Co-exposure to pathogens in wild ungulates from Doñana National Park, South Spain.

Multiple infections or co-exposure to pathogens should be considered systematically in wildlife to better understand the ecology and evolution of host-pathogen relationships, so as to better determine the potential use of multiple pathogens as indicators to guide health management. We describe the pattern of co-exposure to several pathogens (i.e. simultaneous positive diagnosis to pathogens in an individual considering Mycobacterium tuberculosis complex lesions, and the presence of antibodies against Toxoplasma gondii, bluetongue virus, and hepatitis E virus) and assessed their main drivers in the wild ungulate community from Doñana National Park (red deer, fallow deer, and wild boar) for a 13-years longitudinal study. The lower-than-expected frequency of co-exposure registered in all species was consistent with non-mutually exclusive hypotheses (e.g. antagonism or disease-related mortality), which requires further investigation. The habitat generalist species (red deer and wild boar) were exposed to a greater diversity of pathogens (frequency of co-exposure around 50%) and/or risk factors than fallow deer (25.0% ± CI95% 4.9). Positive relationships between pathogens were evidenced, which may be explained by common risk factors favouring exposure. The specific combination of pathogens in individuals was mainly driven by different groups of factors (individual, environmental, stochastic, and populational), as well as its interaction, defining a complex eco-epidemiological landscape. To deepen into the main determinants and consequences of co-infections in a complex assemblage of wild hosts, and at the interface with humans and livestock, there also is needed to expand the range of pathogens and compare diverse assemblages of hosts under different environmental and management circumstances.

Serum supplementation during in vitro fertilization of sheep oocytes influences blastocyst quality through the differential abundance of mRNA transcripts.

Incubation with estrous sheep serum (ESS) is required to induce in vitro capacitation of spermatozoa during in vitro fertilization of small ruminants. However, the effect of adding different serum concentrations in the fertilization media on the quality of resulting blastocysts has not yet been studied. Here, 298 sheep oocytes were co-incubated with capacitated spermatozoa with either 10% or 2% ESS. There were no differences between treatments in cleavage (10% ESS: 63.81 ± 5.87% and 2% ESS: 45.31 ± 5.87%) and blastocyst rates (10% ESS: 20.83 ± 2.12% and 2% ESS: 15.93 ± 2.12%). Nonetheless, in vitro-produced blastocysts from the 10% ESS treatment showed a higher transcript abundance of mRNAs involved in apoptosis (ITM2B and BCL2), antioxidant defence (GPX1) and growth-related imprinting (IGF2R). Our data suggest that ESS supplementation during in vitro fertilization can influence the quality of sheep embryos at later stages of development by increasing the transcription of developmentally important genes.

Impact of Cryopreservation on Motile Subpopulations and Tyrosine-Phosphorylated Regions of Ram Spermatozoa during Capacitating Conditions.

The heterogeneous nature of ejaculates highlights the relevance of studying the behavior of different sperm subpopulations. Changes in sperm motility and the increase in tyrosine phosphorylation are key events that usually occur during capacitation and can be modified by the cryopreservation process. However, the relationship between both events remains poorly defined throughout capacitation in the different sperm subpopulations. Fresh and frozen-thawed spermatozoa were incubated in capacitating (CAP) and non-capacitating (NC) media up to 240 min. Sperm kinematics, tyrosine phosphorylation and mitochondrial activity were measured by the CASA system and imaging flow cytometry. Four motile sperm subpopulations (SP) were identified in fresh and frozen-thawed ram semen after the cluster analysis. Incubation under CAP conditions over time led to greater changes in the percentage of spermatozoa included in each subpopulation compared to NC conditions, being different between fresh and frozen-thawed spermatozoa. The SP1, characterized by slow spermatozoa, progressively increased after 15 min in frozen-thawed samples incubated in both media but not in fresh ones. The SP4, characterized by fast and non-linear spermatozoa, showed a marked increase during CAP, but not under NC conditions, occurring more rapidly in frozen-thawed spermatozoa. This subpopulation (SP4) was also the only one positively and strongly correlated with mitochondrial activity and all phosphorylated sperm regions during capacitation, either in fresh or frozen-thawed samples. Our results indicated that in vitro capacitation induced significant changes in the distribution of motile sperm subpopulations, being affected by cryopreservation. Notwithstanding, the subpopulation which probably represents hyperactivated-like spermatozoa (SP4) also increased in frozen-thawed samples, occurring faster and simultaneously to the increment of mitochondrial activity and tyrosine phosphorylation of different sperm regions.

