Wildlife-Livestock Host Community Maintains Simultaneous Epidemiologic Cycles of Mycoplasma conjunctivae in a Mountain Ecosystem.

Infectious keratoconjunctivitis (IKC) is an eye disease caused by Mycoplasma conjunctivae that affects domestic and wild caprines, including Iberian ibex (Capra pyrenaica), a medium-sized mountain ungulate. However, its role in IKC dynamics in multi-host communities has been poorly studied. This study assessed M. conjunctivae in Iberian ibex and seasonally sympatric domestic small ruminants in the Natural Space of Sierra Nevada (NSSN), a mountain habitat in southern Spain. From 2015 to 2017, eye swabs were collected from 147 ibexes (46 subadults, 101 adults) and 169 adult domestic small ruminants (101 sheep, 68 goats). Mycoplasma conjunctivae was investigated through real-time qPCR and statistically assessed according to species, sex, age category, year, period, and area. The lppS gene of M. conjunctivae was sequenced and phylogenetically analysed. Mycoplasma conjunctivae was endemic and asymptomatic in the host community of the NSSN. Three genetic clusters were shared by ibex and livestock, and one was identified only in sheep, although each host species could maintain the infection independently. Naïve subadults maintained endemic infection in Iberian ibex, with an epizootic outbreak in 2017 when the infection spread to adults. Wild ungulates are epidemiologically key in maintaining and spreading IKC and other shared diseases among spatially segregated livestock flocks.

Dynamics of Crimean-Congo hemorrhagic fever virus in two wild ungulate hosts during a disease-induced population collapse.

Identifying the role that host species play in pathogen transmission and maintenance is crucial for disease control, but it is a difficult task, in particular for vector-borne and multi-host pathogens, and especially when wildlife species are involved. This is the case for a Crimean-Congo hemorrhagic fever virus (CCHFV) hotspot in north-eastern Spain, where Iberian ibex (Capra pyrenaica) and wild boar (Sus scrofa) are involved, but their roles in disease transmission are unclear. In this context, we studied the dynamics of CCHFV transmission in these two species during the collapse of an Iberian ibex population due to a sarcoptic mange outbreak. We carried out a repeated cross-sectional study measuring the trends of CCHFV seroprevalence in Iberian ibex and wild boar and their abundances. In addition, we identified the tick species present in this area on the vegetation and on wild boars, and evaluated relevant meteorological factors. Results show that while the trends in CCHFV seroprevalence in Iberian Ibex and density of wild boars remained constant (p = 1.0 and p = 0.8, respectively), both the trends in Iberian ibex census and CCHFV seroprevalence in wild boars decreased significantly (p = 0.003 and p = 0.0001, respectively), and were correlated (Spearman's rank, 0.02 < p-adjusted<0.05). The correlation between the patterns of reduction of Iberian ibex abundance and the decrease of seroprevalence in wild boars suggests some sort of shared transmission cycle between the two species. Data from tick species in the area suggest a possible role of Rhipicephalus bursa in CCHFV transmission. The dynamics of CCHFV were unlikely caused by changes in meteorological variables such as temperature or water vapor pressure deficit. Further studies will be needed to confirm these hypotheses.

The local skin cellular immune response determines the clinical outcome of sarcoptic mange in Iberian ibex (Capra pyrenaica).

