Abstract
As global reptile populations continue to decline, improving reproductive success in managed populations of listed species, such as Phrynosoma cornutum (the Texas horned lizard) has become increasingly critical for species survival. One understudied area of reproductive research in reptile species is gamete collection and storage, a crucial component for maintaining genetic diversity. Semen was collected from wild P. cornutum (n = 20) collected from Cottle County, Texas, in June 2025. Semen collection was performed via electroejaculation (EEJ) under alfaxalone anesthesia. Prior to semen collection, snout-vent-lengths (SVLs) and weights were recorded and testes measurements were taken using a portable ultrasound. Semen samples were extended in a commercial equine preservation medium (INRA96) and divided for use in cold-storage longevity or cryopreservation trials. Samples under cold-storage conditions were assessed for motility daily for 10 days. For cryopreservation, samples were diluted 1:1 in INRAFreeze cryopreservation media and frozen in liquid nitrogen, then immediately thawed. Average fresh sperm motility and concentration across all lizards was 83.7% and 85.7 x 106 sperm/mL, respectively. While lizards with longer SVLs had higher sperm motility, weight and testis size did not affect sperm parameters. During cold storage, sperm motility was not significantly reduced until 48 hours post-collection and maintained 19% motility until day 10. Following cryopreservation trials, the average post-thaw sperm motility was 13.9%, with the highest post-thaw motility recorded at 38.2%. This is the first report of semen storage and cryopreservation in Phrynosoma and provides valuable insight into semen storage potential in reptile species.
1 Introduction
With over 21% of reptile species today threatened with extinction (; ), the importance of maintaining assurance populations across zoological and conservation facilities has risen with an impetus on increasing reproductive output for species sustainability and reintroduction (; ). However, due to their diverse reproductive strategies, complex gametic dynamics, and specific biotic and abiotic requirements (; ; ; ), breeding reptiles within managed populations can be challenging. Much of the research into the reproductive biology of reptile species has been largely focused on female endocrinology and reproductive function. Female follicular dynamics and the induction of ovulation and oviposition have been studied since the 1970s (; ; ), with a focus on the treatment of dystocia and follicular stasis, also called egg binding, wherein oviposition is obstructed or impeded from a number of potential causes. Conversely, male reproductive research has been widely neglected. To date, few studies into the topics of semen collection, analysis, and storage have been conducted, and of these, fewer have related to application within lizard species (). This limited scale of study in comparison to other vertebrate classes () has slowed the development of reliable methodologies for techniques such as semen collection, sperm analysis, and preservation, impeding efforts for application in improving captive population management and reproductive output.
There are several benefits to sperm analysis within a breeding population. Primarily, the establishment of male reproductive health and fecundity is a critical component for breeding pairings and population management. This has been difficult to standardize across reptile species, however, due to the lack of study into sperm parameters. Establishing safe and successful protocols for semen collection has been a focal point for the development and improvement of sperm analysis (). Commonly in reptiles, semen collection is achieved through euthanasia of the animal and collection of semen from the vas deferens (; ), a method largely incompatible with managed populations of threatened and endangered species. As an alternative, manual collection of semen in living reptiles through massage has gained popularity in several species, including the crevice swift and Mexican horned lizard (e.g., ) and the saltwater crocodile () and is the primary method for semen collection in snake species (; ). Across species, however, successful collection and semen sample quality has been variable using this non-terminal method (), limiting application of this methodology in breeding programs for non-snake species.
