Assessment of runs of homozygosity, heterozygosity-rich regions and genomic inbreeding estimates in a subpopulation of Guzerá (Bos indicus) dual-purpose cattle.

For decades, inbreeding in cattle has been evaluated using pedigree information. Nowadays, inbreeding coefficients can be obtained using genomic information such as runs of homozygosity (ROH). The aims of this study were to quantify ROH and heterozygosity-rich regions (HRR) in a subpopulation of Guzerá dual-purpose cattle, to examine ROH and HRR islands, and to compare inbreeding coefficients obtained by ROH with alternative genomic inbreeding coefficients. A subpopulation of 1733 Guzerá animals genotyped for 50k SNPs was used to obtain the ROH and HRR segments. Inbreeding coefficients by ROH (FROH ), by genomic relationship matrix based on VanRaden's method 1 using reference allele frequency in the population (FGRM ), by genomic relationship matrix based on VanRaden's method 1 using allele frequency fixed in 0.5 (FGRM_0.5 ), and by the proportion of homozygous loci (FHOM ) were calculated. A total of 15,660 ROH were identified, and the chromosome with the highest number of ROH was BTA6. A total of 4843 HRRs were identified, and the chromosome with the highest number of HRRs was BTA23. No ROH and HRR islands were identified according to established criteria, but the regions closest to the definition of an island were examined from 64 to 67 Mb of BTA6, from 36 to 37 Mb of BTA2 and from 0.50 to 1.25 Mb of BTA23. The genes identified in ROH islands have previously been associated with dairy and beef traits, while genes identified on HRR islands have previously been associated with reproductive traits and disease resistance. FROH was equal to 0.095 ± 0.084, and its Spearman correlation with FGRM was low (0.44) and moderate-high with FHOM (0.79) and with FGRM_0.5 (0.80). The inbreeding coefficients determined by ROH were higher than other cattle breeds' and higher than pedigree-based inbreeding in the Guzerá breed obtained in previous studies. It is recommended that future studies investigate the effects of inbreeding determined by ROH on the traits under selection in the subpopulation studied.

Insights on sperm assays and cryopreservation in six Neotropical pit vipers.

Breeding snakes in captivity has become more and more relevant due not only to the growing interest on their venoms but also to the increasing number of endangered species worldwide. Unfortunately, studies on the formation of germplasm banks for these reptiles do not follow the same pace, and literature on sperm cryopreservation remains in its infancy when compared to other taxa. Herein, we first validated a sperm-egg binding assay (using chicken egg perivitelline membrane - EPM) and some nonfluorescent staining techniques for semen analysis of two pit viper genera (Bothrops and Crotalus), and then we investigated the protective effects of dimethylacetamide (DMA), dimethylformamide (DMF), and dimethylsulfoxide (DMSO) at different concentrations (3, 6 and 12%) throughout the freezing process in five species of lancehead and one of rattlesnake. Our validation process showed high correlations among sperm functional tests (including sperm-binding to EPM) and motion parameters. A total of 166 fresh ejaculates were acquired from 233 collection attempts, and 63.9% of these samples exhibited minimal motility for freezing (≥20%). During cryopreservation we observed that post-thaw motility and quality was improved by higher levels of cryoprotectants (CPA), regardless the CPA type. Lower concentrations of CPA were less harmful to sperm motility and progressive motility following the equilibrium phase, but were ineffective in protecting these cells from the freeze-thaw cycle. Likewise, higher CPA concentrations increased post-thaw integrity of the acrosome and plasma membrane for most species, except for rattlesnakes in which only 12% DMSO produced better outcomes.

Physical and ecological isolation contribute to maintain genetic differentiation between fire salamander subspecies.

