Dry storage of mammalian spermatozoa and cells: state-of-the-art and possible future directions.

This review provides a snapshot of the current state-of-the-art of drying cells and spermatozoa. The major successes and pitfalls of the most relevant literature are described separately for spermatozoa and cells. Overall, the data published so far indicate that we are closer to success in spermatozoa, whereas the situation is far more complex with cells. Critical for success is the presence of xeroprotectants inside the spermatozoa and, even more so, inside cells to protect subcellular compartments, primarily DNA. We highlight workable strategies to endow gametes and cells with the right combination of xeroprotectants, mostly sugars, and late embryogenesis abundant (LEA) or similar 'intrinsically disordered' proteins to help them withstand reversible desiccation. We focus on the biological aspects of water stress, and in particular cellular and DNA damage, but also touch on other still unexplored issues, such as the choice of both dehydration and rehydration methods or approaches, because, in our view, they play a primary role in reducing desiccation damage. We conclude by highlighting the need to exhaustively explore desiccation strategies other than lyophilisation, such as air drying, spin drying or spray drying, ideally with new prototypes, other than the food and pharmaceutical drying strategies currently used, tailored for the unique needs of cells and spermatozoa.

Bronchoalveolar lavage for diagnosis of tuberculosis infection in elephants.

Tuberculosis (TB) has been known to affect elephants for thousands of years. It was put into spotlight when few circus elephants were diagnosed carrying Mycobacterium (M.) tuberculosis. Because of the zoonotic risk and high susceptibility to M. tuberculosis, periodic testing was enacted since, in captive breeding programmes. Presently, trunk wash is the recommended diagnostic procedure for TB. Trunk wash, however, puts the operator at risk, has low sensitivity, and is prone to contamination. Here, bronchoalveolar lavage is described for the first time for TB diagnosis in elephants. Bronchial, trunk and mouth fluids were investigated using bacterial culture, M. tuberculosis complex (MTC)-specific real-time quantitative PCR (qPCR) and mycobacterial genus-specific qPCR for overall presence of mycobacteria or mycobacterial DNA including bacteria or DNA of closely related genera, respectively, in 14 elephants. Neither bacteria of the MTC nor their DNA were identified in any of the elephants. Yet, 25% of the cultures grew non-tuberculous mycobacteria (NTM) or closely related bacterial species. Furthermore, 85% of the samples contained DNA of NTM or closely related bacterial genera. This finding might explain continued false-positive results from various serological tests. From a zoonotic point of view, bronchoalveolar lavage is safer for the testing personal, has higher probability of capturing MTC and, through PCR, identifies DNA NTM in elephants. Yet, necessary endoscopic equipment, animal sedation and access to a TB reference laboratory might pose challenging requirements in remote conditions in some elephant range countries.

Cryopreservation of captive roe deer (Capreolus capreolus) semen.

To address the need to preserve current genetic diversity before it is lost forever; further studies to adapt assisted reproductive technologies to various endangered species are needed, among other things. Roe deer (Capreolus capreolus), an over abundant wild deer, can serve as model species to develop or improve sperm cryopreservation of threatened or endangered deer species. The aim of this study was to compare the ability of three diluents (Berliner Cryomedium [BC]; Tris, citric acid, glucose [TCG]; TES, Tris, glucose) to support chilling, cryopreservation (with 5% glycerol; G) and postthaw incubation (at 22 °C and 37 °C) of roe deer spermatozoa collected by electroejaculation. Berliner Cryomedium was the diluent that better preserved roe deer spermatozoa during refrigeration, able to maintain motility for at least 14 days, longer than the other extenders. BC + G was the extender of choice for cryopreservation, showing higher viability compared with TCG + G (66.7 ± 3.4 vs. 54.5 ± 6.5; P < 0.05) and higher level of acrosome integrity compared with TES, Tris, glucose + G (79.4 ± 3.4 vs. 67.9 ± 5.0; P < 0.05). Maintaining the samples at 22 °C after thawing presented higher values in various parameters compared with 37 °C. The knowledge gained through this study can potentially act as a preliminary step toward development of new protocols to help increase the reproductive success of biologically similar, yet endangered, wild species.

