge from a single nest on the same day to carry out the physical tests. Approximately 15 min before the quality tests, all of the hatchlings were sprayed lightly with sea water so that they entered a state of frenzy (i.e., rapid flipper movement).
Malformations
All live hatchlings and dead embryos were examined for the presence of malformations. Visual inspections were carried out systematically following cranial-caudal and dorsal-ventral orientations. If an embryo or hatchling showed multiple malformations, each type of malformation was recorded and photographed separately (e.g., Figs. 1-3). The malformations were classified by anatomical region, type of malformation, and intensity.
The incidence of malformations was estimated using the prevalence and intensity indices described by Margolis et al. (1982). In our study, prevalence is defined as the number of organisms that registered at least 1 malformation divided by the number of organisms studied. Intensity is the number of malformations found per organism; it is obtained by dividing the number of malformations found in the study by the number of organisms with malformations (Bárcenas and Maldonado 2009).
Morphometry and weight
Ten hatchlings were selected randomly from each nest. The carapace length and width (cm) of the hatchlings was measured using a conventional metallic vernier, obtaining means of 3 measurements of the straight carapace length and 3 measurements of the straight carapace width. A pre-calibrated scale was used to obtain the weight (g) of the hatchlings; each was weighed 3 times and a final mean was obtained.
Locomotor performance
With the 10 hatchlings that were measured and weighed, 2 measurements were made for hatchling locomotor performance (righting response and crawling speed) following the methods of Maulany et al. (2012) and Hart et al. (2016). For righting response, the time it took for the hatchlings to flip over was taken; hatchlings were placed upside down on their carapace and the mean of 5 trials (s) was taken. If any hatchling took more than 60 s to turn, a 5-s rest period was established before the next attempt (Hart et al. 2016).
For crawling speed, the time it took for a hatchling to crawl 3 m on the beach was recorded. To do this, a track was built in the sand by placing two 3-m-long wooden boards 10 cm apart. For the nocturnal tests, a LED lamp was placed at the end of the tables to guide the hatchlings towards the end. A time limit of 10 min was established for each hatchling to finish crawling the 3 m, and if the hatchlings did not finish covering that distance within the time limit, the centimeters advanced were recorded (cm·s-1). However, if the hatchlings did not move within the first 5 min of being placed at the top of the tables, they were assigned to the failure category (0 cm·s-1).
Hatching success
All hatchlings were released within 24 h of hatching. Cleaning of each nest was carried out on the third day after releasing the hatchlings. The total number of dead hatchlings was recorded and grouped into 3 categories: (1) eggs without visible embryonic development, (2) dead hatchlings outside the egg, (3) dead embryo inside the egg. Hatching success was calculated using the number of live hatchlings over the total eggs in the nest.
Statistical analysis
Chi-square (χ2) tests were used to test the hypothesis that there were no differences between the treatments-boxes and hatchery-for hatching success and malformation parameters (prevalence, anatomical region, intensity). The data were normalized by the mean number of inspected organisms, and for counts below 10 we used the Yates correction. For the discrete data (temperatures, days of incubation, locomotor performance, weight, and morphometry [straight carapace length and straight carapace width]), the Ryan-Joiner normality test was used first, and if the distributions were normal, the Student-t test was used (t). For non-normal distributions, we used the Wilcoxon signed rank (W) test to check for differences between the means of the 2 groups (boxes and hatchery). Data were analyzed in Excel or Minitab 17.1.0 (Minitab; State College, Pennsylvania, USA), and all tests used an α = 0.05 significance level.
Results
Hatching success
A significant difference was found between the 2 methods (boxes and hatchery) for the mean hatching success. The eggs incubated in the hatchery obtained a higher hatching percentage (mean = 77.40%, n = 1,368) than those incubated in boxes (mean = 59.91%, n = 1,160) (χ2 = 70.97, P < 0.001, n = 1,160) (Table 1). Rain entered one box and the majority of embryos died, skewing the overall mean hatching success for the boxes. Removing that box’s data from the analysis increased the mean hatching success to 64.60% (n = 1,050) but did not change the significant difference between the incubation methods (χ2 = 33.9, P < 0.001) (Table 1).
