Production of live offspring following unilateral (left) ovariectomized Potamotrygon rays (Potamotrygon castexi, Potamotrygon leopoldi, and Potamotrygon motoro).

OBJECTIVE: To evaluate the surgical technique and subsequent clinical observations (reproductive and ultrasound findings) of left unilateral ovariectomy in 3 species of Potamotrygon rays-Potamotrygon castexi, Potamotrygon leopoldi, and Potamotrygon motoro-for reproductive management. ANIMALS: Between 2018 and 2019, multiple Potamotrygon rays (P castexi, n = 1; P leopoldi, 1; P motoro, 6) underwent left ovariectomies to evaluate this technique for reproductive management. PROCEDURES: At time of surgery, patient age ranged from juvenile to adult. Rays were anesthetized with MS222 buffered with sodium bicarbonate, and a left craniodorsal surgical approach was made to isolate and excise the left ovary. All rays had uneventful recoveries. Eight unilateral ovariectomized females and 6 males were combined in a mixed-species freshwater touch pool of Potamotrygon rays and teleost species. RESULTS: In December 2020, 3 live and 1 premature autolyzed pup were noted in the habitat. The following day, the adult females were examined via ultrasound and separated from the males. Four dams were identified that produced 8 viable offspring and 4 premature abortions. A large right ovary was observed in all females, with no evidence of left ovarian tissue present via ultrasound. CLINICAL RELEVANCE: Previous histologic evaluation of freshwater ray ovarian tissue suggests both ovaries may be functionally active yet maintain left dominance like some other elasmobranch species. This manuscript provides proof the right ovary alone can produce live offspring. Furthermore, the enlarged right ovary observed in these females suggests that removal of the left ovary may result in compensatory enlargement of the right ovary.

Cryo-scanning electron microscopy demonstrates that ice morphology is not associated with the post-thaw survival of domestic boar (Sus domesticus) spermatozoa: A comparison of directional and conventional freezing methods.

Directional freezing (in 2 or 10 ml hollow glass tubes) has been reported to improve post-thaw sperm survival parameters compared to conventional methods (in 0.5 ml straws). However, the biophysical properties that increase post-thaw survival are poorly understood. Therefore, the aim for the current study was to investigate the effect of ice morphology on the post-thaw survival of domestic boar spermatozoa directionally and conventionally cryopreserved in 0.5 ml straws. Ice morphology was quantitatively analyzed using a combination of cryo-scanning electron microscopy and Fiji Shape Descriptors. Multivariate analysis found a significant, non-linear effect (p < 0.05) of interface velocity on ice morphology, with an increase in both ice-lake size, as indicated by area and in aspect ratio, at an interface velocity of 0.2 mm/s. By contrast, post-thaw sperm survival (defined as spermatozoa with both intact plasma membranes and acrosomes) was biphasic, with peaks of survival at interface velocities of 0.2 mm/s (54.2 ± 1.9%), and 1.0 or 1.5 mm/s (56.5 ± 1.5%, 56.7 ± 1.7% respectively), and lowest survival at 0.5 (52.1 ± 1.6%) and 3.0 mm/s (51.4 ± 1.9%). Despite numerical differences in Shape Descriptors, there was no difference (p > 0.05) in the post-thaw survival between conventionally and directionally cryopreserved samples at optimal interface velocities of 1.0 or 1.5 mm/s. These findings suggest that: 1) ice morphology has little impact on post-thaw survival of boar spermatozoa, and 2) directional freezing in 0.5 ml straws (rather than 2 or 10 ml hollow glass tubes) may attenuate benefits of directional freezing.

Resurrecting biodiversity: advanced assisted reproductive technologies and biobanking.

