Reviving vacuum-dried encapsulated ram spermatozoa via ICSI after 2 years of storage.

Introduction: Freeze-drying techniques give alternative preservation mammalian spermatozoa without liquid nitrogen. However, most of the work has been conducted in the laboratory mouse, while little information has been gathered on large animals that could also benefit from this kind of storage. Methods: This work adapted a technique known as vacuum-drying encapsulation (VDE), originally developed for nucleic acid conservation in anhydrous state, to ram spermatozoa, and compared it to canonical lyophilization (FD), testing long-term storage at room temperature (RT) and 4°C. Results and discussion: The results demonstrated better structural stability, namely lipid composition and DNA integrity, in VDE spermatozoa than FD ones, with outcomes at RT storage comparable to 4°C. Likewise, in VDE the embryonic development was higher than in FD samples (12.8% vs. 8.7%, p < 0.001, respectively). Our findings indicated that in large mammals, it is important to consider dehydration-related changes in sperm polyunsaturated fatty acids coupled with DNA alterations, given their crucial role in embryonic development.

Reproductive biotechnology and critically endangered species: Merging in vitro gametogenesis with inner cell mass transfer.

A fifth of mammalian species face the risk of extinction. A variety of stresses, and lack of sufficient resources and political endorsement, mean thousands of further extinctions in the coming years. Once a species has declined to a mere few individuals, in situ efforts seem insufficient to prevent its extinction. Here we propose a roadmap to overcome some of the current roadblocks and facilitate rejuvenation of such critically endangered species. We suggest combining two advanced assisted reproductive technologies to accomplish this task. The first is the generation of gametes from induced pluripotent stem cells, already demonstrated in mice. The second is to 'trick' the immunological system of abundant species' surrogate mothers into believing it carries conceptus of its own species. This can be achieved by transferring the inner cell mass (ICM) of the endangered species into a trophoblastic vesicle derived from the foster mother's species. Such synthesis of reproductive biotechnologies, in association with in situ habitat conservation and societal changes, holds the potential to restore diversity and accelerate the production of animals in the most endangered species on Earth.

Dry biobanking as a conservation tool in the Anthropocene.

Species are going extinct at an alarming rate, termed by some as the sixth mass extinction event in the history of Earth. Many are the causes for this but in the end, all converge to one entity - humans. Since we are the cause, we also hold the key to making the change. Any change, however, will take time, and for some species this could be too long. While working on possible solutions, we also have the responsibility to buy time for those species on the verge of extinction. Genome resource banks, in the form of cryobanks, where samples are maintained under liquid nitrogen, are already in existence but they come with a host of drawbacks. Biomimicry - innovation inspired by Nature, has been a huge source for ideas. Searching methods that Nature utilizes to preserve biological systems for extended periods of time, we realize that drying rather than freezing is the method of choice. We thus argue here in favor of preserving at least part of the samples from critically endangered species in dry biobanks, a much safer, cost-effective, biobanking approach.

Exploring dry storage as an alternative biobanking strategy inspired by Nature.

Biobanking is a rapidly growing industry, covering diverse fields such as human medicine, farm animal production, laboratory animals record keeping, and wildlife conservation. Presently, biobanking is done almost exclusively by cryopreservation, followed by maintenance of the samples under liquid nitrogen. Cryopreservation has satisfactory efficiency but it comes with a host of problems, and the process is highly species-specific. Like in many other walks of life, we turn to Nature in search for better alternatives. Nature opted for controlled drying rather than water preservation via freezing when long-term preservation is desired, a strategy known as 'anhydrobiosis'. To achieve reversible drying, anhydrobiotic organisms utilise an assortment of protective materials, including disaccharides, late embryogenesis abundant proteins, anhydrin, heat shock protein, and more. Once dry, desiccation-tolerant organisms can survive extended periods of time and be resistant to extreme environmental stressors. Over the past 70 years researchers attempted applying this idea to preserve desiccation-sensitive mammalian cells in the dry form. At present dried cells mostly do not resume biological activity upon rehydration. The DNA, however, is often well preserved to allow utilisation in advanced reproductive techniques. Spermatozoa are by far the most commonly dried cell type, primarily from mice and bulls. A number of drying approaches have been applied, with freeze-drying taking the lead. To date offspring have been produced from dried spermatozoa in mouse, rat, hamster, rabbit, and horse. No offspring were produced from dried somatic cells. Desiccation experiences a sharp increase in interest and research output in recent years. Presented here is an overview of dry preservation, its possible applications, the open questions the field is still facing, and some suggested directions for the future.

