Evaluation of Methods to Determine Sperm Density for the European eel,Anguilla anguilla

European eel, Anguilla anguilla, is a target species for future captive breeding, yet best methodology to estimate sperm density for application in in vitro fertilization is not established. Thus, our objectives were to evaluate methods to estimate European eel sperm density including spermatocrit, computer‐assisted sperm analysis (CASA) and flow cytometry (FCM), using Neubauer Improved haemocytometer as benchmark. Initially, relationships between spermatocrit, haemocytometer counts and sperm motility were analysed, as well as the effect of sperm dilution on haemocytometer counts. Furthermore, accuracy and precision of spermatocrit, applying a range of G‐forces, were tested and the best G‐force used in method comparisons. We found no effect of dilution on haemocytometer sperm density estimates, whereas motility associated positively with haemocytometer counts, but not with spermatocrit. Results from all techniques, spermatocrit, CASA and FCM, showed significant positive correlations with haemocytometer counts. The best correlation between spermatocrit and haemocytometer counts was obtained at 6000 ×  g (r = 0.68). Of two CASA variants, one or three photographic fields (CASA‐1 and CASA‐2), CASA‐2 showed a very high accuracy to haemocytometer counts (r  =  0.93), but low precision (CV: CASA‐2 = 28.4%). FCM was tested with and without microfluorospheres (FCM‐1 and FCM‐2), and relationships to haemocytometer counts were highly accurate (FCM‐1: r  =  0.94; FCM‐2: r  =  0.88) and precise (CV: FCM‐1 = 2.5; FCM‐2 = 2.7%). Overall, CASA‐2 and FCM‐1 feature reliable methods for quantification of European eel sperm, but FCM‐1 has a clear advantage featuring highest precision and accuracy. Together, these results provide a useful basis for gamete management in fertilization protocols.     

Changes in fox squirrel anti-predator behaviors across the urban–rural gradient

Predator stimuli created by humans in the urban environment may alter animals’ anti-predator behaviors. I hypothesized that habituation would cause anti-predator behaviors to decrease in urban settings in response to humans. Additionally, I hypothesized that populations habituated to humans would show reduced responses to other predator stimuli. I observed three populations of squirrels (urban, suburban and rural) responses to human approaches, red-tailed hawk vocalizations (Buteo jamaicensis) and coyote (Canis latrans) vocalizations. Mahalanobis distances of anti-predator behaviors in response to human approaches were consistent with the urban–rural gradient. Flight initiation distances (X ² = 26.33, df = 2, P < 0.001) and amount of time dedicated to anti-predator behavior (X ² = 10.94, df = 2, P = 0.004) in response to human approaches were also consistent with the urban–rural gradient. Supporting the habituation hypothesis, naive juvenile squirrels increased flight initiation distances (X ² = 35.89, df = 1, P < 0.001) and time dedicated to anti-predator behaviors (X ² = 9.46, df = 1, P = 0.002) relative to adult squirrels in the same urban environment. Time dedicated to anti-predator behaviors differed among all three sites in response to both coyote (X ² = 9.83, df = 2, P = 0.007) and hawk (X ² = 6.50, df = 2, P = 0.035) vocalizations. Responses to both vocalizations on rural sites (coyote = 45%, hawk = 55%) greater than twice that found on the urban sites (coyote = 11%, hawk = 20%). This is possibly the first case of a transfer of habituation demonstrated under field conditions.     

Seasonal and temporal changes in species use of the landscape: how do they impact the inferences from multi-scale habitat modeling?

Context

Multi-scale approaches to habitat modeling have been shown to provide more accurate understanding and predictions of species-habitat associations. It remains however unexplored how spatial and temporal variations in habitat use may affect multi-scale habitat modeling.

Objectives

We aimed at assessing how seasonal and temporal differences in species habitat use and distribution impact operational scales, variable influence, habitat suitability spatial patterns, and performance of multi-scale models.

Methods

We evaluated the environmental factors driving brown bear habitat relationships in the Cantabrian Range (Spain) based on species presence records (ground observations) for the period 2000–2010, LiDAR data on forest structure, and seasonal estimates of foraging resources. We separately developed multi-scale habitat models for (i) each season (spring, summer, fall and winter) (ii) two sub-periods with different population status: 2000–2004 (with brown bear distribution restricted to the main population nuclei) and 2005–2010 (with expanding bear population and range); and (iii) the entire 2000–2010 period.

Results

Scales of effect remained considerably stable across seasonal and temporal variations, but not the influence of certain environmental variables. The predictive ability of multi-scale models was lower in the seasons or periods in which populations used larger areas and a broader variety of environmental conditions. Seasonal estimates of foraging resources, together with LiDAR data, appeared to improve the performance of multi-scale habitat models.

