Heterogeneity after harmonisation: A retrospective cohort study of bleeding and stroke risk after the introduction of direct oral anticoagulants in four Western European countries.

PURPOSE: Database heterogeneity can impact effect estimates. Harmonisation provided by common protocols and common data models (CDMs) can increase the validity of pharmacoepidemiologic research. In a case study measuring the changes in the safety and effectiveness of stroke prevention therapy after the introduction of direct oral anticoagulants (DOACs), we performed an international comparison. METHODS: Using data from Stockholm, Denmark, Scotland and Norway, harmonised with a common protocol and CDM, two calendar-based cohorts were created: 2012 and 2017. Patients with a diagnosis code of atrial fibrillation 5 years preceding the 1-year cohort window were included. DOAC, vitamin K antagonist and aspirin treatment were assessed in the 6 months prior to the start of each year while strokes and bleeds were assessed during the year. A Poisson regression generated incidence rate ratios (IRRs) to compare outcomes from 2017 to 2012 adjusted for changes in individual-level baseline characteristics. RESULTS: In 280 359 patients in the 2012 cohort and 356 779 in the 2017 cohort, treatment with OACs increased on average from 45% to 65%, while treatment with aspirin decreased from 30% to 10%. In all countries except Scotland, there were decreases in the risk of stroke and no changes in bleeding risk, after adjustment for changes in baseline characteristics. In Scotland, major bleeding (IRR 1.09, 95% confidence interval [CI] [1.00; 1.18]) and intracranial haemorrhage (IRR 1.31, 95% CI [1.13; 1.52]) increased from 2012 to 2017. CONCLUSIONS: Stroke prevention therapy improved from 2012 to 2017 with a corresponding reduction in stroke risk without increasing the risk of bleeding in all countries, except Scotland. The heterogeneity that remains after methodological harmonisation can be informative of the underlying population and database.

Turn up the power - pharmacological activation of mitochondrial biogenesis in mouse models.

The oxidative phosphorylation (OXPHOS) system in mitochondria is responsible for the generation of the majority of cellular energy in the form of ATP. Patients with genetic OXPHOS disorders form the largest group of inborn errors of metabolism. Unfortunately, there is still a lack of efficient therapies for these disorders other than management of symptoms. Developing therapies has been complicated because, although the total group of OXPHOS patients is relatively large, there is enormous clinical and genetic heterogeneity within this patient population. Thus there has been a lot of interest in generating relevant mouse models for the different kinds of OXPHOS disorders. The most common treatment strategies tested in these mouse models have aimed to up-regulate mitochondrial biogenesis, in order to increase the residual OXPHOS activity present in affected animals and thereby to ameliorate the energy deficiency. Drugs such as bezafibrate, resveratrol and AICAR target the master regulator of mitochondrial biogenesis PGC-1α either directly or indirectly to manipulate mitochondrial metabolism. This review will summarize the outcome of preclinical treatment trials with these drugs in mouse models of OXPHOS disorders and discuss similar treatments in a number of mouse models of common diseases in which pathology is closely linked to mitochondrial dysfunction. In the majority of these studies the pharmacological activation of the PGC-1α axis shows true potential as therapy; however, other effects besides mitochondrial biogenesis may be contributing to this as well.

SNP marker detection and genotyping in tilapia.

We have generated a unique resource consisting of nearly 175 000 short contig sequences and 3569 SNP markers from the widely cultured GIFT (Genetically Improved Farmed Tilapia) strain of Nile tilapia (Oreochromis niloticus). In total, 384 SNPs were selected to monitor the wider applicability of the SNPs by genotyping tilapia individuals from different strains and different geographical locations. In all strains and species tested (O. niloticus, O. aureus and O. mossambicus), the genotyping assay was working for a similar number of SNPs (288-305 SNPs). The actual number of polymorphic SNPs was, as expected, highest for individuals from the GIFT population (255 SNPs). In the individuals from an Egyptian strain and in individuals caught in the wild in the basin of the river Volta, 197 and 163 SNPs were polymorphic, respectively. A pairwise calculation of Nei's genetic distance allowed the discrimination of the individual strains and species based on the genotypes determined with the SNP set. We expect that this set will be widely applicable for use in tilapia aquaculture, e.g. for pedigree reconstruction. In addition, this set is currently used for assaying the genetic diversity of native Nile tilapia in areas where tilapia is, or will be, introduced in aquaculture projects. This allows the tracing of escapees from aquaculture and the monitoring of effects of introgression and hybridization.

