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The Dama Gazelle, Nanger dama ruficollis, is critically endangered largely due to over-hunting and poaching. Fewer than 500 animals remain in the wild and ∼134 individuals are managed in captivity in North American zoos as a ’hedge’ against extinction. The ability to preserve and transport gene diversity could be enhanced through the use of frozen-thawed sperm and artificial insemination. Our aim was to assess the beneficial effects of cholesterol-loaded cyclodextrin (CLC) on dama gazelle sperm cryosurvival. Electroejaculates from three male dama gazelle were evaluated for total motility, forward progressive status, concentration, and acrosomal membrane integrity. Ejaculates were diluted (120×106 sperm/ml) with Ham’s F10 supplemented with Hepes (Irvine Scientific), aliquoted into five 1.5ml eppendorf centrifuge tubes and incubated (37°C; 15min) with 0, 0.5, 1.5, 3.0 and 6.0mg/ml CLC, respectively. Aliquots were centrifuged (300g; 8min) and the resulting sperm pellets were resuspended (250×106sperm/ml) in TEST-Yolk Buffer Refrigeration Medium (Irvine Scientific) containing 6% (v/v) glycerol. Sperm suspensions were loaded in straws (0.25ml), cooled to 5°C and placed 4cm over liquid nitrogen (LN2) for 10min before being plunged into LN2. Straws were thawed at 37°C (30s), stored protected from light at 25°C and evaluated for total motility, progressive status, and acrosomal integrity at 0 (immediately post-thaw), 1, 2 and 3h. Data were analyzed using generalized linear regression models and significance was defined as P <0.05. All males produced ejaculates with good initial motility (∼80%), progressive status (∼3.3; scale 0–5; 5=best), intact acrosomes (∼92.5%) and morphologically normal spermatozoa (∼65%). Immediately after thawing, sperm motility was unaffected (P <0.05) in the presence of 0.5mg/ml CLC (73.3±2.6%) but declined (P <0.05) in other groups (0, 56.7±3.1; 1.5, 60±0.8; 3.0, 41.7±6.9; and 6.0, 40±0.8%). At 1 h post-thaw, there were no differences in sperm motility amongst 0, 0.5, 1.5 and 3.0mg/ml CLC but 6.0mg/ml CLC was lower (P <0.05) than 0, 0.5, and 1.5mg CLC/ml. At 2h post-thaw, aliquots treated with 0.5 CLC exhibited the highest (P <0.05) motility. At time 3h post-thaw, sperm motility was higher in 0.5mg/ml CLC than non-treated control and 6mg/ml CLC but comparable to 1.5 and 3.0mg/ml CLC treated spermatozoa. There were no differences amongst 0, 1.5, 3.0 or 6.0mg/ml CLC. Progressive status was unaffected by treatment at all times evaluated, but declined (P <0.05) over time (0h, 3.8±0.2; 3h, 2.8±0.3) within all treatments. Acrosome integrity did not differ from raw ejaculates amongst treatment or time. Data demonstrate that pre-treatment of dama gazelle spermatozoa with CLC (0.5mg/ml) prior to cryopreservation protects spermatozoa from cryo-injury. These data could facilitate the development of artificial insemination technologies and establishment of a frozen repository for this species to assist with establishing self-sustaining populations and supporting reintroduction projects. Source of funding: None declared. Conflict of interest: None declared. pukazhenthib@si.edu [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

The maned wolf is a threatened canid species native to South America. Previous studies have suggested the species exhibits induced ovulation. In captive breeding facilities, reproductive success is low while rates of neonatal mortality are high. Females that are not recommended for breeding are frequently housed together. However there has never been a systematic study of the reproductive consequences of co-housing females. This study was conducted for three purposes, to: (1) corroborate the presence of induced ovulation, (2) determine whether elevated cortisol is implicated in neonatal pup mortality, and (3) evaluate the endocrine correlates of group housed females. Using fecal hormone monitoring for estrogen, progesterone, and cortisol, 43 cycles from 33 female maned wolves were studied from 2002 to 2015. Females were categorized by their reproductive status: pregnant and successfully raised pups (PR; n = 11), pregnant with neonatal pup demise within 3 days (PL; n = 7), housed with a male but no signs of breeding or pregnancy (PP; n = 10), housed singly (S; n = 8), or housed with related females (F; n = 7). Estrogen and progestagen remained at baseline for all females not housed with a male (S, F), while elevations consistent with ovulation were seen in females housed with a male (PP, PL, PR). Compared to PR females, PL individuals showed similar cortisol levels throughout the cycle and slightly lower progesterone levels during gestation. As for the effect of co-housing related females, F females showed estrogen and progesterone levels lower even than S females while cortisol levels were elevated compared to all other groups. These findings support the previous evidence of induced ovulation in the maned wolf. Although elevated cortisol does not seem to be implicated in pup loss, a non-significant trend towards lower progesterone during gestation could be implicated. Future studies should assess depressed progesterone levels as a correlate to neonatal pup mortality. Female maned wolves housed with related females experience suppressed reproductive hormones and elevated adrenal hormones. Therefore, a more systematic study of hormonal and behavioral correlates to co-housing with related females is warranted. [ABSTRACT FROM AUTHOR]

