A large population study to assess the magnitude of prenatal programming in dairy cattle.

The performance of an adult dairy cow may be influenced by events that occur before her birth. The present study investigated potential effects of 2 prenatal groups of factors, Assisted Reproductive Techniques (ART) and maternal characteristics (e.g., dam parity), on offspring performance during their first lactation, in populations of 2 dairy cow breeds: French Holstein and Montbéliarde. The different ART studied included the type of semen (conventional or X-sorted) used for Artificial Insemination (AI) and the technology of conception used (AI, embryo transfer, or in vitro fertilization). Three maternal characteristics were considered: (1) the dam age at first calving, (2) dam parity number, and (3) indicators of dam udder health during gestation (somatic cell score and events of clinical mastitis). First, we investigated whether heifer survival from 3d to 18 mo old was associated with any of the prenatal factors considered. We then estimated the associations of these prenatal factors with 8 traits of commercial interest: (1) stature, (2-4) milk, fat, and protein yields, (5) somatic cell score, (6) clinical mastitis, and (7-8) heifer and cow conception rate, all measured on genotyped cows. Linear models were used for this study with the prenatal factors as covariates in the model, and for the 8 traits, phenotypes were adjusted for their corresponding genomic estimated breeding value. The results indicated that the survival rate of heifers born from embryo transfer was significantly higher than that of heifers born from AI (probably due to preferential management practices), while the other prenatal factors did not explain differences in heifer survival. Among the Montbéliarde cows born from AI, those born from X-sorted semen showed a lightly but significantly lower milk yield than those born without X-sorting of the semen (-52 kg of milk in the first lactation). Among the Holstein cows, those born from embryo transfer presented significantly lower milk performance than cows born from AI. Regarding the maternal characteristics, none or very weak associations were found between the dam age at first calving and the offspring performance in both breeds. Dam parity, on the other hand, was associated with offspring performance for milk, fat, and protein yield in both breeds, however not in the same direction. In the Holstein breed, an increase in dam parity was favorable for offspring performance for milk, fat, and protein yield, whereas in the Montbéliarde breed, an increase in dam parity was associated with lower milk and protein yield and no association was found for fat yield. The udder health of the dam during gestation was not or only weakly associated with the traits studied in the offspring. Although some significant associations were identified due to the large sample size, the effects were modest, typically less than 1% of the phenotypic mean, and were not consistently observed across the 2 breeds.

Invited review: Good practices in genome-wide association studies to identify candidate sequence variants in dairy cattle.

Genotype data from dairy cattle selection programs have greatly facilitated GWAS to identify variants related to economic traits. Results can enhance the accuracy of genomic prediction, analyze more complex models that go beyond additive effects, elucidate the genetic architecture of a trait, and finally, decipher the underlying biology of traits. The entire process, comprising data generation, quality control, statistical analyses, interpretation of association results, and linking results to biology should be designed and executed to minimize the generation of false-positive and false-negative associations and misleading links to biological processes. This review aims to provide general guidelines for data analysis that address data quality control, association tests, adjustment for population stratification, and significance evaluation to improve the reliability of conclusions. We also provide guidance on post-GWAS strategy and the interpretation of results. These guidelines are tailored to dairy cattle, which are characterized by long-range linkage disequilibrium, large half-sib families, and routinely collected phenotypes, requiring different approaches than those applied in human GWAS. We discuss common limitations and challenges that have been overlooked in the analysis and interpretation of GWAS to identify candidate sequence variants in dairy cattle.

Opportunities for genomic selection of cheese-making traits in Montbéliarde cows.

