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Nephronophthisis (NPHP), an autosomal recessive kidney disease, is the most frequent genetic cause of end-stage renal failure in the first three decades of life. Positional cloning of the six known NPHP genes has linked its pathogenesis to primary cilia function. Here we identify mutation of GLIS2 as causing an NPHP-like phenotype in humans and mice, using positional cloning and mouse transgenics, respectively. Kidneys of Glis2 mutant mice show severe renal atrophy and fibrosis starting at 8 weeks of age. Differential gene expression studies on Glis2 mutant kidneys demonstrate that genes promoting epithelial-to-mesenchymal transition and fibrosis are upregulated in the absence of Glis2. Thus, we identify Glis2 as a transcription factor mutated in NPHP and demonstrate its essential role for the maintenance of renal tissue architecture through prevention of apoptosis and fibrosis.

In order to investigate the reliability of muscle fibre trait estimations of pig longissimus muscle and to derive the minimum number of samples required per muscle cross-section and animal, intraclass correlation coefficients (ICC, ϑˆ) were obtained by one-way analysis of variance. From each of 23 market weight pigs five samples, evenly distributed over the muscle cross-sectional area at the 12th/13th rib level, were taken and analyzed for various muscle fibre traits. The number of samples required per muscle cross-section was found to be different between selected fibre traits, ranging from a minimum of three (for number of muscle fibres) to a maximum of five or more (for mean fibre area, fibre type composition and relative area occupied by each fibre type). These findings should be taken as a recommendation, but their usefulness will depend upon the goal and conditions of future experiments.

We have identified a consanguineous family from Morocco segregating autosomal recessive congenital progressive hearing loss (ARNSHL) and retinal degeneration. Detailed clinical investigation of the six siblings revealed combined severe cone-rod dystrophy (CORD) and severe/profound hearing impairment in two of them, while there is isolated CORD in three and nonsyndromic profound hearing loss in one. We therefore assumed a partial overlap of two nonsyndromic autosomal recessive conditions instead of a monogenic syndrome and performed genomewide linkage analysis. The disease loci were mapped to chromosome 2q31.1-2q32.1 for ARNSHL and to 2q13-2q14.1 for CORD, respectively. The retinal phenotype was shown to be due to homozygosity for a novel splice site mutation, c.2189+1G>T, in the retinitis pigmentosa gene MERTK. The ARNSHL interval comprised the DFNB59 locus. The DFNB59 gene has been identified recently, and two missense mutations (p.R183W and p.T54I) have been shown to cause auditory neuropathy in both humans and transgenic mice. Mutation screening in the DFNB59 gene in our family revealed homozygosity for a 1-bp insertion in exon 2 (c.113_114insT), predicting a truncated protein of 47 amino acids, in all three hearing impaired subjects. This is the first description of biallelic putative loss-of-function of the DFNB59 gene. Detailed audiological investigation clearly indicated hair cell dysfunction and, in contrast to cases reported previously, excluded auditory neuropathy. We show that besides otoferlin (OTOF), DFNB59 is the second known gene in which mutations can result in these two distinct forms of hearing impairment. Moreover, all patients in our family with homozygosity for the DFNB59 mutation display central vestibular dysfunction., ((c) 2007 Wiley-Liss, Inc.)

The influence of social rank and social environment on visual discrimination learning of small groups of Nigerian dwarf goats (Capra hircus, n = 79) was studied using a computer-controlled learning device integrated in the animals' home pen. The experiment was divided into three sections (LE1, LE1 u, LE2; each 14d). In LE1 the goats learned a discrimination task in a socially stable environment. In LE1u animals were mixed and relocated to another pen and given the same task as in LE1. In LE2 the animals were mixed and relocated again and given a new discrimination task. We used drinking water as a primary reinforcer. The rank category of the goats were analysed as alpha, omega or middle ranking for each section of the experiment. The rank category had an influence on daily learning success (percentage of successful trials per day) only in LE1 u. Daily learning success decreased after mixing and relocation of the animals in LE1 u and LE2 compared to LE1. That resulted in an undersupply of drinking water on the first day of both these tasks. We discuss social stress induced by agonistic interactions after mixing as a reason for that decline. The absolute learning performance (trials to reach the learning criterion) of the omega animals was lower in LE2 compared to the other rank categories. Furthermore, their absolute learning performance was lower in LE2 compared to LE1. For future application of similar automated learning devices in animal husbandry, we recommend against the combination of management routines like mixing and relocation with changes in the learning task because of the negative effects on learning performance, particularly of the omega animals.

We observed two unrelated consanguineous families with malformation syndromes sharing anophthalmia and distinct eyebrows as common signs, but differing for alveolar capillary dysplasia or complex congenital heart defect in one and diaphragmatic hernia in the other family. Homozygosity mapping revealed linkage to a common locus on chromosome 15, and pathogenic homozygous mutations were identified in STRA6, a member of a large group of "stimulated by retinoic acid" genes encoding novel transmembrane proteins, transcription factors, and secreted signaling molecules or proteins of largely unknown function. Subsequently, homozygous STRA6 mutations were also demonstrated in 3 of 13 patients chosen on the basis of significant phenotypic overlap to the original cases. While a homozygous deletion generating a premature stop codon (p.G50AfsX22) led to absence of the immunoreactive protein in patient's fibroblast culture, structural analysis of three missense mutations (P90L, P293L, and T321P) suggested significant effects on the geometry of the loops connecting the transmembrane helices of STRA6. Two further variations in the C-terminus (T644M and R655C) alter specific functional sites, an SH2-binding motif and a phosphorylation site, respectively. STRA6 mutations thus define a pleiotropic malformation syndrome representing the first human phenotype associated with mutations in a gene from the "STRA" group.

Autosomal recessive inheritance of non-syndromic mental retardation (ARNSMR) may account for approximately 25% of all patients with non-specific mental retardation (NSMR). Although many X-linked genes have been identified as a cause of NSMR, only three autosomal genes are known to cause ARNSMR. We present here a large consanguineous Turkish family with four mentally retarded individuals from different branches of the family. Clinical tests showed cognitive impairment but no neurological, skeletal, and biochemical involvements. Genome-wide mapping using Human Mapping 10K Array showed a single positive locus with a parametric LOD score of 4.92 in a region on chromosome 1p21.1-p13.3. Further analyses using polymorphic microsatellite markers defined a 6.6-Mb critical region containing approximately 130 known genes. This locus is the fourth one linked to ARNSMR.

The aim of the study was to investigate the effect of the GnRH agonist Buserelin given on day 10 after ovulation on pregnancy rate and concentrations of progesterone and LH. Altogether 191 warmblood mares were used for two trials. Fresh or frozen/thawed semen from 27 stallions was used for A.I. In trial A 171 mares received either Buserelin (Receptal, Hoechst, Germany, 40 microg/animal) or 10 ml 0.9% NaCl (placebo). On day 16 after A.I. pregnancy diagnosis was performed by ultrasound scanning of the uterus. For statistical analysis, data were analyzed by a mixed model, with four fixed factors (treatment, type of spermatozoa, A.I. number, reproductive status of the mare) and a random factor (stallion). Least Square Means (LSM) for pregnancy rate were 46.0% in GnRH agonist treated mares and 36.4% in the control group (P=0.22). In trial B 20 lactating and cycling mares were used for endocrine studies. Blood samples were recovered for analyses of progesterone and LH from days 0 to 11. The mean progesterone concentrations increased continuously from days 0 to 8 after ovulation in both groups (GnRH group: from 0.81+/-0.48 to 5.47+/-0.48 ng/ml, control group: from 0.63+/-0.68 to 5.83+/-0.68 ng/ml). Moreover, the progesterone concentrations from days 9 to 11 were not different between the GnRH and the control group. In contrast to this LH concentrations were markedly influenced by the GnRH agonist. On day 10 LH concentrations were significantly higher in GnRH agonist treated than in placebo treated animals. From the data obtained from individual animals it can be concluded that GnRH agonist, given during luteal phase may have different effect on luteal function.