Landscape features shape patterns of gene flow among populations, ultimately determining where taxa lay along the continuum between panmixia to complete reproductive isolation. Gene flow can be restricted, leading to population differentiation in two non-exclusive ways: "physical isolation", in which geographic distance in combination with the landscape features restricts movement of individuals promoting genetic drift, and "ecological isolation", in which adaptive mechanisms constrain gene flow between different environments via divergent natural selection. In central Iberia, two fire salamander subspecies occur in parapatry across elevation gradients along the Iberian Central System mountains, while in the adjacent Montes de Toledo Region only one of them occurs. By integrating population and landscape genetic analyses, we show a ubiquitous role of physical isolation between and within mountain ranges, with unsuitable landscapes increasing differentiation between populations. However, across the Iberian Central System, we found strong support for a significant contribution of ecological isolation, with low genetic differentiation in environmentally homogeneous areas, but high differentiation across sharp transitions in precipitation seasonality. These patterns are consistent with a significant contribution of ecological isolation in restricting gene flow among subspecies. Overall, our results suggest that ecological divergence contributes to reduce genetic admixture, creating an opportunity for lineages to follow distinct evolutionary trajectories.

Inbreeding depression in Zebu cattle traits.

The productivity of herds may be negatively affected by inbreeding depression, and it is important to know how intense is this effect on the livestock performance. We performed a comprehensive analysis involving five Zebu breeds reared in Brazil to estimate inbreeding depression in productive and reproductive traits. Inbreeding depression was estimated for 13 traits by including the individual inbreeding rate as a linear covariate in the standard genetic evaluation models. For all breeds and for almost all traits (no effect was observed on gestation length), the performance of the animals was compromised by an increase in inbreeding. The average inbreeding depression was -0.222% and -0.859% per 1% of inbreeding for linear regression coefficients scaled on the percentage of mean (ßm ) and standard deviation (ßσ ), respectively. The means for ßm (and ßσ ) were -0.269% (-1.202%) for weight/growth traits and -0.174% (-0.546%) for reproductive traits. Hence, inbreeding depression is more pronounced in weight/growth traits than in reproductive traits. These findings highlight the need for the management of inbreeding in the respective breeding programmes of the breeds studied here.

Detrimental effect of selection for milk yield on genetic tolerance to heat stress in purebred Zebu cattle: Genetic parameters and trends.

In an attempt to determine the possible detrimental effects of continuous selection for milk yield on the genetic tolerance of Zebu cattle to heat stress, genetic parameters and trends of the response to heat stress for 86,950 test-day (TD) milk yield records from 14,670 first lactations of purebred dairy Gir cows were estimated. A random regression model with regression on days in milk (DIM) and temperature-humidity index (THI) values was applied to the data. The most detrimental effect of THI on milk yield was observed in the stage of lactation with higher milk production, DIM 61 to 120 (-0.099kg/d per THI). Although modest variations were observed for the THI scale, a reduction in additive genetic variance as well as in permanent environmental and residual variance was observed with increasing THI values. The heritability estimates showed a slight increase with increasing THI values for any DIM. The correlations between additive genetic effects across the THI scale showed that, for most of the THI values, genotype by environment interactions due to heat stress were less important for the ranking of bulls. However, for extreme THI values, this type of genotype by environment interaction may lead to an important error in selection. As a result of the selection for milk yield practiced in the dairy Gir population for 3 decades, the genetic trend of cumulative milk yield was significantly positive for production in both high (51.81kg/yr) and low THI values (78.48kg/yr). However, the difference between the breeding values of animals at high and low THI may be considered alarming (355kg in 2011). The genetic trends observed for the regression coefficients related to general production level (intercept of the reaction norm) and specific ability to respond to heat stress (slope of the reaction norm) indicate that the dairy Gir population is heading toward a higher production level at the expense of lower tolerance to heat stress. These trends reflect the genetic antagonism between production and tolerance to heat stress demonstrated by the negative genetic correlation between these components (-0.23). Monitoring trends of the genetic component of heat stress would be a reasonable measure to avoid deterioration in one of the main traits of Zebu cattle (i.e., high tolerance to heat stress). On the basis of current genetic trends, the need for future genetic evaluation of dairy Zebu animals for tolerance to heat stress cannot be ruled out.

Utilisation of sperm-binding assay combined with computer-assisted sperm analysis to evaluate frozen-thawed bull semen.