Conclusions

We highlight that the understanding of multi-scale behavioral responses of species to spatial patterns that continually shift over time may be essential to unravel habitat relationships and produce reliable estimates of species distributions.

     

Skin and coat traits in sheep in Brazil and their relation with heat tolerance

The objective of this study was to evaluate and compare physical parameters in groups of sheep with different phenotypic characteristics in the Centre-west region of Brazil. Five groups of sheep, with nine animals per group, were selected, three groups of Santa Inês animals with different coat colours (white, brown and black), one group with crossbred animals (Santa Inês × Bergamasca) and one group with animals of the Bergamasca breed. The following traits were evaluated: coat thickness, number and length of hair, pigmentation level in the coat and the skin as well as the percentage area of sweats glands in the skin tissue, carried out by histological analysis. The number of hairs and the area of sweats glands were not significantly different between the evaluated groups. The Bergamasca breed showed low pigmentation of the skin and long hairs. The levels of pigmentation of the hair and of the skin were highly correlated. Between the Santa Inês groups, the group with white hair showed the better parameters for heat adaptation, while the brown hair group showed the lower heat adaptation when compared with another hair breed groups.     

Effects of three-dimensional spheroid culture on equine mesenchymal stem cell plasticity

Mesenchymal stem cells (MSCs) are useful candidates for tissue engineering and cell therapy fields. We optimize culture conditions of equine adipose tissue-derived MSCs (eAD-MSCs) for treatment of horse fractures. To investigate enhancing properties of three-dimensional (3D) culture system in eAD-MSCs, we performed various sized spheroid formation and determined changes in gene expression levels to obtain different sized spheroid for cell therapy. eAD-MSCs were successfully isolated from horse tailhead. Using hanging drop method, spheroid formation was generated for three days. Quantitative real-time PCR was performed to analyze gene expression. As results, expression levels of pluripotent markers were increased depending on spheroid size and the production of PGE₂ was increased in spheroid formation compared to that in monolayer. Ki-67 showed a remarkable increase in the spheroid formed with 2.0?×?10⁵ cells/drop as compared to that in the monolayer. Expression levels of angiogenesis-inducing factors such as VEGF, IL-6, IL-8, and IL-18 were significantly increased in spheroid formation compared to those in the monolayer. Expression levels of bone morphogenesis-inducing factors such as Cox-2 and TGF-β1 were also significantly increased in spheroid formation compared to those in the monolayer. Expression levels of osteocyte-specific markers such as RUNX2, osteocalcin, and differentiation potential were also significantly increased in spheroid formation compared to those in the monolayer. Therefore, spheroid formation of eAD-MSCs through the hanging drop method can increases the expression of angiogenesis-inducing and bone morphogenesis-inducing factors under optimal culture conditions.     

Estimating the spatial scales of landscape effects on abundance

Context

Spatial variation in abundance is influenced by local- and landscape-level environmental variables, but modeling landscape effects is challenging because the spatial scales of the relationships are unknown. Current approaches involve buffering survey locations with polygons of various sizes and using model selection to identify the best scale. The buffering approach does not acknowledge that the influence of surrounding landscape features should diminish with distance, and it does not yield an estimate of the unknown scale parameters.

Objectives

The purpose of this paper is to present an approach that allows for statistical inference about the scales at which landscape variables affect abundance.

Methods

Our method uses smoothing kernels to average landscape variables around focal sites and uses maximum likelihood to estimate the scale parameters of the kernels and the effects of the smoothed variables on abundance. We assessed model performance using a simulation study and an avian point count dataset.

Results

The simulation study demonstrated that estimators are unbiased and produce correct confidence interval coverage except in the rare case in which there is little spatial autocorrelation in the landscape variable. Canada warbler abundance was more highly correlated with site-level measures of NDVI than landscape-level NDVI, but the reverse was true for elevation. Canada warbler abundance was highest when elevation in the surrounding landscape, defined by an estimated Gaussian kernel, was between 1300 and 1400 m.

Conclusions

Our method provides a rigorous way of formally estimating the scales at which landscape variables affect abundance, and it can be embedded within most classes of statistical models.

