Search

The main goal of the current study was to investigate the molecular systematics and host–vector interactions of sandfly species (Phlebotomus spp.) in Iran. Sandflies were collected using CDC light traps, sticky traps, and BG-Sentinel traps at seven trapping sites in Iran during 2019 and 2020. The source of blood meals was identified using primer pairs specific to either the 16S rRNA or the mitochondrial cytochrome b (cytb) genes. A total of 736 blood-fed female sandflies were collected. Six species of the genus Phlebotomus (Ph.) and four species of the genus Sergentomyia (Se.) were identified using molecular identification techniques. The most abundant sandfly species were Ph. papatasi (38.45 %) and Se. sintoni (36.28 %). Blood meal analysis identified 19 different host species, specifically, 15 mammalian and four bird species. The most common blood meal sources were Rattus norvegicus , followed by Homo sapiens , Bos primigenius f. taurus , Ovis orientalis f. aries , Meriones persicus , Gallus gallus f. domestica , Rhombomys opimus , and Canis lupus; other host species had incidences of less than 5 %. We observed that Ph. major complex and Se. sintoni had mostly mammalophilic blood-feeding behavior, while Ph. papatasi and Ph. sergenti had mostly anthropophilic and ornithophilic blood-feeding preferences, respectively. The current results confirmed cytb is a suitable gene for regular species level identification, and that phylogenetic analyses of cytb allowed for clear demarcation of sandfly species. [ABSTRACT FROM AUTHOR]

Highlights • Mycoplasma conjunctivae is detected for the first time in ear canals and flies. • It was detected in 7.2 % ear and 9.5% nasal swabs from different species. • Concurrent detection in eye and nasal swabs, but independent to external ear swabs. • External ear canals may have a role in mycoplasma persistence. • Mycoplasma was detected in 7.1% Musca spp. associated with a disease outbreak. Abstract 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; r 2 = 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. [ABSTRACT FROM AUTHOR]

This study examines the effectiveness of two methods for cryopreserving post-mortem epididymal sperm - conventional slow freezing employing a short equilibration time with glycerol, and ultra-rapid freezing - from the wild ruminant species Ovis musimon (mouflon) and Dama dama (fallow deer). A Tris-citric acid-glucose (TCG) + 12% egg yolk-based medium was used for the conventional slow freezing of the fallow deer sperm, whereas a Tes-Tris-glucose (TEST) + 6% egg yolk-based medium was used for the mouflon sperm. Glycerol was added to a final concentration of 5% to both media. The same diluents were used for ultra-rapid freezing but replacing the glycerol with 100 mM of sucrose. Sperm variables (motility, viability, acrosome integrity, membrane integrity, and morphological abnormalities) were analyzed before and after cryopreservation. Although values were generally better after the thawing of the conventionally cryopreserved sperm, total sperm motility (38.40 ± 4.44% in mouflon and 31.25 ± 3.37% in fallow deer) and total live sperm (47.19 ± 5.18% in mouflon and 43.13 ± 2.43% in fallow deer) were acceptable for the ultra-rapidly cooled sperm. Independent of the cryopreservation method, membrane integrity, acrosome integrity and the percentages of dead sperm and sperms with a damaged acrosome were better for the cryopreserved mouflon sperm than the fallow deer sperm (P < 0.05). Despite exerting a more harmful effect on sperm variables than conventional freezing, ultra-rapid freezing may be a useful alternative for the cryopreservation of these species' epididymal sperm in the field, as this simple technique does not require sophisticated equipment and expertise. [ABSTRACT FROM AUTHOR]

The mouflon’s traditional classification as a grazer has recently been questioned, therefore new insight into the species’ feeding and digestion is needed. The presence and share of different ciliate genera in the rumen may contribute to an understanding of a species’ feeding physiology, therefore we analysed the composition of mouflon rumen ciliates and correlated their density with the percentage of different food types eaten by the animals. We found that the dominant ciliates were Entodinium spp., with a small proportion of Anoplodinium denticulatum, Eudiplodinium maggii, Epidinium ecaudatum caudatum and Dasytricha ruminantium . The main food types taken by the animals were deciduous tree leaves, grass and bramble. The density of ciliates from the Entodinium genus rose with an increase in deciduous leaves in the diet and fell with an increase of graminoids in the diet. The density of ciliates from the Anoplodinium and Eudiplodinium sub-genera increased with a rise in tree shoots and was reduced with a rise of deciduous leaves in the diet. These results confirm that the protozoal fauna vary with current food composition in intermediate feeders. [ABSTRACT FROM AUTHOR]

Anaplasmosis caused by gram-negative bacteria poses a great threat to the wildlife and livestock population in most tropical and subtropical regions of the world. Among these, Anaplasma capra (A. capra) is an emerging zoonotic pathogen. The present study aimed to determine the molecular prevalence of A. capra in mouflon sheep (Ovis gmelini) and domestic sheep (Ovis aries) of Pakistan. A total of 210 mouflon and domestic sheep blood samples were collected and analyzed by microscopy and PCR. The study reported an overall prevalence of A. capra at 20.48 % (43/210) based on PCR. However, the prevalence of A. capra was found at 11.43 % (12/105) in wild and 29.52 % (31/105) in domestic sheep respectively. Phylogenetic analysis of 16S rRNA gene of Anaplasma capra, revealed that four isolates of the current study were clustered together and showed a close resemblance with the Anaplasma capra isolates of Korea, China, Iran, and Russia; however, two isolates showed close resemblance with Indian isolate of A. capra. Hematobiochemical analysis of diseased (n = 10) and healthy animals (n = 10) revealed a significant (p < 0.05) decrease in WBCs, RBCs, PCV, hemoglobin, mean calcium levels, and total protein, while an increased amount of AST, ALP, total bilirubin, creatinine, and the BUN levels were observed in animals suffering from anaplasmosis. This is the first report regarding the molecular characterization of Anaplasma capra in captive mouflon and domestic sheep of Pakistan. The current study provides information about epidemiology of A. capra and hematobiochemical changes in mouflon and domestic sheep of Pakistan. • First identification of novel zoonotic tick-borne Anaplasma sp. infecting captive mouflon and domestic sheep of Pakistan. • An overall prevalence of Anaplasma capra (20.48 %) has been reported in mouflon and domestic sheep of Pakistan. • Phylogenetic analysis of Anaplasma capra showed a close resemblance in isolates of captive mouflon and domestic sheep. • Hematobiochemical changes observed in sheep infected with Anaplasma capra. [ABSTRACT FROM AUTHOR]

The objective was to characterize the stress response and the seminal parameters obtained with electroejaculation (EE) or transrectal ultrasound-guided massage of the accessory sex glands (TUMASG) in two captive but nondomestic ruminants, the mouflons and the Iberian ibex under general anesthesia. In mouflons, the physiological responses (heart and respiratory rate, rectal temperature, cortisol, creatine kinase, potassium and glucose concentrations) changed similarly with both procedures. The TUMASG procedure was faster than EE in mouflons (21.7 ± 1.4 vs. 12.4 ± 1.2 minutes, P < 0.01). In ibexes, respiratory rate, cortisol and creatine kinase concentration changes were greater with EE than with TUMASG (final respiratory rate: 62.7 ± 5.5 vs. 38.1 ± 5.6 breaths/min [P < 0.05]; final cortisol: 51.4 ± 5.1 vs. 25.3 ± 5.6 ng/mL [P < 0.001]; and final creatine kinase: 300.9 ± 99.9 vs. 87.1 ± 16.9 U/L [P < 0.001]). Electroejaculation provided better results in some sperm parameters (mouflons: sperm score: 3.4 ± 0.3 vs. 2.6 ± 0.2 [P < 0.01]; total number of sperm ejaculated: 982.4 ± 299 vs. 710.0 ± 542.2 [P < 0.05]; ibexes: sperm with progressive motility: 47.7 ± 6.2 vs. 20.5 ± 8.3 [P < 0.05]). The transrectal ultrasound-guided massage of the accessory sex glands appears to be an alternative technique to collect sperm from wild ruminants, reducing the need for electrical stimuli and thus decreasing the undesired responses of EE in the more sensitive species. On the other hand, better fresh sperm may be collected with EE. However, TUMASG provides practical advantages in animal welfare, firstly in these wild species more sensible to stress management and capture myopathy. [ABSTRACT FROM AUTHOR]