Nephrotic syndrome, a malfunction of the kidney glomerular filter, leads to proteinuria, edema and, in steroid-resistant nephrotic syndrome, end-stage kidney disease. Using positional cloning, we identified mutations in the phospholipase C epsilon gene (PLCE1) as causing early-onset nephrotic syndrome with end-stage kidney disease. Kidney histology of affected individuals showed diffuse mesangial sclerosis (DMS). Using immunofluorescence, we found PLCepsilon1 expression in developing and mature glomerular podocytes and showed that DMS represents an arrest of normal glomerular development. We identified IQ motif-containing GTPase-activating protein 1 as a new interaction partner of PLCepsilon1. Two siblings with a missense mutation in an exon encoding the PLCepsilon1 catalytic domain showed histology characteristic of focal segmental glomerulosclerosis. Notably, two other affected individuals responded to therapy, making this the first report of a molecular cause of nephrotic syndrome that may resolve after therapy. These findings, together with the zebrafish model of human nephrotic syndrome generated by plce1 knockdown, open new inroads into pathophysiology and treatment mechanisms of nephrotic syndrome.

Anonychia is an autosomal recessive disorder characterized by the congenital absence of finger- and toenails. In a large German nonconsanguineous family with four affected and five unaffected siblings with isolated total congenital anonychia, we performed genomewide mapping and showed linkage to 20p13. Analysis of the RSPO4 gene within this interval revealed a frameshift and a nonconservative missense mutation in exon 2 affecting the highly conserved first furin-like cysteine-rich domain. Both mutations were not present among controls and were shown to segregate with the disease phenotype. RSPO4 is a member of the recently described R-spondin family of secreted proteins that play a major role in activating the Wnt/ beta -catenin signaling pathway. Wnt signaling is evolutionarily conserved and plays a pivotal role in embryonic development, growth regulation of multiple tissues, and cancer development. Our findings add to the increasing body of evidence indicating that mesenchymal-epithelial interactions are crucial in nail development and put anonychia on the growing list of congenital malformation syndromes caused by Wnt-signaling-pathway defects. To the best of our knowledge, this is the first gene known to be responsible for an isolated, nonsyndromic nail disorder.

Congenital nephrotic syndrome is clinically and genetically heterogeneous. The majority of cases can be attributed to mutations in the genes NPHS1, NPHS2, and WT1. By homozygosity mapping in a consanguineous family with isolated congenital nephrotic syndrome, we identified a potential candidate region on chromosome 3p. The LAMB2 gene, which was recently reported as mutated in Pierson syndrome (microcoria-congenital nephrosis syndrome; OMIM #609049), was located in the linkage interval. Sequencing of all coding exons of LAMB2 revealed a novel homozygous missense mutation (R246Q) in both affected children. A different mutation at this codon (R246W), which is highly conserved through evolution, has recently been reported as causing Pierson syndrome. Subsequent LAMB2 mutational screening in six additional families with congenital nephrotic syndrome revealed compound heterozygosity for two novel missense mutations in one family with additional nonspecific ocular anomalies. These findings demonstrate that the spectrum of LAMB2-associated disorders is broader than previously anticipated and includes congenital nephrotic syndrome without eye anomalies or with minor ocular changes different from those observed in Pierson syndrome. This phenotypic variability likely reflects specific genotypes. We conclude that mutational analysis in LAMB2 should be considered in congenital nephrotic syndrome, if no mutations are found in NPHS1, NPHS2, or WT1.

In two large Turkish consanguineous families, a locus for autosomal recessive nonsyndromic hearing loss (ARNSHL) was mapped to chromosome 6p21.3 by genome-wide linkage analysis in an interval overlapping with the loci DFNB53 (COL11A2), DFNB66, and DFNB67. Fine mapping excluded DFNB53 and subsequently homozygous mutations were identified in the lipoma HMGIC fusion partner-like 5 (LHFPL5) gene, also named tetraspan membrane protein of hair cell stereocilia (TMHS) gene, which was recently shown to be mutated in the "hurry scurry" mouse and in two DFNB67-linked families from Pakistan. In one family, we found a homozygous one-base pair deletion, c.649delG (p.Glu216ArgfsX26) and in the other family we identified a homozygous transition c.494C>T (p.Thr165Met). Further screening of index patients from 96 Turkish ARNSHL families and 90 Dutch ARNSHL patients identified one additional Turkish family carrying the c.649delG mutation. Haplotype analysis revealed that the c.649delG mutation was located on a common haplotype in both families. Mutation screening of the LHFPL5 homologs LHFPL3 and LHFPL4 did not reveal any disease causing mutation. Our findings indicate that LHFPL5 is essential for normal function of the human cochlea., (Copyright 2006 Wiley-Liss, Inc.)

Lacrimo-auriculo-dento-digital (LADD) syndrome is characterized by lacrimal duct aplasia, malformed ears and deafness, small teeth and digital anomalies. We identified heterozygous mutations in the tyrosine kinase domains of the genes encoding fibroblast growth factor receptors 2 and 3 (FGFR2, FGFR3) in LADD families, and in one further LADD family, we detected a mutation in the gene encoding fibroblast growth factor 10 (FGF10), a known FGFR ligand. These findings increase the spectrum of anomalies associated with abnormal FGF signaling.

The present study was conducted to assess effects of the gonadotropin-releasing hormone agonist (GnRHa) triptorelin in dairy heifers. The peptide was released from a commercial 4-week depot formulation (Decapeptyl Depot) administered at animals' estrus (day 0). First experiment (EXP I, n=5), which was aimed to explore the availability of peptide, detected a maximum of triptorelin concentration between day 2 and 5 after depot injection, and the peptide remained detectable by RIA in peripheral blood for about 3 weeks. In further experiments, the peptide release was terminated on day 9 (EXP II, n=16) or day 21 (EXP III, n=47). Treatment effects were studied on follicular development, the characteristics of cumulus-oocyte complexes (COCs) (EXP II; EXP IIIa) and secretions of LH and progesterone (EXP IIIb). Results showed that the occurrence of the pre-ovulatory LH surge was more uniform in treated heifers than that in controls. The duration of ovulation periods was similar amongst the heifers of EXP II, but more compact amongst those of EXP III each compared with the respective controls. Post-ovulatory, the number of LH pulses was significantly reduced by treatment, whereas both basal LH and progesterone concentrations were elevated on a few days. Follicular growth was reduced only by the prolonged influence of the GnRHa. There were increased proportions of both degenerated COCs and immature oocytes from small follicles (<3mm in diameter), and meiotic configuration and quality of oocytes isolated from follicles 3-5mm were changed after the prolonged, 21-day treatment. These results indicate that a continuous influence of a GnRHa over more than 1 week may increasingly impair the development of bovine follicles and oocytes. This may have some significance for the development of novel GnRH-based techniques in regulating the reproductive function in cattle.

The aim of this study was to prove if oxidation-reduction levels in the follicular fluid were new functional indices of follicular health and whether there was a high level of accordance with endocrinological parameters and with the growth stage as detected by ultrasound monitoring of individual follicles during the oestrous cycle in mares. Follicles were classified as growing and regressing follicles using ultrasonography. Altogether 48 follicles with a diameter from 20 to 56 mm were aspirated by transvaginal ultrasound guided follicular aspiration. Follicular concentration of oestradiol and progesterone in relation to the diameter of growing follicles showed correlations of r = 0.64 and r = 0.57, respectively. The redox potential derived index D2 varied from -448 to +431 in the collected fluids of the follicles. The accordance of the judgement of all follicles using both complexes of methods - endocrinological and ultrasonographic parameters vs. analysis of oxidation and reduction levels - reached 72.5%. This finding has shown that parameters of redox reactions do not correlate closely with the stage of follicular growth or regression as determined by in vivo scanning of ovaries or by assessment of follicular steroid concentrations. However, the measurement of redox potentials offers an opportunity to examine the whole process of metabolism in follicular cells and to forecast impairments of cellular performances. Changes of redox parameters in growing follicles enable an earlier prediction of their further development. The data demonstrate that growing and regressing follicles do not represent nonatretic, early atretic and atretic follicles, respectively.

In most studies on animal learning, individual animals are tested separately in a specific learning environment and with a limited number of trials per day. An alternative approach is to test animals in a familiar environment in their social group. In this study, the authors--applying a fully automated learning device--investigated voluntary, self-controlled visual shape discrimination learning of group-housed dwarf goats (Capra hircus). The majority of the tested goats showed successful shape discrimination, which indicates the adaptive value of an effective learning strategy. However, in each group, a few individual goats developed behavioral strategies different from shape discrimination to get reward. Relocation impairs memory retrieval (probably by attention shifting) only temporarily for previously learnt shapes. The results demonstrate the usefulness of a self-controlled learning paradigm to assess learning abilities of social species in their normal social settings. This may be especially relevant for captive animals to improve their welfare., (((c) 2006 APA, all rights reserved).)

