Stabenau and Vietti: The physiological effects of multiple forced submergences of Caretta caretta 



891 



following the same rest interval between the first and 

 second submergence episodes, and pre- and postsubmer- 

 gence blood samples were collected as described above. A 

 seventh serial blood sample was collected 180 min after 

 the final submergence in all turtles. Blood samples were 

 also collected from nonsubmerged control turtles over the 

 same time intervals to ensure that repetitive handling and 

 blood sampling did not alter blood homeostasis. The blood 

 sampling technique and volume collected was identical to 

 that described for the laboratory component of the study. 



Blood and plasma analyses 



In the laboratory study, blood PcOj and pH were analyzed 

 immediately following collection by using a clinical blood 

 gas analyzer with electrodes thermostatted at 37°C. Both 

 variables were corrected to turtle cloacal temperature 

 using requisite correction factors for sea turtle blood and 

 plasma (Stabenau and Heming, 1994). In the field study, 

 blood gases (Po., and PcOr,) and pH were analyzed on the 

 trawl vessel immediately following collection using a 

 blood gas analyzer with electrodes thermostatted to turtle 

 body temperature (27-28. 5°C). The remaining analyses 

 were comparable for both the laboratory and field compo- 

 nents of the submergence study. Packed red cell volume 

 (hematocrit) was determined by following centrifugation 

 of heparinized microcapillary tubes. Two hundred micro- 

 liters of whole blood were then added to 10% trichloro- 

 acetic acid for lactate analysis. The deproteinized samples 

 were centrifuged, and the supernatant removed and stored 

 at -70°C. Lactate was determined spectrophotometrically 

 by using standard enzymatic techniques (Sigma, kit 826- 

 B, Saint Louis, MO). The remaining whole blood was then 

 centrifuged, the plasma removed and stored at -70°C. 

 Plasma Na+ and K* were measured with flame photometry 

 ( Jenway, model PFP7, Essex, England), and plasma CL was 

 determined with electrometric titration (Haake-Bucher, 

 model 4425000, Saddle Brook, NJ). Plasma glucose was 

 measured spectrophotometrically (Sigma, kit 16-20), and 

 plasma osmolality was determined with a vapor pressure 

 osmometer (Wescor, model 5500, Logan, UT). For the labo- 

 ratory study, plasma norepinephrine was analyzed with 

 HPLC (BAS, model LC-300, West Lafayette, IN). 



All data are expressed as means ±SE. Where appropri- 

 ate, the data was analyzed with one-way ANOVA. Post-hoc 

 comparisons between means were analyzed with Tukey's 

 multiple comparison test. A fiduciary level of P<0.05 was 

 regarded as significant. 



Results 



Blood pH, PcOj, and lactate 



The initial submergence of loggerhead sea turtles under 

 laboratory and field conditions produced a dramatic and 

 severe acidosis in all experimental turtles. Blood pH fell 

 an average of 0.54 ±0.03 (range 0.49 to 0.59 pH units) 

 and 0.63 ±0.06 (range 0.53 to 0.73 pH units) m labora- 

 tory turtles and field turtles, respectively, following ini- 



tial submergence (Figs. lA and 2A). The blood acidosis 

 was derived from respiratory and metabolic components 

 as evident from a positive proton-lactate deficit (Buffer 

 capacityx4pH-/l[Iactatel), and from significant increases 

 in blood PcOj and lactate (Figs. 1 and 2). The initial sub- 

 mergence also produced significant decreases in blood Pog 

 and increases in plasma norepinephine (P<0.05, n=24 for 

 Po., and n = ll for norepinephrine). In contrast, minimal 

 changes in blood pH, PcOg, and lactate were observed fol- 

 lowing collection of the first two blood samples in nonsub- 

 merged control turtles (Figs. 1 and 2). 



Recovery of the respiratory and metabolic derangements 

 in submerged turtles was dependent on the interval be- 

 tween successive submergences. A 10-min in-water rest 

 interval between the first and second submergence (treat- 

 ment-! and -4 turtles) permitted partial recovery of blood 

 pH (Figs. lA and 2A) and Pco,, (Figs. IB and 2B), but blood 

 pH remained significantly different from presubmergence 

 values. Washout of additional lactate was also detected in 

 these animals, whereby blood lactate concentration in- 

 creased higher than the postsubmergence value (Figs. IC 

 and 2C ). Turtles with a 42-min surface interval (treatment- 

 2 and -5 turtles) between the first and second submergence 

 had partial to complete recovery of blood pH (Figs. lA and 

 2A), complete recovery of blood Pco.^ (Figs. IB and 2B), and 

 slight recovery of blood lactate (Figs. IC and 2C). Only 

 the blood lactate remained significantly different from 

 the initial presubmergence value after the 42-min rest in- 

 terval. Turtles with a 180-min in-water recovery interval 

 (treatment-3 and -6 turtles) showed complete recovery of 

 blood pH and PcOr,, although the lactate concentration was 

 slightly higher than baseline levels (Figs. 1 and 2). Blood 

 Pog and plasma norepinephrine recovered completely re- 

 gardless of the surface interval (P>0.05, n=24 and n = ll 

 for Poj and norepinephrine, respectively). Nonsubmerged 

 control turtles in the laboratory and the field exhibited few 

 significant changes in blood pH, Pcoo, or lactate, whether 

 the interval between the second and third serial blood 

 sample was 10, 42, or 180 min (Figs. 1 and 2). 



The second 7.5-min submergence produced a drop in 

 blood pH and an increase in Pco,^ (Figs. 1 and 2) in all of the 

 experimental animals, and significant differences occurred 

 in treatment 2-6 turtles. It is noteworthy, however, that 

 the severity of the acid-base imbalance was not as drastic 

 as the acidosis measured following the first submergence. 

 The mean pH difference (4pH) between the second pre- and 

 postsubmergence ranged from 0.11 and 0.16 in treatment-1 

 and -4 turtles (animals with a 10-min interval between 

 submergences), respectively, to 0.50 in treatment-3 turtles 

 and 0.66 in treatment-6 turtles (animals with a 180-min 

 interval between submergences). The acidosis in treat- 

 ment-1 and -4 turtles resulted, in part, from the continual 

 elevation in blood lactate. In contrast, the longer surface 

 interval between the two submergence episodes resulted 

 in enhanced recovery of acid-base variables. Therefore, 

 the turtles with a surface interval of 42 or 180 min had in- 

 creased production of CO2 and lactate in relation to turtles 

 with a brief surface interval (Figs. 1 and 2). Comparable 

 changes in blood Poj and norepinephrine were measured 

 following the second submergence (P<0.05, n=24 and n=9 



