Cryopreservation of Piau‐Breed Wild Boar Sperm: Assessment of Cooling Curves and Centrifugation Regimes

This study aimed to assess the effects of different cooling curves and centrifugation regimes used in cryopreservation protocols on the post‐thaw viability of Piau‐breed wild boar (Sus scrofa) sperm using in vitro assessment tests. Two centrifugations (800  g for 10 min and 2400  g for 3 min) and two cooling curves (conventional cooling using nitrogen vapour – freezing 1 and automated cooling using a programmed freezing machine – freezing 2) were tested. Therefore, the treatments were divided into M3 – centrifugation at 2400  g for 3 min and freezing 2; M10 – centrifugation at 800  g for 10 min and freezing 2; R3 – centrifugation at 2400  g for 3 min and freezing 1; and R10 – centrifugation at 800  g for 10 min and freezing 1. No significant differences (p > 0.05) between treatments occurred post‐thawing regarding the total sperm motility means recorded. The mean values of the different treatments were not different from each other regarding the supravital staining (SV), hypo‐osmotic test (HO), sperm–egg binding assay or sperm morphology. This study showed that both the cooling curve and the centrifugation regime affected the quality of post‐thaw sperm, and centrifugation for shorter times and cooling curves using automated cooling are the most suitable for minimizing sperm injury.     

Immunohistochemical Expression of Growth Factors in the Follicular Wall of Normal and Cystic Ovaries of Sows

The expression of growth factors was evaluated immunohistochemically in normal and cystic ovaries of sows. The immunohistochemically stained area (IHCSA) was quantified by image analysis to analyse the expression of these proteins in the follicular wall of secondary, tertiary and cystic follicles. IGF‐I immunoreactivity was strong in the granulosa cell layer (GC), moderate in the theca interna (TI) and mild in the theca externa (TE) of the normal follicles. There was severe reduction of the labelling to IGF‐I in the GC of the follicular and luteinized cysts. In the normal follicles, the reactivity for IGF‐II was very similar to pattern noted in IGF‐I. There was reduction of the IHCSAs in the GC of the follicular and luteinized cysts, but the decrease was not significant. The staining of the IGF‐II in the TI and TE of the cysts was increased, in comparison with normal follicles. The IHCSAs for VEGF were higher in the GC and TE of the normal follicles in contrast to TI, but this difference was noted only in the tertiary follicle. The VEGF reactivity increased in the GC of the cysts, in relation to normal follicles. The results of the current study show that the formation of ovarian cysts in sows is associated with alterations in the immunohistochemical expression of some growth factors.     

Evidence of multiple divergent mitochondrial lineages within the southern African diplopod genus Bicoxidens Attems, 1928 (Spirostreptida)

Two recent studies have suggested that divergent mitochondrial lineages may be present within spirostreptid genera such as Bicoxidens Attems, 1928. Bicoxidens, similar to many other endemic soil invertebrates, exhibits low dispersal capabilities and strict microclimate habitat preferences, attributes that often lead to geographic isolation. Given that prolonged geographic isolation often lays the foundation for population genetic differentiation, genetic divergence and possibly speciation, there was good reason to suspect that Bicoxidens may consist of several distinct lineages. On this basis, the mitochondrial cytochrome c oxidase subunit 1 (COI) was used to reconstruct the phylogeny of Bicoxidens and reveal divergent lineages within the genus. Maximum likelihood and Bayesian inference analyses recovered a paraphyletic Bicoxidens phylogram with divergent lineages present in three species – B. friendi, B. flavicollis and B. brincki – suggesting high genetic diversity within the genus. Bayesian genetic cluster analyses suggested the presence of multiple distinct mitochondrial lineages within the genus with four identified in B. flavicollis alone. It was therefore concluded that the divergent lineages observed among Bicoxidens populations may suggest the presence of hidden species.     

