Interaction effect of tegmen tympani and superior semicircular canal statuses on the thickness of the roof of the glenoid fossa: a cross-sectional descriptive study.

PURPOSE: Homogeneous development of temporal bone structures is explained by their ontogenic origin; tegmen tympani (TT) and superior semicircular canal (SSC) are related with the glenoid fossa at the temporomandibular joint (TMJ). Therefore, our objective was to determine a possible relationship between TT status (dehiscence or integrity) and the roof of the glenoid fossa (RGF) thickness; SSC status has also been considered. METHODS: This cross-sectional descriptive study was conducted in two tertiary hospitals on 95 patients (109 ears) presenting hypoacusia, facial palsy, vertigo, tinnitus, and other single or combined symptoms, and submitted to a thin-section multidetector-row computed axial tomography (CT) scan. RESULTS: A significant interaction effect of TT × SSC statuses on RGF thickness was found (p = 0.049). A significant difference in RGF thickness was found only for SSC integrity status between TT integrity and TT dehiscence (p = 0.004). The TT dehiscence increased the risk for RGF dehiscence 12.047 times (p = 0.002). CONCLUSIONS: There is an interaction effect of the statuses of both TT and SSC on the thickness of the RGF, instead of an independent effect of the TT status. When RGF dehiscence is found, TT and SSC statuses should be assessed, to discard associated dehiscences.

Influence of Sex and Age on Posterior Semicircular Canal Thickness.

OBJECTIVE: The aim of this study is to determine whether sex and age influence posterior semicircular canal (PSC) thickness. METHODS: This observational study was conducted in 3 tertiary hospitals. The minimal distance between the PSC and the posterior cranial fossa (PSC thickness) was estimated by thin-section multidetector row computed axial tomography (CAT) scan of the temporal bones. Nonselected consecutive patients of all ages (607 temporal bones) were considered. RESULTS: A significant effect was only detected for sex (F = 5.418, p = 0.020); PSC thickness showed a higher mean value in women (mean difference ± SE: 0.224 ± 0.096 mm). A significant and negative r value was detected for males aged >45 years (-0.173, p = 0.026); in that group of patients, PSC thickness decreased as age increased (0.018 ± 0.008 mm/year). For females aged ≤45 years, a significant and positive r value was found (0.198, p = 0.022); in that group, PSC thickness increased as age increased (0.020 ± 0.008 mm/year). CONCLUSIONS: PSC thickness did not significantly evolve with age in young males (≤45 years) but it decreased from age 45 years onwards. On the other hand, PCS thickness increased with age in women until the age of 45 years and it did not significantly change in older females.

In silico identification and three-dimensional modelling of the missense mutation in ADAMTS2 in a sheep flock with dermatosparaxis.

BACKGROUND: Dermatosparaxis (Ehlers-Danlos syndrome in humans) is characterized by extreme fragility of the skin. It is due to the lack of mature collagen caused by a failure in the enzymatic processing of procollagen I. We investigated the condition in a commercial sheep flock. HYPOTHESIS/OBJECTIVES: Mutations in the ADAM metallopeptidase with thrombospondin type 1 motif, 2 (ADAMTS2) locus, are involved in the development of dermatosparaxis in humans, cattle and the dorper sheep breed; consequently, this locus was investigated in the flock. ANIMALS: A single affected lamb, its dam, the dam of a second affected lamb and the rams in the flock were studied. METHODS: DNA was purified from blood, PCR primers were used to detect parts of the ADAMS2 gene and nucleotide sequencing was performed using Sanger's procedure. Skin samples were examined using standard histology procedures. RESULTS: A missense mutation was identified in the catalytic domain of ADAMTS2. The mutation is predicted to cause the substitution in the mature ADAMTS2 of a valine molecule by a methionine molecule (V15M) affecting the catalytic domain of the enzyme. Both the 'sorting intolerant from tolerant' (SIFT) and the PolyPhen-2 methodologies predicted a damaging effect for the mutation. Three-dimensional modelling suggested that this mutation may alter the stability of the protein folding or distort the structure, causing the protein to malfunction. CONCLUSIONS AND CLINICAL IMPORTANCE: Detection of the mutation responsible for the pathology allowed us to remove the heterozygote ram, thus preventing additional cases in the flock.

