Using genotyping-by-sequencing to predict gender in animals.

Gender assignment errors are common in some animal species and lead to inaccuracies in downstream analyses. Procedures for detecting gender misassignment are available for array-based SNP data but are still being developed for genotyping-by-sequencing (GBS) data. In this study, we describe a method for using GBS data to predict gender using X and Y chromosomal SNPs. From a set of 1286 X chromosomal and 23 Y chromosomal deer (Cervus sp.) SNPs discovered from GBS sequence reads, a prediction model was built using a training dataset of 422 Red deer and validated using a test dataset of 868 Red deer and Wapiti deer. Prediction was based on the proportion of heterozygous genotypes on the X chromosome and the proportion of non-missing genotypes on the Y chromosome observed in each individual. The concordance between recorded gender and predicted gender was 98.6% in the training dataset and 99.3% in the test dataset. The model identified five individuals across both datasets with incorrect recorded gender and was unable to predict gender for another five individuals. Overall, our method predicted gender with a high degree of accuracy and could be used for quality control in gender assignment datasets or for assigning gender when unrecorded, provided a suitable reference genome is available.

Efficiency of genomic prediction for boar taint reduction in Danish Landrace pigs.

Genetic selection against boar taint, which is caused by high skatole and androstenone concentrations in fat, is a more acceptable alternative than is the current practice of castration. Genomic predictors offer an opportunity to overcome the limitations of such selection caused by the phenotype being expressed only in males at slaughter, and this study evaluated different approaches to obtain such predictors. Samples from 1000 pigs were included in a design which was dominated by 421 sib pairs, each pair having one animal with high and one with low skatole concentration (≥0.3 µg/g). All samples were measured for both skatole and androstenone and genotyped using the Illumina SNP60 porcine BeadChip for 62 153 single nucleotide polymorphisms. The accuracy of predicting phenotypes was assessed by cross-validation using six different genomic evaluation methods: genomic best linear unbiased prediction (GBLUP) and five Bayesian regression methods. In addition, this was compared to the accuracy of predictions using only QTL that showed genome-wide significance. The range of accuracies obtained by different prediction methods was narrow for androstenone, between 0.29 (Bayes Lasso) and 0.31 (Bayes B), and wider for skatole, between 0.21 (GBLUP) and 0.26 (Bayes SSVS). Relative accuracies, corrected for h(2) , were 0.54-0.56 and 0.75-0.94 for androstenone and skatole respectively. The whole-genome evaluation methods gave greater accuracy than using only the QTL detected in the data. The results demonstrate that GBLUP for androstenone is the simplest genomic technology to implement and was also close to the most accurate method. More specialised models may be preferable for skatole.

Comparison of greenhouse gas emissions from sheep measured using both respiration and portable accumulation chambers.

Methane (CH4) is a potent gas produced by ruminants, and new measurement techniques are required to generate large datasets suitable for genetic analysis. One such technique are portable accumulation chambers (PAC), a short-term sampling method. The objectives of the current study were to explore the relationship between CH4 and carbon dioxide (CO2) output measured using both PAC and respiration chambers (RC) in growing lambs, and separately investigate the relationship among CH4, CO2 and measured ad libitum DM intake (DMI). Methane, CO2 and DMI were measured on 30 Suffolk and 30 Texel ewe lambs (age 253 ± 12 days) using the RC and PAC sequentially. The experiment was conducted over a 14-day period, with DMI measured from days 1 to 14; measurements in RC were conducted from days 10 to 12, while measurements in PAC were taken twice, the day immediately prior to the lambs entering the RC (day 9; PAC Pre-RC) and on the day lambs exited the RC (day 13; PAC Post-RC). Greater CH4 and CO2 output was measured in the RC than in the PAC (P < 0.01); similarly mean CH4 yield was greater when measured in the RC (15.39 ± 0.452 g CH4/kg DMI) compared to PAC (8.01 ± 0.767 g CH4/kg DMI). A moderate correlation of 0.37 was found between CH4 output measured in PAC Pre-RC and the RC, the corresponding regression coefficient of CH4 output measured in the RC regressed on CH4 output measured in PAC Pre-RC was close to unity (0.74; SE 0.224). The variance of CH4 and CO2 output within the measurement technique did not differ from each other (P > 0.05). Moderate to strong correlations were found between CH4 and CO2 per kg of live weight and CH4 and CO2 yield. Results from this study highlight the suitability of PAC as a ranking tool to rank animals based on their gaseous output when compared to the RC. However, repeated measurements separated by several days may be beneficial if precise rankings are required. Given the close to unity regression coefficient of CH4 output measured in the RC regressed on CH4 output measured in PAC Pre-RC suggests that PAC could also be potentially used to estimate absolute CH4 output; however, further research is required to substantiate this claim. When DMI is unknown, CH4 and CO2 per kg of live weight are a suitable alternative to the measurement of CH4 and CO2 yield.

