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Ex situ conservation breeding for species recovery often must address small founder number. Here we investigated the long‐term effects of small founder size in the endangered black‐footed ferret Mustela nigripes using 20 years of reproductive data. Specifically, we studied seminal traits, pregnancy success and litter size in the context of the population's low genetic diversity. Detailed evaluation of 539 electroejaculates from 262 pedigreed donors with varied inbreeding coefficients (F = 0.000–0.250) revealed a wide range in sperm motility (0.0–100.0%), forward progression (type of forward movement on a scale 0–5; 5 = best; 0.0–4.5), cell concentration (7.2–6348.0 × 106 mL−1 of ejaculate), structural normality (0.0–89.0%) and normal apical ridges (critical for fertilization; 4.8–100.0%). Over this two decade interval, there was an increase (P < 0.05) in F and a decrease (P < 0.05) in sperm forward progression and structurally normal cell morphology. From analysis of artificial insemination (AI) data, higher values for sperm motility, forward progression and structural normality or spermatozoa that retained a normal apical ridge had a positive influence (P < 0.05) on litter size. Males producing higher values for these four traits also sired more kits. Results demonstrate that sperm motility and morphology are decreasing in black‐footed ferrets managed intensively ex situ. These two metrics appear associated with an increasing F and likely are at least partially responsible for lowered reproductive success in this closed, endangered population. These results illustrate the potential physiological and offspring production impact of having few founders available in an ex situ conservation breeding program. [ABSTRACT FROM AUTHOR]

One of the most significant challenges in the recovery of threatened species is the ability to maintain genetic diversity, avoid inbreeding and sustain population health and reproduction. Assisted reproductive techniques, including artificial insemination ( AI), have been touted for decades as approaches that could contribute to the demographic and genetic management of rare species. Here, we report the first successful integration of AI with frozen semen into a formal recovery program and the positive impact on genetic diversity for the critically endangered black-footed ferret M ustela nigripes. Techniques developed in the taxonomically related domestic ferret Mustela putorius furo and Siberian polecat M ustela eversmannii were applied over time to selected black-footed ferrets, including semen banking from six of the last 18 survivors. After evaluation, processing and storage in liquid nitrogen (−196° C/−321° F), for as long as 20 years, sperm samples were thawed and transabdominally inseminated into the uterine horns of female conspecifics. Eight black-footed ferret offspring were produced using thawed sperm samples (including after two decades of cryopreservation) with inseminates containing as few as 3.4 × 106 motile spermatozoa. The incorporation of these offspring and/or their descendants into the ex situ breeding program prevented heterozygosity loss in the population and actually enhanced gene diversity ( GD) significantly by 0.2% and lowered measures of inbreeding by 5.8%. This study demonstrates the utility and genetic diversity benefits of applying AI with cryopreserved spermatozoa 20 generations removed from the contemporary population for a wild animal revival program. [ABSTRACT FROM AUTHOR]

Hormonal analysis provides information about wildlife populations, but is difficult to conduct in the field. Our goal was to develop a rapid and effective field method for fecal steroid analysis by comparing: (1) three extraction methods (laboratory (LAB), homogenize (HO) and handshake (HS)) and (2) two storage methods (solid-phase extraction (SPE) tubes vs. plastic tubes (PT)). Samples ( n=23) from captive African wild dogs ( Lycaon pictus) were thoroughly mixed, three aliquots of each were weighed (∼0.5 g) and 5 ml of 90% ethanol was added. For LAB, samples were agitated (mixer setting 60; 30 min), centrifuged (1,500 rpm; 20 min) and poured into glass tubes. Or aliquots were HO (1 min) or HS (1 min) and poured through filter paper into glass tubes. Samples were split, analyzed for corticosterone (C) and testosterone (T) metabolites using enzyme immunoassays or stored in SPE or PT. Samples were stored (room temperature) for 30, 60 or 180 days, reconstituted in buffer and analyzed. Mean C and T recoveries of HO were greater ( P=0.03) than HS compared with LAB, which was similar to HO ( P>0.05). After 30 days <21% of C and T was recovered from SPE, but ∼100% of each was recovered from HO-PT and HS-PT. Similarly, after 60 and 180 days, ∼100% of C and T was recovered from HO-PT and HS-PT. Results demonstrated that, for C and T, HO was more comparable ( P<0.001) to LAB than HS and PT storage was more efficient than SPE ( P<0.001). Zoo Biol 29:289-302, 2010. © 2009 Wiley-Liss, Inc. [ABSTRACT FROM AUTHOR]

