Transit along the vas deferens results in a high percentage of filiform spermatozoa with compacted chromatin in the rooster (Gallus domesticus).

The present work aimed to evaluate the chromatin compaction of rooster spermatozoa along the male reproductive tract, and to study the vas deferens lining cells, potentially involved in sperm maturation. Chromomycin A3 (CMA3) was used to determine the chromatin compaction of spermatozoa from testis (T), proximal (including epididymis, V1), intermediate (V2) and distal (V3) vas deferens, and ejaculate (E). Six Birchen Leonesa roosters were used. E was obtained in vivo by dorso-ventral massage. V1, V2 and V3 sperm were obtained post mortem (six pairs of vasa deferentia), by flushing. T was obtained by washing the testes, cut in halves. The fixed cells were stained with CMA3 and propidium iodide for flow cytometry assessment. Results showed higher (P P P.

Birchen and Blue Leonesa sperm cryopreservation: a new technique for evaluating the integrity of cockerel sperm membranes.

1. Birchen and Blue Leonesa are two endangered chicken breeds mainly raised in Curueño Valley in North Spain. The establishment of a germplasm bank to guarantee the preservation of these breeds is needed. However, cockerels from different breeder flocks can show variance in semen cryoresistance.2. The following work focused on the sperm characterisation and cryopreservation of Birchen and Blue Leonesa cockerels from four different breeders. A total of 30 semen pools were analysed. Besides conventional sperm analysis, including motility by computer-aided sperm analysis (CASA) and DNA fragmentation by TUNEL, the present study tested a double staining method (MitoTrackerTM Green FM/propidium iodide). This gave simultaneous assessment of plasma and acrosomal and mitochondrial membranes, which were previously validated by SYBR-14/PI, CASA, aniline blue and TUNEL.3. No significant differences were found among fresh semen variables between breeds and breeders. For post-thawed variables, significant differences (P < 0.05) were found between breeders in sperm viability (58.0 ± 1.90 breeder D vs. 35.2 ± 7.41 breeder A, 37.2 ± 4.09 breeder B and 22.3 ± 5.92 breeder C) and DNA fragmentation (62.4 ± 9.91 breeder C vs. 31.8 ± 7.08 breeder B and 24.5 ± 5.49 breeder D). The lowest DNA fragmentation values for semen from breeder D birds were coincident with higher integrity of the mitochondrial membrane.4. The results revealed higher sperm cryoresistance in the cockerels from one of the breeders, possibly due to differences in management system (e.g. diet, housing, control of stress elements and pathogens, reproduction practices or maintenance of genetic diversity). These differences may determine the sperm freezability, and thus the effectiveness of developing a germplasm bank.

Effect of supplementation of valine to chicken extender on sperm cryoresistance and post-thaw fertilization capacity.

Recent reports showed a positive correlation between frozen-thawed rooster sperm DNA integrity and the concentrations of valine in seminal plasma. The present study evaluated the effect of supplementing valine to semen extender for freezing sperm of 2 endangered local Spanish chicken breeds with different sperm cryoresistance: Red Villafranquina (VF) showing low sperm DNA integrity after cryopreservation and Quail Castellana that shows higher DNA integrity. One pool of semen per breed was obtained twice a week for 10 wk (n = 40, 20 per breed). Each pool was divided into 2 fractions. One of these fractions was frozen in presence of valine as additive in the extender (concentration 10 mmol), whereas the other was used as control. The evaluation of the samples before and after freezing-thawing included motility (CASA-Mot system), viability (propidium iodide and SYBR-14), DNA integrity (terminal deoxynucleotidyl transferase dUTP nick end labeling), and fertility rate (percentage of eggs with blastoderm development after artificial insemination). Supplementation of valine increased several motility variables of fresh semen. In VF breed, valine increased percentage of progressive motile sperm (P = 0.025), curvilinear velocity (P = 0.033), straight-line velocity (P = 0.040), and average path velocity (P = 0.033), whereas progressive motile sperm (P = 0.019), curvilinear velocity (P = 0.006), straight-line velocity (P = 0.003) and average path velocity (P = 0.004) were improved in the Quail Castellana breed. Valine addition increased the DNA integrity of cryopreserved semen (decreased post-thaw DNA fragmentation) in both breeds, with a significant effect (P = 0.002) in VF (36.3% VF-control vs 31%VF-valine). As expected, Quail Castellana cryopreserved sperm control showed higher fertility rate (34.4% ± 12.1) than VF cryopreserved sperm control (16.1% + 6.2). Supplementing valine to the freezing extender doubled (P = 0.026) the fertility rate of VF (32.6% ± 12.2) compared with the control (16.1% + 6.2). In conclusion, supplementation of valine to chicken freezing extenders shows a positive effect on DNA fragmentation and fertilizing ability of frozen-thawed sperm, with a better response in a breed considered as the lowest freezer in our conservatory.

