Effect of acepromazine and haloperidol in male Iberian Ibex (Capra pyrenaica) captured by box-trap

Short-acting neuroleptic drugs are used to prevent adverse effects of stress in wildlife. We compared the effect of acepromazine and haloperidol in Iberian ibex (Capra pyrenaica) captured with box-traps. We captured 23 male Iberian ibex at the National Game Reserve of Ports de Tortosa i Beseit, northeastern Spain, March 2003-June 2005. Seven animals received 0.1 mg/kg of acepromazine maleate, eight received 0.33 mg/kg of haloperidol and eight animals acted as controls. Clinical, hematologic, and serum biochemical parameters were analyzed. Both treatments decreased rectal temperature, white blood cells, lymphocytes, and concentrations of creatinine, creatine kinase, and lactate dehydrogenase. Acepromazine also decreased red blood cells, packed cell volume, hemoglobin concentration, neutrophils, and concentrations of glucose and cholesterol. Haloperidol also decreased heart rate and concentrations of urea and potassium. Our results demonstrate the suitability of using acepromazine and haloperidol in capture operations to reduce stress and prevent its adverse effects.     

Epidemiology of Mycoplasma agalactiae infection in free-ranging Spanish ibex (Capra pyrenaica) in Andalusia, southern Spain

Mycoplasma agalactiae is the main causal agent of contagious agalactia syndrome in Spain. It is a severe disease of small ruminants, endemic in Mediterranean countries, that is characterized by mastitis, arthritis, and keratoconjunctivitis. This paper investigates the temporal, spatial, and host-related factors in the distribution of M. agalactiae infection from October 1996 to November 1998 and March 2002 to May 2003 in Spanish ibex (Capra pyrenaica) populations from Andalusia, in southern Spain. The predisposing factors to infection among previously selected factors (year of sampling, climatic season, geographic origin according to province, mountain range and metapopulation, sex, year of life, presence of scabies, and phase of the reproductive cycle) were established. We collected conjunctival and ear-canal swabs from 411 free-ranging ibexes. The frequency of infected ibexes was 11.2%. The peak frequency of infection occurred in 1998 and in summer. Granada was the province with greatest risk (odds ratio = 2.6) of carriers (18.8% infected). The predisposing factors were sex (females), age (young animals), and metapopulation (Sierra Nevada). We identified a higher number of infected ibexes in the metapopulation "Sierra Nevada" (34/ 256) and significant differences among the three established metapopulations (P<0.01). Mycoplasma agalactiae infection represents a risk for population density and maintenance of these wild populations; infections can result in blindness, malnutrition, and polyarthritis leading to numerous deaths.     

Horn quality and postmortem sperm parameters in Spanish ibex (Capra pyrenaica hispanica)

The horns are secondary sexual characteristics used by males of many ungulate species for intra-sexual fights during the rut. Thus, the dominant males with most developed horns are naturally selected for reproduction. Several studies have suggested that the quality of the horn, in many wild ruminants, may be correlated with semen quality. The aim of the present study was to determine whether inter-individual differences in levels of horn asymmetry and horn size are related to differences in sperm quality in a wild population of Spanish ibex by the assay of epididymal spermatozoa collected postmortem. In order to test this hypothesis we collected morphometric horns data from a total of 59 mature males (9-15 years of age) that were legally hunted during rutting season. The testicles were recovered, and the collection of epididymal spermatozoa was done at different times after death (2-60 h). The percentage of motile spermatozoa, motility rate, plasma membrane integrity, sperm viability, sperm morphology, and acrosome integrity were evaluated. Our findings showed that viable epididymal spermatozoa may be retrieved from dead animals many hours after death. However, sperm parameters were affected by the elapsed time between the death of the animal and spermatozoa collection. The study revealed that the horn quality was firstly associated with sperm motility.     

Potential pathogens carried by Spanish ibex (Capra pyrenaica hispanica) in southern Spain