     

Relating tradable credits for biodiversity to sustainability criteria in a dynamic landscape

Tradable biodiversity credit systems provide flexible means to resolve conflicts between development and conservation land-use options for habitats occupied by threatened or endangered species. We describe an approach to incorporate the influence of habitat fragmentation into the conservation value of tradable credits. Habitat fragmentation decreases gene flow, increases rates of genetic drift and inbreeding, and increases probabilities of patch extinction. Importantly, tradable credit systems will change the level of fragmentation over time for small and/or declining populations. We apply landscape equivalency analysis (LEA), a generalizable, landscape-scale accounting system that assigns conservation value to habitat patches based on patch contributions to abundance and genetic variance at landscape scales. By evaluating habitat trades using two models that vary the relationship between dispersal behaviors and landscape patterns, we show that LEA provides a novel method for limiting access to habitat at the landscape-scale, recognizing that the appropriate amount of migration needed to supplement patch recruitment and to offset drift and inbreeding will vary as landscape pattern changes over time. We also found that decisions based on probabilities of persistence alone would ignore changes in migration, genetic drift, and patch extinction that result from habitat trades. The general principle of LEA is that habitat patches traded should make at least equivalent contributions to rates of recruitment and migration estimated at a landscape scale. Traditional approaches for assessing the “take” and “jeopardy” standards under the Endangered Species Act based on changes in abundance and probability of persistence may be inadequate to prevent trades that increase fragmentation.     

Exploration of plant genomes in the FLAGdb++ environment

Background

Efficient high throughput screening systems of useful mutants are prerequisite for study of plant functional genomics and lots of application fields. Advance in such screening tools, thanks to the development of analytic instruments. Direct analysis in real-time (DART)-mass spectrometry (MS) by ionization of complex materials at atmospheric pressure is a rapid, simple, high-resolution analytical technique. Here we describe a rapid, simple method for the genetic discrimination of intact Arabidopsis thaliana mutant seeds using metabolic profiling by DART-MS.

Results

To determine whether this DART-MS combined by multivariate analysis can perform genetic discrimination based on global metabolic profiling, intact Arabidopsis thaliana mutant seeds were subjected to DART-MS without any sample preparation. Partial least squares-discriminant analysis (PLS-DA) of DART-MS spectral data from intact seeds classified 14 different lines of seeds into two distinct groups: Columbia (Col-0) and Landsberg erecta (Ler) ecotype backgrounds. A hierarchical dendrogram based on partial least squares-discriminant analysis (PLS-DA) subdivided the Col-0 ecotype into two groups: mutant lines harboring defects in the phenylpropanoid biosynthetic pathway and mutants without these defects. These results indicated that metabolic profiling with DART-MS could discriminate intact Arabidopsis seeds at least ecotype level and metabolic pathway level within same ecotype.

Conclusion

The described DART-MS combined by multivariate analysis allows for rapid screening and metabolic characterization of lots of Arabidopsis mutant seeds without complex metabolic preparation steps. Moreover, potential novel metabolic markers can be detected and used to clarify the genetic relationship between Arabidopsis cultivars. Furthermore this technique can be applied to predict the novel gene function of metabolic mutants regardless of morphological phenotypes.     

Effect of sow vaccination against Mycoplasma hyopneumoniae on sow and piglet colonization and seroconversion, and pig lung lesions at slaughter

The objectives of the present study were to compare Mycoplasma hyopneumoniae (Mh) colonization and serologic status on Mh vaccinated and non-vaccinated sows and to assess the effect of sow vaccination on colonization and serologic status of their piglets at weaning as well as presence of enzootic pneumonia (EP) lung lesions at slaughter. Fifty sows (25 vaccinated and 25 unvaccinated) as well as five of their piglets were included in the study. Blood samples and nasal swabs from sows at 7 weeks pre-farrowing and 1 week post-farrowing and from piglets at 3-4 weeks of age were taken. Nasal swabs and sera were tested by a nested polymerase chain reaction (nPCR) to detect Mh DNA and by an enzyme-linked immunosorbent assay (ELISA) test to detect antibodies to the pathogen, respectively. Finally, at 23 weeks of age, pigs were sent to the slaughter where the extension of EP-compatible gross lesions was assessed. Vaccination with two doses of Mh vaccine resulted in a significantly higher (p<0.05) percentage of seropositive sows than in the non-vaccinated group at 1 week post-farrowing. On the contrary, no statistical significant differences were found in the number of nasal nPCR positive sows among different treatments (p>0.05). At 3-4 weeks of age, a significantly higher percentage (p<0.001) of seropositive piglets came from vaccinated than from non-vaccinated sows. Although the number of Mh infected piglets coming from non-vaccinated sows was higher than the one from vaccinated sows, the difference was not statistically significant (p>0.05). Overall, piglets from vaccinated sows had a significant lower (p<0.05) mean of EP-compatible lung lesions (1.83+/-2.8) than piglets from non-vaccinated sows (3.02+/-3.6). Under the conditions described in this study, sow vaccination did not affect sow or piglet colonization but increased the percentage of seropositive sows and piglets at weaning and reduced significantly the mean EP-compatible lung lesion scoring at slaughter.