Anti-predator behavior in pure and hybrid red-legged partridges.

The purpose of this study was to examine the anti-predator behavior in pure red-legged partridges (Alectoris rufa) and hybrids with chukar partridges (A. rufa × (A. rufa × A. chukar)). A total of 117 birds (66 pure and 51 hybrid partridges; 74 females and 43 males) maintained in outdoor cages were used at a range of 52 to 55 wk of age. Four active fear tests were performed (novel object test, fear of humans test, emergence test, and restraint test) in order to analyze differences between pure and hybrid populations regarding the escape response type (novel object test and fear of humans test), the withdrawal distance (fear of humans test), and the reaction time (emergence test and restraint test). These variables could determine the ability of pure partridges captive to survive when released into the wild for hunting. There were no significant differences in anti-predator behavior in pure and hybrid birds, only one trend was observed in withdrawal distance (P = 0.09), being greater in pure than in hybrids partridges. There was a significant difference between sexes in the response type in the novel object test (P = 0.05); whereby the females came closer to the object than the males. The results of the present study indicate that active anti-predatory behavior was not associated with the genotype. However, since captivity could reduce the active anti-predator behavior, the results could be different if the wild birds that were tested had been sourced from their natural environment. Both sexes had similar active anti-predator behavior, although the males showing less approach behavior to a novel object that could be related to the sex-dimorphic behavior in natural social structure.

Access to pasture in an outdoor housing system affects welfare indicators and improves rooster sperm quality in two native Mediterranean breeds.

The aim of the present work was to examine the influence of access to pasture in an outdoor housing system on rooster sperm quality and response to cryopreservation and to examine the possible correlation between values for sperm quality variables and welfare indicators. Two groups of Black-barred Andaluza and Red-barred Vasca roosters were housed in an outdoor system, with one group given daily access to a grazing area containing plant species that typically grow on uncultivated Mediterranean land. Semen was collected once per week from each group, and the following sperm quality variables were assessed: sperm volume, appearance, concentration, motility, membrane integrity, acrosome integrity, and morphological abnormalities. In addition, two welfare indicators were examined: the heterophil/lymphocyte (H/L) ratio, and the duration of tonic immobility (TI). Ejaculates from the birds with access to pasture had higher percentages of sperm showing progressive motility (P = 0.019), and returned a higher motility index (P = 0.035). Unexpectedly, the H/L ratio was also higher in these birds. Virtually no differences were seen between the treatment groups with respect to sperm quality after freezing-thawing, although the semen of the Red-barred Vasca birds with access to pasture did show a higher percentage of progressive motility (P = 0.023) than the birds of the same breed with no such access. Significant correlations were detected between the H/L ratio and sperm motility (r = 0.420, P = 0.038), the sperm motility index (r = 0.526, P = 0.002), and progressive motility (r = 0.467, P = 0.003). No differences were seen between the treatment groups with respect to the duration of TI. In conclusion, access to pasture improved fresh sperm motility.

Successful chilling of red-legged partridge (Alectoris rufa) sperm for use in artificial insemination.

The fertilizing capacity of pure, fresh avian semen may disappear in just half an hour, hindering its successful use in artificial insemination (AI) projects. Longer storage requires the use of infra-physiological temperatures and of semen diluents that help preserve the spermatozoa but that do not interfere with their fertilizing capacity. This study examines the effect on sperm quality of storing red-legged partridge sperm for 3 h at 5°C with 2 different semen extenders: 1) a medium referred to as L&R-84, composed of sodium glutamate, glucose, magnesium acetate, potassium acetate, and polyvinylpyrrolidone, and 2) Lake 7.1 medium, composed of sodium glutamate, glucose, magnesium acetate, potassium citrate, and N,N-Bis(2-hydroxyethyl)taurine (BES). Extending with L&R-84 returned better curvilinear velocity (P < 0.01), straight-line velocity (P < 0.01), average path velocity (P < 0.01), linearity (P < 0.05), straightness (P < 0.05), and wobble (P < 0.05) values, while extending with the Lake 7.1 medium was associated with higher percentages (P < 0.001) of motile sperm. The fertility rate was higher (P < 0.05) when birds were inseminated with L&R-84-extended sperm than with Lake 7.1-extended sperm. The mean number of penetrations of perivitelline layer samples (taken from above the germinal disc) was also higher for the L&R-84-extended sperm (P < 0.05). These results show L&R-84 can be recommended as an extender for red-legged partridge semen to be stored for at least 3 h at 5°C.