As part of the From'MIR project, traits related to the composition and cheese-making properties (CMP) of milk were predicted from 6.6 million mid-infrared spectra taken from 410,622 Montbéliarde cows (19,862 with genotypes). Genome-wide association studies of imputed whole-genome sequences highlighted candidate SNPs that were then added to the EuroG10K BeadChip, which is routinely used in genomic selection. In the present study, we (1) assessed the reliability of single-step genomic BLUP breeding values (ssEBVs) for cheese yields, coagulation traits, and casein and calcium content generated from test-day records of the first 3 lactations, (2) estimated realized genetic trends for these traits over the last decade, and (3) simulated different cheese-making breeding objectives and estimated the responses for CMP as well as for other traits currently selected in the Montbéliarde breed. To estimate the reliability of ssEBVs, the available data were split into 2 independent training and validation sets that respectively contained cows with the oldest and the most recent lactation data. The training set included 155,961 cows (12,850 with genotypes) and was used to predict ssEBVs of 2,125 genotyped cows in the validation set. We first tested 4 models that included either lactation (LACT) or test-day (TD) records from the first (1) or the first 3 (3) lactations, giving equal weight to all 50K SNP effects. Mean reliabilities were 61%, 62%, 63%, and 64% for the LACT1, LACT3, TD1, and TD3 models, respectively. Using the most accurate model (TD3), we then compared the reliabilities of 3 scenarios with: SNPs from the Illumina BovineSNP50 BeadChip only, equally weighted (50K); 50K SNPs plus additional candidate SNPs, equally weighted (50K+); and 50K and candidate SNPs with additional weight given to 7 to 14 candidate SNPs, depending on the trait (CAND). The 50K+ and CAND scenarios led to similar mean reliabilities (67%) and both outperformed the 50K scenario (64%), whereas the CAND scenario generated the less biased ssEBVs. To assess genetic trends, SNP effects were estimated with a single-step GBLUP based on the TD3 model and the 50K scenario applied to the whole population (2.6 million performance records from 190,261 cows and 423,348 animals in the pedigree, of which 21,874 were genotyped) and then applied to 50K genotypes of 21,171 males and 311,761 females. We detected a positive genetic trend for all CMP during the last decade, probably due to selection for an increase in milk protein and fat content in Montbéliarde cows. Finally, we compared the selection responses to 3 different breeding objectives: the current Montbéliarde total merit index (TMI) and 2 alternative scenarios that gave a weight of 70% to TMI and the remaining 30% to either milk casein content (TMI-COMP) or a combination of 3 CMP (TMI-Cheese). The TMI-Cheese scenario yielded the best responses for all the CMP analyzed, whereas values in the TMI-COMP scenario were intermediate, with a slight effect on other traits currently included in TMI. Based on these results, a program of genomic evaluation for CMP predicted from mid-infrared spectra was designed and implemented for the Montbéliarde breed.

Frequencies of milk protein variants and haplotypes estimated from genotypes of more than 1 million bulls and cows of 12 French cattle breeds.

Due to their major effects on milk composition and cheese-making properties and their putative effects on human health, there is a great deal of interest in bovine milk protein variants. The objectives of this study were to estimate frequencies of milk protein variants and haplotypes in 12 cattle breeds as well as their trends over time to assess the effect of selection on milk traits. Milk protein variants and haplotypes were identified from SNP genotype data from more than 1 million animals from 12 dairy, beef, or dual-purpose cattle breeds that had been genotyped for genomic selection. We examined a total of 15 loci in the genes that encode ß-lactoglobulin (ß-LG) and 3 caseins (αS1-CN, ß-CN, and κ-CN); genotypes were directly called from customized SNP chips (50.6%) or imputed (49.4%). Variants A and B of ß-LG were frequent in the 12 breeds. For the caseins, we found 3 variants for αS1-CN (B, C, and D), 6 for ß-CN (A1, A2, A3, B, C, and I), and 5 for κ-CN (A, B, C, D, and E). For αS1-CN, the B variant was the most frequent in all breeds except Jersey. For ß-CN, the A2 variant was the most abundant in all breeds except Tarentaise, although in Normande animals, the I variant (30.9%) was almost as common as A2 (39.7%). The C variant was very rare except in the Tarentaise sample (4.8%). The most frequent variant for κ-CN was A in 5 breeds (including Holstein), and B in the 7 other breeds. The B variant was present at a particularly high frequency in Jersey (82.6%) and Normande (85.5%) animals. The C and E variants of κ-CN appeared to be particularly frequent in the Tarentaise (12.7%) and Holstein (9%) breeds, respectively. We found 20 haplotype combinations of αS1-ß-κ CN that were present at a frequency >0.1% in at least one breed; however, only 6 to 9 haplotypes were found in any given breed, demonstrating a strong degree of linkage disequilibrium. The most frequent haplotypes were B-A1-A, B-A2-A, B-A2-B, B-I-B, C-A2-A, and C-A2-B. Some alleles were predominantly found in only one haplotype, such as the E and C variants of κ-CN and the I variant of ß-CN, which were mainly found in the B-A1-E, B-A1-C, and B-I-B haplotypes, respectively. We observed changes in the frequency of certain variants over time in several breeds, such as an increase in the frequency of variants A of ß-LG, I of ß-CN, and B of κ-CN. With these results, we update and complete frequency data that were first estimated 30 to 50 yr ago, and, for the first time in these breeds, we assess the effect of selection on milk protein variants.

Comparison of Bayesian and partial least squares regression methods for mid-infrared prediction of cheese-making properties in Montbéliarde cows.

