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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.

Human–wildlife conflicts are a growing problem in Northern Hemisphere where wild ungulates are one of the taxonomic groups most frequently involved. To mitigate these conflicts, it is essential to develop preventive actions able to avoid encounters between wildlife and human (activities). We here employed photo-trapping to evaluate the behaviour of red deer (Cervus elaphus) when confronted with dissuasive portable deterrents that function on the basis of changing patterns of light and ultrasound. This was done by following a before/after experimental design, with two phases: (i) a test phase, with active deterrents, and (ii) a control phase, without deterrents. When deterrents were activated they achieved a 48.96% reduction in the frequency of use by red deer (up to 66.64% when it was assessed on a thinner Sect. 10 m wide from the line of deterrents) and produced a reduction of 67.71% in the frequency of deterrent-line crossings. However, a habituation effect was detected since the use by red deer of the treatment area increased as time since treatment. These results indicate that these portable devices are effective as regards dissuading deer, mainly on short time scales. The deterrents tested here could be suitable for use at focal points for short periods or in combination with other methods to improve their effectiveness in vulnerability points. This device could potentially be used to mitigate conflicts caused by wildlife species and in response to relevant and timely situations, such as vehicle collisions and damage to crops, among others., Open Access funding provided thanks to the CRUE-CSIC agreement with Springer Nature., This research was funded by VISEVER S.L. E. Laguna is supported by a predoctoral grant (Programa Talento Formación) funded by the European Social Fund (ESF) and the Castilla-La Mancha regional government (JCCM) (ref: SBPLY/16/180501/000269). P. Palencia is supported by MINECO (FPU16/00039). AJ. Carpio is supported by a ‘Juan de la Cierva’ contract (FJCI-2017–33114) from MINECO-UCLM. C. Herraiz is supported by ESF and JCCM (ref: SUPLY/19/180501/000487).

4 Pág. Departamento de Reproducción animal AGL2017‐89017‐R, 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., This work was supported by the Ministry of Economy and Competitiveness (.AGL2017‐89017‐R)PP-F was supported by a University of Castilla-La Mancha scholarship

This article belongs to the Special Issue Factors Affecting In Vitro Assessment of Sperm Quality., 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., This research was supported by the Spanish Ministry of Economy and Competitiveness (AGL2013-48421-R and AGL2017-89017-R). A.M.M. was supported by a Ministry of Economy and Competitiveness scholarship and P.P.-F. was supported by a University of Castilla-La Mancha scholarship [2015/4062].

Se optimizo un protocolo de criopreservacion de esperma de morueco mediante ensayos que implican diferentes velocidades de enfriamiento a traves de un congelador biologico programable. Noventa y ocho eyaculados de semen de doce moruecos Merino adultos fueron criopreservados usando ya sea Protocolo 1 (velocidad de enfriamiento de desaceleracion en tres pasos) de 5 a −35 ° C (40 ° C / min), de −35 a −65 ° C (17 ° C / min), y luego de -65 a -85 ° C (3 ° C / min); Protocolo 2 (velocidad de enfriamiento acelerado de tres pasos), de 5 a −5 ° C (4 ° C / min), de −5 a −110 ° C (25 ° C / min), y luego de −110 a −140 ° C (35 ° C / min); o Protocolo 3 (velocidad de enfriamiento acelerado en dos pasos), de 5 a −10 ° C (5 ° C / min), y luego de −10 a −130 ° C (60 ° C / min). Los resultados mostraron que el Protocolo 3 produjo mayores valores de motilidad espermatica despues de la descongelacion que el Protocolo 2 (p

COVID‐BCG Collaborative Working Group: Consuelo Almazán, Isabel G. Fernández de Mera, Margarita Villar, Marinela Contreras, Elisa Ferreras‐Colino, Jānis Ancāns, Alejandro Cabezas‐Cruz, José M. Urra, Carmen M. Cabrera, Francisco J. Rodríguez del Río, Lorena Torres, Andrei Daniel Mihalca, Fredrick Kabi, Dibesh Karmacharya, Espiridión Ramos, Vidal Montoro, Katherine M. Kocan, Edmour F. Blouin, Hugo Fragoso, Sergio Bermúdez, Agustín Estrada‐Peña, Luis M. Hernández‐Triana, Michalis Kotsyfakis, Libor Grubhoffer, Gabriela de la Fuente, Karelia Deulofeu, Leticia Deulofeu, Anna Zelinska‐Fedorovych, Zorica Zivkovic, Ana Domingos, Anna Papa, Ard M. Nijhof, Mónica Florin‐Christensen, Lucas Domínguez, Mercedes Domínguez, Juan Mosqueda, Octavio Merino, Ala E. Tabor, Albert Mulenga, Ernesto Doncel‐Pérez, Gervacio H. Bechara, Srikant Ghosh, Alessandra Torina, Guido Sireci, Rodrigo Nova, Joao Queirós, Current results do not provide conclusive evidence on the effect of BCG vaccination on COVID‐19 alone or in combination with other factors. To address this limitation, in this study we used a citizen science initiative on the COVID‐19 pandemic to collect data worldwide during 2 October 2020–30 October 2020 (1,233 individuals) in a structured way for analysing factors and characteristics of affected individuals in relation to BCG vaccination. For the first time, the results of our study suggested that vaccination with BCG may increase the risk for COVID‐19 at certain age, particularly in individuals vaccinated at childhood. Childhood BCG vaccination increased the likelihood of being diagnosed with COVID‐19 fivefold in COVID‐19 low‐incidence countries and threefold in high‐incidence countries. A reasonable explanation for this effect is the activation of certain innate immunity mechanisms associated with inflammatory reactions. These factors should be considered when analysing the risks associated with this global pandemic., The authors thank responders to the survey and colleagues and rela-tives who assisted in the distribution of the survey- associated link. We thank members of our laboratories and collaborators for fruitful discussions and acknowledge support of the University of Castilla La Mancha (UCLM, Spain) to COVID- 19- related studies