Due to homologies between the chicken egg perivitelline membrane with mammalian zona pellucida proteins, spermatozoa of several species are able to bind to this membrane. However, adequate standardisation is required to attest possible applications of this technique for semen evaluation of a given species. Therefore, we thawed and divided cryopreserved semen samples into two aliquotes, one kept in water bath at 37 °C (thawed) and the other submitted to snap-freezing to damage sperm cells (dead spermatozoa). Aliquotes were mixed into different ratios of thawed:dead cells and analysed for motility, membrane and acrosomal integrity, and mitochondrial activity. In parallel, chicken egg perivitelline membranes were inseminated with these ratios, and the number of spermatozoa bound per mm(2) of membrane was assessed by conventional microscopy (CM) and computer-assisted sperm analysis (CASA). Linear regression showed high correlation between thawed:dead sperm ratio and number of spermatozoa bound to the membrane (CM: r(2) = 0.91 and CASA: r(2) = 0.92 respectively). Additionally, positive correlations were found between the number of spermatozoa bound to the membrane and acrosomal integrity, membrane integrity, mitochondrial activity and motility. These findings indicate that sperm-egg-binding assay associated with CASA is a reliable, practical and inexpensive method for examining the fertilising capacity of cryopreserved bull semen.

Random regression analysis of test-day milk yields in the first and second lactations of Brazilian Gyr cows.

The objective of the present study was to estimate the genetic parameters for test-day milk yields (TDMY) in the first and second lactations using random regression models (RRM) in order to contribute to the application of these models in genetic evaluation of milk yield in Gyr cattle. A total of 53,328 TDMY records from 7118 lactations of 5853 Gyr cows were analyzed. The model included the direct additive, permanent environmental, and residual random effects. In addition, contemporary group and linear and quadratic effects of the age of cows at calving were included as fixed effects. A random regression model fitting fourth-order Legendre polynomials for additive genetic and permanent environmental effects, with five classes of residual variance, was applied. In the first lactation, the heritabilities increased from early lactation (0.26) until TDMY3 (0.38), followed by a decrease until the end of lactation. In the second lactation, the estimates increased from the first (0.29) to the fifth test day (0.36), with a slight decrease thereafter, and again increased on the last two test days (0.34 and 0.41). There were positive and high genetic correlations estimated between first-lactation TDMY and the remaining TDMY of the two lactations. The moderate heritability estimates, as well as the high genetic correlations between half the first-lactation TDMY and all TDMY of the two lactations, suggest that the selection based only on first lactation TDMY is the best selection strategy to increase milk production across first and second lactations of Gyr cows.

Genotype by environment interaction for post-weaning weight gain, scrotal circumference, and muscling score of composite beef cattle in different regions of Brazil.

The objectives of this study were to characterize and define homogenous production environments of composite beef cattle in Brazil in terms of climatic and geographic variables by using multivariate exploratory techniques; to evaluate the presence of genotype by environment interaction (GxE) for post-weaning weight gain (PWG), yearling scrotal circumference (SC), and yearling muscling (MUS). Hierarchical and nonhierarchical cluster analysis was used to group farms located in regions with similar environmental variables into clusters. Six clusters of farms were formed. The effect of sire-cluster interaction was tested by single-trait analysis. Genetic parameters were estimated by multi-trait analysis considering the same trait to be different in each cluster. The effect of sire-cluster interaction was significant (P <0.01) for PWG and MUS. Estimates of genetic correlations among clusters ranged from 0.31 to 0.93 for PWG, 0.64 to 0.89 for SC, and 0.18 to 0.80 for MUS. These results indicate the need for a genetic analysis on a regional basis or inclusion of the GxE effect in the statistical model to permit appropriate evaluation of the animals.

Longitudinal genetic dynamics of weaning index and implications for cow-calf production efficiency.