Subtype Distribution of Blastocystis spp. in Patients with Gastrointestinal Symptoms in Northern Spain.

Limited molecular data exist on the prevalence and subtype distribution of Blastocystis spp., the most prevalent parasite in human and animal feces worldwide. A total of 44 different subtypes (STs) of Blastocystis are currently recognized based on the sequence of the small subunit ribosomal RNA (SSU-rRNA) gene. This is a molecular study of Blastocystis spp. in hospitalized patients with gastrointestinal symptoms in northern Spain. We analyzed 173 Blastocystis-positive patients with gastrointestinal symptoms by using nested PCR for molecular detection, subtype identification, phylogenetic analyses, and genetic diversity assessment. ST2 (34.1%) and ST3 (34.7%) predominated, followed by ST1 (15.6%) and ST4 (15.6%). Mixed infections with different subtypes were observed in some patients. Sequence analysis revealed for the first time in European humans the allele 88 (a variant of ST1). In other cases, alleles commonly found in animal samples were detected (allele 9 in ST2, allele 34 in ST3, and allele 42 in ST4). Phylogenetic analysis showed high variability in ST1 and ST2, suggesting a polyphyletic origin, while both ST3 and ST4 exhibited higher genetic homogeneity, indicating a possible monophyletic origin and recent transmission to humans. These data confirm Blastocystis spp. subtype diversity and may help in understanding the evolutionary processes and potential zoonotic transmission of this parasite.

Molecular Diagnosis of Footrot and Contagious Ovine Digital Dermatitis in Small Ruminants in the Iberian Peninsula.

Contagious ovine digital dermatitis (CODD) and footrot (FR), a sub-acute or acute necrotic (decaying) infectious disease involving the hoof and underlying tissues, pose economic challenges to herds in Spain and worldwide. The aetiological agent for FR is Dichelobacter nodosus, while CODD is caused by pathogenic Treponema phylogroups. We detail the findings derived from the analysis by qPCR of 105 pooled samples from 100 ovine and five caprine herds in Spain and Portugal, alongside 15 samples from healthy flocks in order to identify Dichelobacter nodosus, Fusobacterium necrophorum, Treponema spp., and three pathogenic Treponema phylogroups (T. phagedenis, T. medium, and T. pedis). Treponema spp. were detected in all 120 pools, including samples from the 15 healthy flocks where only one positive result for F. necrophorum was recorded. Mixed infections by agents different from Treponema spp. were identified in 68.57% of samples. Positive results for F. necrophorum and/or D. nodosus, were obtained for 91.4% of the pools, whereas the presence of the three pathogenic Treponema phylogroups was rare: each of them appeared in isolation in a single pool, while they were found in 18 pools in combination with other agents. While F. necrophorum was the sole finding in 16.2% of samples from affected herds, D. nodosus (the footrot causative agent) was only detected in 61% of affected farms. An improved qPCR protocol was implemented to determine the serogroups of D. nodosus in the samples and found all of them (except the G serogroup), often in combined infections (35.1%). This report concludes with comprehensive proposals for diagnosing, preventing, and treating hoof ailments, remarking the interest of the information about D. nodosus serogroups in order to improve the efficiency of immunization by choosing appropriate vaccine protocols.

Host association of Cryptosporidium parvum populations infecting domestic ruminants in Spain.