In this study, we examined in Sardinia the brain of 555 autochthonous sheep, 50 goats, and 4 mouflons which were found affected by neurological signs. We found 6 goats and one mouflon with meningoencephalitis caused by Cryptococcus sp. There was no evidence of cryptococcal infections in any of the examined sheep. MLST genotyping on Cryptococcus sp. isolates identified Cryptococcus gatti genotype AFLP4/VGI and Cryptococcus neoformans var. neoformans genotype AFLP2/VNIV . Phylogenetically, all Cryptococcus gattii isolates fell within the autochthonous animal, human and environmental Mediterranean isolate cluster, forming a distinct branch along with environmental strains from Alicante, in the southern Mediterranean coast of Spain. [ABSTRACT FROM AUTHOR]

• In current study FMD outbreak in the wild ungulate species in the Jallo Wildlife Park was investigated with possible risk factors. • ELISA was performed to detect antibodies against the non-structural protein of FMD virus. • Possible cause of FMD could be spillover from domestic livestock from animal handlers having FMD. infected animals in their houses and transport vehicles coming from adjacent disease-affected villages. The potential spillover of foot and mouth disease (FMD) virus at the wildlife-livestock interface is mainly responsible for the outbreaks in captive wild ungulates. The current study was planned to investigate an FMD outbreak in the wild ungulate species in the Jallo Wildlife Park and breeding facility, Lahore, Pakistan from Mar 2021– Jun 2021. The disease was confirmed based on ELISA through the detection of antibodies against the non-structural protein of FMD virus and typical clinical signs of oral and feet lesions, and even partial or complete hoof shedding in severe cases. To investigate the possible cause of FMD spillover and its pattern, a series of interviews were conducted with wildlife practitioners, animal handlers, and the local veterinarians in villages adjacent to the park. The data revealed neither vaccination nor any FMD outbreak in the last 10 years. The epidemic curve and time-series analysis showed a mixed outbreak characterized by the introduction of disease and then propagative spread from infected to susceptible animals. After the first reports of clinical signs in Blackbuck, Urial and Mouflon sheep were infected while in the end, the cases were reported from the enclosures of Sambar deer and Spotted deer. The morbidity rate among all the wild ungulate species was 92% and the mean mortality rate was 27%. The study concluded that possible sources for primary disease incidence might be animal handlers having FMD infected animals in their houses and transport vehicles coming from adjacent disease-affected villages. For the disease spread between enclosures, another possibility of wind-borne FMD virus spread could also be considered. This is the first report regarding the FMD outbreak and its spillover pattern in captive wild ungulates in Pakistan. These findings will be helpful in understanding the importance of wildlife livestock interface in FMD transmission and to design effective strategies to control the disease. [ABSTRACT FROM AUTHOR]

The presence of Ixodes ricinus and their associated Borrelia infections on large grazers was investigated. Carcases of freshly shot red deer, mouflon and wild boar were examined for the presence of any stage of I. ricinus . Questing ticks were collected from locations where red deer and wild boar are known to occur. Presence of Borrelia burgdorferi s.l. DNA was examined in a fraction of the collected ticks. Larvae, nymphs and adult ticks were found on the three large grazers. Red deer had the highest tick burden, with many of the nymphs and adult females attached for engorgement. Most larvae had not attached. The mean number of ticks on the animals varied from 13 to 67. Ticks were highly aggregated amongst the animals: some animals had no ticks, while others had high numbers. Larvae and nymphs were mostly found on the ears, while adult ticks were attached to the axillae. The Borrelia infection rate of questing nymphs was 8.5%. Unengorged wandering nymphs on deer had a Borrelia infection rate of 12.5%, while only 0.9% of feeding nymphs carried a Borrelia infection. The infection rate of unengorged adult male ticks was 4.5%, and that of feeding female ticks was 0.7%. The data suggest that ticks feeding on red deer and wild boar lose their Borrelia infections. The implications of the results are discussed with respect to Borrelia epidemiology and maintenance of a Borrelia reservoir as well as the role of reproductive hosts for Ixodes ricinus . [ABSTRACT FROM AUTHOR]

Abstract: The control region of mtDNA (D-loop) was used for hair samples of the five hunting game species identification: red deer (Cervus elaphus), roe deer (Capreolus capreolus), fallow deer (Dama dama), mouflon (Ovis aries musimon), and wild boar (Sus scrofa). For D-loop multilevel PCR detection scheme was applied in six primers (CE CVZV 1=5′-GATCACGAGCTTGATCACCA-3′; CE CVZV 2=5′-AGGAGTGGGCGATTTTAGGT-3′; DD CVZV 3=5′-CGCGTGAAACCAACAACCCGC-3′; DD CVZV 4=5′-CCGGGTCGGGGCCTTAGACG-3′; SSW CVZV 5=5′-ACACGTGCGTACACGCGCATA-3′; SSW CVZV 6=5′-GGTGCCTGCT T TCGTAGCACG-3′) designed to identify unknown biological samples of the hunting game animals. The PCR reaction volume was 25μl at conditions 95°C for 2min, 94°C for 30s, 60°C for 30s, 72°C for 30s, 35cycles, with last extension at 72°C for 10min. D-loop mtDNA amplicons of the game animals are characterized with specific PCR product sizes depending on species: red deer=163bp and 140bp, fallow deer=280bp and 138bp, roe deer=303bp, 280bp, 160bp and 138bp, mouflon=299bp and 178bp, wild boar=137bp and 229bp. [Copyright &y& Elsevier]

• SNP markers were used to study genetic diversity of domestic and wild Iranian sheep and goats. • Inbreeding coefficients (FGRM, FHOM and FROH), linkage disequilibrium (LD), and effective population size (Ne) were used. • Wild species had lower genetic diversity than domestic species. • Lower genetic diversity for wild sheep and goat species was due to decrease in Ne in recent generations. • Wild sheep population is in a more critical conditions from genetic diversity point of view. In present study, genetic diversity of domestic and wild Iranian sheep and goats were evaluated using inbreeding coefficients (F GRM , F HOM and F ROH), linkage disequilibrium (LD), and effective population size (Ne). Genomic data of Iranian sheep and goat species were obtained from the NEXTGEN project. Animals consisted of 20 domestic sheep (Ovis aries), 14 wild sheep (Ovis orientalis), 20 domestic goats (Capra hircus) and 18 wild goats (Capra aegagrus). Mutations equivalent to the IlluminaSNPchip50 K were extracted to study mutations for each animal. Based on F GRM , F HOM and F ROH , higher rates of inbreeding were observed in wild species than domestic species. Wild sheep had the highest rate of inbreeding among studied populations. Our results showed a decreasing in LD with increasing in distance between pairs of SNPs so that the average LD in short distance was in the range of 0.11−0.12, and in long distance was in the range of 0.091−0.093. The analysis of LD also demonstrated higher genetic diversity in domestic sheep and goat than wild species and wild sheep had the lowest genetic diversity among studied populations. The results indicated that Ne in 13 generations ago was higher in domestic species compared to wild species. In general, present results revealed that wild species had lower genetic diversity compared to the domestic species in Iran. Meanwhile, wild sheep population is in critical conditions from genetic diversity point of view and required more attention. Designing appropriate breeding programs to improve genetic diversity in wild species, especially in wild sheep in Iran is necessary to protect them from extinction. [ABSTRACT FROM AUTHOR]

Abstract: Wild sheep (Ovis orientalis) as the true ancestor of domestic sheep (Ovis aries) is currently listed as vulnerable (VU) by IUCN. Effective conservation of this species requires collecting all available ecological knowledge into a single framework that could be used for management decision making. Use of Bayesian Belief Networks for such purposes have been advocated over recent decades as this approach can integrate different sources of knowledge and perform comprehensive analyses. We built a decision support tool using BBN to assist in habitat management of wild sheep populations throughout the species’ geographical range. The behaviour of the model was tested using scenario and sensitivity analysis. Habitat security, food and water suitability, and thermal cover were recognised as the main habitat variables determining wild sheep habitat suitability. Integrating the complex interactions between variables, the model can be applied for both diagnostic and predictive analyses answering “what if” and “how” questions. This approach can assist wildlife conservationists for building similar models for other poorly-studied threatened species. [Copyright &y& Elsevier]