Assessing the cheese-making properties (CMP) of milks with a rapid and cost-effective method is of particular interest for the Protected Designation of Origin cheese sector. The aims of this study were to evaluate the potential of mid-infrared (MIR) spectra to estimate coagulation and acidification properties, as well as curd yield (CY) traits of Montbéliarde cow milk. Samples from 250 cows were collected in 216 commercial herds in Franche-Comté with the objectives to maximize the genetic diversity as well as the variation in milk composition. All coagulation and CY traits showed high variability (10 to 43%). Reference analyses performed for soft (SC) and pressed cooked (PCC) cheese technology were matched with MIR spectra. Prediction models were built on 446 informative wavelengths not tainted by the water absorbance, using different approaches such as partial least squares (PLS), uninformative variable elimination PLS, random forest PLS, Bayes A, Bayes B, Bayes C, and Bayes RR. We assessed equation performances for a set of 20 CMP traits (coagulation: 5 for SC and 4 for PCC; acidification: 5 for SC and 3 for PCC; laboratory CY: 3) by comparing prediction accuracies based on cross-validation. Overall, variable selection before PLS did not significantly improve the performances of the PLS regression, the prediction differences between Bayesian methods were negligible, and PLS models always outperformed Bayesian models. This was likely a result of the prior use of informative wavelengths of the MIR spectra. The best accuracies were obtained for curd yields expressed in dry matter (CYDM) or fresh (CYFRESH) and for coagulation traits (curd firmness for PCC and SC) using the PLS regression. Prediction models of other CMP traits were moderately to poorly accurate. Whatever the prediction methodology, the best results were always obtained for CY traits, probably because these traits are closely related to milk composition. The CYDM predictions showed coefficient of determination (R2) values up to 0.92 and 0.87, and RSy,x values of 3 and 4% for PLS and Bayes regressions, respectively. Finally, we divided the data set into calibration (2/3) and validation (1/3) sets and developed prediction models in external validation using PLS regression only. In conclusion, we confirmed, in the validation set, an excellent prediction for CYDM [R2 = 0.91, ratio of performance to deviation (RPD) = 3.39] and a very good prediction for CYFRESH (R2 = 0.84, RPD = 2.49), adequate for analytical purposes. We also obtained good results for both PCC and SC curd firmness traits (R2 ≥ 0.70, RPD ≥1.8), which enable quantitative prediction.

Use of meta-analyses and joint analyses to select variants in whole genome sequences for genomic evaluation: An application in milk production of French dairy cattle breeds.

As a result of the 1000 Bull Genome Project, it has become possible to impute millions of variants, with many of these potentially causative for traits of interest, for thousands of animals that have been genotyped with medium-density chips. This enormous source of data opens up very interesting possibilities for the inclusion of these variants in genomic evaluations. However, for computational reasons, it is not possible to include all variants in genomic evaluation procedures. One potential approach could be to select the most relevant variants based on the results of genome-wide association studies (GWAS); however, the identification of causative mutations is still difficult with this method, partly because of weak imputation accuracy for rare variants. To address this problem, this study assesses the ability of different approaches based on multi-breed GWAS (joint and meta-analyses) to identify single-nucleotide polymorphisms (SNP) for use in genomic evaluation in the 3 main French dairy cattle breeds. A total of 6,262 Holstein bulls, 2,434 Montbéliarde bulls, and 2,175 Normande bulls with daughter yield deviations for 5 milk production traits were imputed for 27 million variants. Within-breed and joint (including all 3 breeds) GWAS were performed and 3 models of meta-analysis were tested: fixed effect, random effect, and Z-score. Comparison of the results of within- and multi-breed GWAS showed that most of the quantitative trait loci identified using within-breed approaches were also found with multi-breed methods. However, the most significant variants identified in each region differed depending on the method used. To determine which approach highlighted the most predictive SNP for each trait, we used a marker-assisted best unbiased linear prediction model to evaluate lists of SNP generated by the different GWAS methods; each list contained between 25 and 2,000 candidate variants per trait, which were identified using a single within- or multi-breed GWAS approach. Among all the multi-breed methods tested in this study, variant selection based on meta-analysis (fixed effect) resulted in the most-accurate genomic evaluation (+1 to +3 points compared with other multi-breed approaches). However, the accuracies of genomic evaluation were always better when variants were selected using the results of within-breed GWAS. As has generally been found in studies of quantitative trait loci, these results suggest that part of the genetic variance of milk production traits is breed specific in Holstein, Montbéliarde, and Normande cattle.

Genetic parameters for cheese-making properties and milk composition predicted from mid-infrared spectra in a large data set of Montbéliarde cows.