Canine tarsal architecture as revealed by high-resolution computed tomography.

Central tarsal bone (CTB) fractures are well documented and are a subject of increasing importance in human, equine and canine athletes although the mechanism of these fractures in dogs is not fully understood and an extrapolation from human medicine may not be accurate. This study reports the use of high-resolution computed tomography (CT) of 91 tarsal joints from 47 dogs to generate a more detailed in situ anatomical description of the CTB architecture in order to obtain a better understanding of the pathogenesis of CTB fractures in this species. The dogs studied represented a wide range of ages, breeds and levels of habitual physical activity and the angles of the tarsal joints studied ranged between maximal flexion (16.4°) and maximal extension (159.1°). Regardless of tarsal angle, the CTB articulated with the calcaneus exclusively at the level of its plantar process (PPCTB) in all dogs. The PPCTB presented two distinct parts in all dogs, a head and a neck. The calcaneus tended to rely on the PPCTB neck during flexion and on the PPCTB head during extension. This study describes new tarsal elements for the first time, including the calcaneal articular process, the fourth tarsal bone plantar articular process and the talar plantar prominence of the CTB. Based on calcaneo-PPCTB architecture, it is postulated that the PPCTB is a keystone structure and that at least some of CTB fractures in dogs could either commence at or are induced at this level due to the impingement forces exercised by the calcaneus.

Enriching the captive elephant population genetic pool through artificial insemination with frozen-thawed semen collected in the wild.

The first successful AI in an elephant was reported in 1998, using fresh semen. Since then almost 40 calves have been produced through AI in both Asian and African elephants worldwide. Following these successes, with the objective of enriching the captive population with genetic material from the wild, we evaluated the possibility of using frozen-thawed semen collected from wild bulls for AI in captivity. Semen, collected from a 36-yr-old wild African savanna elephant (Loxodonta africana) in South Africa was frozen using the directional freezing technique. This frozen-thawed semen was used for four inseminations over two consecutive days, two before and two after ovulation, in a 26-yr-old female African savanna elephant in Austria. Insemination dose of 1200 × 10(6) cells per AI with 61% motility resulted in pregnancy, which was confirmed through ultrasound examination 75, 110 and 141 days after the AI procedure. This represents the first successful AI using wild bull frozen-thawed semen in elephants. The incorporation of AI with frozen-thawed semen into the assisted reproduction toolbox opens the way to preserve and transport semen between distant individuals in captivity or, as was done in this study, between wild and captive populations, without the need to transport stressed or potentially disease-carrying animals or to remove animals from the wild. In addition, cryopreserved spermatozoa, in combination with AI, are useful methods to extend the reproductive lifespan of individuals beyond their biological lifespan and an important tool for genetic diversity management and phenotype selection in these endangered mammals.

Cooling and freezing of epididymal sperm in the common hippopotamus (Hippopotamus amphibius).

Knowledge concerning reproduction in common hippopotamus is scarce and in particular very little is known about male reproductive physiology and sperm cryopreservation. Testes were obtained from nine castrated bulls and sperm extracted from the epididymides of eight of these individuals. Mean ± SEM values of reproductive parameters were: testicular weight (including epididymis and tunicas)--275.9 ± 54.1 g, total sperm motility--88.1 ± 4.2%, total cells extracted--11.0 ± 3.6 × 10(9), intact acrosome--87.7 ± 1.8%, intact sperm morphology--51.6 ± 4.1%, and, for 3 individuals, hypoosmotic swelling test for membrane integrity-83.3 ± 1.8%. Chilled storage extenders tested were Berliner Cryomedium (BC), Biladyl(®), modification of Kenney modified Tyrode's medium (KMT), and Human Sperm Refrigeration Medium (HSRM). Extender had significant effect on post-dilution motility and motility and intact morphology after 4h and 24h at 4°C (P ≤ 0.007 for all). Berliner Cryomedium and HSRM were superior to Biladyl(®) and KMT. Freezing extenders tested were BC with either 6% dimethyl sulfoxide (Me(2)SO), or 5%, 7%, or 10% glycerol. Post-thaw motility was < 5% in 3/7 bulls in all extenders. When frozen in BC with 6% Me(2)SO, one bull had 15% post-thaw motility and 3/7 had 20 to 60%. In glycerol, 3/7 had 15-30% post-thaw motility in 5%, 2/7 in 7%, and 1/7 in 10%. The extender had significant effect on post-chilling motility (P = 0.008), post-thaw morphology (P = 0.016), and motility 30 min after thawing (P = 0.015). Berliner Cryomedium with 6% Me(2)SO or 7% glycerol were the freezing extenders of choice. Information obtained in this study allows initiation of cryobanking of sperm from the common hippopotamus which is of particular importance for genetically valuable individuals.

