Evolutionary Patterns of Modularity in the Linkage Systems of the Skull in Wrasses and Parrotfish.

The concept of modularity is fundamental to understanding the evolvability of morphological structures and is considered a central framework for the exploration of functionally and developmentally related subsets of anatomical traits. In this study, we explored evolutionary patterns of modularity and integration in the 4-bar linkage biomechanical system of the skull in the fish family Labridae (wrasses and parrotfish). We measured evolutionary modularity and rates of shape diversification of the skull partitions of three biomechanical 4-bar linkage systems using 205 species of wrasses (family: Labridae) and a three-dimensional geometric morphometrics data set of 200 coordinates. We found support for a two-module hypothesis on the family level that identifies the bones associated with the three linkages as being a module independent from a module formed by the remainder of the skull (neurocranium, nasals, premaxilla, and pharyngeal jaws). We tested the patterns of skull modularity for four tribes in wrasses: hypsigenyines, julidines, cheilines, and scarines. The hypsigenyine and julidine groups showed the same two-module hypothesis for Labridae, whereas cheilines supported a four-module hypothesis with the three linkages as independent modules relative to the remainder of the skull. Scarines showed increased modularization of skull elements, where each bone is its own module. Diversification rates of modules show that linkage modules have evolved at a faster net rate of shape change than the remainder of the skull, with cheilines and scarines exhibiting the highest rate of evolutionary shape change. We developed a metric of linkage planarity and found the oral jaw linkage system to exhibit high planarity, while the rest position of the hyoid linkage system exhibited increased three dimensionality. This study shows a strong link between phenotypic evolution and biomechanical systems, with modularity influencing rates of shape change in the evolution of the wrasse skull.

Ecomorphology of Neotropical Electric Fishes: An Integrative Approach to Testing the Relationships between Form, Function, and Trophic Ecology.

The relationship between form and function is thought to play an integral role in structuring broad-scale patterns of morphological evolution and resource utilization. In ecomorphological studies, mechanical performance is widely understood to constrain the evolution of form and function. However, the relationship between form, function, and resource utilization is less clear. Additionally, seasonal fluctuations in resource availability may further complicate patterns of resource use. How organisms cope with these complexities, and the effect of these factors on broadscale patterns of morphological evolution is also poorly understood. Here we use three-dimensional geometric morphometrics, biomechanics, stable isotope analysis, and gut-content analysis to study trophic evolution in a clade of riverine-adapted electric fishes from a region with high seasonal variability; the Amazon River. We find significant and phylogenetically structured relationships among measures of trophic ecology and skull shape. We also recover a significant relationship between the mechanical advantage of the mandible and trophic position, where species feeding at higher trophic levels have narrower jaws with lower mechanical advantages, and species feeding at lower trophic levels have deeper jaws with higher mechanical advantages. Our results indicate that selection is driving the evolution of mandible shape and performance toward specialization on different trophic ecologies.

Ecomorfologia de Peixes Elétricos Neotropicais: Uma Abordagem Integrativa para Testar as Relações Entre Forma, Função, e Ecologia Trófica Acredita-se que a relação entre forma e função desempenhe um papel integral na estruturação de padrões de larga-escala de evolução morfológica e utilização de recursos. Em estudos ecomorfológicos, se é amplamente atribuído ao desempenho mecânico a restrição da evolução da forma e da função. Em estudos de sistemas complexos, como mandíbulas de peixes, a ideia de que a morfologia está relacionada ao desempenho é frequentemente estabelecida, mas sua relação com a utilização de recursos é menos clara. Aqui, nós usamos morfometria geométrica tridimensional, biomecânica, análise de isótopos estáveis e análise de conteúdo intestinal para estudar a evolução trófica em um clado de peixes elétricos adaptados a rios, da região da bacia Amazônica. Encontramos relações significativas e estruturadas filogeneticamente entre medidas de ecologia trófica e forma do crânio. Também recuperamos uma relação significativa entre a vantagem mecânica da mandíbula e posição trófica, onde as espécies que se alimentam em níveis tróficos superiores têm mandíbulas mais estreitas com menores vantagens mecânicas, e as espécies que se alimentam em níveis tróficos inferiores têm mandíbulas mais profundas com maiores vantagens mecânicas. Nossos resultados indicam que a seleção está conduzindo a evolução da forma e desempenho da mandíbula para a especialização em diferentes ecologias tróficas. Translated to Portuguese by J.P. Fontenelle (jp.fontenelle@mail.utoronto.ca).

