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 the interaction between cryoprotectant concentration and cryopreservation method on frozen/thawed chicken sperm variables.

This work examines the effect of the interaction between different concentrations of two cryoprotectants - glycerol (GLY) and dimethylacetamide (DMA) - and two methods of cryopreservation - pellets produced by plunging into liquid nitrogen and gradual in-straw freezing - on frozen/thawed chicken sperm variables. Sperm was cryopreserved using: (i) 6% DMA, following the in-straw and the pellet methods (ii) 11% GLY, following the in-straw and the pellet methods; and (iii) 8% GLY in the in-straw method and 3% DMA in the pellet method (i.e. reduced cryoprotectant concentrations). When 6% DMA was used as the cryoprotectant, no differences were seen between the in-straw and pellet methods in terms of frozen/thawed sperm variables or fertility (10.8% and 12.8%, respectively). The viability and motility variables of the frozen/thawed sperm produced using the in-straw method with 11% GLY were higher (p < 0.05) than those recorded for the sperm preserved using the same cryoprotectant and concentration in the pellet method. However, fertility was extremely low in both groups (2.1% and 4.2% for the in-straw and pellet methods, respectively). Finally, the use of 8% GLY in the in-straw method returned higher sperm viability, intact acrosome and motility values than the use of 3% DMA in the pellet method (p < 0.01). No differences were seen, however, in the fertility results obtained (28.8% and 25.0%, respectively). These results suggest that cryoprotectant concentrations can be reduced and still provide acceptable fertility rates.

Influence of Staining Method on the Values of Avian Sperm Head Morphometric Variables.

Computer-assisted systems for the assessment of sperm morphometry (ASMA systems) have been used successfully with several mammalian species. Unfortunately, they have so far been of little use for assessing bird semen, a consequence of the filiform shape of avian spermatozoa. This study compares two staining techniques (Hemacolor(®) and aniline blue staining) for the morphometric analysis of rooster and red-legged partridge spermatozoa as part of a computer-assisted light microscopy method. For both species, Hemacolor(®) staining provided a significantly higher percentage of measurable cells (93.7 ± 11.7% in roosters and 71.9 ± 15.3% in red-legged partridges). Hemacolor(®) also showed greater repeatability (lower coefficients of variation) for length and area in roosters' sperm and for width in the case of red-legged partridge's sperm. In the roosters, the Hemacolor(®) technique returned significantly (p < 0.05) larger sperm head width and area values than did the aniline blue technique, while the latter resulted in greater sperm head length values (p < 0.05). In the red-legged partridge, no differences were seen in the results for sperm head width and area provided by the two techniques, but aniline blue staining was associated with longer length measurements. In conclusion, the morphometric values recorded differed depending on the staining method and species. However, the Hemacolor(®) technique might be deemed the more appropriate for computerized sperm assessment systems as it provides larger percentages of measureable cells and shows greater repeatability.

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.

Characterization of red-legged partridge (Alectoris rufa) sperm: Seasonal changes and influence of genetic purity.

The general decline in wild Iberian populations of the red-legged partridge (Alectoris rufa) has been accompanied by an increase in game-farm facilities producing hybrids with chukar partridges (Alectoris chukar). Genetic introgression from chukar partridges is thought to modify male red-legged partridge reproductive indicators. The aim of the present study was to determine the effects of such genetic introgression on seasonal reproductive patterns by comparing the sperm and plasma testosterone concentrations of males from pure red-legged and hybrid red-legged/chukar populations. Semen was collected twice monthly over a 12-mo period using a massage technique. Both types of bird showed a clear seasonal pattern of spermatogenic activity. The proportion of males ejaculating sperm was higher (P<0.05) among the pure red-legged birds. The greatest sperm production was recorded in March to May among the pure birds and April to May among the hybrids. Reproductive activity in both groups decreased in June, to reach a minimum in August to December among the hybrids and in September to December among the pure birds. Spermatogenic activity resumed in January in both groups. The sperm concentration produced by the pure birds was smaller than that of the hybrids (P<0.001), but the percentage of motile sperm was higher in the pure birds (P<0.001). The sperm of the hybrids showed greater straight-line velocity (P<0.05), linearity (P<0.001), straightness (P<0.001), sperm wobble (P<0.05), and beat-cross frequency values (P<0.001). The length and area of the sperm head were smaller in the pure birds (P<0.05). The seasonal plasma testosterone concentration pattern followed a trend roughly parallel to the ejaculatory response. The present results suggest that genetic introgression influences the reproductive variables of the red-legged partridge.