cAMP Modulators before In Vitro Maturation Decrease DNA Damage and Boost Developmental Potential of Sheep Oocytes.

To date, the underlying mechanisms by which cAMP modulators act during in vitro maturation to improve oocyte developmental competence are poorly understood. Here, we sought to fill this knowledge gap by evaluating the use of phosphodiesterase inhibitor 3-isobutyl-1-methylxanthine (IBMX) and adenylyl cyclase activator forskolin during a culture period of 2 h before in vitro maturation (pre-IVM) on the nuclear and cytoplasmic maturation features in essential organelles, cumulus cells activity, and in vitro developmental potential of sheep oocytes. Results showed that pre-IVM treatment significantly decreased (p < 0.05) the DNA damage of mature oocytes (pre-IVM = 2.08% ± 3.51% vs. control = 20.58% ± 3.51%) and increased (p ≤ 0.05) expanded blastocyst rates compared to the control (from the total of oocytes: pre-IVM = 23.89% ± 1.47% vs. control = 18.22% ± 1.47%, and from the cleaved embryos: pre-IVM = 45.16% ± 1.73% vs. control = 32.88% ± 1.73%). Considering that oocytes are highly vulnerable to the accumulation of DNA damage because of exposure to in vitro culture conditions, our results suggest that the modulation of intra-oocyte cAMP levels with forskolin and IBMX before IVM might afford oocytes a more effective DNA repair mechanism to overcome damage obstacles and ultimately improve developmental competence. This previously unappreciated action of cAMP modulators could help to develop improved methods for assisted reproduction technologies in animal and clinical research.

Long-Term Determinants of the Seroprevalence of the Hepatitis E Virus in Wild Boar (Sus scrofa).

The hepatitis E virus (HEV) is an emerging zoonotic pathogen whose main reservoir is suids. Most of the ecological and epidemiological aspects of its sylvatic cycle remain unknown. Thus, in this work, we study the drivers of HEV exposure in the wild boar population of Doñana National Park (DNP, southwest Spain) operating in the medium and long-term (2005-2018). Anti-HEV antibodies are widely distributed throughout the wild boar (46.7 ± 3.8%, 327 out of 700 sampled), showing a statistically significant age-increasing pattern. The temporal pattern displayed important interannual fluctuations. This could be mediated by marked variations in the population control of the wild boar, and subsequent changes in abundance rates, and its interplay with climatic conditions; as wet years together with a low abundance of wild boar led to the lowest seroprevalence. The fact that seroprevalence is high during conditions of high abundance, and not affected by rainfall level, is probably due to the increased interactions among the animals, and possibly, the subsequent higher environmental contamination with HEV particles. The proximity to the marshland (the main water body of the study area) is associated with a higher risk of testing positive, which is probably mediated by the preferential use of this area during the dry season and the favourable environmental conditions for the survival of HEV particles. A deeper understanding of the epidemiology of HEV in host communities deserves future research concerning other susceptible species. Most importantly, wild boar population control remains a challenge at the international level, and an increase of shared pathogen-related conflicts associated with this species is expected, as exemplified by HEV. Therefore, surveillance of wild boar diseases, including integrated population monitoring and sustainable population control programmes, will be essential to control the associated risks.

Long-term determinants of the seroprevalence of the bluetongue virus in deer species in southern Spain.