Longevity and mortality in Kennel Club registered dog breeds in the UK in 2014.

BACKGROUND: The domestic dog is one of the most diverse mammalian species, exhibiting wide variations in morphology, behaviour and morbidity across breeds. Therefore, it is not unexpected that breeds should also exhibit variation in mortality and longevity. While shorter longevity per se may not necessarily be a welfare issue, a generally foreshortened lifespan in a breed that is accompanied by a high prevalence of a particular cause of death may reveal potentially serious welfare concerns and highlight scope to improve breed welfare. Survey data gathered directly from owners offer useful insights into canine longevity and mortality that can support the overall evidence base for welfare reforms within breeds. RESULTS: Mortality data on 5663 deceased dogs registered with the UK Kennel Club were collected from an owner-based survey. The most commonly reported causes of death were old age (13.8%), unspecified cancer (8.7%) and heart failure (4.9%); with 5.1% of deaths reported as unknown cause. Overall median age at death was 10.33 years (interquartile range: 7.17-12.83 years). Breeds varied widely in median longevity overall from the West Highland Terrier (12.71 years) to the Dobermann Pinscher (7.67 years). There was also wide variation in the prevalence of some common causes of death among breeds, and in median longevity across the causes of death. CONCLUSION: Substantial variation in the median lifespan and the prominent causes of death exists across breeds. This study has identified some breeds with both a low median lifespan and also a high proportional mortality for one or more specific causes of death that should be considered as both potential welfare concerns as well as opportunities for improvement.

Preventing skeletal and neurological problems in guide dogs for the blind and other working dogs.

Skeletal and neurological problems consistently rank among the top health conditions necessitating the withdrawal or retirement of guide dogs and other working dogs from their service roles. Management practices such as weight management and physical conditioning may help to reduce the incidence of such conditions. However, where there is at least partial genetic aetiology, selective breeding strategies are the only way to produce a lasting reduction in their incidence in future generations. The management of single gene disorders is relatively simple through the judicious use of DNA test results. Breeding strategies to tackle complex diseases, into which category many skeletal and neurological diseases fall, are more challenging but can be successful through the use of data collected through screening schemes and, most efficiently, through the use of estimated breeding values.

Les troubles squelettiques et neurologiques comptent parmi les principaux problèmes de santé imposant la réforme ou la mise à la retraite des chiens guide d'aveugles et autres chiens de travail. Certaines pratiques de bonne gestion veillant notamment à contrôler le poids et la condition physique des chiens peuvent contribuer à réduire l'incidence de ce type de problèmes. Toutefois, lorsque l'étiologie des problèmes de santé est totalement ou partiellement déterminée par la génétique, les stratégies de sélection constituent le seul moyen de réduire durablement cette incidence pour les générations futures. La gestion de certains troubles dus à un gène unique est relativement aisée, grâce à un emploi judicieux des résultats de tests ADN. Lorsqu'il s'agit de maladies plus complexes, catégorie recouvrant nombre d'infections squelettiques et neurologiques, les stratégies de sélection sont plus difficiles à mettre en oeuvre mais elles peuvent réussir si l'on procède à une collecte de données via des dispositifs de dépistage, ou en recourant aux valeurs d'élevage estimées, qui déterminent encore plus efficacement le potentiel génétique d'un chien.