Heat Shock Protein 70 and Sex Steroid Receptors in the Follicular Structures of Induced Ovarian Cysts

The purpose of this study was to estimate the expression and relative amounts of estrogen (ER) and progesterone receptors (PR) and their isoforms as well as heat shock protein 70 (HSP70) in ovaries of rats with induced cystic ovarian disease (COD). Primary, secondary, tertiary, atretic and cystic follicles were evaluated by immunohistochemistry and total ovarian proteins were analyzed by Western blot. In the granulosa layer, growing and cystic follicles in the treated group have a higher expression of ERα than growing follicles of control individuals. In the theca interna layer, tertiary follicles presented a significantly higher expression of ERα in the treated group. An increase in total ERα protein was detected in the treated group. Granulosa cells of all growing, atretic and cystic follicles show a lower expression of ERβ in animals with COD, and the total protein expression of ERβ was lower in this group. The expression of PR was lower in the granulosa cell layer of tertiary and cystic follicles in treated animals, and theca interna layer had less intense immunostaining in this group. Although there were no differences in the expression of PR-B by Western blotting, the expression of PR-A was higher and the expression of PR-C was smaller in the treated group. An intense HSP70 immunostaining was observed in the cells of cystic follicles. By Western blotting, higher protein expression of HSP70 was detected in the ovarian samples of the control group than those of the treated ones. Ovaries of animals with COD exhibited an altered steroid receptor expression and subtype balance as compared with control animals, and an increase in HSP70 immunoexpression.     

The use of ultrasonography in the reproductive evaluation of boars

The objective was to study the use of ultrasound as a complementary test in the breeding soundness evaluation in male pigs and study the pattern of echogenicity of the testicular parenchyma in boars of different racial groups. Twenty‐six adult boars from four different racial groups were used, 10 from the Piau breed (group 1), four from the commercial and finishing group (group 2), six Pietrain breed (group 3) and six from the Duroc breed (group 4). All animals were evaluated for breeding soundness evaluation and the ultrasound examination of the testicles. The groups of animals that were evaluated showed no difference in the main semen parameters that were evaluated, except for the sperm volume, concentration of the ejaculated sperm and the supravital staining; the lowest figures were for the animals from the Piau breed (group 1). In relation to the testicular biometrics, Duroc animals (group 4) had a greater scrotal width compared to the other groups. But when we assessed the intensity of pixels of the testicles, there was a difference between groups. The groups 2 (finishing animals), 3 (Pietrain) and 4 had no difference between themselves. Group 3 had greater pixel intensity in relation to group 1. Of the 26 animals studied, five showed an abnormality during ultrasound evaluation, like hydrocele, hyperechoic mass in the testicular parenchyma, cyst in the head of the epididymis and the presence of fluid in the head and tail of the epididymis. The various animal groups studied did not differ in the principal reproductive parameters evaluated, showing that despite the great variability of reproductive traits between breeds and within the same breed, the breeding soundness evaluation, the more complete it is, is essential for the selection of breeders and the ultrasonography of the reproductive system becomes an important addition in this examination.     

The role of movement restrictions and pre-emptive destruction in the emergency control strategy against CSF outbreaks in domestic pigs

Classical swine fever (CSF) outbreaks in domestic pig herds lead to the implementation of standard control measures according to legislative regulations. Ideal outbreak control entails the swift and efficient culling of all pigs on premises detected positive for CSF virus. Often all pig holdings around the detected cases are pre-emptively destroyed to exclude transmission into the neighbourhood. In addition to these measures, zones are defined in which surveillance and protection measures are intensified to prevent further distant disease spread. In particular, all movements are prohibited within standstill areas. Standstill also excludes the transport of fattened pigs to slaughter. Historical outbreaks provide evidence of the success of this control strategy. However, the extent to which the individual strategy elements contribute to this success is unknown. Therefore, we applied a spatially and temporally explicit epidemic model to the problem. Its rule-based formulation is tailored to a one-by-one model implementation of existing control concepts. Using a comparative model analysis the individual contributions of single measures to overall control success were revealed. From the results of the model we concluded that movement restrictions had the dominant impact on strategy performance suggesting a reversal of the current conceptual thinking. Additional measures such as pre-emptive culling only became relevant under imperfect compliance with movement restrictions. The importance of movement restrictions for the overall control success illustrates the need for explicit consideration of this measure when contingency strategies are being amended (e.g. emergency vaccination) and associated risks assessed.     

Efficiency of spatio-temporal vaccination regimes in wildlife populations under different viral constraints

ABSTRACT: Classical Swine Fever (CSF) is considered an endemic disease in European wild boar populations. In view of the high economic impact of the introduction of the virus into domestic pig units, huge efforts are invested in the preventive control of CSF in wild boar populations. Recent European Community guidelines favour oral mass vaccination against CSF in wild boar populations. The guidelines are explicit on the temporal structure of the vaccination protocol, but little is known about the efficacy of different spatial application schemes, or how they relate to outbreak dynamics.We use a spatially explicit, individual-based wild boar model that represents the ecology of the hosts and the epidemiology of CSF, both on a regional scale and on the level of individual course of infection. We simulate adaptive spatial vaccination schemes accounting for the acute spread of an outbreak while using the temporal vaccination protocol proposed in the Community guidelines.Vaccination was found to be beneficial in a wide range of scenarios. We show that the short-term proactive component of a vaccination strategy is not only as decisive as short-term continuity, but also that it can outcompete alternative practices while being practically feasible. Furthermore, we show that under certain virus-host conditions vaccination might actually contribute to disease persistence in local populations.     