Lower urinary tract obstruction by posterior urethral valves (PUV) is an important cause of congenital renal failure in male infants. Though population-based studies point to a role of genetic factors in the etiology of PUV, no clear evidence for a specific gene defect for PUV has been observed so far. Here we present a consanguineous family with four male descendants affected by PUV and a healthy girl, suggestive of autosomal recessive inheritance. One boy presented with prune-belly syndrome (PBS) in addition to PUV. Using a DNA chip-based genome-wide linkage analysis, we identified a region of homozygosity for the affected boys in an interval of 35 cM on chromosome 1q41-44 with a maximum multipoint LOD score of Z(max) = 3.134 at theta = 0 for single nucleotide polymorphisms (SNPs) rs158724-rs720163. By applying a second genetic model based on the assumption of a male-limited phenotype and the girl being carrier of the genetic defect without expressing the phenotype, a second alternative locus of 9 cM on chromosome 11p11 was identified with a LOD score of Z(max) = 3.61 at theta = 0. Equal significance for both loci with a LOD score of Z(max) = 3.01 at theta = 0 was obtained after the affection status of the female descendant was set "unknown". We suppose that most probably, only one of the two identified loci harbours the disease-causing gene. As the interpretation of the girl's status remains uncertain, we are not able to exclude one of the two loci. Analyses of additional informative families will be important to exclude one of the two loci and to restrict the critical interval.

An experiment was conducted to examine the response to wheat gluten (WG)-based diets at two lysine levels in adult minipigs (23 kg BW) using the indicator AA oxidation (IAAO) approach and N balance. Twenty minipigs (n = five per group), fitted with reentrant ileoileal cannulas allowing collection of ileal effluents, were fed restrictively two WG-based diets (WG and WG + Lys; 2.7 and 6.6 g of lysine/kg, respectively) for adaptation periods of 10 and 100 d. On d 7 and 9, for pigs fed the diets for 10 d, and on d 97 and 99, for pigs fed the diets for 100 d, primed i.v. fasted/fed tracer protocols with [(13)C]bicarbonate, and [(13)C]leucine were performed. With the WG diet, [(13)C]bicarbonate recoveries (%) were lower irrespective of the adaptation period, and higher during the fed period (fasted: WG + Lys = 82.5, and WG = 69.1; fed: WG + Lys = 90.6, and WG = 85.9; P < 0.05). Leucine oxidation rate was higher with the lower lysine intake (WG = 194.6 vs. 109.5 mg/[kg BW x d]; P < 0.05). Wheat gluten feeding resulted in a negative leucine balance independent of the adaptation period (WG = -29.1, and WG + Lys = 48.2 mg/[kg BW x d]; P < 0.05). In contrast with the IAAO method, N balance did not differ between the two lysine intakes, possibly because of an underestimation of N losses. The finding of a lower (13)C bicarbonate recovery with the lower dietary lysine intake suggests that caution should be taken in using a single recovery factor for all AA oxidation studies.

This study was conducted to examine stress-induced effects on gene expression of specific markers for HPA axis and neuronal activity in fetuses and neonatal pigs. Brain, pituitary gland, and adrenal gland were obtained to determine the mRNA levels for corticotropin-releasing hormone (CRH), CRH receptor 1 (CRHR1), pro-opiomelanocortin (POMC), ACTH receptor (MC2R), c-jun and c-fos. The suitability of these molecular markers was determined in neonatal pigs which were maternally deprived for two hours. It was found that maternal deprivation caused significantly higher transcript levels of c-fos and CRH in brain accompanied by a down-regulation of CRHR1 mRNA and an up-regulation of c-jun in the pituitary gland. To determine the effect of elevated maternal cortisol levels on gene expression of these molecular markers in fetuses, pregnant sows were treated with 100 IU ACTH (Synacthen Depot) s.c. every two days between Day 49 and Day 75 of gestation (normal gestation length 114 days). Animals were killed 48 hours after the last ACTH administration and fetuses of each sow were isolated. The ACTH treatment of sows significantly increased mRNA expression of c-fos but not of CRH in the fetal brain, and significantly decreased MC2R mRNA expression in the adrenal gland. However, HPA axis seems not to be fully developed in Day 77-fetuses because fetal pituitary CRHR1 and POMC mRNA expression was low in most of the fetuses. Although the expression of endocrine regulatory factors was partially incomplete in fetuses at the beginning of the third-trimester, ACTH dependent activation of c-fos mRNA in brain indicates a stress-related increase of neuronal activity. Based on these results it is assumed that prenatal stress in pigs may also have effects on the activity of the HPA axis in the offspring.

We studied visual discrimination learning in a group of Nigerian dwarf goats using a computer-based learning device which was integrated in the animals' home pen. We conducted three consecutive learning tasks (T1, T2 and T3), each of which lasted for 13 days. In each task, a different set of four visual stimuli was presented on a computer screen in a four-choice design. Predefined sequences of stimulus combinations were presented in a pseudorandom order. Animals were rewarded with drinking water when they chose the positive stimulus by pressing a button next to it. Noninvasive measurements of goats' heartbeat intervals were carried out on the first and the last 2 days of each learning task. We analysed heart rate (HR) and heart rate variability (HRV) of resting animals to study sustained physiological effects related to general learning challenge rather than acute excitement during an actual learning session. The number of trials to reach the learning criterion was 1000 in T1, when visual stimuli were presented to the goats for the first time, but decreased to 210 in T2 and 240 in T3, respectively. A stable plateau of correct choices between 70% and 80% was reached on Day 10 in T1, on Day 8 in T2 and on Day 6 in T3. We found a significant influence of the task and of the interaction between task and day on learning success. Whereas HR increased throughout T1, this relationship was inverted in T2 and T3, indicating different effects on the HR depending on how familiar goats were with the learning task. We found a significant influence of the task and the interaction between task and time within the task on HRV parameters, indicating changes of vagal activity at the heart. The results suggest that changes in HR related to learning were predominantly caused by a withdrawal of vagal activity at the heart. With regard to nonlinear processes in heartbeat regulation, increased deterministic shares of HRV indicated that the animals did not really relax until the end of T3. Comparing changes of HR and HRV in T3 and in a subsequent postexperiment (PE), we assume a positive effect of such cognitive challenges once the task had been learned by the animals., (Copyright 2004 Elsevier Inc.)

We explored whether CLA isomers and other C18 FA affect (i) lipid content and FA concentrations in total adipocyte lipids, (ii) FA synthesis from glucose in TAG and phospholipids of primary brown (BAT) and white adipocytes (WAT), and (iii) mRNA expression of uncoupling protein 1 (UCP1) in primary brown adipocytes of Djungarian hamsters (Phodopus sungorus). c9,t11-CLA, oleic, linoleic, and alpha-linolenic acid increased whereas t10,c12-CLA decreased lipid accumulation in both adipocyte types. t10,c12-CLA treatment affected FA composition mainly in BAT cells. CLA incorporation into lipids, in particular c9,t11-CLA, was higher in BAT. In both cell types, t10,c12-CLA treatment reduced the incorporation of glucose 13C carbon into FA of TAG and phospholipids, whereas c9,t11-CLA, linoleic, and alpha-linolenic acid either did not influence or dose-dependently increased glucose carbon incorporation into FA. UCP1 mRNA expression was inhibited by t10,c12-CLA but increased by c9,t11-CLA, linoleic, and alpha-linolenic acid. It is concluded that c9,t11-CLA and t10,c12-CLA have distinctly different effects on lipid metabolism in primary adipocytes. The effects of c9,t11-CLA are similar to those of other unsaturated C18 FA. The opposite effects of c9,t11-CLA and t10,c12-CLA are evident in both WAT and BAT cultures; however, brown adipocytes seem to be more susceptible to CLA treatment.

