Stress and recovery of american bullfrog after biometry management: biochemistry and erythrogram responses

The aim was to evaluate the recovery of bullfrog homeostasis after biometry, a routine management. An experiment in a completely randomized design was conducted with six treatments and 10 repetitions: the bullfrog being the experimental unit. Five treatments consisted of evaluating the bullfrog recovery times after biometry (0 - immediately after biometry, 6, 12, 24 and 48 h) and a control (animals in the pen before biometry). Sixty bullfrogs (285.33 ± 10.00 g) were subjected to a 12-h fasting. Subsequently, 50 animals underwent biometry and 10 were used for blood collection before biometry (control). A significant increase was observed in all variables analyzed for animals subjected to density stress, except for total proteins, globulin, and hemoglobin. There was a significant increase in blood glucose and erythrocyte numbers in the subjects immediately after biometry. Plasma proteins and globulin had no significant difference in any of the groups that underwent biometry. Lactate, albumin, and triglycerides levels were significantly elevated in animals shortly after biometry and remained elevated until 12 h after management. The results showed that 24 h after biometry stress, all the analyzed variables were already at similar levels as to the levels of control group animals.


Introduction
The bullfrog (Lithobates catesbeianus) is among the most cultivated frog species in the world for human consumption. Bullfrog meat has a protein content between 16 and 19%, which is compared to other lean white meats, but it has less fat, around 0.6 to 0.7% (Cribb et al. 2013), while chicken meat has 14% fat.
For commercial production, a bullfrog farming needs three distinct sectors to meet the needs of bullfrogs during their lifetime: the reproduction, the tadpole rearing, and the fattening sectors (Lima 2012). During the cultivation, several managements are carried out with the animals, such as transfers between sectors, transport to other properties, biometry, and screening of the animals for classification by size. In the fattening sector, biometry and screening are important for adjusting the feed supply and to guarantee the uniformity of the lots, thus avoiding the cannibalism of larger animals over smaller ones (Cribb et al. 2013;Seixas-Filho et al. 2017). In the fattening sector, biometry and screening should be performed weekly (Seixas-Filho et al. 2017) or at most biweekly (Cribb et al. 2013), to avoid cannibalism losses and improve animal performance. However, biometry and screening can be considered stressful and traumatic procedures for exposing the animals to a series of managements such as catching, handling and densification.
In amphibians, stress promotes responses from the sympathetic nervous system and the hypothalamic-pituitary-interrenal axis (Andrade et al. 2016). These responses can be divided into three phases: primary, secondary, and tertiary. In the primary response, stress hormones, catecholamines and glucocorticoids are produced. In the secondary response, the action of these hormones occurs at the tissue and blood level, causing physiological, metabolic, structural, and hematological changes as an attempt to restore homeostasis. If the stressor persists, in the tertiary response there is a state of exhaustion of the organism and the population in which growth, reproduction, immune response and the ability to tolerate new stress are reduced. (Rollins-Smith 2017).
Thus, even in situations in which there is no mortality, knowledge of the restoration of homeostasis after stressful operations, such as biometry and screening, can bring important results for the establishment of adequate management practices in bullfrog farming and for the prevention of losses. Therefore, the purpose of the study was to evaluate the recovery of bullfrog homeostasis after performing biometry.

Local and experimental design
The experiment was carried out in the Bullfrog Farming Sector of the Aquaculture Laboratory of the Veterinary School of the Federal University of Minas Gerais, Belo Horizonte, Minas Gerais, Brazil. The project was approved by the Ethics Committee on the Use of Animals according to the protocol # 275/2019.
The experiment was carried out in a completely randomized design with six treatments and 10 repetitions, with the bullfrog (Lithobates catesbeianus) being the experimental unit. The treatments consisted of five evaluation times to assess the recovery of bullfrogs' homeo-stasis after biometry (0 -immediately after biometry, 6, 12, 24 and 48 h after biometry) and a control (condition of the animal in the pen before biometry).