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., This research was funded by the Ministerio de Economía y Competitividad (MINECO; AEI/FEDER, UE; AGL2016-76358-R). S.J. is co-supported by the UCLM and the European Social Fund (2018/12504). P.P. received support from the MINECO (FPU/16/00039).

The interfaces among different compartments have been spaces of transition and contact where pathogens have adapted to new hosts. The domestication and establishment of local settlements modelled multiple wildlife–livestock–human interfaces, characterized by the maintenance of “crowd diseases” (both by humans and/or by animals) and the continuous presence of a domestic pathogen reservoir. Through the course of human history, our impact on the natural environment has increasingly magnified, including our ability to alter, shape, and reshape nature at different rates, often varying by epochs, civilizations, and regions. Human expansion, technical development, trade, population growth, and growing interdependence of the global economy have both increased livestock spatial ranges and opportunities for animal pathogens to cross geographic boundaries. Correspondingly, there is a greater risk of disease re-emergence and potential spillback into wildlife populations that may act as disease reservoirs, which has determined the characteristics of wildlife–livestock interfaces and subsequent opportunities for pathogen transfer that exist today. The wildlife–livestock–human interface has also intensified as a result of human encroachment into historically wilderness areas, especially in tropical ecosystems. Thus, the wildlife–livestock interface has advanced to become more interconnected than ever as humans and animals (domestic and wild) increasingly share a common global infectious disease ecology (i.e., the wildlife–livestock interface is becoming global). However, the severity of disease related human–wildlife conflicts has a complex nature. While wildlife is becoming less abundant and more fragmented, wildlife–livestock interfaces have become more frequent and interconnected. For example, although there are catastrophic wild species extinctions in certain regions (e.g., tropical forests), we also observe the recovery or expansion of some wildlife populations over expansive areas that are associated with increased sanitary risks at the human–livestock interface (e.g., ungulates in temperate regions of the Northern hemisphere). Therefore, mitigating disease risk at wildlife–livestock–human interfaces must be a crucial component of modern wildlife management and it is essential to understand local, regional, and international social, economic, and cultural systems to better focus strategic disease control programs. Integral population and disease monitoring are also necessary to improve knowledge of the role of wildlife and the mode of transmission and emergence of new infections. Climate and associated anthropogenic and environmental change are impacting the distribution, abundance, behavior, and population dynamics of pathogens and hosts, threatening human health, and livelihoods. The current context urgently requires a better understanding of global warming impacts on the wildlife–livestock–human interface, particularly in complex host/pathogen/vector systems. Transdisciplinary integrative approaches that enable countries and international organizations to better target shared disease control programs and move toward disease eradication, which includes strategic research in collaboration with the medical sector, will be required to ensure decisions are scientifically informed for current challenges in the interest of humanity.

This article belongs to the Special Issue Wildlife Diseases., 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., This research was funded by the Ministerio de Economía y Competitividad (MINECO; AEI/FEDER, UE; AGL2016-76358-R). S.J. is co-supported by the UCLM and the European Social Fund (2018/12504). J.C. received support from the Ministerio de Ciencia, Innovación y Universidades (FPU/17/01319). A.R.J. is the recipient of a Miguel Servet Research Contract by the Ministerio de Ciencia, Promoción y Universidades of Spain (CP18/00111).

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., The present work has benefitted from the financial aid of the Spanish Ministry of Economy and Competitiveness (MINECO) research grants Ref. AGL2013‐49159‐C2‐2‐R and AGL2016‑76358‑R; and from the research grant by the Regional Ministry of Economy and Infrastructure of the Board of Extremadura Ref. TB‐PORCEX. RTO received support from UCLM through a pre‑doctoral scholarship and from the Community of Madrid through a post‐doctoral contract (PEJD‐2019‐POST/BIO‐16805). SJR holds a predoctoral contract from UCLM. EL is supported by a JCCM predoctoral contract. JAB is supported by the EU project Ref. H2020 VACDIVA 862874. PA received support from UCLM through an extension of Ramón y Cajal contract Ref. RYC‐2012‐11970.

