A fine structure genetic analysis evaluating ecoregional adaptability of a Bos taurus breed (Hereford).

Ecoregional differences contribute to genetic environmental interactions and impact animal performance. These differences may become more important under climate change scenarios. Utilizing genetic diversity within a species to address such problems has not been fully explored. In this study Hereford cattle were genotyped with 50K Bead Chip or 770K Bovine Bead Chip to test the existence of genetic structure in five U.S. ecoregions characterized by precipitation, temperature and humidity and designated: cool arid (CA), cool humid (CH), transition zone (TZ), warm arid (WA), and warm humid (WH). SNP data were analyzed in three sequential analyses. Broad genetic structure was evaluated with STRUCTURE, and ADMIXTURE software using 14,312 SNPs after passing quality control variables. The second analysis was performed using principal coordinate analysis with 66 Tag SNPs associated in the literature with various aspects of environmental stressors (e.g., heat tolerance) or production (e.g., milk production). In the third analysis TreeSelect was used with the 66 SNPs to evaluate if ecoregional allelic frequencies deviated from a central frequency and by so doing are indicative of directional selection. The three analyses suggested subpopulation structures associated with ecoregions from where animals were derived. ADMIXTURE and PCA results illustrated the importance of temperature and humidity and confirm subpopulation assignments. Comparisons of allele frequencies with TreeSelect showed ecoregion differences, in particular the divergence between arid and humid regions. Patterns of genetic variability obtained by medium and high density SNP chips can be used to acclimatize a temperately derived breed to various ecoregions. As climate change becomes an important factor in cattle production, this study should be used as a proof of concept to review future breeding and conservation schemes aimed at adaptation to climatic events.

Female sex selection using clomiphene citrate and albumin separation of human sperm.

BACKGROUND: This study was undertaken to assess whether the use of clomiphene citrate in conjunction with albumin-separated sperm would alter the sex ratio (expressed as the proportion of males) towards females and, if so, whether this skewing was due solely to the induction of ovulation. METHODS: The sex ratios of 184 single and 42 twin births at five assisted reproduction biology clinics were determined. The normal approximation to the binomial distribution was used to determine significant differences between these sex ratios and the established sex ratios for single, twin and combined (single and twin) non- and ovulation-induced births. RESULTS: The non-ovulation-induced sex ratios for singletons (51.4%) and twins (50.2%) were greater than the treatment singleton (27.7%; P < 0.001) and twin (33.3%; P < 0.01) sex ratios respectively. Correspondingly, the non-induced sex ratio for combined births (51.4%) was greater than the treatment sex ratio (28.8%; P < 0.001). The previously established induced singleton and twin sex ratios (48.1%) were lower than the non-induced sex ratio (51.4%), but higher than the treatment singleton (27.7%; P < 0.001) or twin (33.3%; P < 0.03) sex ratios. The ovulation-induced combined ratio (48.1%) was less than the non-induced combined (51.4%) sex ratio, although greater than the treatment combined sex ratio (28.8%; P < 0.001). CONCLUSION: Clomiphene citrate in conjunction with albumin-separated sperm decreased the sex ratio; a reduction that was not exclusively due to induction of ovulation.

The use of buck and ram extenders and two packaging systems to cryopreserve aoudad (Ammotragus lervia) spermatozoa.

Because the aoudad has been hunted to near extinction, cryopreservation of their semen would be useful for DNA conservation and for the possible re-establishment of captive bred animals to their former ranges. This study was conducted to investigate the effectiveness of cryopreserving aoudad spermatozoa. Semen samples from four post-pubertal animals were collected using electro-ejaculation. Microscopic analysis was performed to assess the percentages of progressively and non-progressively motile spermatozoa as well as intact acrosomes in samples prior to freezing and post-thaw. Extended samples (0.2 mL) were frozen using 2 different extenders and packaging systems and stored in LN2 Post-thaw data were arcsine-transformed and analyzed using ANOVA, 2 x 2 factorial. Samples that were processed using the ram/straw method had a significantly higher percentage (P < 0.05) of spermatozoa with intact acrosomes than did any other system. In addition, samples that were processed with the buck/pellet system had significantly greater percentages (P < 0.05) of progressive and non-progressively motile spermatozoa than the samples processed using either extender and packaged in straws. This study illustrates that some aoudad spermatozoa may be cryopreserved using the extender/processing systems developed for the domestic buck and ram.