The gut microbiome of mammals engages in a dynamic relationship with the body and contributes to numerous physiological processes integral to overall health. Understanding the factors shaping animal-associated bacterial communities is therefore paramount to the maintenance and management in ex situ wildlife populations. Here, we characterized the gut microbiome of 48 endangered black-footed ferrets (Mustela nigripes) housed at Smithsonian's National Zoo and Conservation Biology Institute (Front Royal, Virginia, USA). We collected longitudinal fecal samples from males and females across two distinct reproductive seasons to consider the role of host sex and reproductive physiology in shaping bacterial communities, as measured using 16S rRNA amplicon sequencing. Within each sex, gut microbial composition differed between breeding and non-breeding seasons, with five bacterial taxa emerging as differentially abundant. Between sexes, female and male microbiomes were similar during non-breeding season but significantly different during breeding season, which may result from sex-specific physiological changes associated with breeding. Finally, we found low overall diversity consistent with other mammalian carnivores alongside high relative abundances of potentially pathogenic microbes such as Clostridium, Escherichia, Paeniclostridium, and (to a lesser degree) Enterococcus-all of which have been associated with gastrointestinal or reproductive distress in mammalian hosts, including black-footed ferrets. We recommend further study of these microbes and possible therapeutic interventions to promote more balanced microbial communities. These results have important implications for ex situ management practices that can improve the gut microbial health and long-term viability of black-footed ferrets., (© The Author(s) 2023. Published by Oxford University Press on behalf of The American Genetic Association. All rights reserved. For commercial re-use, please contact reprints@oup.com for reprints and translation rights for reprints. All other permissions can be obtained through our RightsLink service via the Permissions link on the article page on our site—for further information please contact journals.permissions@oup.com.)

Reproductive microbiomes contribute to reproductive health and success in humans. Yet data on reproductive microbiomes, and links to fertility, are absent for most animal species. Characterizing these links is pertinent to endangered species, such as black-footed ferrets (Mustela nigripes), whose populations show reproductive dysfunction and rely on ex-situ conservation husbandry. To understand microbial contributions to animal reproductive success, we used 16S rRNA amplicon sequencing to characterize male (prepuce) and female (vaginal) microbiomes of 59 black-footed ferrets at two ex-situ facilities and in the wild. We analyzed variation in microbiome structure according to markers of fertility such as numbers of viable and non-viable offspring (females) and sperm concentration (males). Ferret vaginal microbiomes showed lower inter-individual variation compared to prepuce microbiomes. In both sexes, wild ferrets harbored potential soil bacteria, perhaps reflecting their fossorial behavior and exposure to natural soil microbiomes. Vaginal microbiomes of ex-situ females that produced non-viable litters had greater phylogenetic diversity and distinct composition compared to other females. In males, sperm concentration correlated with varying abundances of bacterial taxa (e.g., Lactobacillus), mirroring results in humans and highlighting intriguing dynamics. Characterizing reproductive microbiomes across host species is foundational for understanding microbial biomarkers of reproductive success and for augmenting conservation husbandry., (© 2024. The Author(s).)

Environmental variation can influence the reproductive success of species managed under human care and in the wild, yet the mechanisms underlying this phenomenon remain largely mysterious. Molecular mechanisms such as epigenetic modifiers are important in mediating the timing and progression of reproduction in humans and model organisms, but few studies have linked epigenetic variation to reproductive fitness in wildlife. Here, we investigated epigenetic variation in black-footed ferrets ( Mustela nigripes ), an endangered North American mammal reliant on ex situ management for survival and persistence in the wild. Despite similar levels of genetic diversity in human-managed and wild-born populations, individuals in ex situ facilities exhibit reproductive problems, such as poor sperm quality. Differences across these settings suggest that an environmentally driven decline in reproductive capacity may be occurring in this species. We examined the role of DNA methylation, one well-studied epigenetic modifier, in this emergent condition. We leveraged blood, testes, and semen samples from male black-footed ferrets bred in ex situ facilities and found tissue-type specificity in DNA methylation across the genome, although 1360 Gene Ontology terms associated with male average litter size shared functions across tissues. We then constructed gene networks of differentially methylated genomic sites associated with three different reproductive phenotypes to explore the putative biological impact of variation in DNA methylation. Sperm gene networks associated with average litter size and sperm count were functionally enriched for candidate genes involved in reproduction, development, and its regulation through transcriptional repression. We propose that DNA methylation plays an important role in regulating these reproductive phenotypes, thereby impacting the fertility of male ex situ individuals. Our results provide information into how DNA methylation may function in the alteration of reproductive pathways and phenotypes in artificial environments. These findings provide early insights to conservation hurdles faced in the protection of this rare species., Competing Interests: The authors have no known conflicts of interest., (© 2024 The Authors. Evolutionary Applications published by John Wiley & Sons Ltd.)