This article belongs to the Special Issue Reproductive Biotechnology in Wildlife., [Simple Summary]: The development of in vitro embryo production (IVP) in wild species, such as Iberian red deer, can become a daunting challenge since prolonged ovary transport times to the laboratory are often unavoidable. This may have detrimental effects on the quality and developmental capacity of oocytes. We evaluated the effect of supplementing the ovary transport medium with the antioxidant melatonin and observed an increased level of oocyte intracellular reduced glutathione content. Moreover, melatonin enhanced cleavage and blastocyst rates and had a positive effect on embryo quality in terms of the expression of essential embryo development-related genes. In conclusion, the addition of melatonin to the ovary storage medium could mitigate the negative impacts that long transport times may have on oocyte developmental competence and quality of the resulting blastocysts in Iberian red deer., 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., This research was funded by the Spanish Ministry of Economy and Competitiveness (AGL2017-89017-R) and Regional Government (SBPLY/17/180501/000500). M.I.-C. and A.M.-M. were supported by a Ministry of Economy and Competitiveness scholarship. P.P.-F. and D.-A.M.-C. were supported by a University of Castilla-La Mancha scholarship.

This article belongs to the Special Issue Advances in Molecular Regulation of Spermatozoa Function., 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., Spanish Ministry of Economy and Competitiveness (AGL2016-81905-REDT). MIC and AMM were supported by a Ministry of Economy and Competitiveness scholarship and PPF was supported by a University of Castilla-La Mancha scholarship.

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 H2O2/800 μM FeSO4•7H2O) 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., This work has been supported by the Spanish Ministry of Economy, Industry and Competitiveness, MINECO (AGL2017‐85603‐P) and by the Education and Science Council of Junta de Comunidades de Castilla‐La Mancha (SBPLY/17/180501/000369).

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., RC-G had a JCCM fellowship during the study period. The present work has benefited from financial aid from the following research grants: Ministerio de Economía y Competitividad (MINECO; AGL2016-76358-R).

Reactive oxygen species (ROS) are fundamental for intracellular signalling. In spermatozoa, they are involved both to apoptosis and to capacitation, and changes in ROS levels can alter the balance between these two processes. Oestrous sheep serum (OSS) is considered an efficient agent for in vitro capacitation of ram spermatozoa. We have explored the effects of OSS on ram sperm physiology, especially on ROS production, during in vitro capacitation. Semen samples from 15 rams were cryopreserved. After thawing, samples were submitted to four treatments: control (CTL), 10% OSS supplementation for in vitro sperm capacitation, caspase inhibitor (INH, Z-VAD-FMK 100 μM) and OSS (10%) plus caspase inhibitor (I + E). Sperm samples were incubated for 30 min at 38.5°C and 5% CO and evaluated motility and kinetic parameters by computer-assisted semen analysis (CASA) and viability (propidium iodide), apoptotic-like membrane changes (YO-PRO-1), acrosomal status (PNA-FITC), intracellular calcium (FLUO-3), membrane fluidity (M540) and ROS production (CM-HDCFDA) by flow cytometry. OSS induced changes in kinetic parameters compatible with capacitation, with a decrease in the percentage of progressive motility and linearity, and an increase in the amplitude of the lateral displacement of the sperm head (p, This research was supported by Education and Science Council (JCCM), Spain, grant (PAI09-0006-3806). Enrique del Olmo is supported by a grant for training of technical personnel associated with PTA2008-0858-P project (Spanih ministry of science and Innovation, MICINN).

In a system based on a test-day alternate recording, a total of 1,227,949 test-day records for somatic cell counts (SCC) and milk yield obtained throughout 12 years (1998-2009) from 196,496 ewes in 156 Manchega flocks belonging to the National Association of Manchega Breeders (AGRAMA, Spain) were used to study the environmental variations of the SCC and its relationship with milk yield. Two subsets containing test-day SCC and milk yield data were also analyzed: SCC≤300×10 3cells/ml, and SCC>300×10 3cells/ml.Analysis of variance showed significant effects for the ewe, season, age of ewe, lactation stage, number of lambs born, milking shift, and for the flock×year random effect on variables studied. Average values obtained for somatic cell score (SCS) and milk yield were 4.09 and 1156ml/d, respectively. The SCS increased 7.6% through lactation and was highest in winter. The age of ewe effect was highly significant for SCS and milk yield, both increasing with the age, even for the group of ewes with a SCC≤300×10 3cells/ml. Twin-birth ewes showed higher SCS (5.0%) and milk yield (9.8%) than single-birth ewes. SCS for p.m. milking was significantly higher than for the a.m. milking. Phenotypic correlation between milk yield and SCS was negative (-0.09), which reflects the reduction in the production ability of ewes with higher SCS. Average differences in milk yield between test-day subsets studied were 11.8%; the greatest differences were in autumn (14.5%) and the lowest in spring (9.8%). Differences in milk yield between the ewes with SCC≤300×10 3cells/ml and ewes with SCC>300×10 3cells/ml also increased through lactation. Thus, in the first test-day record differences in milk yield reached 5.4%, and the difference increased up to 11.8% in the 4th test-day record. Our results suggest that it seems necessary to take into account the effect of non-infectious factors to interpret the SCC results of recording systems.

