Freezing chicken semen: Influence of base medium osmolality, cryoprotectants, cryoprotectant concentration, and cooling rate on post-thaw sperm survival.

A classical chicken semen diluent (Lake's 7.1 diluent) was modified to have lowered osmolalities (ranging from 290 to 410 mOsm/kg). The modified medium with physiological osmolality of 325 mOsm/kg allowed cold storage of fresh semen for several days with very little loss of membrane integrity and motility, while high osmolalities inhibited motility. This modified medium was then used as base for freezing medium to test effects of the type and concentration of cryoprotective agent (CPA), and the cooling rate (CR). A number of CPAs (methylformamide, methylacetamide, dimethylformamide (DMF), dimethylacetamide (DMA), diethylformamide, and propylene glycol) were first compared by freezing semen with 0.6 mol/l of the respective CPA at a cooling rate of 250 °C/min. Post-thaw motility and membrane integrity were highest with DMA and DMF. Finally, in more detailed factorial experiments, semen from individual cocks or pooled semen was frozen using CRs of 4, 50, 250, and 440 °C/min and DMA concentrations ([DMA]) of 0.4, 0.6, 1.0, and 1.5 mol/l. Straws from each semen sample x treatment combination were divided for semen assessment at three different research groups for sperm motility, membrane integrity, kinked tails, and DNA fragmentation, using microscopy, computer assisted motility analysis, and flow cytometry. There were clear effects of both CR and [DMA] and their interaction. CRs 50 and 250 °C/min gave best post-thaw sperm performance. Higher DMA concentrations gave better post-thaw membrane integrity, but concentrations above 1.0 mol/l can decrease sperm velocity or even inhibit sperm motility. Therefore [DMA] may best be 0.6-1.0 mol/l at a CR of 50-250 °C/min.

Relatedness between numerically small Dutch Red dairy cattle populations and possibilities for multibreed genomic prediction.

Red dairy breeds are a valuable cultural and historical asset, and often a source of unique genetic diversity. However, they have difficulties competing with other, more productive, dairy breeds. Improving competitiveness of Red dairy breeds, by accelerating their genetic improvement using genomic selection, may be a promising strategy to secure their long-term future. For many Red dairy breeds, establishing a sufficiently large breed-specific reference population for genomic prediction is often not possible, but may be overcome by adding individuals from another breed. Relatedness between breeds strongly decides the benefit of adding another breed to the reference population. To prioritize among available breeds, the effective number of chromosome segments (Me) can be used as an indicator of relatedness between individuals from different breeds. The Me is also an important parameter in determining the accuracy of genomic prediction. The Me can be estimated both within a population and between 2 populations or breeds, as the reciprocal of the variance of genomic relationships. We investigated relatedness between 6 Dutch Red cattle breeds, Groningen White Headed (GWH), Dutch Friesian (DF), Meuse-Rhine-Yssel (MRY), Dutch Belted (DB), Deep Red (DR), and Improved Red (IR), focusing primarily on the Me, to predict which of those breeds may benefit from including reference animals of the other breeds. All of these breeds, except MRY, are under high risk of extinction. Our results indicated high variability of Me, especially between Me ranging from ∼3,500 to ∼17,400, indicating different levels of relatedness between the breeds. Two clusters are especially important, one formed by MRY, DR, and IR, and the other comprising DF and DB. Although relatedness between breeds within each of these 2 clusters is high, across-breed genomic prediction is still limited by the current number of genotyped individuals, which for many breeds is low. However, adding MRY individuals would increase the reference population of DR substantially. We estimated that between 11 and 133 individuals from other breeds are needed to achieve accuracy of genomic prediction equivalent to using one additional individual from the same breed. Given the variation in size of the breeds in this study, the benefit of a multibreed reference population is expected to be lower for larger breeds than for the smaller ones.

Conservation priorities for the different lines of Dutch Red and White Friesian cattle change when relationships with other breeds are taken into account.

From a genetic point of view, the selection of breeds and animals within breeds for conservation in a national gene pool can be based on a maximum diversity strategy. This implies that priority is given to conservation of breeds and animals that diverge most and overlap of conserved diversity is minimized. This study investigated the genetic diversity in the Dutch Red and White Friesian (DFR) cattle breed and its contribution to the total genetic diversity in the pool of the Dutch dairy breeds. All Dutch cattle breeds are clearly distinct, except for Dutch Friesian breed (DF) and DFR and have their own specific genetic identity. DFR has a small but unique contribution to the total genetic diversity of Dutch cattle breeds and is closely related to the Dutch Friesian breed. Seven different lines are distinguished within the DFR breed and all contribute to the diversity of the DFR breed. Two lines show the largest contributions to the genetic diversity in DFR. One of these lines comprises unique diversity both within the breed and across all cattle breeds. The other line comprises unique diversity for the DFR but overlaps with the Holstein Friesian breed. There seems to be no necessity to conserve the other five lines separately, because their level of differentiation is very low. This study illustrates that, when taking conservation decisions for a breed, it is worthwhile to take into account the population structure of the breed itself and the relationships with other breeds.