Abstract: The heart fatty acid-binding protein gene (h-fabp) plays an important role in intracellular fatty acid transport. h-fabp had been cloned and sequenced and is considered a functional candidate gene for assessing the meat quality traits in pig, chicken, and cattle. However, there have only been few studies reported on this gene in sheep. In the present study, we cloned the full-length cDNA of h-fabp in the Lanzhou fat-tailed sheep using RACE technique. Its tissue specific expression profile in the skeletal muscle, liver and 4 fat depots at seven developmental stages was determined by real-time quantitative PCR. A 425bp 5′-RACE cDNA, 231bp 3′-RACE cDNA and 177bp intermediate fragment were spliced to obtain a 748bp full-length cDNA of h-fabp (GenBank accession number JQ780322). The phylogenetic analysis revealed that the Lanzhou fat-tailed sheep is more closely related to the goat, Capra hircus. In skeletal muscle, h-fabp was expressed at a very low level in one-month-old sheep. The expression level increased in three- and five-month-old sheep, and a peak expression level was observed in seven-month-old sheep. In nine- and eleven-month-old sheep, the gene expression level was similar to the mRNA level observed in three- and five-month-old sheep. In thirteen-month-old sheep, the h-fabp mRNA level was similar to the low expression level observed in one-month-old sheep. The expression pattern observed in the skeletal muscle at different developmental stages in sheep indicates that h-fabp expression declines with age. A similar conclusion on the h-fabp expression pattern in the liver was drawn from our findings in this study. The amount of h-fabp mRNA in the subcutaneous (s.c.) and visceral (v.s.) depots at each developmental stage was very low, and there was no significant difference in the expression levels among these depots. When we added the data from the seven developmental stages together, however, the highest h-fabp expression level was observed in the s.c. fat near the wither (s.c.W), followed by the v.c. perirenal (v.c.P), v.c. omental (v.c.O), and s.c. fat near the base of tail (s.c.T). These findings provide basic data that may be useful in future studies that investigate the role h-fabp plays in fat deposition and the regulation of fatty acid metabolism in sheep. [Copyright &y& Elsevier]

Genetic studies on taste sensitivity, and bitter taste receptors (T2R) in particular, are an essential tool to understand ingestive behavior and its relation to variations of nutritional status occurring in ruminants. In the present study, we conducted a data-mining search to identify T2R candidates in sheep by comparison with the described T2R in cattle and using recently available ovine genome. In sheep, we identified eight orthologs of cattle genes: T2R16, T2R10B, T2R12, T2R3, T2R4, T2R67, T2R13 and T2R5. The in silico predicted genes were then confirmed by PCR and DNA sequencing. The sequencing results showed a 99% to 100% similarity with the in silico predicted sequence. Moreover, we address the chromosomal distribution and compare, in homology and phylogenetic terms, the obtained genes with the known T2R in human, mouse, dog, cattle, horse and pig. The eight novel genes identified map either to ovine chromosome 3 or 4. The phylogenetic data suggest a clustering by receptor type rather than by species for some of the receptors. From the species analyzed, we observed a clear proximity between the two ruminant species, sheep and cattle, in contrast with lower similarities obtained for the comparison of sheep with other mammals. Although further studies are needed to identify the complete T2R repertoire in domestic sheep, our data represent a first step for genetic studies on this field. [ABSTRACT FROM AUTHOR]

Abstract: Two consecutive randomized double-blind field fertility experiments were conducted over a 4-month period and aimed at evaluating the association of two commercial soybean lecithin-based extenders (AndroMed [Minitub, Tiefenbach, Germany] and BioXcell [IMV Technologies, L''Aigle, France]) with pregnancy rates of chilled-stored (CS) and frozen-thawed (FT) ram semen. Semen samples with more than 2 × 109 sperm per mL and 70% progressive motile spermatozoa were collected via an artificial vagina from twelve proven fertile Chios rams, split-diluted with the above mentioned extenders, packaged in 0.25 mL straws and either stored at 5 ± 1 °C for 30 to 36 hours or frozen and thawed. Non-lactating multiparous ewes were inseminated in progestagen-synchronized estrus either with CS (AndroMed: N = 212 and BioXcell: N = 206; intracervical AI) or with FT (AndroMed: N = 114 and BioXcell: N = 92; laparoscopic intrauterine AI) semen. Ovulation was confirmed in all ewes based on determination of blood plasma progesterone (>1 ng/mL) 8 days post AI. Ewes were screened for pregnancy diagnosis by transabdominal ultrasonography 65 days post AI. BioXcell was superior to AndroMed in preserving the fertilizing potential of CS (P < 0.05) and FT (P < 0.005) semen. In AndroMed-stored semen, young rams (1.5–2.5 years old, N = 8) had a pregnancy rate (59.1%; 124/210) lower than that (72.4%; 84/116) of mature rams (4.5 to 5.5 years, N = 4; P < 0.025). Compared with AndroMed extender, processing of young ram semen in BioXcell extender improved pregnancy rates of CS (66.7%; 88/132 vs. 83.9%; 94/112; P < 0.005) and FT (46.2%; 36/78 vs. 71.0%; 44/62; P < 0.01) spermatozoa. Both extenders were similarly effective in preserving pregnancy rates of mature ram semen (P > 0.05). Ram-by-extender interactions were significant for pregnancy rates of CS and FT semen. Irrespective of extenders, overall pregnancy rates after intracervical and intrauterine AI were 75.1% and 62.2%, respectively (P < 0.001). In conclusion, BioXcell is a suitable extender for short- and long-term storage of ram semen. Selection of the ewes, farms, and extenders for intracervical AI programs can contribute to satisfactory fertility rates with semen preserved more than 24 hours at 5 °C. [Copyright &y& Elsevier]

Abstract: This study was undertaken to investigate the occurrence and public health significance of Cryptosporidium species/genotypes and subtypes in a newborn lambs. A total of 175 diarrheic fecal samples from lambs (younger than 21 days) were collected in seven sheep flocks located in western Romania, and were microscopically examined for the presence of Cryptosporidium oocysts after staining with modified Ziehl–Neelsen technique. Twenty-four (13.7%) fecal samples were tested Cryptosporidium positive by microscopy and were subjected for molecular characterization. All positive samples were successfully amplified through a nested polymerase chain reaction (PCR) of the small subunit (SSU) rRNA gene (18S). Cryptosporidium species were determined by restriction fragment length polymorphism (RFLP) analysis of the secondary PCR products using the conventional SspI and VspI restriction enzymes. The identified species were: Cryptosporidium parvum (20/24), C. ubiquitum (2/24) and C. xiaoi (2/24), respectively. PCR-RFLP results for C. ubiquitum and C. xiaoi isolates were confirmed by DNA sequencing. Subsequently, subtyping of seven randomly selected C. parvum isolates, based on sequence analysis of the GP60 gene, revealed the presence of five different subtypes (IIaA17G1R1, IIaA16G1R1, IIdA20G1, IIdA24G1 and IIdA22G2R1) belonging in two zoonotic subtype families (IIa and IId). These findings may suggest the potential role of the newborn lambs as a source for human cryptosporidiosis. This is the first published report about the presence of C. ubiquitum and C. xiaoi in lambs from Romania. [Copyright &y& Elsevier]

Abstract: We hypothesized that decreased diversity and/or unique polymorphisms in MHC class II alleles of bighorn sheep (BHS, Ovis canadensis) are responsible for lower titer of antibodies against Mannheimia haemolytica leukotoxin, in comparison to domestic sheep (DS, Ovis aries). To test this hypothesis, DRA and DRB transcripts from 24 captive BHS (Ovca-DRA and Ovca-DRB) were sequenced. Based on exon 2 (β1 domain) sequences, eight different Ovca-DRB cDNA sequences were identified in BHS. Six of them were 100% identical to previously reported Ovca-DRB genomic DNA sequences. The new alleles DRB*23 and DRB*24, were closely related to two other Ovca-DRB exon 2 genomic DNA sequences. Nineteen out of 24 BHS (79%) Ovca-DRB exon 3 (β2 domain) sequences were 100% identical to exon 3 sequence of DRB1 of DS (Ovar-DRB1). Ovca-DRA full length cDNA sequences exhibited >99% identity. Based upon exon 2 sequences, this BHS herd yielded higher Ovca-DRB allelic diversity than that reported in the previous study. Positively selected amino acid positions were identified in the peptide-binding groove of BHS and DS, but BHS showed more such sites. This highlights differing population histories, and may suggest differing needs for DR peptide-binding specificities. Presence of glutamine at position 52 (52Q) in some of the desert and captive BHS is predicted to alter the efficiency of DR dimerization, which may influence antigen presentation and Th cell activation. Functional assays with unique alleles should reveal whether the presentation of M. haemolytica leukotoxin peptides to Th cells by Ovca-DRB alleles is equivalent to that of Ovar-DRB1 alleles. [Copyright &y& Elsevier]