Los problemas esqueléticos y neurológicos se sitúan sistemáticamente entre las principales dolencias que hacen necesario retirar o jubilar a los perros lazarillos y otros perros de trabajo de las tareas que cumplen al servicio del ser humano. Hay procedimientos de gestión, como la regulación del peso o el condicionamiento físico, que pueden ayudar a reducir la incidencia de este tipo de trastornos. Sin embargo, en los casos en que hay, cuando menos parcialmente, una etiología genética, las estrategias de cría selectiva son el único modo de conseguir una reducción duradera de su incidencia en las generaciones futuras. La gestión de trastornos ligados a un solo gen es relativamente sencilla, pues pasa por un uso juicioso de los resultados de las pruebas de ADN. Las estrategias de cría selectiva para combatir enfermedades complejas, categoría en la que entran muchas enfermedades esqueléticas y neurológicas, plantean mayor dificultad, pero pueden deparar buenos resultados cuando se utilizan datos obtenidos con programas de cribado sistemático y ser de lo más eficaces cuando se aplican empleando valores estimados de cría.

In vitro sperm quality and DNA integrity of SexedULTRA™ sex-sorted sperm compared to non-sorted bovine sperm.

SexedULTRA™ is an improved method of sex-sorting sperm creating a less damaging environment to retain sperm integrity through the sorting process. The aim of this study was to evaluate the in vitro characteristics of fresh and frozen bovine sperm using the SexedULTRA™ method, and compare it to conventional (non-sorted) sperm. For both methods, percent total sperm motility was estimated visually and also classified into total and progressively motile using a computer assisted sperm analyzer (CASA). Percent sperm with intact plasma membranes (VIA) and acrosomes (PIA) were assessed using flow cytometry and sperm DNA fragmentation index (DFI) was estimated using the Bull sperm Halomax® Kit. Two contemporaneous ejaculates from 10 bulls were processed and cryopreserved using one of the two procedures (SexedULTRA™ and conventional). Sperm motility, VIA and PIA were assessed post-thaw (0 h) and post-incubation (3 h at 37 °C, 8 h and 24 h at 18 °C). DFI was analyzed post-thaw (0 h) and after 6, 24, 48 and 72 h of incubation at 37 °C. In a second experiment, ejaculates from 7 bulls were split sampled into the two types of processing (SexedULTRA™ and conventional) and diluted using a fresh semen extender developed for sex-sorted bovine sperm. Sperm quality was assessed after dilution (0 h) and after incubation for 12, 24, 48, 72 h at 18°, and the same time points of incubation at 37 °C for DFI. Frozen-thawed SexedULTRA™ sperm was significantly (P < 0.05) better than conventional semen after a 3 h incubation at 37 °C for PIA, and after a 24 h incubation at 18 °C for percent visual motility and PIA. DFI was significantly lower for SexedULTRA™ compared to conventional at all time points of incubation (37 °C). Fresh SexedULTRA™ sperm showed improved quality compared to conventional at all time points of incubation at 18 °C for percent visual and total motile sperm, VIA, PIA, and DFI. Significant differences were also found in progressive motile sperm immediately after dilution (0 h), but not at any time point after incubation. The results show that the SexedULTRA™ process maintains the quality of sex-sorted sperm and, in many cases, has better in vitro longevity than conventional semen.

Large-scale survey to estimate the prevalence of disorders for 192 Kennel Club registered breeds.

BACKGROUND: Pedigree or purebred dogs are often stated to have high prevalence of disorders which are commonly assumed to be a consequence of inbreeding and selection for exaggerated features. However, few studies empirically report and rank the prevalence of disorders across breeds although such data are of critical importance in the prioritisation of multiple health concerns, and to provide a baseline against which to explore changes over time. This paper reports an owner survey that gathered disorder information on Kennel Club registered pedigree dogs, regardless of whether these disorders received veterinary care. This study aimed to determine the prevalence of disorders among pedigree dogs overall and, where possible, determine any variation among breeds. RESULTS: This study included morbidity data on 43,005 live dogs registered with the Kennel Club. Just under two thirds of live dogs had no reported diseases/conditions. The most prevalent diseases/conditions overall were lipoma (4.3%; 95% confidence interval 4.13-4.52%), skin (cutaneous) cyst (3.1%; 2.94-3.27%) and hypersensitivity (allergic) skin disorder (2.7%; 2.52-2.82%). For the most common disorders in the most represented breeds, 90 significant differences between the within breed prevalence and the overall prevalence are reported. CONCLUSION: The results from this study have added vital epidemiological data on disorders in UK dogs. It is anticipated that these results will contribute to the forthcoming Breed Health & Conservation Plans, a Kennel Club initiative aiming to assist in the identification and prioritisation of breeding selection objectives for health and provide advice to breeders/owners regarding steps that may be taken to minimise the risk of the disease/disorders. Future breed-specific studies are recommended to report more precise prevalence estimates within more breeds.

