Jörns Fickel

Semen cryopreservation and the conservation of endangered species

Currently, more than 16,000 plant and animal species are imminently threatened by extinction, often as a direct consequence of anthropogenic influences. One of the measures to halt that process is genetic resource banking. This short review focuses on mammal sperm cryopreservation in combination with assisted reproduction techniques. It summarizes general problems, recent developments, and currently applied protocols and gives an overview of hitherto existing successes of assisted reproduction measures in wild animals in the light of conservation efforts.

Captive roe deer (Capreolus capreolus) select for low amounts of tannic acid but not quebracho: fluctuation of preferences and potential benefits

Browsing ruminants have been shown to tolerate a certain amount of tannins in their natural diet, and preference trials with captive roe deer (Capreolus capreolus) have suggested an active selection for a low dose of hydrolysable tannins. In this study, we investigated the preference patterns for tannic acid, a source of hydrolysable tannins, and quebracho, a source of condensed tannins, in a series of preference trials with captive roe deer over time, using a pelleted feed that differed only in the respective tannin content. Additionally, two groups of four hand-raised roe deer fawns were fed either a control or a 3% tannic-acid containing diet and physiological parameters were compared after 7.5 months. There were large differences in preference patterns between the individual roe deer groups; quebracho was mostly avoided, whereas tannic acid was actively included in the diet in differing, low proportions. However, one group consistently preferred the quebracho diet over both the control or the tannic acid diet. For the tannic acid, the preference pattern often revealed an initial period of high preference, followed by a stable period of a moderate preference. The fawns on the tannic acid diet had a lower pellet intake and a higher relative mass gain than the fawns on the control diet; differences in salivary tannin-binding capacity and in blood antioxidant status were below significance. These results are the first indications of potential benefits of a low-dose tannin diet, which need further confirmation. The results of the preference trials demonstrate that the time pattern of tannin intake is not constant, and pose the question about the validity of short-term preference trials in general.

Epizootiological investigations of canine distemper virus in free-ranging carnivores from Germany

Canine distemper virus (CDV) infects a broad range of carnivores. To assess whether wild carnivores may play a role in the epidemiology of CDV in domestic dogs in Germany, the seroprevalence of CDV was determined. In sera from red foxes (30 of 591 (5%)) and stone martens (2 of 10 (20%)) antiviral antibodies were detected using a neutralization assay, whereas sera of raccoons, two mink, one pine marten and one raccoon dog were negative. In foxes, there was a significantly higher prevalence in urban and suburban compared to rural regions. When testing lung and spleen tissue samples (fox, badger, stone marten, polecat, raccoon dog) 13 of 253 (5.1%) foxes, 2 of 13 (15.4%) stone martens and 2 of 6 (33%) badgers were virus positive using RT-PCR. Phylogenetic analysis based on partial sequences of the F gene revealed a distinct relatedness to canine CDV isolates. Together, the data support the concept of transmission of CDV between domestic dogs and wild carnivores.

The intraruminal papillation gradient in wild ruminants of different feeding types: implications for rumen physiology

Browsing and grazing ruminants are thought to differ in the degree their rumen contents are stratified—which may be due to different characteristics of their respective forages, to particular adaptations of the animals, or both. However, this stratification is difficult to measure in live animals. The papillation of the rumen has been suggested as an anatomical proxy for stratification—with even papillation indicating homogenous contents, and uneven papillation (with few and small dorsal and ventral papillae, and prominent papillae in the atrium ruminis) stratified contents. Using the surface enlargement factor (SEF, indicating how basal mucosa surface is increased by papillae) of over 55 ruminant species, we demonstrate that differences between the SEFdorsal or SEFventral and the SEFatrium are significantly related to the percentage of grass in the natural diet. The more a species is adapted to grass, the more distinct this difference, with extreme grazers having unpapillated dorsal and ventral mucosa. The relative SEFdorsal as anatomical proxy for stratification, and the difference in particle and fluid retention in the rumen as physiological proxy for stratification, are highly correlated in species (n = 9) for which both kind of data are available. The results support the concept that the stratification of rumen contents varies among ruminants, with more homogenous contents in the more browsing and more stratified contents in the more grazing species. J. Morphol., 2009.