Collection and preservation of pygmy hippopotamus (Choeropsis liberiensis) semen.

Knowledge about the reproduction of the endangered pygmy hippopotamus is almost non-existent. This study takes the first step toward changing this by devising a protocol for the collection, evaluation, and short-term preservation of semen of this endangered species. Semen was collected successfully from seven bulls by electroejaculation, using a specially designed rectal probe. Mean +/- SEM values of native sperm parameters from combined best fractions were: motility-80.0 +/- 4.1%, concentration-2421 +/- 1530 x 10(6) cells/mL, total collected cell number-759 +/- 261 x 10(6) cells, intact acrosome-87.8 +/- 1.2%, intact morphology-52.7 +/- 4.3%, and, for some, hypoosmotic swelling test-79.3 +/- 4.4% and seminal plasma osmolarity-297.5 +/- 3.3 mOsm. Seven different extenders were tested for sperm storage under chilling conditions: Berliner Cryomedium (BC), Biladyl, modification of Kenney modified Tyrode's medium (KMT), MES medium, Androhep((R)), boar M III() extender and Human Sperm Refrigeration Medium. While differences between males were apparent, the BC was consistently superior to all other extenders in sperm motility and facilitated storage for 7 d with up to 30% motility and some motility even after 3 weeks. With this knowledge in hand, the obvious two directions for future research are to conduct artificial insemination and to develop a technique for sperm cryopreservation.

Ovarian superstimulation, transrectal ultrasound-guided oocyte recovery, and IVF in rhinoceros.

Numerous reports on reproductive pathology in all rhinoceros species illustrate the abundance of female infertility in captive populations. In infertile rhinoceroses, oocyte collection and embryo production could represent the best remaining option for these animals to reproduce and to contribute to the genetic pool. We report here on superstimulation, repeated oocyte recovery, and attempted in vitro fertilization (IVF) in white and black rhinoceroses. Four anestrous rhinoceroses (two white, two black) with unknown follicular status were treated with gonadotropin-releasing hormone analogue, deslorelin acetate, for 6 to 7 d. Number and size of follicles in superstimulated females was significantly higher and larger compared with those in nonstimulated anestrous females (n=9). Ovum pick-up was achieved by transrectal ultrasound-guided follicle aspiration. Up to 15 follicles were aspirated per ovary. During six ovum pick-ups, a total of 29 cumulus-oocyte complexes (COCs) were harvested with a range of 2 to 9 COCs per collection. No postsurgical complications were noted on the rhinoceros ovaries using this minimally invasive approach. Various in vitro maturation (IVM) and IVF protocols were tested on the collected COCs. Despite the low total number of COCs available for IVM and IVF in this study, we can report the first rhinoceros embryo ever produced in vitro. The production of a 4-cell embryo demonstrated the potential of transrectal ultrasound-guided oocyte recovery as a valuable tool for in vitro production of rhinoceros embryos from otherwise infertile females.

Protective effects of iodixanol during bovine sperm cryopreservation.

