Genetic variation of the endangered Puerto Rican crested toad (PRCT;Peltophryne lemur) has dwindled over time in both wild and captive populations, leading to long-term sustainability issues for the recovery program. To address this challenge, we propose that PRCTs can be used as a model species to show how in-situ and ex-situ populations can be linked through sperm biobanking and gamete transfer, expanding genetic variability of both populations. Male toads (n = 10) in Guayanilla, Puerto Rico were administered human chorionic-gonadotropin (hCG) and luteinizing-hormonereleasing-hormone analog (GnRHa) to stimulate spermiation. Sperm was collected noninvasively from 9/10 males, cryopreserved in 10% trehalose with either 10% N,N-dimethylformamide (DMFA) or 10% dimethyl sulfoxide (DMSO), and transported in liquid nitrogen vapor to the National Amphibian Genome Resource Bank at Mississippi State University. Ultrasonography was used to identify females (n = 3) with mature oocytes for GnRHa-induced oviposition of eggs for in-vitro fertilization (IVF). Post-thaw sperm motility was 28% and 25% for sperm cryopreserved with DMFA or DMSO, respectively. Of the 9,672 eggs used for IVF, 4% (n = 306/6,981) were fertilized with frozenthawed sperm, compared with 20% (n = 525/2691) fertilized with fresh sperm controls. Overall, 46 toadlets were produced from frozen-thawed sperm. After 4.5 months of headstarting, 14 juvenile toads produced from various genetic crosses using frozen-thawed sperm were released to new sites in the wild, introducing unique genetic representation and new founder lines. After 1.5 years, 24 adult toads produced using frozen-thawed sperm continue to thrive in the captive collection, and one of these males has now produced an F2 generation of offspring with 5,085 tadpoles released to the wild. This transformational study is the first to produce reproductively viable adult PRCTs using cryopreserved sperm from hormonally-induced wild males and captive females, introducing innovative methods that link in-situ and ex-situ populations of endangered amphibians to revolutionize genetic management.

No abstract

No abstract

The capability of near-infrared spectroscopy (NIR) to detect biomolecules in aqueous solutions, a sub-field of NIR called aquaphotomics, has yet to be fully explored. Aquaphotomics references water absorbance patterns and wavelength shifts in the 1st overtone of the water spectrum as they change patterns with solute composition and concentrations. Recently, NIR was used as a rapid method for detecting oestrus in Holstein raw milk and for monitoring reproductive stages in urine of the Bornean Orangutan (Pongo pygmaeus) and the giant panda (Ailuropoda melanoleuca). Near-infrared spectroscopy detects bond vibrations from organic molecules and water to create unique absorbance patterns, which are used to profile complex mixtures of biomolecules. The objectives of this study are to (1) characterise serum NIR spectral profiles for oestrus, metestrus, and diestrus in mares, and (2) determine if NIR can accurately decipher these reproductive phases from serum due to the biochemical effects of reproductive hormones. Mare oestrus cycles were assessed every other day by ultrasound and serum hormone analysis. Serum was collected via jugular venipuncture on day 0, 2, 4, 8, and 16 of each cycle for 3 consecutive cycles from each mare. Radioimmunoassay analysis of reproductive hormones E2 and P4 was used to validate and characterise the NIR spectra. Spectra were collected from triplicate samples of 200 μL of serum in a 1-mm-path-length quartz cuvette with an ADS FieldSpec®3 spectrophotometer, at room temperature (22°C). Chemometric analysis (Unscrambler® X v.10.4; CAMO Software) included pretreatment with a Savitsky-Golay second-derivative function for visual inspection of spectral features and principal component analysis (PCA), after mean centering, for distinction of reproductive status. Spectral peaks at 1347, 1367, and 1465 nm were unique to serum collected from mares in oestrus exhibiting high E2 (11.87 to 16.88 pg mL−1). Early metestrus is characterised by prominent spectral peaks at 1383 and 1437 nm, corresponding to E2 levels (0.02 to 5.29 pg mL−1) and P4 levels (0.02 to 3.61 ng mL−1). Diestrus peaks were found at 1342, 1426, and 1473 nm, when P4 levels ranged from 5.14 to 9.60 ng mL−1. In our PCA models, 98% of the total variance in serum spectra between any pair of reproductive phases was described in only 3 spectral PCs, (PC1 = 83–86%, PC2 = 9–10%, PC3 = 2–5% of total variance). The PCA scores separated into distinct groups, indicating clear spectral profiles describing each reproductive phase uniquely. In conclusion, PCA analysis and spectral variances indicate that NIR, specifically aquaphotomics, has the theoretical capability to discriminate complex mixtures of biomolecules present in mare serum during oestrus, metestrus, and diestrus reproductive states. Study was supported by USDA-ARS Biophotonics Initiative grant #58-6402-3-018.

The Chiricahua leopard frog (Lithobates chiricahuensis) is a threatened species endemic to the southwestern United States and Northern Mexico. Captive breeding programs were established to support reintroduction efforts, yet reproductive output has been lower than needed for recovery of the species. This study aimed to evaluate the effects of captivity on amphibian reproduction by (1) determining if captive, semi‐captive, and wild male L. chiricahuensis produce sperm at similar rates and concentration in response to hormone treatment; and (2) evaluating the quality of sperm obtained over time from these populations. Males from captive, semi‐captive, and wild locations were administered a combination of human chorionic gonadotropin and gonadotropin‐releasing hormone to stimulate sperm production and release. A high percentage of males in the captive (60%), semi‐captive (100%), and wild (95.3%) populations produced sperm following treatment. Sperm quality (forward progressive motility and total sperm motility) did not differ between groups. However, sperm quantity (sperm/ml) differed (p < .05) between populations, with semi‐captive and wild males producing higher concentrations of sperm than captive males. These results suggest that Chiricahua leopard frog sperm quantity, but not quality, may become negatively impacted by long‐term captivity in indoor, controlled settings.