Cheese-making properties of pressed cooked cheeses (PCC) and soft cheeses (SC) were predicted from mid-infrared (MIR) spectra. The traits that were best predicted by MIR spectra (as determined by comparison with reference measurements) were 3 measures of laboratory cheese yield, 5 coagulation traits, and 1 acidification trait for PCC (initial pH; pH0PPC). Coefficients of determination of these traits ranged between 0.54 and 0.89. These 9 traits as well as milk composition traits (fatty acid, protein, mineral, lactose, and citrate content) were then predicted from 1,100,238 MIR spectra from 126,873 primiparous Montbéliarde cows. Using this data set, we estimated the corresponding genetic parameters of these traits by REML procedures. A univariate or bivariate repeatability animal model was used that included the fixed effects of herd × test day × spectrometer, stage of lactation, and year × month of calving as well as the random additive genetic, permanent environmental, and residual effects. Heritability estimates varied between 0.37 and 0.48 for the 9 cheese-making property traits analyzed. Coagulation traits were the ones with the highest heritability (0.42 to 0.48), whereas cheese yields and pH0 PPC had the lowest heritability (0.37 to 0.39). Strong favorable genetic correlations, with absolute values between 0.64 and 0.97, were found between different measures of cheese yield, between coagulation traits, between cheese yields and coagulation traits, and between coagulation traits measured for PCC and SC. In contrast, the genetic correlations between milk pH0 PPC and CY or coagulation traits were weak (-0.08 to 0.09). The genetic relationships between cheese-making property traits and milk composition were moderate to high. In particular, high levels of proteins, fatty acids, Ca, P, and Mg in milk were associated with better cheese yields and improved coagulation. Proteins in milk were strongly genetically correlated with coagulation traits and, to a lesser extent, with cheese yields, whereas fatty acids in milk were more genetically correlated with cheese yields than with coagulation traits. This study, carried out on a large scale in Montbéliarde cows, shows that MIR predictions of cheese yields and milk coagulation properties are sufficiently accurate to be used for genetic analyses. Cheese-making traits, as predicted from MIR spectra, are moderately heritable and could be integrated into breeding objectives without additional phenotyping cost, thus creating an opportunity for efficient improvement via selection.

Short communication: Confirmation of candidate causative variants on milk composition and cheesemaking properties in Montbéliarde cows.

In a previous study, we identified candidate causative variants located in 24 functional candidate genes for milk protein and fatty acid composition in Montbéliarde, Normande, and Holstein cows. We designed these variants on the custom part of the EuroG10K BeadChip (Illumina Inc., San Diego, CA), which is routinely used for genomic selection analyses in French dairy cattle. To validate the effects of these candidate variants on milk composition and to estimate their effects on cheesemaking properties, a genome-wide association study was performed on milk protein, fatty acid and mineral composition, as well as on 9 cheesemaking traits (3 laboratory cheese yields, 5 coagulation traits, and milk pH). All the traits were predicted from midinfrared spectra in the Montbéliarde cow population of the Franche-Comté region. A total of 194 candidate variants located in 24 genes and 17 genomic regions were imputed on 19,862 cows with phenotypes and genotyped with either the BovineSNP50 (Illumina Inc.) or the EuroG10K BeadChip. We then tested the effect of each SNP in a mixed linear model including random polygenic effects estimated with a genomic relationship matrix. We confirm here the effects of candidate causative variants located in 17 functional candidate genes on both cheesemaking properties and milk composition traits. In each candidate gene, we identified the most plausible causative variant: 4 are missense in the ALPL, SLC26A4, CSN3, and SCD genes, 7 are located in 5'UTR (AGPAT6), 3' untranslated region (GPT), or upstream (CSN1S1, CSN1S2, PAEP, DGAT1, and PICALM) regions, and 6 are located in introns of the SLC37A1, MGST1, CSN2, BRI3BP, FASN, and ANKH genes.

Initial autophagic protection switches to disruption of autophagic flux by lysosomal instability during cadmium stress accrual in renal NRK-52E cells.