In Longissimus muscle from a F(2) population of Duroc×Berlin Miniature Pigs, micro-structural fibre traits and fatty acid composition were investigated to calculate correlation coefficients between these traits and meat quality. The animals of the F(2) population exhibited low carcass weight (55.7±11.2 kg), low meat percentage (35.0±8.4%) but a relatively high intramuscular fat content (3.52±1.44%) compared to pure bred animals (F(0)). No unacceptable meat quality was observed. The variation coefficients of carcass composition, muscle fibre traits, and fat traits were high enough to allow the analysis of candidate genes which influence the growth of muscle fibres, fat cells, and meat quality. Phenotypic correlation coefficients between muscle fibre characteristics and meat quality traits were low whereas fatty acid composition and meat quality were more closely related. The correlation coefficients between muscle fibre traits and fatty acid composition ranged from 0.10 to 0.40. The relationship between a low quotient of n-6/n-3 fatty acids in muscle and greater fibre sizes, higher percentages of the oxidative fibre type and higher capillary density was noteworthy indicating good conditions for muscle growth and meat quality.

Objective of the study was to investigate growth characteristics and susceptibility to apoptosis in different murine muscle cell lines (selected for high body weight, DU-6; randomly mated control, DU-Ks; immortalized myoblast cell line, C2C12). Apoptosis was induced by serum deprivation. At days 4, 5, and 6 of cultivation, protein, DNA and the frequency of apoptotic cells were determined. Until day 4, C2C12 accumulated more DNA and protein compared with DU-Ks and DU-6, while exhibiting a lower percentage of apoptotic cells. Serum deprivation impaired the growth of each cell line. C2C12 continued to accumulate DNA and protein after serum deprivation, whereas reductions, indicative of cell death, were apparent in DU-Ks and DU-6. Serum deprivation did not enhance apoptosis in C2C12. Higher percentages of apoptosis were observed in DU-Ks and DU-6 after 2 days of serum deprivation with greater responsiveness of DU-6 to serum deprivation. The results suggest that cell loss in response to serum deprivation is in part due to induction of apoptosis. C2C12 are less sensitive to sub-optimal culture conditions compared with DU-Ks and DU-6 which are at a closer distance to the in vivo status. Moreover, long-term selection for growth decreases the basic frequency of apoptosis of muscle satellite cells, but increases their susceptibility to apoptosis induction.

Satellite cell cultures were derived from mice selected long-term over 70 generations for body weight (DU-6, growth), carcass protein amount (DU-6P, protein) and an index combining body weight and endurance treadmill performance (DU-6+LB, growth + fitness) at 42 days of age and from an unselected control line (DU-Ks). They were grown under identical environmental conditions to examine intrinsic cellular differences in proliferation, protein metabolism and responsiveness to growth factors. Growth kinetics (DNA and protein amounts) were determined over a 12-day period. During exponential growth, all growth-selected cultures grew faster than the control culture: (DU-6+LB=DU-6P)>DU-6>DU-Ks. The differences in DNA and protein levels were maintained until day 8. DU-Ks cultures reached similar levels as the growth (DU-6) and protein (DU-6P) cultures in terms of DNA at day 12 of cultivation. Thus, the cultures from the growth and protein lines, but not from the growth + fitness line, exhibited larger protein:DNA ratios (cell size) than the control cultures. Cell cultures from the selected lines were more responsive to serum and epidermal growth factor in terms of [(3)H] thymidine incorporation into DNA, whereas no stimulation by insulin or insulin-like growth factor-I was detectable in cultures from selected lines or controls. During differentiation, protein metabolism in cultures from selected lines was characterised by higher rates of protein synthesis (PS) and degradation (PD), as measured by [(3)H] phenylalanine incorporation or release, respectively, than in control cells. The ratios of the relative differences from the control in PS and PD were only >1.0 in the growth and protein lines. In conclusion, long-term selection for growth therefore modifies the intrinsic capability of satellite cells for proliferation and protein metabolism, with changes being dependent on the selection trait.

Background: We previously reported microbial lysine contribution to plasma lysine homeostasis in humans with an adequate lysine intake., Objective: We sought to explore whether the low lysine intake from a wheat gluten-based diet is balanced by enhanced microbial lysine contribution in a pig model., Design: Twenty miniature pigs (minipigs) fitted with ileo-ileal cannulas were fed 2 wheat gluten-based diets. One diet provided 2.7 g lysine/kg diet (WG diet) and one diet was supplemented with crystalline lysine to provide 6.6 g lysine/kg diet (WG+Lys diet). Both diets were fed for 10 or 100 d (n = 5 per group): 10WG+Lys, 10WG, 100WG+Lys, and 100WG diets. Ileal microbial lysine, which we considered to be the precursor pool for absorption, was labeled by oral administration of (15)NH(4)Cl for the final 10 d. On days 10 and 100, a 10-h fast-fed tracer protocol with [1-(13)C]lysine was performed., Results: Lysine rates of appearance decreased by 25% with the WG diet in the fed state but increased by 50% with the WG+Lys diet in the fasted state (P < 0.05). Daily gross microbial lysine contribution was lower (P < 0.05) with the WG diet (205.3 micro mol. kg(-) (1). d(-)(1)) than with the WG+Lys diet (370.7 micro mol. kg(-) (1). d(-)(1)), irrespective of the adaptation period and was similar to the ileal lysine loss with the WG diet. In the WG groups, incorporation of microbial lysine increased in the duodenum and liver (P < 0.05) but not in whole-body and muscle proteins., Conclusion: Minipigs fed the WG diet did not adapt by showing an enhanced absorption of microbial lysine to the extrasplanchnic tissues, presumably because microbial lysine continues to be used for splanchnic protein synthesis.

The objective of this study was to examine the effects of somatotropin (ST) on porcine reproductive and metabolic statuses during early pregnancy. Four pregnant crossbred gilts received 6 mg of recombinant porcine somatotropin (rpST) daily from days 10 to 27 after artificial insemination while six pregnant gilts served as controls. Blood samples were taken on days 8, 10, 12, 14, 18, 22, and 27 prior to rpST injections (8:00 h) and subsequently at 9:00, 10:00, 12:00, 14:00, 16:00, 18:00, and 20:00 h. On all remaining days of treatment, samples were taken once daily before injections (8:00 h). The samples were assayed for the metabolic hormones: ST, insulin-like growth factor I (IGF-I), insulin, thyroxine (T(4)), triiodothyronine (T(3)), and cortisol; for metabolites: free fatty acids (FFA) and glucose; and for the reproductive hormones: luteinizing hormone (LH), progesterone, estradiol-17beta, estrone sulfate, and prostaglandin F(2alpha). Delivery of rpST daily induced a 20- to 40-fold increase in plasma ST concentrations. Moreover, repeated administration of rpST resulted in a continuous increase in plasma IGF-I concentration (P < 0.001), from 191.0 +/- 22.3-340.0 +/- 15.3 ng/mL 24 h after initial injection to 591.3 +/- 46.8 ng/mL after final injections. Mean serum insulin tended to be greater in rpST-treated gilts. Blood concentrations of T(4) were reduced (P < 0.05) from day 14 of gestation in treated gilts while T(3) concentrations remained unchanged. Concentrations of both glucose and FFA were greater (P < 0.01) and cortisol concentrations were unchanged in treated gilts. Changes in reproductive steroid hormones were minimally affected. Circulating progesterone (P = 0.078), and estradiol-17beta (P = 0.087) concentrations tended to be lower in treated animals. These data show that treatment of pregnant gilts with rpST during early gestation mainly impacts metabolic rather than reproductive status., (Copyright 2002 Elsevier Science Inc.)

This study on heifers (n = 27) compared the effects of a GnRH antagonist (Antarelix) and those of an agonist (Triptorelin) on gonadotropin release during the periovulatory period of the oestrous cycle. In three experiments (EXP I-III), an initial injection of GnRH analogs was given 48 h after a single PGF 2alpha pretreatment during the luteal phase, with a further five at 12 h intervals. A challenge by a GnRH agonist (Gonavet) was performed six hours after the last application of analogs. In EXP I (n = 9), heifers received six times 1.5 mg of Antarelix, 0.5 mg of Triptorelin, and mannitol (5%; control), respectively. In EXP II (n = 12), identical Antarelix and Triptorelin treatments were followed by a single injection of estradiol-17beta valerate (6 h after Gonavet). The dosage of Antarelix was increased to 5 mg for each injection in EXP III (n = 6). Measurement of LH in blood plasma frequently sampled was performed parallely by a competitive RIA method (EXP I + II) and by a sandwich-type electro-chemiluminescence-immunoassay (ECLIA) in EXP III. This non-isotopic technique was also used to additionally analyse FSH levels. Results of EXP I showed that the GnRH antagonist equally suppressed LH surges and ovulation. On the contrary, prior to suppression of LH levels due to down-regulation of pituitary GnRH receptors the agonist Triptorelin induced an initial increase in LH concentration which was followed by ovulation. In the control animals we observed endogenous LH surges as well as smaller elevations after the agonist (Gonavet) challenge. An increase was also observed in antagonist, but not in Triptorelin treated heifers. Pituitary GnRH receptors were not detectable in animals previously treated by the analogs, whereas concentrations between 2.2-21.0 fmol/mg protein were measured in controls. Results of EXP II confirmed the described effects of GnRH analogs. Additionally, it was shown that exogenous estradiol is able to release LH from the pituitary, although a previous treatment by a GnRH agonist had dropped the pulsatile gonadotropin secretion. Contrary to the LH pattern and despite elevated amounts of the antagonist, the mean concentration and pulse number of FSH were not influenced by the antagonist treatment (EXP III). These data confirmed that (a) the reversibly blocked pituitary function induced by a potent GnRH antagonist may be a useful tool to study gonadotropin-dependent final follicular growth as well as ovulation in cyclic heifers and (b) the novel non-isotopic ECLIA methods for the determination of FSH and LH provided practical alternatives to other immunoassay types.