Animals and experimental conditions
Sixty specimens of bullfrog with an average weight of 285.33 ± 10.00 g were used. The animals were randomly distributed in two pens in a flooded system (1.15 × 1.06 m) at a density of 30 bullfrogs per pen, where they underwent an acclimatization period of 15 days. The bullfrogs were subjected to a 14 L:10D photoperiod (L -hours of light, D -hours of dark). Air (26.5 ± 2.5 ° C) and water (20.4 ± 0.6 ° C) temperatures were measured daily, three times a day (8:00, 12:00 and 16:00), using a digital thermometer. The maximum (29.3 ± 0.8 ° C) and minimum (21.9 ± 0.7 ° C) air temperature and the maximum (21.2 ± 0.4 ° C) and minimum (19. 5 ± 0.5 ° C) water temperature were also measured daily. During this period, the bullfrogs were fed until apparent satiety with commercial feed for carnivorous fish (40% crude protein, 8% ether extract, 3% fibrous matter and 13% mineral matter), three times a day (8:00, 12:00 and 16:00). The pens were cleaned twice a day, one hour before the first and the last feeding, with total drainage and replacement of water equal to the initial volume (5 cm water depth). After the acclimatization period, the animals were fasted for 12 h. Soon after, 10 bullfrogs were randomly selected in the pens to collect blood samples from. The data from the collected blood samples evaluated would serve as a reference for animals kept in the pen (control group animals). Immediately after collecting samples from the control group animals, the remaining 50 animals were placed in a raffia bag and weighed on a portable scale with a capacity of 50 kg and an accuracy of ± 10 g. Biometry handling started in the morning at 7:50 and ended at 8:15. After biometry, the bullfrogs were returned to their original flooded pens. Then, 10 bullfrogs were randomly selected for a blood draw from the 0 h evaluation time (immediately after biometry). The other blood draws took place in the same manner for times 6, 12, 24 and 48 h after the animals' biometry. In each evaluation period, the time to collect blood from the first and tenth frogs was approximately 5 min. After blood collection, the bullfrogs were housed in a third pen so there was no risk of collecting blood from the same bullfrog more than once.

Blood collection and blood analysis
Blood collection was performed through the posterior limb by puncture with 3 mL syringes and needles previously moistened in 10% EDTA. Approximately 2 mL of blood were collected per animal. Lidocaine (50 mg g − 1 ) was used as a local topical anesthetic. With a 10 µL aliquot, the glycemia of the animals was evaluated using a digital glucometer (ACON, On-Call® Plus, San Diego, USA). The remaining blood was stored in 2 mL microtubes under 4 °C refrigeration for further analysis. The hematocrit (Ht) was determined by the microhematocrit method, by means of centrifugation (12,000 rpm for 5 min) in a microhematocrit centrifuge (Microspin® model SPIN 1000). Total hemoglobin (Hb) was determined using the Drabikin reagent, by the cyanomethahemoglobin method, with a reading on a Biochrom spectrophotometer (Libra S22) with a wavelength of 540 nm. With the aid of a ProWay® light microscope (XSZ-PW206BT), the total erythrocyte count (Er) was performed in the Neubauer chamber. Hematimetric indices were calculated as follows: Mean corpuscular volume -MCV (fL) = Ht x 10/ Er; Mean corpuscular hemoglobin -MCH (pg) = Hb x 10/ Er; Mean corpuscular hemoglobin concentration -MCHC (g dL − 1 ) = Hb x 100/ Ht. The rest of the collected blood was centrifuged at 3000 rpm for 15 min in a microtube centrifuge (Spinlab® model SL-5AM) and then the plasma was collected and stored at − 80 ° C. Subsequently, analyses were performed to determine the concentrations of lactate (

Statistical analysis
At the end, the data was analyzed in Software R 3.5.3, and submitted to Shapiro Wilk and Bartlett's tests to assess the normality and homoscedasticity of the variances, respectively. To meet normality, the albumin and triglyceride variables were transformed into a natural log (ln), the A / G ratio was transformed into an inverse square root (x) − 0,5 . The data was then submitted to an ANOVA which, when significant (P < 0.05), was followed by a 5% Dunnett test to verify the difference in the variables of each time after the biometry in comparison the control group. The cholesterol was subjected to the Kruskal Wallis nonparametric test, as it did not meet normality and homoscedasticity.

