Previous morphological work on lizards suggests that the volume of growing eggs may result in a significant decrease in lung volume during gravidity. Iguanid lizard lungs are located within continuous thoracic and abdominal cavities and are highly distensible. Because of their distensible nature and lack of a diaphragm, both naturally occurring and introduced structures within the abdominal and thoracic cavities (i.e. organs, food, eggs) compress them and potentially reduce available lung volume for gas exchange. During reproduction, this decrease comes at a time of increased energetic demands, due to the cost of provisioning eggs and the physical burden of transporting the extra mass before laying. This means that females must increase the oxygen/carbon dioxide exchange with effectively smaller lung capacity than when they are not reproductive. Therefore, the way species compensate for this decrease affects performance, and ultimately the survival of individuals and their offspring.
This research has been focused on investigating how the burden of carrying eggs (gravidity) affects the respiratory physiology of two species of lizard, Crotaphytus collaris and Gambelia wislizenii. This work has been centered around two questions: 1) What is the respiratory response to morphological reductions in lung volume during reproduction in these two lizard species and, 2) Are these responses the same for two species of similar size and morphology but with differing activity levels?
Lizards were housed outside in screen cages (LLL Reptile) to allow access to natural light and climatic conditions. Cages were outfitted with sand, wood and rock refugia, and one end of the cage was covered with 18 by 12” board to provide shade. Lizards were fed crickets every other day throughout the period of the study. Crotaphytus collaris were grouped as two females to every male and G. wislizenii as one male to every female, when possible, to provide mating opportunities for both species. Females estimated to be close to oviposition were placed in smaller cages filled with moist perlite to provide an appropriate substrate for laying.
CO2 Production, Breathing Frequency and Tidal Volume Estimates:
Lizards were fasted for 48 hours prior to all measurements and placed in individual chambers in the dark at 33ºC for two hours to acclimate before recording. All procedures were performed between 2300 and 0400 during their normal resting phase, to minimize activity. We measured CO2 production using a flow-through respirometry system (Sable Systems, LI-COR). Following the recording of metabolic rate, tubing from each chamber was connected to a differential pressure transducer (Sable Systems), and breathing frequency and tidal (expired breath) volume were recorded. Tidal volumes were calibrated using a syringe to inject known volumes of air into an empty chamber and recording the resulting signal. Volumes approximating the lizards’ expected tidal volume range (from Templeton and Dawson 1963) were used for the calibration. The following day we recorded post-exercise tidal volumes (PETV) by placing an individual in a 3x2x3 plastic tub and encouraging the lizard to run for approximately one minute. Individuals who were reluctant to run were repeated placed on their back to force them to right themselves. These activities were used to induce active, forced breathing. Animals were immediately placed into chambers and tidal volume was recorded as above. PETV was measured at room temperature, which averaged 24.1 ± .12ºC (mean and SEM). Once all respiratory parameters were measured lizards were weighed, scanned with ultrasound imaging to determine reproductive stage (see Gilman and Wolf 2007 for procedures), fed, and returned to their respective cages.
For analyses, females were placed into one of five categories, based on the size and stage off the eggs (early follicles, late-stage follicles, early egg, shelled egg, post laying). Statistical analyses were performed on non-gravid-mass-specific (when appropriate) values using Kruskal-Wallis and Mann-Whitney non-parametric tests and significance was determined as P<0.05. Tests of significance for CT estimates of lung volumes were performed using 2-sample and paired t-tests and Mann-Whitney non-parametric tests (P<0.05).
Total Lung Volume:
We used Computed-Tomography (NanoSPECT/CT®) imaging to estimate lung capacity at two points during the reproductive cycle (gravid, post-laying) in four females (two of each species), and one point in the males. Two females and one male from each species were imaged. Individuals were fasted for 48 hours at 30 ºC and placed within cloth bags in a standard lab refrigerator for ~20 minutes and/or freezer for ~5 minutes until cool, to reduce activity. Lizards were then be taped to a cardboard restraint board with surgical tape (about the head body and limbs), to allow respiration but restrict other movement, and placed within a cloth bag on the imaging tray. Lung volume was estimated using the reconstructed CT data (NanoSPECT/CT, Bioscan, Washington, DC) by fitting 3-dimensional volumes to the interior of the lungs. A series of smaller volumes were fitted to the inside of the lungs and then summed to determine an estimate of total volume. Each sub-region spanned 8 slices of the reconstructed CT volume. Within each 8-slice sub-region, the estimation volume was drawn using a sum of the 8 slices as a guide. The region followed the border between the lungs and the body cavity of the subject. The axial length of each region was 3.6 mm (0.4 mm x 8).
Lizards were hand-caught or noosed and placed in cloth bags for transport to the lab.
* Instrument Name: CO2/H2O analyzer
* Manufacturer: LI-COR Biotechnology, Lincoln, NE
* Model Number: LI-7000
* Instrument Name: Respirometry Multiplexer, Universal Interface, Pressure Meter
* Manufacturer: Sable Systems, Las Vegas, NV
* Model Number: V2.0, UI2, PT-100B
*Instrument Name: Omega flowmeters,
* Manufacturer: Omega Engineering, Inc., Stamford, CT
* Model Number: FL-3402C and FL-3403G
The range of data values were double-checked.