The renal proximal tubule (PT) is the major target of cadmium (Cd2+) toxicity where Cd2+ causes stress and apoptosis. Autophagy is induced by cell stress, e.g., endoplasmic reticulum (ER) stress, and may contribute to cell survival or death. The role of autophagy in Cd2+-induced nephrotoxicity remains unsettled due to contradictory results and lack of evidence for autophagic machinery damage by Cd2+. Cd2+-induced autophagy in rat kidney PT cell line NRK-52E and its role in cell death was investigated. Increased LC3-II and decreased p62 as autophagy markers indicate rapid induction of autophagic flux by Cd2+ (5-10 µM) after 1 h, accompanied by ER stress (increased p-PERK, p-eIF2α, CHOP). Cd2+ exposure exceeding 3 h results in p62/LC3-II accumulation, but diminished effect of lysosomal inhibitors (bafilomycin A1, pepstatin A +E-64d) on p62/LC3-II levels, indicating decreased autophagic flux and cargo degradation. At 24 h exposure, Cd2+ (5-25 µM) activates intrinsic apoptotic pathways (Bax/Bcl-2, PARP-1), which is not evident earlier (≤6 h) although cell viability by MTT assay is decreased. Autophagy inducer rapamycin (100 nM) does not overcome autophagy inhibition or Cd2+-induced cell viability loss. The autophagosome-lysosome fusion inhibitor liensinine (5 µM) increases CHOP and Bax/Bcl-2-dependent apoptosis by low Cd2+ stress, but not by high Cd2+. Lysosomal instability by Cd2+ (5 µM; 6 h) is indicated by increases in cellular sphingomyelin and membrane fluidity and decreases in cathepsins and LAMP1. The data suggest dual and temporal impact of Cd2+ on autophagy: Low Cd2+ stress rapidly activates autophagy counteracting damage but Cd2+ stress accrual disrupts autophagic flux and lysosomal stability, possibly resulting in lysosomal cell death.

Short communication: Genetic parameters for milk protein composition predicted using mid-infrared spectroscopy in the French Montbéliarde, Normande, and Holstein dairy cattle breeds.

Genetic parameters for the major milk proteins were estimated in the 3 main French dairy cattle breeds (i.e. Montbéliarde, Normande, and Holstein) as part of the PhénoFinlait program. The 6 major milk protein contents as well as the total protein content (PC) were estimated from mid-infrared spectrometry on 133,592 test-day milk samples from 20,434 cows in first lactation. Lactation means, expressed as a percentage of milk (protein contents) or of protein (protein fractions), were analyzed with an animal mixed model including fixed environmental effects (herd, year × month of calving, and spectrometer) and a random genetic effect. Genetic parameter estimates were very consistent across breeds. Heritability estimates (h2) were generally higher for protein fractions than for protein contents. They were moderate to high for αS1-casein, αS2-casein, ß-casein, κ-casein, and α-lactalbumin (0.25 < h2 < 0.72). In each breed, ß-lactoglobulin was the most heritable trait (0.61 < h2 < 0.86). Genetic correlations (rg) varied depending on how the percentage was expressed. The PC was strongly positively correlated with protein contents but almost genetically independent from protein fractions. Protein fractions were generally in opposition, except between κ-casein and α-lactalbumin (0.39 < rg < 0.46) and κ-casein and αS2-casein (0.36 < rg < 0.49). Between protein contents, rg estimates were positive, with highest values found between caseins (0.83 < rg < 0.98). In the 3 breeds, ß-lactoglobulin was negatively correlated with caseins (-0.75 < rg < -0.08), in particular with κ-casein (-0.75 < rg < -0.55). These results, obtained from a large panel of cows of the 3 main French dairy cattle breeds, show that routinely collected mid-infrared spectra could be used to modify milk protein composition by selection.

Genomic evaluation of regional dairy cattle breeds in single-breed and multibreed contexts.

An important prerequisite for high prediction accuracy in genomic prediction is the availability of a large training population, which allows accurate marker effect estimation. This requirement is not fulfilled in case of regional breeds with a limited number of breeding animals. We assessed the efficiency of the current French routine genomic evaluation procedure in four regional breeds (Abondance, Tarentaise, French Simmental and Vosgienne) as well as the potential benefits when the training populations consisting of males and females of these breeds are merged to form a multibreed training population. Genomic evaluation was 5-11% more accurate than a pedigree-based BLUP in three of the four breeds, while the numerically smallest breed showed a < 1% increase in accuracy. Multibreed genomic evaluation was beneficial for two breeds (Abondance and French Simmental) with maximum gains of 5 and 8% in correlation coefficients between yield deviations and genomic estimated breeding values, when compared to the single-breed genomic evaluation results. Inflation of genomic evaluation of young candidates was also reduced. Our results indicate that genomic selection can be effective in regional breeds as well. Here, we provide empirical evidence proving that genetic distance between breeds is only one of the factors affecting the efficiency of multibreed genomic evaluation.

Whole-genome scan to detect quantitative trait loci associated with milk protein composition in 3 French dairy cattle breeds.