Biochemical variables
The glucose of the bullfrogs evaluated immediately after biometrics (time 0 h) was significantly higher (P < 0.01) than that measured in the animals of the control group. On the other hand, the glycemia of the bullfrogs evaluated 6, 12, 24 and 48 h after biometry did not differ (P > 0.05) from the specimens in the control group (Fig. 1).
The lactate of the bullfrogs evaluated immediately after biometry was significantly higher (P < 0.01) than that measured in the animals of the control group and remained high (P < 0.05) in the bullfrogs evaluated at times 6 and 12 h after the biometry. However, the lactate of the bullfrogs evaluated 24 and 48 h after biometry did not differ (P > 0.05) from the control group (Fig. 1).
Both plasma proteins and globulins did not undergo significant changes (P > 0.05) at any time evaluated after biometry in relation to the values of the animals in the control group (Fig. 1).
The albumin of the bullfrogs evaluated at time 0 and 12 h after the biometry suffered a significant increase (P < 0.01) in relation to the values of the animals in the control group, as well as the albumin of the bullfrogs evaluated 6 h after the biometry (P < 0.05). The albumin of the bullfrogs measured in the evaluation time 24 h after the biometry did not differ in relation to the animals in the control group (P > 0.05). However, 48 h after biometrics, there was a significant decrease (P < 0.01) in albumin levels compared to the animals in the control group (Fig. 1).
The albumin / globulins ratio of the bullfrogs assessed at 0 and 6 h after biometry increased significantly (P < 0.01) in relation to the animals in the control group and remained high (P < 0.05) in the bullfrogs assessed at 12 h after biometry. The albumin / globulins ratio of the bullfrogs evaluated 24 h after biometry did not differ in relation to the animals in Fig. 1 Bars represent mean ± standard deviation of the variables glucose, lactate, plasma proteins, albumin, globulin, and ratio albumin/globulin of bullfrogs after biometry. CT -control treatment (animals in homeostasis sampled before biometry); 0 -animals sampled immediately after biometry; 6 -animals sampled 6 h after biometry; 12 -animals sampled 12 h after biometry; 24 -animals sampled 24 h after biometry; 48 -animals sampled 48 h after biometry. ** Significant difference (P < 0.01) in relation to the control treatment by the Dunnett's test. * Significant difference (P < 0.05) in relation to the control treatment by the Dunnett's test the control group (P > 0.05). By comparison, 48 h after biometry there was a significant decrease in this variable (P < 0.05) in relation to the animals in the control group (Fig. 1).
The triglycerides of the bullfrogs evaluated at times 0, 6 and 12 h after biometrics were significantly higher (P < 0.01) than the levels of the animals measured in the control group. Alternatively, the triglycerides of the frogs evaluated 24 and 48 h after biometrics did not differ (P > 0.05) from the levels of the specimens in the control group (Fig. 2).
The total cholesterol concentration of the bullfrogs evaluated immediately after the biometry was significantly higher (P < 0.05) than that of the animals measured in the control group. By contrast, the total cholesterol levels of the bullfrogs evaluated 6, 12, 24 and 48 h after biometry did not differ (P > 0.05) from the control treatment specimens (Fig. 2).

Erythrogram variables
The number of erythrocytes and hematocrit of the bullfrogs evaluated immediately after the biometry showed a significant increase (P < 0.01) in relation to the values of the bullfrogs in the control group. On the other hand, these two variables evaluated at times 6, 12, 24 and 48 h after biometry did not differ (P > 0.05) from the control treatment specimens (Fig. 3).
Hemoglobin only changed at 48 h after biometry, where it suffered a significant decrease (P < 0.05) in relation to the frogs in the control group. The MCV changed only in the bullfrogs evaluated 6 h after biometrics, showing a significant decrease (P < 0.01) in relation to the animals in the control group (Fig. 3).
The bullfrogs evaluated 6 and 48 h after biometry, experienced a significant decrease (P < 0.01) in MCH compared to animals in the control group. The bullfrogs in the other evaluation periods did not show a significant difference (P > 0.05) compared to the control treatment specimens (Fig. 3).
Soon after biometrics and 48 h after biometrics, there was a significant decrease (P < 0.05) in MCHC in relation to the bullfrogs in the control group. The animals in the other evalua- Fig. 2 Bars represent mean ± standard deviation of the variables triglycerides and total cholesterol of bullfrogs after biometry. CT -control treatment (animals in homeostasis sampled before biometry); 0 -animals sampled immediately after biometry; 6 -animals sampled 6 h after biometry; 12 -animals sampled 12 h after biometry; 24 -animals sampled 24 h after biometry; 48 -animals sampled 48 h after biometry. Significant difference ** Significant difference (P < 0.01) in relation to the control treatment by the Dunnett's test. * Significant difference (P < 0.05) in relation to the control treatment by the Dunnett's test tion times did not show significant difference (P > 0.05) to the control treatment specimens (Fig. 3).