Genomic prediction of dry matter intake in dairy cattle from an international data set consisting of research herds in Europe, North America, and Australasia.

With the aim of increasing the accuracy of genomic estimated breeding values for dry matter intake (DMI) in Holstein-Friesian dairy cattle, data from 10 research herds in Europe, North America, and Australasia were combined. The DMI records were available on 10,701 parity 1 to 5 records from 6,953 cows, as well as on 1,784 growing heifers. Predicted DMI at 70 d in milk was used as the phenotype for the lactating animals, and the average DMI measured during a 60- to 70-d test period at approximately 200 d of age was used as the phenotype for the growing heifers. After editing, there were 583,375 genetic markers obtained from either actual high-density single nucleotide polymorphism (SNP) genotypes or imputed from 54,001 marker SNP genotypes. Genetic correlations between the populations were estimated using genomic REML. The accuracy of genomic prediction was evaluated for the following scenarios: (1) within-country only, by fixing the correlations among populations to zero, (2) using near-unity correlations among populations and assuming the same trait in each population, and (3) a sharing data scenario using estimated genetic correlations among populations. For these 3 scenarios, the data set was divided into 10 sub-populations stratified by progeny group of sires; 9 of these sub-populations were used (in turn) for the genomic prediction and the tenth was used for calculation of the accuracy (correlation adjusted for heritability). A fourth scenario to quantify the benefit for countries that do not record DMI was investigated (i.e., having an entire country as the validation population and excluding this country in the development of the genomic predictions). The optimal scenario, which was sharing data, resulted in a mean prediction accuracy of 0.44, ranging from 0.37 (Denmark) to 0.54 (the Netherlands). Assuming near-unity among-country genetic correlations, the mean accuracy of prediction dropped to 0.40, and the mean within-country accuracy was 0.30. If no records were available in a country, the accuracy based on the other populations ranged from 0.23 to 0.53 for the milking cows, but were only 0.03 and 0.19 for Australian and New Zealand heifers, respectively; the overall mean prediction accuracy was 0.37. Therefore, there is a benefit in collaboration, because phenotypic information for DMI from other countries can be used to augment the accuracy of genomic evaluations of individual countries.

Functional analysis of inter-individual transcriptome differential expression in pig longissimus muscle.

Selection of pigs for increased meat production or improved meat quality changes muscle mass and muscle composition. This will be related to transcriptome expression profile changes in muscle tissue, generating inter-individual differences. This study investigated the differentially expressed genes in the transcriptome profiles of the longissimus muscle of 75 Large White-Duroc cross sows and castrates. The use of a common reference design enabled to investigate the inter-individual transcriptome expression profile differences between the animals as compared with the means of all animals. The aim of the study was to identify the biological processes related to these inter-individual differences. It was expected that these processes underlie the selection effects. In total, 908 transcripts were differentially expressed. Among them, 762 were mainly downregulated and 146 were mainly upregulated. Gene Ontology and Pathways analyses indicated that the differentially expressed genes belong to three groups of processes involved in protein synthesis and amino acid-protein metabolism, energy metabolism and muscle-specific structure and activity processes. Comparing the functional biological analysis results with previously reported data suggested that the protein synthesis, energy metabolism and muscle-specific structure would contribute to meat production and the meat quality.

A time-series approach for clustering farms based on slaughterhouse health aberration data.

A large amount of data is collected routinely in meat inspection in pig slaughterhouses. A time series clustering approach is presented and applied that groups farms based on similar statistical characteristics of meat inspection data over time. A three step characteristic-based clustering approach was used from the idea that the data contain more info than the incidence figures. A stratified subset containing 511,645 pigs was derived as a study set from 3.5 years of meat inspection data. The monthly averages of incidence of pleuritis and of pneumonia of 44 Dutch farms (delivering 5149 batches to 2 pig slaughterhouses) were subjected to 1) derivation of farm level data characteristics 2) factor analysis and 3) clustering into groups of farms. The characteristic-based clustering was able to cluster farms for both lung aberrations. Three groups of data characteristics were informative, describing incidence, time pattern and degree of autocorrelation. The consistency of clustering similar farms was confirmed by repetition of the analysis in a larger dataset. The robustness of the clustering was tested on a substantially extended dataset. This confirmed the earlier results, three data distribution aspects make up the majority of distinction between groups of farms and in these groups (clusters) the majority of the farms was allocated comparable to the earlier allocation (75% and 62% for pleuritis and pneumonia, respectively). The difference between pleuritis and pneumonia in their seasonal dependency was confirmed, supporting the biological relevance of the clustering. Comparison of the identified clusters of statistically comparable farms can be used to detect farm level risk factors causing the health aberrations beyond comparison on disease incidence and trend alone.