In the context of the PhénoFinLait project, a genome-wide analysis was performed to detect quantitative trait loci (QTL) that affect milk protein composition estimated using mid-infrared spectrometry in the Montbéliarde (MO), Normande (NO), and Holstein (HO) French dairy cattle breeds. The 6 main milk proteins (α-lactalbumin, ß-lactoglobulin, and αS1-, αS2-, ß-, and κ-caseins) expressed as grams per 100g of milk (% of milk) or as grams per 100g of protein (% of protein) were estimated in 848,068 test-day milk samples from 156,660 cows. Genotyping was performed for 2,773 MO, 2,673 NO, and 2,208 HO cows using the Illumina BovineSNP50 BeadChip (Illumina Inc., San Diego, CA). Individual test-day records were adjusted for environmental effects and then averaged per cow to define the phenotypes analyzed. Quantitative trait loci detection was performed within each breed using a linkage disequilibrium and linkage analysis approach. A total of 39 genomic regions distributed on 20 of the 29 Bos taurus autosomes (BTA) were significantly associated with milk protein composition at a genome-wide level of significance in at least 1 of the 3 breeds. The 9 most significant QTL were located on BTA2 (133 Mbp), BTA6 (38, 47, and 87 Mbp), BTA11 (103 Mbp), BTA14 (1.8 Mbp), BTA20 (32 and 58 Mbp), and BTA29 (8 Mbp). The BTA6 (87 Mbp), BTA11, and BTA20 (58 Mbp) QTL were found in all 3 breeds, and they had highly significant effects on κ-casein, ß-lactoglobulin, and α-lactalbumin, expressed as a percentage of protein, respectively. Each of these QTL explained between 13% (BTA14) and 51% (BTA11) of the genetic variance of the trait. Many other QTL regions were also identified in at least one breed. They were located on 14 additional chromosomes (1, 3, 4, 5, 7, 15, 17, 19, 21, 22, 24, 25, 26, and 27), and they explained 2 to 8% of the genetic variance of 1 or more protein composition traits. Concordance analyses, performed between QTL status and sequence-derived polymorphisms from 13 bulls, revealed previously known causal polymorphisms in LGB (BTA11) and GHR (BTA20 at 32 Mbp) and excluded some other previously described mutations. These results constitute a first step in identifying causal mutations and using routinely collected mid-infrared predictions in future genomic selection programs to improve bovine milk protein composition.

Successful cryopreservation in biodegradable containers of sperm from aquaculture Mediterranean fishes.

We aimed to evaluate the efficiency of hard-gelatin and hard-hydroxypropyl methylcellulose (HPMC) capsules as biodegradable alternative containers to plastic straws in European eel (Anguilla anguilla), gilthead seabream (Sparus aurata) and European sea bass (Dicentrarchus labrax) sperm cryopreservation. Sperm samples from each European eel (n = 12) were diluted 1:8:1 (sperm: extender P1+5 % egg yolk: methanol). Gilthead seabream (n = 12) samples were individually diluted in a cryoprotectant solution of 5 % Me2SO + NaCl 1 % plus BSA (10 mg mL-1) at a ratio of 1:6 (sperm: cryoprotectant solution). European sea bass (n = 10) sperm from each male was diluted in non-activating medium (NAM) at a ratio of 1:5.7 (sperm: NAM), and 5 % of Me2SO was added. The diluted European eel and sea bass sperm aliquots (0.5 mL) were individually filled in plastic straws (0.5 mL), hard-gelatin, and HPMC capsules (0.68 mL). Gilthead seabream diluted sperm (0.25 mL) were filled in plastic straws (0.25 mL) and identical capsules described. All samples were frozen in liquid nitrogen vapor and stored in a liquid nitrogen tank. Sperm kinetic parameters were evaluated by CASA-Mot software. Sperm membrane integrity was performed using a Live and Dead KIT and an epifluorescence microscope. To quantify DNA damage, the alkaline comet assay was performed and TailDNA (TD-%) and Olive Tail Moment (OTM) were evaluated by CaspLab software. Sperm cryopreservation of the three Mediterranean species in straws, gelatin, or HPMC capsules reduced the kinetic parameters and cell membrane integrity. Generally, the post-thawing samples cryopreserved in straws and capsules did not differ for the kinetic parameters and cell membrane integrity, except for European sea bass sperm, where the samples stored in gelatin capsules showed higher velocities (VCL - 100; VSL - 76; VAP - 90 µm s-1) than the sperm stored in HPMC capsules (VCL - 87; VSL - 59; VAP - 73 µm s-1). The cryopreservation process did not damage the sperm DNA of European eel and European sea bass, regardless of the containers used. On the other hand, gilthead seabream sperm cryopreserved in gelatin (TD - 9.8 %; OTM - 9.7) and HPMC (TD - 11.1 %; OTM - 11.2) capsules showed higher DNA damage than fresh samples (TD - 3.6 %; OTM - 2.7) and the sperm stored in straws (TD - 4.4 %; OTM - 5.2). The hard-gelatin and HPMC biodegradable capsules can be used as an alternative to straws for European eel, gilthead seabream, and European sea bass sperm cryopreservation.