High levels of corticosterone, and gene expression of star, cyp17a2, hsd3b, cyp21, hsd11b2 during acute stress in common carp with interrenal hyperplasia.

We investigated the acute stress response in a common carp strain (E5) with interrenal hyperplasia due to 17α-hydroxylase deficiency, and in an isogenic standard (STD) carp strain. Cortisol, corticosterone and the head kidney-somatic index were measured during and after a 3 h net confinement stress. Star, cyp17a2, hsd3b, cyp21, hsd11b2 mRNA levels were measured in head kidneys using real-time qPCR. The results show very high corticosterone levels and enlargement of the head kidney in E5 fish. This is the first report in a teleost fish showing a significant increase of corticosterone levels in response to stress due to interrenal hyperplasia. The high levels of corticosterone in E5 suggest that corticosterone is not converted to aldosterone in common carp. star and hsd3b mRNA levels were significantly higher in E5 compared to STD fish, while cyp17a2 levels were significantly lower in E5. In contrast to E5, star levels did not change during stress and recovery in STD, suggesting that the enzyme is regulated in a different manner in E5 and STD fish. In E5, the levels of cyp17a2 dropped below control values after 20 min stress. These findings strongly suggest that cyp17a2 is impaired at (post)-transcriptional level. As a consequence the accumulated precursor (pregnenolone) is not converted to cortisol, but to corticosterone. In contrast to STD, significant levels of cortisol could not be detected in E5. Finally, hsd11b2 mRNA levels were significantly lower in E5 compared to STD, and did not change during stress and recovery. These results support the idea that hsd11b2 is involved in the conversion of physiologically active cortisol to inactive cortisone, as reported earlier for STD carp. In conclusion our results show high levels of corticosterone in E5 and differences in star and mRNA levels of steroidogenic genes between E5 and STD carp during net confinement stress.

Transcript levels of five enzymes involved in cortisol synthesis and regulation during the stress response in common carp: relationship with cortisol.

In this study the expression of five genes involved in cortisol synthesis and regulation in the head kidneys of common carp (Cyprinus carpio L.) has been investigated in response to 3h net confinement stress, followed by 22h recovery. Cortisol, glucose, lactate and free fatty acid levels were measured in blood plasma. StAR, P450c17a2, 3betaHSD, P450c21 and 11betaHSD2 transcript sequences were identified based on Cyprinidae homologs and quantified by real-time PCR. Results showed that the plasma cortisol level reached a peak at one hour post-stress (85-fold higher than in control) and quickly returned to normal after 4h recovery. 11betaHSD2 transcripts were for the first time identified in interrenals. Changes in cortisol levels during and after confinement were correlated in a time-delayed relationship with increase and decrease in mRNA levels of 11betaHSD2, respectively. These results suggest that cortisol may be involved in the control or activation of 11betaHSD2. StAR and P450c21 mRNA levels did not change during net confinement stress and recovery, but P450c17a2 levels were significantly increased 4 and 22h after recovery. Since plasma cortisol levels increased by 68-fold within 5min net confinement stress, it seems that transcriptional activation of this enzyme is not directly involved in acute cortisol production.

Organization and integration of biomedical knowledge with concept maps for key peroxisomal pathways.

