The members of the Chlamydiaceae family are important pathogens that infect a wide range of vertebrate hosts, including humans. Among them, Chlamydia psittaci, historically considered as an avian agent, has recently been identified in livestock, primarily sheep and cattle, but also in horses, with the infection being linked to reproductive disorders, such as abortion, absorption of embryos, stillbirth, and the birth of weak foals. Much less is known about chlamydial infections in the Sardinian equine population. This study aimed to identify the chlamydial diversity in genital samples from asymptomatic Sardinian horses. However, some horses had a previous history of reproductive disorders, i.e., abortion and infertility. A total of 60 horses (39 mares and 21 stallions) were opportunistically recruited from 17 equine farms in central-northern Sardinia. Vaginal and uterine swabs from mares and urethral swabs and seminal fluid from stallions were sampled for the presence of chlamydial DNA. Samples from environments where the horses lived were also tested for the detection of Chlamydia spp. Eight vaginal swabs (8/39; 20%), two uterine swabs (2/27; 7%), two seminal fluid samples (2/20; 10%), and one urethral swab (1/21; 4.7%) were found to be positive for Chlamydia spp. by PCR analysis. In addition, results from environmental samples showed the presence of Chlamydia spp. in three environmental swabs (3/8; 37.5%) and five water samples (5/16; 31.2%). Sequencing results revealed that strains here identified were 99-100% similar to members belonging to the Chlamydiaceae family, including C. abortus, C. psittaci, and uncultured Chlamydia genotypes. ompA species-specific PCR performed on samples was found to be positive after 16S rRNA amplification gave positive results for C. psittaci. These results reveal the first presence of C. psittaci in the genital tract of horses and in the environment in Sardinia and indicate that this pathogen could be the prevailing cause of infertility and abortion in the tested equines. However, these findings need further proof and highlight the importance of adopting a 'One Health' approach to control the presence of this zoonotic bacteria in domestic animals in order to understand its impact on people exposed to the infection risk.
A viable tool for the monitoring of the systemic condition of the pregnant jenny may be the determination of serum protein fraction (SPF) levels, including metabolic profiling. Tissue development and composition of the growing fetus requires the mother to provide adequate nutrients to its body parts and organs. In this regard, body fluid distribution and strategic molecule transportation can be screened using SPF electropherograms and analysis of intermediate metabolites. The nutritional and health status of 12 jennies (age: 5-8 years; BW at the start: 135-138 kg; Body Condition Score, BCS [1 to 5 points] = 2.25-2.50; 4th month of gestation) were monitored throughout gestation (approximate gestation period 350-356 d). All animals were pasture-fed and were offered hay ad libitum. Individual blood samples were collected within the 4th, 7th, and 10th month following conception (ultrasound scanning). Serum biochemistry, in particular, the analysis of 6 fractions of serum proteins was carried out. The significant decrease in circulating albumin in jennies from mid- to late-gestation (p < 0.001) suggests a considerable role of dietary amino acids in the synthesis of protein for fetal tissue formation as well as body fluid distribution and blood pressure control of the jenny in those stages. Moreover, α1-globulin decreased significantly in late gestation (p < 0.047), corresponding to major organ development in the terminal fetus and supported by lipid transportation in the bloodstream of the jenny. Similarly, α2-globulin decreased in late gestation (p < 0.054) as haptoglobin, an important component for the transport of free circulating hemoglobin, is likely used for fetal synthesis. Mid-gestation, appears to be a crucial moment for adequate dietary nutrient supplementation in order to prevent homeostasis perturbation of jennies, as observed in this trial.
The pattern of selected metabolites for interpreting homeostasis during the growth of foals can be used as an indicator of energy balance state and liver health. Against this background, the literature on circulating parameters of foals across growth stages is scanty. We hypothesized that circulating metabolites indicating energy distribution such as non-esterified fatty acids (NEFA), ß-hydroxy-butyric acid (BHBa), UREA and liver enzyme-like γ-glutamyl-transferase (γ-GT) [interpreted in the light of circulating total bilirubin (TBIL), alanine aminotransferase (ALT) and aspartate aminotransferase (AST)] may be used to monitor the energy balance of growing foals. A total of 12 Anglo-Arab (AA) foals from the same stable were enrolled in this trial. All foals were serially weighed on a digital scale and sampled for total blood at weaning, at 12 and 18 months of age. Feeding and keeping conditions were similar for all the foals involved. Animals appeared healthy and no signs of poor growth performance were pointed out. The peak of circulating NEFA mobilized from body depots was reached at one year of age but markedly dropped at 18 months, when BHBa increased (p < 0.001) alongside with liver enzyme. BHBa and γ-GT levels turned out to positively correlate (p = 0.051). However, at 6, 12 and 18 months, γ-GT dropped in the physiological reference range for the horse, thus showing no prognostic value. ALT and UREA significantly increased (p = 0.008 and p = 0.006, respectively) when NEFA also increased (p = 0.001). Liver enzyme increase could be associated with fat mobilization and ketone bodies production meanwhile amino acid transamination for energy purposes led to the increase of UREA in the bloodstream. However, no prognostic value to liver enzyme could be attributed in this trial.
