Short communication. In vitro oocyte maturation and fertilization rates in the Spanish Lidia bovine breed

The Lidia bovine breed is the most successful cattle breed on the Iberian Peninsula, also considered a hallmark of Spanish tradition and image around the world. The aims of the study were to characterize the oocyte recovery rates and to evaluate the effect of two standard in vitro maturation protocols on oocyte maturation (cumulus expansion and nuclear maturation) and fertilization rates after in vitro fertilization in this breed. For this purpose, 261 ovaries from Lidia cows were processed obtaining 1,125 viable cumulus oocyte complexes (COCs). The oocyte recovery rate obtained (4.31 viable COCs per ovary) was lower than those described previously in other studied breeds. Maturation rates were evaluated in two different oocyte maturation media with (M1) and without (M2) hormonal supplementation. The percentage of COCs with expanded cumulus cells was significantly lower in M1 (74.35%) compared with M2 (82.25%). Metaphase II (MII) rates (67.75% in M1 and 73.18% in M2) were similar to previous studies in different cattle populations. M2 significantly improved the percentage of COCs with their cumulus cells expanded (p < 0.01) and nuclear maturation rates (p < 0.05), but it did not affect the fertilization percentages obtained in this experiment. In conclusion, our study suggests that oocytes of the Lidia cattle breed can be obtained, matured and fertilized following standard protocols previously used in other cattle breeds

Genomic Population Structure of the Main Historical Genetic Lines of Spanish Merino Sheep

According to historiographical documentation, the Romans first began to select Merino sheep in the Iberian Peninsula during the first century, with the aim of obtaining a breed appreciated for the quality of its wool. This process continued locally during the Middle Ages, when Spanish sheep were protected, and their export to foreign countries was banned. It was during the 16th century when individual Merino sheep were allowed to spread around the world to be used to improve the wool quality of local breeds. However, the wool crisis of the 1960s shifted the selection criteria of the Merino breed towards meat production at the expenses of wool. Consequently, individuals that display the genetic and phenotypic characteristics of those sheep originally bred in the kingdom of Spain in the Middle Ages are extremely difficult to find in commercial herds. In this study, we characterized the genetic basis of 403 individuals from the main historical Spanish Merino genetic lines (Granda, Hidalgo, Lopez-Montenegro, Maeso, Donoso and Egea), which were bred in isolation over the last 200 years, using a genomic approach based on genotyping data from the Axiom™ Ovine 50 K SNP Genotyping Array. Our analysis included measuring population structure, genomic differentiation indexes, runs of homozygosity (ROH) patterns, and an analysis of molecular variance (AMOVA). The results showed large genetic differences between the historical lines, even though they belong to the same breed. In addition, ROH analysis showed differences due to increased inbreeding among the ancient generations compared with the modern Merino lines, confirming the breed’s ancestral and closed origin. However, our results also showed a high variability and richness within the Spanish historical Merino lines from a genetic viewpoint. This fact, together with their great ability to produce high-quality wool, suggests that ancestral Merino lines from Spain should be considered a valuable genetic population to be maintained as a resource for the improvement of wool-producing sheep breeds all around the world

Nutraceutic Potential of Two Allium Species and Their Distinctive Organosulfur Compounds: A Multi-Assay Evaluation

This study aimed to evaluate the biological activities of two Allium species (garlic and onion) as well as diallyl disulphide (DADS) and dipropyl disulphide (DPDS) as their representative bioactive compounds in a multi-assay experimental design. The genotoxic, antigenotoxic, and lifespan effects of garlic, onion, DADS, and DPDS were checked in Drosophila melanogaster and their cytotoxic, pro-apoptotic, and DNA-clastogenic activities were analyzed using HL60 tumoral cells. All compounds were non-genotoxic and antigenotoxic against H2O2-induced DNA damage with a positive dose-response effect and different inhibition percentages (the highest value: 95% for DADS) at all tested concentrations. Daily intake of Allium vegetables, DADS, or DPDS had no positive effects on flies’ lifespan and health span. Garlic and DADS exerted the highest cytotoxic effects in a positive dose-dependent manner. Garlic and DADS exerted a DNA-internucleosomal fragmentation as an index of induced proapoptotic activity on HL60 cells. Allium vegetables and DADS were able to induce clastogenic strand breaks in the DNA of HL60 cells. This study showed the genomic safety of the assayed substances and their protective genetic effects against the hydrogen peroxide genotoxine. Long-term treatments during the whole life of the Drosophila genetic model were beneficial only at low-median concentrations. The chemo-preventive activity of garlic could be associated with its distinctive organosulfur DADS. We suggest that supplementary studies are needed to clarify the cell death pathway against garlic and DADS