Bayesian QTL analyses using pedigreed families of an outcrossing species, with application to fruit firmness in apple.

KEY MESSAGE: Proof of concept of Bayesian integrated QTL analyses across pedigree-related families from breeding programs of an outbreeding species. Results include QTL confidence intervals, individuals' genotype probabilities and genomic breeding values. Bayesian QTL linkage mapping approaches offer the flexibility to study multiple full sib families with known pedigrees simultaneously. Such a joint analysis increases the probability of detecting these quantitative trait loci (QTL) and provide insight of the magnitude of QTL across different genetic backgrounds. Here, we present an improved Bayesian multi-QTL pedigree-based approach on an outcrossing species using progenies with different (complex) genetic relationships. Different modeling assumptions were studied in the QTL analyses, i.e., the a priori expected number of QTL varied and polygenic effects were considered. The inferences include number of QTL, additive QTL effect sizes and supporting credible intervals, posterior probabilities of QTL genotypes for all individuals in the dataset, and QTL-based as well as genome-wide breeding values. All these features have been implemented in the FlexQTL(™) software. We analyzed fruit firmness in a large apple dataset that comprised 1,347 individuals forming 27 full sib families and their known ancestral pedigrees, with genotypes for 87 SSR markers on 17 chromosomes. We report strong or positive evidence for 14 QTL for fruit firmness on eight chromosomes, validating our approach as several of these QTL were reported previously, though dispersed over a series of studies based on single mapping populations. Interpretation of linked QTL was possible via individuals' QTL genotypes. The correlation between the genomic breeding values and phenotypes was on average 90 %, but varied with the number of detected QTL in a family. The detailed posterior knowledge on QTL of potential parents is critical for the efficiency of marker-assisted breeding.

DNA fragmentation kinetics and postthaw motility of flow cytometric-sorted white-tailed deer sperm.

This study examined DNA damage and postthaw motility of white-tailed deer sperm (n = 28) before and after sex selection and conventional sorting using MoFlo XDP SX flow cytometry. Semen samples from the same individuals were treated in 4 different ways: 1) chilled-extended sperm samples (without glycerol); 2) cryopreserved conventional samples, samples directly cryopreserved after the addition of extenders; 3) cryopreserved conventionally sorted samples, sorted samples to remove the dead sperm subpopulation; and 4) cryopreserved sex-sorted samples; sorted samples to remove the dead sperm subpopulation and separation of X- and Y-chromosome-bearing sperm. In all the cases (n = 6), conventional samples showed decreased postthaw motilities (43 ± 26%) when compared with X-sorted samples (59 ± 20%; P < 0.05) and Y-sorted samples (54 ± 20%; P > 0.05). The DNA fragmentation baseline was <5% for frozen-thawed conventional samples, but even less after sex sorting and conventional sorting: 2.4 and 1.7%, respectively. On the other hand, conventional samples showed greater (P < 0.05) DNA fragmentation than the sex-sorted sperm (n = 6) at 96 h (average of 4.8 ± 4.5% and 5.3 ± 4%, respectively). Conventionally sorted samples (n = 8) did not have greater (P > 0.05) DNA fragmentation when compared with the sex-sorted samples. Fragmentation of DNA on X-chromosome and Y-chromosome-bearing sorted sperm were not significantly different (n = 10, P > 0.05) after 96 h (2.6 ± 3.6% and 2.2 ± 0.5%, respectively). Future research should be implemented for examining the fertilizing potential of sex-sorted white-tailed deer sperm (e.g., AI fertility trials).

Sex-sorted bovine spermatozoa and DNA damage: I. Static features.