Role of sperm velocity variables associated with poultry breed in 'last male precedence'.

It is well known that when a hen mates with multiple roosters, it is the sperm of the last male that usually fertilizes most of the eggs ('last male precedence'). Sperm quality varies between males within a breed, but also between breeds, and thus, sperm competitiveness after mating may depend on the breeds of the roosters involved. The aim of the present work was to identify differences in sperm competitiveness between breeds, especially with respect to motility. A multibreed mating model was used. Blue Andaluza (BA) and Black Castellana (BC) hens left for 21 days with BA and BC roosters, respectively, were then left with Black-barred Andaluza (Bb) roosters for another 21 days (experimental groups hBA-rBC-rBb and hBC-rBA-rBb). Bb roosters (as the second breed replacing the first) fertilized the majority of eggs in both the hBC-rBA-rBb and hBA-rBC-rBb groups. The percentage of offspring sired by BA roosters (8.0%) was higher (p < 0.05) than the percentage of chicks sired by BC roosters (2.1%). The fertility of the BC hens in the hBC-rBA-rBb group was higher (p < 0.01) than that of the BA hens in the hBA-rBC-rBb group. No difference in sperm concentration was seen between the breeds. Within the rapid sperm subpopulation (sperm velocity, >50 µm/s), Bb sperm showed a higher straight-line velocity (VSL) and average path velocity (VAP) (p < 0.05) than BC sperm. The VSL and VAP values for Bb and BA sperm were similar. In conclusion, the present results show that the sperm of the BA breed, traditionally regarded as of moderate fertility, compensates for this drawback via sperm movement characteristics that afford it an advantage in competition scenarios involving males of other breeds. The VSL and VAP of the rapid sperm subpopulation may play the most important role in securing last male precedence.

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.

Influence of season on the freezability of free-range poultry semen.

The purpose of the present study was to examine the seasonal variation in freezing damage in free-range rooster sperm. Over a period of 1 year, heterospermic semen samples were collected weekly by massage from the roosters of 14 Spanish chicken breeds, all housed under natural photoperiod and climatic conditions. All samples were frozen in straws using DMA as a cryoprotectant, placing them first in nitrogen vapour and then plunging them into liquid nitrogen. No seasonal effects on fresh sperm quality were found. Neither did season affect the percentage of viable frozen-thawed spermatozoa nor the percentage with an intact acrosome. However, the collection season influenced (p < 0.05) most frozen-thawed sperm motility values. The percentage of immotile frozen-thawed spermatozoa was lower (p < 0.05) in spring-collected sperm than in summer- or autumn-collected samples. The percentage of spermatozoa showing progressive motility was higher in spring-collected sperm compared with winter-, summer- or autumn-collected samples (p < 0.05). The curvilinear velocity (VCL), straight-line velocity (VSL) and average path velocity (VAP) values of spring-collected sperm were also higher (p < 0.05). In conclusion, spring would appear to be the best season for collecting and freezing the semen of free-range Mediterranean chicken breeds.

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.

Relationship among fluctuating asymmetry, morphological traits, and sperm quality in layers.

The aim of the present study was to analyze the relationship between fluctuating asymmetry and the mean value of several bilateral traits (toe, leg, wing, and wattle lengths, and leg width) and sperm quality variables (volume, appearance, concentration, motility, morphological abnormalities, and acrosome and membrane integrities) in different breeds of layers. In total, 165 roosters were sampled at 36 wk of age. There was no significant correlation between fluctuating asymmetry and sperm quality variables. There was a positive association (P < 0.01) between sperm motility and concentration, and between sperm concentration and volume. Sperm motility was positively correlated (P < 0.001) with morphologically normal sperm. The membrane integrity did not correlate with motility. This study shows different results for the association between sperm quality variables and morphological traits. There was a negative association (P < 0.05) between toe length and membrane integrity and between leg length and sperm motility. In disagreement with the expected results, wattle length did not correlate with any sperm quality parameter. In general, roosters with longer legs had longer toes and wings, whereas those with longer wattles had longer toes, legs, and wings. Breed effect was significant for the sperm motility index (P < 0.001), concentration and membrane integrity (P < 0.05), and morphologically normal sperm (P < 0.01). Spanish breeds had similar sperm quality variables to the White Leghorn population, although the Blue Andaluza (a Spanish breed) showed smaller values for sperm motility index and concentration and membrane integrity. Breed effect was not significant for any fluctuating asymmetry, with values ranging from 1 to 6% of the trait value; however, breed effect was significant for all of the morphological traits (P < 0.001). The CV was higher for ejaculate volume and concentration, intermediate for semen appearance, motilities, and morphologically normal sperm, and smaller for membrane and acrosome integrity. In conclusion, the results suggest that fluctuating asymmetry is of limited use for assessment of rooster sperm quality. Some morphological traits (toe length, leg length, and leg width) might be associated with some sperm quality variables (motility and membrane integrity).