Trace element status and metabolic milieu are sometimes overlooked in common veterinary clinical practice across animal species. The evaluation of requirements of trace elements, in fact, may be useful to prevent the perturbation of tissue-specific metabolic impair. In particular, essential trace elements in the diet play key roles within sub-cellular metabolic patterns with macro effects at the systemic level, like blood cell stability and semen quality. This effect was studied in breeding stallions, in which semen quality and haemogram are important for reproduction. A case-control feeding trial involved 40 stallions (age: 8-21 years; body weight, BW: 510-531 kg) of one stud centre, allotted to two experimental groups (n = 20 control, CON vs. n = 20 supplemented, SUPPL100), following a matched-pairs approach based on age. Supplemented stallions (SUPPL100) received a mixed mineral and vitamin supplement of Zn/Se and α-tocopherol (α-TOH) (100 g/day stallion) to compound feed, fed as control diet to horses of the control group (CON). Horses resulted deficient in circulating α-TOH and Zn at the start, though clinically healthy. After supplementation, different plasmatic levels of α-TOH, Zn and Se were found between groups. Circulating basophils (BASO) and mean cell haemoglobin concentration (MCHC) were affected by the dietary treatment (p < 0.05). Plasmatic Se affected monocyte count, haematocrit, mean cell volume and mean cell haemoglobin concentration. Semen traits were not affected by the dietary treatment per se, except for mobile/progressive sperm cells (%) of stallions aged > 13 years marginal circulating levels of α-TOH (p = 0.04). Ameliorating the micromineral status showed to improve the haemogram of stallions in view of circulating levels of Cu. Semen quality appeared to be strongly dependent on animal effects.
Semen Analysis , Semen , Animals , Breeding , Diet/veterinary , Dietary Supplements , Horses , Male , Zinc/pharmacology , alpha-Tocopherol/pharmacology
The dependence of a stallion's spermatozoa on oxidative phosphorylation for energy requirements results in an unconventional relationship between reactive oxygen species (ROS) production and fertility. In such a scenario, antioxidant activity must be finely controlled and not affect the essential functions of ROS. Some in vivo evidence suggests that the naturally occurring antioxidant ergothioneine (ERT) interferes with the critical roles of ROS/reactive nitrogen species (RNS) in pro-oxidant states but not in healthy tissues. The measurement of ERT in seminal plasma collected from 14 stallions (five Anglo-Arab, five Sella Italiano and four Thoroughbreds of which three are Arabian and one English) aged 16 ± 6 years (range 6-25 years) confirms that ERT is present at high concentrations in this biological fluid, between 16.80 and 971.48 µmol/L. Although the presence of high ERT concentrations in the seminal plasma of a stallion has long been known, its exact biological role is uncertain. This might be due to the peculiar antioxidant cycle of ERT, specifically its rapid recovery, which potentially masks concentration fluctuations and, therefore, the extent of its physiological effects. The measurement of the ERT precursor and redox metabolite hercynine (ERY) may overcome such issues, as ERY does not undergo regeneration processes. ERY was detectable and measurable in the seminal plasma of all stallions at a median concentration of 7.50 (IQR 15.26) nmol/L. The analysis of the association between the ERT and ERY, as well as with other established antioxidants such as glutathione and cysteine, suggests that ERT may play a major role in the antioxidant machinery of seminal plasma, and that ERY might serve as a new combined marker of oxidative stress and semen quality.
