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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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).

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.

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.

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.

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.

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.

Directional freezing of spermatozoa and embryos.

Directional freezing is based on a simple thermodynamic principle whereby the sample is moved through a predetermined temperature gradient at a velocity that determines the cooling rate. Directional freezing permits a precise and uniform cooling rate in small- and large-volume samples. It avoids supercooling and reduces mechanical damage caused by crystallisation.

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.

One small step for rhinos, one giant leap for wildlife management--imaging diagnosis of bone pathology in distal limb.

Chronic foot disease poses a threat to the general health, represents a tremendous clinical challenge, and often is a reason for euthanasia in captive megaherbivores, among them the elephant and rhinoceros. Nevertheless, apart from the elephant, foot pathology is handled as being confined only to soft tissues whereas bone pathology is often overlooked. As a case in point, the authors selected the second largest mammal on land, the rhinoceros.

Freezing African elephant semen as a new population management tool.

Background: The captive elephant population is not self-sustaining and with a limited number of breeding bulls, its genetic diversity is in decline. One way to overcome this is to import young and healthy animals from the wild. We introduce here a more sustainable alternative method - importation of semen from wild bulls without removing them from their natural habitat.

Use of a medetomidine-ketamine combination for anesthesia in captive common hippopotami (Hippopotamus amphibius).

Objective: To establish an anesthetic protocol suitable for surgical interventions in hippopotami (Hippopotamus amphibius).

Design: Prospective case series.

Animals: 10 adult male hippopotami undergoing castration.