Fig. 3
Bars represent mean ± standard deviation of the variables number of erythrocytes (RBC), hematocrit, hemoglobin, mean corpuscular volume (MCV), mean corpuscular hemoglobin (MCH), and mean corpuscular hemoglobin concentration (MCHC) of bullfrogs after biometry. CT -control treatment (animals in homeostasis sampled before biometry); 0 -animals sampled immediately after biometry; 6 -animals sampled 6 h after biometry; 12 -animals sampled 12 h after biometry; 24 -animals sampled 24 h after biometry; 48 -animals sampled 48 h after biometry. ** Significant difference (P < 0.01) in relation to the control treatment by the Dunnett's test. * Significant difference (P < 0.05) in relation to the control treatment by the Dunnett's test

Discussion
As with all other vertebrates, amphibians develop physiological reactions to promote their survival during stressful events (Davis and Maerz 2011). In situations of stress in bullfrogs, adrenaline is released, and one of the roles of this hormone is the rapid rise in plasma glucose levels in response to the stressor (Herman 1977). In the present study, the increase in plasma glucose levels of the bullfrogs evaluated immediately after biometry indicates that the animals had an increased energy demand shortly after stress, mobilizing glycogen and releasing glucose into the bloodstream. The increase in glycemia was also observed by MbangKollo and deRoss (1983) when bullfrogs were submitted to different levels of adrenaline and noradrenaline; Harri (1981), when specimens of Rana temporaria were subjected to agitation in the laboratory, using an automatic stirrer (60 cps / min) for 1 h; Santos et al. (2021) when bullfrog specimens were transported for approximately 10 h; and Alves et al. (2022) when bullfrogs of the same species were densified for 1h30min. in raffia bag.
The decrease in glycemia to the same levels of the animals in the control group from the evaluation at 6 h after biometry indicates that, in relation to this variable, the animals recovered quickly from the management stress. A similar behavior was observed in a bullfrog study in which the application of 100 µl of adrenaline promoted an increase in glucose levels, returning to baseline levels 6 h after application (Herman 1977). A study of the transport of bullfrogs showed a return to baseline blood glucose levels just 24 h after transport (Santos et al. 2021). In this case, the longer recovery time of the frogs' basal glucose levels after transport was probably due to the longer exposure of these animals to the stressor.
Anaerobic glycolysis results in lactate production through muscle glycogen mobilization by the enzyme lactate dehydrogenase (LDH). LDH catalyzes the conversion of pyruvate to lactate, an important source of energy for the muscles (Alves et al. 2022;Bennett and Licht, 1974;Santos et al. 2021). In the present study, the lactate levels of the frogs analysed immediately after biometrics increased more than three times the concentrations of the control animals, proving to be a good stress indicator for these animals. Increased lactate levels were also observed in wild specimens of Rana temporaria captured during the winter in Finland and subjected to an acclimatization in the laboratory for 1 h at 28 ºC (Harri 1981); in bullfrogs when they received an infusion of 500 µg / kg of norepinephrine and with the same dose of epinephrine (MbangKollo and deRoos, 1983); in bullfrog specimens analyzed immediately after transport for approximately 10 h (Santos et al. 2021); and after densification of bullfrogs for 1h30min. in nylon bags (Alves et al. 2022).
In the present study, 24 h after biometry, the bullfrogs had already restored baseline plasma lactate standards. On the other hand, a study with bullfrog transport showed that bullfrog lactate levels returned to baseline levels 6 h after transport (Santos et al. 2021). Hutchison and Turney (1975), demonstrated that the lactate levels in Rana pipiens submitted to electrical stimulus are restored to baseline levels 4 h after treatment. The same period required for oxygen consumption to return to baseline levels after stimulus. This difference in recovery time is probably associated with the type and intensity of the stressor agent. In the present study, the frogs submitted to biometric management, in addition to exposure to a high density in the nylon bag, remained in a condition of lower oxygen availability until the management was carried out. This condition probably contributed to a greater mobilization of lactate and, consequently, a longer recovery time for this variable. In this circumstance the lactate can be preferentially oxidized, instead of converted into forms of storage, like hepatic glycogen. However, recovering muscle glycogen stores after a period of activity is important for anurans since they may be needed as a substrate in the production of anaerobic energy in case of future need (Withers et al. 1988).