Sardinia, an island located to the west of Italy in the Mediterranean Sea, boasts three native horse breeds: Giara, Sarcidano, and Sardinian Anglo-Arab. Here, we have investigated for the first time three loci of the non-recombining region of the Y chromosome (NRY) in 34 stallions from these breeds and performed a phylogenetic analysis of the maternal relationships among 178 previously published mitochondrial control regions. We found that the current NRY diversity of Sardinian horse breeds is linked to three haplotypes (HT), all identified within Sarcidano. Each breed showed a typical HT: HT1 (ancestral) was the most represented in Sarcidano, HT2 (Neapolitan/Oriental wave) in Giara, and HT3 (Thoroughbred wave) in Sardinian Anglo-Arab. The specificity of each haplotype suggests the influence of independent breeding strategies and the effect of genetic drift in each Sardinian population. The female counterpart, extended to 178 horses, showed a low genetic variability and a common maternal origin for Giara and Sarcidano. The higher variability of the Sardinian Anglo-Arab indicates multiple mare lineages in its current population. Further genetic analyses will be crucial to understand the paternal history of male horses, preserve the endangered mares' and stallions' lineages, and improve the enhancement of autochthonous genetic resources on this island.
This first survey on Sardinian Anglo-Arab horse (SAA) race traits highlights important aspects for the breeding purpose of this population. The heritability of the race traits were estimated through a trivariate model; the estimates were 0.39, 0.33, and 0.30 for the number of placings, total earnings and Elo rating, respectively. The genetic progress could be improved by using an MT genetic evaluation of stallions and mares, combining information from competition traits.
This study pointed to explore if variations in circulating levels of metabolites in the blood stream of no. 25 feral donkeys occur in view of the different coat color between specimens of Asinara (albino, no. 8) vs. Sardo (dun-grey, no. 17) breed. All individuals involved in this investigation are living in the nature, at Mediterranean latitudes and roam in the same areas all over the National Park of Capo Caccia, where they feed on spontaneous vegetation sources. The study was conducted during the positive photoperiod of the boreal hemisphere (peak in the month of June, 2019) to maximize the effect of exposure to the natural sun radiation and thus elicit the coping ability of albino (Asinara) in comparison with pigmented donkeys (Sardo). The biochemical profile of all donkeys was used in a Discriminant Analysis (DA) to explore if circulating levels of metabolites could point to metabolic markers for breed assignment of individuals following a canonical discriminant analysis (CANDISC). The biochemical investigation included also the determination of the circulating Vitamin E (alpha tocopherol, α-TOH), as an essential biologically active compound involved in antioxidant mechanisms, and its respective status (circulating α-TOH to total triglycerides and total cholesterol ratio). In the CANDISC, the distance between the two breeds was not significant. However, it pointed to different metabolites (UREA, total protein, total triglycerides, Zn) capable of describing biochemical patterns on each respective breed (Asinara vs. Sardo). The multivariate analysis DA carried out using 22 metabolites correctly assigned individuals to the two breeds in the 100% of cases. In view of such metabolic background, circulating α-TOH found in the bloodstream of Asinara vs. Sardo donkeys under free grazing conditions turned out to reach similar values (2.114 vs. 1.872 µg/ml, respectively, p = 0.676). It is worth noting that significant differences were observed as to circulating lactate dehydrogenase (LDH, p = 0.022) levels, in association with increased creatine phosphokinase (CPK, p = 0.076), both above the upper limit of the physiological range reported in other donkey breeds, and found in the totality of Asinara (albino) donkeys solely, still apparently clinically healthy.
The Sardinian Anglo Arab (SAA) is a famous horse breed in Italy, with a significant historical background in the island of Sardinia. The aim of the study is to perform an evaluation of genetic variability in SAA using pedigree and mitochondrial data. In the current population, pedigree completeness was observed to be close to 100%, while the inbreeding coefficient and the average relatedness were lower than 3%. The ratio of effective founders/numbers of ancestors was 3.68 for the whole pedigree. The effective population size (Ne) computed by an individual increase in inbreeding (Ne_1) was 456.86, the Ne on equivalent generations (Ne_2) was 184.75, and this value slightly increased to 209.31 when computed by log-regression on equivalent generations (Ne_3). These results suggest the presence of crossbreeding and bottleneck phenomena, and they were compared with other Italian horses (reported in bibliography) to present the SAA among the Italian horse breeds scenario. Furthermore, the noteworthy mitochondrial variability reflects the use of a considerable number of founder mares; the contribution of L lineage was very important, probably because of the re-colonization from the Iberian Peninsula after the Last Glacial Maximum.