We studied a Syrian family with 3 children who had low-density lipoprotein cholesterol (LDL) concentrations of 13.3, 12.2, and 8.6 mmol/L, respectively. Three other siblings and the parents all had LDL values <4.52 mmol/L, suggesting an autosomal-recessive mode of inheritance. The extended pedigree had 66 additional persons with normal LDL values. A genome-wide scan in the core family with 427 markers showed support for linkage on both chromosomes 1 and 13. Markers on chromosome 1 revealed a 3.07 multipoint LOD score between 1p36.1-p35, an 18-cM interval. Surprisingly, we also found linkage to 13q22-q32, a 14-cM interval, with a 3.08 LOD score. We had identified this locus earlier as containing a gene strongly influencing LDL in another Arab family with autosomal-dominant familial hypercholesterolemia and in normal dizygotic twins. We found evidence for an interaction between these loci. We next genotyped our twin panel and confirmed linkage of the 1p36.1-p35 locus to LDL (P<0.002) in this normal population. Elucidation of ARH, the LDL receptor adaptor protein at chromosome 1p35, caused us to sequence that gene. We first identified the genomic structure of ARH gene and then sequenced the gene in our family. We found an intron 1 acceptor splice-site mutation. This mutation was not found in any other family members, in 31 nonrelated Syrian persons, or in 30 Germans. Our results underscore the importance of ARH on chromosome 1 and the chromosome 13q locus to LDL, not only in families with unusual illnesses, but also to the general population.

The aim of this study was to evaluate the usefulness of heart rate variability (HRV) and its specific parameters as a new approach to assess stress load in cattle. We recorded HRV in 52 calves in three groups and in 31 cows in two groups. In calves we divided Group 1 with no obvious stress load (n=18), Group 2 with external stress load (n=17), and Group 3 with internal stress load from sickness (n=17). In cattle we divided lactating cows (n=21) and nonlactating cows (n=10). HRV parameters were analyzed in the time domain and in the frequency domain. Moreover, we applied Recurrence Quantification Analysis (RQA) to quantify nonlinear components of HRV. In calves, linear HRV parameter decreased from Group 1 to Group 3 (P<.05). However, not a single parameter showed significant differences regarding all three groups. The value of all nonlinear measurements increased at the same time (P<.05). The only parameter that exhibited significant differences between all three groups was the longest diagonal line segment in the recurrence plot (L(MAX)) which is inversely related to the Lyapunov exponent. We did not find differences concerning the linear HRV parameters between the two groups in the cows. The nonlinear parameter Determinism showed significant higher values in lactating cows compared to nonlactating cows. The importance of particular HRV-parameters was tested by applying a discriminant analysis approach. In calves and cattle nonlinear parameters were most important to indicate the level of stress load on the animals. Based on the results we assume HRV to be a valuable physiological indicator for stress load in animals. Whereas linear parameters of HRV are supposed to be useful to separate qualitative different level of stress, nonlinear components of HRV distinguish quantitative different challenges for the animals.

In the following investigations, the LH secretion of cells from pituitaries in heifers on days 16-18 of their oestrous cycle (n = 14) was analysed. Cells were dissociated with trypsin and collagenase and maintained in a static culture system. For the estimation of LH release, the cells were incubated with various concentrations of mammalian GnRH (Lutrelef) for 6 h. To determine the action of Antarelix (GnRH antagonist), the cells were preincubated for 1 h with concentrations of 10(-5) or 10(-4) M Antarelix followed by 10(-6) M GnRH coincubation for a further 6 h. At the end of each incubation, the medium was collected for LH analysis. Parallel, intracellular LH was qualitatively detected by immunocytochemistry. Changes in the intensity of LH staining within the cells in dependence of different GnRH concentrations were not observed, but a significant increase LH secretion in pituitary cells was measured at 10(-6) M GnRH. Antarelix had no effect on basal LH secretion at concentrations of 10(-4) and 10(-5) M. After coincubation of pituitary cells with Antarelix and GnRH, Antarelix blocked the GnRH-stimulated LH secretion with a maximal effect of 10(-4) M, but the staining of immunoreactive intracellular LH was detected at approximately the same level compared to the pituitary cells treated with exogenous GnRH alone. These data demonstrate that Antarelix is effective in influencing the GnRH-stimulated LH secretion of pituitary cells in vitro. After administration of Antarelix in vivo, the GnRH-stimulated LH secretion of cultured pituitary cells was not inhibited.

The epidermal growth factor receptor (EGF receptor) system is involved in regulation of proliferation and differentiation in oviductal and endometrial tissues. In this study the influence of ovarian steroids and EGF on the expression and activity of specific markers of transcription (mitogen-activated protein kinase; MAP42k) and translation (a potential repressor of eukaryotic initiation factor 4E; 4E-BP1) in pig oviducts was investigated. Furthermore, determination of the distribution of translationally active (polysomal) and repressed (free) mRNA, and cell cycle analysis were performed. Oviductal tissue collected at two points of the oestrous cycle (days 12 and 20) from gilts and tissues from ovariectomized gilts with or without steroid replacement treatment were analysed. The influence of EGF was detected by culture of oviductal explants. MAP42k activity was stimulated by oestrogen treatment, whereas progesterone treatment appeared to decrease its activity. High oestrogen but not high progesterone concentrations resulted in reduced mobility of 4E-BP1 on polyacrylamide gels, indicating its inactivation. EGF and oestrogen treatment of oviductal explants further reduced the mobility of 4E-BP1 on polyacrylamide gels. High concentrations of oestrogen in the plasma promoted cell cycle activity. Progesterone treatment alone did not stimulate the rate of DNA synthesis. There were no significant differences in the distribution of free oviductal poly (A+) mRNA, but the amount of polysomal mRNA was downregulated by oestrogen and progesterone. Increased oestrogen concentrations are involved in the regulation of MAP42k and 4E-BP1 activation in the oviductal tissue of pigs. The effect of oestrogen and EGF in reducing the mobility of 4E-BP1 on gels in oviductal explants indicates that EGF may mediate the effect of oestradiol in the oviducts.

The gene encoding the neuronal nicotinic acetylcholine receptor alpha7 subunit (CHRNA7) is located on chromosome 15q13.2. This region was suggested to be involved in the etiopathogenesis of: (a) schizophrenia combined with a neurophysiological deficit; (b) lithium-responsive bipolar disorder; and (c) familial catatonic schizophrenia (periodic catatonia). Therefore, members of a large family with periodic catatonia strongly supporting the chromosome 15q13-22 region were genotyped with polymorphic markers localized around the CHRNA7 locus. A recombination event distally of marker D15S144 leading to the exclusion of the CHRNA7 locus from this candidate region was detected in one branch of the pedigree. This result provides strong evidence that a gene located telomeric to CHRNA7 is causative for the pathogenesis of catatonic schizophrenia in this family.