Abstract: Molecular DNA techniques in combination with appropriate reference population database and statistical methods are fundamental tools to forensic wildlife investigations. This is even more relevant when taxa with uncertain systematics are involved, as is the case of the genus Ovis (Bovidae), whose evolution has been influenced by multiple events of domestication. The Cypriot mouflon, Ovis orientalis ophion, a protected subspecies endemic to Cyprus, is threatened by poaching. This study deals with a case of alleged poaching that occurred in Cyprus (September, 2010). A car did not stop at a checkpoint and when finally blocked by the police, several bloodstained exhibits (n =12) were recovered. Three recently deceased mouflons were found by game wardens at the roadside. The Cyprus Veterinary Services established that these animals had been killed by gunshot. As part of the investigation, DNA testing was performed to establish if there was a link between the dead mouflons and the bloodstained exhibits. The mitochondrial Cytochrome-b gene (Cyt-b) and 12 loci of microsatellite DNA were used as markers. The Cyt-b sequences were obtained from 11 exhibits. They were the same as each other and the same as the single haplotype obtained from the three dead mouflons and all the investigated wild Cypriot mouflons (20 individuals). A database of wild mouflons (47 individuals) from which the unknown samples may have originated was generated. The probability of identity (P ID) of the microsatellite panel, computed by genotyping all 47 wild mouflons (10 selected loci, P ID =10−5), allowed us to assign nine exhibits to two out of the three carcasses (seven with very strong support: Likelihood Ratio, LR>3000 and Random Match Probability, RMP, <10−3). This study represents the first genetic reference for the Cypriot mouflon and the first published material of forensic wildlife investigations in Cyprus. [Copyright &y& Elsevier]

Abstract: Actinomyces hyovaginalis, an organism initially described from pigs, was recovered from nine sheep and a moufflon. Further strains of A. hyovaginalis were recovered from five samples from pigs over the same period. 16S rRNA sequencing and extensive phenotyping demonstrated high similarity between the ovine and porcine isolates; however differences with respect to erythritol, adonitol and l-arabitol fermentation were detected. Ovine isolates were made from various sample sites including abscesses and highlight the importance of the accurate identification of the various coryneform isolates which affect sheep. A. hyovaginalis can be added to the growing list of coryneforms which can cause disease in sheep including Corynebacterium pseudotuberculosis, Trueperella pyogenes and Arcanobacterium pluranimalium. [Copyright &y& Elsevier]

Abstract: The families of TGF-β proteins are the most important growth factors in the ovary for growth and differentiation of early ovarian follicles. Three related oocyte-derived members of the transforming growth factor-β superfamily, namely GDF9, BMP15 and BMPR-IB have been shown to be essential for follicular growth and ovulation. The objective of the present study was to detect the incidence of mutation in intronic portion of BMP 15 gene in Corriedale and local Kashmir valley sheep breeds. Blood samples were collected from 85 ewes and genomic DNA was extracted using the modified phenol chloroform method. The quantity and quality of extracted DNA was examined using spectrophotometry and gel electrophoresis, respectively. A fragment with the size of 356bp was amplified using polymerase chain reaction (PCR) with a pair of specific primers. The amplified PCR products were digested with Mph11031 restriction enzyme. In the presence of mutation at this locus, the Mph11031 enzyme cannot recognize the restriction site. However, here in the absence of mutations, the enzyme recognizes one restriction site and divides the amplified fragment into two fragments of 152 and 204bp. The 356bp fragment was also analyzed for polymorphism by SSCP technique. The results indicated two different banding patterns AA and AB for this fragment. Later on two different allelic forms A and B were confirmed by nucleotide sequencing. The 356bp nucleotide sequence was subjected to alignment analysis and it was observed that sequence similarity of this fragment with that of other sheep and Jining grey goat was more than 97.8%. Phylogenetic analysis revealed that both designated A and B alleles as well as published sequence of sheep form a common cluster indicating their evolutionary closeness. The origin of Jining grey goat was located some distance away from the sheep. The overall frequencies of AA and AB genotypes were 0.79 and 0.21. The breed wise frequencies were 0.78 and 0.22 in Corriedale sheep and the frequencies in Kashmir valley sheep were 0.80 and 0.20 for AA and AB genotypes, respectively. The overall allelic frequencies of A and B alleles were 0.89 and 0.11 whereas allelic frequencies Corriedale sheep was 0.89 and 0.11 and that of Kashmir valley sheep were 0.90 and 0.10. [Copyright &y& Elsevier]

Abstract: Mouflon and domestic Manchega sheep differ in the timing of their reproductive season under natural photoperiod (NP) conditions. This study examines whether they also differ in their reproductive responses to artificial photoperiod cues. For this, mouflons (n =24) and ewes (n =24) were exposed between the winter and summer solstices to artificial long days (LD: 16h light/day), to short days (SD) simulated via the use of melatonin implants, or to NP conditions (controls), and their ovulatory activity monitored. The effects of these treatments on annual changes in prolactin concentration were also recorded. In the LD mouflon ewes, the offset and onset (7 March±5 and 2 October±4, respectively) of cyclic ovulatory activity occurred earlier (P <0.001) than in the NP animals (26 April±6 and 20 October±2, respectively), but no differences were seen (P >0.05) between the SD and NP mouflon ewes in either the onset of anoestrus (12 May±6 and 26 April±6, respectively) or the onset of subsequent ovulatory activity (13 October±8 and 20 October±2, respectively); however the duration of the anoestrus period was significantly reduced in the SD. In LD Manchega ewes, the onset of anoestrus was advanced (2 February±5 vs 15 March±11), but ovulatory activity started at the same time as in NP Manchega ewes (16 July±4 vs 5 July±8). In the SD Manchega ewes, two animals showed continuous cyclic ovulatory activity over the course of the experiment while the remainder entered anoestrus two months later (16 May±6, P <0.001) than their NP counterparts. In these SD ewes, the onset of cyclic ovarian activity was very variable. An annual rhythm of plasma prolactin concentration was seen in both the mouflon and Manchega ewes under all three photoperiod conditions. However, the amplitudes of the changes seen in prolactin concentration were smaller in both the LD and SD animals than in the corresponding NP animals (P <0.001). In conclusion, the results show that these two types of Mediterranean sheep differ in their ovulatory response when subjected to artificial photoperiods. The results also suggest that refractoriness to SDs may be the most important physiological mechanism regulating the onset of anoestrus in highly seasonal breeds, but not in less seasonal breeds. [Copyright &y& Elsevier]

Abstract: The aim of this study was to optimise a two-tube reverse transcription triplex quantitative real time PCR (qRT-PCR) combining amplification of two loci with an internal amplification control (IAC) for detection and quantitation of hepatitis E virus (HEV) RNA and to validate its performance on a pool of biological samples. Optimisation was performed on serially diluted “home-made” RNA standards. The limit of detection was determined experimentally as 10copies/μl of the RNA standard for both amplification targets. The qRT-PCR was validated on a cohort of samples originating from 48 wild boars (Sus scrofa), 17 fallow deer (Dama dama) and 28 mouflons (Ovis musimon), with nested RT-PCR used as a reference method. qRT-PCR was found to be more specific for the detection of HEV RNA in examined samples. HEV RNA was detected in samples of five more animals (one wild boar and four mouflons) in comparison with nested RT-PCR. [Copyright &y& Elsevier]

Abstract: A highly species-specific polymerase chain reaction (PCR) was developed for authentic identification of raw and heat treated sheep meat (mutton). Oligonucleotide primers were designed against a unique target region of sheep mitochondrial D-loop, that yielded four PCR products upon gel electrophoresis of which two amplicons of molecular size 329 and 404bp were found highly diagnostic. Chances of cross-amplification were precluded by considering as many as 25 species and suitability of the developed sheep-specific PCR was confirmed in raw (n =20), cooked (60, 80 and 100°C for 30min), autoclaved (121°C for 30min) and microwave processed meats. Further, the sensitivity of the assay was established to be 0.1% for the detection of adulteration, while limit of detection of ovine DNA was as low as 1pg. This PCR technique has applications in solving problems related to meat adulteration, quality assurance, forensics, mutton authentication and traceability. [Copyright &y& Elsevier]