A stock of 148 Cryptosporidium parvum DNA extracts from lambs and goat kids selected from a previous study examining the occurrence of Cryptosporidium species and GP60 subtypes in diarrheic lambs and goat kids in northeastern Spain was further characterized by a multilocus fragment typing approach with six mini- and microsatellite loci. Various degrees of polymorphism were seen at all but the MS5 locus, although all markers exhibited two major alleles accounting for more than 75% of isolates. A total of 56 multilocus subtypes (MLTs) from lambs (48 MLTs) and goat kids (11 MLTs) were identified. Individual isolates with mixed MLTs were detected on more than 25% of the farms, but most MLTs (33) were distinctive for individual farms, revealing the endemicity of cryptosporidial infections on sheep and goat farms. Comparison with a previous study in calves in northern Spain using the same six-locus subtyping scheme showed the presence of host-associated alleles, differences in the identity of major alleles, and very little overlap in MLTs between C. parvum isolates from lambs and those from calves (1 MLT) or isolates from lambs and those from goat kids (3 MLTs). The Hunter-Gaston index of the multilocus technique was 0.976 (95% confidence interval [CI], 0.970 to 0.982), which supports its high discriminatory power for strain typing and epidemiological tracking. Population analyses revealed the presence of two host-associated subpopulations showing epidemic clonality among the C. parvum isolates infecting calves and lambs/goat kids, respectively, although evidence of genetic flow between the two subpopulations was also detected.

Feline osteochondromatosis in a 12-year-old feline leukaemia virus-negative cat.

Feline osteochondromatosis is a spontaneous osteocartilaginous exostosis associated with feline leukaemia virus (FeLV) infection or due to a frameshift variant in the exostosin glycosyltransferase 1 (EXT1) gene. Osteochondromatosis was diagnosed in an indoor-only, 12-year-old, neutered female, Russian Blue cat. Radiographs revealed bilateral calcified proliferations in the elbow, costochondral and sternochondral joints, which distorted the normal skeletal structure. Grossly, the proliferated joints presented with consistent, rounded masses, causing complete ankylosis. The main histopathological finding was an osteocartilaginous proliferation composed of multiple irregular islands of well-differentiated hyaline cartilage surrounded and delimited by osteoid tissue. Immunohistochemistry of the osteochondromas, bone marrow and mediastinal lymph nodes, using a primary anti-FeLV gp70 antibody, and FeLV proviral DNA real-time polymerase chain reaction on bone marrow were negative. Sequencing of exon 6 of the EXT1 gene was performed and nucleotide BLAST analysis demonstrated the absence of a frameshift variant. This study reports the only case of spontaneous feline osteochondromatosis in an animal more than 10 years old.

Phylogeographic and population genetic structure of hound-like native dogs of the Mediterranean Basin.

The dog was probably the first domesticated animal. Despite extensive archaeological and genetic investigations, the origin and the evolution of the extant dogs are still being debated. Dog breeds that have over time been selected for hunting share common ancestral traits. This study represents the first comprehensive attempt to survey at the genomic and mitochondrial level eight hound-like dogs breeds indigenous to the Mediterranean Basin to determine if they share common ancient origins. Results from the microsatellite analysis indicate that all the dog populations have a low inbreeding value.The Kelb tal-Fenek has a high divergence from the current Egyptian street population, however there is not enough evidence from this study to exclude completely the potential of an ancient common relationship. Overall, the mitochondrial results indicate high frequencies of haplogroups A and B and a low representation of haplogroup C, while only one Egyptian dog could be assigned to haplogroup D. Results reveal identities and shared clades, suggesting the conservation of ancient European mitotypes in the Mediterranean hound-like breeds, especially in the Egyptian population. Although none of the dog populations/breeds participating in this study indicate to be direct descendants of the Egyptian dogs, they still have a very close morphologically resemblance to those iconic Egyptian dogs often depicted in ancient art forms and share some genetic links with the current Egyptian population. Further research is required with other markers such us complete mitogenomes and SNP panels to confirm the complex history of the Mediterranean dogs involved in this study.

Prevalence and Associated Factors of Blastocystis sp. Infection in Patients with Gastrointestinal Symptoms in Spain: A Case-Control Study.

