Transport of American Bullfrogs with Moistened Foam and without Foam: Plasma Biochemistry and Erythrogram Responses

Abstract

This study aimed to evaluate two transportation methods (with moistened foam and without foam) for 10 h on blood parameters of bullfrogs 0, 6, 12, 24, and 48 h after transportation. There was no mortality. The glucose increased at 0 and 12 h after transportation and returned to baseline at 24 h in both transportations. Triglycerides increased at 0 and 6 h in both transportations and were restored 12 h after transport with foam and 24 h in transport without foam. Plasma proteins and globulins increased at 0 h after transportation under both transportations. After 48 h, there was a reduction in transport without foam. Globulins decreased 48 h under both transportations. Albumin increased at 12, 24, and 48 h after both transportations. Transport with foam had high albumin. The albumin/globulin ratio increased 24 and 48 h after both transportations. The number of erythrocytes increased at 0 h and recovered after 6 h in transport with foam and 12 h in transport without foam. Hematocrit and hemoglobin increased at 0 h and recovered at 6 h in both transportations. MCV increased 48 h after transportation with foam. MCHC decreased 12, 24, and 48 h after both transportations. MCH was lower in the transport carried out with foam.

1. Introduction

The American bullfrog (Aquarana catesbeiana, Shaw, 1802) is an amphibian of the order Anura, naturally distributed in the eastern part of North America, Canada, and Mexico [1]. It is considered the largest frog species in North America, reaching up to 15 cm in length in the adult phase, from snout to cloaca [2]. Beyond its substantial size in both sexes, the American bullfrog is of great interest for commercial breeding due to its short life cycle, rapid growth, and resilience as a species, allowing it to adapt well to different captive settings and management practices [3,4].

Among the practices in commercial production, the transport of live frogs is a common procedure in frog farming, both for sending animals to slaughterhouses and fish processing plants and for acquiring animals from other farms for the formation and renewal of the breeding stock. However, this practice acts as a potent stressor due to the diversity of procedures involved, ranging from capturing frogs in pens, crowding these animals, to the transportation itself [5]. Recommendations for frog transportation suggest that this management should be carried out during the cooler hours of the day and preferably in enclosed vehicles. During transportation, animals should be housed in enclosed containers that allow minimal movement but prevent overcrowding [3]. In practice, the transportation of these animals occurs in various ways. Among the housing types used for the transportation of adult frogs are perforated polyvinyl chloride (PVC) tubes of 100 mm in diameter, nylon bags, and perforated plastic boxes [5]. The duration of the transportation of these animals from one facility to another is highly variable.

Despite the existence of diversified forms of transporting adult bullfrogs and their known hardiness during this handling, this does not imply that these animals are not susceptible to stress and its consequences. Within this context, little is still known about the effects that these different transportation management conditions may have on the well-being of the American bullfrog [5]. Inefficient transportation, in addition to compromising the well-being and the quality of the final product, can lead to high mortality rates [6,7], resulting in significant losses for the producer. Thus, although many potential sources of stress are inevitable during transportation, it is possible that some practices can reduce the intensity of stress experienced by the animals [6].

Water is an essential resource for the proper development and well-being of the American bullfrog, given its necessity for reproduction, maintenance of electrolyte balance, hydration, respiration, thermoregulation, and elimination of excreta and skin remnants [3]. However, despite the importance of water for this organism, during transportation handling, bullfrogs are typically subjected to water restriction, which can last for several hours. In this condition, frogs employ behavioral and physiological strategies to avoid excessive dehydration. To avoid water loss, semiaquatic frogs, such as the bullfrog, tuck their limbs under their bodies and compress their entire ventral surface onto a supporting substrate. This behavior reduces the surface area of contact between the animal’s body and the environment, reducing water loss through evaporation. Furthermore, in situations of dehydration, the antidiuretic hormone arginine vasotocin is released, which reduces the rate of urine formation and stimulates the absorption of water stored in the bladder, which maintains adequate levels of solutes in the plasma [8].