An alternative method for semen collection is electroejaculation (EEJ). Briefly, a rectal probe is inserted into the cloaca of the male, and an electrical current is used to stimulate the nerves surrounding the vas deferens, resulting in the expulsion of mature semen (; ). The application of EEJ in reptile species has been validated across large-bodied species such as turtles (; ), crocodilians (), and the green iguana (; ). Due to the standard sizes of widely available commercial EEJ probes and the body size of medium- and small-bodied lizards, concerns for animal welfare and safety presented challenges to application in smaller species. In 2018, however, Juri et al. performed EEJ as a safe and successful method for semen collection in the medium-bodied spiny lava lizard (Tropidurus spinulosus) for a single timepoint. One year later, published successful EEJ-induced semen collection in the crevice swift (Sceloporus torquatus). In the same year, the safety of repeated EEJ-induced semen collection was assessed by using health parameters in veiled chameleons (Chamaeleo calyptratus; ). Additionally, semen from veiled and panther chameleons (Furcifer pardalis) was repeatedly collected for a year to determine associations between semen production and hormonal cyclicity (). Today, however, these methods have seen little expansion into other, smaller species, and their use is not widespread in conservation breeding programs despite repeated successes reported in the literature.
Due in part to the lack of established non-fatal semen collection and analysis protocols in reptiles, techniques for both short- and long-term semen storage remain one of the least developed assisted reproductive technologies (ART) for this taxon. Short-term refrigerated semen storage and cryopreservation are critical tools in conservation science and population management as both techniques allow for the salvage, transportation, and storage of valuable male genetics between and within managed populations (; ; ; ). Both techniques also have immediate utility in procedures such as artificial insemination (AI) and for the preservation of semen, ranging from short term (refrigeration) to perpetuity (cryopreservation). In order to safeguard reptile species against current and future threats of extinction and reduction in genetic diversity, it is imperative that these tools be developed for application in conservation breeding programs.
The Texas horned lizard (Phrynosoma cornutum) is a medium-bodied lizard endemic to the south-central United States and northern Mexico (; ). In recent decades, populations have drastically declined in Texas (; ), leading to the species’ state listing as “threatened” in Texas. Assurance colonies and breeding populations have been established across several Texas zoological institutions as a method for mitigating further decline through the release of captive-bred offspring. Unfortunately, breeding and hatch rates across programs remain relatively low, hindering sustainability and reintroduction efforts. Due to the lack of established protocols for investigating and addressing reproductive failure in reptiles, efforts to improve fertility rates in managed care have been slow. Here, we aim to validate electroejaculation as a safe and reliable means of semen collection, determine cold-storage longevity of collected semen for future application, and investigate the current cryopreservation potential of collected semen from P. cornutum.
2 Materials and methods
2.1 Animals
Wild adult P. cornutum (n= 20) were collected and sampled at the Matador Wildlife Management Area (MWMA) in Cottle County, Texas, in June of 2025 under Texas Park and Wildlife Permit #1091-455. Males were collected during road surveys and captured by hand. All animals were acclimated indoors overnight in plastic holding containers. Weights (g) and snout-vent length (SVL) measurements (mm) were taken for each male prior to anesthesia and semen collection. All research was conducted following review by Fort Worth Zoo’s Institutional Animal Care and Use Committee (IACUC) under case number FWZ-25-R-002.
2.2 Anesthesia
Each individual was weighed and administered alfaxalone (Alfaxan® Multidose, Zoetis, Kalamazoo, MI 49007 USA) at a dose of 15–18 mg per kg body weight, rounded to the nearest hundredth by volume, in the left forelimb. Injections were administered intramuscularly, though some subcutaneous administration is expected given limited muscle mass of the forelimbs. Lizards were monitored via visual and reflex assessment for respirations and anesthetic depth. A Butterfly portable ultrasound (Butterfly iQ/iQ+™) linear probe (1–10 MHz) compatible with Android was used to assess heart rate. Lizards were monitored for return of spontaneous reflexes and movements and were considered recovered when an immediate righting reflex returned.