A genome scan for chromosomal regions influencing body conformation traits was conducted for a population of Spanish Churra dairy sheep following a daughter design. A total of 739 ewes from 11 half-sib sire families were included in the study. The ewes were scored for the 5 linear traits used in the breeding scheme of the Churra breed to assess body conformation: stature, rear legs-rear view, foot angle, rump width, and general appearance. All the animals, including the 11 sires, were genotyped for 181 microsatellite markers evenly distributed across the 26 sheep autosomes. Using the yield deviations of the raw scores adjusted for fixed factors as phenotypic measurements, a quantitative trait loci (QTL) analysis was performed on the basis of a multi-marker regression method. Seven suggestive QTL were identified on chromosomes Ovis aries (OAR)2, OAR5, OAR16, OAR23, and OAR26, but none reached a genome-wise significance level. Putative QTL were identified for all of the traits analyzed, except for general appearance score. The suggestive QTL showing the highest test statistic influenced rear legs-rear view and was localized on OAR16, close to the growth hormone receptor coding gene, GHR. Some of the putative linkage associations reported here are consistent with previously reported QTL in cattle for similar traits. To the best of our knowledge, this study provides the first report of QTL for body conformation traits in dairy sheep; further studies will be needed to confirm and redefine the linkage associations reported herein. It is expected that future genome-wide association analyses of larger families will help identify genes underlying these putative genetic effects and provide useful markers for marker-assisted selection of such functional traits. [ABSTRACT FROM AUTHOR]

Abstract: A forensic short tandem repeat (STR) typing test using a population database was developed to investigate an instance of poaching on the protected Sardinian mouflon. The case study involves a suspected poacher found in possession of a carcass, which he claimed was that of a sheep from his flock and had died accidentally. His claim was refuted by the molecular forensic analyses as DNA typing and the Bayesian assignment test revealed the carcass to be mouflon-derived; the genetic profile of the carcass matched also that of additional trace evidence collected by forestry officers at the scene of the kill. The matching evidence led to the poacher being charged with the illegal harvest of protected wildlife. Molecular techniques, in combination with a reference population database, and the appropriate statistical evaluation of genetic information, are fundamental to wildlife forensics. This approach allows DNA testing to be accepted in court as submissible evidence in the fight against poaching and other crimes involving wildlife. [Copyright &y& Elsevier]

Abstract: Intake induction refers to the phenomenon by which animals increase consumption of a less-valued meal when followed by a highly-preferred food relative to when followed by no food or by the same less-preferred food. In the Training phase of the present experiment, we assessed the induction effect in sheep using a within-subject design where learning could be tested while controlling for digestive state. Results showed that, once intake reached stability, subjects ate more low-nutritious food (oat hay) when followed than when preceded by a preferred food (soybean meal), supporting the learning hypothesis of induction. The objective of the second, Revaluation, phase of the experiment was to explore the associative mechanism of induction, for which we paired gastrointestinal malaise caused by lithium chloride intoxication with consumption of soybean meal or a control food (wheat bran). Despite subjects partially rejecting soybean meal relative to controls after the aversive conditioning protocol, oat hay consumption seemed unaffected by soybean meal devaluation. We conclude that intake induction in sheep may rely on changes in hedonic properties of the low-nutritious food based on its association with post-ingestive feedback from the preferred food (hedonic hypothesis), but not on an explicit anticipation of the latter (signalling hypothesis). [Copyright &y& Elsevier]

Abstract: The fundamental precondition to the survival and development of lambs is the formation of a close and exclusive bond between the ewe and her lambs. Maternal behaviour plays an important role in the establishment of this bond, and it is influenced by several factors including genotype. The aim of the present study was to detect differences in maternal behaviour in ewes (n =602) of five different breeds during periods of separation from their lambs (n =1003) and to relate the behaviour measurements in ewes to productivity traits of the lambs. Sheep belonged to one of the following German sheep breeds that have evolved under different husbandry intensities (in order from most to least intensive): Merino Landschaf (ML), Deutsches Schwarzköpfiges Fleischschaf (BH), Weißes Deutsches Bergschaf (MS), Rhönschaf (RS), and Graue Gehörnte Heidschnucke (GS). Within 12–24h after birth, lambs were separated from their dams, and dams were scored for maternal behaviour such as vocalising and activity during separation and sniffing and licking after reunification. GS showed highest rates (P <0.001) of vocalisation during separation, whereas BH showed lowest rates. Both BH and RS showed lower rates of activity during separation than ML and GS. Behaviour after reunification did not differ significantly (P >0.05) between breeds. Older ewes showed more interest in their offspring (P <0.05) and had lambs with higher birth weights (P <0.05). Significant (P <0.01) correlations and relationships were found between maternal behaviour and breed, mortality rate and birth weights. Therefore, it can be concluded that maternal behaviour differs between the observed breeds, potentially implying animal-welfare benefits under the given husbandry conditions for some breeds. Further research is required to establish to what extent within-breed selection for maternal behaviour may further improve animal-welfare and economy of sheep farming. [Copyright &y& Elsevier]

Abstract: The aim of the present study was to estimate the genetic intra-breed variability of Churra tensina and Churra lebrijana endangered breeds and to establish genetic relationships with Churra, Latxa and Merino breeds, as well as Spanish mouflon, by using 28 microsatellite markers, to provide useful information for their conservation. Allele frequencies and heterozygosity revealed high genetic variation in the two endangered breeds despite their small population size. Estimates of inbreeding coefficient (F IS) were significant for all breeds studied, except for Churra lebrijana breed. The highest inbreeding coefficient (F IS =0.143) was found in the Spanish mouflon. Genetic differentiation tests (F ST =0.121) and assignment of individuals to populations indicated the existence of defined breed populations, and low genetic flow between these breeds. The highest pairwise Reynolds distance (D R) values were observed between Mouflon and the domestic sheep breeds. Considering only domestic sheep breeds, the Churra lebrijana breed showed the highest pairwise D R values. The lowest values were found between Latxa and the other domestic sheep, except for Churra lebrijana. Results of pairwise D R values, as well as phylogenetic tree and bottleneck analysis showed an important genetic isolation of the Churra lebrijana breed from the other Churra types, and genetic signatures of a demographic bottleneck. Finally, structure analysis of populations detected a population subdivision in the Latxa sheep breed. In conclusion, this study presents valuable insight into the existing genetic variability of two Spanish endangered breeds, as well as the first study in Spanish mouflon based on microsatellite analysis. The high degree of variability demonstrated in Churra tensina and Churra lebrijana implies that these populations are rich reservoirs of genetic diversity. [ABSTRACT FROM AUTHOR]

Abstract: The aim of this study was to investigate the influence of light/dark (L/D) cycle, associated with restricted feeding, on total daily locomotor activity in sheep. Eight clinically healthy Comisana ewes were housed individually in a soundproof box and subjected to three different light and feeding periods. During period 1 (light on 8:00; light off 20:00) and during period 2 (light on 20:00; light off 8:00) hay and water were available ad libitum. During period 3 the animals were subjected to constant light and restricted feeding. Total locomotor activity was recorded by means of an Actiwatch-Mini actigraphy-based data loggers. The application of one-way for repeated measures ANOVA showed a significant effect of 24h (P <0.0001) on locomotor activity. The highest daily amount of locomotor activity was observed during the photophase than the scotophase (P <0.001). During all three periods locomotor activity exhibited robust daily rhythmicity with diurnal acrophases. During period 3 locomotor activity started before food supplement and it was concentrated during food availability. In conclusion, we can claim that the daily rhythm of locomotor activity in sheep is entrained by the light/dark cycle in couple with food availability. [Copyright &y& Elsevier]

Abstract: This study examines the patterns of melatonin secretion during postnatal development in wild (mouflon; n =7) and domestic (Spanish Merino; n =6) female lambs under their natural photoperiod conditions. The aim was to determine whether these types of sheep which differ in the timing of puberty, also differ in the establishment of daily melatonin secretory rhythms and/or in the nocturnal plasma melatonin secretion. In addition, the time when the lambs first reached nocturnal levels of melatonin similar to adults was also evaluated. For this purpose, the melatonin secretion in lambs was compared with those of their mothers. A day/night (D/N) difference in plasma melatonin concentration was noticed as early as 1 day after birth in the Merino female lambs (D: 5.9±1.0pg/ml compared to N: 22.0±3.3pg/ml; p <0.05), and by 1 week after birth in the mouflon female lambs (D: 4.9±0.3pg/ml compared to N: 56.9±15.3pg/ml; p <0.05). An effect of the genotype (p <0.05) and age (p <0.01) was detected on mean nocturnal plasma melatonin concentrations, which were lower in the Merino than in the mouflon lambs. Night-time plasma concentration of melatonin was also high in mouflon than in Merino mothers (p <0.05). No differences were detected between the wild genotype and the domestic one in the time when the lambs first reached nocturnal levels of melatonin similar to those of their mothers. [Copyright &y& Elsevier]