Towards candidate genes affecting body fatness at the SSC7 QTL by expression analyses.

A quantitative trait locus (QTL) affecting fatness in a way opposite to expectations based on breed means was mapped to swine chromosome 7 (SSC7) using crosses between Large White (LW) and Meishan (MS) founders. Defining the molecular fatness trait more explicitly would allow deducing positional candidate genes, for which expression differences must be analysed in experimental populations. First, mRNA levels of genes representing sequential steps in adipogenesis or involved in lipid metabolism were studied in backfat of pigs having homozygous LW(QTL7)/LW(QTL7) or heterozygous LW(QTL7)/MS(QTL7) alleles and considered at two ages. mRNA level of DLK1 expressed in preadipocytes was greater in MS(QTL7)/LW(QTL7) pigs than in homozygous pigs at 28 days. Transcript abundances of CEBPA involved in differentiation, the prolipogenic FASN gene and the adipocyte-specific marker FABP4 were lower in MS(QTL7)/LW(QTL7) pigs compared with LW(QTL7)/LW(QTL7) pigs at 150 days. Because these results suggest a lag time in terminal differentiation associated with the MS allele, seven genes in the QTL interval were deduced as promising candidates for the QTL effect by bioinformatics analysis. Among them, PPARD and CDKN1A had lower expression levels in MS(QTL7)/LW(QTL7) pigs at both ages. Genotype-related differences were observed in mRNA levels of PPARD target genes involved in cell differentiation (FZD7) or fatty acid oxidation (ACADL and ACOX1) at 150 days. These results re-evaluate the potential of PPARD to explain part of variation in pig adiposity.

Impact of nucleosome dynamics and histone modifications on cell proliferation during Arabidopsis development.

Eukaryotic chromatin is a highly structured macromolecular complex of which DNA is wrapped around a histone-containing core. DNA can be methylated at specific C residues and each histone molecule can be covalently modified at a large variety of amino acids in both their tail and core domains. Furthermore, nucleosomes are not static entities and both their position and histone composition can also vary. As a consequence, chromatin behaves as a highly dynamic cellular component with a large combinatorial complexity beyond DNA sequence that conforms the epigenetic landscape. This has key consequences on various developmental processes such as root and flower development, gametophyte and embryo formation and response to the environment, among others. Recent evidence indicate that posttranslational modifications of histones also affect cell cycle progression and processes depending on a correct balance of proliferating cell populations, which in the context of a developing organisms includes cell cycle, stem cell dynamics and the exit from the cell cycle to endoreplication and cell differentiation. The impact of epigenetic modifications on these processes will be reviewed here.

Portable gamma-camera for the diagnosis of brain death diagnosis. / Minigammacámara portátil para el diagnóstico de muerte encefálica.

OBJECTIVE: To evaluate the feasibility of using the Sentinella® portable gamma-camera for the diagnosis of brain death (BD). DESIGN: A prospective, observational feasibility study was carried out. SETTING: Intensive Care Unit of a third level hospital. PATIENTS: Consecutive recording was made of the adults diagnosed with brain death based on clinical criteria following admission to the Intensive Care Unit in the period from January to December 2017. INTERVENTIONS: The procedure was performed at the patient bedside with the intravenous administration of technetium 99 metastable hexamethylpropylene amine oxime. The absence of perfusion in the cerebral hemispheres and brainstem was described as a pattern consistent with BD. The diagnosis was correlated to the transcranial Doppler and / or electroencephalographic findings. RESULTS: A total of 66.1% of the patients were men with an average age of 60 years [IQR: 51-72]. The most frequent causes resulting in BD were hemorrhagic stroke (48.2%, n=27), followed by traumatic brain injury (30.4%, n=17), ischemic stroke (10.7%, n=6) and post-cardiac arrest anoxic encephalopathy (7.1%, n=4). A clinical diagnosis of BD was made in all cases, and the portable gamma-camera confirmed the diagnosis in 100% of the patients with a pattern characterized by the absence of brain perfusion. In addition, the results were compared with the transcranial Doppler findings in 46 patients, confirming the presence of diastolic reverberation and / or systolic peaks. The electroencephalographic tracing was obtained in 10 cases, with the appearance of electrical silence, due to the absence of an acoustic window in the transcranial Doppler study. CONCLUSIONS: A portable gamma-camera could be a useful and feasible tool for the diagnosis of BD.