MOTIVATION: One important area of clinical genomics research involves the elucidation of molecular mechanisms underlying (complex) disorders which eventually may lead to new diagnostic or drug targets. To further advance this area of clinical genomics one of the main challenges is the acquisition and integration of data, information and expert knowledge for specific biomedical domains and diseases. Currently the required information is not very well organized but scattered over biological and biomedical databases, basic text books, scientific literature and experts' minds and may be highly specific, heterogeneous, complex and voluminous. RESULTS: We present a new framework to construct knowledge bases with concept maps for presentation of information and the web ontology language OWL for the representation of information. We demonstrate this framework through the construction of a peroxisomal knowledge base, which focuses on four key peroxisomal pathways and several related genetic disorders. All 155 concept maps in our knowledge base are linked to at least one other concept map, which allows the visualization of one big network of related pieces of information. AVAILABILITY: The peroxisome knowledge base is available from www.bioinformaticslaboratory.nl (Support-->Web applications). SUPPLEMENTARY INFORMATION: Supplementary data is available from www.bioinformaticslaboratory.nl (Research-->Output--> Publications--> KB_SuppInfo)

Effects of mixed housing of birds from two genetic lines of laying hens on open field and manual restraint responses.

Birds from Rhode Island Red (RIR) origin show a lower fear response and less feather pecking than birds from White Leghorn (WL) origin. This study investigated whether responses in fear eliciting tests were affected if RIR and WL birds were housed together. Experimental groups contained either birds from one line only ('pure' groups) or an equal number of RIR and WL birds ('mixed' groups). These arrangements were maintained from hatch onwards, throughout the rearing and laying period. Birds were subjected to open field tests at 5-6 weeks and 17-18 weeks of age and to manual restraint tests at 7-8 weeks and 24 weeks of age. RIR birds were more active in both open field tests and in the manual restraint test at 24 weeks of age as compared with WL birds. RIR birds from pure groups were more active in the open field test at 17-18 weeks and in the manual restraint test at 24 weeks of age than RIR birds from mixed groups. These results suggest that otherwise low fearful RIR birds may adopt a higher fear response if they are housed together with more fearful conspecifics. These effects do not emerge until after 8 weeks of age.

Phytanic acid impairs mitochondrial respiration through protonophoric action.

Refsum disease is a rare, inherited neurodegenerative disorder characterized by accumulation of the dietary branched-chain fatty acid phytanic acid in plasma and tissues caused by a defect in the alphaoxidation pathway. The accumulation of phytanic acid is believed to be the main pathophysiological cause of the disease. However, the exact mechanism(s) by which phytanic acid exerts its toxicity have not been resolved. In this study, the effect of phytanic acid on mitochondrial respiration was investigated. The results show that in digitonin-permeabilized fibroblasts, phytanic acid decreases ATP synthesis, whereas substrate oxidation per se is not affected. Importantly, studies in intact fibroblasts revealed that phytanic acid decreases both the mitochondrial membrane potential and NAD(P)H autofluorescence. Taken together, the results described here show that unesterified phytanic acid exerts its toxic effect mainly through its protonophoric action, at least in human skin fibroblasts.

Peroxisomes, Refsum's disease and the alpha- and omega-oxidation of phytanic acid.

In the present paper, we describe the current state of knowledge regarding the enzymology of the phytanic acid alpha-oxidation pathway. The product of phytanic acid alpha-oxidation, i.e. pristanic acid, undergoes three cycles of beta-oxidation in peroxisomes after which the products, including 4,8-dimethylnonanoyl-CoA, propionyl-CoA and acetyl-CoA, are exported from the peroxisome via one of two routes, including (i) the carnitine-dependent route, mediated by CRAT (carnitine acetyltransferase) and CROT (carnitine O-octanoyltransferase), and (ii) the free acid route, mediated by one or more of the peroxisomal ACOTs (acyl-CoA thioesterases). We also describe our recent data on the omega-oxidation of phytanic acid, especially since pharmacological up-regulation of this pathway may form the basis of a new treatment strategy for ARD (adult Refsum's disease). In patients suffering from ARD, phytanic acid accumulates in tissues and body fluids due to a defect in the alpha-oxidation system.

Consequences of selection for yield traits on calving ease performance.