Millions of people globally depend on camelids, which demands an increased knowledge of their reproduction. We used zoo-housed Bactrian camels (Camelus bactrianus) to better understand camelid reproductive physiology. Our specific objectives were to: 1) validate the use of fecal hormone metabolite analysis to characterize camel reproductive physiology during sexual maturity and pregnancy; and 2) determine the influence of season on male and female reproduction. We collected fecal samples from 1 male and 3 females housed at Lincoln Park Zoo (Chicago, IL, USA) 1 to 2 times per week for 3.5 years. Extracted hormones were analyzed using enzyme immunoassays for progestogen (FPM), estrogen (FEM), and androgen (FAM) metabolite concentrations. One female sexually matured during our study as evidenced by increased FEM baseline. Results demonstrated seasonal effects on male androgen production with FAMs higher (P < 0.05) January to June (mean ± SEM: 664.6 ± 22.6 ng/g wet feces), compared to July to December (401.6 ± 17.5 ng/g wet feces). One female experienced a persistent corpus luteum, a reproductive abnormality, which was identified by prolonged elevated FPM. FPMs increased during pregnancy for two females (452.9 ± 24.9 and 294.4 ± 19.8 ng/g wet feces) with a gestation of 404 d and 442 d, respectively. The third female never conceived. The FEMs varied (P < 0.05) during the year with no clear seasonal patterns (monthly mean range: 213.1-371.0 ng/g wet feces). Fecal hormone metabolite analysis is a validated method for assessing male seasonality and female pregnancy in the Bactrian camel and can for their management and conservation in zoos and the wild., (Copyright © 2022 Elsevier Inc. All rights reserved.)

Infectious disease outbreaks pose a significant threat to the conservation of chimpanzees (Pan troglodytes) and all threatened nonhuman primates. Characterizing and mitigating these threats to support the sustainability and welfare of wild populations is of the highest priority. In an attempt to understand and mitigate the risk of disease for the chimpanzees of Gombe National Park, Tanzania, we initiated a long-term health-monitoring program in 2004. While the initial focus was to expand the ongoing behavioral research on chimpanzees to include standardized data on clinical signs of health, it soon became evident that the scope of the project would ideally include diagnostic surveillance of pathogens for all primates (including people) and domestic animals, both within and surrounding the National Park. Integration of these data, along with in-depth post-mortem examinations, have allowed us to establish baseline health indicators to inform outbreak response. Here, we describe the development and expansion of the Gombe Ecosystem Health project, review major findings from the research and summarize the challenges and lessons learned over the past 16 years. We also highlight future directions and present the opportunities and challenges that remain when implementing studies of ecosystem health in a complex, multispecies environment., (© 2021 Wiley Periodicals LLC.)

Living in a rapidly changing environment can alter stress physiology at the population level, with negative impacts on health, reproductive rates, and mortality that may ultimately result in species decline. Small, isolated animal populations where genetic diversity is low are at particular risks, such as endangered Virunga mountain gorillas ( Gorilla beringei beringei ). Along with climate change-associated environmental shifts that are affecting the entire population, subpopulations of the Virunga gorillas have recently experienced extreme changes in their social environment. As the growing population moves closer to the forest's carrying capacity, the gorillas are coping with rising population density, increased frequencies of interactions between social units, and changing habitat use (e.g., more overlapping home ranges and routine ranging at higher elevations). Using noninvasive monitoring of fecal glucocorticoid metabolites (FGM) on 115 habituated Virunga gorillas, we investigated how social and ecological variation are related to baseline FGM levels, to better understand the adaptive capacity of mountain gorillas and monitor potential physiological indicators of population decline risks. Generalized linear mixed models revealed elevated mean monthly baseline FGM levels in months with higher rainfall and higher mean maximum and minimum temperature, suggesting that Virunga gorillas might be sensitive to predicted warming and rainfall trends involving longer, warmer dry seasons and more concentrated and extreme rainfall occurrences. Exclusive use of smaller home range areas was linked to elevated baseline FGM levels, which may reflect reduced feeding efficiency and increased travel efforts to actively avoid neighboring groups. The potential for additive effects of stress-inducing factors could have short- and long-term impacts on the reproduction, health, and ultimately survival of the Virunga gorilla population. The ongoing effects of environmental changes and population dynamics must be closely monitored and used to develop effective long-term conservation strategies that can help address these risk factors.