Blastocystis sp. is known to be the most prevalent parasite in fecal samples of humans worldwide. In the present report, a case-control study (1:9.89 (≈10)) was performed, by analyzing data from 3682 patients who attended a public hospital in the northern area of Spain showing gastrointestinal symptoms. Diagnosis was performed in human fecal samples by means of optical microscopy. The prevalence of Blastocystis sp. in patients with gastrointestinal symptoms was 9.18% (338/3682). Most of the Blastocystis sp.-infected patients tested negative for protozoa and helminths, and were underweight and foreign-born (26.4%), mainly from Africa and Central/South America. Gastrointestinal symptoms, such as abdominal pain, anorexia, halitosis, plus relative eosinophilia, as well as co-infections with pathogenic bacteria were associated with Blastocystis sp. infection. Both type 2 diabetes and treatment with immunosuppressive medicines at the time of Blastocystis sp. detection were associated with a higher proportion of infected patients. This is the first case-control study of Blastocystis sp. in humans in northern Spain and may contribute to surveillance and intervention strategies by public health authorities.

Occurrence of Rotavirus A Genotypes and Other Enteric Pathogens in Diarrheic Suckling Piglets from Spanish Swine Farms.

Species A rotavirus (RVA) is a major viral pathogen causing diarrhea in suckling piglets. Studies on its genetic heterogeneity have implications for vaccine efficacy in the field. In this study, fecal samples (n = 866) from diarrheic piglets younger than 28 days were analyzed over a two-year period (2018-2019). Samples were submitted from 426 farms located in 36 provinces throughout Spain and were tested using real-time PCR (qPCR) and reverse transcription real-time PCR (RT-qPCR) for five enteric pathogens. The individual prevalence was 89.4%, 64.4%, 44.9%, 33.7% and 4.4% for Clostridiumperfringens, Clostridioides (formerly Clostridium) difficile, species A rotavirus, species C rotavirus and porcine epidemic diarrhea virus, respectively. Most specimens (96.9%) were positive for at least one of the target pathogens, and more than 80% of samples harbored mixed infections. Nucleotide sequencing of 70 specimens positive for RVA revealed the presence of the VP7 genotypes G4, G9, G3, G5, G11 and the VP4 genotypes P7, P23, P6 and P13, with the combinations G4P7 and G9P23 being the most prevalent, and especially in the areas with the highest pig population. The study shows the extensive genetic diversity of RVA strains as well as discrepancies with the genotypes contained in the vaccine available in Spain, and multiple amino acid differences in antigenic epitopes of different G- and P- genotypes with the vaccine strains. Further investigations are needed to determine the efficacy of the vaccine to confer clinical protection against heterologous strains.

Coxiella burnetii and Co-Infections with Other Major Pathogens Causing Abortion in Small Ruminant Flocks in the Iberian Peninsula.

Coxiella burnetii is an intracellular bacterium causing human Q fever and reproductive disorders in domestic ruminants. We analyzed the occurrence of C. burnetii and co-infections with six other major pathogens causing abortion in sheep (1242 cases) and goat (371 cases) flocks from Spain and Portugal. After real-time PCR detection, co-infections were established by principal component and cluster analysis that grouped cases based on the joint presence/absence of several microorganisms. C. burnetii and Chlamydia abortus were the most common abortifacient agents with approximately 75% of cases from both hosts testing positive, followed by Toxoplasma gondii, Campylobacter sp., Salmonella enterica, border disease virus and Neospora caninum. C. burnetii was significantly more common than C. abortus in goat abortions (p < 0.001). Co-infections with at least two pathogens were found in more than 66% cases of ovine abortions and 36% cases of caprine abortions testing positive for C. burnetii, mostly including mixed infections with only C. abortus. These findings indicate that both pathogens are the most significant ones to be readily prevented by vaccination in this geographical area. Biosecurity and biocontainment measures are also steadfastly recommended to prevent both the economic losses and public health risks associated with most of these abortifacient agents.

Multilocus fragment typing and genetic structure of Cryptosporidium parvum Isolates from diarrheic preweaned calves in Spain.