The Spanish ibex (Capra pyrenaica hispanica) population of southern Spain was surveyed for potential pathogens associated with the conjunctiva, external ear canal, as well as reproductive and upper respiratory tracts. We sampled 321 ibex (131 adult males, 100 adult females, and 90 yearlings); these included 271 apparently healthy animals and 50 that were naturally infected with Sarcoptes scabiei. A total of 688 bacterial isolates were identified (377 gram-negatives, 225 gram-positives, and 86 Mycoplasma spp.); sex, age, location, infection with S. scabiei, and disposition of the animal (free-ranging versus captive) were evaluated as risk factors for infection. Infections with Mycoplasma agalactiae and Mycoplasma arginini were associated with age, having a higher frequency of isolation in young animals. With Escherichia coli, Mannheimia haemolytica, Pasteurella multocida biotype A, and Staphylococcus aureus, significantly higher isolation rates were associated with adults. The isolation frequency for E. coli was higher in females, whereas Moraxella bovis isolations were mostly associated with males. The presence of mange increased the risk of infection with both Streptococcus equi subsp. zooepidemicus and M. haemolytica. The geographic origin of sampled animals was related to the isolation of Branhamella ovis, M. agalactiae, and all Pasteurella sp. Isolations of M. haemolytica, P. multocida biotype A, E. coli, and B. ovis were more prevalent in samples from free-ranging rather than captive animals. Of the gram-positive bacteria, S. aureus represented the predominant species isolated from nasal, vaginal, and ocular samples. Mycoplasma agalactiae and M. arginini were the predominant Mycoplasma spp., and both were associated most often with the external ear canal. The most frequently isolated gram-negative bacteria included E. coli, M. haemolytica, P. multocida biotype A, and B. ovis. Isolation rates of gram-negative species varied by source. In nasal samples, M. haemolytica and P. multocida biotype A were isolated most frequently, whereas in ocular and vaginal samples, B. ovis and E. coli, respectively, were most frequently isolated.     

Rumen protozoal diversity in the Spanish ibex (Capra pyrenaica hispanica) as compared with domestic goats (Capra hircus)

Rumen protozoal diversity in the Spanish ibex (SI) was studied in males (n=4), females (n=7) and young (n=4) from the Maestrazgo (Spain) and contrasted with domestic goats (n=3; DG) of the same region. There were no differences among SI types in protozoal concentration or in the number of protozoal species. Only protozoa from the genus Entodinium were observed in SI (seven species), the highest numbers corresponding to E. damae, E. ovibos and E. parvum. DG harboured threefold more species than SI. Nine to 10 Entodinium spp. were observed, but E. ovibos was absent from the rumen of DG, and E. damae was in only one animal. E. caudatum (caudatum, dubardi and lobospinosum morphotypes) occurred in the highest percentage, and E. dubardi, E. exiguum and E. parvum were quite abundant. Four genera of the subfamily Diplodiniinae and the genera Isotricha and Dasytricha from the family Isotrichidae were detected in DG. Epidinium (two DG) and Ophryoscolex (one DG) were also observed. Denaturing gradient gel electrophoresis analysis agreed with microscopic classification, showing up to 8 and 16 bands in SI and DG samples, respectively. The three DG clustered together (similarity index over 0.84), and separately from SI (similarity index over 0.86), with only 0.58 similarity between host species.     

A survey of the gastrointestinal nematodes of Spanish ibex (Capra pyrenaica) in a high mountain habitat

We analyzed the content of the abomasum (n = 79) and small intestine (n = 83) of Spanish ibex from Sierra Nevada Natural Park, southern Spain. Fifteen species of trichostrongylid nematodes were identified, 4 of which were found for the first time in this host, i.e., Nematodirus fillicollis, N. oiratianus, Ostertagia lyrata, and O. ostertagi. Teladorsagia circumcincta and Marshallagia marshalli were the most abundant abomasal species, whereas N. abnormalis, N. davtiani, and N. oiratianus were dominant in the small intestine. Counts of both abomasal and intestinal nematodes were generally low (year-round-median = 292 and 94 worms, respectively), and significantly lower numbers of M. marshalli, N. davtiani, and N. oiratianus were found in summer. No sex-related differences in helminth abundance were found, but young ibex harbored significantly more N. davtiani and N. oiratianus than adults. The presence of scabies was not related to increased nematode counts.     

Effect of post-mortem time on post-thaw characteristics of Spanish ibex (Capra pyrenaica) spermatozoa

Viable epididymal sperm can be obtained in the Spanish ibex during 24h after death, but it has been observed a significant effect of the post-mortem time on fertility success, so only goats inseminated with semen recovery during the first 8h became pregnant. The aim of this study was to determine the effect of post-mortem time on epididymal semen samples from of Spanish ibex. For this purpose, sperm samples from 36 males were collected at different post-mortem times, from 2 to 24h, and cryopreserved. Thawed samples were incubated for 2h at 37°C without dilution or after dilution in a modified Tyrode medium, in order to study the sperm resistance to dilution. Moreover, flow cytometry was used to assess the sperm viability (PI), phospolipid disorder of the plasma membrane (M540), mitochondrial membrane potential (Mitotracker Deep Red), indirect apoptosis markers (YOPRO-1) and sperm chromatin stability (SCSA(?)). Sperm motility was evaluated by computer-assisted sperm analysis (CASA). Our results have shown that post-mortem time caused a reduction in mitochondrial membrane potential. In this regard, the loss of energy could be responsible for the loss of maintenance of the membrane with a consequent increase in permeability leading to a decrease in sperm viability and motility, losing linearity and speed. Moreover, the loss of maintenance of the membrane influence the extent to which sperm will survive the cryopreservation process, as it shows the results obtained from the dilution-incubation resistance test. Finally, one important finding of this study is the demonstration of no effect of post-mortem time on post-thaw DNA integrity, giving us the possibility of using sperm samples from valuable males, even if it was not possible to process during the first 8h.     

