Genetic variances and covariances of live weight traits in Charolais cattle by multi-trait analysis.

Live weight traits are economically important for beef cattle production systems. Genetic analysis of live weight traits frequently presents a problem due to animal records, in that matter, not all the animals have complete records as many young animals leave the herd because of sale, transfer to another herd, or culling reasons. Therefore, the use of multi-trait genetic analysis might be of assistance to help overcome any possible loss of information for those animals with incomplete records. In this study, genetic variances and covariances were obtained to estimate genetic parameters for birth (BW), weaning (WW), and yearling (YW) live weights in a registered Charolais beef cattle population using a multivariate model, where a considerable reduction of data from birth weight to year weight was observed. Direct and maternal heritabilities for BW, WW, and YW were 0.50, 0.30, and 0.28, and 0.31, 0.25, and 0.14, respectively. Direct and maternal genetic correlations were negative in all live weight traits. Genetic correlations among direct BW with direct WW and YW were low, while genetic correlations among maternal traits were medium or high (r>0.39). Comparison between univariate and multi-trait models with substantial reduction of information revealed important differences, implying that multi-trait analysis is better for the structure of data allowing a better fitting of genetic effects by covariance among evaluated traits. Results support multi-trait analysis implementation for genetic evaluations for live weight traits of Charolais cattle.

Physical activity of university students with disabilities: accomplishment of recommendations and differences by age, sex, disability and weight status.

OBJECTIVES: This article aims to study physical activity and the achievement of World Health Organization physical activity recommendations in university students with disabilities, and to examine differences by sex, age, disability characteristics and weight status. STUDY DESIGN: Cross-sectional data from a wider research project conducted at the Spanish universities from Autumn 2016 to Autumn 2017 were analysed. METHODS: The International Physical Activity Questionnaire-Short Form was administered to 1103 Spanish university students with different disabilities. Nonparametric tests were performed to examine the differences in physical activity based on the interest variables. RESULTS: The mean metabolic equivalent (MET)-minutes/week was 1772.75 (±2161.00) for total physical activity, 642.93 (±1303.08) for vigorous physical activity, 344.31 (±699.53) for moderate physical activity and 785.50 (±1053.31) for walking intensity physical activity. Overall, 72.2% of the participants did not meet the recommendation of 75 min/week of vigorous physical activity, 80.3% did not meet the recommendation of 150 min/week of moderate physical activity and 63.1% did not meet any of these recommendations. Nonparametric tests revealed that students with multiple disabilities, chronic illnesses, acquired disabilities, older students, obese students and women were less active than their counterparts. CONCLUSIONS: A high number of participants did not meet the World Health Organization physical activity recommendations, and some subgroups were especially inactive. Public health policies should implement interventions to encourage people with disabilities to engage in physical activity, paying extra attention to the most inactive subgroups.

The breed and season effects on scrotal circumference and semen characteristics of hair sheep rams under tropical conditions.

The aim of the present work was to evaluate the breed and season effects on scrotal circumference (SC) and semen characteristics of 28 mature hair sheep rams kept under tropical conditions. SCs, sperm concentration (SPC) and abnormal sperm were significantly affected by breed effect (p < 0.001). The season effect was significant in SPC (p < 0.0001) while ejaculate volume, mass motility and SPC were affected by breed × season interaction effect (p < 0.001). It can be concluded that the magnitude of the breed and season effects were not sufficient to affect the reproductive capacity of hair sheep rams throughout the year.

The effects of metabolizable energy intake on body fat depots of adult Pelibuey ewes fed roughage diets under tropical conditions.