In the current global health climate, many conservation biologists are managing crisis situations, including increased species extinction rates. One strategy for securing wildlife populations into the future is to preserve biomaterials in genome resource banks (GRB; or 'biobanks'). However, for GRBs to be successful we must understand the fundamental reproductive biology of species, along with developing assisted reproductive techniques (ARTs), including AI and semen cryopreservation. ART has been successfully used for several taxa, from amphibians to mammals, including ungulates, carnivores and primates. Not all these success stories implemented the use of a biobank, but one example that discussed herein is the black-footed ferret (Mustela nigripes) GRB. From a founder population of seven individuals, this species has been breeding in a managed setting for nearly 30 years. The goal of the breeding program is to maintain genetic integrity by ensuring each individual has the opportunity to pass his/her genes onto the next generation, while simultaneously providing animals for release into the wild. Scientists have used ART (e.g. AI) in the recovery program. Recently, semen from an individual of the founder population that was cryopreserved for up to 20 years was used successfully for AI, which improved the genetic diversity of the population. The black-footed ferret recovery program can serve as a model for other endangered species and demonstrates the usefulness of ART and GRBs to maintain highly endangered species into the future.

Despite the growing body of literature examining the welfare of zoo-housed animals, little standardized work has been published on the effect of construction and environmental disruption on the physiology and behavior of affected animals. When Lincoln Park Zoo (Chicago, IL), embarked on a renovation project for its Kovler Lion House, the opportunity was taken to perform a scientific study of behavioral and physiological markers in the resident felids to determine the effect of construction and environmental disruption. Fecal samples and behavioral observations were collected on four felid species (five individuals) before, during, and after the period of construction. As a group, the average z-score for fecal glucocorticoid metabolite concentration increased during construction relative to baseline. Levels remained elevated after construction, but trended toward baseline. All individuals demonstrated a significant decrease in the frequency of pacing and time spent visible during construction. Overall activity levels also showed a significant decrease relative to baseline measures. As zoological institutions continue to recognize the importance of habitat design, construction and renovation become inevitable. It is important to be aware of the potential consequences this can have on animals in the vicinity and to work toward minimizing negative effects. One recommendation is the availability of ample retreat and hiding space for felids during disruption to their environment., (© 2014 Wiley Periodicals, Inc.)

Pied tamarins are an endangered Amazonian primate that has limited breeding success in zoos. Unfortunately, little is known about their reproductive biology and adrenocortical activity. Objectives were: (1) determine if fecal hormones could be utilized to monitor gonadal and adrenocortical activity; (2) characterize male and female gonadal and adrenocortical hormones; and (3) determine if there were differences between adrenocortical activity and behavior in a nonbreeding, on-exhibit (NB-ON) pair compared to a breeding, off-exhibit (B-OFF) pair. Fecal samples were collected from four (two males; two females) individuals. Hormones were analyzed for fecal progesterone (FPM), androgen (FAM), and glucocorticoid (FGM) metabolites by enzyme immunoassay. Behavioral observations were conducted for 6 months. Data were collected on instantaneous behavior, location, and all occurrences of intraspecific behaviors. Fecal progesterone metabolites were validated by pregnancy (mean ± SE, pregnant: 28.47 ± 1.60 μg/g; nonpregnant: 8.63 ± 0.89 μg/g). Fecal androgen metabolites were higher (T = 31,971, P < 0.05) in the B-OFF male (863.66 ± 46.30 μg/g) than the NB-ON male (838.63 ± 60.70 μg/g). Fecal glucocorticoid metabolites were validated by response to veterinary procedure with elevated values (7.31 ± 1.48 μg/g) seven times the baseline (0.37 ± 0.04 μg/g) at 24-hr postphysical. Females had higher baseline FGM than the males (P < 0.05). Baseline FGM were higher (P < 0.05) in the NB-ON female (0.93 ± 0.03 μg/g) compared to the B-OFF female (0.38 ± 0.02 μg/g). Similarly, the NB-ON male's FGM baseline (0.71 ± 0.03 μg/g) were higher (P < 0.05) than the B-OFF male (0.21 ± 0.01 μg/g). Behavioral data revealed stereotypical behaviors in the NB-ON pair but no stereotypical behaviors in the B-OFF pair. Fecal hormone monitoring and behavioral analysis may provide insight on the limited breeding success of pied tamarins in zoos., (© 2012 Wiley Periodicals, Inc.)

