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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.

Purpose: Increasing intracellular energy storage by chemically activating adenosine monophosphate-activated protein kinase (AMPKα) prior to sperm cryopreservation may improve post-thawed sperm function. Using the domestic cat as a biomedical model, the objectives were to (1) confirm the expression of AMPKα and its regulatory kinases in epididymal spermatozoa and (2) assess the influence of AMPK activator, 5'-aminoimidasole-4-carboxamide-1-β-d-ribofuranoside (AICAR) on epididymal sperm function before and after cryopreservation., Methods: In study I, sperm samples of different qualities were obtained from cauda epididymides of domestic cats and evaluated for AMPKα expression. In study II, epididymal spermatozoa were equilibrated for either 30 or 60 min in the presence of 0 (control), 0.5, 2.0, and 5.0 mM AICAR and sperm functions were assessed before and after cryopreservation. In study III, epididymal spermatozoa were treated as in study II and evaluated for AMPKα signaling protein expressions (phospho-AMPKα Thr172 and GLUT1) as well as ATP levels., Results: AMPKα protein expression was higher in high-motility vs poor-motility samples. Thirty-minute equilibration with 0.5 mM AICAR improved motion characteristics and fertilizing ability of cryopreserved sperm to the control. Increased expressions of phospho-AMPKα Thr172 and GLUT1 as well as intracellular ATP level were confirmed in sperm samples equilibrated with 0.5 or 2.0 mM AICAR for 30 min., Conclusions: Presence and role of AMPKα protein in cat regulating sperm function were demonstrated before and after cryopreservation. Findings could be used to potentially enhance cryopreserved sperm function in sub-fertile men.

The capacity to cryopreserve semen from captive cranes facilitates production of offspring from behaviorally incompatible or geographically separated pairs, and allows for long-term preservation of valuable genetic materials. The present study sought to develop effective cryopreservation protocols for whooping (Grus americana) and white-naped (Grus vipio) cranes, through examining the influences of two permeating (DMA and Me 2 SO) and one non-permeating (sucrose) cryoprotectants, as well as vitamin E on post-thaw sperm survival. In Study 1, ejaculates (whooping: n = 10, white-naped: n = 8) were collected and cryopreserved in one of six cryo-diluents (crane extender with: DMA; DMA+0.1M sucrose; Me 2 SO; Me 2 SO+0.1M sucrose; 0.1M sucrose; 0.2M sucrose) using a two-step cooling method. Frozen samples were thawed and assessed for overall motility, motion characteristics, membrane integrity, morphology, and ability to bind to the inner perivitelline membrane (IPVM). In Study 2, whooping crane ejaculates (n = 17) were frozen in crane extender containing Me 2 SO alone or with vitamin E (5 μg/mL or 10 μg/mL). Frozen samples were thawed and assessed as in Study 1, except the binding assay. White-naped crane sperm were more tolerant to cryopreservation than whooping crane (15% vs 6% post-thawed motility). In both species, sperm cryopreserved in medium containing Me 2 SO alone displayed higher post thaw survival and ability to bind to IPVM than the other cryodiluent treatments. Vitamin E supplementation exerted no benefits to post thaw motility or membrane integrity. The findings demonstrated that there was species specificity in the susceptibility to cryopreservation. Nevertheless, Me 2 SO was a preferred cryoprotectant for sperm from both whooping and white-naped cranes., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Primordial follicles dictate a female's reproductive life span and therefore are central to fertility preservation for both endangered species and individuals with fertility-threatening conditions. Ovarian tissue containing primordial follicles can be cryopreserved and later thawed and transplanted back into individuals to restore both endocrine function and fertility. Importantly, increasing numbers of human live births have been reported following ovarian tissue cryopreservation and transplantation. A current limitation of this technology is patient access to sites that are approved or equipped to process and cryopreserve ovarian tissue - especially in larger countries or low resource settings. Here, we review empirical evidence from both animal models and human studies that suggest that ovarian tissue can be transported at cold temperatures for several hours while still maintaining the integrity and reproductive potential of the primordial follicles within the tissue. In fact, several human live births have been reported in European countries using tissue that was transported at cold temperatures for up to 20 h before cryopreservation and transplantation. Ovarian tissue transport, if implemented widely in clinical practice, could therefore expand both patient and provider access to emerging fertility preservation options., Competing Interests: Declaration of Interest The authors declare no conflicts of interest., (© 2016 Society for Reproduction and Fertility.)