Effect of egg yolk concentration on cryopreserving Spanish ibex (Capra pyrenaica) epididymal spermatozoa

Tris-egg yolk based diluents provide adequate cryoprotection for the sperm of most wild species in which they have been tested. The objective of the current study was to evaluate various Tris-based diluents containing different concentrations of egg yolk, for the fertilizing ability of epididymal spermatozoa of the Spanish ibex (Capra pyrenaica) after freezing and thawing. For this purpose, we used heterologous in vivo fertilization by intrauterine insemination of domestic goats (Capra hircus). In Experiment 1, a Tris-citric acid-glucose (TCG) diluent containing 6% (v/v) egg yolk and a TCG extender containing 20% egg yolk were compared. In Experiment 2, a TCG-6% egg yolk extender was compared with Triladyl-20% egg yolk. Diluted samples were cooled slowly to 5 degrees C over 1 h and equilibrated at that temperature for 2 h. At that point, aliquots of samples were loaded into 0.25 ml straws, and frozen in nitrogen vapor for 10 min. The fertility of spermatozoa frozen in TCG-6% egg yolk was higher (P<0.05) than for those extended with TCG-20% egg yolk, and tended to be higher than for those frozen with Triladyl-20% egg yolk. From the results of this study, the use of Tris-based extenders containing low concentrations of egg yolk (6%) is recommended for cryopreserving Spanish ibex epididymal spermatozoa.     

Hematologic and biochemical values for Spanish ibex (Capra pyrenaica) captured via drive-net and box-trap

Between October 2002 and September 2004, 70 free-ranging Spanish ibex (Capra pyrenaica) were captured in Catalonia, northeastern Spain, using two different physical methods, drive-net (n=26) and box-trap (n=44). Blood samples were taken to determine 20 hematologic and 23 biochemical variables. Values obtained fell within already published reference intervals, with the following exceptions: higher values for red blood cells (RBC), packed cell volume (PCV), white blood cells (WBC), eosinophil count, triglyceride concentration, creatine kinase (CK; in box-trap), chloride, sodium, alpha-1, alpha-2, and gamma electrophoretic fractions of serum proteins; and lower values for hemoglobin, mean corpuscular volume (MCV), mean corpuscular hemoglobin concentration (MCHC), urea concentration, CK (in drive-net), albumin, and albumin:globulins ratio (A:G). Published values for aspartate aminotransferase (AST) concentration are both higher and lower than observed in this study. In our study, monocyte and eosinophil counts, as well as triglyceride and potassium concentrations, were lower in animals captured via box-trap than those captured via drive-net. Conversely, MCHC, neutrophil count, total bilirubin concentration, urea, and AST were higher in animals captured via box-trap. Hematologic and biochemical values obtained from Spanish ibexes show that the drive-net is a newer, less-stressful method of capture than the box-trap.     

Interspecies pregnancy of Spanish ibex (Capra pyrenaica) fetus in domestic goat (Capra hircus) recipients induces abnormally high plasmatic levels of pregnancy-associated glycoprotein