Table 1 Average and range of biological parameters in 2 incubation methods.
| Incubation method | ||||||
| Hatchery | Box | |||||
| Parameter | Mean ± SD | Range | Mean ± SD | Range | Statistical test | |
| Hatching success (%) | 77.44 (±16.86) | 33.90–91.07 | 59.91 (±30.24) | 3.60–92.31 | χ2 = 70.97 P < 0.001 | |
| Incubation days | 46 (±0.99) | 45–48 | 51 (±1.71) | 48–54 | t = –9.42 P < 0.001 | |
| Mean daily nest temperature (ºC) | 32.62 (±0.36) | 32.08–33.30 | 30.58 (±0.83) | 28.97–31.62 | t = –7.88 P < 0.001 | |
| Mean daily min temperature (ºC) | 27.61 (±0.92) | 26.02–28.79 | 26.11 (±0.48) | 24.90–26.70 | t = 5.18 P < 0.001 | |
| Mean daily max temperature (ºC) | 36.29 (±1.20) | 34.38–38.99 | 35.70 (±1.85) | 32.23–39.14 | t = 0.94 P = 0.360 | |
| Daily temperature variance (ºC) | 1.46 (±0.33) | 1.06–2.14 | 2.69 (±0.27) | 2.19–3.07 | t = –10.26 P < 0.001 | |
| Straight carapace length (cm) | 4.02 (±0.12) | 3.85–4.24 | 4.04 (±0.15) | 3.85–4.32 | t = –0.28 P = 0.784 | |
| Straight carapace width (cm) | 3.28 (±0.13) | 3.01–3.45 | 3.35 (±0.13) | 3.14–3.61 | t = –1.26 P = 0.219 | |
| Weight (g) | 14.88 (±1.09) | 13.25–16.29 | 15.56 (±1.23) | 14.00–18.83 | W = 155.00 P = 0.305 | |
| Crawl speed (cm·s–1) | 1.48 (±1.10) | 0.67–4.41 | 1.22 (±0.88) | 0.36–3.67 | W = 186.00 P = 0.640 | |
| Righting response (Sg) | 1.82 (±0.57) | 1.10–3.38 | 3.05 (±2.84) | 1.33–11.69 | W = 144.00 P = 0.119 | |
Average incubation days: n = 13, hatchery; n = 13, boxes. Average temperatures: n = 13, hatchery; n = 12, boxes. Average morphometric measurements, weight, and locomotor performance: n = 130, hatchery; n = 124, boxes.
Nest temperatures
Results were obtained from 25 out of the 26 thermal sensors placed in the 26 nests because 1 sensor failed (hatchery, n = 13; boxes, n = 12). A significant difference in mean temperature over the incubation period was observed, with mean temperature being higher in the hatchery (32.62 ºC) than in boxes (30.58 ºC) (t = -7.88, P < 0.001). The mean daily minimum temperature during the incubation period was higher in the hatchery (27.61 ºC) than in the boxes (26.11 ºC), presenting a significant difference (t = 5.18, P < 0.001). No significant difference was found in the mean daily maximum temperature (t = 0.94, P = 0.360). The boxes were exposed to significantly fewer hours above the thermal tolerance limit (>35.00 ºC) (box mean = 47.7 h, hatchery mean = 114.3 h; W = 127, P = 0.020) and a shorter duration (i.e., continuous hours) above 35.00 ºC (box mean = 5.1 h [range = 0 to 42], hatchery mean = 14.6 h [range = 0 to 141]; W = 119, P < 0.010). Regarding the variability in mean temperature over 24 h, there was a significant difference (t = -10.26, P < 0.001), with greater daily fluctuations in boxes (mean = 2.69 ºC) than in the hatchery (mean = 1.46 ºC) (Table 1).