Effect of the presence of hens on roosters sperm variables.

This study examines the effect of the presence of hens on sperm variables in Black Barred Andaluza roosters (which respond well to the massage technique). Between 8 and 22 weeks of age, roosters (n = 60) were housed separate from hens in straw litter pens (4 birds/m(2)). Two groups of roosters were then formed to study the effect of hen presence on sperm variables at 36 weeks of age. The first group (n = 11 roosters) was housed with hens (n = 55; sex ratio 1:5), while the second (n = 27 roosters) was again housed separate from hens. Twenty four sperm pools were obtained over 12 weeks (12 pools from each group, each pool produced by mixing semen from 11 males) for analysis. Compared to the no-female-contact group, the roosters living with hens showed significantly (P < 0.05) reduced percentages of non-progressive motile sperm and slow sperm, and significantly increased VCL and VSL values; they also showed trends (P < 0.1) towards increased progressive motility and a higher percentage of rapid sperm. The presence of hens had no significant effect on the number of spermatozoa per ejaculate or sperm concentration. Positive correlations were recorded between VSL and VCL, and between both of these and progressive motility, and the percentage of rapid sperm. In addition, both VSL and VCL correlated negatively with non-progressive motility and the percentage of slow sperm. These results indicate that rooster sperm quality, but not quantity, improves in the presence of hens.

Association between polymorphism in the melanocortin 1 receptor gene and E locus plumage color phenotype.

The purpose of this study was to investigate the effect of the melanocortin 1 receptor (MC1R) gene on plumage color in chickens. The gene was sequenced in 77 males and 77 females from 13 Spanish breeds, carrying 6 different alleles in the E locus (E*E, E*R, E*WH, E*N, E*B, E*BC), a recessive wheaten (yellowish-white) tester line (E*Y), and a White Leghorn population (heterozygous E*E). A total of 11 significant SNP were detected. Nine of them were nonsynonymous (T212C, G274A, G376A, T398AC, G409A, A427G, C637T, A644C, and G646A, corresponding to amino acid changes Met72Thr, Glu92Lys, Val126Ile, Leu133GlnPro, Ala137Thr, Thr143Ala, Arg213Cys, His215Pro, and Val216Ile), and 2 were synonymous (C69T and C834T). With respect to the significant SNP, 7 had an allelic frequency of 0.5 or greater for some of the alleles at the E locus. These results indicated a significant correlation between MC1R polymorphism and the presence of different alleles at the E locus. All the populations carrying the E*E or E*R alleles, except the Birchen Leonesa, had the G274A polymorphism. Eleven haplotypes were made with 7 of the significant SNP. The distribution of these haplotypes in the different alleles of the E locus showed that each haplotype was predominantly associated to one allele. The number of haplotypes was greatest for the Black Menorca, Birchen Leonesa, and Blue Andaluza breeds, whereas the Quail Castellana and Red-barred Vasca breeds were monomorphic. Our results suggested that the Glu92Lys mutation may be responsible of the activation of the receptor for eumelanin production, being necessary but not sufficient to express the extended black phenotype. They also suggested that the Arg213Cys mutation may be the cause of the loss or the decrease of function of the receptor to produce eumelanin, and the Ala137Thr mutation may be a candidate to attenuate the Glu92Lys effect. The observed co-segregation of the E locus alleles and polymorphisms in MC1R confirms that the E locus is equivalent to MC1R.

Associations among fluctuating asymmetry, tonic immobility duration, and flight distance or ease of capture in chickens.