Interspecies embryo transfer could be a valuable tool in preservation programs of endangered species. In this work the results of both interspecific-monospecific (ibex-in-goat) and interspecific-bispecific (mixed-species; ibex+goat-in-goat) embryo transfers in the capra genus are reported. The aim of this work was to compare the PAG plasmatic profiles occurring in these interspecific gestations to those encountered in normal (i.e. intraspecies) pregnancies of Spanish ibex and domestic goat. Spanish Ibex females were superovulated with 9 mg NIADDK-oFSH-17 and embryos were surgically collected 5.5 d after estrus. Two embryos were transferred per recipient. Domestic goat recipients were previously mated either to vasectomized domestic bucks (n=17 females; interspecific-monospecific gestations) or to fertile ones (n=9 females; interspecific-bispecific gestations). Intraspecific pregnancies were obtained by natural mating between males and females of the same species (Spanish ibex: n=6; domestic goat: n=1). Pregnancy rate diagnosed by progesterone was low in both interspecific-monospecific (7/17) and interspecific-bispecific (3/9) transfers. None of the monospecific (0/7) and 2 (2/3) of the bispecific established pregnancies developed to term. Ibex-in-ibex PAG profile showed 2 similar peaks of 60 to 70 ng/mL on Days 34 and 153 of pregnancy, while goat-in-goat had the maximum value (60 to 70 ng/mL) at Day 50, decreasing slightly afterwards until parturition. Mixed-species gestations (ibex+goat in goat) showed a first peak of 500 to 1000 ng/mL on Day 70 and a second one (200 to 500 ng/mL) on Day 140 of pregnancy. Four ibex-in-goat gestations that terminated with the expulsion of dead fetuses at Days 110 to 170 had their maximum PAG values (100 to 700 ng/mL) on Days 60 to 90. We conclude that it is possible to achieve pregnancies after transfer of ibex embryos into domestic goats, but this requires a great change of the PAG profiles, which increase significantly. Live ibex kids can be produced when embryos from both species share the uterus. This is the first report of successful interspecific pregnancies in the capra genus.     

Seasonal ovulatory activity and plasma prolactin concentrations in the Spanish ibex (Capra pyrenaica hispanica) maintained in captivity

Seasonal changes in ovulatory activity, assessed by the measurement of plasma progesterone and plasma prolactin concentrations were monitored in 10 Spanish ibex females captured in the National Wildlife Reserve of Sierra Nevada (South Spain, 37 degrees N). Five of the 10 female ibex showed ovulatory activity with a mean (+/- s.e.m.) duration of the oestrous cycle of 19.4 +/- 1 days (range: 17-23 days). On average, the five cyclic females weighed 28 +/- 0.9 kg. Progesterone cycles occurred only in animals older than 4 years of age. Ovulatory activity extended from December to January. The duration of the breeding season was 43.2 +/- 7.7 days. Ibex females younger than 4 years of age had a body weight lower (P < 0.01 ) than that of adults and none of them displayed ovulations. Plasma concentrations of prolactin levels were significantly affected by season (P < 0.05), following a trend that was roughly parallel to daylength. The highest values occurred in the spring (119.7 +/- 21.4 ng x mL(-1)) and the summer (139.3 +/- 19.8 ng x mL(-1)), the lowest values in the autumn (26 +/- 7.4 ng x mL(-1)) and in the winter (19.7 +/- 3.2 ng x mL(-1)). Our results showed a very restricted breeding season, despite the fact that the Spanish ibex originates from and lives in a temperate latitude, revealing a remarkably good adaptation to the harsh climatic and nutritional conditions of their mountainous habitat.     

Birth of live Spanish ibex (Capra pyrenaica hispanica) derived from artificial insemination with epididymal spermatozoa retrieved after death

As a consequence of increasing limitations to maintaining genetic variability in endangered wildlife species, methods of assisted reproduction widely used in domestic animals are being applied to nondomestic species. However, practical efforts have met limited success to date. The Spanish ibex (Capra pyrenaica hispanica) is a wild caprine originating exclusively in the mountains of Spain. This study was designed to evaluate the fertilizing capability of cryopreserved Spanish ibex epididymal spermatozoa recovered postmortem. For this purpose, we have previously evaluated the effect of time elapsed between death and sperm recovery on spermatic parameters, and the fertilization ability of frozen-thawed spermatozoa using heterologous in vivo fertilization by intrauterine insemination in domestic goat (Capra hircus). The time of death significantly affected most sperm quality parameters (motility, viability and intact acrosomes). The fertility obtained by heterologous artificial insemination was 18.7%, and only goats inseminated with spermatozoa recovered within 8h after death became pregnant. Our findings showed that heterologous in vivo fertilization is a useful method to evaluate the fertilizing capacity of sperm samples in rare or wild species. Sperm samples, with verified fertilization ability in the previous trial, were used to inseminate a total of six ibex females. Inseminations resulted in one pregnancy. The study demonstrated for the first time the feasibility of applying artificial insemination in Spanish ibex.     

