Speciation with gene flow in equids despite extensive chromosomal plasticity.

Horses, asses, and zebras belong to a single genus, Equus, which emerged 4.0-4.5 Mya. Although the equine fossil record represents a textbook example of evolution, the succession of events that gave rise to the diversity of species existing today remains unclear. Here we present six genomes from each living species of asses and zebras. This completes the set of genomes available for all extant species in the genus, which was hitherto represented only by the horse and the domestic donkey. In addition, we used a museum specimen to characterize the genome of the quagga zebra, which was driven to extinction in the early 1900s. We scan the genomes for lineage-specific adaptations and identify 48 genes that have evolved under positive selection and are involved in olfaction, immune response, development, locomotion, and behavior. Our extensive genome dataset reveals a highly dynamic demographic history with synchronous expansions and collapses on different continents during the last 400 ky after major climatic events. We show that the earliest speciation occurred with gene flow in Northern America, and that the ancestor of present-day asses and zebras dispersed into the Old World 2.1-3.4 Mya. Strikingly, we also find evidence for gene flow involving three contemporary equine species despite chromosomal numbers varying from 16 pairs to 31 pairs. These findings challenge the claim that the accumulation of chromosomal rearrangements drive complete reproductive isolation, and promote equids as a fundamental model for understanding the interplay between chromosomal structure, gene flow, and, ultimately, speciation.

KETAMINE-MEDETOMIDINE AND KETAMINE-MEDETOMIDINE-MIDAZOLAM ANESTHESIA IN CAPTIVE CHEETAHS (ACINONYX JUBATUS)-COMPARISON OF BLOOD PRESSURE AND KIDNEY BLOOD FLOW.

Six clinically healthy captive cheetahs ( Acinonyx jubatus ) were anesthetized twice using two different drug combinations to investigate if blood pressure and kidney blood flow are affected by medetomidine dosage. Protocol KM (2.0 mg/kg ketamine and 0.05 mg/kg medetomidine) was compared with protocol KMM (2.0 mg/kg ketamine, 0.02 mg/kg medetomidine, and 0.1 mg/kg midazolam). Heart rate (HR), respiratory rate (RR), body temperature, end-tidal carbon dioxide pressure (ETCO2), and anesthetic depth were monitored every 10 min. Noninvasive mean (MAP), systolic (SAP), and diastolic (DAP) arterial blood pressure were measured, and Duplex Doppler ultrasonography was performed on the kidneys. The mean arterial resistive index (RI) was determined and the pulse pressure index (PPI) was calculated, as indicators for kidney blood flow. There were no significant differences in induction and recovery times. MAP was significantly higher with KM than KMM at 35 min, and in both protocols decreased significantly after atipamezole administration. DAP was significantly higher at 25 and 35 min in animals anesthetized with KM; it also decreased significantly with both protocols after atipamezole administration. The PPI was significantly lower throughout the procedure with KM, and with both protocols increased significantly after atipamezole administration. Both the higher blood pressure and the reduced PPI with KM were likely a direct effect of the higher medetomidine dosage, and these findings indicate that lower medetomidine dosages might reduce hypertension and lead to a better PPI in cheetah immobilization.

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.

Current State of In Vitro Embryo Production in African Lion (Panthera leo).

In the last 30-40 years, in vitro maturation (IVM) and fertilization (IVF) of domestic cat oocytes have been established as part of the panel of assisted reproduction technologies. As a representative of wild felids, the African lion is not yet considered endangered. Nevertheless, the zoo population management of the African lion itself as well as other closely related felids would benefit from the establishment of an IVF system. Here, we aimed to investigate the transferability of domestic cat IVF technology to the African lion. From the ovaries of 42 lionesses aged between 0.75 and 15 years, a total of 933 IVF-suitable oocytes were retrieved and subjected to IVM and IVF. The overall maturation rate was 40.6% and 18.9% of these oocytes cleaved after fertilization, respectively. Embryos were generated by intracytoplasmic sperm cell injection as well as co-culture with epididymal sperm. Improvements in the model system also led to an improved outcome with in vitro produced embryos in the lion. Compared to domestic cats, the transportation of gonads to a specialized laboratory was time-consuming and influenced oocyte quality negatively. In conclusion, the domestic cat IVF system is adoptable for the African lion, although success rates are still lower.

Intraspecific macroscopic digestive anatomy of ring-tailed lemurs (Lemur catta), including a comparison of frozen and formalin-stored specimens.