The purpose of this study was to analyze the associations among fluctuating asymmetry, duration of tonic immobility, and flight performance or ease of capture in chickens. Cocks (n = 220; 36 wk old) from 10 Spanish breeds and a White Leghorn population were used. The quantitative/qualitative relationship among fluctuating asymmetry, duration of tonic immobility, and landing accuracy (good or bad) or landing distance (long or short), and the quantitative/quantitative relationship among fluctuating asymmetry, duration of tonic immobility, and time of capture or number of attempts of capture were studied. Relative fluctuating asymmetry of toe length, wing length, and the combined relative asymmetry of toe length, leg length, wing length, and leg width was significantly greater (P < 0.05, P < 0.01, and P < 0.001, respectively) in cocks with bad landing accuracy. There was no significant difference for the duration of tonic immobility between groups of cocks with bad or good landing accuracy. Relative asymmetry of wing length and the combined relative asymmetry of the 4 traits was significantly greater (P < 0.05) in cocks with short landing distance and smaller in cocks with long landing distance. There was a significant difference for the duration of tonic immobility between groups of cocks with short or long landing distance (P < 0.001), with that of cocks with short landing distance being longer. There was no significant correlation among relative fluctuating asymmetry, duration of tonic immobility, and time of capture or attempts of capture. Associations between morphological values of toe, leg, and wing and flight performance or ease of capture were not significant. Thus, birds with less asymmetry and fear level have better flight performance than more asymmetric and fearful birds, whereas the ease of capture is not associated with fearfulness and asymmetry.

Effects of auditory and physical enrichment on 3 measurements of fear and stress (tonic immobility duration, heterophil to lymphocyte ratio, and fluctuating asymmetry) in several breeds of layer chicks.

The purpose of this study was to analyze the effect of auditory enrichment (by means of classical music) or physical enrichment (by means of hanging colored string bunches and barley grains on the floor) on tonic immobility duration, heterophil to lymphocyte ratio, and fluctuating asymmetry (FA) in chicks of several layer breeds. In experiment 1, 192 chicks from 8 Spanish breeds and 1 White Leghorn population were reared in cages with or without music auditory enrichment until 8 wk of age. The effect of music auditory enrichment was significant for heterophil to lymphocyte ratio (P < 0.05). The ratios were higher in chicks reared without music than in those reared with music, suggesting that auditory enrichment from classical music reduces stress in chicks. There were significant differences in morphological trait measurements (relative asymmetry of wing length, leg width, and combined asymmetry; P < 0.05), being greater in chicks reared without music. This result suggests that FA is a good indicator for stress level in chicks, given that it follows the same trend as that found for heterophil to lymphocyte ratio. There was a significant treatment by breed interaction (P < 0.05) for tonic immobility duration, indicating no consistent effect by auditory enrichment on tonic immobility duration across breeds. In experiment 2, 180 chicks from 3 Spanish breeds were housed in pens with or without physical enrichment (colored plastic string bunches and barley grains on the floor) until 6 wk of age. The effect of physical enrichment on tonic immobility duration, heterophil to lymphocyte ratio, and FA was not significant, indicating no effect on fear and stress in layer chicks. In conclusion, auditory enrichment by means of classical music is a reliable method for reducing stress levels in several breeds of layer chicks. However, music auditory enrichment was not effective in reducing fearfulness in any of the layer breeds. Physical enrichment by means of colored plastic string bunches and floor barley grains does not appear to be an effective method for reducing stress and fear in layer chicks.

Evaluation of diversity between different Spanish chicken breeds, a tester line, and a White Leghorn population based on microsatellite markers.