Comparison of tests for assessment of Helicobacter pylori eradication: results of a multi-centre study using centralized facility testing.

BACKGROUND: The accuracy of diagnostic tests to assess Helicobacter pylori eradication has rarely been performed. AIM: To compare the tests most commonly used for this purpose, i.e. histology, culture and (13)C urea breath test performed in centralized facilities. METHODS: Prospective study where patients were included in four centres and diagnostic tests performed centrally by biologists experienced in the field. Gastric biopsies were obtained from antrum and corpus (two for histology, two for culture from each site) 4-6 weeks after an eradication treatment. The definition of a gold standard for H. pylori-positive patients was either a positive culture or both positive histology and urea breath test results. RESULTS: Ninety-seven patients for whom data on histology, culture and (13)C urea breath test were available were included. The majority were females (60%) suffering from non-ulcer dyspepsia (52%) and having received proton-pump inhibitor-based triple therapy (62%). Forty-one per cent of the patients were H. pylori-positive according to the gold standard. The sensitivities were 90%, 95% and 92.5% and the specificities 100%, 98.2% and 100% for culture, histology and (13)C urea breath test, respectively. CONCLUSION: All the methods had excellent specificity but the sensitivity ranged between 90 and 95%. The combination of two techniques which increases the sensitivity to virtually 100% is recommended in situations where the eradication treatment requires a precise evaluation such as in clinical trials.

Evidence of circadian rhythm in low-density lipoprotein apoB catabolism and its impact on the estimation of kinetic parameters.

BACKGROUND: Compartmental models with constant parameters are commonly used in kinetic analysis of low-density lipoproteins (LDLs). Recent studies in animals have demonstrated the existence of circadian rhythms (CRs) in cholesterol synthesis and LDL catabolism. In this study, we investigated the possible existence of a CR in the fractional catabolic rate (FCR) of LDL apoB in man. MATERIALS AND METHODS: Radioactivity data from 45 turnover studies using 125I-labelled LDL apoB were analysed. In a preliminary analysis the pattern of radioactivity decay was investigated. Kinetic analysis was performed by using one- and two-compartment models with constant parameters (steady-state, SS, analysis). Parameters were estimated by the use of the whole data set, which included frequent sampling during the first day of the turnover study, or the once-a-day data, taken at 08.00 h. The selection of once-a-day data allowed elimination of the impact of a CR on parameter evaluation. Furthermore, non-steady-state (NSS) analysis was performed in which the FCR of LDL apoB was calculated as a function of time. In one additional subject, the FCR of LDL apoB was calculated separately for the day and the night using the urine-to-plasma (U/P) radioactivity ratio. RESULTS: The presence of a CR in LDL apoB catabolism, with higher FCR values during the day than during the morning, was demonstrated by the NSS analysis and confirmed by LDL apoB calculation from the U/P ratio. The SS analysis with the whole and the once-a-day data sets resulted in similar average FCR of apoB values (0.329 +/- 0.076 and 0.321 +/- 0.071 respectively) when the two-compartment model was used. Thus, a CR appeared to have little impact on the average FCR of apoB estimation. However, frequent sampling used in the hope of improving parameter estimation accuracy actually resulted in deterioration of the intercompartmental parameter estimators. CONCLUSION: The fractional catabolic rate of LDL apoB exhibited a circadian rhythm with higher FCR values during the day than during the morning. The presence of a CR had, however, a limited impact on the overall FCR of apoB values.

Assessing the fertility potential of equine semen samples using the reducible dyes methylene green and resazurin.