Understanding the basic reproductive biology and limitations to successful breeding of the southern three-banded armadillo (Tolypeutes matacus) is necessary to maintain viable zoo populations. Our objectives were to: 1) describe the reproductive biology using non-invasive, fecal hormone analysis; 2) assess influence of season on gonadal hormonal patterns in both the sexes; 3) characterize reproductive cyclicity and pregnancy in the female; and 4) characterize the onset of sexual maturity in males. Nineteen armadillos were monitored including: 13 (7 males, 6 females) from Lincoln Park Zoo and six (3 males, 3 females) from San Antonio Zoological Garden. Fecal samples (n=5220; 275/animal/yr) were collected 5 to 7 times a week for 1 year. Hormones were extracted from feces and analyzed for progestagen (females) and androgen (males) metabolite concentrations using enzyme immunoassays. Mean estrous cycle length (26.4±1.3 days) did not vary (P<0.05) among individuals (n=9). Mean gestation length (n=3) was 114.0±0.6 days long with mean fecal progestagen metabolites increasing 10-fold during pregnancy. Seasons did not influence (P<0.05) fecal androgen or progestagen metabolites. These data can assist with management decisions, which will directly affect the success of this species in zoos., (Published by Elsevier B.V.)

Study Objectives: The objectives of this study were to characterize sleep patterns and determine factors, including sex, age, season, and environmental pressures, that influence sleep in the endangered black rhinoceros (rhino; Diceros bicornis bicornis)., Design: To noninvasively observe sleep behavior of wild rhinos, digital infrared cameras were erected on poles at two bedding sites from September 2009 to March 2010., Setting: The study site was located in South Africa's Addo Elephant National Park (AENP) in the Main Camp (Addo) and Nyathi sections., Participants: A total of 2,417 photos captured rhino sleep behavior on eight individual rhinos during 40 separate sleeping bouts (Addo, n = 15; Nyathi, n = 25)., Inventions: N/A., Results: Results demonstrated that age and season did not affect rhino sleep behavior (P > 0.05); however, sex did influence the length of sleep bouts with males (n = 27; mean, 105.6 ± 11.3 min; range, 14.0-202.0 min) sleeping longer (F(1,48) = 6.93, P = 0.01) than females (n = 13; mean, 58.6 ± 10.4 min; range, 11.0-132.0 min). Park section did not influence the length of sleep episodes, but did affect (rw(40) = 0.88; P < 0.025) the time at which rhinos slept (Addo, 20:00-24:00; Nyathi, 20:00-04:00)., Conclusions: This is the first study to characterize sleep behavior in wild black rhinos. This study resulted in a greater understanding of the biologic factors that affect sleep in wild rhinos and can provide information to assist their management and conservation.

The Sichuan takin (takin; Budorcas taxicolor tibetana) is distributed in the Gansu and Sichuan providences of southern China and along eastern Tibet. Because of their ecology, few data on takin reproductive biology exist, with the exception of its mating season in the Sichuan province, which occurs from July through August. Therefore, the objectives were to: 1) characterize reproductive hormones in zoo-housed male and female takin, including pregnancy in the female, using non-invasive fecal steroid hormonal monitoring; 2) characterize behaviors of zoo-housed takin, emphasizing reproductive behaviors and activity budget; and 3) assess the influence of season on births in North America and reproductive hormonal and behavioral activity. Fecal samples were collected 3 to 5 times per week from two adult males and three adult females. Extracted hormones were analyzed using an enzyme immunoassay for progestagen and androgen concentrations. Behavioral observations were collected for 2 yrs using an ethogram. In this study, season affected reproduction, specifically birth occurrences, reproductive cyclicity in females and androgen production in males. The duration of the estrous cycle was approximately 35 d and cycles occurred June through December. Androgen concentrations peaked in May through August. Season did not influence behavior; however, age and sex may affect some behaviors, including activity level, foraging and drinking, social affiliative behavior, and visibility from the visitor's viewpoint. In conclusion, fecal hormonal and behavioral analyses can provide information for management and conservation of this herd species., (Copyright © 2012 Elsevier Inc. All rights reserved.)