The impact of different breeding goals on the genetic response for calving ease (CE) and yield traits was studied in the Basque Holstein cattle population. The economic value for CE was estimated with a bioeconomic model, using Basque production and market circumstances and taking into account the categorical nature of CE. The economic value for CE was -18.03 euros/cow per calving interval per liability unit. This value was relatively insensitive to changes in the market price of animals but was more sensitive to changes in the incidence of dystocia. Records from parities between 1995 and 2002 were used for the estimation of genetic parameters for yield (actual milk, fat, and protein yield) and CE using a multivariate model. Linear sire models for yield traits and a threshold sire-maternal grandsire model for CE were used. A Holstein population was simulated to determine the consequences of including CE in the breeding goal. Three selection strategies were considered: 1) selection only on yield traits, 2) selection on yield and direct CE (DCE), and 3) selection on yield, DCE, and maternal CE (MCE). Selection on yield traits only resulted in a slight reduction of dystocia. Selection strategies in which DCE or DCE and MCE were included in the breeding goal did not improve the genetic response for DCE and MCE obtained with the first selection strategy. Genetic responses were also calculated using the 2.5th, 50th, and 97.5th percentiles of posterior densities of genetic correlations between DCE and MCE and yield traits. Because responses in CE were sensitive to deviations in estimates of genetic parameters, the inclusion of CE in the monitoring scheme is recommended. Genetic evaluation of bulls for CE is of considerable value because it provides farmers with the opportunity to use assortative matings of sires with favorable estimated breeding values for DCE to primiparous cows.

Stress-induced facilitation of the cortisol response in 17alpha-hydroxylase deficient XX mas-1/mas-1 carp (Cyprinus carpio).

Facilitation of the stress response results from a reduction of the inhibitory effects of circulating corticosteroids, allowing an animal to respond to a novel stressor. In this study, the existence of a facilitated cortisol stress response in normal (STD) and 17alpha-hydroxylase deficient XX mas-1/mas-1 (E5) carp was investigated. E5 carp had previously been characterized as having a low cortisol response to stress. Fish were subjected to either cortisol feeding or daily-acute stress, from 45 until 140 days post-hatch (dph) and were then subjected to a novel net-confinement stressor at 141 dph. Growth of E5 fish was reduced in both the daily-acute stress and cortisol-fed groups, but STD fish were only affected by daily-acute stress. Cortisol feeding had no effect on the stress response of STD fish but daily-acute stress significantly inhibited the response to a subsequent novel stressor. In contrast, daily-acute stress facilitated the cortisol stress response of E5 fish to a novel stressor, while cortisol feeding inhibited the cortisol response. Facilitation was accompanied by significant enlargement of the head-kidney tissue (which contains the steroidogenic interrenal tissue) in E5 fish. To our knowledge this is the first report of stress-induced facilitation in a lower vertebrate.

Identification of the cytochrome P450 enzymes responsible for the omega-hydroxylation of phytanic acid.

Patients suffering from Refsum disease have a defect in the alpha-oxidation pathway which results in the accumulation of phytanic acid in plasma and tissues. Our previous studies have shown that phytanic acid is also a substrate for the omega-oxidation pathway. With the use of specific inhibitors we now show that members of the cytochrome P450 (CYP450) family 4 class are responsible for phytanic acid omega-hydroxylation. Incubations with microsomes containing human recombinant CYP450s (Supersomes) revealed that multiple CYP450 enzymes of the family 4 class are able to omega-hydroxylate phytanic acid with the following order of efficiency: CYP4F3A>CYP4F3B>CYP4F2>CYP4A11.

Hypocorticism and interrenal hyperplasia are not directly related to masculinization in XX mas(-1)/mas(-1) carp, Cyprinus carpio.