The objective of this work was to evaluate the effect of metabolizable energy intake (MEI) on changes in fat depots of adult Pelibuey ewes fed roughage diets under tropical conditions. Eighteen 3-year-old Pelibuey ewes with similar body weight (BW) of 37.6 ± 4.0 kg and body condition score (BCS) of 2.5 ± 0.20 were randomly assigned to three groups of six ewes each in a completely randomized design. Ewes were housed in metabolic crates and fed three levels of MEI: low (L), medium (M), and high (H) for 65 days to achieve different BW and BCS. At the end of the experiment, the ewes were slaughtered. Data recorded at slaughter were: weights of viscera and carcass. Internal fat (IF, internal adipose tissue) was dissected, weighed, and grouped as pelvic (around kidneys and pelvic region), omental, and mesenteric regions. Carcass was split at the dorsal midline in two equal halves, weighed, and chilled at 6°C during 24 h. After refrigeration, the left half of the carcass was completely dissected into subcutaneous and intermuscular fat (carcass fat). Dissected carcass fat (CF) of the left carcass was adjusted as whole carcass. At low levels of MEI, proportion of IF and CF was approximately 50%; however, as the MEI was increased, the proportion of IF was increased up to 57% and 60% for M and H, respectively. Omental and pelvic fat depots were those which increased in a larger proportion with respect to the mesenteric fat depot. Regression equations between the weight of each body fat depot and BW had a coefficient of determination (r (2)) that ranged between 0.37 for mesenteric fat and 0.87 for CF. The regression with BCS had a r (2) that ranged between 0.57 for mesenteric and 0.71 for TBF. BW was the best predictor for TBF, CF, omental fat, and pelvic fat; whereas, BCS was better than BW in predicting IF and mesenteric fat. Inclusion of both BW and BCS in multiple regressions improved the prediction for all fat depots, except for pelvic fat, which was best estimated by BCS alone. The greater slope of the regression for the pelvic fat depot equation, relative to TBF (1.40), EBW (4.02), and BCS (2.36), suggested that pelvic fat has a greater capacity to accumulate and mobilize fat. These results indicated that adult Pelibuey ewes seem to store a considerable proportion of absorbed energy in the IF depots rather than in the carcass.

Productive performance of Creole chickens and their crosses raised under semi-intensive management conditions in Yucatan, Mexico.

1. The productive performance of 4 chicken breed groups managed under semi-intensive conditions in Yucatan, Mexico was evaluated. Thirty-six mixed chickens, one week of age of each of the 4 breed groups (Creole, F1 Hubbard x Creole, 7/8 Hubbard x 1/8 Creole and Hubbard) were used. 2. During 1 to 3 weeks of age all birds were fed on a diet containing 210 g/kg crude protein (CP) and 12.95 MJ/kg metabolisable energy (ME). From weeks 4 to 7, they were given a diet with 190 g/kg CP and 12.55 MJ/kg ME. Food consumption and live body weight were recorded weekly. Statistical analysis of the data was performed according to complete randomised design and means comparison using Tukey test was carried out when necessary. 3. Creole chicks had the lightest weights at all ages as compared to their crosses and the Hubbard birds. At 7 weeks of age, Creole chicks were 2.11, 2.44 and 2.90 times lighter than their contemporary F1, 7/8 Hubbard x 1/8 Creole and Hubbard birds, respectively. Hubbard birds were heavier than F1 and 7/8 commercial x 1/8 Creole birds, and the latter group heavier than the F1 birds. Similarly, Creole chicks had the lowest growth rate compared to the other genetic groups. Heterosis for body weight up to 7 weeks of age was 8.2%. 4. Feed consumption was also lower for the Creole chicks at all ages. Food:gain ratio, however, was higher for the Creole chicks at all ages. Food:gain ratios from weeks 2 to 7 were 2.18, 2.65, 3.04 and 4.36 for the Hubbard, 7/8 Hubbard x 1/8 Creole, F1 and Creole birds, respectively. Heterosis for food:gain ratio from 2 to 7 weeks of age was -7.0%. 5. Crossbreeding of Mexican Creole birds with commercial type broilers might improve productive performance.

Single nucleotide polymorphism determination using primer extension and time-of-flight mass spectrometry.