Relationship between bronchopulmonary nematode larvae and relative abundances of Spanish ibex (Capra pyrenaica hispanica) from Castilla-La Mancha, Spain

The excretion of bronchopulmonary nematode infective larvae was evaluated in 160 faecal samples of Spanish ibex (Capra pyrenaica hispanica) collected from 13 populations in Castilla-La Mancha, south-central Spain in September 2003. Intensities and prevalences were compared with pasture availability, abundances of wild and domestic ungulates at both levels, i.e. for populations and for faeces in a two-step procedure. Protostrongylid larvae showed similar infection rates (mean intensity: 1.56+/-0.12, n=94; mean prevalence: 25.62+/-6.86%, n=160) to Dictyocaulus spp. (mean intensity: 1.03+/-0.11, n=48; mean prevalence: 30.00+/-7.11%, n=160). At the population level, positive correlations were found between the prevalences of both bronchopulmonary taxa. The prevalence in both groups, but not intensity, also correlated positively with Spanish ibex abundance indexes both for the populations and individual faeces. These findings suggest that: (i) parasite spreading across Spanish ibex populations in Castilla-La Mancha could respond to host density-dependent processes; and (ii) these populations may have similar exposition and/or susceptibility to both bronchopulmonary taxa resulting in similar host-parasite patterns, despite their different life cycles. Bronchopulmonary outputs in the Spanish ibex from Castilla-La Mancha seems not to represent a health risk for this endemic wild ungulate but may be useful in any health surveillance scheme for the increasing populations of Spanish ibex.     

Low diversity in the major histocompatibility complex class II DRB1 gene of the Spanish ibex, Capra pyrenaica

During the last two centuries, the Spanish ibex (Capra pyrenaica) has shown a significant demographic decline as a result of the progressive destruction of its natural habitat, disease epidemics, and uncontrolled hunting. Partial sequencing of the class II MHC DRB1 gene revealed that the Spanish ibex has remarkably low levels of genetic variation at this locus, with only six different DRB1 alleles and an observed heterozygosity of 0.429-0.579. The rates of nonsynonymous vs synonymous substitutions were significantly different in the peptide-binding region (dN/dS=5.347, P=0.002), a feature that indicates that the DRB1 gene is under positive selection. A phylogenetic analysis of the Spanish ibex and a set of domestic goat DRB1 alleles revealed that the reported sequences represent four major allelic lineages. The limited allelic repertoire of the DRB1 gene in the Spanish ibex is likely the direct result of the recent history of population bottlenecks and marked demographic decline of this species. A genetic survey of 13 microsatellite loci was consistent with this idea. The Spanish ibex subspecies C. p. hispanica and C. p. victoriae consistently showed considerably lower levels of microsatellite heterozygosity (Ho=0.184-0.231) and allelic diversity (mean number of alleles per locus=2-2.4) than those reported in other wild ruminants. This study demonstrates the significance of both natural selection and the demographic history of populations in determining patterns of genetic variation at MHC loci. In addition, our results emphasize the importance of locally adapted populations for the preservation of genetic diversity.     

Bluetongue virus serotype 8 (BTV-8) infection reduces fertility of Dutch dairy cattle and is vertically transmitted to offspring

In 2007, BTV-8 re-emerged for the second year in the Netherlands and caused morbidity and increased mortality in cattle herds. In addition, cattle farmers reported reduced fertility in their cows. For this study, fifteen herds that were not vaccinated were selected. These were matched to 10 vaccinated herds by geographic region. At the start of the study, in July 2008, all cattle in the non-vaccinated herds >1 year old were sampled. All seronegative cows entered the study program and blood samples from these cows were tested for antibodies against BTV-8 in an ELISA. Cows were sampled at intervals of three weeks and sampling was stopped once a cow tested seropositive. Sampling ceased in all remaining cows in December 2008.Newborn calves originating from infected dams or from vaccinated dams were tested by PCR for BTV-8. Fertility data were obtained from the Royal Dutch Cattle Syndicate (CRV). Multi-level generalized latent and linear models were used for analyses.In 2008, 185 (17.2%) out of 1,074 initially seronegative non-vaccinated cattle seroconverted and were assumed to be infected with BTV-8. Infected cows were 5 (95% CI: 1.9-14.3) times more likely to return for insemination within 56 days after first insemination. In addition, these cows needed 1.7 (95% CI: 1.4-2.0) times more inseminations for an assumed pregnancy, and needed 2.5 (95% CI: 2.4-2.6) times more days between first and last insemination compared to the period prior to seroconversion and compared to cows not infected by BTV-8 in 2008. No association between BTV-8 infection and the chance to abort between 100 and 260 days after last insemination was found.In total, 48 calves originating from infected cows were tested by PCR for the presence of BTV-8. Ten (20.8%) out of these 48 calves were born PCR-positive. None of 256 calves from vaccinated dams tested PCR-positive. Further, cows infected during the second half of gestation had a 15.5 times (95% CI: 1.3-190.4) higher chance of a PCR-positive newborn calf compared to cows infected in the first half of gestation. This study showed that BTV-8 has a negative effect on fertility of dairy cattle.