2.3 Testis measurements
Prior to electro-ejaculation, trans-abdominal ultrasound scans of each male were performed to assess testis size using the Butterfly portable ultrasound. Males were held in dorsal recumbency with their abdomens accessible. Ultrasound gel was applied to the abdomen of each individual and images were taken using the “nerve” setting at a gain (signal amplification) between 60% - 70% at a depth of 3 cm. Two to five images were taken per individual, and testis length and diameter were measured using the Butterfly Network Cloud Software (Figure 1). Testes volumes (V) were then calculated using the following standardized equation from and has been used repeatedly in reptiles:
Figure 1
2.4 Semen collection and analysis
Once a sufficient plane of sedation was achieved, males were laid on their dorsum and a 2 mm wide electroejaculation probe was inserted approximately 12 mm into the cloaca. Each male underwent a stimulation cycle consisting of 5–7 five-second discharges with a five second rest period in between each discharge. Stimulations were 3.0 - 5.0 V. After the stimulation cycle, the probe was removed from the cloaca and gentle pressure was applied to the abdomen in downward sweeping motion to draw semen down to the cloaca (Figure 2). Once semen was observed pooling at the cloacal opening, the semen was drawn up using a 20 µl pipette and the original volume recorded. The semen was then placed into a bullet tube containing 100 µl of INRA96 semen extender (IMV Technologies), regardless of semen volume, and was placed in storage at 4 °C for subsequent analysis. After electroejaculation and during recovery monitoring following anesthesia, lizards were also monitored for deleterious effects from probe stimulation such as bleeding or cloacal inflammation ().
Figure 2
Following collection, semen volume was recorded before extender addition and sperm was analyzed after extension for motility and concentration using an Olympus CX41 phase contrast microscope. Sperm motility counts were conducted by counting individual sperm cells by hand using a multi-cell counter. Sperm motility was categorized as motile (M; sperm exhibiting flagellar movement but no forward movement), forward progressive motile (FPM; sperm exhibiting flagellar movement and progressing forward), and non-motile (NM; sperm exhibiting no flagellar movement). Motile and forward progressive motile values were added to describe total motility (TM). Sperm cells were counted until reaching 100 individual cells, giving the proportion of each cell motility type. Sperm concentration was counted on a Neubauer hemacytometer (Hausser Scientific) using a hand cell counter. Additional extender was added to the semen-INRA96 mixture to further dilute the sample to increase visibility of individual sperm cells in the hemacytometer. This additional dilution varied across samples depending on sperm cell prevalence and initial semen volume. Dilutions ranged from 1:1 to 1:20, with the median dilution being 1:10. Briefly, cells within the top outer four Neubauer ruling grids are counted and this number was then then multiplied by a 2500 correction factor and each sample’s dilution factor to determine sperm per milliliter.
2.5 Short term storage and cryopreservation
An additional 400 µl of INRA96 extender was added to each sample tube and gently mixed by pipetting the sample up and down. Samples were then divided in half with half reserved for testing cold storage longevity and half left for cryopreservation trials. To determine short term cold storage longevity, samples were stored in INRA96 semen extender at 4 °C. A volume of 12 µl was removed daily for 10 days (240 hours) for microscopy to assess changes in motility over storage time.
Cryopreservation trials occurred the same day as initial sample collection. Samples diluted in INRA96 for cryopreservation trials were further diluted 1:1 in INRA Freeze (IMV Technologies) as a cryoprotectant media without a glycerol additive and similarly mixed using a pipette. Samples were then loaded into ¼ cc CASSOU semen straws (IMV Technologies) with a total volume of 200 µl (100 µl of extended semen: 100 µl INRAFreeze) and frozen using a three-step slow freezing method. In step 1, loaded straws were left to equilibrate on an ice pack for 10 minutes. In step 2, equilibrated straws were placed into a Styrofoam freezing box containing liquid nitrogen at a level of 5 cm above the liquid nitrogen for an additional 10 minutes to allow slow vapor freezing. Straws were then plunged into liquid nitrogen.