This study examined the static response of Spermatozoa DNA Fragmentation (SDF) after sex selection in bulls using a MoFlo(®) SX (Beckman Coulter, Miami FL) spermatozoa sorter to produce three different subpopulations: 1) Spermatozoa bearing X- chromosomes with a purity of 95%, 2) Spermatozoa bearing Y-chromosomes with a purity of 95%, and 3) non-viable spermatozoa. The static response of SDF refers to the baseline values observed for DNA damage when analyzed pre- and post sex-sorting. Results showed that while the baseline level SDF in pre-sorted bull spermatozoa samples ranged from 5.3% to 11% with an average of 7.9% ± 2.1%, the level of SDF obtained in X- and Y-chromosome sorted samples was much lower (3.1% ± 1.9%) and statistical differences were obtained after comparing both groups (P < 0.01). Spermatozoa containing a fragmented DNA molecule tend to be accumulated in the non-viable subpopulation. The baseline SDF level in X- and Y-chromosome sorted subpopulations is reduced, by 63% on average when compared to the values obtained in the neat semen sample. Different bulls exhibit unique SDF reduction efficiencies via the X- and Y-chromosome sex selection process.

Sex-sorted bovine spermatozoa and DNA damage: II. Dynamic features.

This study examined the dynamic response of Spermatozoa DNA Fragmentation after sex selection in bulls using a MoFlo(®) SX (Beckman Coulter, Miami FL) spermatozoa sorter. The dynamic response of spermatozoa DNA fragmentation refers to the changing values of SDF, i.e., rate of SDF (rSDF), when analyzed periodically over a set incubation time at 37 °C. A dynamic assessment of SDF using non-sorted and sex-sorted spermatozoa samples during 72 h of incubation at 37 °C was performed. Results showed a reduced DNA longevity in sex-sorted frozen-thawed spermatozoa, with spermatozoa DNA damage appearing between 24 h and 48 h. The baseline SDF level was higher in conventional frozen-thawed than in sex-sorted frozen-thawed spermatozoa samples; while the reverse occurred for the rSDF. The afore-mentioned result produced a crossover point between both dynamic tendencies of SDF for sex-sorted versus conventional samples. We defined this crossover point as the Crossover Positioning Time (CPT) or the time (in hours) where both curves crossover after a period of spermatozoa incubation at 37 °C. The point at which the CPT occurs could be used as an indicator of the rSDF for individual bulls after X- and Y-chromosome bearing spermatozoa selection. CPT values produced a window of SDF ranging between 24 h and 48 h in the present experiment. It is proposed that higher values for CPT are indicative of bulls presenting chromatin that is more resistant to the external stressors affecting spermatozoa DNA after spermatozoa sorting.

Methods and mortality results of a health survey of purebred dogs in the UK.

OBJECTIVES: To collect information on the cause of death and longevity of dogs owned by members of the numerically largest breed clubs of 169 UK Kennel Club-recognised breeds. METHODS: A cross-sectional study was carried out. Approximately 58,363 questionnaires were sent out to breed club members in 2004 (nine clubs failed to report the exact number of questionnaires sent out). Owners reported age at death and cause(s) of death for all dogs that had died within the previous 10 years. RESULTS: A total of 13,741 questionnaires (24% response rate) containing information on 15,881 deaths were included in the analysis. Breed-specific response rates ranged from 64·7 to 4·5%. The median age at death was 11 years and 3 months (minimum=2 months, maximum=23 years and 5 months) and it varied by breed. The most common causes of death were cancer (n=4282, 27%), "old age" (n=2830, 18%) and cardiac conditions (n=1770, 11%). CLINICAL SIGNIFICANCE: This survey shows breed differences in lifespan and causes of death, and the results support previous evidence that smaller breeds tend to have longer lifespan compared with larger breeds. Although many of the breeds in the study may not be representative of the general pedigree dog population in the UK, the results do contribute to the limited information currently available.

Advances in flow cytometry for sperm sexing.