This study reports on a homozygous XX male strain of common carp (E5), which fail to mount a normal cortisol stress response. Earlier classical genetic analysis had indicated that masculinization of E5 fish was caused by a putative recessive mutation (mas(-1)/mas(-1)). Hypocorticism in E5 fish was studied to investigate if it was related to masculinization. Head-kidney tissues isolated from E5 fish showed a low cortisol-producing capacity in vitro, and also demonstrated a reduced sensitivity to stimulation with ACTH, when compared with an isogenic XY male carp strain (STD). There was no strain difference in androgen production by head-kidney tissues in vitro. E5 fish exhibited significant hyperplasia of the interrenal tissue (adrenal homologue of teleost fish) located in the head-kidney. Conversion of pregnenolone was significantly lower in E5 head-kidney homogenates, compared to STD homogenates, however, no strain difference was found in the conversion of 17alpha-hydroxyprogesterone into cortisol. Gonad homogenates incubated with pregnenolone showed no strain difference in conversion to corticosteroids and androgens. Results indicate that the interrenal hyperplasia and hypocorticism in this strain of carp may be due to a dysfunction of the 17alpha-hydroxylase activity of the enzyme P450c17 in the interrenal, but that this defect may not be the primary factor resulting in masculinization of these XX genotypes.

Characterization of phytanic acid omega-hydroxylation in human liver microsomes.

Phytanic acid is a 3-methyl branched-chain fatty acid which originates from dietary sources. Since the 3-methyl group blocks regular beta-oxidation, it is broken down by peroxisomal alpha-oxidation. Adult Refsum disease patients accumulate phytanic acid as a result of an impairment in peroxisomal alpha-oxidation, caused by the deficient activity of the enzyme phytanoyl-CoA hydroxylase in the majority of patients. In this paper, we studied an alternative degradation route for phytanic acid, namely omega-oxidation. During omega-oxidation a fatty acid is hydroxylated at its omega-end by a member of the cytochrome P450 multi-enzyme family. Subsequently, an alcohol dehydrogenase converts the formed hydroxyl group into an aldehyde, which is then converted into a carboxyl-group by an aldehyde dehydrogenase. In case of phytanic acid omega-hydroxylation would lead to the formation of phytanedioic acid, which can be degraded by beta-oxidation from the omega-end. Here, we show that phytanic acid indeed undergoes omega- and (omega-1)-hydroxylation in pooled human liver microsomes in an NADPH-dependent manner with a ratio of 15:1. Studies with imidazole antimycotics indicate that these reactions are catalyzed by one or more cytochrome P450 enzymes. Induction of the cytochrome P450 involved in phytanic acid omega-hydroxylation may increase the flux through the omega-oxidation pathway, causing increased clearance of phytanic acid in ARD patients. Hence, this alternative catabolic pathway is of potential therapeutic relevance.

Omega-hydroxylation of phytanic acid in rat liver microsomes: implications for Refsum disease.

The 3-methyl-branched fatty acid phytanic acid is degraded by the peroxisomal alpha-oxidation route because the 3-methyl group blocks beta-oxidation. In adult Refsum disease (ARD), peroxisomal alpha-oxidation is defective, which is caused by mutations in the gene coding for phytanoyl-CoA hydroxylase in the majority of ARD patients. As a consequence, phytanic acid accumulates in tissues and body fluids. This study focuses on an alternative route of phytanic acid degradation, omega-oxidation. The first step in omega-oxidation is hydroxylation at the omega-end of the fatty acid, catalyzed by a member of the cytochrome P450 multienzyme family. To study this first step, the formation of hydroxylated intermediates was studied in rat liver microsomes incubated with phytanic acid and NADPH. Two hydroxylated metabolites of phytanic acid were formed, omega- and (omega-1)-hydroxyphytanic acid (ratio of formation, 5:1). The formation of omega-hydroxyphytanic acid was NADPH dependent and inhibited by imidazole derivatives. These results indicate that phytanic acid undergoes omega-hydroxylation in rat liver microsomes and that an isoform of cytochrome P450 catalyzes the first step of phytanic acid omega-oxidation.

Effects of oxytocin on semen release response in African catfish (Clarias gariepinus).