In beef cattle, the selection for higher weights at young ages has been questioned with the argument that this criterion may increase the adult weight of cows, resulting in higher costs. Therefore, selection criteria should be employed to increase weights at young ages with minimal impact on the adult weight of cows. Additionally, the relationship between measures of cow production efficiency and other well-established selection criteria in breeding programs remains poorly understood. The objective of this study was to longitudinally evaluate the relationship between the weaning index (WIndex) as a measure of efficiency and growth traits of the cows. Possible changes over time in WIndex due to selection applied for yearling weight (YW) were also investigated. The WIndex was proposed to maximize genetic response in the weaning weight of the calf while maintaining genetic gain in BW of the cow at zero. A random regression model was adopted to estimate correlations between WIndex, BW, hip height (HH), and body condition score (BCS) using records of Nelore cows from three lines. Genetic trends were calculated for the control line (NeC) and lines selected for greater YW (NeS and NeT). The age of 3 years was the most critical for the weaning efficiency of the cows. At this stage, young cows are still growing and wean lighter calves than their adult counterparts. The genetic correlation estimates between WIndex and BW (-0.58 to 0.04), HH (-0.05 to -0.34), and BCS (-0.51 to -0.17) were close to zero or negative. BW and HH were strongly correlated genetically across all ages (0.73-0.76). In general, HH exhibited a weak and negative genetic relationship with BCS. The genetic correlation between BW and BCS was stronger for advanced ages (0.45-0.68). In lines selected for YW, important increases in WIndex were observed. However, NeS has been selected since the 1980s until the present for YW, and thus, it showed a more pronounced trend of increasing BW and, consequently, a more modest trend of increasing WIndex compared to NeT. In contrast, WIndex exhibited a trend close to zero for NeC. In this context, monitoring HH and BCS can be useful to avoid losses in the weaning efficiency of cows. Furthermore, we suggest that one way to mitigate efficiency losses in calf production could involve stabilizing the BW of cows and increasing the weaning weight of calves using the WIndex.

Random regression models using Legendre polynomials or linear splines for test-day milk yield of dairy Gyr (Bos indicus) cattle.

Studies investigating the use of random regression models for genetic evaluation of milk production in Zebu cattle are scarce. In this study, 59,744 test-day milk yield records from 7,810 first lactations of purebred dairy Gyr (Bos indicus) and crossbred (dairy Gyr × Holstein) cows were used to compare random regression models in which additive genetic and permanent environmental effects were modeled using orthogonal Legendre polynomials or linear spline functions. Residual variances were modeled considering 1, 5, or 10 classes of days in milk. Five classes fitted the changes in residual variances over the lactation adequately and were used for model comparison. The model that fitted linear spline functions with 6 knots provided the lowest sum of residual variances across lactation. On the other hand, according to the deviance information criterion (DIC) and bayesian information criterion (BIC), a model using third-order and fourth-order Legendre polynomials for additive genetic and permanent environmental effects, respectively, provided the best fit. However, the high rank correlation (0.998) between this model and that applying third-order Legendre polynomials for additive genetic and permanent environmental effects, indicates that, in practice, the same bulls would be selected by both models. The last model, which is less parameterized, is a parsimonious option for fitting dairy Gyr breed test-day milk yield records.

Comparison of factor-analytic and reduced rank models for test-day milk yield in Gyr dairy cattle (Bos indicus).

We analyzed 46,161 monthly test-day records of milk production from 7453 first lactations of crossbred dairy Gyr (Bos indicus) x Holstein cows. The following seven models were compared: standard multivariate model (M10), three reduced rank models fitting the first 2, 3, or 4 genetic principal components, and three models considering a 2-, 3-, or 4-factor structure for the genetic covariance matrix. Full rank residual covariance matrices were considered for all models. The model fitting the first two principal components (PC2) was the best according to the model selection criteria. Similar phenotypic, genetic, and residual variances were obtained with models M10 and PC2. The heritability estimates ranged from 0.14 to 0.21 and from 0.13 to 0.21 for models M10 and PC2, respectively. The genetic correlations obtained with model PC2 were slightly higher than those estimated with model M10. PC2 markedly reduced the number of parameters estimated and the time spent to reach convergence. We concluded that two principal components are sufficient to model the structure of genetic covariances between test-day milk yields.

Linear and Poisson models for genetic evaluation of tick resistance in cross-bred Hereford x Nellore cattle.