Thus, considering the challenge these animals face under water restriction during transportation, strategies to maintain a humid environment may be interesting to improve the welfare of these animals. In this regard, the aim of this study was to evaluate the effect of two types of transportation, in ventilated boxes and in boxes with foam moistened with water, on the survival and responses of plasma biochemistry and erythrogram of adult bullfrogs.

2. Material and Methods

2.1. Pre-Experimental Conditions

A total of 160 specimens of American bullfrogs (Lithobates catesbeianus), males and females, with an average weight of 468.22 ± 42.22 g, was acquired from the frog farming of Glória Farm at the Federal University of Uberlandia, Uberlandia, Minas Gerais, Brazil. These animals, randomly selected, were kept in two 10 m² pens (2.5 × 4.0 m), in a flooded system, with a density of 50 frogs/m². These pens were in a covered masonry shed, and the photoperiod was maintained at approximately 12L:12D (L—light period and D—dark period). The frogs were fed three times a day, at a rate of 1.5% of body weight, with extruded feed for carnivorous fish measuring 8 to 10 mm, containing 42% crude protein, 9% ether extract, and 4% crude fiber.

2.2. Experimental Design and Transport

An entirely randomized design was used, conducted in a factorial scheme with one additional treatment [5 × 2 + (1)], and 10 repetitions, with the bullfrog as the experimental unit. Parameters related to stress were evaluated at five time points after transportation (0—immediately after transportation, 6, 12, 24, and 48 h after transportation) and under two different transport conditions (with and without foam). One transport condition, referred to as “without foam”, involved transporting frogs in perforated polyethylene boxes measuring 38 × 58 × 22 cm (width × length × height). The second transport condition, referred to as “with foam moistened”, involved transporting frogs in similar boxes, but with the bottom covered with canvas and laminated polyurethane foam (30 mm—density 28). The foam was premoistened with one liter of water from the same source as the pens. The additional treatment (control) corresponded to the frogs’ condition in the pens before transportation.

Prior to transportation, the frogs underwent a 36-h fasting period, and then the animals were loaded. For transportation, 150 frogs were randomly captured and placed in six perforated polyethylene boxes, three without foam and three with moistened foam. The density used was 25 frogs/box, i.e., 11.71 kg/box. To prevent the frogs from escaping, the boxes were closed with 50% light transmission monofilament screens. The transportation was carried out in a closed vehicle and lasted approximately 10 h. The origin was the Gloria farm in Uberlandia, Minas Gerais, Brazil, with the destination being the Bullfrog farming Sector of the Aquaculture Laboratory at the School of Veterinary Medicine of Federal University of Minas Gerais, in Belo Horizonte, MG, Brazil, totaling 535.7 km. The frog transport took place during the day and part of the night, with the minimum temperature recorded at 22 °C and the maximum at 33 °C. Frog survival was determined immediately after transportation and continued to be assessed during the blood collection times (6, 12, 24, and 48 h after transportation). The animals were not fed for up to 60 h after transportation (12 h after the end of the experiment) to avoid interference from feeding in blood analyses.

2.3. Blood Collection and Blood Analyses

Before transportation, 10 bullfrogs from the control group (animal condition in the pen) were randomly chosen for blood collection. These animals were chosen from the two pens separated for the experiment, immediately before transportation. Blood collection and blood glucose analysis for these animals were performed immediately after capturing the animals in the pen. Blood samples from the control group animals were stored in 2 mL microtubes and kept in a cooler with ice for further analyses after transportation.

Immediately after transportation (0 h), blood was collected from 20 animals, 10 from boxes without foam and 10 from boxes with foam. The animals from the other evaluation times were housed in eight 1.22 m² (1.15 × 1.06 m) polyethylene pens, maintained in a flooded system, with a density of 15 frogs/pen, four for each transport type (with and without foam). The pens were identified according to the transport type and evaluation times (6, 12, 24, and 48 h after transportation).