The present study was conducted to evaluate the genetic variability and the genetic divergence of 13 Spanish chicken breeds, a tester line, and a White Leghorn population, using 24 microsatellite markers. A total of 150 alleles were detected across all population. The number of alleles by locus ranged from 2 to 13, with the mean value being 6.25. The mean polymorphic information content was 0.591, ranging from 0.847 to 0.172. The combined parentage exclusion probability of excluding 1 parent or 2 parents was 99 and 100%, respectively. The observed heterozygosity was lower than the expected heterozygosity for all loci, the mean values being 0.461 and 0.637. The observed and expected heterozygosity ranged from 0.003 to 0.735 and 0.181 to 0.863, respectively. Mean deficit of heterozygotes within populations (F(IS)) was 0.056 and mean fixation index of each population (F(ST)) was 0.244. The mean global deficit of heterozygotes across populations (F(IT)) was 0.286. A total of 15 private alleles in 10 microsatellites were observed, and in some populations, fixed alleles were found for 7 microsatellites. A total of 300 birds (83%) were properly assigned to the source population. The average observed heterozygosity for each population was 0.461, ranging from 0.328 (Quail Castellana) to 0.538 (Red Villafranquina), and the average expected heterozygosity was 0.488, ranging from 0.320 (Quail Castellana) to 0.550 (White-Faced Spanish). All of the Spanish breeds except the Quail Castellana were more polymorphic than the White Leghorn population. The mean value of the deviation of heterozygote number was 0.052. Nei's genetic distance showed a range from 0.109 (between White-Faced Spanish and Black Menorca) to 0.437 (between Buff Prat and White Leghorn). A phylogenetic tree constructed by the neighbor-joining method, based on Nei's genetic distance, showed a clear separation between the White Leghorn and the remaining breeds. The results indicate that the panel of microsatellite markers was useful in studying the genetic diversity of chicken breeds.

Effect of lighting stress on fluctuating asymmetry, heterophil-to-lymphocyte ratio, and tonic immobility duration in eleven breeds of chickens.

The purpose of the present study was to analyze the effect of a lighting stress on the fluctuating asymmetry (FA), the heterophil-to-lymphocyte ratio, and the tonic immobility duration of chickens. The experiment (440 birds) measured the FA of several traits (outer, middle, inner, and hind toe lengths and leg, wing, second primary feather, and spur lengths), the heterophil-to-lymphocyte ratio, and the tonic immobility duration in 36-wk-old hens and cocks of 8 Spanish breeds of chickens (Black-Barred Andaluza, Black-Red Andaluza, Black Castellana, Buff Prat, Red-Barred Vasca, Red Villafranquina, Birchen Leonesa, and Blue Leonesa), a synthetic breed (Quail Castellana), a White Leghorn population, and the e(y) tester line, which had been housed in continuous light (24L:0D) or in a light-dark regimen (14L:10D) for 16 wk. There was a significant difference between lighting treatments in both females and males on the combined FA of the 4 toes (P < 0.01) and the combined FA of toe, leg, wing, feather, and spur (in males) lengths (P < 0.05), the FA of birds housed under continuous light being greater than that of control birds. There was a significant difference (P < 0.001) for the heterophil-to-lymphocyte ratio and the tonic immobility duration between lighting treatments, the ratio being higher and the duration being longer in the group of birds housed under continuous light. Thus, birds exposed to continuous light were more stressed and fearful than control hens. Results were consistent across the breeds and indicate that a continuous light regimen seriously negatively affects the welfare of birds.

Vinorelbine plus trastuzumab combination as first-line therapy for HER 2-positive metastatic breast cancer patients: an international phase II trial.

The aim of this international phase II trial was to determine the efficacy and safety profile of weekly vinorelbine plus trastuzumab as first-line chemotherapy for women with HER 2-overexpressing metastatic breast cancer. Sixty-nine patients with tumours overexpressing HER 2 received vinorelbine: 30 mg m-2 week-1 and trastuzumab: 4 mg kg-1 on day 1 as a loading dose followed by 2 mg kg-1 week-1 starting on day 8. Sixty-two patients were evaluable for response and 69 patients were evaluable for toxicity. The overall response rate was 62.9%. The median time to response was 8.4 weeks, the median duration of response was 17.5 months, the median progression-free survival was 9.9 months (95% CI, 5.6-12.1) and the one-year progression-free survival was 39.1%. The median survival for all patients was 23.7 months (95% CI, 18.4-32.6). This regimen was safe: grade 3-4 neutropenia were observed over 17.7% of courses in 83.8% of patients, with only two episodes of febrile neutropenia (0.1%) in two patients (2.9%). Only one patient discontinued treatment due to grade 3 symptomatic cardiac dysfunction that resolved with therapy. Vinorelbine plus trastuzumab is one of the most active treatment regimens for patients with HER 2-positive metastatic breast cancer and demonstrates a very favourable safety profile allowing prolonged treatment with long-term survival. This study has been presented in part at the following conferences: The San Antonio Breast Cancer Symposium, San Antonio, TX, USA, 2003; The American Society of Clinical Oncology, Orlando, FL, USA, 2005.

Influence of perches and footpad dermatitis on tonic immobility and heterophil to lymphocyte ratio of chickens.