Techniques for analyzing hormone metabolites in animal excreta have created many opportunities for noninvasive monitoring of health, reproduction, and welfare in zoo animals, but can be difficult to implement when individual samples are not readily identifiable in animal groups. A common approach to this problem is to feed animals an indigestible marker that subsequently appears in feces, but there has been little systematic research on the use of such "fecal markers." First, we used an online survey to assess the prevalence of fecal marker use in North American zoological institutions. Second, we conducted a series of experimental tests utilizing commonly employed fecal markers in a variety of typical zoo taxa to determine the: (1) effectiveness of several markers to accurately distinguish samples in a variety of species, (2) minimum quantity of marker needed for detection, and (3) length of time between ingestion and detection in the feces. The majority of the 45 institutions that completed the survey reported using fecal markers with their collections. The survey also revealed that the most frequently used markers are seeds/grains and food colorants, with the former generally used in Carnivora and the latter in Primates. Our experimental data confirmed the success of these taxa/marker combinations and also revealed that food colorants function as markers in a variety of avian, reptilian, and mammalian species. Our data describe successful fecal markers for a wide variety of zoo taxa and should, therefore, be useful for zoological managers and researchers needing to employ fecal markers in future investigations., (© 2010 Wiley-Liss, Inc.)

Introductions of sable antelope (Hippotragus niger) can be difficult due to the potential ensuing aggression compounded by their large horns. The goal was to use hormonal assays and behavioral analyses to evaluate the success of an introduction of 2 adult females at Lincoln Park Zoo. The objectives were to (a) document behavioral and hormonal changes in 2 female sable antelope during the introduction, (b) compare fecal glucocorticoid metabolites (FGM) in each individual during the introduction stages, (c) measure fecal androgen metabolites (FAM) during introduction and compare with dominance rank and observed aggression, and (d) monitor estrous cycle synchronization. Results demonstrate that FGM were higher before than during and after the introduction. Behavioral observations indicated limited aggression between females, although the keeper survey results revealed that the new female was more dominant and had higher mean FGM and FAM than the resident. Both sable antelope were reproductively active throughout the year. Results indicate that fecal hormone analysis can provide zoo management with valuable information to minimize the risk of aggression, injury, and stress during introductions of nonhuman animals.

The science of cryobiology is essential to the effective, practical use of semen for assisted breeding to help manage small populations of rare wildlife species. In this review, we describe challenges associated with cryopreserving gametes from wild fauna. Based on more than 25 years of experience across a diversity of mammals, it appears that the primary driving force dictating cryo-survival of a spermatozoon is its initial pre-freeze quality and morphology, especially having a morphologically normal, intact acrosome. This assertion is supported through extensive studies of three animal groups that routinely ejaculate semen containing (1) normal sperm/acrosomal quality (examples, Eld's deer, Cervus eldi and giant panda, Ailuropoda melanoleuca), (2) normal acrosomal quality, but from teratospermic donors (>70% pleiomorphic sperm; cheetah, Acinonyx jubatus and black-footed ferret, Mustela nigripes) and (3) abnormal acrosomal quality and general teratospermia (clouded leopard, Neofelis nebulosa). Data revealed that species producing high quality sperm with > 70% normal, intact acrosomes were best able to survive cryopreservation (-80% intact acrosomes post-thaw). Species that were teratospermic, but with high proportions of intact acrosomes (72 to 88%) in ejaculates varied significantly (4 to 55% intact acrosomes post-thaw) in sperm survival to freeze-thawing. Spermatozoa from the clouded leopard (that was both teratospermic while producing only 11% normal acrosomes in fresh semen) failed to survive cryopreservation despite using an array of conventional and unconventional freezing approaches. These observations (combined with zona penetration assays and artificial insemination results) suggest that proportions of malformed sperm and especially initial structural integrity of the acrosome are more important predictors of sperm survivability post-thaw than initial sperm motility scores.