Differential detection of tuberculous and non-tuberculous mycobacteria by qPCR in lavage fluids of tuberculosis-suspicious white rhinoceros.

Tuberculosis (TB) occurs in a wide range of mammalian species and thus poses a health risk to humans living or working in close proximity with TB infected animals. Despite a high incidence of M. bovis infections in domestic or wildlife species tuberculosis infections in rhinoceros have so far been very limited. Over the past 53 years, tuberculosis of the respiratory tract has been confirmed in just 22 rhinoceros, most of those infected not by M. bovis but M. tuberculosis. However, because of the zoonotic risk TB testing is recommended or becomes even mandatory in endangered species. The dilemma in rhinoceros and many other wildlife species; non-validated tests are highly inconsistent in their ability to identify TB infection. Current lack of TB diagnostics may result in TB positive rhinoceros living with the infection, transmitting it to those around them or in euthanasia of animals found unconfirmed at necropsy. This is an unacceptable diagnostic status considering that some species are critically endangered and therefore should not be euthanized in order to confirm suspicion of disease. To overcome this shortcoming we used bronchoscopy to detect mycobacteria in respiratory fluids of TB suspicious rhinoceros. Fluids from seven, TB suspicious white rhinoceros were harvested during 21 bronchoscopies. Our new approach: In addition to bacterial culture a dual quantitative PCR system tested for the general presence of DNA from NTM and more specifically for DNA from MTC. Both, bacterial culture and qPCR were negative for MTC in respiratory fluids of all rhinoceros (7/7). At the same time, respiratory fluids from six rhinoceros tested positive for the presence of NTM or other closely related bacteria (6/7). M. tuberculosis was found only once in an oesophageal aspirate. The high incidence of mycobacterial DNA in the respiratory tract suggests that white rhinoceros, as strict grazers, are immensely exposed to environmental bacteria of this genus. Presence of NTM in the respiratory or intestinal system could possibly cause false positive results in intradermal tests. A wider use of bronchoalveolar lavage is warranted to further elucidate immunologic response to NTM and exposure to, incidence and prevalence of MTC infections in rhinoceros.

DNA fragmentation in epididymal freeze-dried ram spermatozoa impairs embryo development.

Sperm freeze-drying is a revolutionary technique, which has been gaining prominence in recent years. The first related significant result was Wakayama and Yanagimachi's demonstration in 1998 of the birth of healthy mouse offspring by Intracytoplasmic Sperm Injection (ICSI), using epididymal freeze-dried spermatozoa. Mouse, rat, and hamster models were the first small mammals born from lyophilized epididymal spermatozoa, whereas most other studies in this field used ejaculated spermatozoa. In this work, we applied this technique to ram epididymal spermatozoa, checking the correlation between DNA integrity and embryo development following ICSI. To do this, epididymal sperm from four rams was lyophilized in a trehalose, glucose, KCl, HEPES, and Trolox media. To evaluate DNA damage and fragmentation after rehydration, samples were processed for Sperm Chromatin Dispersion test (SCD), Two-Tailed Comet Assay, and were used for ICSI. Ram #2 had a higher rate of spermatozoa with intact DNA compared with rams #1, #3, and #4 (28% vs. 3.8%, 2.8%, and 5%, respectively) and the lowest rate of Single-Strand Breaks (SSBs) (70% vs. 95.9%, 92.6%, and 93% respectively). Ram #3 had a higher level of Double-Strand Breaks (DSBs) compared to Ram #1 (4.6% vs. 0.33%, respectively). Embryo development to the blastocyst stage following ICSI was only reached from rams whose sperm had higher level of intact DNA - Rams #2 and #4 (6%, 5/147 and 6.3%, 4/64, respectively). Definitively, the impact of sperm DNA damage on embryonic development depends on the balance between sperm DNA fragmentation extent, fragmentation type (SSBs or DSBs), and the oocyte's repair capacity.

Follicular size predicts success in artificial insemination with frozen-thawed sperm in donkeys.