The purpose of this study was to estimate the heritability and genetic correlations of four biometric measurements and an overall score (OS) in the Sardinian Anglo-Arab horse (SAA); moreover, the effect of inbreeding on these traits was investigated. A dataset with 43,624 horses (27,052 females and 16,572 males) was provided by the Agricultural Research Agency of Sardinia (AGRIS). Cannon bone circumference (BC), chest girth (CG), shoulder length (SL), and withers height (WH) were measured on 6033 SAA horses born in Sardinia between 1967 and 2005; beside the measurements, an overall score (OS) was taken comparing the morphology of each horse to an "ideal type" that is scored out of 100. The mean value is 20.5 cm for BC, 185.9 cm for CG, 67.6 cm for SL, 160.8 cm for WH, and 73.2 for the OS. The heritability estimates ranged from 0.78 to 0.23. The results allow to foresee high genetic progress through the breeding programs. The most affected trait by the inbreeding rate seems to only be the withers height.
Physical exercise is universally recognized as stressful. Among the "sport species", the horse is probably the most appropriate model for investigating the genomic response to stress due to the homogeneity of its genetic background. The aim of this work is to dissect the whole transcription modulation in Peripheral Blood Mononuclear Cells (PBMCs) after exercise with a time course framework focusing on unexplored regions related to introns and intergenic portions. PBMCs NGS from five 3 year old Sardinian Anglo-Arab racehorses collected at rest and after a 2000 m race was performed. Apart from differential gene expression ascertainment between the two time points the complexity of transcription for alternative transcripts was identified. Interestingly, we noted a transcription shift from the coding to the non-coding regions. We further investigated the possible causes of this phenomenon focusing on genomic repeats, using a differential expression approach and finding a strong general up-regulation of repetitive elements such as LINE. Since their modulation is also associated with the "exonization", the recruitment of repeats that act with regulatory functions, suggesting that there might be an active regulation of this transcriptional shift. Thanks to an innovative bioinformatic approach, our study could represent a model for the transcriptomic investigation of stress.
Gene Expression Regulation , Genome , Introns/genetics , Physical Conditioning, Animal , RNA, Messenger/genetics , Stress, Physiological , Transcriptome , Animals , Female , Horses , Leukocytes, Mononuclear/metabolism , Male
Donkeys have played an important role in agricultural land practices and in human historical periods of recent past and, still today, are used as a working power in several world areas. The objective of this study was to identify genetic variability in six Italian donkey breeds using mtDNA D-loop. Fifteen haplotypes, grouped in three haplogroups, were identified. The genetic indices were informative and showed a high population genetic variability. The results of AMOVA analyses based on geographic structuring of Italian populations highlighted that the majority of the observed variance is due to differences among samples within breeds. Comparison among Italian haplotypes and mtDNA D-loop sequences belonging to European domestic and Ethiopian donkeys and wild asses, clearly define two clades referred to Nubian lineage. The results can be useful to complement safeguard planes for donkey breeds that are considered to extinction endangered.
DNA, Mitochondrial/genetics , Equidae/classification , Genetic Variation , Sequence Analysis, DNA/methods , Animals , Breeding , DNA, Mitochondrial/chemistry , Equidae/genetics , Ethiopia , Europe , Genetics, Population , Haplotypes , Italy , Phylogeny , Phylogeography
BACKGROUND: The climatic and cultural diversity of the Italian Peninsula triggered, over time, the development of a great variety of horse breeds, whose origin and history are still unclear. To clarify this issue, analyses on phenotypic traits and genealogical data were recently coupled with molecular screening. METHODOLOGY: To provide a comprehensive overview of the horse genetic variability in Italy, we produced and phylogenetically analyzed 407 mitochondrial DNA (mtDNA) control-region sequences from ten of the most important Italian riding horse and pony breeds: Bardigiano, Esperia, Giara, Lipizzan, Maremmano, Monterufolino, Murgese, Sarcidano, Sardinian Anglo-Arab, and Tolfetano. A collection of 36 Arabian horses was also evaluated to assess the genetic consequences of their common use for the improvement of some local breeds. CONCLUSIONS: In Italian horses, all previously described domestic mtDNA haplogroups were detected as well as a high haplotype diversity. These findings indicate that the ancestral local mares harbored an extensive genetic diversity. Moreover, the limited haplotype sharing (11%) with the Arabian horse reveals that its impact on the autochthonous mitochondrial gene pools during the final establishment of pure breeds was marginal, if any. The only significant signs of genetic structure and differentiation were detected in the geographically most isolated contexts (i.e. Monterufolino and Sardinian breeds). Such a geographic effect was also confirmed in a wider breed setting, where the Italian pool stands in an intermediate position together with most of the other Mediterranean stocks. However, some notable exceptions and peculiar genetic proximities lend genetic support to historical theories about the origin of specific Italian breeds.
Breeding , DNA, Mitochondrial/genetics , Genes, Mitochondrial , Horses/genetics , Animals , Climate , Female , Italy , Male
