Influence of sperm fertilizing concentration, sperm selection method and sperm capacitation procedure on the incidence of chromosomal numeric abnormalities in in vitro fertilized early bovine embryos

The occurrence of numerical chromosomal aberrations, widely described as a major cause of mortality in in vitro-produced (IVP) embryos, has been linked to several factors. In the present study we investigated the effect of sperm fertilising concentration and semen handling (sperm selection and capacitation) before IVF on the rate of numerical chromosomal abnormalities in bovine embryos. In all, 466 IVP cattle embryos were karyotyped throughout three sequential experiments, analysing the effects of sperm fertilising concentration (0.1, 1.0 or 10×106 spermatozoa mL-1), selection method (unselected or Percoll-selected spermatozoa) and capacitation medium (bovine serum albumin (BSA), heparin or their combination). The percentage of normal (diploid) and aberrant (haploid, polyploid or aneuploid) embryos was noted in each experiment. The rate of numerical chromosomal abnormalities was mainly affected by sperm fertilising concentration (P0.05). Finally, aneuploidy rates were not affected during the experiments (P>0.05), which suggests that they are not related to sperm-related factors. On the basis of these results, we conclude that sperm fertilising concentration is the 'paternal' key factor that affects the rate of numerical chromosomal abnormalities in IVP bovine embryos. By making small adjustments to fertilising protocols, the rate of cytogenetically aberrant embryos can be markedly reduce

Reproductive traits in Pura Raza Española mares manifest inbreeding depression from low levels of homozygosity

Inbreeding depression is a genetic phenomenon associated with the loss of fitness and mean phenotypic performance due to mating between relatives. Historically, inbreeding coefficients have been estimated from pedigree information. However, the onset of genomic selection programs provides large datasets of individuals genotyped using SNP arrays, enabling more precise assessment of an individual's genomic-level inbreeding using genomic data. One of the traits most sensitive to issues stemming from increased inbreeding is reproduction. This is particularly important in equine, in which fertility is only moderate compared to other livestock species. To explore this further, we evaluated the effect of inbreeding on five reproductive traits (age at first foaling (AFF), average interval between foalings (AIF), total number of foalings (NF), productive life (PL) and reproductive efficiency (RE)) in Pura Raza Español mares using genomic data. Residual predicted phenotypes were obtained by purging these traits through the REML (wgResidual) and ssGREML (gResidual) approaches in reproductive data of 29,847 PRE mares using the BLUPF90+ program. Next, we used pedigree-based (Fped) and ROH-based genomic (FROH) inbreeding coefficients derived from 1018 animals genotyped with 61,271 SNPs to estimate the inbreeding depression (linear regression). Our results indicated significant levels of inbreeding depression for all reproductive traits, with the exception of the AIF trait when Fped was used. However, all traits were negatively affected by the increase in genomic inbreeding, and FROH was found to capture more inbreeding depression than Fped. Likewise, REML models (ssGREML) using genomic data for estimated predicted residual phenotypes resulted in higher variance explained by the model compared with the models not using genomics (REML). Finally, a segmented regression analysis was conducted to evaluate the effect of inbreeding depression, revealing that the levels of genealogical and genomic homozygosity do not manifest uniformly in reproductive traits. In contrast, the levels of inbreeding depression ranged from low to high as homozygosity increased. This analysis also showed that reproductive traits are very sensitive to inbreeding depression, even with relatively low levels of homozygosity

In vitro induction of the acrosome reaction in spermatozoa from endangered Spanish bulls: Effect of breed, culture media and incubation time

Current conservation programs for endangered species have been widely benefited from the use of modern assisted reproductive techniques. However, at the present time, there is little information available regarding the use of locally adapted cattle breeds on IVF programs, and even less about their sperm behavior. The aims of this study were to (1) evaluate standard acrosome reaction (AR) protocols in cryopreserved semen doses of Andalusian endangered cattle breeds, and (2) investigate the effect of breed, culture medium supplementation and incubation time on AR test results. To this end, 80 frozen semen doses from 16 bulls (5 semen samples per bull) of five different cattle breeds (3 Berrendo en Colorado, 6 Berrendo en Negro, 2 Cardena Andaluza, 1 Pajuna and 4 Retinta) were in vitro incubated in three test media (Heparin, BSA or their combina-tion), using Sp-TALP as a control, to induce in vitro AR. The percentage of acrosome-reacted spermatozoa was evaluated using fluorescence techniques (FITC-PNA and Propidium Iodide) at four time-points (0, 30, 60 or 120 min.). Throughout the whole experiment, spermatozoa incubated with a combination of both supplements (Heparin and BSA) showed significantly (Po0.05) higher results than those processed with Heparin or BSA alone. In contrast, the control media (without supplementation) showed the lowest results. We also found a significant (Po0.05) influence of cattle breed on the dynamics of the in vitro induced AR using different protocols. Based on our results, we can conclude that the response pattern of in vitro induced AR depends not only on culture media and incubation time but also on the cattle breed. Therefore, all these factors must be taken in consideration to assess the rate of AR sperm in bulls

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

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

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

The use of molecular and cytogenetic methods as a valuable tool in the detection of chromosomal abnormalities in horses: a case of sex chromosome chimerism in a spanish purebred colt

Chromosomal abnormalities associated to sex chromo-somes are reported as a problem more common than be-lieved to be in horses. Most of them remain undiagnosed due to the complexity of the horse karyotype and the lack of interest of breeders and veterinarians in this type of diagno-sis. Approximately 10 years ago, the Spanish Purebred Breed-ers Association implemented a DNA paternity test to evalu-ate the pedigree of every newborn foal. All candidates who showed abnormal or uncertain results are routinely submit-ted to cytogenetical analysis to evaluate the presence of chromosomal abnormalities. We studied the case of a foal showing 3 and even 4 different alleles in several loci in the short tandem repeat (STR) -based DNA parentage test. To confirm these results, a filiation test was repeated using fol-licular hair DNA showing normal results. A complete set of conventional and molecular cytogenetic analysis was per-formed to determine their chromosomal complements. C-banding and FISH had shown that the foal presents a sex chimerism 64,XX/64,XY with a cellular percentage of approximately 70/30, diagnosed in blood samples. The use of a di-agnostic approach combining routine parentage QF-PCR-based STR screening tested with classical or molecular cyto-genetic analysis could be a powerful tool that allows early detection of foals that will have a poor or even no reproductive performance due to chromosomal abnormalities, saving time, efforts and breeders' resources

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