Straws were thawed one at a time immediately following freezing. Straws were removed from liquid nitrogen and thawed in a 35 °C water bath for approximately 3–5 seconds. Once removed from the water bath, straws were gently dried and emptied into Eppendorf tubes for analysis. Samples were analyzed for post-thaw motility using methods outlined above. Post-thaw motility values were calculated as follows:
2.6 Statistical analysis
To determine the effects of testes volume on sperm parameters, we ran three different analyses for (i) total motility, (ii) forward progressive motility, and (iii) concentration. Because total motility and forward progressive motility were proportional, we used beta regressions for these models. We first ran a correlation analysis between SVL and weight to test for collinearity. We found that these two variables exhibited collinearity (r > 0.7; Dorman et al., 2013); thus, we ran two models for each analysis: one with SVL as a covariate and one with weight as a covariate, followed by AICc to determine which variable was the better predictor. For concentration, we used a generalized linear model with a Gamma distribution and a log-link since the data was positive continuous. For each model, testes volume was a fixed effect and SVL or weight was a covariate.
We were also interested in the effect of cold storage duration on motility. Therefore, we ran two beta regressions with (i) total motility or (ii) forward progressive motility as the response, time in cryostorage as the fixed effect, and male ID as a random effect to account for repeated measures. Lastly, we were interested in the effect of cryopreservation on motility. We conducted a beta regression with either (i) total motility or (ii) forward progressive motility as the response and freezing state (pre-freeze versus post-thaw) as the fixed effect, with male ID as a random effect. We conducted all analyses in R (v 4.4.1). We tested for assumptions of normality using residual diagnostic plots.
3 Results
3.3 Effects of individual and testis size on sperm quality
Average male weight, snout-vent length, and testis volumes were 29.2 ± 0.9 g, 79.7 ± 0.9 mm, and 0.86 ± 0.01 mm3, respectively. Snout-vent length was a better predictor of total motility (ΔAICc = 11.94) and forward progressive motility (ΔAICc = 2.28) compared to weight. Thus, we only discuss the results of the SVL analysis. SVL had a significant effect on total motility (β = 0.08 ± 0.02, p < 0.001), with larger individuals having more motile sperm, but not forward progressive motility (β = 0.05 ± 0.02, p = 0.06). Additionally, testes volume did not have an effect on total motility (β = 1.49 ± 1.49, p = 0.32) or forward progressive motility (β = 0.95 ± 1.97, p = 0.63). We found that SVL and weight had similar AICc values for the concentration analysis (ΔAICc = 0), yet we found no effect of either covariate or testes volume on concentration (SVL: β = 0.005 ± 0.05, p = 0.92; weight: β = -0.0003 ± 0.05; testes volume in SVL analysis: β = 1.41 ± 4.08, p = 0.73; testes volume in weight analysis: β = 1.42 ± 4.08, p = 0.73).
3.2 Semen collection and analysis
Semen was successfully collected from all 20 sampled males. Ejaculate sizes were between 5 – 40 µl. The average semen volume was 18.9 ± 2.6 µl. Though sperm morphological staining analysis was not conducted in the current study, sperm cells were visualized using phase contrast microscopy and characterized by long pointed heads with distinct distal droplets at the base of the head, likely signifying spermatozoa maturation. Tails were approximately 3x the length of the heads (Figure 3). Average total sperm motility across all sperm samples was 85% ± 1.8%, with an average forward progressive motility of 75.4% ± 2.6%. The highest percentages of total motility and forward progressive motility were 96.0% and 87.0%, respectively, from separate individuals. The lowest forward progressive motility was 38% and the lowest total motility observed was 58%. The average sperm concentration across all sampled males was 81.5 x 106 ± 11.7 x 106 sperm/mL, with the highest concentration at 156.9 x 106 sperm/mL and lowest at 1.0 x 106 sperm/mL.
Figure 3
3.3 Short-term storage and cryopreservation
We found that sperm total motility and forward progressive motility significantly decreased with the duration of cold storage (TM: β = -0.009 ± 0.0008, p < 0.001; FPM: β = -0.009 ± 0.0009, p < 0.001), with values approaching zero after 240 hours. Total and forward progressive motility of sperm in cold storage were similar to fresh sperm up to 24 hours post-collection (Figure 4). Average total motility and forward progressive motility dropped below 50% after 192 hours and 144 hours, respectively (Figure 4).
