Fig 1.
Experimental design for studying the effect of orally-delivered GnRH on tiger salamander sperm production.
Male tiger salamanders (Ambystoma tigrinum) (n = 11) were cycled through Prime-Only, Low, Medium, and High GnRH oral administration hormone treatments. Response variables included both sperm quantity and quality measures and parameters were measured at 1, 3, 6, 9, 12, and 24 hours after the resolving dose.
Fig 2.
Diagram outlining the protocol for delivering hormones orally to caudates.
Below: A) Cricket being emptied using a pipette; B) Tiger salamander (Ambystoma tigrinum) being fed a treatment cricket; C) Collection of spermic material from the cloaca of a male A. tigrinum; and D) A. tigrinum sperm cell under 200x magnification.
Fig 3.
Live/dead stain of salamander sperm using SYBR14/propidium iodide to indicate sperm sample viability.
Damaged cells fluoresce red whereas intact cells fluoresce green.
Fig 4.
Comparison of sperm sample concentrations between treatments.
(A) Number of spermic urine (blue) versus milt (red) samples for each hormone treatment. The dashed line highlights the average milt concentration between each treatment, whereas the dotted line highlights the average spermic urine concentration. Letters indicate significant differences within and between sample types. (B) Concentration of sperm samples averaged across sample type and time points for each hormone treatment. Boxes represent the interquartile range, whiskers show the range of the data, the horizontal bars indicate the median, the orange squares represent the mean, and different letters indicate significant differences between treatments (p < 0.05).
Table 1.
Sperm characteristics compared between oral hormone treatments.
Fig 5.
(A) Total motility; (B) quality of movement; (C) viability; and (D) normal morphology of samples collected from male tiger salamanders (Ambystoma tigrinum) comparing Prime-Only, Low, Medium, and High GnRH oral hormone treatments. Boxes represent the interquartile range, whiskers show the range of the data, the horizontal bars indicate the median, the orange squares represent the mean, and different letters indicate significant differences (p < 0.05).
Fig 6.
Time course showing the change in mean ± SEM in (A) sperm concentration, (B) total motility, (C) quality of movement, and (D) normal morphology over time across treatments. Samples were collected at 1, 3, 6, 9, 12, and 24 hours post-resolving dose. Note the episodic surges in sperm concentration.
Fig 7.
Application of the medium (0.25 μg/g prime + 1.0 μg/g resolving dose) oral hormone dose in a smaller plethodontid species, Aneides lugubris.
Time course showing the change in mean sperm concentration and total motility over time. Samples were collected 1, 3, 5, 7, and 24 hours post-resolving dose. Data points with error bars indicate time points where samples from two individuals were collected. Data points without error bars indicate data were recorded for only one male.