In silurid fishes, semen collection is practically impossible, even after hormonal stimulation. Instead, males are killed and testes macerated to obtain sperm. To understand the endocrine control of semen release in catfishes, we investigated the role of smooth muscle contractors in semen release and semen quality of African catfish (Clarias gariepinus). In in vitro experiments, testis slices were incubated with oxytocin (1 and 10 IU), isotocin (2 and 20 ug), vasopressin (0.2 and 2 ug), epinephrine (1 and 10 ug), PGF2alpha (1 and 10 ug), purified Clarias LH (300 ng) and partly purified Clarias pituitary extract (containing 300 ng LH). Only oxytocin increased sperm concentration of the medium (assessed by optical density measurements) compared to control incubations. Oxytocin was then tested in vivo in two groups of fish: normal males, and males that had been treated with 17alpha-methyltestosterone during larval stages to inhibit seminal vesicle development (MT males). Both groups of fish received two doses of carp pituitary suspension (8 and 10 mg/kg, respectively i.m.) with or without subsequent oxytocin treatment (5 IU/kg i.v.; cPS-OT treatment and cPS treatment, respectively). There was no effect of oxytocin on the number of strippable males. Of cPS and cPS-OT treated fish, 87% MT males and 60% normal males were strippable. The stripped semen volume was low in both groups but MT males produced higher (P < 0.001) hatching rates (63.1%) than did normal males (2.1%).

Sex steroids and their involvement in the cortisol-induced inhibition of pubertal development in male common carp, Cyprinus carpio L.

The onset and regulation of puberty is determined by functional development of the brain-pituitary-gonad (BPG) axis. Sex steroids produced in the gonads play an important role in the onset of puberty. Stress interferes with reproduction and the functioning of the BPG axis, and cortisol has frequently been indicated as a major factor mediating the suppressive effect of stress on reproduction. Prolonged elevated cortisol levels, implicated in stress adaptation, inhibited pubertal development in male common carp (Cyprinus carpio). Cortisol treatment caused a retardation of pubertal testis development and reduced the LH pituitary content and the salmon GnRHa-stimulated LH secretion in vitro. A reduced synthesis of androgens also was observed. These findings suggest that the cortisol-induced inhibition of testicular development and the maturation of pituitary gonadotrophs are mediated by an effect on testicular androgen secretion. In this study, we combined cortisol treatment with a replacement of the testicular steroid hormones (testosterone and 11-oxygenated androgens) to investigate the role of these steroids in the cortisol-induced suppression of pubertal development. The effect of cortisol on spermatogenesis was independent of 11-ketotestosterone, whereas the effect on the pituitary was an indirect one, involving the testicular secretion of testosterone.

First Report of Virulence in Puccinia graminis f. sp. tritici to Wheat Stem Rust Resistance Genes Sr8b and Sr38 in South Africa.

During the 2000 to 2001 season, 27 stem rust samples were collected from wheat (Triticum aestivum), barley (Hordeum vulgare), and triticale (× Triticosecale) cultivars and lines in the Western Cape, South Africa. Following inoculation and multiplication on McNair 701 seedlings, 40 single pustule isolates of P. graminis f. sp. tritici were established. Twenty-six isolates obtained from wheat, barley, or triticale that produced a similar reaction pattern on a set of differentiating host lines, were designated as pathotype Pgt-2SA55. Pgt-2SA55 is avirulent to Sr5, -6, -7b, -8b, -9b, -9e, -9g, -23, -24, -27, -30, -38, and -Gt, and virulent to Sr11, and -Agi. The remaining 14 isolates, all from wheat and designated as pathotype Pgt-2SA88, were avirulent to Sr24, -27, and -Agi, and virulent to Sr5, -6, -7b, -8b, -9b, -9e, -9g, -11, -23, -30, -38, and -Gt. On an expanded differential set, representative isolates of each pathotype were all avirulent to Sr13, -15, -21, -22, -25, -26, -29, -31, -32, -33, -35, -39, -43, and -Em, and virulent to Sr7a, -8a, -9a, -9d, -9f, -10, -12, -14, -16, -19, -20, -34, and Lc. Pgt-2SA55 was avirulent on cv. Renown (Sr2, -7b, -9d, and -17), whereas Pgt-2SA88 was virulent on this cultivar and Line R Sel carrying only Sr17. Both pathotypes differ from those identified previously in South Africa (1) and to our knowledge, Pgt-2SA88 is the first local isolate to have virulence towards Sr8b and the T. ventricosum-derived gene Sr38. Virulence to Sr38 has been reported in a P. graminis f. sp. tritici isolate collected in Uganda (2). Pathotype Pgt-2SA88 is virulent to seedlings of the previously resistant South African cvs. SST 57 (heterogeneous), Tugela, Tugela DN, and PAN 3377. Furthermore, 20% of the elite breeding lines in the spring and winter wheat breeding program of the Small Grain Institute expressed susceptible seedling reactions to Pgt-2SA88. Triticale cvs. Rex and Kiewiet were heterogeneous in their seedling reaction towards Pgt-2SA55. Seedling and field reactions recorded for the barley cvs. Sterling and SSG 532 and the experimental varieties Puma and Jaguar, showed an increase in stem rust susceptibility to Pgt-2SA55 when compared with existing South African pathotypes. The higher incidence of stem rust in commercial fields and experimental plots of wheat and barley in the Western Cape may be attributed to a recent increase in the cultivation of stem rust-susceptible cultivars in the region. The detection of two new pathotypes of P. graminis f. sp. tritici is of concern to the local small grain industry and requires continued resistance breeding. References: (1) W. H. P. Boshoff et al. S. Afr. J Plant Soil 17:60, 2000. (2) Z. A. Pretorius et al. Plant Dis. 84:203, 2000.