Abstract: Bluetongue serotype 4 (BTV4) has been detected for the first time in tissue samples from 2 mouflons (Ovis aries musimon) from the South of Spain, in a retrospective study. The samples included in this study had been fixed and paraffin-embedded for over a year prior to their analysis using a BTV group-specific and a BTV4-specific RT-PCR test. Lung and lymphatic nodes were found positive in both specimens. The amplified DNA was confirmed to be BTV4 by sequencing the RT-PCR products and comparing them with other sequences from GenBank. The combination of RNA extraction from paraffin-embedded samples and serotype-specific real-time RT-PCR assays provides the tools for the detection of BTV from samples stored for a long time. The results shown in this study set out the basis for a greater survey with fixed samples from different species of wild ruminants that the veterinary services have been collecting for years. [Copyright &y& Elsevier]

Abstract: In order to investigate the polymorphism of α-globin chain of hemoglobin amongst caprines, the linked Iα and IIα globin genes of Barbary sheep (Ammotragus lervia), goat (Capra hircus), European mouflon (Ovis aries musimon), and Cyprus mouflon (Ovis aries ophion) were completely sequenced, including the 5′ and 3′ untranslated regions. European and Cyprus mouflons, which do not show polymorphic α globin chains, had almost identical α globin genes, whereas Barbary sheep exhibit two different chains encoded by two nonallelic genes. Four different α genes were observed and sequenced in goat, validating previous observations of the existence of allelic and nonallelic polymorphism. As in other vertebrates, interchromosomal gene conversion appears to be responsible for such polymorphism. Evaluation of nucleotide sequences at the level of molecular evolution of the Iα-globin gene family in the caprine taxa suggests a closer relationship between the genus Ammotragus and Capra. Molecular clock estimates suggest sheep-mouflon, goat-aoudad, and ancestor-caprine divergences of 2.8, 5.7, and 7.1 MYBP, respectively. [Copyright &y& Elsevier]

Abstract: In domestic ruminants, the stratification of forestomach contents – the results of flotation and sedimentation processes – is an important prerequisite for the selective particle retention in this organ. A series of anatomical and physiological measurements suggests that the degree of this stratification varies between browsing and grazing wild ruminants. We investigated the forestomach contents of free-ranging mouflon and roe deer shot during regular hunting procedures. There was no difference between the species in the degree by which forestomach ingesta separated according to size due to buoyancy characteristics in vitro. However, forestomach fluid of roe deer was more viscous than that of mouflon, and no difference in moisture content was evident between the dorsal and the ventral rumen in roe deer, in contrast to mouflon. Hence, the forestomach milieu in roe deer appears less favourable for gas or particle separation due to buoyancy characteristics. These findings are in accord with notable differences in forestomach papillation between the two species. In roe deer, particle separation is most likely restricted to the reticulum, whereas in mouflon, the whole rumen may pre-sort particles to a higher degree. The results suggest that differences in forestomach physiology may occur across ruminant species. [Copyright &y& Elsevier]

Abstract: The objective of this study was to compare slaughtering performance, carcass characteristics, and meat quality in lambs of two different genotypes. Sixteen crossbred Mouflon×Sarda (M×S) and sixteen pure Sarda breed (S×S) suckling lambs were analysed. Chemical and fatty acid composition were determined on semitendinosus and longissimus dorsi muscles and perirenal and pelvic fats. Dressing percentage was higher (P <0.01) in M×S group and after histological dissection M×S carcasses had more muscle tissue (P <0.01) and less separable fat (P <0.05). Muscles of M×S had less cholesterol than the S×S lambs (P <0.01). Polyunsaturated/saturated and n−6/n−3 fatty acids ratios of the intramuscular lipids were optimal in both groups. Indexes of atherogenicity and of thrombogenicity in muscles varied between 0.9 and 1.1. The results indicated several good qualities in both the genotypes, but M×S lambs have some traits which could provide a higher market price. [Copyright &y& Elsevier]

Abstract: Observations of the flight response in mammals are commonly used to test the behavioural response to predation risk. Such a response is likely to be stronger in more sensitive individuals such as females, and females with offspring in particular, as well as during such periods as the birth season. From August 2005 to July 2006 we recorded the flight distances of different mouflon group types in a natural population in Sardinia. The comparison of the flight distances of the groups (n groups =375, n mouflons =1303) provided evidence for the key role of lamb presence. Female groups with lambs fled at greater distances than male groups and female groups without lambs. This difference was linked to the females’ priority to secure the survival of their offspring. Indeed, the uniformity of the flight distances of males and non-reproductive females was in contrast to the general expectations regarding ungulate species. In fact, smaller females were expected to be more sensitive to predation risk than larger males. The absence of major differences is probably due to the low sexual dimorphism of mouflon. Flight distances were shorter during the rut than during the lambing season, arguably because during the mating less time and energies were available for anti-predator behaviour. However, during the rut females with lambs still fled at greater distances than females without lambs, arguably because mothers took it as a priority to protect their parental investment rather then to find a potential mate. [Copyright &y& Elsevier]

Abstract: An interspecific embryo transfer program was conducted for genetic improvement and increasing the number of offspring from a flock of mouflon sheep in Argentina. The female donor mouflons were divided into three groups, G1 (n =5), G2 (n =4) and G3 (n =5). The total NIH-FSH-P1 dose given to each donor on the superovulatory treatment was 260, 200 and 160mg for G1, G2 and G3, respectively. The mouflons in G3 were maidens, while the others were multiparous. Domestic Corriedale ewes (n =60) were synchronized and used as recipients. The embryo recovery and transfer was performed by a surgical method. Mouflons (n =13) responded to the superovulatory treatment with an average of 9.1±2.8 ovulations. A low incidence of early luteal regression was found (1 out of 14 donors). Embryo recovery rates were 60, 31 and 76% in groups G1, G2 and G3, respectively. The percentage of transferable embryos obtained in G1 and in G2 exceeded 80%. None of the embryos obtained from G3 were of transferable quality. In G1, 25 transferable embryos were recovered and transferred to 13 recipients, resulting in a pregnancy rate of 76.9% (10/13). In G2, 10 embryos were transferred to 5 recipients, resulting in a 60% pregnancy rate (3/5). Lambing rate was 60% (15/25) and 30% (3/10) for G1 and G2, respectively. Thirteen lambs were born to the 14 donors following natural service after the embryo recoveries. This study demonstrates that the application of IET technology would have great reproductive impact, especially when the donor mouflon hinds are selected according to age and reproductive history. [Copyright &y& Elsevier]

Abstract: A polymerase chain reaction (PCR) assay was developed for the identification of meats from chamois (Rupicapra rupicapra), pyrenean ibex (Capra pyrenaica), and mouflon (Ovis ammon) by using oligonucleotides targeting mitochondrial D-loop sequences. A D-loop region (∼700–1000 bp) was firstly amplified and sequenced from various game and domestic meat DNAs, and three primer sets were then designed on the basis of nucleotide multialignment of the generated D-loop sequences. As expected from sequence analysis, PCR amplification of the targeted D-loop fragments was successfully achieved from chamois (88 bp), pyrenean ibex (178 bp), and mouflon (155 bp) meats, showing adequate specificity and reproducibility against a number of game and domestic meats. Mouflon and sheep meats were amplified together in accordance to the high nucleotide identity of their mt D-loop sequences. In this work, satisfactory amplification was also accomplished in the analysis of experimentally pasteurized (72°C for 30min) and sterilized (121°C for 20min) meats, with a detection limit of ∼0.1% for each of the targeted species. The proposed PCR assay represents a rapid and straightforward method for the detection of possible adulterations in game meat products. [Copyright &y& Elsevier]