This review presents the key technological developments that have been implemented in the 20 years since the first reports of successful measurement, sorting, insemination and live births using flow cytometry as a proven physical sperm separation technique. Since the first reports of sexed sperm, flow technology efforts have been largely focused on improving sample throughput by increasing the rate at which sperm are introduced to the sorter, and on improving measurement resolution, which has increased the proportion of cells that can be reliably measured and sorted. Today, routine high-purity sorting of X- or Y-chromosome-bearing sperm can be achieved at rates up to 8000 s(-1) for an input rate of 40,000 X- and Y-sperms(-1). With current protocols, straws of sex-sorted sperm intended for use in artificial insemination contain approximately 2 x 10(6)sperm. The sort rate of 8000 sperms(-1) mentioned above corresponds to a production capacity of approximately 14 straws of each sex per hour per instrument.

Targeting invasion and egress: from tools to drugs?

A recent resurgence in the use of compounds to study essential biological processes raises important questions concerning the link between fundamental research and drug development. This article discusses many of the issues involved, in the context of host cell invasion and egress by parasites of the Phylum Apicomplexa. In addition, an overview of the key steps in invasion and egress is provided with a particular emphasis on potential parasite protein drug targets.

A challenging case of malignant hypercalcaemia.

The investigation and management of patients presenting with hypercalcaemia is not always straightforward. We describe the case of a middleaged man presenting with severe symptomatic hypercalcaemia. The difficulties encountered here - in striving for biochemical and symptom control, and in establishing a definite diagnosis - are fortunately rare. The case illustrates the range of options in the treatment of severe hypercalcaemia, and highlights the use of haemodialysis in treatmentresistant cases.

Flow cytometric sorting of fresh and frozen-thawed spermatozoa in the western lowland gorilla (Gorilla gorilla gorilla).

We adapted flow cytometry technology for high-purity sorting of X chromosome-bearing spermatozoa in the western lowland gorilla (Gorilla gorilla gorilla). Our objectives were to develop methodologies for liquid storage of semen prior to sorting, sorting of liquid-stored and frozen-thawed spermatozoa, and assessment of sorting accuracy. In study 1, the in vitro sperm characteristics of gorilla ejaculates from one male were unchanged (P > 0.05) after 8 hr of liquid storage at 15 degrees C in a non-egg yolk diluent (HEPES-buffered modified Tyrode's medium). In study 2, we examined the efficacy of sorting fresh and frozen-thawed spermatozoa using human spermatozoa as a model for gorilla spermatozoa. Ejaculates from one male were split into fresh and frozen aliquots. X-enriched samples derived from both fresh and frozen-thawed human semen were of high purity, as determined by fluorescence in situ hybridization (FISH; 90.7%+/-2.3%, overall), and contained a high proportion of morphologically normal spermatozoa (86.0%+/-1.0%, overall). In study 3, we processed liquid-stored semen from two gorillas for sorting using a modification of methods for human spermatozoa. The sort rate for enrichment of X-bearing spermatozoa was 7.3+/-2.5 spermatozoa per second. The X-enriched samples were of high purity (single-sperm PCR: 83.7%) and normal morphology (79.0%+/-3.9%). In study 4 we examined frozen-thawed gorilla semen, and the sort rate (8.3+/-2.9 X-bearing sperm/sec), purity (89.7%), and normal morphology (81.4%+/-3.4%) were comparable to those of liquid-stored semen. Depending on the male and the type of sample used (fresh or frozen-thawed), 0.8-2.2% of gorilla spermatozoa in the processed ejaculate were present in the X-enriched sample. These results demonstrate that fresh or frozen-thawed gorilla spermatozoa can be flow cytometrically sorted into samples enriched for X-bearing spermatozoa.

Identification of molecular markers linked to the mildew resistance gene Pl-d in apple.