The objective of this study was to determine if spermatozoal reduction of the dyes methylene green to colorless and resazurin to pink or colorless was associated with the fertility potential of an equine semen sample. Fifty samples from 38 stallions were evaluated for the number of spermatozoa per milliliter and number of motile sperm per milliliter. Methylene green (20 micrograms/mL of semen) or resazurin (85 micrograms/mL of semen) was added to 3-mL aliquots of semen. Semen samples were identified as having low fertility potential (< 200 x 10(6) total cells/mL and < 140 x 10(6) motile cells/mL) or high fertility potential (> or = 200 x 10(6) total cells/mL and > or = 140 x 10(6) motile cells/mL). The sensitivities were 80% for the methylene green, 68% for the resazurin to pink, and 60% for the resazurin to colorless tests. The specificities were 80% for the methylene green, 79% for the resazurin to pink, and 92% for the resazurin to colorless tests. The overall accuracies were 80% for the methylene green test, 74% for the resazurin to pink test, and 76% for the resazurin to colorless tests. The methylene green and resazurin reduction tests can provide valuable information on the fertility potential of an equine semen sample.

The influence of age on low density lipoprotein metabolism: effects of cholestyramine treatment in young and old healthy male subjects.

OBJECTIVE: The plasma concentration of low density lipoproteins (LDL) increases with age, mainly as the result of a reduced clearance of LDL. Because the conversion of cholesterol to bile acids is reduced with age, we hypothesized that the response of plasma LDL to stimulation of bile acid production would be different in young and old subjects. DESIGN, SUBJECTS AND SETTING: Comparison of the response to cholestyramine treatment in two groups of normolipidaemic, normal weight males: seven young (23-34 years) and eight old (64-73 years). Outpatients at the metabolic ward of a university hospital given a standardized diet of natural type. INTERVENTION: Cholestyramine was given 8 g b.i.d. for 3 weeks and continued during the second LDL turnover study. MAIN OUTCOME MEASURES: Kinetics of autologous radiolabelled LDL before and during treatment. Mean values of lipoprotein lipid levels obtained during the two turnover studies. Changes in LDL particle composition and LDL receptor affinity between the two study periods. RESULTS: Both age groups responded with decreased levels of LDL cholesterol and apolipoprotein-B (apoB), the change being more pronounced in the old subjects. The LDL apoB fractional catabolic rate was increased by approximately 11% in both groups. In contrast, there was a reduced ability in the old subjects to sustain their production rates for LDL apoB with resin therapy, resulting in a 23% reduction in LDL input. No effect on the apparent LDL apoB synthesis rate was observed in the young subjects. LDL particles isolated from cholestyramine-treated subjects were triglyceride-enriched and cholesterol-depleted, and showed a lowered affinity for the LDL receptor in tissue culture studies. CONCLUSIONS: The results demonstrate that stimulation of bile acid production by cholestyramine treatment decreases LDL cholesterol levels in both young and old subjects. This therapy increases LDL apoB elimination in both age groups, but reduction of apparent LDL apoB production is only seen in old subjects.

Low-density lipoprotein metabolism and its association to plasma lipoprotein(a) in the nephrotic syndrome.

Patients with nephrotic syndrome have multiple abnormalities of lipoprotein metabolism, but the cause and exact nature of these abnormalities have not been established. In the present study we have determined the kinetics of plasma low-density lipoprotein (LDL) apoB in seven nephrotic patients demonstrating an elevated LDL apoB production rate (25.7 +/- 6.4 vs. 13.1 +/- 0.3 mgkg-1 day-1; P < 0.001) but a normal LDL apoB fractional catabolic rate (FCR) (0.31 +/- 0.04 vs. 0.33 +/- 0.008 pools day-1; NS) compared with 41 healthy control subjects. However, two out of the seven patients had a markedly low LDL apoB-FCR. Serum albumin was inversely correlated with the LDL apoB production rate (R = -0.82; P < 0.05). Plasma lipoprotein (a) [Lp(a)] levels were significantly (P < 0.001) increased in the nephrotic patients compared with control subjects. Significant correlations were observed between log Lp(a) and LDL apoB production rate (R = 0.90; P < 0.01), VLDL-cholesterol (R = 0.95; P < 0.001) and VLDL-triglycerides (R = 0.80; P < 0.05) respectively. In summary, the present study suggests that nephrotic hyperlipidaemia may be caused by at least two independent mechanisms. The elevated LDL apoB production rate is highly correlated with the prevailing levels of serum albumin, whereas some nephrotic patients seem to have a decreased LDL apoB clearance, suggesting impaired LDL receptor-mediated clearance. The present results also suggest that the elevated plasma Lp(a) levels in nephrosis are related to an increased hepatic synthesis rather than a decreased catabolism of lipoproteins.