• Follicular development of in the whooping crane female (Grus americana) can be monitored by weekly ultrasound examination. • Follicular dynamics and hormonal patterns of the female whooping crane are similar to those of other avian species. • The failure of small, yolky follicles to enter the follicular hierarchy that are responsive to follicle stimulating hormone was the likely cause of no egg production in the whooping crane. • Small yolk laden follicles are responsive to follicle stimulating hormone. However, their failure to enter the follicular hierarchy during non-laying cycles of female whooping cranes was considered causative of a lack of egg production. For endangered species managed ex situ , production of offspring is a key factor to ensure healthy and self-sustaining populations. However, current breeding goals for the whooping crane (Grus americana) are impeded by poor reproduction. Our study sought to better understand mechanisms regulating ovarian function in ex situ managed whooping cranes and the regulatory function of the hypothalamic-pituitary–gonadal (HPG) axis in relation to follicle formation and egg laying. To characterize hormonal regulation of follicular development and ovulation, we collected weekly blood samples from six female whooping cranes during two breeding seasons, for a total of 11 reproductive cycles. The plasma samples were assessed for follicle stimulating hormone, luteinizing hormone, estradiol, and progesterone and the yolk precursors vitellogenin and very low-density lipoprotein. Ultrasonographic examination of the ovary was conducted at the time of blood collection. Preovulatory follicles (>12 mm) were present in laying cycles (n = 6) but absent in non-laying cycles (n = 5). The patterns of plasma hormone and yolk precursor concentrations corresponded to the stage of follicle development. Specifically, gonadotropin and yolk precursor concentrations increased as follicles transitioned from the non-yolky to yolky stage but did not increase further as the follicle advanced to preovulatory and ovulatory stages. Estrogen and progesterone concentrations increased as follicle size increased and reached peak concentrations (P < 0.05) when follicles developed to ovulatory and preovulatory stages, respectively. While overall mean circulating gonadotropin, progesterone, and yolk precursor concentrations did not differ for laying versus non-laying cycles, mean plasma estradiol in laying cycles was significantly higher than that in non-laying cycles. In summary, the findings suggested that disruption of mechanisms regulating follicle recruitment is likely responsible for the oviposition failure of the captive female whooping crane. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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 (Me 2 SO) 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% Me 2 SO, 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 Me 2 SO 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. [ABSTRACT FROM AUTHOR]

Reproductive success of endangered whooping cranes ( Grus americana ) maintained ex situ is poor. As part of an effort to identify potential causes of poor reproductive success in a captive colony, we used non-invasive endocrine monitoring to assess gonadal and adrenal steroids of bird pairs with various reproductive outcomes and evaluated the relationships of hormones and behaviors to reproductive performance. Overall, reproductively successful (i.e., egg laying) females had significantly higher mean estrogen levels but lower mean progestogen concentrations than did unsuccessful females. Other hormones, including glucocorticoids and androgens, were not significantly different between successful and unsuccessful individuals. Observations of specific behaviors such as unison calling, marching, and the number of copulation attempts, along with overall time spent performing reproductive behaviors, were significantly higher in successful pairs. Our findings indicate that overall reproductive performance of whooping crane pairs is linked to female gonadal hormone excretion and reproductive behaviors, but not to altered adrenal hormone production. [ABSTRACT FROM AUTHOR]

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 10min. 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. [ABSTRACT FROM AUTHOR]

Abstract: 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. [Copyright &y& Elsevier]

Abstract: In this study we measured excreted fecal corticoid metabolites (FCM) in maned wolves (Chrysocyon brachyurus) living within a protected reserve, on farmlands or in a boundary zone between the two habitats, and determined the impacts of season and reproductive status on adrenal activity. Feces were collected within a national park (n =191 samples), a park boundary zone (n =39) and on nearby farmlands (n =27), processed and analyzed by enzyme immunoassay. FCM amounts from samples collected on farmlands were higher (P <0.05) than in those collected inside the reserve and from the boundary zone. In relation to seasonality, FCM were elevated (P <0.05) in spring (September–November) when wolf pairs were raising young. We then divided the samples collected during breeding season (March–August) into cycling females and male/non-cycling females based on fecal progesterone: fecal testosterone ratio. FCM concentrations of the former collected inside the park were higher than (P <0.05) than the latter group. However, there were no differences in FCM levels between the two groups for samples collected in the boundary zone and on farmlands. Furthermore, FCM concentrations of male/non-cycling females samples collected on farmlands were 2- to 5-fold higher (P <0.05) than in counterparts collected inside the park. The consistently high FCM concentrations in samples collected on farmlands indicate that, in addition to seasonality, gender and reproductive status, anthropogenic pressures also contribute to elevating adrenal steroid for individuals living in altered habitat. [Copyright &y& Elsevier]