Incubation duration
Nests incubated in boxes (mean = 51 d) took 5 d longer to hatch than those in the beach hatchery (mean = 46 d), and a significant difference was observed between the techniques (W = 92, P < 0.001, n = 26) (Table 1).
Neonate quality
Regarding locomotor performance (crawling and righting speed), morphometry (straight carapace length and straight carapace width), and weight, no significant differences were found between the incubation techniques (t and W tests, P > 0.050, n = 254) (Table 1). Of the hatchery nests, a total of 1,171 organisms were examined for malformations. Seventeen hatchlings presented at least 1 malformation, resulting in a prevalence rate of 1.45%. Overall, 26 malformations were found, for an intensity index of 1.52 malformations per organism. Of the eggs incubated in boxes, out of a total of 829 studied organisms, 9 malformed hatchlings were observed with a total of 17 malformations, representing a prevalence rate of 1.09% and an intensity index of 1.88 malformations per organism. No significant differences were found in the prevalence between the box and hatchery methods (χ2 = 3.88, P = 0.140, n = 2,000) (Table 2).
Table 2 Prevalence (%) and intensity of malformations in Lepidochelys olivacea hatchlings.
| Incubation method | Total nests (N) | Nests with malformations (N) | Total eggs (N) | Inspected eggs (N) | Malformed hatchlings (N) | Malformations (N) | Total prevalence of inspected eggs (%) | Intensity: malformations/organism (N) |
| Hatchery | 13 | 11 | 1,368 | 1,171 | 17 | 26 | 1.45% | 1.52 |
| Boxes | 13 | 5 | 1,160 | 829 | 9 | 17 | 1.08% | 1.88 |
| Statistical test | χ2 = 3.88 | |||||||
| P = 0.140 | ||||||||
| d.f. = 2 | ||||||||
| n = 2,000 |
An irregular carapace was the most frequent malformation type observed in both incubation methods, with at least 1 occurrence in 23.5% of the boxes (n = 13) and in 50% of hatchery nests (n = 13) (Table 3, Fig. 3). The incubation technique was not found to influence the anatomical region of malformations (χ2 = 4.42, d.f. = 4, P = 0.112, n = 43 malformations) (Table 3).
Table 3 Number (N) and proportion (%) of type of malformation by anatomical region and incubation method (beach hatcheries and polystyrene boxes).
| Anatomical region | Type of malformation | Boxes (N) | Boxes (%) | Hatchery nests (N) | Hatchery nests (%) |
| General | Albinism | 2 | 7.7% | ||
| Identical twins | |||||
| Unequal twins | 1 | 5.90% | |||
| Complete aplasia | 2 | 11.80% | 1 | 3.85% | |
| Head | Exencephaly | ||||
| Eyes | Cyclopia | ||||
| Sinophtalmia | |||||
| Monophtalmia | 1 | 5.90% | 1 | 3.85% | |
| Anophtalmia | 1 | 5.90% | 2 | 7.70% | |
| Nose | Arhinia | ||||
| Rhinodimo | |||||
| Jaw | Lower Prognatia | 3 | 11.50% | ||
| Agnathia | |||||
| Flippers | Monoamelia | ||||
| Biamelia | 1 | 5.90% | |||
| Tetramelia | |||||
| Hypoplasia | 3 | 17.50% | |||
| Bifurcation | 2 | 11.80% | 2 | 7.70% | |
| Shell | Irregular | 4 | 23.50% | 13 | 50.00% |
| Kyphosis | 1 | 5.90% | 1 | 3.85% | |
| Hypoplasia | 1 | 5.90% | 1 | 3.85% | |
| Total | 17 | 100% | 26 | 100% | |
Discussion
In our study, a greater hatching success was found in the beach hatchery compared to polystyrene boxes. In other studies of ex situ incubation methods of sea turtles (box and beach) and their effect on hatching success, discrepancies have been reported, where some did not find significant differences between methods (Wyneken et al. 1988, Chan 1989, Abd-Mutalib and Fadzly 2015, Hart et al. 2016) and others have obtained similar results to the present study (Revuelta et al. 2014, Santos et al. 2019). Differences in findings may be attributed to the studied species, environmental conditions, nesting season, type of transport from the beach to the ex situ site, and even the economic resources of the project. For example, at our study site with limited financial resources, rain infiltrated the deteriorated tent, causing the drowning of the majority of embryos in one of the boxes.