The effects of maternal treatment with somatotropin during early gestation on fetal muscle development were determined. Crossbred gilts received daily injections of either 3 ml of a placebo ( n=31) or of 6 mg porcine somatotropin ( n=31) from day (d) 10 to 27 of gestation and samples were collected from d 28 embryos, d 37 and 62 fetuses, and from neonates. Administration of somatotropin increased the total number of fibres (primary and secondary fibres) in neonatal semitendinosus muscle of middle- and low-weight littermates, whilst no increase was observed in psoas major muscle. Somatotropin induced increases in muscular protein concentration, creatine kinase activity, muscle fibre girth, as well as type II to type I fibre conversion which revealed an advanced degree of differentiation at birth. Treatment effects on prenatal development preceded these changes. Increased DNA concentrations at d 28 of gestation indicate stimulation of cellular proliferation during the embryonic stages. Thereafter, the withdrawal of somatotropin caused a transient delay of differentiation as indicated by lower protein concentrations and creatine kinase activity compared with controls at d 37 of gestation. This was compensated again at d 62, and the number of semitendinosus primary fibres was increased in middle-weight fetuses, whereas secondary or total fibre number did not yet differ. However, enhanced expression of Myf5 and MyoD indicates higher numbers of initially determined, proliferating myoblasts that may have contributed to increased formation of secondary fibres. In conclusion, maternal somatotropin is an influential factor in early pregnancy capable of affecting the basic events of myogenesis.

Little information is available on methods of sperm capacitation for IVF in the horse. In this study, we summarized results of several independent trials that compared acrosome reaction, hyperactivation and chromatin integrity of fresh or cryopreserved stallion spermatozoa after treatment with heparin or with calcium ionophore. We also examined the influence of spermatozoa storage (fresh vs. cryopreserved), capacitation treatment, oocyte maturation time and cumulus morphology on the penetration rate and fertilization rate. We recovered cumulus-oocyte-complexes (COCs) from ovaries by ultrasound guided follicle aspiration or by scraping of follicles from ovaries obtained at a slaughterhouse. Upon recovery, we evaluated the cumulus morphology, and the COCs were matured in vitro for 18 to 24 or 26 to 40 h. Fresh semen and cryopreserved semen were treated either with heparin (200 microg/mL) or calcium ionophore (7.14 microM). Overall, 28.4% (99/349) of the oocytes were penetrated, and 12.9% (45/349) were fertilized. Fresh spermatozoa treated with calcium ionophore showed a higher penetration rate than cryopreserved spermatozoa (36.0 vs. 0%). Fresh and heparin-treated spermatozoa showed a penetration rate of 29.1%, and the same treatment for cryopreserved spermatozoa showed a penetration rate of 33.7%; none of these differences was significant (P>0.05). Fertilization rates after the calcium and heparin treatment followed the same trend and also showed no significant differences. Prolonged maturation period resulted in higher penetration (P<0.05) and fertilization rates in compact (26 to 40 h: 37.7 and 13.1% vs. 18 to 24 h: 13.1 and 2.8%) and in tendency in expanded COCs (26 to 40 h: 40.0 and 30.3% vs. 18 to 24 h: 29.4 and 13.5%). In oocytes with only a few cumulus cells, the rates tended to be higher after the shorter incubation (18 to 24 h: 33.5 and 18.8% vs. 26 to 40 h: 17.2 and 6.5%). We observed hyperactivation more frequently in fresh than in cryopreserved semen after different treatments (43.2, 39.1 and 35.4% for heparin, calcium ionophore and control vs. 15.7, 10.8 and 5.7%, respectively). We observed significant changes in the acrosome reaction of fresh spermatozoa after heparin treatment (62.6 vs. 48.2%, P<0.05), as well as in cryopreserved spermatozoa after calcium ionophore treatment (31.7 vs. 17.6%, P<0.05). The chromatin integrity was significantly reduced after heparin treatment of fresh spermatozoa, in comparison to control and calcium ionophore (81.0 vs. 87.3 and 86.6, P<0.02). We also observed a similar reduction of chromatin quality after heparin treatment in cryopreserved spermatozoa, but the difference was significant only between heparin and calcium ionophore treatment [77.4 vs. 86.4 (P<0.02) and 84.9]. The results in the this retrospective study show that capacitating fresh spermatozoa with calcium ionophore, or using heparin in cryopreserved spermatozoa, results in higher penetration and fertilization rates of in vitro matured horse oocytes. A prolonged maturation time of 26 to 40 h is necessary for compact cumulus oocyte complexes to achieve the fertilization capacity. Further investigation is needed to show the developmental capacity of these fertilized oocytes.

The objective of this study was to determine the effects of maternal treatment with porcine somatotropin (pST) during early gestation on embryonic survival, fetal development, and internal environment for fetal growth. Sixty-two crossbred gilts received daily injections of either 3 mL of a placebo (control, n = 31) or 6 mg of pST (n = 31) from d 10 to 27 of gestation. Representative gilts were slaughtered on d 28, 37, and 62 of gestation. The remaining gilts were allowed to farrow. It was found that embryonic survival was not influenced by pST treatment (P > 0.10). However, pST affected the growth and composition of the maternal (endometrium) and fetal (chorion) parts of the placenta. Thus, endometrial RNA concentration tended to be increased by pST at d 37 (P = 0.15), and it was increased at d 62 (P < 0.05) of gestation, which is indicative of increased capacity for protein synthesis. At birth, placental chorion weight (P < 0.10) and contents of DM and protein (P < 0.05) were increased due to pST treatment, but no effects were detectable up to d 62 of gestation. Maternal pST treatment was effective at increasing nutrient supply to the embryo as suggested from elevated glucose concentrations in amniotic and allantoic fluids (P < 0.05) at d 28 of gestation. With regard to prenatal growth, embryonic DNA concentration was slightly elevated at d 28 (P < 0.10), but pST did not induce any changes in average embryonic, fetal, or neonatal weights. However, within litters, the birth weights of piglets in the 25% lowest weight group (LW) were increased by pST treatment vs control LW pigs (1,241+/-55 vs 1,099+/-59 g, P < 0.10). Thirty-eight neonates from 15 litters divided among the three weight groups were examined for body composition. The weight of the intestinal tract was increased above average after maternal pST treatment (P < 0.01). Additionally, the amounts of tissues such as bone (P = 0.12) and s.c. fat (P = 0.06), and of protein, fat (P = 0.10), and ash (P < 0.05) were increased, whereas the relative body composition remained unchanged by pST (P > 0.10). On average, muscle protein concentration was elevated due to pST (P < 0.01), and, in LW piglets, plasma IGF-I concentration was increased (P < 0.10). The results suggest that maternal somatotropin is a critical factor in early pregnancy capable of influencing placental nutrient transfer and placental growth. It thereby selectively improves the growth conditions for the smaller littermates.

The present study was undertaken to determine the tissue-specific expression of estrogen receptor beta (ERbeta), and the effects of a daidzein supplement to the diet of pregnant sows on the expression of ERbeta, and type 1 insulin-like growth factor receptor (IGF-1R) genes in newborn piglets by using semi-quantitative RT-PCR. Eight sows received a dietary supplement of daidzein at a dosage of 8 mg per kg feed from day 85 of gestation, and six sows were used as controls. After parturition, 2 male neonatal piglets were selected from each litter for sampling. ERbeta mRNA was detected in intestine, lung, thymus, kidney, pituitary and hypothalamus tissues, but not in heart, adrenal, skeletal muscle, liver or placental tissues. Daidzein treatment significantly increased the birth weight of male piglets and markedly reduced the level of ERbeta mRNA in the hypothalamus, but not in the pituitary. An up-regulation of IGF-1R gene transcription was observed in skeletal muscles of newborn piglets. In addition, the IGF-1R mRNA was found to be most abundant in pituitary and hypothalamus, followed by skeletal muscle, thymus, and liver tissues in decreasing order. Our results demonstrate that (1) ERbeta is expressed in a tissue-specific manner in newborn piglets, (2) daidzein down-regulates ERbeta gene expression in the hypothalamus, possibly indicating central effects of daidzein, and (3) daidzein influences fetal growth associated with higher IGF-IR gene expression in skeletal muscle.

Background: We have previously described an autosomal recessive syndrome of macrocephaly, multiple epiphyseal dysplasia (MED), and distinctive facies in a large, extended Omani family. The MED observed seems to be part of a larger malformation syndrome, since both craniofacial and central nervous system changes were present in the family. We performed a whole genome scan in this family in order to identify the gene locus for this malformation syndrome., Methods and Results: Using homozygosity mapping, we show linkage to the telomeric region of the long arm of chromosome 15. The position of both the disease gene and the principal glycoprotein, chondroitin sulphate proteoglycan (aggrecan, AGC1) on chromosome 15q26, suggested that the aggrecan gene is a candidate for the disease in this family. However, three of the four affected children were heterozygous for a polymorphism at position 831 of the coding sequence of AGC1, providing strong evidence against its involvement., Conclusion: We have identified a gene locus for a recessive syndrome of macrocephaly, MED, and distinctive facies in a large Omani family. Aggrecan located on chromosome 15q26, within the critical region determined for this syndrome in this family, was excluded as a candidate gene.

