Abstract
Artificial insemination (AI) is a valuable tool for ex situ wildlife conservation, allowing the re-infusion and dissemination of genetic material, even after death of the donor. However, the application of AI to species conservation is still limited, due mainly to the poor survival of cryopreserved sperm. Recent work demonstrated that oviductal extracellular vesicles (oEVs) improved cat sperm motility and reduced premature acrosomal exocytosis. Here, we build on these findings by describing the protein content of dog and cat oEVs and investigating whether the incubation of cryopreserved red wolf and cheetah sperm with oEVs during thawing improves sperm function. Both red wolf and cheetah sperm thawed with dog and cat oEVs, respectively, had more intact acrosomes than the non-EV controls. Moreover, red wolf sperm thawed in the presence of dog oEVs better maintained sperm motility over time (>15%) though such an improvement was not observed in cheetah sperm. Our work demonstrates that dog and cat oEVs carry proteins important for sperm function and improve post-thaw motility and/or acrosome integrity of red wolf and cheetah sperm in vitro. The findings show how oEVs can be a valuable tool for improving the success of AI with cryopreserved sperm in threatened species.
Highlights
The red wolf (Canis rufus) is a critically endangered American canid [1], with fewer than 25 wild individuals remaining in a reintroduced population in North Carolina, United States, and approximately 262 managed individuals in the Species Survival Plan population [2]
Extracellular vesicles (EVs) are membrane-encapsulated particles containing regulatory molecules that contribute to cell–cell communication by carrying proteins, peptides, RNA species, lipids, and DNA fragments [39,40,41]
EVs secreted by the oviduct facilitate gamete function, fertilization and embryo development [31,32,44,45,46,50]
Summary
The red wolf (Canis rufus) is a critically endangered American canid [1], with fewer than 25 wild individuals remaining in a reintroduced population in North Carolina, United States, and approximately 262 managed individuals in the Species Survival Plan population [2]. While natural breeding is the gold standard, assisted reproductive technologies (ARTs), especially artificial insemination (AI), have played a pivotal role in the management of many endangered species, including the black-footed ferret, giant panda and whooping crane [7,9,10,11,12,13,14]. The value of AI with cryopreserved sperm in endangered species conservation has been demonstrated in the black-footed ferret, African elephant, southern white rhinoceros, gray wolf, giant panda, clouded leopard and cheetah [11,15,16,17,18]. The use of AI in the black-footed ferret (including by sperm that had been cryopreserved for 20 years) in the captive breeding program has increased population gene diversity by 0.2% and reduced inbreeding by 5.8% when compared with natural mating alone [12]
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