The aim of the present study was to evaluate the effect of selecting a sperm subpopulation by means of a discontinuous density gradient centrifugation (DGC) on the quality of ram thawed semen, and the relationships between sperm parameters assessed in unselected and in selected sperm samples with in vivo fertility after intrauterine artificial insemination (IUI) using unselected sperm samples. Semen samples from twenty males were collected by artificial vagina and cryopreserved following a standard protocol. After thawing, unselected sperm samples were used in an in vivo fertility trial and sperm motility (subjective and objective, assessed by means of CASA) and membrane and acrosomal integrities (microscopy) were evaluated on unselected and selected sperm samples. In addition, plasmalemma integrity (YO-PRO-1/PI), membrane fluidity (Merocyanine 540/YO-PRO-1), mitochondrial activity (Mitotracker Deep Red/YO-PRO-1), and DNA fragmentation index (%DFI) assessed by Sperm Chromatin Structure Assay (SCSA®) were evaluated by flow cytometry before and after sperm processing using DGC. Results showed that DGC improved all sperm parameters significantly, except the %DFI, which increased after the selection procedure. No relationships were found between sperm parameters evaluated in unselected sperm samples and in vivo fertility. However, we found a positive correlation between spermatozoa with high membrane fluidity within the viable sperm population (VIABMerocyanine+) evaluated in selected sperm samples and in vivo fertility (r = 0.370, P = 0.019). In conclusion, our results suggest that selected spermatozoa represent a sperm subpopulation different to the unselected one that could be related with the in vivo fertility., This work was supported by Spanish Ministry of Education and Science (RTA2005-00091-CO2) and by Education and Science Council of Junta de Comunidades de Castilla-La Mancha (PCC08-0105). Olga García-Álvarez and Pilar Jiménez-Rabadán were recipients of scholarships from INIA and Alejandro Maroto-Morales from JCCM.

Computer-assisted sperm morphometry analysis (CASMA) was used in this study to identify sperm morphometric subpopulations in Iberian red deer epididymal sperm samples. Epididymal sperm samples were collected from 37 mature stags and were divided. One portion was diluted in a Tris–citrate–egg yolk medium. A microscope slide was prepared from single extended sperm samples prior to freezing. The remainder of each sample was frozen in nitrogen vapours using a conventional protocol. After thawing, sperm smears were prepared as described for extended samples. All slides were air-dried and stained with Hemacolor®. The sperm-head dimensions for a minimum of 145 sperm-heads were analyzed from each sample by means of the Sperm-Class Analyser®, and the mean measurements recorded. Each sperm-head was measured for four primary sperm-head parameters, and five parameters of head shape. All sperm morphometric parameters evaluated were placed in a statistical database and a multivariate cluster analysis was performed. The clustering analyses, based on 10 867 individual spermatozoa, revealed the existence of three subpopulations (SP1, SP2, SP3) of spermatozoa with different morphometric characteristics (p 0.05) through the cryopreservation process. Pre-freeze and post-thaw sperm quality was in vitro evaluated by microscopic assessments of individual sperm motility and of plasma membrane and acrosome integrities. In conclusion, our results show that applying the CASMA techniques and multivariate cluster analyses, it was possible to determine that three subtle subpopulations of spermatozoa with different morphometric characteristics coexist in red deer semen., This study was sponsored by Grant AGL-2007-60271/GAN from the Spanish Ministry of Education and Science. Felipe Martínez-Pastor was supported by the Juan de la Cierva programme from Spanish Ministry of Education and Science.

Semen collection for cryopreservation is a key step for small ruminant conservation programs. While in these species semen is mainly collected via artificial vagina (AV), electroejaculation (EE) provides a viable alternative for untrained males. Herein we investigated the effect of semen collection method on post-thaw sperm quality by comparing two small ruminant species, sheep and goats. Semen from Blanca-Celtibérica bucks and Manchega rams was collected by AV and EE on the same day and cryopreserved using a standard protocol. At thawing, sperm motion parameters were evaluated by CASA, whereas membrane stability (YO-PRO-1), sperm viability (propidium iodide, PI) and mitochondrial activity (Mitotracker Deep Red) were analyzed using flow cytometry. The semen collection method negatively affected post-thaw sperm quality in bucks but not in rams. Thus, in bucks, post-thaw sperm motility was higher for samples collected by AV as compared to those obtained via EE. Similarly, post-thaw sperm parameters evaluated by flow cytometry were worse for buck samples collected by EE than those collected by AV in the same species, or than ram samples regardless of collection method. These results suggest that ovine and caprine spermatozoa have a different response to the cryopreservation process depending upon the semen collection method used. We hypothesize that the EE procedure may lead to changes in the composition of the ejaculate in bucks that would make spermatozoa more susceptible to the cryopreservation process, whereas this procedure would have had no effect on ram spermatozoa. This assumption requires further investigation., This research was supported by INIA (RZ2008-00009-CO2). The authors thank AGRACE, their farmers and CERSYRA staff for their collaboration in the collection of the samples. Manuel Ramón is supported by the DOC-INIA program.