Among the roles of plasma proteins are the transport of hormones, vitamins, and lipids in the bloodstream. They are also responsible for maintaining the balance between blood and tissue fluids, maintaining blood volume through osmoregulation, functions mainly attributed to albumin (Coppo et al. 2005;Frieden 1961). In the bullfrogs in which biometry were performed, there was no increase in the levels of plasma proteins or globulins. However, the albumin concentration increased immediately after biometry and remained elevated for up to 12 h, restoring normal conditions in just 24 h after handling. In this case, the increase in albumin levels probably occurred to promote the transport of free fatty acids (Coppo et al. 2005) resulting from the mobilization of triglycerides from adipose tissue for use as an energy source under stress conditions. The increase in the concentration of albumin can also be attributed to an adaptation in the osmoregulatory process (Alves et al. 2022) in a situation of preventing water loss during biometry. According to the same authors, an increase in albumin levels was also demonstrated in bullfrogs submitted to densification stress in a nylon bag for 1h30min. A similar response occurs during metamorphosis, a time considered critical for these animals, with the increase in the concentration of albumin, responsible for increasing the osmotic pressure of the blood and its water retention capacity, an important adaptation considering the transition of amphibians in the environment aquatic to terrestrial (Duellman and Trueb, 1986;Frieden 1961). A study with the species Rana temporaria, Bombina variegata and Bufo bufo found a rapid increase in the concentration of total proteins and albumin as the metamorphosis occurred (Chen 1970). In addition, albumin is an excellent indicator of protein biosynthesis and functions as an excellent reserve of amino acids (Coppo et al. 2005). Therefore, the decrease in albumin of the bullfrogs evaluated 48 h after biometry may be related to a mobilization of amino acid reserves for gluconeogenesis due to the long fasting period that these animals were submitted to before biometry until the end of blood collection. Similar behavior was observed in a study with bullfrog transport, in which albumin levels also decreased 48 h after transport (Santos et al. 2021).
The maintenance of the A / G ratio indicates a balance between the plasma protein fractions, this balance being maintained until the stressors' compensation mechanisms fail (Gras, 1983). The increase in the ratio at times 0, 6 and 12 h after the biometry stress is due to the increase in the albumin concentration at the same evaluation times. Likewise, the decrease in the A / G ratio 48 h after biometry is attributed exclusively to the decrease in albumin levels that probably served as an amino acid reserve for gluconeogenesis.
During circumstances of increased energy demand, such as a stressful situation, animals can mobilize energy through triglycerides (Byrne and White 1975), releasing fatty acids and glycerol into the bloodstream (Alves et al. 2022). In addition, in these circumstances, the liver increases the production of triglycerides, making this metabolite available in the bloodstream (Brindley et al. 1993). Increase that was observed in present study for up to 12 h after the biometry of bullfrogs. In these circumstances, cortisol, catecholamines and glucagon stimulate phosphatidate phosphohydrolase, promoting an increase in the synthesis of triglycerides in the liver, making this metabolite available in the bloodstream (Alves et al. 2022;Brindley et al. 1993;Santos et al. 2021). The presence of cortisol, norepinephrine and fatty acids, also promotes a reduction in insulin sensitivity (Alves et al. 2022;Brindley et al. 1993;Niaura et al. 1992;Santos et al. 2021), which results in a decrease in the activ-ity of lipoprotein lipase, decreasing the uptake of triglycerides in peripheral tissues (Rizza et al. 1982). Under stress, there is also a reduction in hepatic lipase activity, which results in increased triglyceride transporter in the bloodstream (Niaura et al. 1992). An increase in triglycerides levels was also observed in salamander Batrachupems tibetanus exposed to low temperatures (Xia and Li 2010) and adults of bullfrog that were restraint in nylon bag for 1h30min and in frogs exposed to low temperatures (Alves et al. 2022).