Powdery mildew poses a serious problem for apple growers, and resistance to the disease is a major objective in breeding programmes for cultivar improvement. As selective pressure allows pathogens to overcome previously reliable resistances, there is a need for the introduction of novel resistance genes into new breeding lines. This investigation is concerned with the identification of the first set of molecular markers linked to the gene for mildew resistance, Pl-d, from the accession 'D12'. As no prior information on the map position or markers for Pl-d were available, a bulked-segregant approach was used to test 49 microsatellite primers, 176 amplified fragment length polymorphism (AFLP) primers and 80 random amplified polymorphic DNA (RAPD) primers in a progeny segregating for Pl-d resistance, 'Fiesta' (susceptible) x A871-14 ('Worcester Pearmain' x 'D12'). The segregations of the markers identified in the resistant and susceptible bulks were scored in the progeny, then the recombination fractions between Pl-d and the most tightly linked markers were calculated and a map prepared. Three AFLP, one RAPD and two microsatellite markers were identified. One AFLP was developed into a sequence-characterised amplified region marker, while the microsatellites CH03C02 and CH01D03 were flanking markers, 7 and 11 recombination units, respectively, from Pl-d. Two more distant microsatellites on the same linkage group, CH01D09 and CH01G12, confirmed the orientation of the markers on the linkage group. These microsatellites place Pl-d on the bottom of linkage group 12 in published apple maps, a region where a number of other disease resistance genes have been identified.

Birth of lambs of a pre-determined sex after in vitro production of embryos using frozen-thawed sex-sorted and re-frozen-thawed ram spermatozoa.

UNLABELLED: The characteristics and functional capacity of ram spermatozoa frozen-thawed prior to and after flow cytometric sorting was assessed after incubation (37 degrees C; 6 h), in vitro fertilisation (IVF), and transfer of fresh and vitrified in vitro produced embryos. Frozen-thawed spermatozoa from two rams were allocated to four treatment groups: (i) non-sorted (CONTROL); (ii) sorted (FS); (iii) sorted then re-frozen (FSF) and (iv) re-frozen control (FCF). Frozen-thawed samples were separated into X- and Y-chromosome bearing spermatozoa using a high-speed sperm sorter after density gradient centrifugation (X: 88 +/- 1.5% and Y: 87 +/- 1.1% purity). After 6 h incubation (37 degrees C), the percentage of motile spermatozoa was higher (P < 0.001) for FS (84 +/- 2.0%) compared with all other treatments ( CONTROL: 36 +/- 3.3%, FSF: 28 +/- 3.1%, FCF: 20 +/- 2.0%). In a sperm migration test greater numbers of FS spermatozoa penetrated 5 mm into the artificial cervical mucus compared with spermatozoa from all other treatments (152 +/- 39.4 vs 31 +/- 9.2 spermatozoa respectively; P < 0.05). Fertilisation and cleavage rates were higher (P < 0.05) for in vitro matured oocytes inseminated with CONTROL compared with FSF spermatozoa. However, the Day 7 blastocyst development rate was higher for oocytes inseminated with FSF (62.2%) than FS and CONTROL spermatozoa (52.7 and 50.0%; P < 0.05). The number of ewes pregnant (Day 60), lambing and the in vivo embryo survival rate was greater (P < 0.01) after the transfer of fresh embryos rather than vitrified embryos derived from X- and Y-spermatozoa (67.6, 64.7 and 41.2% vs 29.6, 25.9 and 14.8% respectively). Twenty-six of the 30 (86.7%) lambs derived from sex-sorted spermatozoa were of the correct sex. These results demonstrate that frozen-thawed ram spermatozoa can be sex-sorted for immediate or future use after re-cryopreservation and, in conjunction with IVF and embryo transfer, can be used to efficiently produce offspring of pre-determined sex.

Identification of SCAR markers linked to Pl-w mildew resistance in apple.

Resistance to powdery mildew is an important objective for cultivar improvement programmes of apple and several different major genes for resistance to mildew are available. Molecular markers linked to such key traits can be used to screen progenies for resistant individuals. A progeny derived from the crab apple 'White Angel' (the source of Pl-w) was screened for resistance to mildew for two seasons in the glasshouse and four seasons in the field. DNA bulks of resistant and susceptible seedlings were screened with 176 AFLP primer combinations. Seven AFLP markers were identified that differentiated the bulks, and two of these markers were developed into SCARs, EM M01 and EM M02, mapping at 4.6 and 6.4 recombination units from Pl-w.

Flow cytometric sorting of frozen-thawed spermatozoa in sheep and non-human primates.