The high frequency of single nucleotide polymorphisms (SNPs) in the human genome makes them a valuable source of genetic markers for identity testing, genome mapping, and medical diagnostics. Conventional technologies for detecting SNPs are laborious and time-consuming, often prohibiting large-scale analysis. A rapid, accurate, and cost-effective method is needed to meet the demands of a high-throughput DNA assay. We demonstrate here that analysis of these genetic markers can now be performed routinely in a rapid, automated, and high-throughput fashion using time-of-flight mass spectrometry and a primer extension assay with a novel cleavable primer. SNP genotyping by mass spectrometry involves detection of single-base extension products of a primer immediately adjacent to the SNP site. Measurement of the mass difference between the SNP primer and the extension peak reveals which nucleotide is present at the polymorphic site. The primer is designed such that its extension products can be purified and chemically released from the primer in an automated format. The reduction in size of the products as a result of this chemical cleavage allows more accurate identification of the polymorphic base, especially in samples from a heterozygotic population. All six possible heterozygotes are resolved unambiguously, including an A/T heterozygote with extension products differing by only 9 Da. Multiplex SNP determination is demonstrated by simultaneously probing multiple SNP sites from a single polymerase chain reaction (PCR) product as well as from multiplexed PCR amplicons. Samples are processed in parallel on a robotic workstation, and analyzed serially in an automated mass spectrometer with analysis times of only a few seconds per sample, making it possible to process thousands of samples per day.

Reliable genotyping of short tandem repeat loci without an allelic ladder using time-of-flight mass spectrometry.

DNA separations which traditionally have been performed by slab gel or capillary electrophoresis, may now be conducted via time-of-flight mass spectrometry (TOF-MS). The advantages of using a mass spectrometry approach for short tandem repeat (STR) characterization include a dramatic increase in both the speed of analysis and the accuracy of mass measurements. We report here typing of the STR loci TH01, TPOX, and CSF1PO as well as the sex-typing marker amelogenin using TOF-MS. Allelic ladders, which are typically used with electrophoretic separation systems to correct for mobility differences of DNA fragments under various conditions, are not needed for accurate genotyping with TOF-MS. A mass precision of 0.1% RSD, which corresponds to approximately 0.1 nucleotide, was routinely observed. Mass accuracies were better than a fraction of a single nucleotide when a daily mass calibration was used. STR microvariants, such as the TH01 allele 9.3, could be detected and resolved from alleles which differ by as little as a single base. In addition, the smaller PCR product sizes (55-125 bp) examined in this study have the potential advantage of being more successful when amplifying forensic samples with degraded DNA.

Determination of tissue distribution of an intramuscular plasmid vaccine using PCR and in situ DNA hybridization.

The increasing use of nucleic acid-based therapeutics has created a need for new methods of determining tissue distribution and levels. Radiolabel methods may not always be appropriate because nucleic acids are easily degraded. Quantitation using the polymerase chain reaction (PCR) has the advantage that only continuous stretches of DNA will be amplified. In situ hybridization allows detection of specific sequences in histological preparations. We have used quantitative PCR and in situ hybridization techniques to study the pharmacokinetics and distribution of PGagPol (a potential anti-HIV plasmid vaccine) in rabbits. Samples were obtained 4 hr, 24 hr, 7 days, and 28 days after intramuscular injection of 100 micrograms or 400 micrograms of plasmid. A simplified procedure for collecting and processing tissues for PCR that minimizes the risk of contamination was developed. Using PCR, plasmid was found principally in the skin and muscle of the injection site and in blood plasma. At 4 hr after dosing with 400 micrograms, the plasmid was detected at the injection site with mean copy numbers of 10(6) (in muscle) and 4 x 10(4) (in skin) per microgram of tissue. Plasmid copy number declined rapidly in muscle during the first 24 hr and was undetectable at 7 and 28 days after injection. The decline was slower in the skin, and the plasmid was still detectable at 28 days. With in situ hybridization, plasmid was detected in muscle, mainly in the perimysium and to a lesser degree in the endomysium and within the muscle fibers. These data indicate that quantitative PCR and in situ hybridization are sensitive methods for examining tissue distribution of DNA used for gene therapy.