Baiting intervals and duration of control of the small fox tapeworm: a simulation study

Field trials to control the small fox tapeworm Echinococcus multilocularis failed to reduce the prevalence of the parasite persistently. With the experience from this empirical work, a simulation model for the population dynamics of the small fox tapeworm was developed. This model is used to examine the effect of different control strategies. Sufficient control duration is able to eradicate the parasite in simulation experiments. Baiting intervals of 4 or 6 weeks were found to be more efficient than shorter or longer intervals. The strategy used in field trials showed no sustainable effect in any of the simulation experiments. The applicability of simulation models for the planning of control measures for wildlife diseases is discussed.     

Controlling Echinococcus multilocularis-ecological implications of field trials

Two field trials to reduce the prevalence of Echinococcus multilocularis in foxes have been conducted in recent years. Although both trials reduced prevalence considerably, they failed to eradicate the parasite in the study region. Following the control trial in northern Germany, prevalence recovered unexpectedly and rapidly, reaching pre-control levels five quarters (15 months) after the end of control. To understand the internal dynamics of the parasite-host system's reaction to control, we developed a spatially explicit simulation model, Echi. The simulation model incorporates the information available concerning fox tapeworm population dynamics.Using epidemiological parameters to adjust pre-control prevalence, the model predicts the temporal evolution of the prevalence of E. multilocularis in controlled foxes without departing from the range of uncertainty of the field data. However, the model does not predict the rapid pre-control recovery observed in the field trial.The deviation of the model's prediction from field data indicates the involvement of processes not yet taken into account. We modified the model step by step to mimic processes with the potential to cause the rapid post-control recovery of the prevalence of E. multilocularis in foxes.Neither the longevity of tapeworm eggs nor the migratory behaviour of foxes showed any influence on the post-control reaction of the parasite-host system. However, landscape structures leading to a heterogeneous distribution of infected foxes have the potential to alter the system's reaction to control. If infected foxes are concentrated in multiple clusters in the landscape, the model prediction tallied with the range of uncertainty of the field data. Such spatial distribution of infected foxes may be caused by differential abiotic conditions influencing the survival of tapeworm eggs.The model was found to comply best with field data if the foxes acquire partial immunity by being exposed to the fox tapeworm.Both hypotheses explaining the rapid post-control recovery of the prevalence of E. multilocularis observed in the fox population were supported by field data.Both hypotheses have far-reaching consequences for future control trials. The spatial aggregation of infected foxes would enable control efforts to be concentrated on these highly infected areas. However, the acquisition of immunity acts as a buffer to control, necessitating intensified control measures.     

Oestradiol Induced Inhibition of Neuroendocrine Marker Expression in Leydig Cells of Adult Rats

The objectives of this work were to determine the changes in the expression of neuroendocrine markers in Leydig cell by oestradiol treatment, and to determine whether testosterone is able to recover partially the effects of hormonal suppression induced by oestradiol. Adult male rats were injected daily with either 50 microg of oestradiol or oestradiol plus testosterone propionate (25 mg every 3 days) for 15 days. The animals were sacrificed and testicles were dissected and processed by routine histological protocols. FSH and LH serum levels were determined by radioimmunoassay. The visualization of antigens was achieved by the streptavidin-peroxidase immunohistochemical method. Antibodies against chromogranin A (CrA), S-100 protein (S-100), P substance (PS), synaptofisin (SYN), neurofilament protein (NF), gliofibrillary acidic protein (GFAP) and neuron specific enolase (NSE) were used. The mean LH and FSH serum concentrations were consistently suppressed with hormonal treatments. Intermediate filaments (NF and GFAP) showed no difference in their expression. The expression of S-100, NSE and SYN was significantly lower in both hormone-treated groups. In oestradiol-treated rats, the immunoreactivity of CrA and SP decreased significantly but was restored after testosterone supplementation. Although the nature and functions of many of these substances in Leydig cells remain unknown, these results are consistent with the hypothesis that the expression of some neuroendocrine markers is hormonally controlled.