Can we have our steak and eat it: The impact of breeding for lowered environmental impact on yield and meat quality in sheep.

Global agreements in place to reduce methane emissions in livestock are a potential threat to food security. Successful but independent breeding strategies for improved production and lower methane are in place. The unanswered questions are whether these strategies can be combined and how they impact one another, physically and economically. The New Zealand economy is largely dependent on pastoral agriculture from grazing ruminants. The sheep industry produces ∼20 million lamb carcasses for export each year primarily from grass. Methane emitted from the fermentation of forage by grazing ruminants accounts for one-third of all New Zealand's greenhouse gas emissions. Here, we use sheep selection lines bred for divergent methane production and large numbers of their relatives to determine the genetic and phenotypic correlations between enteric methane emissions, carcass yield, and meat quality. The primary objectives were to determine whether previously shown physiological differences between methane selection lines (differing by ∼12% in methane) result in a negative impact on meat production and quality by measuring close relatives. The results show no negative effects of breeding for lowered methane on meat and carcass quality. Gross methane emissions were highly correlated with liveweight and measures of carcass weight and negatively correlated with dressing-out percentage and fat yield (GR). Trends were similar but not significant for methane yield (g CH4/kg DMI). Preliminary evidence, to date, shows that breeding for low methane may result in animals with higher lean yields that are economically favorable even before carbon costs and environmental benefits are taken into account. These benefits were seen in animals measured for methane on fixed intakes and require validation on intakes that are allowed to vary.

Protein kinase C activation antagonizes melatonin-induced pigment aggregation in Xenopus laevis melanophores.

The pineal hormone, melatonin (5-methoxy N-acetyltryptamine) induces a rapid aggregation of melanin-containing pigment granules in isolated melanophores of Xenopus laevis. Treatment of melanophores with activators of protein kinase C (PKC), including phorbol esters, mezerein and a synthetic diacylglycerol, did not affect pigment granule distribution but did prevent and reverse melatonin-induced pigment aggregation. This effect was blocked by an inhibitor of PKC, Ro 31-8220. The inhibitory effect was not a direct effect on melatonin receptors, per se, as the slow aggregation induced by a high concentration of an inhibitor of cyclic AMP-dependent protein kinase (PKA), adenosine 3',5'-cyclic monophosphothioate, Rp-diastereomer (Rp-cAMPS), was also reversed by PKC activation. Presumably activation of PKC, like PKA activation, stimulates the intracellular machinery involved in the centrifugal translocation of pigment granules along microtubules. alpha-Melanocyte stimulating hormone (alpha-MSH), like PKC activators, overcame melatonin-induced aggregation but this response was not blocked by the PKC inhibitor, Ro 31-8220. This data indicates that centrifugal translocation (dispersion) of pigment granules in Xenopus melanophores can be triggered by activation of either PKA, as occurs after alpha-MSH treatment, or PKC. The very slow aggregation in response to inhibition of PKA with high concentrations of Rp-cAMPS, suggests that the rapid aggregation in response to melatonin may involve multiple intracellular signals in addition to the documented Gi-mediated inhibition of adenylate cyclase.

Treatment of organic impotence with penile prosthesis in renal transplant patients.

Seven patients were identified who underwent both renal transplantation and penile prosthesis implantation at our institution between June 1980 and June 1990, and their charts were retrospectively reviewed. A total of nine penile prostheses were placed in these patients, five prior to transplantation and four following transplantation. One patient received two prostheses prior to transplantation. One patient received a prosthesis both before and after transplantation. No complications were seen in the four prostheses placed following transplantation with a follow-up of one to forty months (mean 18 months). Of the five prostheses placed prior to transplantation, two were removed due to periprosthetic infections, a cylinder leak developed in one, and one was complicated by penile and scrotal erythema with sepsis.