Preserving genetic diversity of the critically endangered Addra gazelle (Nanger dama ruficollis) could be enhanced through the use of frozen-thawed sperm and artificial insemination. Our aim was to characterize Addra ejaculate traits and to assess the effects of cholesterol-loaded cyclodextrin (CLC) on sperm cryosurvival. Fresh ejaculates were treated with CLC (0, 0.5, 1.5, 3.0, and 6.0 mg/ml) prior to cryopreservation. All males produced spermic ejaculates with >75% sperm motility. The mean ± SEM seminal volume, sperm concentration, percent motility, forward progression, and percent morphologically normal spermatozoa were 3.2 ± 0.3 ml, 1.2 ± 0.3 × 10 9 , 75.82 ± 2.7%, 3.2 ± 0.3 (0-5 scale; 5 = most progressive), and 57.12 ± 3.8%, respectively. More than 92% contained an intact acrosome. There was no effect of time or in vitro incubation on progression or acrosomal integrity on thawed samples (P > 0.05). Spermatozoa pre-treated with 0.5 mg/ml CLC retained higher (P < 0.05) motility post-thaw than aliquots treated with 0, 3.0, or 6.0 mg/ml of CLC. Spermatozoa pre-treated with 0.5, 1.5, or 3.0 mg/ml CLC exhibited greater viability than counterparts (P < 0.05). Sperm kinetics including beat cross frequency (BCF), average path velocity (VAP), curvilinear velocity (VCL), and straight line velocity (VSL) did not differ among samples (P > 0.05). Linearity (LIN) and straightness (STR) were different among samples after thawing. Results demonstrate treatment with CLC (0.5 mg/ml) protects Addra spermatozoa from cryo-damage. Reported advances will facilitate establishment of a frozen repository and support the genetic management of this critically endangered north African desert antelope., (Published by Elsevier Inc.)

To assist in genetic resource management and recovery efforts of the white-naped crane (Antigone vipio), we conducted two experiments to evaluate the effect of cooling condition, thawing rate, and cryoprotectant concentration on sperm survival post-thaw. Semen was collected from four mature males during breeding season (March and April) and evaluated for volume, sperm concentration, motility, and membrane integrity. In Experiment 1, ejaculates (n = 8) were diluted with Beltsville Poultry Semen Extender (BPSE) containing 10% dimethylsulfoxide (Me2SO) and frozen using either one (average cooling rate = 2.5 °C/min) or two step (average cooling rate = 7 and 9 °C/min, respectively) cooling method. The frozen samples were thawed using one of two thawing rates: 37 °C 30 s vs. 4 °C 1 min. In Experiment 2, samples were diluted with crane semen extender containing either 6% or 10% Me2SO, frozen using two-step method and then thawed at 37 °C for 30 s. Both cooling condition (two-step > one-step) and thawing rate (37 °C 30 s > 4 °C 1 min) impacted sperm motility, progression and kinetic characteristics (P < 0.05), but did not (P > 0.05) affect plasma membrane or acrosomal integrity. Concentration of Me2SO did not impact frozen-thaw survival. We conclude that white-naped crane sperm cryopreserved using a combination of two-step cooling and thawing at 37 °C 30 s was superior to other cooling and thawing combinations regarding to sustaining sperm motility with good motility kinetics. Findings represent the first steps towards the development of effective cryopreservation protocols and establishment of a genome resource bank for this threatened species., (Copyright © 2016. Published by Elsevier Inc.)

We assessed the influences of medium osmolality, cryoprotectant and cooling and warming rate on maned wolf (Chrysocyon brachyurus) spermatozoa. Ejaculates were exposed to Ham's F10 medium (isotonic control) or to this medium plus NaCl (350-1000mOsm), sucrose (369 and 479mOsm), 1M glycerol (1086mOsm) or dimethyl sulfoxide (Me2SO, 1151mOsm) for 10 min. Each sample then was diluted back into Ham's medium and assessed for sperm motility and plasma membrane integrity. Although glycerol and Me2SO had no influence (P>0.05), NaCl and sucrose solutions affected sperm motility (P<0.05), but not membrane integrity. Motility of sperm exposed to <600mOsm NaCl or sucrose was less (P<0.05) than fresh ejaculate, but comparable (P>0.05) to the control. As osmolality of the NaCl solution increased, motility decreased to <5%. In a separate study, ejaculates were diluted in Test Yolk Buffer containing 1M glycerol or Me2SO and cooled from 5°C to -120°C at -57.8°C, -124.2°C or -67.0°C/min, frozen in LN2, thawed in a water bath for 30s at 37°C or 10s at 50°C, and then assessed for motility, plasma- and acrosomal membrane integrity. Cryopreservation markedly (P<0.05) reduced sperm motility by 70% compared to fresh samples. Higher (P<0.05) post-thaw motility (20.0±1.9% versus 13.5±2.1%) and membrane integrity (51.2±1.7% versus 41.5±2.2%) were observed in samples cryopreserved in Me2SO than in glycerol. Cooling rates influenced survival of sperm cryopreserved in glycerol with -57.8°C/min being advantageous (P<0.05). The findings demonstrate that although maned wolf spermatozoa are similar to domestic dog sperm in their sensitivity to osmotic-induced motility damage, the plasma membranes tolerate dehydration, and the cells respond favorably to Me2SO as a cryoprotectant., (Published by Elsevier Inc.)