Research was conducted in sheep to determine an effective preparation method for high-purity sorting of frozen-thawed spermatozoa. The efficacy of sorting frozen-thawed spermatozoa was then investigated in several non-human primate species. An aliquot of each ejaculate (three rams, three ejaculates per ram) was processed as a fresh control (FRESH). Frozen spermatozoa were thawed and prepared for sorting by no further processing (FT-NEAT), washing (FT-WASH) or gradient centrifugation (FT-GRADIENT) and evaluated for motility at 1 h post-staining and motility and acrosomal status at 0 and 4 h post-sorting. Samples were analysed using a high-speed cell sorter. High levels of purity for X- and Y-enriched samples were achieved for all treatments (85-92%). The percentage of motile spermatozoa before sorting was lower (P < 0.05) for frozen-thawed samples (FT-NEAT: 32.7 +/- 2.5%; FT-WASH: 32.2 +/- 3.3%; FT-GRADIENT: 73.9 +/- 3.7%) compared with FRESH (83.3 +/- 1.2%). Post-sorting, the percentage of motile spermatozoa before and after incubation for FT-NEAT (60.0 +/- 5.1% and 27.2 +/- 6.1% for 0 and 4 h, respectively) was lower than that for FRESH (87.8 +/- 0.9% and 83.3 +/- 1.2% for 0 and 4 h, respectively; P < 0.05), FT-WASH (80.0 +/- 2.4% and 71.7 +/- 3.6% for 0 and 4 h, respectively; P < 0.05) and FT-GRADIENT (84.4 +/- 1.3% and 77.2 +/- 1.7% for 0 and 4 h, respectively; P < 0.05). Vanguard sperm migration distance through artificial cervical mucus was lower (P < 0.05) for FT-NEAT (17.7 +/- 1.7 mm) compared with FT-WASH (29.1 +/- 3.8 mm) and FT-GRADIENT (28.4 +/- 2.0 mm) and similar (P < 0.05) to FRESH (23.7 +/- 1.8 mm). Sample preparation using a modified wash method enabled high-purity sorting (range 86-97% purity) of frozen-thawed epididymal spermatozoa in the baboon (Papio hamadryas), common marmoset (Callithrix jacchus) and common chimpanzee (Pan troglodytes). For all non-human primate species, sorted spermatozoa were progressively motile (marmoset: 20.5 +/- 5.5%; baboon: 37.5 +/- 2.5%; chimpanzee: 73.0 +/- 2.0%), acrosome intact (marmoset: 68.5 +/- 7.5%; baboon: 89.5 +/- 1.5%; chimpanzee: 84.0 +/- 1.0%) and able to penetrate an artificial cervical mucus. In summary, high-purity sorting of frozen-thawed ram and non-human primate spermatozoa with recovery of progressively motile, acrosome-intact spermatozoa was possible after processing to remove cryodiluent.

Biomechanical and histopathological changes in the support structures of bovine hooves around the time of first calving.

Hooves were removed from heifers killed two weeks before calving (C-2), and four and twelve weeks post-calving (C+4 and C+12), and from age-matched maiden heifers. Segments were dissected from the anterior walls of lateral hind claws, to include horn, corium and bone, and the biomechanical properties were measured. There was reduced rigidity, both overall and at a physiologically appropriate displacement of 2 mm, and a greater displacement was required to reach initial and maximum support in the hooves of calving, compared with maiden, heifers. Histological examination revealed widened and distorted interdigitating laminae, progressing to C+12 in the hind claws, but with evidence of recovery in the front claws after C+4. Increased connective tissue strength from dorsal to ventral hoof segments was correlated with protein, proteoglycan, pro- and activated matrix metalloproteinase-2, and tissue inhibitors of metalloproteinases, and was inversely correlated with fat, water and collagen content. This implies that mechanical changes reflect alterations in the biochemistry of the connective tissue. This evidence supports the hypothesis that primary causal events associated with calving weaken the connective tissue of the hoof suspensory apparatus, leading to increased susceptibility to clinical lameness associated with sole ulcers and white line disease.