Cattle resistance to ticks is measured by the number of ticks infesting the animal. The model used for the genetic analysis of cattle resistance to ticks frequently requires logarithmic transformation of the observations. The objective of this study was to evaluate the predictive ability and goodness of fit of different models for the analysis of this trait in cross-bred Hereford x Nellore cattle. Three models were tested: a linear model using logarithmic transformation of the observations (MLOG); a linear model without transformation of the observations (MLIN); and a generalized linear Poisson model with residual term (MPOI). All models included the classificatory effects of contemporary group and genetic group and the covariates age of animal at the time of recording and individual heterozygosis, as well as additive genetic effects as random effects. Heritability estimates were 0.08 ± 0.02, 0.10 ± 0.02 and 0.14 ± 0.04 for MLIN, MLOG and MPOI models, respectively. The model fit quality, verified by deviance information criterion (DIC) and residual mean square, indicated fit superiority of MPOI model. The predictive ability of the models was compared by validation test in independent sample. The MPOI model was slightly superior in terms of goodness of fit and predictive ability, whereas the correlations between observed and predicted tick counts were practically the same for all models. A higher rank correlation between breeding values was observed between models MLOG and MPOI. Poisson model can be used for the selection of tick-resistant animals.

Milk yield persistency in Brazilian Gyr cattle based on a random regression model.

With the objective of evaluating measures of milk yield persistency, 27,000 test-day milk yield records from 3362 first lactations of Brazilian Gyr cows that calved between 1990 and 2007 were analyzed with a random regression model. Random, additive genetic and permanent environmental effects were modeled using Legendre polynomials of order 4 and 5, respectively. Residual variance was modeled using five classes. The average lactation curve was modeled using a fourth-order Legendre polynomial. Heritability estimates for measures of persistency ranged from 0.10 to 0.25. Genetic correlations between measures of persistency and 305-day milk yield (Y305) ranged from -0.52 to 0.03. At high selection intensities for persistency measures and Y305, few animals were selected in common. As the selection intensity for the two traits decreased, a higher percentage of animals were selected in common. The average predicted breeding values for Y305 according to year of birth of the cows had a substantial annual genetic gain. In contrast, no improvement in the average persistency breeding value was observed. We conclude that selection for total milk yield during lactation does not identify bulls or cows that are genetically superior in terms of milk yield persistency. A measure of persistency represented by the sum of deviations of estimated breeding value for days 31 to 280 in relation to estimated breeding value for day 30 should be preferred in genetic evaluations of this trait in the Gyr breed, since this measure showed a medium heritability and a genetic correlation with 305-day milk yield close to zero. In addition, this measure is more adequate at the time of peak lactation, which occurs between days 25 and 30 after calving in this breed.

Genetic associations of visual scores with subsequent rebreeding and days to first calving in Nellore cattle.

Records of Nellore animals born from 1990 to 2006 were used to estimate genetic correlations of visual scores at yearling (conformation, C; finishing precocity, P; and muscling, M) with primiparous subsequent rebreeding (SR) and days to first calving (DC), because the magnitude of these associations is still unknown. Genetic parameters were estimated by multiple-traits Bayesian analysis, using a nonlinear (threshold) animal models for visual scores and SR and a linear animal models for weaning weight (WW) and DC. WW was included in the analysis to account for the effects of sequential selection. The posterior means of heritabilities estimated for C, P, M, SR and DC were 0.24 ± 0.01, 0.31 ± 0.01, 0.30 ± 0.01, 0.18 ± 0.02 and 0.06 ± 0.02, respectively. The posterior means of genetic correlations estimated between SR and visual scores were low and positive, with values of 0.09 ± 0.02 (C), 0.19 ± 0.03 (P) and 0.18 ± 0.05 (M). On the other hand, negative genetic correlations were found between DC and C (-0.11 ± 0.09), P (-0.19 ± 0.09) and M (-0.16 ± 0.09). The primiparous rebreeding trait has genetic variability in Nellore cattle. The genetic correlations between visual scores, and SR and DC were low and favourable. The genetic changes in C, P and M were 0.02, 0.03 and 0.03/year, respectively. For SR and DC, genetic trends were 0.01/year and -0.01 days/year, respectively, indicating that the increase in genetic merit for reproductive traits was small over time. Direct selection for visual scores together with female reproductive traits is recommended to increase the fertility of beef cows.