Transgenic animal models for detection of in vivo mutations.

Transgenic rodent models for measuring mutations provide a tool for assessing tissue-specific mutations following in vivo treatment. These systems are based on the insertion into the rodent genome Escherichia coli lacI (lac repressor) or lacZ (beta-galactosidase) genes that serve as targets for mutations. Following in vivo treatment of animals, genomic DNA is isolated from tissues of interest, and the target gene is screened for mutations using either lambda-phage packaging or isolation of the target gene with magnetic affinity capture. In this paper we review the various experimental methods used in the conduct of transgenic mutation assays and discuss critical factors that affect the interpretations of results of these assays.

Mutagenesis of the hairpin ribozyme.

Extensive in vitro mutagenesis studies have been performed on the hairpin ribozyme and substrate in an effort to refine the overall secondary structure of the molecule and provide further insight into what elements are essential for activity. A secondary structure consisting of four helices and five loop regions remains the basic model as originally proposed. Two helices, helix 1 and 2, form between the substrate and ribozyme while helices 3 and 4 are within the ribozyme itself. Our results suggest that helices 3 and 4 are smaller than previously proposed, consisting of four base pairs and three base pairs respectively. Helix 4 can be extended without loss of activity and loop 3 at the closed end of the hairpin model can be varied in sequence with retention of activity. There is an unpaired nucleotide between helices 2 and 3 consisting of a single A base, suggesting the opportunity for flexibility within the tertiary structure at this point. Comparisons are made between the new data and previously published mutagenesis and phylogenetic data. Substrate targeting rules require base pairing between helices 1 and 2 with cleavage (*) occurring in a preferred 5'(g/c/u)n*guc3' sequence of the substrate.

Transgenic animal models for measuring mutations in vivo.

Transgenic animal models for measuring mutations provide a powerful tool for rapidly assessing tissue-specific mutations following in vivo treatment. These models are based on the insertion into the rodent genome of the Escherichia coli lacI (lac repressor) or lacZ (beta-galactosidase) genes that serve as targets for mutations. Following in vivo treatment of animals, genomic DNA is isolated from various tissues and the target gene is packaged into lambda-phage heads; the lambda-phage are used to infect E. coli in order to produce plaques. Mutations in the target gene are then detected using colorimetric or selective procedures. In this review methods are discussed for producing these transgenic models, the target genes used, gene rescue techniques, sequencing of isolated mutants, and parameters that affect dosing regimens and design of studies. We also present a summary of data published to date with these systems and present our conclusions and proposed directions for future research.

RNA folding during transcription by T7 RNA polymerase analyzed using the self-cleaving transcript assay.

We have used a self-cleaving RNA molecule (a "hammerhead") to study the length-dependent folding of RNA produced during transcription by T7 RNA polymerase. Transcript elongation is arrested at defined positions using chain-terminating ribonucleoside triphosphate analogues, 3'-deoxynucleoside triphosphates. When the nascent transcript attains the minimum length required for the "hammerhead" domain of the transcript to fully emerge from the ternary complex, the "hammerhead" structure forms and self-cleaves, producing a truncated product. The experiment yields an RNA sequencing ladder which terminates at the length at which cleavage becomes possible; the sequencing ladder is compared to that generated by using a noncleaving control template. We have shown that 13 nucleotides past the cleavage point must be synthesized before the transcript can self-cleave in the ternary complex whereas RNA freed from the complex by heating can cleave with only 3 or more nucleotides present beyond the cleavage site. The results indicate that the RNA in T7 RNA polymerase is not free of steric interactions in the ternary complex and not available for structure formation until it is at least 10 bases away from the site of polymerization. The results suggest that the maximum possible length of the RNA-DNA hybrid in the ternary complexes is 10. The relevance of the results in comparisons with other RNA polymerases, especially Escherichia coli RNA polymerase, is discussed.

RNA folding during transcription by Escherichia coli RNA polymerase analyzed by RNA self-cleavage.