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.

Semen cryopreservation for the creation of a Spanish poultry breeds cryobank: optimization of freezing rate and equilibration time.

A sperm cryopreservation protocol requiring dimethylacetamide (DMA, 6%) as a cryoprotectant was optimized via assays involving different prefreezing equilibration times (1, 10, 30, 60, and 120 min at 5°C) and different freezing rates achieved by the following: 1) using nitrogen vapor to reduce the temperature from 5°C to -85°C at 10°C/min (slow freezing rate); 2) using a biological freezer unit in a 2-step method to reduce the temperature from 5°C to -35°C at 7°C/min and then from -35°C to -140°C at 60°C/min (medium freezing rate); or 3) using a biological freezer unit in a 1-step freezing method to reduce the temperature from 5°C to -180°C at 60°C/min (rapid freezing rate). Heterospermic semen samples from chicken breeds raised as part of a Spanish genetic resource conservation program were used in all assays. The 1-min equilibration treatment was associated with a lower percentage of viable thawed spermatozoa than the 30-min treatment (P < 0.05). The remaining sperm variables studied were not affected by equilibration time. The medium-rate 2-step freezing method was associated with a higher percentage of motile spermatozoa after thawing and with greater acrosome integrity (P < 0.05) than the slow nitrogen vapor or rapid 1-step methods. Thawed sperm movement quality and plasma membrane integrity (as assessed by the hypoosmotic swelling test) were better (P < 0.05) in samples frozen by the medium-rate 2-step freezing method than in those subjected to the slow nitrogen vapor method. Fertility was not influenced by freezing method, although that achieved with the medium rate 2-step freezing method showed a trend toward being greater than that achieved with the rapid 1-step method (P = 0.07). Together, the present results suggest that slow cooling rates are not recommendable when using dimethylacetamide. The 2-step freezing method may be useful in the establishment of a germplasm bank for Spanish chicken breeds.

Effect of heat and several additives related to stress levels on fluctuating asymmetry, heterophil:lymphocyte ratio, and tonic immobility duration in White Leghorn chicks.

The purpose of this study was to analyze the effects of heat and several additives related to stress on fluctuating asymmetry (groups 1 to 10), heterophil:lymphocyte ratio (groups 1 to 3 and 8 to 10), and tonic immobility duration (groups 1 to 7 and 10) in White Leghorn chicks at 42 d of age. Chicks in group 1 (heat) were reared with temperatures 8°C greater than those of the control group. Groups 2 to 9 consisted of chicks reared with temperatures 8°C greater than those of the control group and addition of capsaicin, allicin, ascorbic acid, tryptophan, brewer's yeast, lactic acid, corticosterone, or cholesterol in diet. Chicks in group 10 (control) were reared with standard temperatures. Heat effect was significant (P<0.05) for the heterophil:lymphocyte ratio, which was greater in heat-stressed chicks without any additives and smaller in control chicks. There were no significant differences for the fluctuating asymmetry and the tonic immobility duration between both groups. Heterophil:lymphocyte ratio for heat-stressed chicks with capsaicin or allicin was significantly lower (P<0.05) than that of heat-stressed chicks without any additives. Capsaicin effect was not significant for the fluctuating asymmetry and the tonic immobility duration, whereas allicin significantly increased fluctuating asymmetry of wing length and tonic immobility duration (P<0.05). The addition of lactic acid or corticosterone resulted in greater fluctuating asymmetry of wing length of heat-stressed chicks (P<0.05). In conclusion, an increased heterophil:lymphocyte ratio was found in heat-stressed chicks without additives, indicating that it is a more reliable indicator of the effect of heat in chicks. In addition, dietary capsaicin or allicin supplementation was effective to alleviate the stress induced by the high temperature, as indicated by a lower heterophil:lymphocyte ratio.