Introduction: Sarcoptic mange, caused by Sarcoptes scabiei, is a disease with implications for wildlife conservation and management. Its severity depends on the host's local skin immune response, which is largely unknown in Iberian ibex (Capra pyrenaica), a mountain ungulate dramatically affected by mange. In this species, the clinical outcome of sarcoptic mange varies among individuals, and the local immune response could be key to controlling the infestation. This study aims to characterize the local cellular immune response and its relationship with the clinical outcome. Methods: Fourteen Iberian ibexes were experimentally infested with S. scabiei and six more served as controls. Clinical signs were monitored, and skin biopsies were collected from the withers at 26, 46, and 103 days post-infection (dpi). The presence and distribution of macrophages (including M1 and M2 phenotypes), T lymphocytes, B lymphocytes, plasma cells, and interleukine 10 were quantitatively evaluated using immunohistochemical techniques. Results: An inflammatory infiltrate that decreased significantly from 26 to 103 dpi was observed in all the infested ibexes. The predominant inflammatory cell population in the skin of the mangy ibexes was formed by macrophages (mainly the M2 phenotype) followed by T lymphocytes, with lower numbers of B lymphocytes and plasma cells. Three clinical courses were identified: total recovery, partial recovery, and terminal stage. The inflammatory infiltrates were less pronounced in the fully recovered ibexes than in those that progressed to the terminal stage throughout the study. Discussion: The results suggest an exacerbated but effective Th1-type cellular immune response controlling mange in Iberian ibex. Furthermore, the local immune response appears to determine the variability of the clinical responses to S. scabiei infestation in this species. This first report on the progression of local skin immune cells is relevant not only for individuals but also for population management and conservation.

Diseases of Iberian ibex (Capra pyrenaica).

Iberian ibex (Capra pyrenaica) is an ecologically and economically relevant medium-sized emblematic mountain ungulate. Diseases participate in the population dynamics of the species as a regulating agent, but can also threaten the conservation and viability of vulnerable population units. Moreover, Iberian ibex can also be a carrier or even a reservoir of pathogens shared with domestic animals and/or humans, being therefore a concern for livestock and public health. The objective of this review is to compile the currently available knowledge on (1) diseases of Iberian ibex, presented according to their relevance on the health and demography of free-ranging populations; (2) diseases subjected to heath surveillance plans; (3) other diseases reported in the species; and (4) diseases with particular relevance in captive Iberian ibex populations. The systematic review of all the information on diseases affecting the species unveils unpublished reports, scientific communications in meetings, and scientific articles, allowing the first comprehensive compilation of Iberian ibex diseases. This review identifies the gaps in knowledge regarding pathogenesis, immune response, diagnostic methods, treatment, and management of diseases in Iberian ibex, providing a base for future research. Moreover, this challenges wildlife and livestock disease and wildlife population managers to assess the priorities and policies currently implemented in Iberian ibex health surveillance and monitoring and disease management.

Ivermectin Plasma Concentration in Iberian Ibex (Capra pyrenaica) Following Oral Administration: A Pilot Study.

Sarcoptic mange is considered the main driver of demographic declines occurred in the last decades in Iberian ibex (Capra pyrenaica) populations. Mass treatment campaigns by administration of in-feed acaricides are used as a measure to mitigate the impact of mange in the affected populations. However, there are no data on ivermectin (IVM) pharmacokinetics in this wild caprine, and the treatment through medicated feed is not endorsed by evidence on its effectiveness. The aim of this study is to determine the pharmacokinetic profile of IVM in plasma samples of ibexes after the experimental oral administration of IVM, using high performance liquid chromatography (HPLC) with automated solid phase extraction and fluorescence detection. A dose of 500 µg of IVM per body weight was orally administered in a feed bolus to nine healthy adult ibexes (seven males and two females). Blood samples were collected by jugular venipuncture into heparin-coated tubes at day 1, 2, 3, 4, 7, 10, 15, and 45 post-administration (dpa). The highest plasma concentration of IVM (Cmax = 3.4 ng/ml) was detected 24 h after the oral administration (T1), followed by a rapid decrease during the first week post-administration. Our results reveal that plasma IVM concentration drops drastically within 5 days of ingestion, questioning the effectiveness of a single in-feed dose of this drug to control sarcoptic mange. To the best of our knowledge, this is the first study on plasma availability of oral IVM in ibexes and in any wild ungulate species.

Expression of Aquaglyceroporins in Spermatozoa from Wild Ruminants Is Influenced by Photoperiod and Thyroxine Concentrations.