The interactions of extensively farmed livestock with wild ungulates contribute to the transmission and maintenance of, among other diseases, tuberculosis (TB) at the interface in South Central Spain (SCS), where farming typically occurs close to wildlife habitats. The aim of this paper is to describe (i) the use of extensive farm resources by wildlife and livestock and (ii) the factors involved. Camera traps (CTs) were placed at a priori risk points (n = 149 points, 2145 CT days) for interspecies interactions: water points, buildings, food points, and acorn fields (which also provide rich pasture), on 11 representative extensive beef cattle (n = 6) and Iberian pig (n = 5) farms. The use of extensive farm resources by wild ungulates was frequent and widespread throughout the study area (over 50 % of daily presence per farm of either wild boar Sus scrofa or red deer Cervus elaphus). This presence increased when there was a high abundance of wild ungulates on nearby hunting estates. No direct interactions were detected, and livestock and wild ungulates partitioned resource use temporally, signifying that the interaction was mainly indirect. The wild species that used all farm resources with by far the most frequency was red deer, although its presence decreased markedly when far from woodland edges. The presence of cattle was positively associated with that of wild boar at all the CTs and specifically with red deer at water points and in acorn fields. Pig presence was negatively associated with that of wild boar, suggesting the existence of competence-mediated avoidance. We propose that interactions at the wildlife-livestock interfaces could be reduced by carrying out specific actions adapted to Mediterranean conditions and farm management: removing and segregating access to risky water points, the wise management of grazing in space and time, and protecting food-rich pasture plots and woodland edges by means of fencing, which is particularly interesting as regards attempting to prevent red deer from accessing areas occupied by livestock and vice versa. Biosecurity programs must be farm-specific, and the effectiveness, costs, and practical value of interventions should be adaptively evaluated in the field in future research., The present work was supported by Ministerio de Economía y Competitividad research grant Ref. AGL2013-48523-C3-1-R and “ASFORCE,” European Union’s Seventh Framework Programme (ref. FP7 - KBBE.2012.1.3-02). Ricardo Carrasco-Garcia had a JCCM fellowship during the study period.

Although there is ample evidence for the effects of sperm head shape on sperm function, its impact on fertility has not been explored in detail at the intraspecific level in mammals. Here, we assess the relationship between sperm head shape and male fertility in a large-scale study in Manchega sheep (Ovis aries), which have not undergone any selection for fertility. Semen was collected from 83 mature rams, and before insemination, head shapes were measured for five parameters: area, perimeter, length, width, and p2a (perimeter2/2×π×area) using a computer-assisted sperm morphometric analysis. In addition, a cluster analysis using sperm head length and p2a factor was performed to determine sperm subpopulations (SPs) structure. Our results show the existence of four sperm SPs, which present different sperm head phenotype: SP1 (large and round), SP2 (short and elongated), SP3 (shortest and round), and SP4 (large and the most elongated). No relationships were found between males' fertility rates and average values of sperm head dimensions. However, differences in fertility rates between rams were strongly associated to the proportion of spermatozoa in an ejaculate SP with short and elongated heads (P < 0.001). These findings show how the heterogeneity in sperm head shape of the ejaculate has an effect on reproductive success, and highlight the important role of modulation of the ejaculate at the intraspecific level.

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. © 2005 Elsevier Inc. All rights reserved., Fran Ruiz-Fons has a grant of the “Ministerio de Educación y Ciencia”. The study was supported by projects AGL2001-3947, Ministerio de Ciencia y Tecnología and FEDER and PREG-04-004, “Junta de Comunidades de Castilla-La Mancha”. This is a contribution to the agreements between Yolanda Fierro and UCLM, and between CSIC and Principado de Asturias.

[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., Carlos Martín, Universidad de Zaragoza, kindly provided the vaccine BCG strain. Many colleagues at SaBio IREC, NEIKER and VISAVET granted valuable technical help. This is a contribution to EU FP7 grant 613779 WildTBVac and to MINECO Plan Nacional grant AGL2011-30041 and FEDER.