Associations among fluctuating asymmetry, duration of tonic immobility, heterophil-to-lymphocyte ratio, and one-legged standing, crooked toes, or footpad dermatitis in chickens.

The purpose of this study was to analyze the associations among fluctuating asymmetry, duration of tonic immobility, heterophil-to-lymphocyte ratio, and one-legged standing, crooked toes, or footpad dermatitis in chickens. In experiment 1, cocks (n = 96; 36 wk old) from 11 Spanish breeds and a White Leghorn population that showed one-legged or normal standing were used. There were no significant differences for the relative fluctuating asymmetry, the duration of tonic immobility, and the heterophil-to-lymphocyte ratio between both groups of cocks, with mean values of one-legged standing birds being similar to those of normal birds. Thus, this pain-related behavior is not associated with some measures of well-being, fear, and stress. In experiment 2, cocks (n = 106; 36 wk old) from 9 Spanish breeds and the White Leghorn population that showed crooked or normal toes were used. Group effect was not significant for the relative fluctuating asymmetry and duration of tonic immobility. Heterophil-to-lymphocyte ratio and heterophil number were significantly greater (P < 0.001) in cocks with crooked toes and smaller in cocks with normal toes, with the opposite being true for lymphocyte number. Thus, this leg problem is associated with the stressfulness of birds and does not contribute to their well-being or fear levels. In experiment 3, cocks (n = 68; 36 wk old) from 5 Spanish breeds with or without footpad dermatitis were used. Group effect was significant for the relative asymmetry of toe length (P < 0.05), the relative asymmetry of cocks with footpad dermatitis being larger. Group x breed interaction was significant for the relative asymmetry of wattle length (P < 0.001), with the difference between cocks with footpad dermatitis and normal cocks being significant in one breed. In this breed (Red-Barred Vasca), fluctuating asymmetry of wattle length was significantly greater in cocks with footpad dermatitis, suggesting that this leg problem negatively affects the well-being of cocks.

Effects of moist litter, perches, and droppings pit on fluctuating asymmetry, tonic immobility duration, and heterophil-to-lymphocyte ratio of laying hens.

The purpose of this study was to analyze the effects of moist litter, perches, and droppings pit on the fluctuating asymmetry, the tonic immobility duration, and the heterophil:lymphocyte ratio of laying hens. In experiment 1, hens from 4 Spanish breeds and a White Leghorn population, that had been housed in moist or dry litter pens from 20 wk of age, were used. Treatment effect was significant for the fluctuating asymmetry of toe length (P < 0.05), the fluctuating asymmetry of hens housed in moist litter pens being larger. The duration of tonic immobility was significantly longer (P < 0.01) in hens housed in moist litter pens. Thus, litter moisture is associated with the well-being or fear levels of hens, as indicated by the fluctuating asymmetry and the tonic immobility duration. In experiment 2, hens from the Black Menorca breed, that had been housed in pens with or without perches from 20 wk of age, were used. The fluctuating asymmetry of wattle length and the combined fluctuating asymmetry were significantly greater (P < 0.001) in hens housed in pens without perches. Thus, the presence of perches is associated with the well-being level of birds, as indicated by the fluctuating asymmetry. In experiment 3, hens from 4 Spanish breeds and a White Leghorn population, that had been housed in pens with or without a droppings pit, were used. There was significant difference for the fluctuating asymmetry of leg length (P < 0.001) between hens housed in pens with or without a droppings pit, the fluctuating asymmetry of hens housed in pens without a droppings pit being larger. There were no significant differences for the duration of tonic immobility and the heterophil:lymphocyte ratio between both treatments. Thus, the presence of a droppings pit is associated with the well-being of hens, as indicated by the fluctuating asymmetry, and does not contribute to their fear or stress levels, as indicated by the tonic immobility duration and the heterophil:lymphocyte ratio.

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.

Use of the hypo-osmotic swelling test and aniline blue staining to improve the evaluation of seasonal sperm variation in native Spanish free-range poultry.