This work identified the presence of AQPs in frozen-thawed sperm of wild ruminants and assessed the influence of the interaction between photoperiod and thyroxine on AQP expression, and on testosterone secretion. Thyroxine and melatonin were administered to ibexes. In a second experiment, performed in mouflons, circulating thyroxine was reduced via treatment with propylthiouracil (PTU), and an artificial long day (LD) photoperiod established. In the ibexes, the melatonin treatment increased the blood plasma testosterone concentration, reduced the cryoresistance ratio (CR) for sperm viability and the presence of an intact acrosome, and increased the percentage of sperm with AQP7 in the acrosome and of AQP3 and AQP10 in the midpiece. In the mouflons, neither the PTU treatment, the LD, nor the combination of both affected the CR of any sperm variable. The percentage of sperm with AQP3 increased in the post-acrosome region but decreased in the tail in the LD+PTU group. The percentage of sperm with AQP10 in the principal piece and endpiece was lower in the PTU+LD group than in the control and LD groups. The influence of photoperiod/melatonin on AQP expression might be indirectly exerted through changes in the testosterone concentration, and thus ultimately affect sperm cryoresistance.

Assessing the levels of intraspecific admixture and interspecific hybridization in Iberian wild goats (Capra pyrenaica).

Iberian wild goats (Capra pyrenaica, also known as Iberian ibex, Spanish ibex, and Spanish wild goat) underwent strong genetic bottlenecks during the 19th and 20th centuries due to overhunting and habitat destruction. From the 1970s to 1990s, augmentation translocations were frequently carried out to restock Iberian wild goat populations (very often with hunting purposes), but they were not systematically planned or recorded. On the other hand, recent data suggest the occurrence of hybridization events between Iberian wild goats and domestic goats (Capra hircus). Augmentation translocations and interspecific hybridization might have contributed to increase the diversity of Iberian wild goats. With the aim of investigating this issue, we have genotyped 118 Iberian wild goats from Tortosa-Beceite, Sierra Nevada, Muela de Cortes, Gredos, Batuecas, and Ordesa and Monte Perdido by using the Goat SNP50 BeadChip (Illumina). The analysis of genotypic data indicated that Iberian wild goat populations are strongly differentiated and display low diversity. Only three Iberian wild goats out from 118 show genomic signatures of mixed ancestry, a result consistent with a scenario in which past augmentation translocations have had a limited impact on the diversity of Iberian wild goats. Besides, we have detected eight Iberian wild goats from Tortosa-Beceite with signs of domestic goat introgression. Although rare, hybridization with domestic goats could become a potential threat to the genetic integrity of Iberian wild goats; hence, measures should be taken to avoid the presence of uncontrolled herds of domestic or feral goats in mountainous areas inhabited by this iconic wild ungulate.

Re-emergence of bluetongue virus serotype 4 in Iberian ibex (Capra pyrenaica) and sympatric livestock in Spain, 2018-2019.

Between early October and mid-December 2018, mortalities were detected in Iberian ibex (Capra pyrenaica) populations in southern Spain. In the same region and period, bluetongue virus (BTV) circulation was also reported in sentinel and clinically affected domestic ruminant herds. Molecular analyses confirmed BTV serotype 4 (BTV-4) infection in eight Iberian ibexes from six hunting areas, and in 46 domestic ruminants from seven herds in close proximity to affected hunting estates. Histopathological analyses revealed vascular changes in several organs, pneumonia, lymphoid depletion, inflammatory mononuclear cell infiltrate and fibrosis as the most frequently observed lesions in the affected Iberian ibexes. Epidemiological and laboratory results indicate that BTV-4 was the main aetiological agent involved in outbreaks detected in Iberian ibex populations during the study period. Sequence analyses indicated that the BTV-4 strain detected in Iberian ibex had high homology (99.4%-100%) with strains isolated in livestock during the same period, and with previous isolates (≥98.9%) from Spain and Mediterranean Basin countries. Further studies are warranted to determine the impact of BTV-4 on the health status of Iberian ibex populations after the outbreaks. The inclusion of this species in the surveillance programme may be useful for early detection of BTV, especially in epidemiological scenarios at the wildlife-livestock interface.