Figure 4
Of the 20 males sampled in this study, semen samples from 19 males were selected for cryopreservation trials. One male was excluded due to low forward progressive sperm motility (52%). The average pre-freeze total motility across samples selected for freezing was 83.7%, with an average forward progressive motility of 75.9%. Out of the 19 males, only 1 male’s samples resulted in zero post-thaw motility, and 5 of the 19 males had over 20% relative post-thaw total motility. The overall average relative post-thaw total motility and forward progressive motility were 13.9% and 10.3% respectively. The highest reported post-thaw return for total motility reached 38.2%, with forward progressive motility having a maximum reported post-thaw motility of 44.6% (Table 1). Following post-thaw analysis, we found that both total (β = 3.70 ± 0.25, p < 0.001) and forward progressive (β = 3.79 ± 0.26, p < 0.001) post-thaw motility was significantly lower than pre-freeze motility (Figure 5). Sperm concentration was not standardized across samples for cryopreservation and the average sperm concentration in each straw was 8.57 x 106 ± 1.16 x 106 sperm/mL.
Table 1
| Male | Sperm/mL (x106) | Forward progressive sperm (%) | Total sperm motility (%) | ||||
|---|---|---|---|---|---|---|---|
| Pre-freeze | Absolute post-thaw | Relative post-thaw | Pre-freeze | Absolute post-thaw | Relative post-thaw | ||
| 1 | 132.8 | 62 | 5 | 7.7 | 69 | 15 | 21.7 |
| 2 | 34.4 | 75 | 5 | 7.7 | 75 | 10 | 13.3 |
| 3 | 9.76 | 73 | 1 | 1.5 | 81 | 6 | 7.4 |
| 4 | 2.55 | 89 | 3 | 4.6 | 91 | 7 | 7.7 |
| 5 | 120 | 73 | 11 | 16.9 | 77 | 20 | 26 |
| 6 | 62.4 | 83 | 21 | 32.3 | 91 | 28 | 30.8 |
| 7 | 107.2 | 70 | 17 | 26.2 | 81 | 27 | 33.3 |
| 8 | 102.4 | 73 | 5 | 7.7 | 78 | 7 | 9 |
| 9 | 156.875 | 90 | 0 | 0 | 92 | 1 | 1.1 |
| 10 | 80 | 83 | 4 | 6.2 | 89 | 8 | 9 |
| 11 | 153.6 | 78 | 3 | 4.6 | 88 | 5 | 5.7 |
| 12 | 137.6 | 38 | 0 | 0 | 58 | 6 | 10.3 |
| 13 | 105.6 | 73 | 5 | 7.7 | 86 | 15 | 17.4 |
| 14 | 28.4 | 85 | 10 | 15.4 | 88 | 15 | 17 |
| 15 | 150.4 | 81 | 29 | 44.6 | 89 | 34 | 38.2 |
| 16 | 86.4 | 84 | 3 | 4.6 | 91 | 7 | 7.7 |
| 17 | 60.8 | 74 | 0 | 0 | 80 | 2 | 2.5 |
| 18 | 96 | 82 | 5 | 7.7 | 90 | 5 | 5.6 |
| 19 | 1 | 76 | 0 | 0 | 96 | 0 | 0 |
| Average | 85.7 | 75.9 | 6.7 | 10.3 | 83.7 | 11.5 | 13.9 |
| SEM | 11.6 | 2.6 | 1.8 | 2.7 | 2.1 | 2.6 | 2.5 |
Male sperm concentration (sperm/mL) and motility values pre- and post- cryopreservation following electroejaculation in the Texas horned lizard (Phrynosoma cornutum).
Absolute post-thaw motility refers to the raw post-thaw motility. Relative post-thaw motility refers to the motility relative to pre-freeze motility. Values are shown as mean ± SEM.