In asses, semen collection, cryopreservation, and artificial insemination (AI) with frozen-thawed semen have been scarcely described and success rate, particularly following AI, is reportedly low. In the absence of reliable protocols, assisted reproductive technologies cannot support the conservation efforts aimed at endangered wild ass species and domestic donkey breeds. Two experiments were conducted in this study. In experiment 1 we evaluated freezing Abyssinian donkey (N = 5, 4 ejaculates each) spermatozoa using three freezing extenders (Berliner Cryomedium + glycerol, BC+G; BotuCrio, BOTU; INRAFreeze, INRA) and two cryopreservation techniques (liquid nitrogen vapour, LNV; directional freezing, DF). Post-thaw evaluation indicated that BOTU and INRA were similar and both superior to BC+G (P ≤ 0.004 for all motility tests), and that DF was superior to LNV (P < 0.002 for all evaluation parameters). In experiment 2, relying on these results, we used Abyssinian donkey sperm frozen in BOTU and INRA by DF for AI (N = 20). Prior to AI, thawed samples were diluted in corresponding centrifugation media or autologous seminal fluids at 1:1 ratio. No difference was found between BOTU and INRA or between the addition of seminal fluids or media, all resulting in ~50% pregnancy, and no differences were noted between males (N = 4). The size of pre-ovulatory follicle was a significant (P = 0.001) predictor for AI success with 9/10 pregnancies occurring when follicular size ranged between 33.1-37.4 mm, no pregnancy when it was smaller, and only one when larger. A number of ass species face the risk of extinction. Knowledge gained in this study on the Abyssinian donkey can be customised and transferred to its closely related endangered species and breeds.

Ovarian down Regulation by GnRF Vaccination Decreases Reproductive Tract Tumour Size in Female White and Greater One-Horned Rhinoceroses.

Reproductive tract tumours, specifically leiomyoma, are commonly found in female rhinoceroses. Similar to humans, tumour growth in rhinoceroses is thought to be sex hormone dependent. Tumours can form and expand from the onset of ovarian activity at puberty until the cessation of sex-steroid influences at senescence. Extensive tumour growth results in infertility. The aim of this study was to down regulate reproductive function of tumour-diseased and infertile females to stop further tumour growth using a Gonadotropin releasing factor (GnRF) vaccine. Four infertile southern white (Ceratotherium simum simum) and three Greater one-horned rhinoceroses (rhinoceros unicornis) with active ovaries and 2.7 ± 0.9 and 14.0 ± 1.5 reproductive tract tumours respectively were vaccinated against GnRF (Improvac®, Zoetis, Germany) at 0, 4 and 16 weeks and re-boostered every 6-8 months thereafter. After GnRF vaccination ovarian and luteal activity was suppressed in all treated females. Three months after vaccination the size of the ovaries, the number of follicles and the size of the largest follicle were significantly reduced (P<0.03). Reproductive tract tumours decreased significantly in diameter (Greater-one horned rhino: P<0.0001; white rhino: P<0.01), presumably as a result of reduced sex-steroid influence. The calculated tumour volumes were reduced by 50.8 ± 10.9% in Greater one-horned and 48.6 ± 12.9% in white rhinoceroses. In conclusion, GnRF vaccine effectively down regulated reproductive function and decreased the size of reproductive tract tumours in female rhinoceros. Our work is the first to use down regulation of reproductive function as a symptomatic treatment against benign reproductive tumour disease in a wildlife species. Nonetheless, full reversibility and rhinoceros fertility following GnRF vaccination warrants further evaluation.

Rewinding the process of mammalian extinction.

With only three living individuals left on this planet, the northern white rhinoceros (Ceratotherium simum cottoni) could be considered doomed for extinction. It might still be possible, however, to rescue the (sub)species by combining novel stem cell and assisted reproductive technologies. To discuss the various practical options available to us, we convened a multidisciplinary meeting under the name "Conservation by Cellular Technologies." The outcome of this meeting and the proposed road map that, if successfully implemented, would ultimately lead to a self-sustaining population of an extremely endangered species are outlined here. The ideas discussed here, while centered on the northern white rhinoceros, are equally applicable, after proper adjustments, to other mammals on the brink of extinction. Through implementation of these ideas we hope to establish the foundation for reversal of some of the effects of what has been termed the sixth mass extinction event in the history of Earth, and the first anthropogenic one. Zoo Biol. 35:280-292, 2016. © 2016 The Authors. Zoo Biology published by Wiley Periodicals, Inc.

Non-Breeding Eusocial Mole-Rats Produce Viable Sperm--Spermiogram and Functional Testicular Morphology of Fukomys anselli.