QTL analysis of body weight and conformation score in commercial broiler chickens using variance component and half-sib analyses.

The aim of the study was to investigate quantitative trait loci (QTL) in previously identified regions of chicken chromosomes 1, 4 and 5 relating to 40-day body weights and conformation scores. Half-sib (HS) and variance component analyses were implemented and compared using QTL Express software. Data were from a two-generation design and consisted of 100 dam families nested in 46 sire families with trait values for 2,708 offspring. Chicken chromosome 4 showed nominal significance for QTL affecting body weight and conformation, and linkage was confirmed for both traits on chromosome 5. Results varied according to method of analysis and with common parent in the HS method.

An evaluation of ABR audiometry for the screening and detection of hearing loss in ex-SCBU infants.

The study aimed to use auditory brainstem response (ABR) audiometry to test ex-SCBU infants born during 1986 in West Berkshire. Two hundred and forty-three babies were tested as out-patients, thus achieving a coverage rate of 86% of the target population. Mean post-conceptional age at test was 48 weeks. Whenever possible, full ABR threshold determination was performed on both ears and the mean ABR threshold was found to be 14 dB nHL. Of those babies attending, 85% showed ABRs at less than or equal to 30 dB nHL bilaterally and the remaining 15% were referred for further assessment. Approximately two-thirds of these were recalled successfully for repeat testing. The estimated prevalence rate for bilateral sensorineural hearing impairment was 1.4% with four confirmed cases. Two additional permanent unilateral hearing losses were also detected. On the basis of recorded ABR data, sensitivity, specificity and positive predictive values were estimated for click intensities which could be used for single-intensity ABR screens. It is concluded that delaying screening until the post-neonatal stage is a viable alternative to screening neonates prior to discharge from the SCBU.

Risk factors for childhood sensorineural hearing loss in the Oxford region.

We have used a comprehensive register of hearing-impaired children born in the former Oxford Health Region to study risk factors for sensorineural hearing loss. The occurrence of a wide variety of risk factors was documented from the case notes of 145 children; these were all the cases known at the time of the study with all degrees of hearing loss born between 1984 and 1988. Comparison with the normal Regional population showed that maternal age over 35 years and Asian ethnic origin were significant risk factors for congenital (non-acquired) hearing loss (odds ratio 1.7 and 2.5 respectively). Black/Asian children were also significantly more likely to have acquired losses. Low birthweight (below 2500 g) also gave a significantly increased risk, with an odds ratio of 4.5, rising to 9.6 for birthweight less than 1500 g. We also found that significantly more hearing-impaired cases were in lower social classes compared with the general population. A high proportion of cases (24%) had cranio-facial abnormalities (CFA), including many non-aural abnormalities and dysmorphic features, which therefore should be counted as high risk. Hearing losses acquired due to perinatal causes were almost all mild or moderate. Four factors-admission to special care baby unit for more than 72 hours, CFA, family history, and meningitis-accounted for 69% of all cases in this study. Targeted neonatal screening based on the first three factors, plus obligatory testing following meningitis, therefore, should be highly efficient at detecting deafness early.

The role of noradrenaline in the differentiation of amphibian embryonic neurons.