For blood collection, frogs were restrained with a damp cloth and anesthetized with a topical anesthetic (4% lidocaine). Blood collection was performed on the hind limb, through sciatic artery puncture, from which approximately 2 mL of blood per frog was taken. The syringes used were 3 mL with 25 × 0.7 mm needles, previously moistened with 10% EDTA. After collection, the blood was stored in 2 mL microtubes, which were kept refrigerated at 4 °C. The frogs used in the procedure were directed to another eight pens, four for each transport type, according to the established assessment times (6, 12, 24, and 48 h).

Blood glucose was determined immediately after blood collection with a 10 μL blood aliquot, using strips and a digital glucometer (Acon, On-Call^® Plus, San Diego, CA, USA). After homogenization of the blood samples stored in the microtubes, 2/3 of the microcapillaries were filled with the samples. Then, the microcapillaries were centrifuged for 5 min at 12,000 rpm to determine hematocrit by reading the microcapillaries on a microhematocrit ruler. The total hemoglobin level was determined by the cyanomethemoglobin method. After homogenizing the microtubes with blood samples, 10 µL of blood was added and mixed in 2.5 mL of Drabkin’s reagent, where it was left for 30 min until reading on a Biochrom spectrophotometer (Libra S22, Cambridge, United King), using a wavelength of 540 nm. Erythrocyte count was determined using a Neubauer chamber and a light microscope (ProWay^® XSZ-PW206BT, Ningbo, China), with a 400× magnification. From the results of hemoglobin (Hb), total number of erythrocytes (Er), and hematocrit (Ht), the following absolute hematometric indices were calculated:

MCV (Mean Corpuscular Volume, fL): MCV (fL) = Ht × 10/Er;

MCH (Mean Corpuscular Hemoglobin, pg): MCH (pg) = Hb × 10/Er;

MCHC (Mean Corpuscular Hemoglobin Concentration, g dL⁻¹): MCHC (g dL⁻¹) = Hb × 100/Ht.

Plasma was obtained after centrifugation of the remaining blood at 3000 rpm in a microtube centrifuge (Spinlab^® model SL-5AM, Ribeirão Preto, Brazil) for 15 min. Subsequently, the plasma was transferred to 2 mL microtubes and stored at −80 °C. Commercial kits (Bioclin^®, Belo Horizonte, Brazil) and a Biochrom spectrophotometer (Libra S22, Cambridge, UK) were used for reading the following biochemical analyses: plasma proteins (Bioclin^® ref. K031), albumin (Bioclin^® ref. K040), triglycerides (Bioclin^® ref. K117), cholesterol (Bioclin^® ref. K083-2), lactate (Bioclin^® ref. K084-2), and urea (Bioclin^® ref. K047-1). Globulins were determined by subtracting albumin from plasma proteins. The albumin/globulin (A/G) ratio was estimated by dividing the albumin fraction value by the globulin fraction value for each sample. To perform the biochemical analyses and erythrogram, 10 frogs (n = 10) were used for each type of transport (with foam and without foam) and evaluation time.

2.4. Statistical Analysis

The data were analyzed in R Software 3.5.3. To assess normality and homoscedasticity of variances, the Shapiro–Wilk and Bartlett tests were used, respectively. A two-way ANOVA (5 × 2) was performed to verify the interaction between the factors, collection time, and transport type, or the isolated effect of each factor. According to the factorial analysis result, the Dunnett test was used to compare the additional treatment (control) with all combinations of the factorial or with all collection times or transport types. Significant differences were considered when p < 0.05. The Tukey test was used to compare the two transport methods at each collection time. To meet normality, the variables MCV and glucose were transformed by exponential (x)^−0.5, and triglyceride and cholesterol data by square root (x)^0.5. The Kruskal–Wallis nonparametric test (p < 0.05) was used for the albumin/globulin ratio.