Growth hormone treatment does not alter biliary lipid metabolism in healthy adult men.

GH is important for the hepatic low density lipoprotein (LDL) receptor induction that occurs after estrogen treatment. GH treatment increases liver LDL receptors and lowers plasma LDL cholesterol in man. Estrogen treatment enhances biliary secretion of cholesterol, resulting in supersaturation of bile and an increased risk of gallstone formation. The present study was undertaken to investigate whether GH treatment also influences biliary lipid metabolism in humans. Twelve healthy male volunteers (mean age, 31 +/- 1 yr) were studied before and during the third week of treatment with recombinant human GH (0.1 IU/kg.day). Plasma lipids, bile acid kinetics, and biliary lipid composition were monitored. Plasma total and LDL cholesterol levels were reduced by 10% in response to therapy. However, no significant changes were observed in the biliary lipid composition or cholesterol saturation of gallbladder bile. Furthermore, there were no changes in chenodeoxycholic acid or cholic acid kinetics. The reduction of plasma LDL cholesterol in response to GH treatment in healthy adult men is not associated with detectable changes in biliary lipid metabolism. Thus, in contrast to estrogen, GH therapy of adults probably does not result in an increased risk of cholesterol gallstone development.

Modified resazurin reduction test for determining the fertility potential of bovine spermatozoa.

The objective of this study was to determine if reduction of the dye resazurin by bovine sperm is associated with the concentration of motile and progressively motile sperm. Metabolically active sperm reduce resazurin (blue color) to resorufin (pink color) and upon further reduction to dihydroresorufin (white color). A total of 68 semen samples from 20 Limousin bulls were collected using electroejaculation. The concentration of motile and progressively motile sperm was determined. Resazurin was added to each semen sample (8.8 microgram/mL semen), samples were then incubated at 37 degrees C for a maximum of 15 min, and visual color changes indicative of dye reduction were noted. Samples were identified as having either low fertility potential (< 200 x 10(6)/mL motile sperm and < 100 x 10(6)/mL progressively motile sperm) or high fertility potential (> or = 200 x 10(6)/mL motile sperm and > or = 100 x 10(6)/mL progressively motile sperm). Assessment of the reduction of resazurin from blue to pink (< or = 3.5 min) allowed identification of 88% of the low and 94% of the high fertility potential samples. In addition, the reduction of resazurin from blue to white (< 15 min) resulted in identification of 82% of the low and 76% of the high fertility potential samples. The modified resazurin reduction test is useful for determining the fertility potential of bovine spermatozoa.

Does migration stabilize local population dynamics? Analysis of a discrete metapopulation model.

A discrete model for a metapopulation consisting of two local populations connected by migration is described and analyzed. It is assumed that the local populations grow according to the logistic law, that both populations have the same emigration rate, and that migrants choose their new habitat patch at random. Mathematically this leads to a coupled system of two logistic equations. A complete characterization of fixed point and two-periodic orbits is given, and a bifurcation analysis is performed. The region in the parameter plane where the diagonal is a global attractor is determined. In the symmetric case, where both populations have the same growth rate, the analysis is rigorous with complete proofs. In the nonsymmetric case, where the populations grow at different rates, the results are obtained numerically. The results are interpreted biologically. Particular attention is given to the sense in which migration has a stabilizing and synchronizing effect on local dynamics.

Apolipoprotein E polymorphism in a healthy Swedish population: variation of allele frequency with age and relation to serum lipid concentrations.