A single-nucleotide polymorphism-based approach for rapid and cost-effective genetic wolf monitoring in Europe based on noninvasively collected samples

Noninvasive genetics based on microsatellite markers has become an indispensable tool for wildlife monitoring and conservation research over the past decades. However, microsatellites have several drawbacks, such as the lack of standardisation between laboratories and high error rates. Here, we propose an alternative single‐nucleotide polymorphism (SNP)‐based marker system for noninvasively collected samples, which promises to solve these problems. Using nanofluidic SNP genotyping technology (Fluidigm), we genotyped 158 wolf samples (tissue, scats, hairs, urine) for 192 SNP loci selected from the Affymetrix v2 Canine SNP Array. We carefully selected an optimised final set of 96 SNPs (and discarded the worse half), based on assay performance and reliability. We found rates of missing data in this SNP set of <10% and genotyping error of ~1%, which improves genotyping accuracy by nearly an order of magnitude when compared to published data for other marker types. Our approach provides a tool for rapid and cost‐effective genotyping of noninvasively collected wildlife samples. The ability to standardise genotype scoring combined with low error rates promises to constitute a major technological advancement and could establish SNPs as a standard marker for future wildlife monitoring.

Geographical variation in and evolutionary history of the Sunda clouded leopard (Neofelis diardi) (Mammalia: Carnivora: Felidae) with the description of a new subspecies from Borneo

Recent morphological and molecular studies led to the recognition of two extant species of clouded leopards; Neofelis nebulosa from mainland southeast Asia and Neofelis diardi from the Sunda Islands of Borneo and Sumatra, including the Batu Islands. In addition to these new species-level distinctions, preliminary molecular data suggested a genetic substructure that separates Bornean and Sumatran clouded leopards, indicating the possibility of two subspecies of N. diardi. This suggestion was based on an analysis of only three Sumatran and seven Bornean individuals. Accordingly, in this study we re-evaluated this proposed subspecies differentiation using additional molecular (mainly historical) samples of eight Bornean and 13 Sumatran clouded leopards; a craniometric analysis of 28 specimens; and examination of pelage morphology of 20 museum specimens and of photographs of 12 wild camera-trapped animals. Molecular (mtDNA and microsatellite loci), craniomandibular and dental analyses strongly support the differentiation of Bornean and Sumatran clouded leopards, but pelage characteristics fail to separate them completely, most probably owing to small sample sizes, but it may also reflect habitat similarities between the two islands and their recent divergence. However, some provisional discriminating pelage characters are presented that need further testing. According to our estimates both populations diverged from each other during the Middle to Late Pleistocene (between 400 and 120 kyr). We present a discussion on the evolutionary history of Neofelis diardi sspp. on the Sunda Shelf, a revised taxonomy for the Sunda clouded leopard, N. diardi, and formally describe the Bornean subspecies, Neofelis diardi borneensis, including the designation of a holotype (BM.3.4.9.2 from Baram, Sarawak) in accordance with the rules of the International Code of Zoological Nomenclature.

NEWCASTLE DISEASE VIRUS AND CHLAMYDIA PSITTACI IN FREE-LIVING RAPTORS FROM EASTERN GERMANY

Organ samples from free-living raptors from the federal states of Berlin and Brandenburg in eastern Germany were tested for Newcastle disease virus (NDV; n=331) and Chlamydia psittaci (n=39) by polymerase chain reaction (PCR). In 18 individuals NDV nucleic acids were detected. These samples originated from barn owls (Tyto alba; n=15, 28%), tawny owl (Strix aluco; n=1, 5%), common buzzard (Buteo buteo, n=1, 1%), and European kestrel (Falco tinnunculus; n=1, 4%). In 29 (74%) of 39 samples C. psittaci was detected. Chlamydia psittaci is common in free-living birds of prey in the investigated area.

Detection of growth factors in the testis of roe deer (Capreolus capreolus).