Ansell's mole-rats (Fukomys anselli) are subterranean rodents living in families composed of about 20 members with a single breeding pair and their non-breeding offspring. Most of them remain with their parents for their lifetime and help to maintain and defend the natal burrow system, forage, and care for younger siblings. Since incest avoidance is based on individual recognition (and not on social suppression) we expect that non-breeders produce viable sperm spontaneously. We compared the sperm of breeding and non-breeding males, obtained by electroejaculation and found no significant differences in sperm parameters between both groups. Here, we used electroejaculation to obtain semen for the first time in a subterranean mammal. Spermiogram analysis revealed no significant differences in sperm parameters between breeders and non-breeders. We found significantly larger testes (measured on autopsies and on living animals per ultrasonography) of breeders compared to non-breeders (with body mass having a significant effect). There were no marked histological differences between breeding and non-breeding males, and the relative area occupied by Leydig cells and seminiferous tubules on histological sections, respectively, was not significantly different between both groups. The seminiferous epithelium and to a lesser degree the interstitial testicular tissue are characterized by lesions (vacuolar degenerations), however, this feature does not hinder fertilization even in advanced stages of life. The continuous production of viable sperm also in sexually abstinent non-breeders might be best understood in light of the mating and social system of Fukomys anselli, and the potential to found a new family following an unpredictable and rare encounter with an unfamiliar female ("provoked or induced dispersal"). Apparently, the non-breeders do not reproduce because they do not copulate but not because they would be physiologically infertile. The significantly increased testes volume of breeding males (compared to non-breeders) is in agreement with previously found higher testosterone levels of breeders.

Directional freezing of sperm and associated derived technologies.

Directional freezing has now completed 30 years of development since it was first introduced to cryobiology. In the field of sperm cryopreservation, directional freezing has been shown to be advantageous over slow freezing for numerous domestic and wildlife species. In particular, it was shown that freezing of large volume is possible. Furthermore, double freezing of sperm and freezing of sex-sorted sperm are possible and became the routine in the sex sorted sperm industry. In wild animals, our labs and others showed that sperm from a wide range of terrestrial and aquatic species can be successfully cryopreserved using directional freezing. Finally, we will describe for the first time the successful freeze-drying of human sperm in an aseptic method. Using a device that produces clean liquid air, we froze human sperm in small droplets and then dried them in a bench top lyophilizer that was sterilized prior to use. More than 80% of DNA integrity was found after rehydration.

Controlled ice nucleation--Is it really needed for large-volume sperm cryopreservation?

Controlled ice nucleation (CIN) is an integral stage of slow freezing process when relatively large volumes (usually 1 mL or larger) of biological samples in suspension are involved. Without it, a sample will supercool to way below its melting point before ice crystals start forming, resulting in multiple damaging processes. In this study, we tested the hypothesis that when freezing large volumes by the directional freezing technique, a CIN stage is not needed. Semen samples collected from ten bulls were frozen in 2.5-mL HollowTubes in a split-sample manner with and without a CIN stage. Thawed samples were evaluated for viability, acrosome integrity, rate of normal morphology, and, using computer-aided sperm analysis system, for a wide range of motility parameters that were also evaluated after 3 hours of incubation at 37 °C. Analysis of the results found no difference between freezing with and without CIN stage in any and all of the 29 parameters compared (P > 0.1 for all). This similarity was maintained through 3 hours of incubation at 37 °C. Possibly, because of its structure, the directional freezing device promotes continuous ice nucleation so a specific CIN stage is no longer needed, thus reducing costs, energy use, and carbon footprint.

CRYOPRESERVATION OF ONAGER (EQUUS HEMIONUS ONAGER) EPIDIDYMAL SPERMATOZOA.

Genetic diversity is a primary component of adaptive evolution, and its loss or reduction can decrease the long-term survival probability of populations. Utilization of cryopreserved semen may be considered a perfect tool to improve genetic diversity, reduce inbreeding, and avoid animal translocation for breeding. The present study aimed at finding a reliable epididymal sperm freezing protocol for the critically endangered onager (Equus hemionus onager). Six testicles from three animals were processed postmortem. The effects of two transportation temperatures (22°C and 4°C; testicles submerged in saline), two cryopreservation techniques (conventional liquid nitrogen vapor freezing in straws and directional freezing in 8-ml HollowTubes(TM)), and two postthaw incubation temperatures (22°C and 37°C; evaluated after 0.5, 1, 2, and 3 hr) were tested in a 2×2×2 experimental design. Sperm samples were evaluated for motility, viability, acrosome integrity, and sperm morphology. The resulting optimal freezing protocol includes transportation of testicles at 4°C, cryopreservation by directional freezing, and, if needed, postthaw incubation at 22°C. With this combination of transportation temperature and cryopreservation technique, the authors obtained the following postthaw values normalized to prefreezing values: 60.3±8.8% motility, 60.7±13.3% viability, 75.3±9.5% acrosome integrity, and 94.7±2.9% normal morphology (excluding defects due to the epididymal origin of the sperm). After incubation at 22°C, motility values for the above combination were 40±5.7%, 30.3±5.2%, 28.3±4.4%, and 16.7±4.4% for 0.5, 1, 2, and 3 hr, respectively. In conclusion, with this protocol, good quality semen can be stored for future use in artificial inseminations when and where needed.