We analyzed blood samples from 407 healthy Swedish individuals, 244 men and 163 women, ages 17 to 86 years, for apolipoprotein (apo) E isoforms and serum triglycerides, low-density lipoprotein (LDL), high-density lipoprotein (HDL), and total cholesterol. Parallel genotyping by means of polymerase chain reaction (PCR)-amplified DNA was performed in 200 subjects. Identical results were obtained by genotyping and phenotyping in 95% of all subjects analyzed. The apo E allelic frequencies were 7.8% for epsilon 2, 71.9% for epsilon 3, and 20.3% for epsilon 4. Compared with other Caucasian populations, the present population had a high relative allelic frequency of epsilon 4. The epsilon 4 frequency decreased with increasing age and was significantly lower in individuals > 60 years of age (14.7%). When controlling for age and sex, there were strong correlations between total serum and LDL cholesterol and the various epsilon alleles. The epsilon 4 and epsilon 3 alleles correlated positively with serum cholesterol and the epsilon 4 allele correlated positively with LDL cholesterol. In contrast, HDL cholesterol and serum triglycerides did not show any correlation to the allele types. Thus, the results demonstrate a considerable age variation of the epsilon allele frequency among healthy Swedes and an influence of apo E alleles on serum and LDL cholesterol concentrations.

Comparison of motility and flow cytometric assessments of seminal quality in fresh, 24-hour extended and cryopreserved human spermatozoa.

Functional differences among fresh 24-hour extended and cryopreserved human spermatozoa were assessed using both computer-assisted semen analysis (CASA) and flow cytometry. The objective was to determine if there were interrelationships among various qualitative parameters of the fresh and treated samples when assessed by these two automated methods. Fertile donor specimens (n = 15) were split and examined for sperm motility and curvilinear velocity using CASA within 1 hour postejaculation, after 24 hours in TEST-yolk buffer at 5 degrees C and after cryopreservation in TEST-yolk-glycerol medium. Flow cytometric analyses were performed on 24-hour extended and cryopreserved (CP) samples after fluorescent staining with rhodamine 123 to quantify mitochondrial function and carboxydimethyl fluorescein diacetate and propidium iodide to assess plasma membrane integrity. The percentages of spermatozoa with functional mitochondria and intact membranes along with the proportion of dead cells were identified and quantified by flow cytometry. Quadrant analyses of these data were used to determine the relative red and green fluorescent intensities. The initial sperm motility was correlated to the motility observed for the 24-hour stored and the CP samples. The sperm velocity of both the initial and the 24-hour extended samples was correlated to the velocity of CP samples. As for the comparison of the two automated methods for assessing seminal quality, the only sperm motion parameter that was correlated with a sperm population identified by flow cytometry (quadrant 4) was the curvilinear velocity of the sperm after 24 hours storage (r = 0.69) and after cryopreservation (r = 0.74). The present findings indicate that additional research is needed to determine if prefreeze analyses of donor sperm could be useful in predicting the post-thaw integrity of CP samples and, thereby, be useful in screening potential semen donors.

Interrelationships among fluorometric analyses of spermatozoal function, classical semen quality parameters and the fertility of frozen-thawed bovine spermatozoa.

Cryopreserved spermatozoa from 8 bulls were used to examine the interrelationships among flow cytometric spermatozoal quality assessments and classical semen quality parameters and nonreturn rate estimates of fertility. The integrity of the sperm cell membrane and the functional capacity of the mitochondria were quantified by flow cytometry after concurrent staining with carboxydimethylfluorescein diacetate (CDMFDA), propidium iodide (PI), and rhodamine 123 (R123). For each sample a total of 10,000 stained spermatozoa were simultaneously quantified for the intensity of their green and red fluorescence. Three straws from each bull were each examined initially and following incubation at 37 degrees C for 3 hours to assess the rate of senescence. The proportion of spermatozoa retaining membrane integrity and having functional mitochondria, as determined by CDMFDA and R123 staining, were compared with classical semen quality assessments (sperm motility, acrosomal status, cellular and head morphology, presence of vacuoles/craters and cytoplasmic droplets) and with fertility (nonreturn to estrus rates). For individual ejaculates nonreturn rates, the range was from 61.8 to 78.8%, whereas the cumulative rates of several ejaculates for each bull ranged from 71.3 to 83.5%. The proportion of spermatozoa with functional membranes and mitochondria were positively correlated with the percentage of spermatozoa with normal morphology (r=0.82; P=0.01) and motility after 4 hours of incubation (r=0.78; P=0.02), but not with the estimates of fertility. The actual number of spermatozoa per straw staining with CDMFDA and R123 after 4 hours of incubation at 37 degrees C was correlated with the percentage of spermatozoa with normal morphology (r=0.73; P=0.04). Multiple regression equations indicated that combinations of semen quality measurements could be useful in estimating fertilizing potential.