Regarding incubation temperatures, in our study significantly higher minimum and mean daily temperatures were recorded in the hatchery compared to the boxes, which is consistent with previous studies (Mrosovsky and Yntema 1980, Mrosovsky 1982, Dutton, Whitmore and Mrosovsky 1985, Arzola-González 2007, Hart et al. 2016). This probably influenced the significantly longer incubation duration in the boxes (mean 5 d), as temperature has been shown to negatively correlate with incubation duration (Ackerman 1997).
The mean temperatures remained within the thermal tolerance range of 25.00-35.00 ºC for survival. No thermometer registered temperatures <25.00 ºC; however, temperatures higher than 35.00 ºC were registered in both methods with different patterns. In the hatchery temperatures >35.00 ºC presented longer durations (continuous hours), while the boxes had more spiked diurnal fluctuations above 35.00 ºC. Sustained temperatures outside the recommended range could cause an increase in embryonic mortality (Ackerman 1997), as reported by studies of in situ olive ridley turtle nests on the Pacific coast of Costa Rica (Valverde et al. 2010). Considering that multiple studies predict that the temperature of many beaches will increase with global warming (Raustiala 1997, Fuentes et al. 2011, Pike 2014), it is essential to carefully monitor artificial incubation techniques.
Regarding neonate quality, our study did not find differences in crawling speed between the treatments. However, another study on L. olivacea in the Boca de Tomates region by Hart et al. (2016) reported that individuals incubated in a hatchery were faster than those incubated in boxes. We observed that the hatchlings in the boxes were in a state of lethargy for several hours, and therefore we waited until the hatchlings entered a state of “frenzy” for the locomotor performance tests. The lethargy is probably due to the lower temperatures recorded in the boxes, as ectothermic species take longer to activate their metabolism (Les et al. 2007).
Regarding neonate malformations, both incubation methods produced hatchlings with malformations, but no significant differences were found. Our results of malformation prevalence and intensity are similar to studies by Bárcenas and Maldonado (2009) and Camacho-Muñoz (2018), in which they concluded that malformations in L. olivacea have low prevalence and low intensity of malformations per organism compared to other studies of different sea turtle species (Kaska and Downie 1999, Gularte 2000, Kaska and Furness 2001). Our results coincide with findings from similar studies in that the most affected anatomical region was the carapace and the most frequent malformation type was an irregular carapace (Gularte 2000, Kaska and Furness 2001, Bárcenas-Ibarra and Maldonado 2009, Bárcenas-Ibarra et al. 2015).
Although the “normal” rates of prevalence and intensity of malformations in in situ olive ridley turtle nests in the region or the possible causes are unknown, in mammals the etiology of malformations has been classified into genetic, environmental (e.g., chemical, infectious, physical agents), and unknown factors (Deltsidou et al. 2000, Rojas and Walker 2012). Therefore, it has been suggested that these causes may be similar in sea turtles, but more research is needed to corroborate the possible causes (Kaska and Furness 2001, Bárcenas-Ibarra and Maldonado 2009).
This is the first study to compare the quality of hatchlings using 2 ex situ incubation methods in the same turtle species, location, and time period. Although our results do not demonstrate that there are serious consequences to neonate quality with these methods, differences in temperatures and hatching success were found. The possible relationship with temperatures, sand humidity (Arzola-González 2007, Lolavar and Wyneken 2019), and the neonate quality deserves further investigation in future studies that cover more of the nesting season. As the world moves towards ex situ incubation of many species due to lack of suitable natural habitat and global climate change, it is crucial to maximize the success of artificial management techniques, especially for endangered species such as the sea turtles.