A collection of 140 Cryptosporidium parvum isolates previously analyzed by PCR-restriction fragment length polymorphism (PCR-RFLP) and sequence analyses of the small-subunit (SSU) rRNA and 60-kDa glycoprotein (GP60) genes was further characterized by multilocus fragment typing of six minisatellite (MSB and MS5) and microsatellite (ML1, ML2, TP14, and 5B12) loci. Isolates were collected from diarrheic preweaned calves originating from 61 dairy cattle farms in northern Spain. A capillary electrophoresis-based tool combining three different fluorescent tags was used to analyze all six satellites in one capillary. Fragment sizes were adjusted after comparison with sizes obtained by sequence analysis of a selection of isolates for every allele. Size discrepancies at all but the 5B12 locus were found for those isolates that were typed by both techniques, although identical size differences were reported for every allele within each locus. A total of eight alleles were seen at the ML2 marker, which contributed the most to the discriminatory power of the multilocus approach. Multilocus fragment typing clearly improved the discriminatory power of GP60 sequencing, since a total of 59 multilocus subtypes were identified based on the combination of alleles at the six satellite loci, in contrast to the 7 GP60 subtypes previously reported. The majority of farms (38) displayed a unique multilocus subtype, and individual isolates with mixed multilocus subtypes were seen at 22 farms. Bayesian structure analysis based on combined data for both satellite and GP60 loci suggested the presence of two major clusters among the C. parvum isolates from cattle farms in this geographical area.

New contributions to the study of common double mutants in the human LDL receptor gene.

Variations in the gene encoding the low-density lipoprotein receptor (LDLR) can cause familial hypercholesterolemia (FH), one of the most common inherited metabolic disorders in humans. The functional effects of the p.Gln92Glu and p.Asn564His alterations are predicted as benign, but the c.313 + 1G>C and p.Lys799_Phe801del changes are believed to cause disease. Although p.Gln92Glu and c.313 + 1G>C have been observed only in Spain, p.Asn564His and p.Lys799_Phe801del are widespread in Western Europe. In order to estimate the ages (t generations) of these four variants of the gene, to determine their possible origin and to consider the influence of age and selective pressure on their spread, we analyzed 86 healthy individuals and 126 FH patients in Spain. Most of the FH patients investigated carried two of these four LDLR variants simultaneously, while only one patient carried three of them simultaneously. Haplotype analyses were based on five LDLR SNPs: c.81T>C, c.1413G>A, c.1725C>T, c.1959T>C and c.2232G>A. The results suggest that p.Gln92Glu and c.313 + 1G>C arose at about the same time (99 and 103 generations ago, respectively) in the CACTG haplotype and that p.Asn564His and p.Lys799_Phe801del appeared in the CGCCG haplotype and might be slightly more recent variations (92 and 95 generations ago, respectively). Low selective pressures could explain the maintenance of these variants in spite of their ages. The origin of p.Gln92Glu and c.313 + 1G>C appears to be in Spain whereas p.Asn564His and p.Lys799_Phe801del could have been introduced in Spain by Celtic migrations in the seventh to fifth centuries BC.

Mitochondrial DNA diversity in Francolinus pondicerianus interpositus (grey francolin, Galliformes) from Pakistan.

Francolinus pondicerianus interpositus (grey francolin, Galliformes) is the only francolin present in the Suleiman Range (central Pakistan), one of the poorest and least developed areas in Pakistan. As a game bird, the francolin is an important income source for the region, but no demographic data are available. Therefore, the aim of this work was to study the polymorphism pattern of the Control Region gene (mitochondrial DNA, mtDNA), in order to obtain some initial information about genetic diversity, possible structure and demographic dynamics in this population. In 29 individuals captured in four sampling areas in the western and the eastern Suleiman Range, we detected nine polymorphic sites in a 511 bp fragment of the mtDNA Control Region gene, resulting in seven haplotypes. Haplotype (h = 0.818 ± 0.032) and nucleotide diversity (π % = 0.308 ± 0.210) values suggested a large population size and a low divergence among the haplotypes. AMOVA (Φ(ST) = 0.005; P = 0.352) did not detect any significant differences among the western and eastern populations; therefore, specimens of both sampled areas could be considered as drawn from a single population. The observed distribution of pairwise mismatches was bimodal, revealing significant departure from a growing-decreasing population model (P = 0.030); these results would point to a demographic equilibrium. Tribal control of hunting might provide an explanation for this situation, but future overhunting would threaten the survival of this population.