The purpose of the present study was to analyze the effect of perches and footpad dermatitis on stress and fear levels of chickens. In experiment 1 heterophil to lymphocyte ratio and tonic immobility duration were measured in 36-wk-old hens (36 birds) from 2 Spanish breeds of chickens (Black Menorca and Quail Castellana) housed in pens with or without perches from 20 wk of age. There was a significant difference in the heterophil to lymphocyte ratio between perch treatments (P < 0.05), the ratio of hens with perches being lower than that of hens without perches. Hens with perches had significant lymphophilia (P < 0.05). There was no significant difference for the duration of tonic immobility between hens with or without perches. Thus, hens with perches were less stressed than hens without perches, as indicated by the heterophil to lymphocyte ratio. Heterophil to lymphocyte ratio and heterophil number for Black Menorcas were significantly smaller (P < 0.001) than for Quail Castellanas, whereas lymphocyte number was greater in Black Menorcas. The Black Menorca hens had significantly (P < 0.01) shorter tonic immobility duration. In experiment 2 heterophil to lymphocyte ratio and tonic immobility duration were measured in 52-wk-old cocks (36 birds) of each of two different Spanish breeds of chickens (Black Barred Andaluza and Red Barred Vasca) housed in cages from 36 wk of age and showing or not showing footpad dermatitis. There was no significant effect of footpad dermatitis on the heterophil to lymphocyte ratio, its numerator, or its denominator, whereas there was significant effect on tonic immobility duration (P < 0.05) with tonic immobility of cocks with footpad dermatitis being longer than that of cocks without footpads. Thus, cocks with footpad dermatitis were more fearful than cocks without footpad dermatitis, as indicated by the tonic immobility duration. Heterophil to lymphocyte ratio and heterophil number were significantly smaller (P < 0.05) for Black Barred Andaluzas than for the Red Barred Vascas.

Estimation of heritability for fluctuating asymmetry in chickens by restricted maximum likelihood. Effects of age and sex.

The purposes of the present study were to estimate the heritability of the fluctuating asymmetry in chickens, using the restricted maximum likelihood procedure, and to evaluate the effects of age and sex on the fluctuating asymmetry. Leg, wing, and feather lengths and ear-lobe and wattle areas were measured. In experiment 1, 1,073 birds were used from 2 generations with complete pedigree of the Quail Castellana breed to estimate the heritability for the fluctuating asymmetry at 36 wk of age. The estimated heritability of absolute fluctuating asymmetry was not significantly different from zero for all 5 traits, and similar estimates were obtained for relative fluctuating asymmetry, directional asymmetry, transformed absolute and relative fluctuating asymmetry, and 3 alternative indexes of fluctuating asymmetry. The heritability of the combined absolute or relative fluctuating asymmetry was still very low, indicating that fluctuating asymmetry was determined solely by environmental sources of variation and that fluctuating asymmetry estimates should not be confounded by appreciable additive genetic contributions. The genetic correlation between sides was not significantly different from one, indicating that differences between sides were purely environmental in origin. Different traits rarely showed much correlation in their level of fluctuating asymmetry, indicating that the level of fluctuating asymmetry in all traits did not reflect equally the quality of animals. In experiment 2, fluctuating asymmetry differences among ages and sexes were investigated at 8, 12, 16, 20, 24, 28, 32, 36, and 40 wk in 360 birds from the same breed. Significant variation with age was observed in leg length, wing length, feather length (females), and wattle area (females), which was mainly related to onset of sexual maturity and adult stage. Females showed significantly greater fluctuating asymmetry for ear-lobe area than males. There were significant differences in fluctuating asymmetry for wing length, feather length, and wattle area near the onset of sexual maturity, with males having significantly greater fluctuating asymmetry than females for wing length and the opposite being true for feather length and wattle area.

Association between plumage condition and fear and stress levels in five breeds of chickens.

Duration of tonic immobility and heterophil to lymphocyte ratio (indices of fear and stress, respectively) were compared in hens having very poor or perfect plumage at 72 wk of age. Five Spanish breeds of chickens (Castellana, Andaluza, Vasca, Villafranquina, and Leonesa) were used. There were significant differences among groups in terms of tonic immobility duration and heterophil to lymphocyte ratio (P < 0.05). Very poorly feathered hens showed shorter tonic immobility (243.03+/-26.81 s) and higher leukocyte ratio (0.42+/-0.02) than did hens with a perfect plumage (322.32+/-26.81 s and 0.35+/-0.02, respectively). Differences were consistent across the breeds. Thus, hens with very poor plumage were less fearful and more stressed than hens with a perfect plumage. The results suggest that very poor plumage is associated with indicators of fearfulness and stress.

