In the present study, both types of transportation proved to be efficient for adult bullfrogs, as no mortality was observed. According to Purbosari et al. (2019), efficient transportation in aquaculture is directly related to maintaining animals in appropriate conditions, allowing for high survival rates upon arrival at the destination. Santos et al. (2021) also demonstrated 100% survival of adult bullfrogs when transported for approximately 10 hours. 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 (Santos et al. 2021). 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 hours 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 (Harri 1981; Romero 2002; Rosenthal and DeRoos 1985). Santos et al. (2021) also observed hyperglycemia in adult bullfrogs in the first hours after transportation when they conducted this management in 100 mm PVC pipes for approximately 10 hours. Like the present study, these authors also observed a recovery in glucose levels, relative to control group animals, starting 24 hours after transportation. Santos et al. (2023) observed an increase in glucose concentrations in adult bullfrogs immediately after biometric management, 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 hours after biometric management. Herman (1977) also found the restoration of basal glucose levels 6 hours after the application of 100 µl of adrenaline. 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 (Peng et al. 2016) and during crowding stress, where adult bullfrogs were kept in a burlap bag for 1 hour and 30 minutes (Alves et al. 2022).
However, despite the decrease in globulin levels in frogs evaluated 48 hours after transportation, there is an increase in albumin levels in these animals 12, 24, and 48 hours after transportation, in both transport conditions (with or without foam). Since the frogs were transported for approximately 10 hours 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. Hillyard (1999) demonstrated that Bufo marinus can rehydrate rapidly after dehydration, gaining a water volume of 30 to 50% of body weight. According to Word and Hilman (2005), 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 (Bell et al. 1965), especially albumin, as this fraction exerts two to three times greater osmotic pressure than globulins due to its lower molecular weight (Frieden 1961). 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, Donald and Trajanovska (2006) 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 hours after transportation occurring in both transport conditions, a higher concentration of this protein fraction was observed in animals transported in foam boxes, 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 foam boxes 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 hours 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 hours after transportation, in both transport conditions. Herner and Frieden (1960) also demonstrated an increase in the A/G ratio in Rana catesbeiana after metamorphosis, due to the increased detection of albumin in the bloodstream. On the other hand, Santos et al. (2021) did not observe changes in the A/G ratio in adult bullfrogs for up to 48 hours after transportation when these animals were transported in 100 mm pipes for approximately 10 hours.
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 hours 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 (Brindley et al. 1993). 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 (Niaura et al. 1992; Brindley et al. 1993) and hepatic lipase, leading to the accumulation of VLDL and low-density lipoprotein (LDL) in circulation (Niaura et al. 1992), 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. Santos et al. (2023) also found an increase in triglyceride levels in adult bullfrogs immediately after biometric management. These authors also demonstrated that triglyceride levels in frogs only decreased 24 hours after transportation, like the present study, with frogs transported without foam. However, frogs transported in foam boxes showed a decrease in triglyceride levels 12 hours 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 (Peng et al. 2016). According to Pakhira et al. (2015), 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 percentage, may indicate a state of dehydration (Voyles et al. 2012). 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 (Santos et al. 2023). However, these authors observed a return to normal levels of these variables 6 hours after biometric management, similar to frogs transported in foam boxes in the present study. Adult bullfrogs transported for approximately 10 hours in PVC pipes also showed an increase in the number of erythrocytes immediately after transportation, returning to normal levels only 12 hours after transportation (Santos et al. 2021), like the animals in the present study, transported in plastic boxes without foam.
After 48 hours 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. Like this study, a decrease in MCHC was demonstrated in adult bullfrogs evaluated 48 hours after biometric management (Santos et al. 2023). However, these authors did not show an increase in MCV and hematocrit in these animals.