Periodontal and Dental Status in Packs of Spanish Dogs.

While periodontal disease (PD) is the most common canine oral pathology, its prevalence varies according to diagnosis methodology, breed, and age. We intended to increase understanding of canine PD by studying dogs that are managed in a specific way: pack dogs in Spain. They received a mixed diet (home-prepared food, commercial dry food, stale bread and bones). Thirty-two conscious individuals from two packs of dogs in Northeastern Spain (30/32 crossbred hunting dogs and 2/32 Siberian Husky; 26 males and 6 females; 27.75 ± 5.807 kgs; 5.48 ± 2.818 years) received visual dental examination for assessment of absent teeth (AT), dental calculus (DC) grade, gingival recession (GR), periodontal disease (PD), tooth fracture (TF), and dental attrition (DA). DC was the most prevalent oral problem (75%), followed by TF/DA (68.75%), AT (34.37%), GR (31.25%), and, finally, PD (15.62%). Low individual affectation values were found for AT, GR, and PD (<1 tooth/individual); mean DC grade per individual was 0.06 ± 0.063; and TF and DA were found in 1.63 and 4.72 teeth/individual, respectively. Low prevalence and extent of PD was attributed to diagnosis methodology, bodyweight effect, breed, and, ultimately, diet. Individuals affected by DC remained under veterinary surveillance due to PD development.

Blood Parameters and Feline Tooth Resorption: A Retrospective Case Control Study from a Spanish University Hospital.

Tooth resorption (TR; progressive destruction of hard dental tissues) varies in prevalence according to population, age, and country (29-66.1%). Our objective was twofold: describing the TR clinical presentation in Northeastern Spain, and studying 34 blood parameters to ascertain potential systemic effects associated with TR. Cases (29; presented from September 2018 to May 2019) and controls (58) were considered. Non-parametric tests were carried out to compare cases and controls for each blood parameter; those showing significant differences were chosen for multiple regression analysis (binomial logistic and hierarchical multiple regressions). In case TR was detected in 130/870 teeth (14.9%), TR stage and type were correlated (p < 0.001). Increasing CREA values (p = 0.034) and decreasing BUN/CREA and ALB/GLOB values were associated with TR presence (p = 0.029 and p = 0.03, respectively). Increasing GLOB was associated with increasing severity of TR (p < 0.01). Type 1 TR (highly related to inflammation and periodontal disease PD) was the most frequently observed type; the association of TR and inflammation biomarkers (ALB/GLOB, GLOB) are explained by this fact. The concomitant presence of PD and TR in old cats would cause TR association with kidney damage biomarkers (CREA, BUN/CREA). When affected by TR, special care in these aspects must be provided to cats.

Occurrence and genetic diversity of rotavirus A in faeces of diarrheic calves submitted to a veterinary laboratory in Spain.

A total of 237 faecal specimens from diarrheic calves younger than two months were collected and submitted for diagnosis of enteropathogens over a two-year period (2017-2018) to a veterinary laboratory. Samples originated from 193 dairy and beef farms in 29 provinces distributed throughout Spain, and were tested for the occurrence of three target enteric pathogens by reverse transcription real-time PCR (RT-qPCR): bovine rotavirus A (RVA), Cryptosporidium parvum and bovine coronavirus (BCoV). RT-PCR and nucleotide sequencing analysis were used to determine the G (VP7 gene) and P (VP4 gene) genotypes of 26 specimens positive for RVA. A total of 188 specimens (79.3 %) were positive for at least one of the three target enteric pathogens, and 101 samples (42.6 %) harbored mixed infections. The individual prevalence was 57.8 %, 50.6 % and 23.6 % for C. parvum, RVA and BCoV, respectively. Molecular analysis of selected RVA strains revealed the presence of the G6, G10, G3, P[5] and P[11] genotypes, with the combinations G6P[5] and G6P[11] being the most prevalent. Alignments of nucleotide sequences of the VP7 and VP4 markers showed a high frequency of single nucleotide polymorphisms (SNPs), with up to 294 SNPs found in 869bp of sequence at the G6 genotype (0.338 SNPs/nt), which reveals the extensive genetic diversity of RVA strains. Phylogenetic analysis of the VP7 gene of the G6 strains revealed four distinct lineages, with most strains clustering in the G6-IV lineage. The discrepancies between the RVA genotypes circulating in the sampled cattle farms and the genotypes contained in commercial vaccines currently available in Spain are discussed. We believe that this is the first study on the molecular characterization of rotavirus infecting cattle in Spain.