We have used a self-cleaving RNA molecule related to a subsequence of plant viroids (a "hammerhead") to study the length-dependent folding of RNA produced during transcription by Escherichia coli RNA polymerase. Transcript elongation is arrested at defined positions using chain-terminating ribonucleoside triphosphate analogues (3'-deoxyNTP's or 3'-O-methylNTP's). When the transcript can form the "hammerhead" structure it self-cleaves to give a truncated product. The experiment yields an RNA sequencing ladder which terminates at the length at which cleavage becomes possible; the sequencing ladder is compared to those generated by using a noncleaving transcript or by using [alpha-thio]ATP in place of ATP. We have shown that 15-18 nucleotides (nt) of RNA past the cleavage point must be synthesized before the transcript can self-cleave within a ternary complex, whereas RNA freed from the complex by heating can cleave with only 3 or more nt present beyond the cleavage point. There are sequence-dependent as well as length-dependent effects. The results suggest that 12 +/- 1 nt are sequestered within the ternary complex and are consistent with the presence of a DNA-RNA hybrid within the transcription bubble, as proposed by others. The results indicate that the "hammerhead" structure does not disrupt the hybrid. It appears that the RNA beyond the hybrid is not restrained by interactions with the enzyme, since the last stem of the self-cleaving structure forms as soon as the RNA composing it emerges from the DNA-RNA hybrid. Self-cleaving of the transcript offers a simple structural probe for studying less well-characterized transcription complexes. The relevance of the results to models for transcription termination is discussed.

Cocaine in Wayne County Medical Examiner's cases.

We determined the incidence of detection of cocaine or its metabolites in Wayne County (Michigan) Medical Examiner's cases from 1984 to 1987. Over this four-year period there was a significant (P less than 0.01) increase each year in evidence of recent cocaine use in this population, reaching 38.3% of all tested cases in 1987. Much of this increase was accounted for by homicide victims, especially those in their third or fourth decade of life, of whom 58.6 and 56.3%, respectively, tested positive in 1987. There was a large increase in recent cocaine use in teenage homicide victims in 1987, having previously changed little from 1984 to 1986. In victims of drug abuse, recent cocaine use also increased significantly (p less than 0.01) each year, reaching 47.6% in 1987, generally in combination with heroin. Although deaths attributed solely to cocaine were not as common, they also increased significantly each year from 4 in 1984 to 25 in 1987. Compared with the general population, those who use cocaine in Wayne County are more likely than those who do not to die prematurely, often as a result of violence.

Fatality from illicit phendimetrazine use.

Phendimetrazine is an anorectic agent which recently has been detected in three medical examiner's cases. In one instance death was attributed to this drug. Methods of detecting and identifying this drug in urine and blood are discussed. In the one instance where death was attributed to this substance, the blood concentration was 300 ng/ml.

A death involving fentanyl.

Selected ion monitoring gas chromatography/mass spectrometry was employed to quantitate fentanyl in blood, liver, and kidney from a death attributed to a fentanyl overdose. The concentration of fentanyl was 3 ng/mL blood, 11 ng/g liver and 14 ng/g kidney.

Immunohistochemical demonstration of homicidal insulin injection site.

Deliberate fatal injection of insulin is apparently a rare event. Its investigation generally requires demonstration of an exogenous insulin injection site and high blood or tissue levels. Interpretation of such results in postmortem samples can be difficult, and the demonstration of an insulin injection site has previously usually involved a tedious extraction procedure. We report a case of infanticide in which insulin was demonstrated immunohistochemically at the fatal injection site and discuss some aspects of interpretation of postmortem insulin levels.

Fatal thyrotoxic crisis.

Graves' disease is a form of hyperthyroidism. A rare complication of Graves' disease is thyrotoxic crisis. Although the crisis is frequently fatal, it is a distinctly unusual cause of sudden death. We investigated the case of a young woman with Graves' disease who died suddenly. Radioimmunoassay tests of thyroid function were used to establish the diagnosis.