This study examined the impact of the gonadotrophin-releasing hormone (GnRH) antagonist Antarelix on LH, FSH, ovarian steroid hormone secretion, follicular development and pituitary response to LHRH in cycling gilts. Oestrous cycle of 24 Landrace gilts was synchronised with Regumate (for 15 days) followed by 800 IU PMSG 24h later. In experiment 1, Antarelix (n=6 gilts) was injected i.v. (0.5mg per injection) twice daily on four consecutive days from day 3 to 6 (day 0=last day of Regumate feeding). Control gilts (n=6) received saline. Blood was sampled daily, and every 20 min for 6h on days 2, 4, 6, 8 and 10. In experiment 2, gilts (n=12) were assigned to the following treatments: Antarelix; Antarelix + 50 microg LHRH on day 4; Antarelix + 150 microg LHRH on day 4 or control, 50 microg LHRH only on day 4. Blood samples were collected daily and every 20 min for 6h on days 2, 4 and 6 to assess LH pulsatility. Ovarian follicular development was evaluated at slaughter. Antarelix suppressed (P<0.05) serum LH concentrations. The amount of LH released on days 4-9 (experiment 1) was 8.80 versus 36.54 ngml(-1) (S.E.M.=6.54). The pattern of FSH, and the preovulatory oestradiol rise was not affected by GnRH antagonist. Suppression of LH resulted in a failure (P<0.05) of postovulatory progesterone secretion. Exogenous LHRH (experiment 2) induced a preovulatory-like LH peak, however in Antarelix treated gilts the LH surge started earlier and its duration was less compared to controls (P<0.01). Furthermore, the amount of LH released from day 4 to 5 was lower (P<0.01) in Antarelix, Antarelix + 50 and Antarelix + 150 treated animals compared to controls. No differences were estimated in the number of LH pulses between days and treatment. Pulsatile FSH was not affected by treatment. Mean basal LH levels were lower (P<0.05) after antagonist treatment compared to controls. Antarelix blocked the preovulatory LH surge and ovulation, but the effects of Antarelix were reduced by exogenous LHRH treatment. The development of follicles larger than 4mm was suppressed (P<0.05) by antagonist treatment. In conclusion, Antarelix treatment during the follicular phase blocked preovulatory LH surge, while FSH and oestradiol secretion were not affected. Antarelix failed to alter pulsatile LH and FSH secretor or pituitary responsiveness to LHRH during the preovulatory period.

We performed a whole genome scan in a family with maternally transmitted paraganglioma (PGL3). The family included five patients with histologically proven paraganglioma and one patient with imaging findings consistent with a paraganglioma. In addition, there were 33 clinically unaffected family members. Of these eight could be examined by magnetic resonance imaging. Our investigations indicate that PGL3 is located in 1q21-q23 for several reasons: 1) two-point linkage analysis yielded the highest LOD score of 2.25 at 1q21-q23 (marker D1S2675); 2) haplotype analysis was most consistent for 1q21-q23 markers; and 3) the locus was excluded from more than 97% of the genome using a total of 381 highly polymorphic markers.

In a genome-wide linkage study, we mapped two major susceptibility loci for periodic catatonia, a phenotype with qualitative disturbances of the psychomotor sphere and a morbidity risk of 26.9% in first-degree relatives of index cases, to chromosome 15q15, and to chromosome 22q13 using nonparametric as well as parametric (autosomal dominant model) analyses. The study included 12 multiplex pedigrees with 135 individuals, among them 57 affected persons. A second genome scan is in progress investigating four families with 21 affected individuals, aiming to confirm linkage results. Age at onset patterns as well as the clinical outcome were similar among affected individuals in both sets of families. Within the pedigrees we observed no physical diseases segregating with periodic catatonia. Under the assumption of genetic homogeneity, the statistical power to detect LOD scores > or = 2.0 was 98.5% in the first set of families, and 57.9% in the second set. Thus, the panel of multiplex pedigrees segregating periodic catatonia seems to represent a homogenous clinical sample, and possesses sufficient statistical power to delineate and confirm linkage to major genetic loci for periodic catatonia.

The nature of subtypes in schizophrenia and the meaning of heterogeneity in schizophrenia have been considered a principal controversy in psychiatric research. We addressed these issues in periodic catatonia, a clinical entity derived from Leonhard's classification of schizophrenias, in a genomewide linkage scan. Periodic catatonia is characterized by qualitative psychomotor disturbances during acute psychotic outbursts and by long-term outcome. On the basis of our previous findings of a lifetime morbidity risk of 26.9% of periodic catatonia in first-degree relatives, we conducted a genome scan in 12 multiplex pedigrees with 135 individuals, using 356 markers with an average spacing of 11 cM. In nonparametric multipoint linkage analyses (by GENEHUNTER-PLUS), significant evidence for linkage was obtained on chromosome 15q15 (P = 2.6 x 10(-5); nonparametric LOD score [LOD*] 3.57). A further locus on chromosome 22q13 with suggestive evidence for linkage (P = 1.8 x 10(-3); LOD* 1.85) was detected, which indicated genetic heterogeneity. Parametric linkage analysis under an autosomal dominant model (affecteds-only analysis) provided independent confirmation of nonparametric linkage results, with maximum LOD scores 2.75 (recombination fraction [theta].04; two-point analysis) and 2.89 (theta =.029; four-point analysis), at the chromosome 15q candidate region. Splitting the complex group of schizophrenias on the basis of clinical observation and genetic analysis, we identified periodic catatonia as a valid nosological entity. Our findings provide evidence that periodic catatonia is associated with a major disease locus, which maps to chromosome 15q15.

Psoriasis is a common chronic inflammatory skin disease with a strong genetic component. Few psoriasis-susceptibility loci have been reported, and only two have been confirmed in independent data sets. This article reports results of a genomewide scan that was performed, using 370 microsatellite markers, for psoriasis-susceptibility loci in 32 German extended families, comprising 162 affected and 195 unaffected individuals. Nonparametric linkage analysis of all families provided strong evidence for a novel psoriasis-susceptibility locus on chromosome 19p (Zlr=3.50; P=.0002). Parametric analysis revealed a heterogeneity LOD score of 4.06, corresponding to a genomewide significance level of.037, under the assumption of a recessive model with high disease-allele frequency and 66% as the proportion of linked families. This study confirms linkage of psoriasis to the HLA region on chromosome 6p and suggests additional regions on chromosomes 8q and 21q for further investigations.

Cumulus-oocyte complexes (COCs) recovered from ovaries of mares killed at abattoirs or after in vivo collection have heterogeneous morphologies and meiotic competence as follicles of variable quality are used. It is thought that it should be possible to recover more uniform COCs, with respect to morphology and nuclear maturation, by repeated follicle aspiration. Therefore, the influence of repeated follicle aspiration on the number and diameter of follicles > or =5 mm in diameter, the morphology and recovery rate of COCs, and the chromatin configuration in oocytes was investigated. Repeated ultrasound-guided aspirations were performed on Warmblood mares (n=6) after either a normal cycle ('cyclic' sessions) or at 4-12 day intervals ('consecutive' sessions). In 88 follicle aspiration sessions, 1268 follicles were aspirated and 280 COCs were recovered: the mean number of follicles aspirated and the number of COCs obtained per session per mare were 14.4 and 3.2, respectively. The mean recovery rate was 22.1%; there was no significant difference in the recovery rate between cyclic and consecutive aspirations. However, the mean number of follicles aspirated was significantly different between cyclic and consecutive aspirations (15.3 versus 10.8, respectively) and, hence, fewer COCs were obtained in consecutive aspirations compared with cyclic aspirations (2.2 versus 3.5, respectively). The proportion of compact COCs was higher for consecutive than for cyclic aspirations (51.9 versus 28.8%, respectively; P < or = 0.02). Within consecutive sessions, the proportion of compact COCs decreased with increasing interval between aspirations. Moreover, the proportion of oocytes with a diffuse germinal vesicle chromatin configuration was higher in COCs collected in consecutive aspirations than in COCs collected in cyclic aspirations. Repeated follicle aspiration can be used to induce a more uniform follicular population and to provide more uniform COCs. The optimum interval between aspirations to provide the greatest number of meiotically competent oocytes must be determined.