Effects of 17alpha-methyltestosterone on seminal vesicle development and semen release response in the African catfish, Clarias gariepinus.

The effects of 17alpha-methyltestosterone on seminal vesicle development in the African catfish, Clarias gariepinus, were investigated in an attempt to improve semen collection from this species. Treatment of larvae with dietary 17alpha-methyltestosterone at 50 mg kg(-1) for days 12-33 or days 12-40 after hatching, or at 20 mg kg(-1) for days 12-26, 12-33, 12-40 or 12-47 after hatching inhibited the development of the seminal vesicle finger-like extensions in male catfish, but did not affect the sex ratio. The minimum effective dose and period of treatment to inhibit seminal vesicle development in all male catfish treated with 17alpha-methyltestosterone was 20 mg kg(-1) for days 12-40 after hatching. Male catfish from this treatment group developed normal testes that, in some cases, contained a few oocytes, which tended to disappear before sexual maturation. After sexual maturation, the semen release response was evaluated in males with incomplete seminal vesicles. Fluid with viable spermatozoa was obtained after two consecutive injections of carp pituitary suspension, from 10 of 19 males that had been fed 20 mg 17alpha-methyltestosterone kg(-1) for days 12-40 or days 12-47 after hatching, but from only 4 of 15 males that did not receive any dietary steroid. Intratesticular semen quality was not affected by 17alpha-methyltestosterone treatment. The results of this study demonstrate that the absence of seminal vesicle extensions induced by treatment with 17alpha-methyltestosterone facilitated the collection of semen by stripping from this species of fish.

Segregation of microsatellite alleles and residual heterozygosity at single loci in homozygous androgenetic common carp (Cyprinus carpio L.).

Thirty-three androgenetic progeny groups of common carp were analysed using 11 microsatellite markers to (i) verify the homozygous status of the 566 androgenetic individuals, (ii) analyse the microsatellite allele segregation, and (iii) study the possible association of microsatellite alleles with phenotypic traits. In total, 92% of the androgenetic individuals proved to be homozygous at all 11 loci. Forty-three of the 47 heterozygous individuals were heterozygous at a single locus only. This heterozygosity was probably due to DNA fragments caused by UV irradiation of the eggs. although the maternal origin of the fragments could not be proved beyond doubt. Screening with 11 microsatellites also revealed two linkage groups, a segregation distortion at two microsatellite loci, and the possible association of some microsatellites with mass, length, stress-related plasma cortisol levels, and basal plasma glucose levels. The success of the linkage and association study could be explained by a low recombination frequency due to high chiasma interference. This would imply a relatively short genetic map for common carp.