Comparison of methods for extraction of tobacco alkaloids.

Ultrasound and microwave techniques were used to extract tobacco alkaloids, and response surface methodology was used to optimize extraction conditions. Ultrasonic technique factors were temperature, 30-85 degrees C; time, 3-45 min; solvent volume, 8-80 mL. Microwave extraction factors were pressure, 15-75 psi; time, 3-40 min; power, 30-90% of the maximum magnetron power of 650 W. Soxhlet and solvent AOAC-modified extraction methods were also applied after some improvements. Nicotine, nornicotine, anabasine, and anatabine were quantified by gas chromatography. A steam distillation International Standards Organization method for total alkaloid evaluation was used as reference. The results obtained by the different methods were compared using a least squares deviation test. The ultrasonic and the proposed modified-AOAC extraction method were the more convenient with regard to practicability and precision. The relative deviations (n = 5) were as follows: For the ultrasonic method in low-level alkaloid tobaccos, 0.7% nicotine and 1.4-14% minor alkaloids; in high-level alkaloid tobaccos, 2.4% nicotine and 4.5-5.1% minor alkaloids. For the modified AOAC method in low-level alkaloid tobaccos, 0.9% nicotine and 2.4-11.6% minor alkaloids; and in high-level alkaloid tobaccos, 1.7% nicotine and 2.0-2.4% minor alkaloids.

Relationships between bilateral asymmetry and tonic immobility reaction or heterophil to lymphocyte ratio in five breeds of chickens.

Bilateral asymmetry of several morphological traits, tonic immobility reaction, and leucocyte ratio were studied in hens and cocks from four Spanish breeds of chickens (Castellana, Buff Prat, Red Villafranquina, and Barred Red Vasca) and an F2 cross between Castellana and Buff Prat (C x BP - F2). Any two or all three types of bilateral asymmetry (fluctuating asymmetry, directional asymmetry, and antisymmetry) occurred together for the same trait in the different breeds. There was a significant difference in relative asymmetry among breeds (P < 0.05) for feather and spur lengths in males, and considerable negative heterosis for relative asymmetry of leg and wing lengths in C x BP - F2 males (-38 and -12%, respectively). The degree of relative asymmetry varied among traits and was between 3 and 12 times greater for spur length in males and for wing bay area in Villafranquina males. There were significant differences among breeds (P < 0.05) for tonic immobility duration. The duration of tonic immobility was positively associated with the mean relative asymmetry of all traits in females from the Villafranquina breed (r = 0.32; P < 0.05), but this finding was not consistent across the different breeds. No significant difference was found among breeds in terms of heterophil to lymphocyte ratio. Although there was no significant association between mean relative asymmetry of all traits and heterophil to lymphocyte ratio, males from the Villafranquina breed had a positive and significant relationship between relative asymmetry for wing bay area and leucocyte ratio (r = 0.34; P < 0.05). These results suggest that relative asymmetry measures did not provide a general tool to assess fear or stress susceptibility.

Oligo(dT)-primed synthesis of cDNA by reverse transcriptase in mycobacteria.

The possibility that mRNA from Mycobacterium tuberculosis and M. bovis BCG may present polyadenylation at the 3' end was investigated. The total RNA, extracted from the bacterial cells and treated with DNase, was used as substrate for reverse transcriptase (RT)-dependent cDNA synthesis. The RT reaction was primed with oligo(dT) and with downstream specific primers for the genes of the antigens 65 KDa and 85-C. PCR probing of the reaction products for cDNAs of the two mycobacterial genes yielded the expected 225 and 307 bp bands when RT synthesis was primed by oligo(dT) and by downstream specific primers. Reaction products from oligo(dT)-primed RT of RNase-treated RNA and untranscribed RNA, probed by PCR, failed to generate the 225 and 307 bp specific bands. These findings support the existence of polyadenylated tracts in mRNA of mycobacteria that can be targeted by oligo(dT) primers to initiate RT-dependent cDNA synthesis. This may result in an advance in the study of gene expression in these and possibly other bacteria.