Spatio-temporal patterns of sarcoptic mange in red deer and Iberian ibex in a multi-host natural park.

The reliance on multiple hosts to survive is what makes the management and control of multi-host infectious agents challenging. Sarcoptes scabiei causes sarcoptic mange in a wide range of mammal species with ungulates being an important host. Little is known about the role different ungulates play in sustaining endemic transmission of the disease and no study has yet to describe the long-term multi-host sarcoptic infestation dynamics in free-ranging wildlife. Here, we explore 24 years of sarcoptic mange infestation data for two Mediterranean ungulate species, red deer and Iberian ibex, living in the Sierras de Cazorla, Segura y Las Villas Natural Park of southern Spain. The temporal analysis showed a clear seasonal pattern of infestation in both ungulates with a peak in early spring and a decline throughout the summer. The spatial analysis, however, showed that caprinae rather than cervidae is the most competent host for sarcoptic mange spreading and persistence. Considering that few studies have described the spatio-temporal pattern of mange outbreaks for long periods of time, the information reported in this work aims to improve our understanding of sarcoptic mange epizootic in wild ruminant populations.

Mycoplasma conjunctivae in insect vectors and anatomic locations related to transmission and persistence.

Mycoplasma conjunctivae is an obligate microparasite that causes Infectious Keratoconjunctivitis (IKC) in Caprinae species. IKC is a long-recognised disease, but little attention has been paid to the mechanisms of transmission of the mycoplasma and its occurrence in locations other than the eyes. In this study, the presence of M. conjunctivae is assessed in the eyes, external ear canals (EEC), nasal cavity, and vagina of host species as well as in potential vectors, which may be involved in the transmission and persistence of infection within the host. M. conjunctivae was detected by qPCR in 7.2 % (CI 95% 4.7-11.0) of the ear swabs and 9.5 % (CI 95% 6.4-13.9) of the nasal swabs from Pyrenean chamois, Iberian ibex, domestic sheep and mouflon without statistical differences between species. Mycoplasma detection in nasal swabs was mostly associated with ocular infection (95.6%), but this was not the case for EEC (52.6%). Among the eye-positive ruminants, 27.3% were positive in ear swabs and 64.7% in nasal swabs, and the threshold cycle values of the qPCR were correlated only between eye and nasal swabs (p < 0.01; r2 = 0.56). M. conjunctivae was detected in 1.7% - 7.1 % of Musca spp. captured during an IKC outbreak in Iberian ibex and in one out of three endemic sheep flocks. The results indicate that the transmission of M. conjunctivae may occur by direct contact with eye or nasal secretions and/or indirectly through flies. The M. conjunctivae DNA detection in EEC suggests that it can colonise the auditory tract, but the significance for its persistence within the host should be further assessed.

Fertilizing capacity of vitrified epididymal sperm from Iberian ibex (Capra pyrenaica).