3. Results

3.1. Survival

The transport condition, with or without foam, did not influence frog survival. No mortalities occurred during transportation or in the subsequent evaluation periods.

3.2. Plasma Biochemistry

There was a significant (p < 0.05) isolated effect of blood collection time on frog blood glucose. Hyperglycemia was observed immediately after transportation (0 h). The animals remained hyperglycemic at 6 and 12 h after transportation. Blood glucose levels returned to baseline 24 and 48 h after transportation (Figure 1A). The transport condition, with or without foam, did not influence (p > 0.05) frog blood glucose.

There was an interaction (p < 0.05) between the factors of the blood evaluation time after transportation and the type of transport (with or without foam) on plasma triglyceride levels.

Bullfrogs evaluated immediately after transportation had increased triglyceride levels compared to control animals, with higher levels in frogs transported in boxes with moistened foam. Frogs evaluated 6 h after transportation still had elevated levels of this variable compared to control animals, with no differences between transport types. However, 12 h after transportation, only frogs transported in perforated boxes remained with elevated triglyceride levels, differing from both control animals and frogs transported in boxes with moistened foam, which had already restored triglyceride levels to those of the control group. After 24 and 48 h of transportation, there were no differences in triglyceride levels between frogs transported in both conditions, showing values similar to those of control animals (Figure 1B).

There was an interaction (p < 0.05) between the factors of blood evaluation time after transportation and the type of transport used on plasma protein concentration. Bullfrogs evaluated immediately after transportation had higher levels of plasma proteins compared to control animals. However, there was no difference (p > 0.05) in this variable between the tested transport conditions. In frogs evaluated 6, 12, and 24 h after transportation, the concentration of plasma proteins remained similar to that of the control group in both transport conditions. However, 24 h after transportation, frogs kept in boxes with moistened foam had higher plasma protein values compared to frogs transported in boxes without foam. After 48 h of transportation, there was a reduction in the amount of plasma proteins in frogs transported in boxes without foam compared to control group frogs. However, there was no difference (p > 0.05) between the transport types, with or without foam, 48 h after transportation (Figure 1C).

There was an interaction (p < 0.05) between the factors of blood evaluation time after transportation and the type of transport (with or without foam) on globulin concentration (Figure 1D). Compared to control animals, bullfrogs evaluated immediately after transportation had higher globulin concentrations. However, there was no difference (p > 0.05) between the tested transport conditions (with or without foam). In frogs evaluated 6 and 12 h after transportation, the concentration of globulins remained similar to that of control group frogs. However, at the 12-h mark after transportation, frogs transported in boxes without foam had higher globulin values compared to those transported in boxes with moistened foam. The globulin values in frogs transported in boxes without foam decreased 24 h after transportation, both compared to control animals and those transported in boxes with moistened foam. There was a reduction in the number of globulins in frogs of both transport types, compared to control frogs, 48 h after transportation (Figure 1D).

There was a significant (p < 0.05) isolated effect of blood collection time on frog albumin concentration. An increase in this protein fraction was observed 12, 24, and 48 h after transportation (Figure 2A). The type of transport also influenced (p < 0.05) the behavior of this variable. Animals transported in boxes with moistened foam had a higher albumin concentration, while frogs transported in boxes without foam maintained values similar to those of control group animals (Figure 2B).

The albumin/globulin (A/G) ratio showed a significant isolated effect (p < 0.05) of the evaluation time. There was an increase in the A/G ratio in frogs evaluated 24 and 48 h after transportation compared to control group animals. No differences (p > 0.05) were observed in the A/G ratio in animals evaluated at other evaluation times (0, 6, and 12 h) compared to control frogs (Figure 1E).

There was no interaction and isolated effect (p > 0.05) of the factors of evaluation time and the type of transport used on plasma cholesterol, urea, and lactate levels.