Digestive tract measurements are often considered species specific, but little information exists on the degree to which they change during ontogeny within a species. Additionally, access to anatomical material from nondomestic species is often limited, with fixed tissues possibly representing the only available source, though the degree to which this material is representative in terms of dimensions and weight is debatable. In the present study, the macroscopic anatomy of the digestive tract (length of intestinal sections, and tissue weights of stomach and intestines) of 58 Lemur catta [ranging in age from 1 month (neonates) to 25 years], which had been stored frozen (n = 27) or fixed in formalin (n = 31), was quantified. Particular attention was paid to the caecum and the possible presence of an appendix. The intraspecific allometric scaling of body mass (BM)0.46[0.40;0.51] for total intestine length and BM0.48[0.41;0.54] for small intestine length was higher than the expected geometric scaling of BM0.33, and similar to that reported in the literature for interspecific scaling. This difference in scaling is usually explained by the hypothesis that, to maintain optimal absorption, the diameter of the intestinal tube cannot increase geometrically. Therefore, geometric volume gain of increasing body mass is accommodated for by more-than-geometric length scaling. According to the literature, not all L. catta have an appendix. No appendix was found in the specimens in the present study. The proportions of length measurements did not change markedly during ontogeny, indicating that the proportions of the foetus are representative of those of the adult animal. By contrast, width and tissue-mass scaling of the caecum indicated disproportionate growth of this organ during ontogeny that was not reflected in its length. Compared to overall intraspecific variation, the method of storage (frozen vs. formalin) had no relevant impact on length or weight measurements.

Toxoplasma gondii in small exotic felids from zoos in Europe and the Middle East: serological prevalence and risk factors.

BACKGROUND: Toxoplasma gondii infections and cases of clinical toxoplasmosis have been recorded in zoo animals. Wild felids in human care can serve as definitive hosts that shed oocysts, but also as intermediate hosts for the parasite. Some felid species, such as the Pallas's cat (Otocolobus manul) or sand cat (Felis margarita), may suffer from clinically apparent toxoplasmosis. In the present study, our main aim was to assess risk factors for T. gondii infections in small exotic felids. METHODS: A seroepidemiological study was conducted using the reduviid bug Dipetalogaster maxima for blood sample collection, a method previously evaluated on domestic cats. A total of 336 samples from 17 felid species were collected in 51 institutions, 48 of which were within Europe and the remaining three in the Middle East (United Arabic Emirates and Qatar). These samples were analyzed for T. gondii antibodies by immunoblotting and an immunofluorescent antibody test. Potential risk factors in zoos for seropositivity regarding T. gondii among members of the European Association of Zoos and Aquaria (EAZA) were evaluated using a questionnaire and individual data from the Zoological Information Management System (ZIMS). RESULTS: The sampled felids showed an overall seroprevalence for T. gondii of 63%. The risk factor study including data of 311 small exotic cats of 10 species resulted in a final generalized linear mixed model comprised of five variables: the likelihood of seropositivity increased statistically significantly with "Age", while feeding "Cattle: frozen" relative to "Cattle: fresh", "Outdoor housing fenced in on all sides", "Mesh size 2-5 cm" relative to "Mesh size > 5 cm" and "Wearing gloves: yes" had statistically significant protective effects. CONCLUSIONS: Wild felids, including endangered species, kept in human care in European and Middle Eastern institutions, are widely exposed to T. gondii. Risk factor analysis revealed that feeding previously frozen tissues, keeping animals in enclosures that are fenced on all sides using fences with small mesh sizes, and wearing gloves when working inside enclosures seem to be the most relevant protective measures to prevent T. gondii infections in these animals .

Seasonal recurrence of cowpox virus outbreaks in captive cheetahs (Acinonyx jubatus).