The aim of cryopreservation is to maintain cellular integrity, thereby enabling resumption of proper biological functioning after thawing. Here we propose OptiPrep (60% iodixanol in water) as a protectant during sperm cryopreservation using pooled bull semen as the model. We evaluated OptiPrep concentration effect and its relation to cryopreservation by comparing frozen-thawed and chilled samples. Semen, extended in Andromed with 0 (control), 1.25%, 2.5%, and 5% OptiPrep, was compared after either chilling or freezing in large volume by directional freezing. Sample evaluation included sperm motility upon thawing and after 3h incubation at 37 degrees C for frozen-thawed samples and after 3h and 6h of chilling for chilled samples; viability, acrosomal integrity, and hypoosmotic swelling were also tested for frozen-thawed and chilled samples. Chilled samples with 5% OptiPrep showed inferior viability (P=0.047) and 3h motility (P=0.017) relative to that for chilled samples with 2.5% OptiPrep and inferior viability (P=0.042), acrosomal integrity (P=0.045), and 0h motility (P=0.024) relative to that for chilled samples with 1.25% OptiPrep. The 1.25%, 2.5%, and control samples did not differ. In frozen-thawed samples, 2.5% OptiPrep was superior to all other concentrations for 3h motility (control, P=0.007; 5% OptiPrep, P=0.005; 1.25% OptiPrep, P=0.004) and to 1.25% OptiPrep for acrosomal integrity (P=0.001). In a search for a protection mechanism, we measured glass transition temperature (T(g)) of Andromed and of Andromed with 1.25%, 2.5%, and 5% OptiPrep. Andromed (-58.78 degrees C) and 1.25% OptiPrep (-58.75 degrees C) groups had lower mean T(g) than that of the 2.5% (-57.67 degrees C) and the 5% (-57.10 degrees C) groups. Directional cryomicroscopy revealed that the presence of iodixanol alters ice crystal formation into an intricate net of dendrites. Thus, iodixanol appears to possess cryoprotective properties by helping spermatozoa maintain motility and membrane integrity, possibly through altering ice crystals formation into a more hospitable environment and increasing the glass transition temperature.

First successful artificial insemination with frozen-thawed semen in rhinoceros.

The first successful artificial insemination (AI) in a rhinoceros was reported in 2007 using fresh semen. Following that success, we decided to evaluate the possibility of using frozen-thawed semen for artificial insemination. Semen, collected from a 35-36 year old Southern white rhinoceros (Ceratotherium simum simum) in the UK was frozen using the directional freezing technique. This frozen semen was used in two intrauterine AI attempts on a 30 years old female rhinoceros in Hungary. The first attempt, conducted 30 days postpartum with an insemination dose of approximately 135 x 10(6) motile cells, failed. The second attempt, conducted two estrus cycles later with an insemination dose of approximately 500 x 10(6) motile cells, resulted in pregnancy and the birth of a healthy offspring. This represents the first successful AI using frozen-thawed semen in a rhinoceros, putting it among very few wildlife species in which AI with frozen-thawed semen resulted in a live birth. The incorporation of AI with frozen-thawed semen into the assisted reproduction toolbox opens the way to preserve and transport semen between distant individuals in captivity or between wild and captive populations, without the need to transport stressed or potentially disease carrying animals. In addition, cryopreserved spermatozoa, in combination with AI, are useful methods to extend the reproductive lifespan of individuals beyond their biological lifespan and an important tool for managing genetic diversity in these endangered mammals.

Pathological findings in dogs with fatal heatstroke.

Eleven dogs with fatal heatstroke were examined grossly and histopathologically post mortem. All showed multi-organ haemorrhagic diathesis with coagulative necrosis. Hypaeremia and diffuse oedema were observed in the skin (eight dogs), lungs (11), brain (11) and bone marrow (one). Congestion of the splenic pulp (10 dogs) and hepatic sinusoids (nine) was also noted. Necrosis was observed in the mucosa of the small intestine (seven dogs), large intestine (eight), renal tubular epithelium (nine), hepatic parenchyma (eight) and brain neural tissue (four). The results showed that naturally occurring, fatal canine heatstroke induces acute multiple organ lesions affecting most body systems, and suggest that the more prevalent lesions include haemorrhagic diathesis, microthrombosis and coagulative necrosis. These are probable sequels of hyperthermia-induced disseminated intravascular coagulation and systemic inflammatory response syndrome, which lead to multi-organ dysfunction and death.