Bluetongue is a vector-borne disease affecting domestic and wild ruminants, with a major socioeconomic impact. Endemic circulation of the bluetongue virus serotype 4 (BTV-4) and BTV-1 have occurred in Spain since 2004 and 2007, respectively. However, epidemiological studies have seldom been approached from a long-term perspective in wild ruminants. A total of 881 deer (red deer and fallow deer) were necropsied from 2005 to 2018 as part of the DNP health-monitoring program. Serum samples were tested for antibodies against BTV with the aims of assessing the temporal trend and to evaluate the modulating factors: individual, populational, environmental, and stochastic. Red deer displayed statistically significant higher seroprevalences of BTV (SBT; 78.6 ± 3.8%) than fallow deer (53.1 ± 4.7%). The detection of BTV-1 and BTV-4 by the serum neutralization test in calves suggested the circulation of both serotypes over the study period. For red deer, wet years together with high densities could provide suitable conditions for vector borne BTV transmission. Moreover, proximity to high suitability habitat for Culicoides, permanent pasturelands, was associated with higher SBT. The differences in the ecology and behaviour of deer species influencing the exposure to the vectors could determine the differences found in the SBT patterns. This study evidences the role that deer species may play in the maintenance of BTV, however, elucidating the epidemiological role of host in different contexts as well as the consequences of climate change on the competent vector populations and its potential effect on the dynamics of BTV infection in hosts communities deserve further research.

The wildlife-livestock interface on extensive free-ranging pig farms in central Spain during the "montanera" period.

The effective management of shared pathogens between wild ungulates and livestock requires the understanding of the processes of interaction between them. In this work, we studied the interspecific frequency of interaction (ifreq) and its spatiotemporal pattern between wild and domestic ungulates that coexist in free-ranging farms. For this purpose, 6 red deer, 6 wild boar, 8 Iberian pigs and 3 cattle were monitored using GPS devices during the "montanera" period (the period in which Iberian pigs are maintained in extensive conditions to feed on acorn). The ifreq was quantified for two spatiotemporal windows: 30 m - 10 min, for inferring potential direct interactions (short window), and 30 m - 12 days for indirect interactions (large window). Secondly, the variation in the ifreq was modelled with regard to 2 temporal (time of the day and week of the year) and 4 environmental factors (distance to water, distance to vegetation cover, Quercus density and distance to feeding points). The interactions at the short window were scarce (N = 13); however, they were very frequent at the large one (N = 37,429), with the red deer as the species with the greatest involvement in the interactions. Models showed that the time of the day and distance to water were the variables that best predicted the ifreq and they were conditioned by differences in the activity pattern of the targeted species. Food resource availability also predicted the ifreq, especially at the short window and between wild species. The results presented here highlight the role that wild ungulates may play in the transmission of pathogens to extensive livestock in general and pigs in particular and show the epidemiological risk of certain areas, periods of time and management practices (for wildlife and livestock) as well as providing useful information in the prevention of the transmission of shared pathogens.

Beneficial Effects of Melatonin in the Ovarian Transport Medium on In Vitro Embryo Production of Iberian Red Deer (Cervus elaphus hispanicus).