Effect of pregnancy on the genetic evaluation of dairy cattle.

We investigated the effect of stage of pregnancy on estimates of breeding values for milk yield and milk persistency in Gyr and Holstein dairy cattle in Brazil. Test-day milk yield records were analyzed using random regression models with or without the effect of pregnancy. Models were compared using residual variances, heritabilities, rank correlations of estimated breeding values of bulls and cows, and number of nonpregnant cows in the top 200 for milk yield and milk persistency. The estimates of residual variance and heritabilities obtained with the models with or without the effect of pregnancy were similar for the two breeds. Inclusion of the effect of pregnancy in genetic evaluation models for these populations did not affect the ranking of cows and sires based on their predicted breeding values for 305-day cumulative milk yield. In contrast, when we examined persistency of milk yield, lack of adjustment for the effect of pregnancy overestimated breeding values of nonpregnant cows and cows with a long days open period and underestimated breeding values of cows with a short days open period. We recommend that models include the effect of days of pregnancy for estimation of adjustment factors for the effect of pregnancy in genetic evaluations of Dairy Gyr and Holstein cattle.

Breeding value accuracy estimates for growth traits using random regression and multi-trait models in Nelore cattle.

We quantified the potential increase in accuracy of expected breeding value for weights of Nelore cattle, from birth to mature age, using multi-trait and random regression models on Legendre polynomials and B-spline functions. A total of 87,712 weight records from 8144 females were used, recorded every three months from birth to mature age from the Nelore Brazil Program. For random regression analyses, all female weight records from birth to eight years of age (data set I) were considered. From this general data set, a subset was created (data set II), which included only nine weight records: at birth, weaning, 365 and 550 days of age, and 2, 3, 4, 5, and 6 years of age. Data set II was analyzed using random regression and multi-trait models. The model of analysis included the contemporary group as fixed effects and age of dam as a linear and quadratic covariable. In the random regression analyses, average growth trends were modeled using a cubic regression on orthogonal polynomials of age. Residual variances were modeled by a step function with five classes. Legendre polynomials of fourth and sixth order were utilized to model the direct genetic and animal permanent environmental effects, respectively, while third-order Legendre polynomials were considered for maternal genetic and maternal permanent environmental effects. Quadratic polynomials were applied to model all random effects in random regression models on B-spline functions. Direct genetic and animal permanent environmental effects were modeled using three segments or five coefficients, and genetic maternal and maternal permanent environmental effects were modeled with one segment or three coefficients in the random regression models on B-spline functions. For both data sets (I and II), animals ranked differently according to expected breeding value obtained by random regression or multi-trait models. With random regression models, the highest gains in accuracy were obtained at ages with a low number of weight records. The results indicate that random regression models provide more accurate expected breeding values than the traditionally finite multi-trait models. Thus, higher genetic responses are expected for beef cattle growth traits by replacing a multi-trait model with random regression models for genetic evaluation. B-spline functions could be applied as an alternative to Legendre polynomials to model covariance functions for weights from birth to mature age.

Genotype by environment interaction due to heat stress during gestation and postpartum for milk production of Holstein cattle.

Remarkable increases in the production of dairy animals have negatively impacted their tolerance to heat stress (HS). The evaluation of the effect of HS on milk yield is based on the direct impact of HS on performance. However, in practical terms, HS also exerts its influence during gestation (indirect effect). The main purpose of this study was to identify and characterize the genotype by environment interaction (G × E) due to HS during the last 60 days of gestation (THI_g) and also the HS postpartum (THI_m) over first lactation milk production of Brazilian Holstein cattle. A total of 389 127 test day milk yield (TD) records from 1572 first lactation Holstein cows born in Brazil (daughters of 1248 dams and 70 sires) and the corresponding temperature-humidity index (THI) obtained between December 2007 and January 2013 were analyzed using different random regression models. Cows in the cold environment (THI_g = 64 to 73) during the last 60 days of gestation produced more milk than those cows in a hot environment (THI_g = 74 to 84), particularly during the first 150 days of lactation (DIM). The heritabilities (h2) of TD were similar throughout DIM for cows in THI_g hot (0.11 to 0.20) or (0.10 to 0.22), while the genetic correlations (rg) for TD between these two environments ranged from 0.11 to 0.52 along the first 250 DIM. The h2 estimates for TD across THI_m were similar for cows in THI_g hot (0.07 to 0.25) and THI_g cold (0.08 to 0.19). The rg estimates ranged from 0.17 to 0.42 along THI_m between TD of cows in cold and hot THI_g. The results were consistent in demonstrating the existence of an additional source of G × E for TD due to THI_g and THI_m. The present study is probably the first to provide evidence of this source of G × E; further research is needed because of its importance when the breeding objective is to select animals that are more tolerant to HS.