3.3. Erythrogram

There was a significant interaction (p < 0.05) between the factors of blood evaluation time after transportation and the type of transport used on the number of erythrocytes. Compared to control frogs, there was an increase in the number of erythrocytes in frogs evaluated 0 h after transportation in both transport conditions. However, 6 h after transportation, frogs transported in perforated boxes without foam still had a high number of erythrocytes compared to control animals. The reestablishment of the number of erythrocytes for these animals occurred 12 h after transportation. On the other hand, frogs transported in boxes with moistened foam restored the number of erythrocytes 6 h after transportation (Figure 3A).

Only the collection time influenced (p < 0.05) the hematocrit of frogs after transportation. The hematocrit increased in frogs evaluated at 0 and 48 h after transportation, compared to animals in the control group. The values of this variable in animals from other collection times did not show differences when compared to control frogs (Figure 3B).

There was a significant interaction (p < 0.05) between the factors of blood evaluation time after transportation and the type of transport used on the mean corpuscular volume (MCV). The MCV of frogs evaluated immediately after transportation did not differ from the values observed for animals in the control group or between transport types. There was also no difference in the MCV values of frogs evaluated 6 h after transportation when compared to animals in the control group. However, in this same evaluation interval, frogs transported in boxes without foam had a reduction in MCV compared to those transported in boxes with moistened foam. At 12 and 24 h after transportation, the MCV of frogs did not differ from the values of animals in the control group or between transport types. However, 48 h after transportation, there was an increase in MCV for animals transported in boxes with moistened foam, compared to control frogs and those transported in boxes without foam. At this same evaluation time, frogs transported in boxes without foam had MCV equal to animals in the control group (Figure 3C).

Hemoglobin was influenced (p < 0.05) only by the evaluation time after transportation of the animals. There was an increase in hemoglobin immediately after transportation. At the evaluation times of 6, 12, 24, and 48 h after transportation, the hemoglobin of frogs was restored, not differing from animals in the control group (Figure 3D).

The mean corpuscular hemoglobin concentration (MCHC) of frogs was influenced (p < 0.05) independently by the time of evaluation after transportation and the type of transport. The MCHC values of animals evaluated 0 and 6 h after transportation remained the same as those of the control frogs. However, there was a reduction in MCHC at 12, 24, and 48 h after transportation (Figure 4A). Regarding the type of transport, frogs transported in boxes with moistened foam had lower MCHC when compared to control frogs. Animals transported in boxes without foam maintained values equal to those of control frogs (Figure 4B).

There was no interaction or isolated effect of the factors time of evaluation and type of transport (p > 0.05) on the mean corpuscular hemoglobin (MCH) of the frogs used in the experiment.

4. Discussion

In the present study, both types of transportation proved to be efficient for adult bullfrogs, as no mortality was observed. According to [7], efficient transportation in aquaculture is directly related to maintaining animals in appropriate conditions, allowing for high survival rates upon arrival at the destination. A 100% survival was also observed for adult bullfrogs transported for approximately 10 h [5]. However, these authors evaluated transportation in 100 mm PVC pipes. The transportation of adult bullfrogs typically occurs during pre-slaughter and the acquisition of animals for breeding stock formation. This process often induces stress in animals, leading to biochemical and erythrogram variables alterations, requiring different periods for the animals’ homeostasis to be restored after transportation [5]. Therefore, understanding the tolerance of bullfrogs and the recovery time from stress after transportation can help improve management protocols and prevent subsequent issues that may affect the health of the animals and the quality of the meat in cases where animals are sent to slaughterhouses.