A major limiting factor for the development of in vitro embryo production (IVP) in wild species, such as Iberian red deer, compared to livestock animals is the poor availability and limited access to biological material. Thus, the use of post-mortem ovaries from slaughtered animals represent a source of oocytes for the large scale production of embryos needed for research and to improve the efficiency of IVP. However, these oocytes are not as developmentally competent as their in vivo counterparts. Moreover, oocytes are usually obtained from ovaries that have been transported for long distances, which may also affect their quality. In order to overcome the issues associated with prolonged storage times of post-mortem material, in this study we examined the effect of melatonin supplementation to the ovary transport medium on oocyte quality, embryo yield, and blastocyst quality in Iberian red deer. When necessary, sheep was used as an experimental model due to the large number of samples required for analysis of oocyte quality parameters. Oocytes were in vitro matured and assessed for early apoptosis; DNA fragmentation; reactive oxygen species (ROS); reduced glutathione (GSH) content, mitochondrial membrane potential, and distribution; and relative abundance of mRNA transcript levels. After in vitro fertilization, embryo rates and blastocyst quality were also investigated. The results revealed that melatonin treatment significantly increased intracellular level of GSH in sheep oocytes. Moreover, the percentage of cleavage and blastocyst yield in red deer was greater compared to the Control group and there was lower abundance of oxidative stress- and apoptosis-related SHC1, TP53, and AKR1B1 mRNA transcripts in blastocysts for the Melatonin group. In conclusion, the supplementation of melatonin to the ovary storage medium had a positive effect on the developmental competence and quality of resulting blastocysts in Iberian red deer.

Sperm Cryodamage in Ruminants: Understanding the Molecular Changes Induced by the Cryopreservation Process to Optimize Sperm Quality.

Sperm cryopreservation represents a powerful tool for livestock breeding. Several efforts have been made to improve the efficiency of sperm cryopreservation in different ruminant species. However, a significant amount of sperm still suffers considerable cryodamage, which may affect sperm quality and fertility. Recently, the use of different "omics" technologies in sperm cryobiology, especially proteomics studies, has led to a better understanding of the molecular modifications induced by sperm cryopreservation, facilitating the identification of different freezability biomarkers and certain proteins that can be added before cryopreservation to enhance sperm cryosurvival. This review provides an updated overview of the molecular mechanisms involved in sperm cryodamage, which are in part responsible for the structural, functional and fertility changes observed in frozen-thawed ruminant sperm. Moreover, the molecular basis of those factors that can affect the sperm freezing resilience of different ruminant species is also discussed as well as the molecular aspects of those novel strategies that have been developed to reduce sperm cryodamage, including new cryoprotectants, antioxidants, proteins, nanoparticles and vitrification.

Ovine sperm DNA oxidation quantification using an 8-OHdG immunodetection assay.

Our aim was to optimize 8-hydroxy-2'-deoxyguanosine (8-OHdG) immunodetection in order to detect DNA damage caused by oxidative stress that may not be detected by other DNA integrity analysis techniques, especially due to the high compaction of DNA in ruminants. Semen samples from 6 rams were cryopreserved. After thawing, samples were subjected to the DNA oxidation quantification using an 8-OHdG immunodetection assay by flow cytometry. We have evaluated two different incubation times (30 min vs. overnight) at 4°C of the primary antibody (monoclonal anti-8-OHdG antibody). We have also compared the results of this technique with the sperm chromatin structure assay (SCSA® ). The analysis revealed that there were no significant differences (p > .05) between different incubation times. However, overnight incubation seems to cause more non-specific binding of the secondary antibody. Significant differences (p < .05) between subjects and oxidation controls (8 M H2 O2 /800 µM FeSO4 •7H2 O) were evident. We can conclude that the 8-OHdG immunodetection assay for DNA oxidation quantification of ram sperm can be performed subjecting sperm samples to a very high oxidative treatment.

Consumption of Big Game Remains by Scavengers: A Potential Risk as Regards Disease Transmission in Central Spain.