Sperm rescue in wild African elephants.

This study investigated ways of improving the usefulness of ~1700mL of poor-quality frozen semen collected from wild African elephant (Loxodonta africana) bulls. Ten semen samples from six bulls, frozen with 5% glycerol in Berliner cryomedium, with or without prior removal of the seminal plasma by centrifugation, were tested. All samples were subjected to the following density-gradient centrifugation treatments: no centrifugation (control), sham centrifugation, Percoll, OptiPrep, Isolate and PureSperm. Sample evaluation included motility, concentration, viability, acrosome integrity and normal morphology after thawing and after gradient centrifugation. Motility was also evaluated 3h after thawing. While all treatments were similar to the Control in acrosome integrity and normal morphology, significant differences were noted in concentration, viability and motility. Samples treated by Percoll showed the best motility, which was maintained unchanged over 3h of incubation (37°C). Correlations between manual and automated evaluations of concentration were high (cytometer; rho=0.92), but were lower for viability (cytometer; rho=0.57) and motility (computer-aided sperm analysis; rho=0.66). By performing density centrifugation, the quality of these sperm samples may be improved to a level suitable for artificial insemination in elephants. Although a sizeable proportion of cells are lost in the process, combining samples may still allow for multiple inseminations.

Directional freezing for large volume cryopreservation.

Cryopreservation is currently the method of choice when it comes to long-term preservation of viable biological samples. The process, and consequently the volume of the sample, however, is limited by the ability to achieve homogenous and efficient heat removal. When this cannot be properly managed, ice crystals will grow uncontrollably resulting in extensive damage to the cryopreserved cells or tissues. Directional freezing is a technique that can be used to precisely control heat dissipation and ice crystal growth and morphology even when freezing large volumes. The technique has been used over the years to cryopreserve spermatozoa, oocytes, embryos, tissue slices and whole organs from a wide variety of domestic and wild species. In this chapter a protocol for directional freezing of spermatozoa is described and its benefits and shortcomings are discussed.

Cloning the mammoth: a complicated task or just a dream?

Recently there has been growing interest in applying the most advanced embryological tools, particularly cloning, to bring extinct species back to life, with a particular focus on the woolly mammoth (Mammuthus primigenius). Mammoth's bodies found in the permafrost are relatively well preserved, with identifiable nuclei in their tissues. The purpose of this chapter is to review the literature published on the topic, and to present the strategies potentially suitable for a mammoth cloning project, with a frank assessment of their feasibility and the ethical issues involved.

Rhinoceros feet step out of a rule-of-thumb: a wildlife imaging pioneering approach of synchronized computed tomography-digital radiography.

Currently, radiography is the only imaging technique used to diagnose bone pathology in wild animals situated under "field conditions". Nevertheless, while chronic foot disease in captive mega-herbivores is widely reported, foot radiographic imaging is confronted with scarcity of studies. Numerous hindrances lead to such limited numbers and it became very clear that the traditional perspective on bone imaging in domestic animals based on extensive studies and elaborated statistical evaluations cannot be extrapolated to their non-domestic relatives. For these reasons, the authors initiated a multi-modality imaging study and established a pioneering approach of synchronized computed tomography (CT) and digital radiography (DR), based on X-ray projections derived from three-dimensional CT reconstructed images. Whereas this approach can be applied in any clinical field, as a case of outstanding importance and great concern for zoological institutions, we selected foot bone pathologies in captive rhinoceroses to demonstrate the manifold applications of the method. Several advances were achieved, endowing the wildlife clinician with all-important tools: prototype DR exposure protocols and a modus operandi for foot positioning, advancing both traditional projections and, for the first-time, species-related radiographic views; assessment of radiographic diagnostic value for the whole foot and, in premiere, for each autopodial bone; together with additional insights into radiographic appearance of bone anatomy and pathology with a unique, simultaneous CT-DR correlation. Based on its main advantages in availing a wide range of keystone data in wildlife imaging from a limited number of examined subjects and combining advantages of CT as the golden standard method for bone diseases' diagnostic with DR's clinical feasibility under field conditions, synchronized CT-DR presents a new perspective on wildlife's health management. With this we hope to provide veterinary clinicians with concrete imaging techniques and substantial diagnostic tools, which facilitate straightforward attainment and interpretation of field radiography images taken worldwide.