Bold values indicate semen parameter averages.
Figure 5
4 Discussion
In the current study, electroejaculation (EEJ) resulted in the successful extraction of semen in all 20 of the sampled male P. cornutum. Furthermore, no deleterious effects such as cloacal inflammation or irritation were observed following observation over 2 hours post-treatment, which is consistent with the findings on the safety of EEJ in other lizards (; ; ). Across all semen samples, average total motility and forward progressive motility was 85% and 75.4%, respectively. These are similar to the 78% motility reported in green iguanas () and spiny lava lizards (). Sperm concentration (sperm/mL) was higher in the current study than in either of these studies; we report an average sperm concentration of 81.5 x 106 sperm/mL, whereas the average concentrations reported in and were 69 x 106 and 2.1 x 106 sperm/mL, respectively. This variation may be due to species-specific differences but may also be due to seasonality. The P. cornutum sampled in this study were sampled at the end of their historic breeding season (April – June; ), while the experimental animals in were sampled outside of their breeding season (October – December; ). The animals in were sampled within the breeding season, but the authors note that recent mating may have reduced sperm quantity. Thus, in both studies the availability of mature spermatozoa may have been reduced compared to animals in the current study. Other possible sources of variation include species-specific differences in sperm production, semen collection and analysis protocols across studies, and the reproductive history of the animals.
In addition to semen quality, testis size may also have been impacted by seasonality. In seasonally-breeding species, testis size can increase during active spermatogenesis and subsequently decrease as mature spermatozoa are released and stored in the seminiferous tubules (). Due to collections in the current study occurring late in the breeding season of P. cornutum, it is likely that this transfer of sperm to the seminiferous tubules had already occurred, and thus testis size was not indicative of sperm quality or quantity.
Following successful semen collection, we tested duration of both short-term and long-term sperm longevity. Short term storage, or refrigerated storage, refers to a cold storage technique wherein semen samples are stored in an extender and kept at low temperatures above freezing. Of the few reptile species wherein short-term storage has been attempted, sperm motility appears to demonstrate longevity spanning days to weeks (; ); however, results appear to be species-specific. Here, we report that sperm from wild P. cornutum remained motile for the entire 10 days (240 hours) of monitoring, with motility remaining above 50% for 8 of the 10 days. Total motility was reduced at an average rate of 7% per day, and forward progressive motility declined at a similar rate of 6.6% per day. The greatest drop in motility occurred for both total motility and forward progressive motility at day 10, wherein average motility decline dropped by over 20%. The longevity described herein provides evidence that horned lizard sperm can be stored and retain motility for up to 10 days, which is a large window during which horned lizard semen can be collected and utilized, making these results promising for the collection and transfer of semen from wild individuals or those that may be located in another breeding facility for use in artificial insemination attempts or cryopreservation.
Following successful short-term storage, we tested a protocol for semen cryopreservation in P. cornutum. Out of the 19 males from which samples were frozen, 18 retained motility following sperm cryopreservation, with an average relative total motility and relative forward progressive motility return of 13.9% and 10.3%, respectively. This is the first instance of successful cryopreservation and post-thaw recovery of sperm in P. cornutum. Until 2016, there had been no published studies on lizard semen cryopreservation (). Since then, successful semen collection in lizards has expanded slightly to include the yellow-spotted monitor (Varanus panoptes; ) and sceloporine lizard species (Sceloporus aeneus, Sceloporus grammicus, and Sceloporus torquatus;). Similar to short-term storage, protocols have also demonstrated species-specificity, limiting transferability and widespread protocol utilization. Our results are similar to those presented by which tested post-thaw recovery of sperm from the yellow-spotted monitor in a DMSO-based cryoprotectant with and without post-thaw incubation in caffeine. Average post-thaw motility without caffeine incubation was reported as 18.1%, while post-thaw motility with caffeine was higher at 48%. Thus, future studies applying caffeine incubation post-thaw may be useful in P. cornutum to increase post-thaw sperm motility.