Understanding the role that facultative scavenger species may play in spreading infectious pathogens, and even becoming reservoirs for humans, domestic and wild ungulates or, on the contrary, preventing the spread of disease, requires a prior understanding of the pattern of carrion scavenging in specific scenarios. The objectives of this paper are (i) to describe the guild of vertebrate scavengers and (ii) to study the species-specific, habitat, and management-related factors involved in the usage of gut piles in South Central Spain (SCS), a tuberculosis (TB) endemic area. We used camera trapping at 18 hunting piles on seven hunting estates. A total of eight bird and five mammal taxa were detected at the remains of hunting piles. The most frequently detected species in terms of number of gut piles visited (78%) and scavenged (61%) was the red fox Vulpes vulpes, followed by the griffon vulture Gyps fulvus (56% as regards both presence and scavenging) and the raven Corvus corax (61 and 39% as regards presence and scavenging, respectively). We evidenced that griffon vultures accounted for most of the scavenging activity in open habitats, while facultative mammal scavengers, red fox, and wild boar Sus scrofa made the highest contribution to scavenging in vegetation-covered habitats. In the case of wild boar, the gut piles deposited during the evening and night favored higher rates of scavenging, while the opposite pattern was observed for griffons. Overall, our findings suggest that when disposing of hunting remains in areas of risk as regards disease transmission it is particularly important to consider the access that facultative mammals, and especially wild boar, have to material, while the presence of the resource needs to be safeguarded to protect specialist scavengers of conservation value. These results are of particular relevance in the case of wild boar in the current context of re-emerging TB and emerging African swine fever (ASF) in Europe.

Oral re-vaccination of Eurasian wild boar with Mycobacterium bovis BCG yields a strong protective response against challenge with a field strain.

BACKGROUND: Field vaccination trials with Mycobacterium bovis BCG, an attenuated mutant of M. bovis, are ongoing in Spain, where the Eurasian wild boar (Sus scrofa) is regarded as the main driver of animal tuberculosis (TB). The oral baiting strategy consists in deploying vaccine baits twice each summer, in order to gain access to a high proportion of wild boar piglets. The aim of this study was to assess the response of wild boar to re-vaccination with BCG and to subsequent challenge with an M. bovis field strain. RESULTS: BCG re-vaccinated wild boar showed reductions of 75.8% in lesion score and 66.9% in culture score, as compared to unvaccinated controls. Only one of nine vaccinated wild boar had a culture-confirmed lung infection, as compared to seven of eight controls. Serum antibody levels were highly variable and did not differ significantly between BCG re-vaccinated wild boar and controls. Gamma IFN levels differed significantly between BCG re-vaccinated wild boar and controls. The mRNA levels for IL-1b, C3 and MUT were significantly higher in vaccinated wild boar when compared to controls after vaccination and decreased after mycobacterial challenge. CONCLUSIONS: Oral re-vaccination of wild boar with BCG yields a strong protective response against challenge with a field strain. Moreover, re-vaccination of wild boar with BCG is not counterproductive. These findings are relevant given that re-vaccination is likely to happen under real (field) conditions.

Influence of various permeating cryoprotectants on freezability of Iberian red deer (Cervus elaphus hispanicus) epididymal spermatozoa: effects of concentration and temperature of addition.

With the aim of finding an ideal cryoprotectant (CPA) in a suitable concentration for red deer epididymal spermatozoa cryopreservation, we evaluated the effects of the 3 most commonly used CPAs, glycerol (GLY), ethylene glycol (EG), and propylene glycol (PG), on sperm cryoresistance. The aim of Experiment 1 was to evaluate the influence of 3 different final concentrations (3%, 6%, and 12%) of each CPA on sperm freezability. Sperm samples were diluted to a final sperm concentration of approximately 400 x 10(6) spermatozoa/mL with a Tris-citrate-fructose-EY extender (TCF) prior to freezing. Sperm cryosurvival was judged in vitro by microscopic assessments of individual sperm motility (SMI), viability, and plasma membrane (by means of the HOS test) and acrosome (NAR) integrities. Thawed samples were incubated at 37 degrees C for 2 hours in the freezing medium. At the end of this incubation period, sperm suspensions were again assessed. Our results showed that 12% of any CPA was toxic to red deer epididymal spermatozoa membrane integrity (P < .05). Moreover, regardless of the level of CPA, results indicated that the cryoprotective effects on red deer epididymal spermatozoa of the 3 CPAs after thawing are in the following sequence: GLY > EG > PG (higher symbols mean P < .001). Furthermore, our results also showed an improvement in sperm parameters when the TCF diluent contained 6% of GLY. In Experiment 2 extenders were prepared using GLY 6%. This experiment was designed to investigate the effect of 2 different temperatures of GLY addition -22 degrees C (ambient temperature) and 5 degrees C- on sperm freezability. Our results showed a differential response (P < .05) of motility (SMI) to temperature of GLY addition before freezing, the best being 22 degrees C (81.94 +/- 2.4% vs 72.38 +/- 2.4%). Although there were no statistically significant differences (P > .05) between the 2 temperatures of GLY addition after thawing in terms of sperm quality, after 2 hours of incubation, results tended to be better when CPAs were added at 22 degrees C. In conclusion, our work showed the efficacy of a TCF diluent with 6% of GLY and its addition at 22 degrees C, as an alternative to the more common 3%-4% of GLY and addition at 5 degrees C, in red deer semen freezing protocols.