Metabolism of plasma low density lipoproteins in familial combined hyperlipidaemia: effect of acipimox therapy.

Ten male patients with familial combined hyperlipidaemia (FCHL) were studied with regard to LDL metabolism and composition. The FCHL patients had higher LDL levels than healthy controls (5.4 +/- 1.4 vs. 3.7 +/- 0.7 mmol l-1; P < 0.005) and a higher rate of production of the lipoprotein (15.8 +/- 3.1 mg kg-1 d-1 in FCHL vs. 13.1 +/- 1.8 mg kg-1 d-1 in the normals; P < 0.005). The fractional catabolic rate of LDL was low-normal in the FCHL patients, with a high level of interindividual variation. The actual individual LDL cholesterol level within the FCHL patient group appeared to be more closely associated with the LDL apoB FCR value than the rate of production of the particle. Analysis of the LDL particles from FCHL patients revealed a relative enrichment in triglycerides, while the cholesterol content of the lipoprotein was normal. Institution of acipimox therapy in 8 patients reversed the high rate of synthesis of LDL (15.2 +/- 3.5 mg kg-1 d-1) to a more normal level (13.9 +/- 4.0 mg kg-1 d-1; P = 0.08), while the FCR did not change significantly. In conclusion, patients with FCHL show an apparent overproduction of LDL apoB, while the actual degree of LDL elevation appears to be dependent on the clearance capacity of the lipoprotein, measured as LDL apoB FCR. The overproduction defect of LDL apoB can, at least in part, be managed by treatment with the nicotinic acid analogue acipimox.

Effects of growth hormone on low-density lipoprotein metabolism.

GH appears to play an important role in mediating the increase in hepatic LDL receptor expression that occurs in response to pharmacological oestrogen treatment. Furthermore, in normal individuals there is evidence of an increased number of hepatic LDL receptors in response to GH, resulting in an accelerated elimination rate of the LDL particle and a subsequent lowering of LDL cholesterol. Further studies on lipoprotein metabolism in patients with GH deficiency, and also in patients with hypercholesterolaemia, would therefore be of interest.

Couples with exclusively female offspring have an increased probability of a male child after using male sex preselection.

The present study was undertaken to determine whether couples with one or more daughters and no sons had an increased probability of having a male child after using male sex preselection. The sex ratio of children born to couples after using 'protocol 3' (n = 70) or 'modified protocol 3' (n = 28) male sex preselection at one of 14 centres was determined. The normal approximation to the binomial distribution was used to determine significant differences between these sex ratios and the established sex ratio for children born to couples with one previous daughter and no sons. The sex ratios of both protocol 3 sex-preselected children (73.0%; P less than 0.0001) and modified protocol 3 sex-preselected children (86%; P less than 0.0001) were significantly different to the established sex ratio (control) for a current child born to parents with one previous daughter and no sons (50.1%). Couples with one or more daughters and no sons will have an increased probability of a male child after using male sex preselection.

Sex ratio of male sex preselected children born to couples with exclusively female offspring.

The present study was undertaken to determine if couples with exclusively female children had a decreased probability of a male child after using male sex preselection. Selection criteria for subjects necessitated that couples have had only female children previously and had produced a child at one of 14 centers after using protocol 3 (n = 70) and modified 3 (n = 28) male sex preselection. Prior to sex selection, protocol 3 couples produced a combined total of 135 female children for an average of 2.01 (range 1-4) females per couple; modified 3 couples produced a combined total of 62 female children for an average of 2.21 (range 1-4) females per couple. The normal approximation to the binomial distribution was used to determine significant differences between the sex ratios prior to and after male sex preselection. For couples using protocol 3 there were significant differences in the sex ratio prior to sex preselection (0%) and after sex preselection (73.0%) (p less than .00003). There were also significant differences between the sex ratio prior to modified 3 (0%) and after sex preselection (86%) (p less than .00003). Couples using male sex preselection do not have a decreased probability of a male child if they have had exclusively female children.