Obstetrics in elephants.

Obstetrics, one of the oldest fields in veterinary medicine, is well described and practiced in domestic and exotic animals. However, when providing care during elephant birth or dystocia, veterinary intervention options differ greatly from any domestic species, and are far more limited due to the dimensions and specific anatomy of the elephant reproductive tract. In addition, aging of captive elephant populations and advanced age of primiparous females make active birth management increasingly important. Intrauterine infection, uterine inertia and urogenital tract pathologies are emerging as major causes for dystocia, often leading to foetal and dam death. This paper reviews the current knowledge on elephant birth and the factors associated with dystocia. It then summarises recommendations for birth and dystocia management. As Caesarean section, the most common ultima ratio in domestic animal obstetrics, is lethal and therefore not an option in the elephant, non-invasive medical treatment, induction of the Fergusson reflex or the conscious decision to leave a retained foetus until it is expelled voluntarily, are key elements in elephant obstetrics. Surgical strategies such as episiotomy and foetotomy are sometimes inevitable in order to try to save the life of the dam, however, these interventions result in chronic post-surgical complications or even fatal outcome. Limited reliable data on serum calcium concentrations, and pharmacokinetics and effect of exogenous oestrogen, oxytocin, and prostaglandins during birth provide the scope of future research, necessary to advance scientific knowledge on obstetrics in elephants.

Directional freezing of equine semen in large volumes.

Despite its potential impact on the horse industry, sperm cryopreservation is not an established technology throughout the industry, for a number of reasons that include a reduction in pregnancy rate and increased cost per pregnancy. We have evaluated a novel directional freezing technique, based on a multi-thermal gradient (MTG), by comparing it with the conventional, controlled-rate cryopreservation method (CRCM). Ninety-seven ejaculates with > or =50% motility, collected from 31 stallions were each divided into two parts and subsequently frozen by either MTG or CRCM. Frozen samples were then stored in liquid nitrogen until thawing. The two treatments were evaluated by three methods: progressive linear motility (PLM), viability stain and hypoosmotic swelling (HOS) test. High correlation was found between the three evaluation methods for all post-thaw samples. Eighty-eight per cent of the ejaculates frozen by MTG had post-thaw PLM > or =35%, whereas only 59% of the ejaculates frozen by CRCM had such motility. Post-thaw evaluations of samples frozen by MTG and CRCM were: PLM - 50.2 +/- 1.5% and 37.4 +/- 1.5%, respectively; viability - 53.6 +/- 1.5% and 39.5 +/- 1.4%, respectively; membrane integrity, as evaluated by HOS - 36.2 +/- 1.3% and 26.5 +/- 1.1%, respectively. The differences according to all the evaluation methods were highly significant (p < 0.001), and the results indicate that freezing stallion semen by MTG is superior to CRCM.

Severe tongue necrosis associated with pine processionary moth (Thaumetopoea wilkinsoni) ingestion in three dogs.

The pine processionary moth (Thaumetopoea wilkinsoni) is an insect of medical significance in the eastern Mediterranean. This report describes three exposure cases in dogs due to ingestion of this moth's caterpillars in Israel. All three dogs were observed in direct contact with caterpillars or pinecones in infested gardens. The disease course and progression of signs were acute in all three cases, and included vomiting and severe tongue swelling. Physical examination findings included hyperthermia, tachypnoea, respiratory distress, cyanosis and tongue oedema, labial angioedema, ptyalism, bilateral submandibular lymphadenomegaly and conjunctivitis. Severe tongue necrosis and sloughing of its distal portion occurred 2-5 days after admission to the hospital. All dogs recovered and were discharged within 2-7 days of admission. Two staff members, attending one of the dogs, experienced an itchy rash and wheals on their arms, wrists and necks. To the best of our knowledge, this should be the first report of severe oral lesions and tongue necrosis due to contact with T. wilkinsoni caterpillars.