The observed hyperglycemia in the first 12 h after transportation, in both evaluated transport conditions, suggests that bullfrogs mobilized energy to meet the increased energy demand caused by transportation stress. The process of aerobic glycolysis is one of the secondary responses observed in various species after stress, occurring due to the breakdown of hepatic glycogen promoted by the action of adrenaline and glucocorticoids [9,10,11]. Hyperglycemia was also observed in adult bullfrogs in the first hours after transport when they were handled in 100 mm PVC tubes for approximately 10 h [5]. Like the present study, these authors also observed a recovery in glucose levels, relative to control animals, starting 24 h after transportation. An increase in glucose concentrations was also observed in adult bullfrogs immediately after biometric management [12], a common practice in bullfrog farms for adjusting feed supply and screening frogs in pens for size classification. However, these authors observed the recovery of glucose levels, compared to the control group, within only 6 h after biometric management. Restoration of basal glucose levels 6 h after application of 100 µL of adrenaline in bullfrogs was also demonstrated by [13]. Therefore, the variation in the time to restore basal glucose levels is likely related to the type and intensity of the stressor.

The elevation of plasma protein levels occurred due to the increase in globulins observed in frogs shortly after transportation in both evaluated conditions. Probably, the increase in globulins is involved in enhancing the immune defense of these animals due to the challenging conditions of transportation, as also observed in adult bullfrogs during the hibernation period [14] and during crowding stress, where adult bullfrogs were kept in a burlap bag for 1 h and 30 min [15].

However, despite the decrease in globulin levels in frogs evaluated 48 h after transportation, there is an increase in albumin levels in these animals 12, 24, and 48 h after transportation, in both transport conditions (with or without foam). Since the frogs were transported for approximately 10 h and then placed in pens flooded with clean and continuously flowing water, it is very likely that they absorbed water to compensate for the period of water restriction during transportation. Ref. [16] demonstrated that Bufo marinus can rehydrate rapidly after dehydration, gaining a water volume of 30 to 50% of body weight. According to [17], this gain occurs through the skin, by osmosis, where water enters directly into the circulation. This rapid and large influx of water creates a situation of hypervolemia, which could lead to the loss of water from the intravascular to the interstitial space. However, as widely known, serum proteins play a role in maintaining osmotic balance between blood and tissue fluids [18], especially albumin, as this fraction exerts two to three times greater osmotic pressure than globulins due to its lower molecular weight [19]. Thus, in this study, the increase in albumin levels in both transport conditions probably occurred as a strategy for frogs to increase osmotic pressure and, consequently, reduce blood pressure. To control the situation and restore blood volume, [20] proposed the hypothesis that the expansion of the vascular compartment during hypervolemia would lead to an increase in the secretion of atrial natriuretic peptide (ANP) and/or brain natriuretic peptide (BNP). The elevation of these hormones would promote a condition of vasodilation and an increase in glomerular filtration rate, resulting in greater capillary permeability and, consequently, fluid passage from blood vessels to the bladder and interstitial space, restoring blood volume in these animals.

Despite the increase in albumin levels in frogs at 12, 24, and 48 h after transportation occurring in both transport conditions, a higher concentration of this protein fraction was observed in animals transported in boxes with foam, regardless of the evaluation time. This increase in albumin is possibly related to a higher oncotic pressure on blood vessels due to greater intravascular water entry in frogs transported in boxes with foam after transportation, when these animals were placed in flooded pens. This hypothesis is supported by the observed increase in mean corpuscular volume (MCV) and the decrease in mean corpuscular hemoglobin concentration (MCHC) in frogs transported in foam boxes.

The albumin-to-globulin ratio (A/G) remained balanced in animals evaluated up to 12 h after transportation, in both transport conditions. However, the decrease in globulin levels and the increase in albumin led to an increase in this ratio in frogs evaluated 24 and 48 h after transportation, in both transport conditions. An increase in the A/G ratio was also demonstrated in Rana catesbeiana after metamorphosis, due to the increased detection of albumin in the bloodstream [21]. On the other hand, [5] did not observe changes in the A/G ratio in adult bullfrogs for up to 48 h after transportation when these animals were transported in 100 mm pipes for approximately 10 h.