Two studies were conducted to understand sperm cryosensitivity in an endangered equid, the Przewalski's horse (Equus ferus przewalski), while testing the cryoprotectant ability of formamides. The first assessed the toxicity of permeating cryoprotectants (glycerol, methylformamide [MF] and dimethylformamide [DMF]) to Przewalski's horse spermatozoa during liquid storage at 4°C. The second examined the comparative influence of three diluents (with or without formamides) on cryosurvival of sperm from the Przewalski's versus domestic horse. When Przewalski's horse spermatozoa were incubated at 4°C in INRA 96 with differing concentrations of glycerol, MF or DMF or a combination of these amides, cells tolerated all but the highest concentration (10% v/v) of MF alone or in combination with DMF, both of which decreased (P<0.05) motility traits. There was no effect of cryoprotectants on sperm acrosomal integrity. In the cryosurvival study, average sperm motility and proportion of cells with intact acrosomes in fresh ejaculates were similar (P>0.05) between the Przewalski's (67%, 84%, respectively) and domestic (66%, 76%) horse donors. Sperm from both species were diluted in lactose-EDTA-glycerol (EQ), Botu-Crio (BOTU; a proprietary product containing glycerol and MF) or SM (INRA 96 plus 2% [v/v] egg yolk and 2.5% [v/v] MF and DMF) and then frozen over liquid nitrogen vapor. After thawing, the highest values recovered for total and progressive sperm motility, acrosomal integrity and mitochondrial membrane potential were 42.4%, 21.8%, 88.7% and 25.4CN (CN=mean JC-1 fluorescence intensity/cell on a channel number scale), respectively, in the Przewalski's and 49.3%, 24.6%, 88.9% and 25.8CN, respectively, in the domestic horse. Although sperm progressive motility and acrosome integrity did not differ (P>0.05) among treatments across species, mitochondrial membrane potential was higher (P<0.05) in both species using EQ compared to BOTU or SM media. Additionally, Przewalski's stallion sperm expressed higher (P<0.05) post-thaw total motility in BOTU and SM compared to EQ, whereas there were no differences among freezing diluents in the domestic horse. In summary, Przewalski's stallion sperm benefit from exposure to either MF or DMF as an alternative cryoprotectant to glycerol. Overt sperm quality appears similar between the Przewalski's and domestic horse, although the total motility of cells from the former appears more sensitive to certain freezing diluents. Nonetheless, post-thaw motility and acrosomal integrity values for Przewalski's horse spermatozoa mimic findings in the domestic horse in the presence of INRA 96 supplemented with 2% (v/v) egg yolk and a combined 2.5% concentration of MF and DMF., (Published by Elsevier Inc.)

To derive a practical procedure to store canine somatic cells, fibroblasts isolated from testicular or ovarian tissues were cryopreserved in 1.2 M ethylene glycol or in 1.2 M dimethylsulfoxide prepared in Dulbecco's Modified Eagle Medium as cryoprotectants, and were frozen either in plastic straws or vials. Thawed cells were cultured for 24 hr at 38.5 degree C in a humidified atmosphere of 5 percent CO2 95 percent air, and then their membrane integrity was assayed with a double fluorescent stain, Fertilight. In addition, frozen-thawed fibroblasts were cultured for 4 days, and then their functional survival was measured after staining small colonies with trypan blue. After freezing and thawing, membrane integrity of testicular fibroblasts was 55-70 percent and functional survival ranged from 20-40 percent. With frozen-thawed ovarian cells, the average membrane integrity was 55-75 percent and the average functional survival was 35-40 percent. When frozen in ethylene glycol, functional survival of ovarian fibroblasts was significantly higher than that of testicular cells (P less than 0.05). These methods should prove useful to preserve cells collected from canids in the wild.