In Vivo and In Vitro Genotoxic and Epigenetic Effects of Two Types of Cola Beverages and Caffeine: A Multiassay Approach

The aim of this work was to assess the biological and food safety of two different beverages: Classic Coca Cola™ (CCC) and Caffeine-Free Coca Cola (CFCC). To this end, we determined the genotoxicological and biological effects of different doses of lyophilised CCC and CFCC and Caffeine (CAF), the main distinctive constituent. Their toxic/antitoxic, genotoxic/antigenotoxic, and chronic toxicity (lifespan assay) effects were determined in vivo using the Drosophila model. Their cytotoxic activities were determined using the HL-60 in vitro cancer model. In addition, clastogenic DNA toxicity was measured using internucleosomal fragmentation and SCGE assays. Their epigenetic effects were assessed on the HL-60 methylation status using some repetitive elements. The experimental results showed a slight chemopreventive effect of the two cola beverages against HL-60 leukaemia cells, probably mediated by nonapoptotic mechanisms. Finally, CCC and CAF induced a global genome hypomethylation evaluated in LINE-1 and Alu M1 repetitive elements. Overall, we demonstrated for the first time the safety of this famous beverage in in vivo and in vitro models

Estimation of the Genetic Components of (Co)variance and Preliminary Genome-Wide Association Study for Reproductive Efficiency in Retinta Beef Cattle

In this study, we analyzed the variation of reproductive efficiency, estimated as the deviation between the optimal and real parity number of females at each stage of the cow’s life, in 12,554 cows belonging to the Retinta Spanish cattle breed, using classical repeatability and random regression models. The results of the analyses using repeatability model and the random regression model suggest that reproductive efficiency is not homogeneous throughout the cow’s life. The h2 estimate for this model was 0.30, while for the random regression model it increased across the parities, from 0.24 at the first calving to 0.51 at calving number 9. Additionally, we performed a preliminary genome-wide association study for this trait in a population of 252 Retinta cows genotyped using the Axiom Bovine Genotyping v3 Array. The results showed 5 SNPs significantly associated with reproductive efficiency, located in two genomic regions (BTA4 and BTA28). The functional analysis revealed the presence of 5 candidate genes located within these regions, which were previously involved in different aspects related to fertility in cattle and mice models. This new information could give us a better understanding of the genetic architecture of reproductive traits in this species, as well as allow us to accurately select more fertile cows

Sex chromosomal abnormalities associated with equine infertility: validation of a simple molecular screening tool in the Purebred Spanish Horse

Chromosomal abnormalities in the sex chromosome pair (ECAX and ECAY) are widely associated with reproductive problems in horses. However, a large proportion of these abnormalities remains undiagnosed due to the lack of an affordable diagnostic tool that allows for avoiding karyotyping tests. Hereby, we developed an STR (single-tandem-repeat)- based molecular method to determine the presence of the main sex chromosomal abnormalities in horses in a fast, cheap and reliable way. The frequency of five ECAXIinked (LEX026, LEX003, TKY38, TKY270 and UCDE0502) and two ECAY-Iinked (EcaYH12 and SRY) markers was characterized in 261 Purebred Spanish Horses to determine the efficiency of the methodology developed to be used as a chromosomal diagnostic tool. All the microsatellites analyzed were highly polymorphic, with a sizeable number of alleles (polymorphic information content > 0.5). Based on this variability, the methodology showed 100% sensitivity and 99.82% specificity to detect the most important sex chromosomal abnormalities reported in horses (chimerism, Turner’s syndrome and sex reversal syndromes). The method was also validated with 100% efficiency in 10 individuals previously diagnosed as chromosomally aberrant. This STR screening panel is an efficient and reliable molecular-cytogenetic tool for the early detection of sex chromosomal abnormalities in equines that could be included in breeding programs to save money, effort and time of veterinary practitioners and breeders.Facultad de Ciencias Veterinaria

Prevalence of Sex-Related Chromosomal Abnormalities in a Large Cohort of Spanish Purebred Horses