Abstract: Parasitic infections can modify the host''s ability to metabolize drugs and other xenobiotics by altering the biotransformation enzymes; these changes may have various pharmacological, toxicological or physiological consequences. In our study, several activities of liver biotransformation enzymes and in vitro metabolism of albendazole (ABZ) were tested and compared in non-infected mouflons (Ovis musimon) and in mouflons infected by lancet fluke (Dicrocoelium dendriticum). Subcellular fractions of liver homogenates were isolated from 5+5 mouflon rams (1-year-old) parasitologically negative or naturally infected by fluke. From the eight enzyme activities that were assayed, only two activities significantly differ in the case of Dicrocoelium-infected versus non-infected animals. In infected mouflons, a significant increase (53%) of thiobenzamide-S-oxidase (TBSO) activity, corresponding mainly to the activity of flavine monooxygenase (FMO), and significant decrease (60%) of glutathione-S-transferase (GST) activity was observed. In addition, dicrocoeliosis caused the enhancement of ABZ hepatic biotransformation. The velocity of the formation of (+)-ABZ sulfoxide and ABZ sulfone was significantly increased. However, the shifts in ABZ biotransformation were very mild that undesirable alterations in ABZ pharmacokinetic are not expected. From this point of view, the use of ABZ in the therapy of mouflon dicrocoeliosis in young animals can be recommended. The treatment of the same mouflons by other drugs that are mainly conjugated with glutathione, seems to be more problematic; hence, all consequences of documented reduced GST activity should be accounted. [Copyright &y& Elsevier]

Annual variations in the growth of horns, and their correlation with seasonal changes of testicular size, and prolactin (PRL) and melatonin secretion were monitored in six pubertal mouflon rams living in their original latitude (40°N). Mouflons born and maintained under captive conditions were classified in two age classes: sub-adult (2 years; n = 3) and adult (≥3 years; n = 3). The rate of horn growth was greater (P < 0.001) in sub-adult than in adult mouflon rams. Horn growth was influenced by season in both adult and sub-adult mouflons (P < 0.05) with largest monthly growth occurring in spring and summer. Seasonal variations of plasma PRL concentrations were correlated with horn growth in adult, but not in sub-adult mouflon rams. The rate of horn growth was inversely correlated with testicular size (r = -0.5, P = 0.07). Seasonal changes in the amplitude of the daily melatonin rhythm in solstices and equinoxes were observed, which were not correlated with variations in the rate of horn growth. These results provide support for a possible role of PRL in the control of growth of horns in the adult mouflon. [Copyright &y& Elsevier]

European mouflon sheep are an endangered species of ovidae residing primarily in the mountenous habitat of the islands of Sardinia and Corsica. The purpose of this study was to assess the fertilizing capacity of cryopreserved European mouflon spermatozoa after AI in synchronized mouflon and domestic ewes and after IVF in in vitro matured mouflon and domestic ewe oocytes collected by OPU technique. Domestic ram (Ovis aries) spermatozoa served as control. Semen was collected by artificial vagina from three mouflons and three domestic rams during the breeding season and was cryopreserved. At thawing, no significant differences in sperm viability were found between the wild and the domestic species (53.1 ± 4.6% versus 56.0 ± 4.7%) whereas the percentage of acrosome-intact sperm was lower in mouflon (55.5 ± 4.6%) than in ram semen (62.7 ± 3.1%; P < 0.05). Lambing rate did not differ between synchronized mouflon and domestic ewes (5/11 versus 8/12) after 150 and 156 days of pregnancy, respectively. After two OPU sessions, 87 and 132 oocytes were collected from three hyperstimulated mouflon and three domestic ewes. Cryopreserved/thawed semen was inseminated with an endoscope into the uterus of corresponding species during the non-breeding season. The oocytes were matured and fertilized in vitro; 61/73 mouflon and 81/101 domestic ewe oocytes were found to be fertilized. From these, we obtained 6/61 and 17/81 blastocysts. After vitrification and thawing, the hatching rate showed no significant difference between mouflon and sheep blastocysts (4/6 versus 14/17). In conclusion, our data showed that cryopreserved mouflon spermatozoa can be successfully used to carry out a genuine and complete program of genetic restoration in small and isolated groups of European mouflons. [Copyright &y& Elsevier]

One of the major problems in serodiagnosis in wild animals is unavailability of specific antiglobulin conjugate. Our study focuses on validation of Protein A/G dependent ELISA in game animals like deer and mouflons as well as in hunting dogs. Binding ability of Protein A/G-conjugate to antibodies was the highest in dogs followed by fallow deer and mouflons. Three different whole cell Borrelia antigens were used to evaluate antigen dependent variation. In new Protein A/G-ELISA the highest sensitivities (90.50%, deer; 85.37%, mouflon & 94.29%, dog) were obtained by B. garinii antigen, with no statistically significant variation (χ2, P>0.05) among all other antigens used. Average seroprevalences observed in deer, mouflons and dogs were 44.90%, 29.41% and 30.43%, respectively. Marked influence of age on seroprevalence was noticed. Protein A/G – ELISA proved to be sensitive and promising diagnostic tool in serodiagnosis of Lyme disease in game ungulates and it can be used effectively for serosurvey in different wild mammals. [Copyright &y& Elsevier]

The aims of this study were to examine whether mouflons exposed to constant long and short day photoperiods are able to exhibit an annual cycle of hair growth and moult, and prolactin (PRL) secretion. Mouflon ewes were assigned to three groups of treatment. Ewes were maintained, either under natural photoperiod (control, n=9), or received a series of subcutaneous melatonin implants from December to April (n=8), or were exposed to a constant long day photoperiod (16-h light:8-h dark; 16L:8D) during 18 months (n=7). Blood was collected weekly to determine PRL concentrations, and hair samples were clipped weekly from the base of the neck to measure the length of predominant hair. Under constant long days and with melatonin implants, mouflons expressed an annual rhythm of PRL secretion, even though these treatments modified the times of rise or falling of PRL concentrations throughout the year. Hair growth initiation was almost coincident with the summer solstice in both control and melatonin-implanted mouflons but occurred two months earlier in long day hold mouflons (P<0.001). Long day hold mouflons had a lower hair growth rate than control and melatonin-implanted mouflons (P<0.001), and at the end of the experiment, a shorter hair length (3.4±0.24 cm; P<0.01) than control (4.3±0.17 cm), and melatonin-implanted mouflons (4.2±0.12 cm). Our data support the conclusion that in mouflon, an endogenous circannual rhythm of PRL secretion exists, and that the seasonal cycle of hair growth and moult appears to depend, at least in part, on circulating levels of PRL. [Copyright &y& Elsevier]

• We analyzed the population diversity and selection signatures of the high altitude sheep breed Changthangi. • Changthangi was compared to three breed groups, namely European commercial, Indian, and breeds inhabiting plains. • Results revealed multiple functional regions on chromosomes 2, 3, 6 and 18 to be under selection in Changthangi. • The UCP gene family, associated with adaptation to cold and hypoxic conditions, was found to be under selection in Changthangi. Changthangi is a high-altitude sheep breed of India that is adapted to cold and hypoxic climate of Himalayas. In the present study, we analysed population structure of Changthangi and contrasted it with selected Indian and European commercial sheep breeds to detect genomic regions under positive selection. The Illumina OvineSNP50v1 genotype data on 292 animals from seven different sheep breeds i.e., Changthangi (n = 29), Garole (n = 26), Deccani (n = 24), Tibetan (n = 37), Rambouillet (n = 102) and Australian Merino (n = 50) was used. European Mouflon (n = 24) was used as an out-group for studying the stratification and phylogenetic lineage. While the principal component analysis (PCA) revealed Changthangi to cluster with Tibetan sheep; TREEMIX and ADMIXTURE results also detected the introgression of lowland Indian sheep inheritance in Changthangi. Changthangi sheep were compared with other breed groups as reference i.e., commercial (Australian Merino and Rambouillet), Indian (Deccani, Garole and Tibetan) and breeds inhabiting plains (Australian Merino, Rambouillet, Deccani and Garole). Genomic comparisons of Changthangi using cross population extended haplotype homozygosity (XP-EHH) showed multiple functional regions present on Ovis aries (Oar) chromosomes 2, 3, 6 and 18 to be under selection in Changthangi sheep. These regions were related with adaptation to climatic and hypoxic stressors, fleece characteristics and functioning of immune and reproductive systems. UCP genes, associated with adaptation to cold and hypoxic conditions, were the main loci under positive selection in Changthangi sheep population. The selection signals in Indian and European commercial sheep breeds were mainly associated with body weight and carcass traits. Furthermore, selection signals found in different comparisons were found to be part of different quantitative trait loci (QTLs) associated with important traits in different breed classes. The genes present in these regions are suitable candidates for future studies on the genetic mechanisms underlying high-altitude adaptation. [ABSTRACT FROM AUTHOR]