Meat quality of pigs is dependent on biochemical and biophysical processes in the time course post mortem (p.m.) and is associated with the intracellular Ca2+ homeostasis. However, there is little known about changes in the Ca2+ transporting proteins controlling the Ca2+ uptake of sarcoplasmic reticulum (SR) in the time course p.m. In this study changes in the Ca2+ transporting proteins were investigated in homogenates of longissimus muscles of 4 malignant hyperthermia susceptible (MHS) and 6 malignant hyperthermia resistant (MHR) Pietrain pigs. Muscle samples were obtained at different time intervals: biopsy 2 h prior slaughtering and from the carcass immediately after exsanguination (0 h), 45 min, 4 h, and 22 h p.m. The SR Ca2+ uptake rate was measured immediately after homogenization with closed calcium release channel (CRC), with opened CRC and without manipulation of CRC. Additionally the SR Ca2+ ATPase activity was determined. The results show: (i) The ability of SR to sequester Ca2+ declined to about 60% in the first 45 min p.m. in MHS samples irrespective of CRC state, whereas in MHR samples this decline was about 5%; (ii) Ca2+ uptake and Ca2+ ATPase activity were not different between the biopsy and 0 h samples, i.e. the stress of slaughter was of no immediate influence; (iii) The Ca2+ ATPase activity of the SR declined at about the same rate as the Ca2+ uptake in both MHS and MHR pig samples in the course of time p.m.; (iv) In samples, taken immediately after exsanguination, the Ca2+ ATPase activity of MHS pigs was higher than that of MHR pigs. However, in samples taken 4 h p.m. Ca2+ ATPase activity of MHS pigs has declined to about 30% of the value at 0 h; (v) The CRC can be closed and opened in all samples up to 22 h p.m. and seems to be fully functional at all sampling times; (vi) The CRC of MHS pigs is almost fully open, whereas the CRC of MHR pigs is only partially open at all sampling times; (vii) The permeability of the SR membrane to Ca2+ (determined as the ratio of SR Ca2+ ATPase with and without ionophore A23187) is the same in both MHS and MHR and did not change with ongoing time; (viii) No uncoupling of uptake from ATP hydrolysis occurred up to 4 h p.m., but the coupling differed between MHS and MHR for all time intervals with lower values for MHS pigs. The results suggest that the decreasing Ca2+ uptake rate of homogenates, sampled at different times p.m., is essentially caused by changes in the Ca2+ pump and not by changes in the CRC or an increased phospholipid membrane permeability to Ca2+.

The effect of dietary n-3 fatty acids on the fatty acid composition and lipid peroxidation of different tissues in pigs were studied. 20 castrated male pigs were included in this investigation, one half was fed daily a diet containing 1.3 g n-3 fatty acids/kg diet (control) and 10 pigs were fed a diet containing 14 g n-3 fatty acids/kg diet (n-3 diet) at the growing-finishing period. The intake of dietary n-3 fatty acids increased the concentration of these fatty acids in backfat, and the neutral and polar fractions of skeletal muscle and heart homogenates. The polar fraction showed an increased relative concentration of n-3 fatty acids in comparison to control, while the n-6 fatty acid content was reduced. In heart homogenates there was an enlargement of n-3 fatty acids both in polar lipids and in neutral lipids whilst n-6 fatty acids were decreased. Feeding n-3 fatty acid enriched diet had no influence on meat quality parameters drip loss, meat colour or pH value. The lipid peroxidation (measured as malondialdehyde equivalents) was in the order liver > heart > skeletal muscle with higher values in the n-3 group. However, by stimulation of oxidation by Fe2+/ascorbate for 3 hours the order of oxidative products in the n-3 group was muscle > liver > heart, whereas in the control group the order was liver > heart = muscle. Summarized, feeding a highly n-3 fatty acid enriched diet caused an incorporation of these fatty acids and increased the susceptibility to peroxidation in all investigated tissues.

Nijmegen breakage syndrome (NBS; Seemanová II syndrome) and Berlin breakage syndrome (BBS), also known as ataxia-telangiectasia variants, are two clinically indistinguishable autosomal recessive familial cancer syndromes that share with ataxia-telangiectasia similar cellular, immunological, and chromosomal but not clinical findings. Classification in NBS and BBS was based on complementation of their hypersensitivity to ionizing radiation in cell-fusion experiments. Recent investigations have questioned the former classification into two different disease entities, suggesting that NBS/BBS is caused by mutations in a single radiosensitivity gene. We now have performed a whole-genome screen in 14 NBS/BBS families and have localized the gene for NBS/BBS to a 1-cM interval on chromosome 8q21, between markers D8S271 and D8S270, with a peak LOD score of 6.86 at D8S1811. This marker also shows strong allelic association to both Slavic NBS and German BBS patients, suggesting the existence of one major mutation of Slavic origin. Since the same allele is seen in both former complementation groups, genetic homogeneity of NBS/BBS can be considered as proved.

Endoscopical ovum pick up (OPU) in swine is a minimal invasive and reliable technique to aspirate oocytes from preovulatory follicles for studying intrafollicular development and oocyte maturation as well as for IVM/IVF-programs. The aim of the present study was to determine the effect of different aspiration vacuum pressures on oocyte recovery and on the morphology of cumulus oocyte complexes (COCs). Oestrus of crossbred Landrace gilts (n = 33) was synchronized by feeding altrenogest and follicular growth was stimulated with 1,000 IU PMSG 24 h after the last altrenogest application. On day 4 after PMSG application preovulatory healthy follicles of > 5 mm diameter were aspirated laparoscopically. Aspiration was carried out using a two-way cannula and an electronic aspiration pump. Five different vacuum pressures were used: 10, 17, 32, 47 and 66 ml water/min, corresponding to 30, 60, 125, 250 and 375 mm Hg, respectively. Fluids from different follicles were pooled per ovary and the morphology of cumulus oocyte complexes (COCs) was determined microscopically immediately after aspiration. COCs were classified into oocytes with intact cumulus (i.e. compact or slightly expanded COC), oocytes with corona radiata and denuded oocytes. A total of 695 follicles were aspirated and 501 COCs recovered. Increasing the aspiration pressure stepwise from 10 ml water/min up to 66 ml water/min resulted in a decrease in oocyte recovery rate. A significant higher (P < 0.05) rate of oocyte recovery (77.4% v.s. 59.8%) was achieved using a vacuum pressure of 17 ml water/min compared to 66 ml water/min, respectively. There was a tendency to reduce the portion of COCs with intact cumulus from 82 to 88% to 77% if the vacuum pressure increased to more than 47 ml water/min. A higher aspiration pressure provoked an increase (P < 0.05) in the number of denuded oocytes: 0 to 3% at 10 to 32 ml water/min, respectively, compared to 10% at 47 ml water/min to 17% at 66 ml water/min. These results demonstrate that variation in aspiration pressure affects oocyte recovery rate and COC quality. Aspiration vacuum pressure of 17 to 32 ml water/min was found to be optimal in swine both for oocyte recovery and COC quality.

Endoscopically and ultrasonographically guided ovum pick up were studied in 15 heifers once per week. Althogether 60 sessions were carried out. The number of aspirated follicles per animal (13.0 vs 10.3) did not differ significantly between both methods. Similar recovery rates (39 % vs 44 %) were achieved after repeated sessions. As a consequence the number of recovered oocytes per animal (5.2 vs 4.7) were not significantly different. Inter-animal- and intra-animal-variations were more important for the results than the applied methods. In contrast to these findings the quality of recovered cumulus-oocyte-complexes (COC) was significantly influenced by the methods. The COC were divided into 4 categories a) oocytes with compact cumulus, b) oocytes with rest of compacted cumulus, c) oocytes with expanded cumulus and d) oocytes without cumulus. There was a higher denudation rate of COC when using the endoscopic aspiration (62.0 % vs 6.6 %) because of turbulent current in this aspiration system. Advantages and disadvantages of both methods are described and discussed. Generally, the ultra sonographic method is the less traumatic procedure for the vagina, fornix and for abdominal organs. The other method is less traumatic for the ovaries.