The mobilization of energy through lipid reserves in bullfrogs transported in both conditions also indicates the attempt of adaptation and tolerance of these animals to transportation-related procedures. In situations of stress, there is an increase in hormone-sensitive lipase, an enzyme responsible for breaking down triglycerides in adipocytes, promoting the release of fatty acids and glycerol into the circulation. In this study, part of the glycerol most likely went into gluconeogenesis, as an increase in glucose levels was observed for up to 12 h after transportation in both conditions. Another part, along with fatty acids, was directed to the liver for the re-esterification process of triglycerides. The increase in triglyceride concentration occurs due to the activity of phosphatidate-phospho-hydrolase in the liver, stimulated by the action of cortisol, catecholamines, and glucagon [22]. After the re-esterification process, triglycerides return to circulation transported by very-low-density lipoproteins (VLDL). In situations of stress and the consequent increase in plasma cortisol levels, there is a reduction in the activity of lipoprotein lipase [22,23] and hepatic lipase, leading to the accumulation of VLDL and low-density lipoprotein (LDL) in circulation [23], resulting in a condition of hypertriglyceridemia and hypercholesterolemia. However, in this study, only an increase in plasma triglyceride levels was observed, with no evidence of an increase in cholesterol concentrations. An increase in triglyceride levels was also found in adult bullfrogs immediately after biometric management [12]. These authors also demonstrated that triglyceride levels in frogs only decreased 24 h after transportation, similar to the present study, with frogs transported without foam. However, frogs transported in boxes with foam showed a decrease in triglyceride levels 12 h after transportation.

In bullfrogs evaluated immediately after transportation, the increase in hematocrit and hemoglobin occurred due to the higher number of erythrocytes. This is a common response in stressful situations, where there is an increased demand for oxygen by tissues [14]. According to [24], the elevation of hematocrit and hemoglobin favors the oxygen transport capacity in the blood and, consequently, the supply of this gas to the main organs in response to increased metabolic demand. In addition, higher concentrations of solutes in the blood, especially erythrocytes contributing to the increased hematocrit, may indicate a state of dehydration [25]. A similar response pattern was observed in adult bullfrogs, where an increase in the number of erythrocytes and hematocrit was immediately observed after biometric management [12]. However, these authors observed a return to normal levels of these variables 6 h after biometric management, similar to frogs transported in boxes with foam in the present study. Adult bullfrogs transported for approximately 10 h in PVC pipes also showed an increase in the number of erythrocytes immediately after transportation, returning to normal levels only 12 h after transportation [5], similar to the animals in the present study, transported in plastic boxes without foam.

After 48 h of transportation, the increase in mean corpuscular volume (MCV) in frogs transported in foam boxes probably occurred due to water entry into erythrocytes, promoting swelling in these cells. This explains the increase in hematocrit and the decrease in MCHC that occurred at the same evaluation time in both transport conditions. Similar to this study, a decrease in MCHC was demonstrated in adult bullfrogs evaluated 48 h after biometric management [12]. However, these authors did not show an increase in MCV and hematocrit in these animals.

5. Conclusions

Both conditions assessed, with foam and without foam, proved effective concerning the well-being of bullfrogs, as there was no mortality, and the restoration of homeostasis in these animals occurred between 12 and 24 h after transportation. The handling methods employed are suitable for the transportation of the species, provided that a density of 25 frogs/box (11.71 kg/box) is adhered to. However, transporting in foam-lined boxes, which is more labor-intensive and costly, did not significantly enhance the animals’ condition. Therefore, due to lower cost and practicality in management, the use of perforated boxes without foam is recommended for the transportation of adult bullfrogs.

We believe that a more limiting factor of the study is that the results are based on transport in a closed vehicle, which is the most recommended and usual. Transport in an open vehicle would certainly promote greater dehydration of the frogs, due to the action of the wind on the body surface of the animals, which could more severely compromise the recovery of their homeostasis and even their survival, depending on the transport time. In this case, a moistened substrate and/or making frequent stops to wet the animals could alleviate dehydration of these animals and would be strongly recommended.