The season may affect the values of fresh semen variables and therefore influence the success of cryopreservation. The aim of this study was to improve the evaluation of seasonal changes in semen quality in Spanish Black Castellana roosters maintained under natural environmental conditions. Semen was collected from 11 Black Castellana roosters (housed under natural photoperiod and temperature conditions) by massage twice every month for 12 mo. In addition to determining ejaculate volume, sperm concentration, and sperm motility (the classic sperm variables), we used the hypo-osmotic swelling test to examine the membrane integrity of the spermatozoa. Further, morphological abnormalities and acrosome integrity were assessed via aniline blue staining. Semen volume (P < 0.05), sperm concentration (P < 0.01), and the percentage of spermatozoa with an intact acrosome (P < 0.01) were significantly affected by the season of the year. The annual profile of the percentage of spermatozoa showing acrosome integrity followed a trend roughly parallel to annual variations in temperature (Spearman rank correlation = 0.77, P < 0.01). According to the hypo-osmotic swelling test, membrane integrity fell in July (P < 0.05 compared with all other months), the month of highest temperatures. Aniline blue staining and the hypo-osmotic swelling test provide an easy and useful means of evaluating sperm abnormalities, including acrosome morphology and membrane integrity, and could be easily introduced into routine avian semen quality assessments. The results show that high semen quality is associated with long day photoperiods. Extreme heat or cold appear to exert a negative influence on sperm quality.

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.

Effect of bad collocation of wing tag on feather amelanosis, heterophil-to-lymphocyte ratio, fluctuating asymmetry, and tonic immobility duration in white-faced black spanish hens.

The purpose of the present study was to analyze the effects of bad collocation of the wing tag on feather amelanosis, the heterophil-to-lymphocyte ratio, fluctuating asymmetry, and tonic immobility duration at 140 d of age in hens from the White-Faced Black Spanish breed. A total of 52 females were used. There was a significant difference (P < 0.05) for the heterophil-to-lymphocyte ratio and the tonic immobility duration between groups of females with bad or good collocation of the wing tag, with the ratio being higher and the duration being longer in the former group. Females with bad collocation of the wing tag had significant heterophilia and lymphopenia (P < 0.05). There was a significant difference (P < 0.05) in the fluctuating asymmetry of the middle and hind toe lengths, the combined asymmetry of the 4 toes, the fluctuating asymmetry of the earlobe area, and the combined asymmetry of toe and leg lengths and earlobe and wattle areas, with the asymmetry of birds with bad collocation of the wing tag being larger than that of birds with good collocation of the wing tag. Results indicate that bad collocation of the wing tag negatively affects measures of stress, such as the heterophil-to-lymphocyte ratio, fluctuating asymmetry, and tonic immobility duration.

Effects of housing system and cold stress on heterophil-to-lymphocyte ratio, fluctuating asymmetry, and tonic immobility duration of chickens.

The purpose of this study was to analyze the effect of housing system and cold stress on the heterophil-to-lymphocyte ratio, the fluctuating asymmetry, and the tonic immobility duration of chickens. In experiment 1, hens (n=120; 36 wk old) from 5 Spanish breeds and a White Leghorn population, which had been housed in pens with or without access to an outdoor area from 20 wk of age, were used. The effect of housing system on heterophil-to-lymphocyte ratio varied from breed to breed, differences between housing systems being significant (P<0.05) in 2 breeds. In these breeds (Red-Barred Vasca and Birchen Leonesa), heterophil-to-lymphocyte ratio was significantly greater in hens housed in deep litter. Housing effect was significant for the relative asymmetry of leg length (P<0.01), wattle length (P<0.05), and the combined relative asymmetry (P<0.05), the relative asymmetry of hens housed in deep litter being larger. There was no significant difference for the duration of tonic immobility between hens housed in deep litter or free range. Thus, hens with access to an outdoor area were less stressed than hens without access to an outdoor area, although the fearfulness was similar in both groups of birds. In experiment 2, cocks (n=120; 36 wk old) from 4 Spanish breeds, a synthetic breed, and the White Leghorn population, which had been housed in cages with or without a cold stress (0 to 10 degrees C) from 24 wk of age, were used. Cold x breed interaction was significant for heterophil-to-lymphocyte ratio (P<0.05), differences between cold-stressed and control birds being significant in 2 breeds. In these breeds (Red-Barred Vasca and Buff Prat), heterophil-to-lymphocyte ratio was significantly greater in cold-stressed birds. Cold stress effect was significant for the relative asymmetry of toe length (P<0.001) and the combined relative asymmetry (P<0.05), the relative asymmetry of birds with cold stress being larger than that of control birds. Thus, cold stress seriously negatively affects the welfare of cocks.