Tuberculosis (TB), a chronic disease caused by infection with the Mycobacterium tuberculosis complex, is endemic in wild boar (Sus scrofa) and red deer (Cervus elaphus) in south-central Spain. Understanding the temporal dynamics of this chronic infection requires long time series data collection over large areas. The aim of this paper was to identify the determinants of TB prevalence and severity in both species in Ciudad Real province, Spain, from 2000 to 2012. Study variables included management, population dynamics, and a range of geographical and climatological factors. The prevalence of TB in wild boar increased from 50% to 63% since the study commenced. This may be due to an increased hunting bag (a proxy for population abundance), which was correlated with TB infection rates. Low rainfall (a stochastic factor) was associated with higher individual risk of TB presence and progression, resulting in an increased proportion of severe cases of wild boar TB in dry years. This was probably a result of increased food restriction leading to a higher susceptibility to TB. In contrast, red deer TB showed an apparent stable trend, which may be a consequence of the species' higher and stable population size. Hunting management, characterized by fencing, was associated with a higher risk of TB in both wild boar and red deer, suggesting that intensive hunting management may have contributed to exacerbated TB figures. This difference was more marked in red deer than in wild boar, probably because fencing imposes less restriction on movement, population mixing and TB spread to wild boar than to deer. Our findings on TB dynamics are fundamental for assessing the impact of future disease-control actions (e.g. field vaccination). Moreover, such control plans must operate in the long term and cover large areas., This study was funded by the MINECO research project AGL2010-20730-C02-01 and JCCM PPEII 10-0262-7673.This is also a contribution to EU FP7 EMIDA (ERA-NET) Grant APHAEA on wildlife disease surveillance in Europeand to FP7 Grants ANTIGONE (project number 278976) and WildTBVac. Jose Angel Barasona enjoyed a scholarshipfrom JCCM and a contract through MINECO Grant AGL2011-30041. We thank the Agriculture Department of JCCM and its Delegation in Ciudad Real for providing data on hunting statistics and sanitary campaigns. Pelayo Acevedo was supported by a Beatriu de Pinòs fellowship funded y ‘Comissionat per a Universitats i Recerca del Departament d’Innovaciò, Universitats i Empresa’ of the Generalitat de Catalunya, and the COFUND Programme–MarieCurie Actions under 7th Framework Programme of the European Community. He is currently funded from the SFRH/BPD/90320/2012 post-doctoral grant provided bythe Portuguese Fundaçao para a Ciencia e a Tecnologia (FCT) and the European Social Fund.

In south-central Spain, the harvest of Eurasian wild boar (Sus scrofa) has increased significantly during recent decades in association with more intensive management actions to increase hunting yields and with consequent effects on the health status of the wild boar populations. We investigated the spatio-temporal trends and the risk factors related to the prevalence of Trichinella spp. in wild boar in order to obtain the annual probability of occurrence for these parasites in the Ciudad Real province of south-central Spain. Based on muscle samples collected during the hunting seasons from 1998/1999 to 2009/2010, the mean prevalence for Trichinella spp. in 95,070 wild boar was 0.2% (95% confidence interval 0.17-0.23). A subsample of 1,432 wild boar was also tested by ELISA. No correlation was observed between the prevalence of infection detected by serology and by the artificial digestion of muscle. The presence of Trichinella infections in wild boar showed a decreasing trend during the study period and was negatively related with fenced wild boar populations. The predicted 'favourability' for Trichinella infections disappeared almost completely after the 2006/2007 hunting season. Risk maps based on biogeographical tools showed, however, that most hunting estates presented favourable risk factors for these parasites during at least one of the hunting seasons studied., This is a contribution to JCCM PPIC10-0226-0243 (Spain) and to the EU EMIDA ERA-NET Grant APHAEA and the Spanish FAU2008-00004-C03 INIA. Studies on diseases shared between domestic animals and wildlife in Castilla – La Mancha are supported by Dirección General de Agricultura y Ganadería (Spain), facilitating access to official data and through the CLM Wildlife Disease Surveillance Scheme. P.A. was supported by the Vicerrectorado de Investigación of the University of Malaga and partially by the project CGL2009-11316/BOS (Spain) – Fondo Social Europeo (European Union).

Big game hunting has been anciently practiced by the humanity as an essential event to survival (Nitecki et al. 1988), and although nowadays man continues to hunt meat, big hunting has expanded to sport. Species included as big game vary with geographical areas and the general range includes mainly medium to large size hoofed ungulates and predators. Such diverse group of species plays essential roles in the ecological dynamics of natural or semi-natural systems (e.g. Putman 1986). Also big game species can often cause conflict with human interests, for example abundant ungulate species damaging to agriculture and conservation habitats or transmitting disease to livestock (e.g. Ammer 1996, Ferroglio et al. 2011). Big game waste consists of solids generated mainly after hunting activity. This waste comprises the whole body of the animals or parts of them, such as the viscera and heads (when are not used as trophies). Much of this ungulate biomass becomes solid waste after hunting events following the removal of the internal and external offal (head, feet, all intestines as well as all internal organs). These materials play an important role in ecosystems, maintaining complex faunal communities extending from invertebrates to large carnivores (DeVault et al. 2003). Therefore a premise is that big game waste must be properly managed to protect the continuation of natural ecological processes (for example just leaving it in situ may be an option), which poses great conservation value. These remains may also be managed to reduce their effect on animal/human health and the environment under certain circumstances, and to a lesser extent, for aesthetics purposes or to recover resources from them. Although there is increasing interest on the ecological role of these materials, the hunting generation and impacts of big game waste has received little attention compared with management of other solid waste. In some contexts this waste may potentially be hazardous for the animal community (including species of high conservation interest), the livestock or the general public. This situation specially arises as a consequence of human interventions in habitats and the natural regulation of wildlife populations (Gortazar et al. 2006). Changes in population density and/or wild host behavior through solid waste may help new pathogens; new hosts or new hazards emerge, favouring disease spread and maintenance. Here we compile existing information on big game waste generation, ecological value, problems and management options under current regulations, remarking the sanitary and environment conservation dilemmas when managing this waste.