Finally, it is also important to note that the samples within the study were stored in the commercial semen media INRA96 and frozen in INRAFreeze media. INRA is a commercial semen extender for stallion sperm, containing milk micellar proteins, antibiotics, and fungicide. While the use of INRA96 as an extender for short-term storage was highly successful in this study, cryopreservation results were low for INRAFreeze as a cryoprotective media. However, the product protocol for INRAFreeze requires the addition of glycerol to the cryoprotective media. Reports in avian reptiles have noted that glycerol is a contraceptive (; ) and could act similarly in non-avian reptiles; however, the direct effect of glycerol on sperm function in reptiles remains uncertain.” As one of the goals of cryopreservation is artificial insemination, we wanted to determine the success of the freezing media without the addition of glycerol. In the study by , however, researchers utilized a Tris–egg yolk (TEY) medium based-cryoprotectant with an addition of glycerol resulting in a post-thaw motility recovery between 11-23%. More study is needed to determine whether the addition of glycerol would improve our methods reported herein. While INRAFreeze did not result in high post-thaw motility rates, it is currently a viable option for cryopreservation in P. cornutum that merits further study. Future studies regarding sperm analysis, storage, and cryopreservation in the species should include sperm morphology and viability assessments, the impact of glycerol on motility and freezing stability, additional extender use, and post-thaw activation media additives such as caffeine. As a semen extender, INRA96 is highly successful and readily accessible tool for short-term sperm storage in the species.
5 Conclusion
Here, we present successful protocols for semen collection, analysis, and short-term storage in P. cornutum. These methods are limited in reptiles, especially lizard species, and the development of successful protocols would drastically expand the horned lizard breeding strategies to include the transfer of semen from the wild or other institutions and/or long-term storage for future breeding. The sperm motility results following storage and cryopreservation presented here are the first to be reported in P. cornutum and are a valuable contribution to the literature surrounding lizard semen storage, cryopreservation, and to captive propagation of lizard species as a whole.
Statements
Data availability statement
The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.
Ethics statement
The animal study was approved by Institutional Animal Care and Use Committee, Fort Worth Zoo. The study was conducted in accordance with the local legislation and institutional requirements.
Author contributions
AJ: Data curation, Methodology, Project administration, Conceptualization, Writing – review & editing, Investigation, Writing – original draft, Formal analysis. JG: Data curation, Conceptualization, Investigation, Methodology, Writing – review & editing. SP: Investigation, Conceptualization, Writing – review & editing, Methodology. RD: Writing – review & editing, Investigation. IB: Writing – review & editing, Data curation, Formal analysis. DB: Methodology, Investigation, Writing – review & editing, Supervision, Resources, Project administration.
Funding
The author(s) declared that financial support was not received for this work and/or its publication.
Conflict of interest
The author(s) declared that this work was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.
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The author(s) declared that generative AI was not used in the creation of this manuscript.
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Summary
Keywords
cold storage, conservation, cryopreservation, Phrynosoma cornutum, reptile, semen analysis, semen collection, squamate
Citation
Julien AR, Griffioen JA, Perry SM, Doege R, Burger I and Barber D (2026) Semen collection, short term storage, and cryopreservation in the Texas horned lizard (Phrynosoma cornutum). Front. Amphib. Reptile Sci. 4:1845399. doi: 10.3389/famrs.2026.1845399
Received
01 April 2026
Revised
27 August 2026
Accepted
28 August 2026
Published
23 September 2026
Volume
4 - 2026
Edited by
Aimee Jade Silla, University of Wollongong, Australia
Reviewed by
Ayanda Maqhashu, University of the Free State, South Africa
Andréia Maria Da Silva, Federal University Rural Semi-Arid, Brazil
Updates
Copyright
© 2026 Julien, Griffioen, Perry, Doege, Burger and Barber.
This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.
*Correspondence: Allison R. Julien, ajulien@fortworthzoo.org
Disclaimer
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