The effects of assortative mating on the genetic change due to linear index selection in Tribolium.

SUMMARY: The effectiveness of the assortative mating of selected individuals in increasing linear index selection response was tested in Tribolium castaneum. The experiment was designed to increase pupal length and adult weight, using selection and assortative mating with respect to a linear index of these traits. The experiment consisted of a randomly (R) and assortatively (A) mated line, with three replicates in each line. The proportion of selection was 25 %. Phenotypic correlations between mates in line A were not significantly different from 1. Average selection responses did not significantly differ in either line, although there was some indication of an effect of the mating system, since the response for the aggregate value was higher in line A (0.78 ± 0.15 as opposed to 0.57 ± 0.13). Average response for the selection goal and adult weight was statistically significant for both lines. ZUSAMMENFASSUNG: Die Auswirkungen assortativer Anpaarung auf genetischen Fortschritt bei Anwendung eines linearen Selektionsindex bei Tribolium Die Wirksamkeit assortativer Anpaarung selektionierter Individuen zur Verstärkung der Selektion mittels linearem Index wurde bei Tribolium castaneum untersucht. Selektion zielte auf längere Puppen und auf Gewichtssteigerung ausgewachsener Tiere. Das Experiment umfaßte eine zufällig angepaarte (R) und eine assortativ angepaarte (A) Linie mit je drei Wiederholungen. Die Remontierung betrug 25%. Die phänotypische Partner-Korrelation bei den in Linie A angepaarten Individuen wich nicht wesentlich von 1 ab. Die durchschnittliche Verbesserung war bei beiden Linien nicht significant verschieden, obwohl es Anzeichen einer Auswirkung des Paarungssystems gab. Die Koeffizienten der Linie A lagen höher (0.78 ± 0.15 gegenüber 0.57 ± 0.13 für Linie R). Die durchnittliche Veränderung des Selektionsziels und des Gewichts der ausgewachsenen Tiere waren statistisch significant. RÉSUMÉ: Les effets de l'accouplement classifié sur le changement génétique dû à un taux linéaire de sélection chez le Tribolium L'effectivité de l'accouplement classifié d'individue sélectionnés pour augmenter la réponse à la sélection d'un taux linéaire, fut prouvée chez le Tribolium castaneum. L'expérience fut conçue pur augmenter la longeur de pupe et le poids de l'adulte, en utilisant la sélection et l'accouplement classifié par rapport à un taux linéaire de ces caractères. L'expérience comprenait une ligne accouplée de façon aléatoire (R) et une autre selon l'accouplement classifié (A), avec trois répétitions dans chaque ligne. La proportion de sélection fut de 25 %. Les corrélations phénotypiques entre individus accouplés dans la ligne A ne furent pas significativement différentes de 1. Les réponses moyennes à la sélection ne présentaient pas de différences significatives dans les deux lignes, bien qu'il y ait des indices d'un effet apparent du systéme d'accouplement car la réponse pour la valeur ajoutée fut plus élevée dans la ligne A (0.78 ± 0.15 contre 0.57 ± 0.13 pour la ligne R). La réponse moyenne pour l'objectif de sélection et le poids de l'adulte fut statistiquement significative pour les deux lignes. RESUMEN: Los efectos del apareamiento clasificado sobre el cambio genético debido a un índice lineal de selección en Tribolium La efectividad del apareamiento clasificado de individuos seleccionados para aumentar la respuesta a la selección de un índice lineal fue comprobada en Tribolium castaneum. El experimento gue disen̄ado para incrementar longitud de pupa y peso de adulto, usando selección y apareamiento clasificado con respecto a un índice lineal de estos caractères. El experimento tenia una linea apareada aleatoriamente (R) y otra con apareamiento clasificado (A), con tres repeticiones en cada línea. La proporción de selección fue del 25%. Las correlaciones fenotípicas entre individuos apareados en la línea A no fueron significativamente diferentes de 1. Las respuestas medias a la selección no diferían significativamente en ambas líneas, aunque había indicios de un efecto aparente del sistema de apareamiento, ya que la respuesta para el valor agregado fue más alta en las línea A (0.78 ± 0.15 frente a 0.57 ± 0.13). La respuesta media para el objetivo de selección y el peso de adulto fue estadísticamente significativa para ambas líneas.