In this study, we successfully described for the first time a vitrification of epididymal Iberian ibex spermatozoa. Spermatozoa from epididymis were obtained from 15 Iberian ibex. The right epididymis' semen sample was vitrified and the left one was frozen. After thawing/warming, samples were selected by density gradient. Sperm characteristics from each treatment were evaluated. To test the spermatozoa fertilization ability, heterologous IVF was carried out using bovine oocytes. Despite of the observation of a decrease of about 40% for motility sperm between pre-freezing and post-thawing (75.0 ±â€¯5.2 and 45.0 ±â€¯6.0) and pre-vitrification and post-warming (78.2 ±â€¯5.2 and 33.9 ±â€¯6.2) (P < .05), after the washing, an improvement of sperm motility was found when using the vitrification treatment compared to frozen-thawed. Heterologous IVF showed that Iberian Ibex spermatozoa, either frozen-thawed or vitrified-warmed, were equally capable of penetrating ZP intact bovine oocytes, leading to pronuclear formation (%) and hybrid embryo cleavage (%), (31.3 ±â€¯27.2 and 45.1 ±â€¯24.4, respectively). As expected, in the homologous IVF group, higher percentages of penetration, pronuclei formation and cleavage were found compared to heterologous groups using Iberian ibex frozen and vitrified sperm (P < 0,5). The highest pronuclei formation was found after 20 h post insemination in both heterologous IVF groups (30.2 ±â€¯6.7 and 31.7 ±â€¯21.5 thawed and vitrified group). Consequently, the cleavage rate (48 h) followed the same results to homologous and thawed and vitrified groups (76.1 ±â€¯15.9; 31.3 ±â€¯27.2 and 45.1 ±â€¯24.4, respectively) (P < .05). In conclusion, Iberian ibex sperm vitrification is a promising and useful alternative to conventional methods resulting in good quality spermatozoa post-thaw, and an adequate in vitro fertilizing ability.

Postepizootic Persistence of Asymptomatic Mycoplasma conjunctivae Infection in Iberian Ibex.

The susceptibility of the Iberian ibex (Capra pyrenaica) to Mycoplasma conjunctivae ocular infection and the changes in their interaction over time were studied in terms of clinical outcome, molecular detection, and IgG immune response in a captive population that underwent a severe infectious keratoconjunctivitis (IKC) outbreak. Mycoplasma conjunctivae was detected in the Iberian ibex, coinciding with the IKC outbreak. Its prevalence had a decreasing trend in 2013 that was consistent with the clinical resolution (August, 35.4%; September, 8.7%; November, 4.3%). Infections without clinical outcome were, however, still detected in the last handling in November. Sequencing and cluster analyses of the M. conjunctivae strains found 1 year later in the ibex population confirmed the persistence of the same strain lineage that caused the IKC outbreak but with a high prevalence (75.3%) of mostly asymptomatic infections and with lower DNA load of M. conjunctivae in the eyes (mean quantitative PCR [qPCR] cycle threshold [CT ], 36.1 versus 20.3 in severe IKC). Significant age-related differences of M. conjunctivae prevalence were observed only under IKC epizootic conditions. No substantial effect of systemic IgG on M. conjunctivae DNA in the eye was evidenced with a linear mixed-models selection, which indicated that systemic IgG does not necessarily drive the resolution of M. conjunctivae infection and does not explain the epidemiological changes observed. The results show how both epidemiological scenarios, i.e., severe IKC outbreak and mostly asymptomatic infections, can consecutively occur by entailing mycoplasma persistence.IMPORTANCEMycoplasma infections are reported in a wide range of epidemiological scenarios that involve severe disease to asymptomatic infections. This study allows a better understanding of the transition between two different Mycoplasma conjunctivae epidemiological scenarios described in wild host populations and highlights the ability of M. conjunctivae to adapt, persist, and establish diverse interactions with its hosts. The proportion of asymptomatic and clinical M. conjunctivae infections in a host population may not be regarded only in response to intrinsic host species traits (i.e., susceptibility) but also to a specific host-pathogen interaction, which in turn influences the infection dynamics. Both epidemic infectious keratoconjunctivitis and a high prevalence of asymptomatic M. conjunctivae infections may occur in the same host population, depending on the circulation of M. conjunctivae, its maintenance, and the progression of the host-pathogen interactions.

Toxic and Essential Element Concentrations in Iberian Ibex (Capra pyrenaica) from the Sierra Nevada Natural Park (Spain): Reference Intervals in Whole Blood.