The possibility that monoamines might act as signalling molecules during the early development of the nervous system has been examined in embryos of the amphibian Xenopus laevis. The distributions of 5-hydroxytryptamine, dopamine, noradrenaline and their precursor, dopa, were determined from the fertilized egg up to the late neurula stages using High Performance Liquid Chromatography, formaldehyde-induced fluorescence and antibody staining. 5-hydroxytryptamine was not detected until the tail bud stage. The fertilized egg contained significant concentrations of dopa (10(-6) M) and dopamine (10(-7) M). Both monoamines persisted with little change in concentration up to the late neurula stage. Early neurula stage embryos contained very low levels of noradrenaline. Aldehyde-induced fluorescence showed that monoamines are localized in dorsal regions of the embryo, in ectoderm and mesoderm cells. Monoamines were not present in endoderm cells. Immunocytochemical staining showed dopamine predominantly in the ectoderm, except in future neural regions where it was found also in the mesoderm. Dopamine staining was always most intense in dorsal regions of the embryo. The consequences for subsequent neuronal differentiation of interfering with the biosynthesis and receptor binding of monoamines during neurulation was assayed. Neuronal differentiation was monitored quantitatively in cultures set up as the neural tube closed and qualitatively in intact tadpoles that were left to develop for two days after washout of test reagent. The number of neurons, the number of muscle cells and the total number of differentiated cells were counted after 18-24 hours of culture. Comparison of the number of neurons that differentiated from control and treated embryos showed that inhibition of dopamine beta-hydroxylase, the enzyme catalysing the conversion of dopamine to noradrenaline, during the neural plate stages reduced substantially subsequent neuronal differentiation. The differentiation of myocytes and the total number of differentiated cells were not affected. Exogenous noradrenaline (10(-6) M) or dopamine (10(-6) M) could increase the number of neurons that differentiated subsequently in culture. Interfering with noradrenaline binding to receptors with receptor antagonists during neurulation showed that alpha-adrenergic receptor antagonists reduced substantially the subsequent differentiation of neurons. The differentiation of myocytes and the total number of differentiated cells were not affected. The effect of alpha-adrenergic receptor antagonists was overcome by the simultaneous inclusion of noradrenaline or alpha-receptor agonists, but not agonists at beta-adrenergic receptors. The quantitative reduction in the differentiation of neurons was paralleled by defects in the Central Nervous System of intact tadpoles.(ABSTRACT TRUNCATED AT 400 WORDS)

Tympanometry and otoacoustic emissions in a cohort of special care neonates.

Otoacoustic emission (OAE) screening and oto-admittance testing (678 Hz probe tone) were performed on both ears of 84 special care neonates, as part of a larger study of middle-ear effusion in neonates and infants. OAE results, tympanometry, and acoustic reflex results are all strongly and significantly associated. No evidence was found of any maturational effects in the results. Based on the findings, a tentative classification scheme for neonatal tympanograms is suggested. We conclude that 678 Hz tympanometry is a useful indicator of middle-ear status in very young babies, and that middle-ear effusion does strongly affect OAEs in neonates. OAEs are also strongly affected by negative middle-ear pressure (MEP), and mean MEP in ears failing OAE screens was significantly more negative than in those passing. The prevalence of abnormal tympanometry, which may indicate middle-ear effusion or dysfunction, was 20% of ears (29% of babies) in this group. It appears that middle-ear effusion could account for about half of the ears failing an OAE screen on the special care baby unit. We also find that length of stay on the special care baby unit is an important risk factor for development of middle-ear effusion: those on the unit for over 30 days have about four times the risk of bilateral abnormal tympanometry.

The neurotransmitter noradrenaline drives noggin-expressing ectoderm cells to activate N-tubulin and become neurons.

Neurotransmitters regulate neuronal function in the nervous system and modulation of their synthesis, release, and binding by immature neurons and their targets is a major part of nervous system development. We propose that the neurotransmitter noradrenaline regulates neuronal fate during neurulation, before neurons have differentiated. The ability of noradrenaline to induce a neural fate was tested in naive ectoderm caps cut from late blastula stage Xenopus embryos. Noradrenaline (10(-6) M) did not switch on otx-2 or NCAM and did not induce the formation of cement glands. We conclude that noradrenaline cannot induce a neural fate. By contrast, 10(-8) M noradrenaline activated N-tubulin in ectoderm caps expressing the neural inducing molecule noggin by the time intact siblings had become mid-neurulae. Methoxamine, a specific alpha-adrenergic receptor agonist, also activated N-tubulin in noggin-expressing caps. The alpha-adrenergic receptor blocker prazosin inhibited both noradrenaline- and methoxamine-induced activation of N-tubulin. The neurotransmitters dopamine and 5-HT did not activate expression of N-tubulin. XA-1, Otx-2, X-Delta, and Xotch transcripts were not sensitive to noradrenaline. HoxB9, which indicates posteriorization, was not activated by noradrenaline. When intact siblings were at stage 27, many cells in noggin-expressing, noradrenaline-treated caps were stained by the neuron-specific mcAb3A10. We propose that noradrenaline is an important endogenous modulator of neuronal fate, driving noggin-expressing cells to become neurons by binding to alpha-adrenergic receptors and activating a cascade that culminates in the expression of the neuronal markers N-tubulin and 3A10.