High levels of population subdivision in a morphologically conserved Mediterranean toad (Alytes cisternasii) result from recent, multiple refugia: evidence from mtDNA, microsatellites and nuclear genealogies.

Pleistocene glaciations often resulted in differentiation of taxa in southern European peninsulas, producing the high levels of endemism characteristic of these regions (e.g. the Iberian Peninsula). Despite their small ranges, endemic species often exhibit high levels of intraspecific differentiation as a result of a complex evolutionary history dominated by successive cycles of fragmentation, expansion and subsequent admixture of populations. Most evidence so far has come from the study of species with an Atlantic distribution in northwestern Iberia, and taxa restricted to Mediterranean-type habitats remain poorly studied. The Iberian Midwife toad (Alytes cisternasii) is a morphologically conserved species endemic to southwestern and central Iberia and a typical inhabitant of Mediterranean habitats. Applying highly variable genetic markers from both mitochondrial and nuclear genomes to samples collected across the species' range, we found evidence of high population subdivision within A. cisternasii. Mitochondrial haplotypes and microsatellites show geographically concordant patterns of genetic diversity, suggesting population fragmentation into several refugia during Pleistocene glaciations followed by subsequent events of geographical and demographic expansions with secondary contact. In addition, the absence of variation at the nuclear beta-fibint7 and Ppp3caint4 gene fragments suggests that populations of A. cisternasii have been recurrently affected by episodes of extinction and recolonization, and that documented patterns of population subdivision are the outcome of recent and multiple refugia. We discuss the evolutionary history of the species with particular interest in the increasing relevance of Mediterranean refugia for the survival of genetically differentiated populations during the Pleistocene glaciations as revealed by studies in co-distributed taxa.

The dietary supplementation of canthaxanthin in combination with 25OHD3 results in reproductive, performance, and progeny quality gains in broiler breeders.

The objective of this study was to evaluate the effects of the dietary supplementation of canthaxanthin and cholecalciferol (25OHD3) to broiler breeders and their progenies on their performance. Eighty 25-wk-old Cobb 500 broiler breeders were distributed according to a completely randomized design in 2 experimental treatments, with 10 replicates of 4 birds each. The treatments consisted of the supplementation or not of a corn-soybean meal diet with canthaxanthin + 25OHD3 to broiler breeders fed from 25 to 62 wk of age. Egg production, fertility, hatchability, hatchability of fertile eggs, embryonic mortality, and egg specific gravity were evaluated. Breeders were inseminated at 35, 45, and 62 wk of age, the eggs were incubated, and the performance of the progenies was evaluated. From the progeny of each breeder age, 300 male chicks were distributed according to a completely randomized design in a 2 × 2 factorial arrangement (chicks from breeders supplemented or not with canthaxanthin + 25OHD3 and chicks supplemented or not with canthaxanthin + 25OHD3), totaling 4 treatments with 5 replicates of 15 birds each. Canthaxanthin + 25OHD3 were supplied to the chicks until 21 D of age. The combination of canthaxanthin and 25OHD3, containing 69 mg of 25OHD3 and 6 g of canthaxanthin, was supplemented at 1 kg/t of feed. Breeders supplemented with canthaxanthin + 25OHD3 showed higher egg production, total hatchability, hatchability of fertile eggs, and lower early embryo mortality compared with those fed the control diet. Broilers from breeders fed canthaxanthin + 25OHD3 and supplemented with this additive up to 21 D of age presented a better feed conversion ratio and higher carcass and breast yields than those derived from nonsupplemented breeders.