Iberian ibex (Capra pyrenaica) blood samples from the Sierra Nevada Natural Park (Spain) were analyzed to establish concentrations of toxic and essential elements. Samples (whole blood from 32 males and 34 females) were taken from wild animals and the concentrations of inorganic elements considered as (1) non-essential and toxic (Pb, Cd and As), (2) essential but potentially toxic (Cu, Zn and Mn) and (3) occasionally beneficial (B, Cr, Al and Ni), as well as (4) essential minerals (Ca, Na, K, P, Mg, S, Co and Fe), were analyzed. The low concentration of Pb and Cd indicated that there is no heavy metal contamination in this geographical area for these elements. The concentration of elements in this ibex population was defined for different genders and ages. Significant differences between genders were only found for Mg and Cu, while significant differences in concentrations of Ca, Cr, Fe, Mn, P, S and Zn were found between ages.

Successful ultrarapid cryopreservation of wild Iberian ibex (Capra pyrenaica) spermatozoa.

A method for cryopreserving wild ibex sperm at high cooling rates was developed. To design a freezing solution based on Tris, citric acid, and glucose (TCG), two preliminary experiments were performed using glycerol (GLY) and dimethyl sulfoxide (DMSO) at different concentrations (5%, 10%, 20%). The 10% GLY + 10% DMSO combination reduced (P < 0.05) frozen-thawed sperm motility, which reached a minimum when 20% GLY + 20% DMSO was used. In the second experiment, sperm tolerance to three sucrose concentrations was evaluated (100-mM sucrose, 300-mM sucrose, 500-mM sucrose). Frozen-thawed sperm motility and sperm viability decreased (P < 0.05) at concentrations above 300 mM. The ultrarapid cooling procedure finally used involved a TCG egg yolk (ey)-based extender with 100-mM sucrose, either alone or with 5% GLY with or without BSA. Two warming procedures (37 °C vs. 60 °C) were also evaluated. The TCG ey with 100-mM sucrose but without GLY/BSA returned the best sperm quality variables. Slow warming at 37 °C strongly affected (P < 0.05) sperm motility and viability in all groups. Sperm selection by density gradient centrifugation produced no motile sperm when slow warming was performed. In contrast, when fast warming was used, sperm selection increased (P < 0.05) percentage of motility, viability, and the percentage of sperms with intact acrosomes. Heterologous in vivo fertilization involving domestic goats was performed to evaluate the in vivo fertilization capacity of the ultrarapidly cooled cryopreserved sperm (in TCG-ey + 100 mM sucrose), with warming undertaken at 60 °C. Inseminations of domestic goats resulted in three pregnancies (3 of 16, 18.7% fertility). In conclusion, ibex spermatozoa are strongly sensitive to high concentrations of permeable cryoprotectants and sucrose. However, the combination of ultrarapid cooling, using TCG-ey + 100-mM sucrose, and fast warming at 60 °C, followed by sperm selection by density gradient centrifugation to collect the motile sperm, has a positive effect on sperm viability.

Acute phase proteins increase with sarcoptic mange status and severity in Iberian ibex (Capra pyrenaica, Schinz 1838).

Sarcoptic mange is a contagious skin disease caused by Sarcoptes scabiei, affecting both domestic and wild mammals, including the Iberian ibex (Capra pyrenaica), a medium-sized mountain ungulate almost endemic to the Iberian Peninsula. Acute phase proteins (APPs) could be an indicator of sarcoptic mange disease and severity in Iberian ibex. Serum samples from 131 healthy and sarcoptic mange-affected Iberian ibexes were collected from 2005 to 2012 in Sierra Nevada Natural Space in southern Spain. Serum alpha-1-acid glycoprotein (AGP), serum amyloid A (SAA) and haptoglobin (Hp) concentrations were quantified, and statistically significant differences according to sarcoptic mange disease and severity were assessed. Both AGP and SAA were significantly higher in the sarcoptic mange-affected ibexes than in the healthy ones as well as in the severely affected ibexes as compared to those with less than 50 % of the body surface affected. For the first time, changes in APP are reported in relation to sarcoptic mange in Iberian ibex. It is also reported for the first time that the intensity of APP increase depends on the severity of sarcoptic mange, which could be related with the pathological secondary amyloidosis, leading to organ dysfunction in severely mange-affected animals. Species and population differences in the increase of APP in response to sarcoptic mange could indicate individual and population differences in the immune capability of each population to deal with mange, population prevalence and mortality being the last indicators of such sensitivity.