Safety of transurethral surgery in the early postoperative period following an open cardiac procedure.

A retrospective investigation was performed to determine whether patients undergoing transurethral surgery soon after cardiac surgery experienced increased morbidity or mortality rates. From 1986 to 1990, 24 patients first underwent open heart surgery and then either transurethral prostatectomy, bladder tumor resection or bladder cup biopsy during the same hospital stay. Postoperative complications included significant hematuria in 2 patients (8%), mild stress incontinence in 1 (4%) and bladder perforation in 1. One patient died of a spontaneous pneumothorax 17 days after the urological operation. None of these patients had had a previous myocardial infarction. The outcome of these patients was compared to that of 115 men who underwent transurethral prostatectomy for presumed benign disease during 1990. Complications of transurethral prostatectomy in this group included significant gross hematuria in 5 men, while 6 experienced urinary retention (1), atrial fibrillation (1), delirium (1), myocardial infarction (1), seizure (1) and intraoperative urethral injury (1). There was 1 death from multiple postoperative complications. Morbidity and mortality rates did not differ significantly between the 2 groups. Transurethral surgery performed after cardiac surgery during the same hospital stay appears to be safe, provided the patient is stable.

Alpha-melanocyte-stimulating hormone reduces impact of proinflammatory cytokine and peroxide-generated oxidative stress on keratinocyte and melanoma cell lines.

We have previously shown that alpha-melanocyte-stimulating hormone (alpha-MSH) can oppose tumor necrosis factor alpha activation of NF-kappaB (1-2 h) and intercellular adhesion molecule 1 up-regulation (mRNA by 3 h and protein by 24 h) in melanocytes and melanoma cells. The present study reports on the ability of four MSH peptides to control intracellular peroxide levels and glutathione peroxidase (GPx) activity in pigmentary and nonpigmentary cells. In human HBL melanoma and HaCaT keratinocytes tumor necrosis factor alpha and H(2)O(2) both activated GPx in a time- and concentration-dependent manner (by 30-45 min). alpha-MSH peptides were found to inhibit the stimulated GPx activity and had biphasic dose-response curves. MSH 1-13 and MSH [Nle(4)-d-Phe(7)] achieved maximum inhibition at 10(-10) and 10(-12) m, respectively. Higher concentrations (10-100 fold) of MSH 4-10 and MSH 11-13 were required to produce equivalent levels of inhibition. alpha-MSH was also capable of reducing peroxide accumulation within 15 min, and again this inhibition was biphasic. The data support a role of alpha-MSH in acute protection of cells to oxidative/cytokine action that precedes NF-kappaB and GPx activation. The rapidity and potency of the response to alpha-MSH in pigmentary and nonpigmentary cells suggest this to be a central role of this peptide in cutaneous cells.

Confirmation of a blocked amino terminus of sulfhydryl oxidase.

The isolation of sulfhydryl oxidase from bovine milk in a suitably pure form for sequencing was carried out by transient covalent affinity chromatography of diafiltered whey using cysteinylsuccinamidopropyl-glass as matrix. The glutathione-eluted proteins were separated by SDS-PAGE. By radiolabeling the affinity chromatography-purified enzyme with [14C]iodoacetate before subjecting to SDS-PAGE, the sulfhydryl oxidase band was identified, because sulfhydryl oxidase is known to be inactivated by alkylation of one sulfhydryl group per mole. The results confirmed that sulfhydryl oxidase corresponds to the 85 (+/- 5)-k-Daband observed on SDS-PAGE. The protein band corresponding to radiolabeled sulfhydryl oxidase was recovered from SDS-PAGE gels by electrophoretic elution and by electroblotting on polyvinylidene difluoride membrane and polyvinylidene difluoride membrane and subjected to gas phase sequencing. Precautions were taken during electrophoretic elution to prevent reactions that result in N-terminal blocking. Both methods of protein recovery yielded negative results when subjected to sequence analysis indicating that the N-terminus of sulfhydryl oxidase is blocked.