Brain atrophy as a marker of cognitive impairment in mildly disabling relapsing-remitting multiple sclerosis.

BACKGROUND AND PURPOSE: We have studied the relationship between neuropsychological impairment and magnetic resonance imaging (MRI) measures in mildly disabling relapsing-remitting multiple sclerosis (RRMS). METHODS: We compared measures of lesion burden and atrophy in 52 patients with Expanded Disability Status Scale

Motor alterations are reduced in mice lacking the PARK2 gene in the presence of a human FTDP-17 mutant form of four-repeat tau.

Independent deletion of the PARK2 gene and hTauVLW over-expression in mice produce mild alterations in the brain. However, the presence of both mutations in a parkin-deficient and hTauVLW double mutant mouse causes a tau neuropathology, reactive astrocytosis, and neuronal loss in the cortex and hippocampus, as well as lesions in nigrostriatal and motor neurons. Moreover, these mutants display some memory and exploratory defects that reflect a functional link between parkin and tau proteins. We have tested the motor activity and coordination of these double mutant mice to determine the effects of parkin deletion in mice over-expressing the hTauVLW transgene. While the loss of parkin alone produces increased exploration and alterations in gait and motor coordination, in hTauVLW transgenic mice the absence of parkin causes less prominent motor impairments. These effects suggest the existence of some compensatory mechanisms that are activated when the hTauVLW transgene is over-expressed in the absence of parkin. This mouse model will hopefully help to study the causes of the motor deficits associated with certain neuropathologies related to the tau and parkin proteins, and to find appropriate treatments.

Evaluating average bioequivalence using methods for high variability drugs: a case study.

The purpose of this study was to determine if different methods for average bioequivalence in high variability drugs coincide or not in their conclusions when applied to the same dataset, and to discuss the method validity and reliability of the conclusions. Different approaches for the evaluation of average bioequivalence were applied to the results of a bioavailability trial on the diuretic drug Furosemide. These methods included widening the bioequivalence limits according to regulatory recommendations, scaling the limits and scaling the bioequivalence statistic, jointly with evaluating alternative bioavailability measures. The methods to establish the bioequivalence limits were also combined with some alternative methods to construct confidence intervals. The decision on bioequivalence depends much more on the bioavailability measures than on the statistical approach. The reliability of the final decision lies mainly in the interpretation of these measures and on the special characteristics of each drug.

Practice Models in Teledermatology in Spain: Longitudinal Study, 2009-2014. / Modelos de práctica de la teledermatología en España. Estudio longitudinal 2009-2014.

INTRODUCTION: Teledermatology is the most advanced clinical specialty in telemedicine. The development of teledermatology in specific countries has not been studied in depth. METHODS: Our objective was to analyze teledermatology models in clinical practice in Spain. We paid special attention to organization, technical aspects, training, and the advantages/disadvantages as seen by teledermatologists. Two surveys were carried out (2009 and 2014). RESULTS: Teledermatology was used at 25 centers in 2009 and at 70 in 2014. The extended survey was completed by 21 centers in 2009 and 41 in 2014. Store-and-forward teledermatology was the main technique (83%) in 2014. Only 12% of centers used the real-time method, and 5% used a hybrid modality. Patients lived less than 25km away in 75% of cases (urban teledermatology). Most centers used mid-range bridge cameras; only 12% used mobile phones. Teledermoscopy and tertiary teledermatology were each used in 15% of centers. Teledermatology was restricted to skin cancer in 25% of cases, and 66% of centers used it to train primary care physicians. The main advantages, assessed on a scale of 1 to 10, were prioritization in cancer screening (8.3), rapid emergency care (7.8), training of and communication with primary care physicians (7.6), screening for trivial conditions (7.6), and reduction in the number of face-to-face visits (7.6). The main disadvantages were poor image quality (6.3), fear of error (5.7), difficulty in coordinating with primary care physicians (3.8), and time commitment (3.3). Between 2009 and 2014, the number of centers using teledermatology and the number of teledermatologists increased, as did use of the store-and-forward and urban models. The technology used also improved. CONCLUSION: Teledermatology is an emerging technology that is becoming well established in Spain. More than 25% of dermatology centers in Spain have implemented a teledermatology model. Store-and-forward in an urban setting is the most widely used modality. Teledermatologists see this technology as an effective option with more advantages than disadvantages. General satisfaction is high, although there is room for significant improvement in some areas.