Seroprevalence of six reproductive pathogens in European wild boar (Sus scrofa) from Spain: the effect on wild boar female reproductive performance.

We studied the seroprevalence of six reproductive pathogens in Spanish hunter-harvested wild boar females. The sample was representative of the hunting harvest in the studied hunting estates. Mean antibody prevalences were: 60.6+/-0.06% for Aujeszky's disease virus (ADV), 56.6+/-0.09% for porcine parvovirus (PPV), 51.8+/-0.06% for porcine circovirus type 2 (PCV2), 29.7+/-0.09% for Brucella spp. and 36.3+/-0.1% for Toxoplasma gondii. We did not detect antibodies against porcine reproductive and respiratory syndrome virus (PRRSv). ADV seroprevalence was associated with PPV and PCV2 seroprevalence in Spanish wild boar females. Ovulation rate in the studied wild boar females was 4.41+/-0.16 (n=120), mean litter size was 3.91+/-0.16 (n=82) and the partial resorption index 0.92+/-0.17 (n=66). Ovulation rate and litter size were statistically associated with age. T. gondii seroprevalence was negatively related to ovulation rate and partial resorption index. Wild boars from managed fenced estates had antibodies against more pathogens than wild boars from open estates. Potential relations between management of wild boar populations and exposure of individuals to different reproductive pathogens are discussed.

The effects of different cryoprotectants and the temperature of addition on the survival of red deer epididymal spermatozoa.

With the aim of finding an ideal cryoprotectant in a suitable concentration for red deer epididymal spermatozoa conservation, we evaluated the effects of four most commonly used cryoprotectants (CPAs), Glycerol (G), Ethylene glycol (EG), Propylene glycol (PG), and Dimethyl sulfoxide (DMSO), on the sperm survival. Besides, the effects of two temperatures of CPA addition--22 degrees C (ambient temperature) and 5 degrees C--on sperm quality were also tested. For each temperature tested, sperm samples were evaluated after 0, 15, 30 and 60 min of spermatozoa exposition to CPAs. Thus, sperm quality was in vitro judged by microscopic assessments of individual sperm motility (SMI), and of plasma membrane (Viability) and acrosome (NAR) integrities. Overall, DMSO showed the highest toxicity for red deer epididymal spermatozoa, and glycerol the lowest. Thus, at 60 min of incubation SMI results showed that the toxicity to red deer epididymal spermatozoa of the four CPAs are in the following sequence: G approximately = EG approximately = PG < DMSO ('less than' symbol means P < 0.05, and approximate symbol means P = 0.08). Furthermore, our results also showed a differential response of acrosome membrane to temperature of CPAs addition. Regardless of the CPA used, statistically significant variations (P < 0.05) were found between the two temperatures of addition of CPAs for acrosome integrity, the best being 22 degrees C (NAR = 83.8% vs. 69.8%). These data indicate that sperm quality of red deer epididymal spermatozoa, in addition to be affected by the cryoprotectant, can also be influenced by the temperature at which CPAs are added prior to freezing.