Chromosomal abnormalities are largely associated with fertility impairments in the domestic horse. To date, over 600 cases of individuals carrying abnormal chromosome complements have been reported, making the domestic horse the species with the highest prevalence. However, studies analyzing the prevalence of chromosomal diseases in whole populations are scarce. We, therefore, employed a two-step molecular tool to screen and diagnose chromosomal abnormalities in a large population of 25,237 Pura Raza Español horses. Individuals were first screened using short tandem repeats parentage testing results and phenotypic evaluations. Those animals showing results suggesting chromosomal abnormalities were re-tested using a single nucleotide polymorphism (SNP)-based diagnostic methodology to accurately determine the chromosomal complements. Thirteen individuals showed a positive screening, all of which were diagnosed as chromosomally abnormal, including five 64,XY mares with sex development disorders (DSD) and four cases of blood chimerism (two male/female and two female/female cases). In addition, we detected one Turner and one Klinefelter syndrome and two individuals carrying complex karyotypes. The overall prevalence in the entire population was ~0.05%, with the prevalence of 64,XY DSD and blood chimerism ~0.02% and ~0.016%, respectively. However, the overall results should be taken with caution since the individuals carrying Turner syndrome (in full (63,X) or mosaic (mos 63,X/64,XX) forms) cannot be detected due to limitations in the methodology employed. Finally, the lack of agreement between populational studies performed using karyotyping or molecular methods is discussed. To our knowledge, this is the largest populational study performed evaluating the prevalence of the most common chromosomal abnormalities in the domestic horse

Genetic Parameters of Somatic Cell Score in Florida Goats Using Single and Multiple Traits Models

A total of 1,031,143 records of daily dairy control test of Spanish Florida goats were used for this study. The database was edited, and only the records of the first three lactations were kept. The final database contained 340,654 daily-test somatic cell counts from 27,749 daughters of 941 males and 16,243 goats. The evolution of this count in the last 14 years was analyzed following French and American international associations’ criteria for the risk of mastitis in goats, and confirmed the slight increase in SCS in the last years and the importance of this problem (50% of dairy control tests show a risk of suffering mastitis). For the genetic analysis, the SCS records were log-transformed to normalize this variable. Two strategies were used for the genetic analysis: a univariate animal model for the SCS assuming that SCS does not vary throughout the parities, and a multi-character animal model, where SCS is not considered as the same character in the different parities. The heritabilities (h2) were higher in the multiple traits models, showings an upward trend from the first to the third parity (h2 between 0.245 to 0.365). The genetic correlations of the same trait, as well as between breeding values (GVs) between different parities, were different from unity. The breeding values (EBVs) obtained for both models were subjected to a PCA: the first eigenvector (λ1) explained most of the variations (between 74% to 90%), while the second λ2 accounted for between 9% to 20% of the variance, which shows that the selection will be proportionally favorable but not equivalent in all parities and that there are some variations in the type of response

Sex chromosomal abnormalities associated with equine infertility: validation of a simple molecular screening tool in the Purebred Spanish Horse

Chromosomal abnormalities in the sex chromosome pair (ECAX and ECAY) are widely associated with reproductive problems in horses. However, a large proportion of these abnormalities remains undiagnosed due to the lack of an affordable diagnostic tool that allows for avoiding karyotyping tests. Hereby, we developed an STR (single-tandem-repeat)- based molecular method to determine the presence of the main sex chromosomal abnormalities in horses in a fast, cheap and reliable way. The frequency of five ECAXIinked (LEX026, LEX003, TKY38, TKY270 and UCDE0502) and two ECAY-Iinked (EcaYH12 and SRY) markers was characterized in 261 Purebred Spanish Horses to determine the efficiency of the methodology developed to be used as a chromosomal diagnostic tool. All the microsatellites analyzed were highly polymorphic, with a sizeable number of alleles (polymorphic information content > 0.5). Based on this variability, the methodology showed 100% sensitivity and 99.82% specificity to detect the most important sex chromosomal abnormalities reported in horses (chimerism, Turner’s syndrome and sex reversal syndromes). The method was also validated with 100% efficiency in 10 individuals previously diagnosed as chromosomally aberrant. This STR screening panel is an efficient and reliable molecular-cytogenetic tool for the early detection of sex chromosomal abnormalities in equines that could be included in breeding programs to save money, effort and time of veterinary practitioners and breeders.Facultad de Ciencias Veterinaria