We have carried out a study on the influence of prolonged cold storage (5 °C) of Iberian red deer epididymides on post-thaw sperm motility and DNA integrity. Twenty-nine pairs of testes, with attached epididymides, were collected during November and December. Spermatozoa from one of each of the pairs were immediately recovered, evaluated and frozen (control). The remaining epididymides were cooled to 5 °C and stored for 24, 96 and 192 h (experimental groups), after which spermatozoa were collected and frozen. Samples were evaluated before freezing, after thawing, and after a 2-h period of incubation at 37 °C. Motility was evaluated by means of a CASA system and chromatin stability was assessed following the Sperm Chromatin Structure Assay (SCSA). Our results showed that, during the first 96 h, the motility (total and progressive) did not significantly decline when assessed after cryopreservation, although there was a significant decline when epididymides had been stored for 192 h at 5 °C (P < 0.001). The present study demonstrates that motility and DNA status of thawed spermatozoa collected from refrigerated epididymes, at least 96 h post-mortem, were good enough to consider their eventual use. Most importantly, sperm DNA integrity after thawing was apparently not affected by storage time, even after 192 h., This work has been supported by the Spanish Ministry of Education and Science (Project AGL2004-05904/GAN). Felipe Martínez-Pastor was supported by the Juan de la Cierva program (Spanish Ministry of Education and Science).

An Aujeszky’s disease (AD) control and eradication campaign started in Spain in 1995, and as a result, AD virus (ADV) seroprevalences have notably diminished in Spanish domestic pig herds, but eradication has not yet been achieved. Since the presence of ADV in Spanish wild boar populations can impede schemes to eradicate ADV in domestic pig, we conducted analyses of risk factors and investigated associations between the patterns of ADV seroprevalence at the municipal level in the wild boar and the domestic pig, respectively, in south-central Spain. There was a clear influence of individual factors on the risk of wild boar to test positive to ADV. Concerning the domestic pig, higher ADV seroprevalences were observed with increasing numbers of pigs per farm. Indoor pig farms showed higher seroprevalences than open-air farms. Our results show that, at least at the study scale, the level of ADV seroprevalence in domestic pig farms is not influenced by the level of ADV seroprevalence in the coexisting wild boar populations. Also, there was no influence of domestic pigs ADV seroprevalence on the risk of wild boars to test seropositive. Finally, there was no evidence for a statistical association between ADV seroprevalence levels in wild boar and domestic pig at the municipal level in the study region. These findings are of interest for both veterinary and public health authorities. Nevertheless, more effort is needed in order to elucidate the nature of particular ADV outbreaks in domestic pigs in the study region due to possible contacts with wild boars, which will probably require a molecular approach., This is a contribution to the contract on ADV epidemiology by “JCCM-Consejería de Agricultura”, to project AGL2005-07401-C03-01 (MEC) and to the agreements between MAPA, Organismo Autónomo de Parques Nacionales (OAPN) and CSIC and between Yolanda Fierro and UCLM.

Computer-automated sperm-head morphometry was used in this study to determine the effects of cryopreservation on red deer sperm-head morphometry. Epididymal sperm samples were collected from 40 mature stags and were divided. One portion was diluted at room temperature in a Tris-citrate egg yolk medium, containing 6% glycerol. A microscope slide was prepared from single extended sperm samples prior to freezing. The remainder of each sample was frozen in nitrogen vapours. After thawing, sperm smears were prepared as described above. All slides were air dried and stained with Hemacolor. The sperm-head dimensions for length, width, area, perimeter and shape factor (length/width), for a minimum of 135 spermatozoa were determined for each slide by means of the Sperm-Class Analyser (SCA). Firstly, our results show that cryopreservation substantially reduced (p < 0.001) sperm motility and plasma membrane and acrosome integrities. In addition, sperm heads were significantly smaller in cryopreserved spermatozoa than in the companion extended samples for area (32.05 microm2 vs 32.56 microm2; p < 0.05), length (8.46 microm vs 8.53 microm; p < 0.0001) and shape factor (1.833 vs 1.849; p < 0.0001) for all stags. These differences were found within 29 of 40 stags (75%) for at least three of the morphometric parameters. The individual variability (CV) of sperm head measurements from extended samples was negatively correlated (p < 0.005) with the per cent of change in sperm head measurements after cryopreservation for area (r = -0.465), width (r = -0.483) and perimeter (r = -0.375). Thus, the lower the sperm head variability in the extended samples, the greater the sperm change as a consequence of the cryopreservation. These results suggest that the variability (heterogeneity) in sperm head dimensions of individual stags may be a good indicator of sperm freezability., This study was sponsored by Grant PBC-02–011 from the Consejería de Ciencia y Tecnología de la Junta de Comunidades de Castilla-La Mancha (JCCM), Spain. A. Quintero-Moreno was sponsored by research funds of the International Cooperation Office from the UCLM (Spain).