• Low genetic variability not associated with intense inbreeding. • Intense gene flow due mostly to mobile male mouflons. • Evidence for a philopatric behaviour of female mouflons. • High habitat heterogeneity and low human disturbance as main drivers of the spatial structure. • A new Ovis haplogroup including the Cyprus and the Anatolian mouflon as sister taxa. The mouflon (Ovis gmelini ophion) is the largest wild terrestrial mammal of Cyprus. Regarded as the local flagship species, its population range is limited to the mountainous Paphos Forest and adjacent areas including part of Troodos National Forest Park (> 700 Km2). This species, protected by both national and international legislation, is classified as Endangered by the IUCN, with livestock intrusion, poaching and habitat loss as the main threatening factors. We determined the spatial genetic structure by investigating sexes separately and shed further light on the Cyprus mouflon placement among Ovis haplogroups (HGs) to enforce its protection within an adaptive conservation framework. We genotyped 108 samples collected across the entire species' range at both14 loci of the microsatellite DNA and the mitochondrial DNA Control Region (CR). Microsatellite genotyping indicated that the wild population retained low genetic variability, which, however, was not associated with a level of inbreeding raising particular concern (F IS = 0.12). An overall weak spatial genetic structure was disclosed, consistently with the limited extension of the mouflon range, the lack of significant physical barriers to dispersal and the intense gene flow mostly occurring along a northeast-southwest axis across the forest. Nevertheless, evidences of structure were found for females in compliance with their philopatric behaviour. The disclosure of unique features compared to Mediterranean and Near East conspecifics, such as the occurrence of a six-fold 76 bp-long repeated motif in the Cyprus mouflon CR, along with the outcome of a phylogenetic reconstruction (based on a far higher number of Ovis GenBank records and Cyprus haplotypes than in previous studies) inspired the proposal of a new haplogroup (HGC2) which included the Anatolian mouflon (O. g. anatolica , former HGX) as sister taxon. While both high habitat heterogeneity and low human disturbance were the main drivers in determining the overall spatial structure, future conservation efforts to preserve this valuable genetic resource should focus on avoiding possible introgressive hybridisation with co-grazing livestock to the edge of its range. [ABSTRACT FROM AUTHOR]

Free-ranging wild ungulates are widespread in Austria, and act as hosts (i.e. feeding hosts) for ticks, including Ixodes ricinus , and as reservoir hosts for pathogens transmitted by I. ricinus. Due to climate change, the abundance of I. ricinus might be increasing, which could potentially lead to higher prevalences of tick-borne pathogens, such as Babesia spp. and Anaplasma phagocytophilum , some known for their zoonotic potential. Human babesiosis is classified as an emerging zoonosis, but sufficient data of these parasites in central Austria is lacking. In order to assess the abundance of vector-borne pathogens, blood of roe deer (Capreolus capreolus ; n = 137), red deer (Cervus elaphus ; n = 37), mouflons (Ovis gmelini ; n = 2) and chamois (Rupicapra rupicapra ; n = 1), was collected and tested for pathogen DNA in two different sampling sites in central Austria. DNA of tick-borne pathogens was detected in 15.5 % (n = 27) of these animals. Babesia capreoli (n = 22 in roe deer; n = 1 in mouflon), Babesia divergens (n = 1, in red deer), and Anaplasma phagocytophilum (n = 4, in roe deer) were detected. DNA sequencing of the 18S rRNA gene of two C. capreolus samples from Upper Austria featured another new genotype of Babesia , which differs in one nucleotide position to B. divergens and B. capreoli , and is intermediate between the main genotypes of B. capreoli and B. divergens within the partial gene sequence analyzed. This study thus confirms that B. capreoli, B. divergens, and A. phagocytophilum are present in free-ranging ungulates in central Austria. Further testing over a longer period is recommended in order to assess the impact of climate change on the prevalence of blood parasites in central Austria. [ABSTRACT FROM AUTHOR]

• Allometric growth by the horns of bovid mammals is said to be biphasic. • The appearance of biphasic growth is an artifact of logarithmic transformation. • Arithmetic distributions are monophasic and well described by power functions. • Studies of bivariate allometry should focus on untransformed data. I used the equivalent of nonlinear analysis of covariance (ANCOVA) to re-examine relative growth by the horns on males and females of alpine ibex (Capra ibex) and mouflon sheep (Ovis gmelini). A prior study of allometric growth by the horns on these animals described a pattern of biphasic allometry for both sexes, with two different mathematical equations being required to capture the pattern of variation over the full range in body size. However, the investigation in question used conventional analytical methods based on logarithmic transformations, which alter bivariate distributions and commonly introduce problems with analysis and interpretation. My new analyses of data for both species revealed that untransformed observations for both males and females are monophasic and that they are described quite well by three-parameter power equations with negative intercepts. Equations for males follow a steep upward trajectory whereas those for females follow much shallower paths. The negative intercepts indicate that males and females of both species must attain a minimum body size before horns begin to develop. Conclusions from the earlier investigation were based on inaccurate perceptions of pattern in the data. Future studies should be based on graphical and analytical analysis of observations expressed on the original arithmetic scale. [ABSTRACT FROM AUTHOR]

• First surveillance on Schmallenberg virus in zoo animals in Spain. • The results evidence SBV circulation in zoo parks in Spain over the last decade. • Seropositivitity was detected before the first Schmallenberg disease outbreak was reported in this country. • Surveillance in zoo species, particularly artiodactyls, could be a complementary approach to monitoring SBV activity. • Further studies are warranted to assess the impact of this virus on the health status of susceptible zoo animals Schmallenberg virus (SBV) is a newly emerged vector-borne pathogen that affects many domestic and wild animal species. A serosurvey was carried out to assess SBV exposure in zoo animals in Spain and to determine the dynamics of seropositivity in longitudinally sampled individuals. Between 2002 and 2019, sera from 278 animals belonging to 73 different species were collected from five zoos (A–E). Thirty-one of these animals were longitudinally sampled at three of these zoo parks during the study period. Seropositivity was detected in 28 (10.1 %) of 278 animals analyzed by blocking ELISA. Specific anti-SBV antibodies were confirmed in 20 (7.2 %; 95 %CI: 4.2–10.3) animals of six different species using virus neutralization test (VNT). The multiple logistic regression model showed that "order" (Artiodactyla) and "zoo provenance" (zoo B; southern Spain) were risk factors potentially associated with SBV exposure. Two (8.7 %) of the 31 longitudinally-sampled individuals showed specific antibodies against SBV at all samplings whereas seroconversion was detected in one mouflon (Ovis aries musimon) and one Asian elephant (Elephas maximus) in 2016 and 2019, respectively. To the best of the author's knowledge, this is the first surveillance conducted on SBV in zoos in Spain. The results confirm SBV exposure in zoo animals in this country and indicate circulation of the virus before the first Schmallenberg disease outbreak was reported in Spain. Surveillance in zoological parks could be a complementary approach to monitoring SBV activity. Further studies are warranted to assess the impact of this virus on the health status of susceptible zoo animals. [ABSTRACT FROM AUTHOR]

Abstract: The introduction of alien species is one of the main threats to conservation. However, the same species may be threatened and be a threat in nearby areas. This is the case of the Barbary sheep (Ammotragus lervia) and mouflon (Ovis orientalis), which greatly hinder the management and conservation of biodiversity outside their native range. To prevent the extinction of many endemic plants, it is necessary to overcome several methodological difficulties. [Copyright &y& Elsevier]

Abstract: This report presents a case of Echinococcus granulosus infection in a Turkish mouflon (Ovis gmelinii anatolica). The mouflon was found dead and postmortem examination was conducted. The diagnosis of liver hydatid cyst was established by pathological and molecular findings. For the purpose of the molecular characterization of cyst material, mitochondrial cytochrome oxidase subunite 1 (mt-CO1) gene region was amplified and sequence analyses were carried out. Sequence corresponding to mt-CO1 gene was identical to a sequence reported for common sheep strain (G1). This is the first report of an occurrence and molecular characterization of E. granulosus in Turkish mouflon. [Copyright &y& Elsevier]