The increase in cholesterol levels in bullfrogs immediately after biometry stress is probably related to three reasons: increased enzyme 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMG-CoA reductase) in the liver, reduced lipoprotein lipase and to prevent water loss. In a stressful situation, the presence of glucocorticoids and free fatty acids in the circulation stimulates the liver to increase the production of the enzyme HMG-CoA reductase, consequently increasing the synthesis of cholesterol. In these circumstances there is also a reduction in the activity of lipoprotein lipase, due to the decrease in insulin sensitivity (Alves et al. 2022;Brindley et al. 1993;Niaura et al. 1992;Santos et al. 2021), which promotes the reduction of uptake of low-density lipoproteins (LDL) in peripheral tissues, increasing cholesterol and triglycerides levels in the circulation (Alves et al. 2022;Rizza et al. 1982;Santos et al. 2021). In addition to an energy source, the increase in total cholesterol levels in bullfrogs immediately after biometry may have been a mechanism to prevent the organism from possible water restriction during biometry management. According to Alyousif (1991), the amount of cholesterol present in the cell membrane is related to the ability of water to diffuse through it, and cholesterol is therefore especially important for the waterproofing of tissues that are in direct contact with body fluids. Increased cholesterol levels have also been reported by Pãunescu and Ponepal (2011) in adults of Pelophylax ridibundus treated with the herbicide Roundup® and in adults of bullfrog exposed low temperatures (Alves et al. 2022).
Erythrocytes are the blood cells responsible for transporting oxygen through hemoglobin (Maekawa and Kato 2015). Therefore, in the present study, the increase in the number of hematocrit and erythrocytes immediately after biometry is likely to promote greater oxygen transport in these circumstances. Hematocrit increase was also observed when bullfrog adults were evaluated 15 min. after a brief manipulation of the animals for 2 min. (Mbang-Kollo and deRoos, 1983) and when were restraint in nylon bag for 1h30min. (Alves et al. 2022). According to Boutilier and Shelton (1986), there is an intense recruitment of young erythrocytes from hematopoietic organs due to increased oxygen demand in a condition of acute stress. The finding of the presence of these young erythrocytes happened 6 h after the biometrics, since the bullfrogs evaluated at this time had smaller volume erythrocytes (MCV). The increase in the number of erythrocytes immediately after biometry is also accompanied by a decrease in MCH, demonstrating a direct relationship between the size of the erythrocyte (MCV) and the amount of hemoglobin present in it. Despite the decrease in the amount of hemoglobin per erythrocyte (MCH) in bullfrogs evaluated 6 h after biometry, the reduced MCV provided an increase in the concentration of hemoglobin per erythrocyte (MCHC). Increased numbers of erythrocytes and decreased MCV have also been demonstrated in adults of L. catesbeianus submitted to low temperatures (Palenske and Saunders 2003) and with adults of same specie after transport (Santos et al. 2021). According to Boutilier and Shelton (1986), the increase in the number of erythrocytes and the reduction in MCV may also be associated with a situation of dehydration. However, the bullfrog biometry management in the present study was performed in a short time interval (25 min.), probably not being enough to cause a significant water loss in these animals. Furthermore, after the biometry management, the frogs returned to the flooded pens, constantly being in contact with the water. Already the decrease in hemoglobin, MCH and MCHC 48 h after biometry may be related to total fasting time was very prolonged, considering the intervals before and after biometry.

Conclusion
The stress caused by biometry in bullfrogs led to increased demand for oxygen and energy in these animals. The results show that these demands were quickly met by adjustments in plasma hematological and biochemical variables, so that 24 h after stress, all the variables analyzed were already at levels like the levels of animals in the control group.