Egg yolk is a common component to sperm refrigeration for most of the deer species, the role of which is to protect sperm membranes against cold shock. In addition, there have been many studies of conservation of ejaculated semen from stags, but few have been reported for epididymal spermatozoa. This work was designed to investigate the combined effects of cooling rates (slow: 0.23°C/min vs rapid: 4.2°C/min) from room temperature to 5°C, and egg-yolk concentration (0, 5 or 20%) in the extender on the survival of Iberian red deer epididymal spermatozoa refrigerated at 5°C. Heterospermic sperm samples were diluted to a final sperm concentration ∼400 × 106 sperm/ml with a Tris–citrate-fructose (TCF)-egg-yolk diluent. Sperm quality was in vitro judged by microscopic assessments of individual sperm motility [sperm motility index (SMI)], and of plasma membrane (hypo-osmotic swelling test) and acrosome (NAR) integrities. Our results first showed that the presence of egg yolk in the extender significantly improves (p = 0.01) the viability and sperm motility after sperm dilution. In addition, acrosome and plasma membrane integrities post-refrigeration did not differ significantly between cooling procedures; however, the SMI differed significantly between cooling procedures (slow: 46.6% vs rapid: 50.0%; p = 0.01). Our results also showed that sperm quality was significantly (p, This study was sponsored by Grant AGL2000-0671 from the Spanish Ministry of Science and Technology. Ana J. Soler and M. R. Fernández-Santos were recipients of scholarships from the Consejería de Ciencia y Tecnología de la Junta de Comunidades de Castilla-La Mancha (Spain).

Two experiments were conducted to determine the effects of egg yolk (EY), glycerol, and cooling rate on the cryosurvival of red deer epididymal spermatozoa. The aim of Experiment 1 was to examine the effects of two EY types (clarified EY, CE, prepared by centrifugation, and whole EY, WE), and four EY concentrations (0, 5, 10 and 20%) on cryosurvival of red deer epididymal spermatozoa. Sperm samples were diluted to a final sperm concentration of ∼200 × 106 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, viability and of plasma membrane (by means of the HOS test) and acrosome (NAR) integrities. Cryopreservation of red deer epididymal spermatozoa frozen in a clarified EY extender, and with a 20% EY resulted in more vigorous post-thaw and post-incubation motilities (P < 0.0001). Moreover, our results showed that regardless of the egg yolk concentration tested, the best sperm quality was obtained with the use of CE. Therefore, the objective of Experiment 2 was to explore the post-thaw effects of four clarified egg yolk concentrations (0, 5, 10 and 20%), two final glycerol concentrations (3 and 6%), and two cooling rates from 22 to 5 °C (slow: 0.23 °C/min; rapid: 4.2 °C/min) on red deer epididymal spermatozoa. At thawing, the effects of CE and glycerol concentrations, and cooling rate, all independently affected post-thaw sperm quality, while there were no effects of interactions on post-thawing sperm quality. Therefore, we studied each variable separately. Differences (P < 0.05) for most of the semen parameters evaluated were found between the two final glycerol concentrations tested, with the high values after thawing found with the use of 6% glycerol (58.8 ± 1.4 versus 46.2 ± 1.4, for sperm motility). Moreover, the cooling rate did not have an effect on the semen characteristics, except for NAR (P < 0.05), with the high values after thawing found with the use of the rapid protocol (64.5 ± 1.4 versus 59.9 ± 1.4). In conclusion, the use of 20% CE and 6% glycerol in combination with a rapid cooling rate, significantly improved red deer epididymal spermatozoa freezability., This study was sponsored by Grant AGL2000-0671 from the Spanish Ministry of Science and Technology. M.R. Fernández-Santos was recipient of a scholarship from the Consejería de Educación y Ciencia de la Junta de Comunidades de Castilla-La Mancha (Spain).

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 ∼400 × 106 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°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°C (ambient temperature) and 5°C2 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°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°C. In conclusion, our work showed the efficacy of a TCF diluent with 6% of GLY and its addition at 22°C, as an alternative to the more common 3%–4% of GLY and addition at 5°C, in red deer semen freezing protocols., Supported by grant AGL2000–0671 from the Spanish Ministry of Science and Technology.

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 = PGDMSO ('less than' symbol means P0.05, 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 (P0.05) 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.