Tooth Resorption in Spanish Domestic Cats: Preliminary Data.

The objective of this study was to collect preliminary data about tooth resorption (TR) from cats treated at the Odontology Service (September 2016-June 2018), part of a University Veterinary Hospital in Spain, with specific emphasis on TR distribution per tooth. Diagnosis was based on visual/tactile inspection and intraoral dental radiographs. This cross-sectional study was carried out on 59 adult cats (27 females, 32 males). TR occurred in 39/59 cats (66.1%; 95% CI: 54.0%-78.2%). The median number of lesioned teeth per TR-affected animal was 3. A highly significant but weak correlation was found for age and number of TR-affected teeth per individual (Spearman´s correlation ρ = 0.381, P = .003, power = 0.853; N = 59). No TR cases were detected in incisors (0/708, 0%) but TR occurred in canines (21/236, 8.9%; 95% CI: 5.4%-12.4%), premolars (78/590, 13.2%; 95% CI: 10.5%-15.9%), and molars (33/236, 14%; 95% CI: 9.7%-18.3%). A significant age influence on TR was found. The greatest TR occurrence corresponded to 307 (21/59; 35.6%; 95% CI: 23.4%-47.8%) followed by 409 (17/59; 28.8%; 95% CI: 17.2%-40.3%), 407 (16/59; 27.1%; 95% CI: 15.7%-38.4%), and 309 (16/59; 27.1%; 95% CI: 15.7%-38.4%). These teeth would be considered as TR-sentinels in the studied population. These findings are relevant for veterinarians working in dental clinics, where the TR prevalence may be high especially in older cats.

Changes in content and localization of proteins phosphorylated at tyrosine, serine and threonine residues during ram sperm capacitation and acrosome reaction.

Previously, we reported the involvement of tyrosine phosphorylation in events that lead to ram sperm capacitation. In this study, we carried out a comparative analysis of the localization of tyrosine, serine and threonine phosphoproteins in different functional stages of ram spermatozoa (after the swim-up procedure, in vitro capacitation, and ionophore-induced acrosome reaction) by immunofluorescence, immunocytochemistry and confocal microscopy. Capacitation increased protein tyrosine, serine and threonine phosphorylation whereas the induction of the acrosome reaction resulted in significantly decreased phosphorylation, mainly in those proteins that increased following capacitation. Control samples showed tyrosine-phosphorylated proteins restricted to the head, mainly distributed at the equatorial region with some cells also displaying an acrosomal and/or post-acrosomal localization. In vitro capacitation promoted both tail and acrosome phosphorylation, and the acrosome reaction induced the loss of labeling on the acrosome and the subsequent increase in the post-acrosomal region and flagellum. The preferential localization of serine- and threonine-phosphorylated proteins in the equatorial and acrosomal regions found in control samples changed during capacitation, which induced tail phosphorylation in a sequential manner. After the acrosome reaction, the labeling of both phosphoamino acids decreased in the acrosome and increased in the post-acrosome. The obtained results were proved by two immunodetection techniques and strengthened by confocal microscopy, and indicate that changes in phosphorylated proteins during capacitation and acrosome reaction of ram spermatozoa may have physiological significance in consolidating certain phosphorylated proteins to specific sperm regions involved in acrosomal exocytosis and zona pellucida recognition, binding and penetration.