Snake populations are facing a significant global decline, with 33% of reptiles being classified as endangered [1]. Assisted reproductive technologies have proven to be fundamental to create gametes banks for species conservation [2, 3]. However, research on sperm collection, characteristics, and cryopreservation in snakes is notably scarce [3]. Beauty rat snakes (Elaphe taenuria) are classified as vulnerable, meaning that this species is considered at risk of extinction, unless the circumstances that are threatening their survival and reproduction change. Moreover, these snakes can serve as a model for assisted reproductive technologies in endangered species belonging to the family of Colubridae. For this reason, we aimed to investigate male anatomical features and sperm characteristics of 18 adult Beauty rat snakes. For each animal both testis and vasa deferentia were collected and measured for weight (testis) and length (testis and vasa deferentia). Furthermore, we assessed the effects of two distinct cryopreservation methods on sperm collected post-mortem. The spermatozoa were retrieved by and float up from vasa deferentia. Specifically, each vas deferens was placed into a 15 mL Falcon tube containing 1 mL of HEPES-TALP solution and incubated at room temperature (22 ◦ C) in the dark for 30 min. When retrieval and sperm analyses were successful (N = 11), spermatozoa were diluted into either a single or double-step TEST-yolk semen extender, with a final glycerol concentration of 12%. The freezing process took place at - 0.3°C/min in 0.5 mL straws, that were subsequently stored in liquid nitrogen for one week, when post-thaw sperm quality was assessed. Statistical analysis was performed using R version 4.3.1 (Vienna, Austria) and significance was considered for P < 0.05. Normally distribution of data was analyzed with Shapiro Wilk Test (P< 0.05). The Wilcoxon sum rank test indicated that the left testicle was smaller (45.77 mm ± 3 vs 51.39 mm ± 3.83; p = 0.007), lighter (1.18 g ± 0.25 vs 1.50 g ± 0.32; p = 0.003), and had a shorter vas deferens (271.94 mm ± 14.68 vs 332.39 mm ± 18.19; p = 0.0002) compared to the right one. However, no significant variations in total sperm count were observed in-between sides (p > 0.05). The total sperm count demonstrated a positive correlation between the number of morphologically normal spermatozoa and the weight of the testicle (p = 0.04 and p = 0.01, respectively; Spearman’s rank correlation test). There was no significant difference in terms of motility and morphology between the two freezing protocols (p > 0.05; Mann-Whitney U test). In conclusion, the present study describes some important andrological features of Beauty rat snakes, including new protocols for post-mortem sperm collection and cryopreservation. Furthermore, these results provide a starting point for studies on male reproductive physiology in Colubridae, not only for future research on this species but also as a model for endangered reptiles. [1] IUCN. 2022. The IUCN Red List of Threatened Species. Version 2022-2. https://www.iucnredlist.org. Accessed on [November 30th, 2023]. [2] Huijsmans T.E.R.G. Postmortem Collection of Gametes for the Conservation of Endangered Mammals: A Review of the Current State-of-the-Art. Animals: an open access journal from MDPI, 13(8), 1360, 2023. [3] Sandfoss M.R. Cryopreservation of Sperm from an Endangered Snake with Tests of Post-Thaw Incubation in Caffeine. Animals: an open access journal from MDPI, 12(14), 1824, 2022.
TESTICULAR MORPHOLOGY AND SEMEN CHARACTERISTICS OF FRESH AND THAWED SEMEN IN BEAUTY RAT SNAKES (ELAPHE TAENIURA): A MODEL FOR PHYSIOLOGY AND SPERM CRYOPRESERVATION OF ENDANGERED COLUBRIDAE.
Cesauri M.;Elmi A.;
2025-01-01
Abstract
Snake populations are facing a significant global decline, with 33% of reptiles being classified as endangered [1]. Assisted reproductive technologies have proven to be fundamental to create gametes banks for species conservation [2, 3]. However, research on sperm collection, characteristics, and cryopreservation in snakes is notably scarce [3]. Beauty rat snakes (Elaphe taenuria) are classified as vulnerable, meaning that this species is considered at risk of extinction, unless the circumstances that are threatening their survival and reproduction change. Moreover, these snakes can serve as a model for assisted reproductive technologies in endangered species belonging to the family of Colubridae. For this reason, we aimed to investigate male anatomical features and sperm characteristics of 18 adult Beauty rat snakes. For each animal both testis and vasa deferentia were collected and measured for weight (testis) and length (testis and vasa deferentia). Furthermore, we assessed the effects of two distinct cryopreservation methods on sperm collected post-mortem. The spermatozoa were retrieved by and float up from vasa deferentia. Specifically, each vas deferens was placed into a 15 mL Falcon tube containing 1 mL of HEPES-TALP solution and incubated at room temperature (22 ◦ C) in the dark for 30 min. When retrieval and sperm analyses were successful (N = 11), spermatozoa were diluted into either a single or double-step TEST-yolk semen extender, with a final glycerol concentration of 12%. The freezing process took place at - 0.3°C/min in 0.5 mL straws, that were subsequently stored in liquid nitrogen for one week, when post-thaw sperm quality was assessed. Statistical analysis was performed using R version 4.3.1 (Vienna, Austria) and significance was considered for P < 0.05. Normally distribution of data was analyzed with Shapiro Wilk Test (P< 0.05). The Wilcoxon sum rank test indicated that the left testicle was smaller (45.77 mm ± 3 vs 51.39 mm ± 3.83; p = 0.007), lighter (1.18 g ± 0.25 vs 1.50 g ± 0.32; p = 0.003), and had a shorter vas deferens (271.94 mm ± 14.68 vs 332.39 mm ± 18.19; p = 0.0002) compared to the right one. However, no significant variations in total sperm count were observed in-between sides (p > 0.05). The total sperm count demonstrated a positive correlation between the number of morphologically normal spermatozoa and the weight of the testicle (p = 0.04 and p = 0.01, respectively; Spearman’s rank correlation test). There was no significant difference in terms of motility and morphology between the two freezing protocols (p > 0.05; Mann-Whitney U test). In conclusion, the present study describes some important andrological features of Beauty rat snakes, including new protocols for post-mortem sperm collection and cryopreservation. Furthermore, these results provide a starting point for studies on male reproductive physiology in Colubridae, not only for future research on this species but also as a model for endangered reptiles. [1] IUCN. 2022. The IUCN Red List of Threatened Species. Version 2022-2. https://www.iucnredlist.org. Accessed on [November 30th, 2023]. [2] Huijsmans T.E.R.G. Postmortem Collection of Gametes for the Conservation of Endangered Mammals: A Review of the Current State-of-the-Art. Animals: an open access journal from MDPI, 13(8), 1360, 2023. [3] Sandfoss M.R. Cryopreservation of Sperm from an Endangered Snake with Tests of Post-Thaw Incubation in Caffeine. Animals: an open access journal from MDPI, 12(14), 1824, 2022.I documenti in IRIS sono protetti da copyright e tutti i diritti sono riservati, salvo diversa indicazione.