Escherichia coli O157:H7 in wild boars (Sus scrofa) and Iberian ibex (Capra pyrenaica) sharing pastures with free-ranging livestock in a natural environment in Spain.

BACKGROUND: Wild ungulates have greatly increased in abundance and range throughout Europe. This new situation presents a concern for public health because many wild ungulates are known reservoirs of zoonotic pathogens. OBJECTIVES: In this work, we tested for the presence of the zoonotic pathogen Escherichia coli O157:H7 in free-ranging livestock and sympatric wild boars (Sus scrofa) and Iberian ibex (Capra pyrenaica) in NE Spain from 2009 to 2011. In addition, antimicrobial resistance and virulence factors were assessed. ANIMALS AND METHODS: In total, individual fecal samples were obtained from 117 hunter-harvested wild boars and 160 Iberian ibexes. Fifty-five samples were obtained from cattle (5 herds, 380 animals in total) and four from the only horse herd in the Natural Park 'Ports de Tortosa i Beseit' (32 animals). Fecal samples were processed according to the ISO 16.654:2001 protocol to obtain E. coli O157 based on immunomagnetic separation. In addition, a multiplex polymerase chain reaction (PCR) targeting nine virulence factors characteristic of human pathotypes was performed. The prevalence was compared between host species with Fisher's exact test. RESULTS: Four wild boars (3.41%, 95% CI = 0.94-8.52) and two Iberian ibexes (1.25%, 95% CI = 0.15-4.4) carried E. coli O157:H7, which was not found in livestock feces (n = 59, 95% CI = 0-8.94). All E. coli O157:H7 isolates were susceptible to all antimicrobial agents tested. CONCLUSIONS: These results indicate that when prevalence in co-habiting livestock is low, wild ungulates do not seem to play an important role as reservoirs of E. coli O157:H7.

Descriptive analysis of sperm head morphometry in Iberian ibex (Capra pyrenaica): optimum sampling procedure and staining methods using Sperm-Class Analyzer ®.

Sperm morphology has been identified as one characteristic which can be useful in prediction of fertility in a species. The development of computer automated sperm morphometry analysis allows for objective analysis of sperm head dimensions. The aim of the current study was to develop an optimum sampling procedure to characterize the Iberian ibex (Capra pyrenaica) sperm head morphometrically. Fresh semen from 11 males was collected using transrectal ultrasonic-guided massage of accessory sex glands and electroejaculation and prepared on slides for morphometric analysis to evaluate technical variation and standardize automated sperm morphometry analysis procedures by Sperm-Class Analyzer(®). Three staining methods (Diff-Quik(®), Hemacolor(®), Spermblue(®)), number of sperm cells necessary to sample and repeatability of the staining technique were assessed. There were significant differences in size of sperm head depending on stain used. Hemacolor(®) was stain most suitable for sperm head morphometry evaluation (length=8.42 µm; width=4.21 µm; area=29.37 µm(2); perimeter=21.93 µm; elongation=0.33; elipticity=2.01; regularity=0.95; rugosity=0.77). Morphometric values obtained from samples of 50, 100, 150, 175 and 200 sperm heads were compared. The most efficient method of analyzing sperm morphometry was to evaluate 100 sperm cells at 60× objective magnification. Thus, this study has allowed for description of optimal sample processing to determine morphometric parameters of sperm heads (size and shape) in Iberian ibex by Sperm-Class Analyzer(®) and provides a basis for future studies on the relationship with freezability and fertility in this species.