Biodiversity is defined as the presence of a variety of living organisms on the Earth that is essential for human survival. However, anthropogenic activities are causing the sixth mass extinction, threatening even our own species. For many animals, dwindling numbers are becoming fragmented populations with low genetic diversity, threatening long-term species viability. With extinction rates 1000-10,000 times greater than natural, ex situ and in situ conservation programmes need additional support to save species. The indefinite storage of cryopreserved (-196°C) viable cells and tissues (cryobanking), followed by assisted or advanced assisted reproductive technology (ART: utilisation of oocytes and spermatozoa to generate offspring; aART: utilisation of somatic cell genetic material to generate offspring), may be the only hope for species' long-term survival. As such, cryobanking should be considered a necessity for all future conservation strategies. Following cryopreservation, ART/aART can be used to reinstate lost genetics back into a population, resurrecting biodiversity. However, for this to be successful, species-specific protocol optimisation and increased knowledge of basic biology for many taxa are required. Current ART/aART is primarily focused on mammalian taxa; however, this needs to be extended to all, including to some of the most endangered species: amphibians. Gamete, reproductive tissue and somatic cell cryobanking can fill the gap between losing genetic diversity today and future technological developments. This review explores species prioritisation for cryobanking and the successes and challenges of cryopreservation and multiple ARTs/aARTs. We here discuss the value of cryobanking before more species are lost and the potential of advanced reproductive technologies not only to halt but also to reverse biodiversity loss. Lay summary: The world is undergoing its sixth mass extinction; however, unlike previous events, the latest is caused by human activities and is resulting in the largest loss of biodiversity (all living things on Earth) for 65 million years. With an extinction rate 1000-10,000-fold greater than natural, this catastrophic decline in biodiversity is threatening our own survival. As the number of individuals within a species declines, genetic diversity reduces, threatening their long-term existence. In this review, the authors summarise approaches to indefinitely preserve living cells and tissues at low temperatures (cryobanking) and the technologies required to resurrect biodiversity. In the future when appropriate techniques become available, these living samples can be thawed and used to reinstate genetic diversity and produce live young ones of endangered species, enabling their long-term survival. The successes and challenges of genome resource cryopreservation are discussed to enable a move towards a future of stable biodiversity.

How can mating systems inform future biobanking strategies? An illustration using two Indonesian bovids, banteng (Bos javanicus) and lowland anoa (Bubalus depressicornis).

Storing cryopreserved spermatozoa in a genome resource bank safeguards against the loss of heterozygosity in endangered species and provides opportunities to reincorporate genes into populations through the application of assisted reproductive technologies. The aim of this study was to demonstrate the effect of breeding strategy on ejaculate characteristics to illustrate how this information may be used to select appropriate methods for the storage and use of cryopreserved sperm. In the present study, ejaculates from a polygynous bovid, banteng (Bos javanicus), were characterized (motility 72.7 ± 4.3%; total sperm count 2,702 ± 764 ×106 sperm; morphologically normal sperm 87.9 ± 3.0%), as well as ejaculates from a monogamous bovid, lowland anoa (Bubalus depressicornis; motility 47.5 ± 5.4%; total sperm count 279 ± 84 ×106 sperm; morphologically normal sperm 69.0 ± 6.1%). As banteng produce an ejaculate with characteristics similar to domestic cattle, translating assisted reproductive technologies from domestic cattle is feasible. By contrast, lowland anoa produce smaller quantities of sperm with a higher prevalence of morphologically abnormal sperm; thus, alternative protocols, optimized for the storage and use of ejaculates containing lower quantities of sperm, is necessary. Sperm tail length was more conserved in banteng (CV 2.7%) than lowland anoa (CV 6.4%) and could be due to differences in levels of sperm competition between species. Additionally, the use of three different diluents (Biladyl, TES-Tris yolk buffer, and whole milk) were investigated for banteng sperm cryopreservation. Sperm cryopreserved in Biladyl and whole milk diluents produced significantly higher post-thaw survival parameters then TES-Tris yolk buffer.

Initial Characterization of Male Southern Stingray (Hypanus americanus) Reproductive Parameters and Preliminary Investigation of Sperm Cryopreservation.

This study investigated the reproductive biology and sperm cryopreservation of ex situ southern stingrays (Hypanus americanus) by semen collection and characterization and the development and validation of an enzyme-linked immunoassay for plasma total testosterone. Semen was collected in March and June using a manual massage technique, and the ejaculates were assessed for volume, pH, osmolarity, motility, status (0-5 scale: 0 = no forward progression, 5 = rapid linear progression) and total sperm count. Semen was extended in Hank's elasmobranch ringer solution containing 10% DMSO, 10% glycerol or 5% glycerol with 5% N-methylformamide and cryopreserved using a conventional freezing method (~-50 °C/min) or a modified slow freezing method (~-3 °C/min). Body condition was scored from 1-5 and was noted to be low in March (1.93 ± 0.07) due to feeding practices and increased by June (2.93 ± 0.05) after dietary corrections were made. A concomitant increase (p < 0.05) in plasma total testosterone concentration and sperm motility was noted between March (8.0 ± 7.2 ng/mL, 5.71 ± 2.77%) and June (97.3 ± 11.3 ng/mL, 51.4 ± 14.3%). Samples cryopreserved using a modified slow freeze method (~-3 °C/min) had higher post-thaw motility and plasma membrane integrity than conventionally cryopreserved samples. Data indicate that southern stingray sperm morphometrics adheres to those of other elasmobranch species and that a slow cooling rate may be an avenue of research to improve southern stingray sperm survival during cryopreservation.