Comparative skull analysis suggests species-specific captivity-related malformation in lions (Panthera leo).

Lion (Panthera leo) populations have dramatically decreased worldwide with a surviving population estimated at 32,000 across the African savannah. Lions have been kept in captivity for centuries and, although they reproduce well, high rates of stillbirths as well as morbidity and mortality of neonate and young lions are reported. Many of these cases are associated with bone malformations, including foramen magnum (FM) stenosis and thickened tentorium cerebelli. The precise causes of these malformations and whether they are unique to captive lions remain unclear. To test whether captivity is associated with FM stenosis, we evaluated 575 lion skulls of wild (N = 512) and captive (N = 63) origin. Tiger skulls (N = 276; 56 captive, 220 wild) were measured for comparison. While no differences were found between males and females or between subadults and adults in FM height (FMH), FMH of captive lions (17.36±3.20 mm) was significantly smaller and with greater variability when compared to that in wild lions (19.77±2.11 mm). There was no difference between wild (18.47±1.26 mm) and captive (18.56±1.64 mm) tigers in FMH. Birth origin (wild vs. captive) as a factor for FMH remained significant in lions even after controlling for age and sex. Whereas only 20/473 wild lions (4.2%) had FMH equal to or smaller than the 5th percentile of the wild population (16.60 mm), this was evident in 40.4% (23/57) of captive lion skulls. Similar comparison for tigers found no differences between the captive and wild populations. Lions with FMH equal to or smaller than the 5th percentile had wider skulls with smaller cranial volume. Cranial volume remained smaller in both male and female captive lions when controlled for skull size. These findings suggest species- and captivity-related predisposition for the pathology in lions.

Reproductive tract tumours: the scourge of woman reproduction ails Indian rhinoceroses.

In Indian rhinoceros, extensive leiomyoma, a benign smooth muscle tumour, was sporadically diagnosed post mortem and commonly thought of as contributing factor for reduced fecundity of this species in captivity. However, to date, the prevalence of reproductive tract tumours and their relevance for fecundity are unknown. Our analysis of the international studbook now reveals that females cease reproducing at the age of 18.1±1.2 years; equivalent to a reproductive lifespan of just 9.5±1.3 years. This short reproductive life is in sharp contrast to their longevity in captivity of over 40 years. Here we show, after examining 42% of the captive female population, that age-related genital tract tumours are highly prevalent in this endangered species. Growth and development of these tumours was found to be age-related, starting from the age of 10 years. All females older than 12 years had developed genital tumours, just 7-9 years past maturity. Tumour sizes ranged from 1.5-10 cm. With age, tumours became more numerous, sometimes merging into one large diffuse tumour mass. These tumours, primarily vaginal and cervical, presumably cause widespread young-age infertility by the age of 18 years. In few cases, tumour necrosis suggested possible malignancy of tumours. Possible consequences of such genital tract tumour infestation are hindered intromission, pain during mating, hampered sperm passage, risk of ascending infection during pregnancy, dystocia, or chronic vaginal bleeding. In humans, leiomyoma affect up to 80% of pre-menopause women. While a leading cause for infertility, pregnancy is known to reduce the risk of tumour development. However, different from human, surgical intervention is not a viable treatment option in rhinoceroses. Thus, in analogy to humans, we suggest early onset and seamless consecutive pregnancies to help reduce prevalence of this disease, better maintain a self-sustained captive population and improve animal welfare.

Surgical castration of the male common hippopotamus (Hippopotamus amphibius).

In a prospective, clinical, surgery study we report here for the first time, in detail, on the surgical castration of 10 captive adult male common hippopotami (Hippopotamus amphibius). The successful procedures, a species-specific modification of standard equine castration techniques, provide valuable insight into the spatially dynamic nature of the common hippopotamus testis. The use of ultrasonography to locate the testis before and during the procedures and species-specific positioning during surgery greatly facilitated this distinctive procedure. Additionally, this surgical method provides an important additional tool for captive management of the common hippopotamus. Castration of individual males not only facilitates population control but can potentially also be employed to limit intermale aggression.