Cowpox virus infections in captive cheetahs (Acinonyx jubatus) with high morbidity and mortality have already been reported in the UK and Russia in the 1970s. However, most of the reported cases have been singular events. Here, we report a total of five cowpox virus outbreaks in cheetahs in the same safari park in Denmark between 2010 and 2014. Nine cheetahs showed varying severity of clinical disease; two of them died (22%). All episodes occurred between August and October of the respective year. No other carnivores kept at the same institution nor the keepers taking care of the animals were clinically affected. The clinical picture of cowpox was confirmed by extensive laboratory investigations including histopathological and molecular analyses as well as cell culture isolation of a cowpox virus. High anti-orthopoxvirus antibody titers were detected in all 9 diseased cheetahs compared to seven contact cheetahs without clinical signs and 13 cheetahs not in direct contact. Additionally, whole genome sequencing from one sample of each cluster with subsequent phylogenetic analysis showed that the viruses from different outbreaks have individual sequences but clearly form a clade distinct from other cowpox viruses. However, the intra-clade distances are still larger than those usually observed within clades of one event. These findings indicate multiple and separate introductions of cowpox virus, probably from wild rodent populations, where the virus keeps circulating naturally and is only sporadically introduced into the cheetahs. Sero-positivity of voles (Arvicola amphibious) caught in zoo grounds strengthens this hypothesis. As a consequence, recommendations are given for medical and physical management of diseased cheetahs, for hygienic measures as well as for pre-shipment isolation before cheetah export from zoo grounds.

Production of lion (Panthera leo) blastocysts after in vitro maturation of oocytes and intracytoplasmic sperm injection.

Assisted reproductive techniques are becoming widely applied to the breeding of endangered species, but establishing reliable protocols for the production of embryos in vitro is challenging because of the scarcity of sample material. In our study, we applied an assisted reproductive technique protocol for IVM and intracytoplasmic sperm injection (ICSI), developed in the domestic cat, to oocytes retrieved from ovaries of four 2-year-old lionesses (Panthera leo) eight hours postmortem. In total, 68 cumulus-oocyte complexes of good quality were randomly distributed and cultured for 32 to 34 hours in two different maturation culture media, consisting of Medium 199 with Earle's salts, 3 mg/mL BSA, 0.1 mg/mL cysteine, 1.4 mg/mL sodium pyruvate, 0.6 mg/mL sodium lactate, 0.15 mg/mL l-glutamine, and 0.055 mg/mL gentamicin. Hormonal supplementation of IVM_1 was 0.02 IU/mL FSH and 0.05 IU/mL LH; IVM_2 consisted of 1.64 IU/mL FSH, 1.06 IU/mL LH, and 1 µg/mL 17ß-estradiol. Differences in hormonal supplementation did not produce significant differences in oocyte maturation rates, which were 39.4% in IVM_1 and 34.3% in IVM_2. Matured oocytes were microinjected with homologous frozen-thawed spermatozoa, and subsequent cleavage rates were 30.8% and 58.3%, respectively. Half of the embryos derived from oocytes matured in IVM_1 developed into blastocysts, whereas only 28.6% of embryos from oocytes matured in IVM_2 reached the blastocyst stage. Morula stages were present from Day 6 onward, and blastocyst stages from Day 9 on, indicating a slower developmental speed in comparison with domestic cats. This is the first report of in vitro-produced blastocysts using ICSI in the lion, and the results report that IVM and ICSI can be successfully performed with cumulus-oocyte complexes retrieved from ovaries after eight hours of shipping, obtaining competent embryos in culture.

Ligation bias in illumina next-generation DNA libraries: implications for sequencing ancient genomes.

Ancient DNA extracts consist of a mixture of endogenous molecules and contaminant DNA templates, often originating from environmental microbes. These two populations of templates exhibit different chemical characteristics, with the former showing depurination and cytosine deamination by-products, resulting from post-mortem DNA damage. Such chemical modifications can interfere with the molecular tools used for building second-generation DNA libraries, and limit our ability to fully characterize the true complexity of ancient DNA extracts. In this study, we first use fresh DNA extracts to demonstrate that library preparation based on adapter ligation at AT-overhangs are biased against DNA templates starting with thymine residues, contrarily to blunt-end adapter ligation. We observe the same bias on fresh DNA extracts sheared on Bioruptor, Covaris and nebulizers. This contradicts previous reports suggesting that this bias could originate from the methods used for shearing DNA. This also suggests that AT-overhang adapter ligation efficiency is affected in a sequence-dependent manner and results in an uneven representation of different genomic contexts. We then show how this bias could affect the base composition of ancient DNA libraries prepared following AT-overhang ligation, mainly by limiting the ability to ligate DNA templates starting with thymines and therefore deaminated cytosines. This results in particular nucleotide misincorporation damage patterns, deviating from the signature generally expected for authenticating ancient sequence data. Consequently, we show that models adequate for estimating post-mortem DNA damage levels must be robust to the molecular tools used for building ancient DNA libraries.