Roe deer is a seasonal breeder characterised by a short rutting season in summer. Mature males show synchronised cycles of testicular involution and recrudescence. Therefore, this species is a valuable model to study seasonal regulation of spermatogenesis in ruminants. It is hypothesised that a time-dependent production of testicular growth factors is required to regulate seasonal changes in testis growth and spermatogenesis. To identify potential candidates, total RNA from roe deer testis tissue was extracted at three different seasonal periods (April, August, December), and using RT-PCR the presence of several growth factors (aFGF, bFGF, IGF-I, IGF-II, TGF-alpha, TGF-beta1, TGF-beta3 and two isoforms of VEGF) was detected. Sequencing of the growth factor PCR fragments revealed a high sequence homology between cattle and roe deer. To further explore the expression patterns of the identified growth factors in roe deer their expression levels were standardised using glyceraldehyde-3-phosphate dehydrogenase (GAPDH) gene expression. The study demonstrates the expression of several growth factors in roe deer testis and supports the assumption of their seasonally diverse regulation. These results provide the basis to investigate the role of growth factors in the regulation of circannual changes of testicular activity.

Comparative investigations on digestion in grazing (Ceratotherium simum) and browsing (Diceros bicornis) rhinoceroses

Rhinoceroses represent the largest extant herbivores with extensive dietary specialization for plant groups like browse (black rhino Diceros bicornis) or grass (white rhino Ceratotherium simum). However, it is not clear to what extent such diet selection patterns are reflected in adaptations of digestive physiology of the respective feeding types. In this study, feeding trials with four black and five white rhinos were conducted in four zoos. The animals had ad libitum access to the same batch of grass hay (second cut; neutral detergent fiber (NDF) 63% dry matter (DM), crude protein 10.2% DM). Total intake, fecal N content, in vitro digestibility of NDF residues of feces, fecal particle size and mean retention time (MRT) of particles (Cr-mordanted fiber; 1-2mm) and fluid (Co-EDTA) were quantified. The average daily DM intake was 70+/-12 g/kg BW(0.75) for white and 73+/-10 g/kg BW(0.75) for black rhinos. In the in vitro fermentation test fecal NDF residues of black rhinos resulted in higher gas productions at fermentation times of 12 to 24h, indicating that white rhinos have a superior capacity to digest NDF. Average MRT for fluids and particles was 28+/-4h and 43+/-5h in white and 34+/-4h and 39+/-4h in black rhinos. The selectivity factor (SF=MRT(particle)/MRT(fluid)) was higher for white (1.5+/-0.2) than for black rhinos (1.2+/-0.1) (p=0.016). In a comparison of 12 ruminant and 3 rhino species, SF was correlated to percentage of grass in diet (R=0.75). Mean fecal particle size was higher in white (9.1+/-1.94 mm) than in black rhinos (6.1+/-0.79 mm) (p=0.016). The results demonstrate differences between white and black rhinos in terms of retention times and fiber digestibility. The more selective retention of particles by the white rhino corresponds with the higher digestion of fiber measured indirectly. Furthermore there is indication for a general pattern of high SF in grazing ruminants and rhinos. The difference in fecal particle size between both rhino species might be due to the considerable difference in body weight.

Evolution of caprine and ovine β-defensin genes

Defensins comprise an important family of anti-microbial peptides. Among vertebrates, numerous defensin genes have been detected, but their evolutionary background is still discussed. We investigated the molecular evolution and variability of β-defensins of Caprini via sequence analyses of defensin introns. Screening of several domestic and wild species of Caprini revealed a total of 13 discrete β-defensin coding sequences, with three of them described before this study. Phylogenetic analyses revealed that the array of newly described defensin genes is of monophyletic origin and has arisen in numerous independent duplication events after separation of the ancestral defensins. As a result of that scenario, recent defensin genes are distributed in a species-specific manner. Values of synonymous and non-synonymous substitutions demonstrated that both modes of evolutionary pressure, positive as well as negative selection, have acted. In addition, conservation of some β-defensin exons is demonstrated. Discrimination of certain β-defensin genes was possible only due to intron-specific differences. Therefore, sequence analyses restricted to the exons would result in underestimation of the number of β-defensin genes. Our study shows that for reconstruction of the phylogenetic history data of defensin introns are more appropriated. Comparisons among the amino acid sequences show moderate substitutions without changing the net charge of the mature peptides.