Effects of rate of pH fall, time of deboning, aging period, and their interaction on veal quality characteristics.

Three groups of veal carcasses were selected on the basis of their pH in the longissimus lumborum muscle at 3 h postmortem (pH3) to study the effects and interaction with time of deboning on quality characteristics of veal. The following groups of 10 calves each were selected: 1) fast pH fall, pH3 < 6.2; 2) intermediate pH fall, 6.2 < pH3 < 6.7; and 3) slow pH fall, pH3 > 6.7. Longissimus thoracis et lumborum (LTL) muscles of sides of the selected carcasses were randomly assigned to be excised at either 24 or 48 h postmortem. Color, water-holding capacity, and shear force measurements were determined after an aging period of 2, 3, 4, 7, and 14 d, respectively. Color of longissimus muscle samples from veal calves with similar preslaughter blood hemoglobin values becomes significantly lighter with a faster pH fall. Muscle color was not affected by time of deboning and overall color of all longissimus samples remained stable during storage and was not affected by rate of pH fall and time of deboning. Cooking losses increased with both a faster rate of pH fall and by deboning at 24 h postmortem. Drip losses during vacuum storage were higher for muscles excised at 24 rather than 48 h postmortem. Both when deboned at 24 and 48 h, postmortem veal carcasses with a lower rate of pH fall had higher shear force (SF) values than did carcasses with a higher rate of pH fall. Deboning at 24 h postmortem resulted in higher SF values than deboning at 48 h postmortem. Differences in SF between 24- and 48-h deboning were larger in slower glycolyzing carcasses. Aging improved tenderness but did not fully reduce the difference in SF values between 24- and 48-h deboning. The results suggest that deboning of veal carcasses before the ultimate pH has been reached may result in muscle contraction, which may exert negative effects on tenderness and water-holding capacity of veal. Effects of time of deboning can, at least partly, be explained by differential effects on shortening of the muscle fibers.

Relationships between behavioral and meat quality characteristics of pigs raised under barren and enriched housing conditions.

In this study the effects of barren vs enriched housing conditions of pigs on their behavior during the lairage period (2-h holding period before slaughter), carcass characteristics, postmortem muscle metabolism, and meat quality were studied. The barren housing system was defined by common intensive housing conditions (i.e., with slatted floors and recommended space allowances), whereas the enriched environment incorporated extra space and straw for manipulation. Salivary cortisol concentrations were measured before transport and at the end of the lairage period. During the lairage period the percentage of time spent walking and fighting by the pigs was registered. Carcass characteristics such as weight, meat percentage, and backfat thickness were determined. At 5 min, 45 min, 4 h, and 24 h postmortem, pH, temperature, and lactate concentrations were determined in the longissimus lumborum (LL) and biceps femoris (BF) muscles. Capillarization of the muscle, mean muscle fiber area, and color and drip loss after 2 and 5 d of storage were determined for both muscle types. Pigs from the barren environment had a significantly higher increase in cortisol from farm to slaughter, but no differences in behavior were observed during the lairage period. Carcass characteristics did not differ between pigs from barren and those from enriched housing conditions. Postmortem lactate formation was significantly lower in LL muscles of enriched pigs at 4 and 24 h postmortem. Capillary density and mean muscle fiber area did not differ between the groups of pigs. The percentage of drip loss at 2 and 5 d after storage of LL muscle samples from enriched-housed pigs was significantly lower than that of the barren-housed pigs. Similar tendencies were found for the BF muscle from pigs kept in an enriched environment, but these were not statistically significant. The housing system did not affect meat color. It is concluded that on-farm improvement of animal welfare by environmental enrichment can also lead to beneficial economic effects after slaughter by improving the water-holding capacity of pork.

Histochemical characteristics in relation to meat quality properties in the Longissimus Lumborum of fast and lean growing lines of Large White pigs.

A selection experiment was carried out to study genetic and physiological factors influencing meat quality in lines of Large White pigs selected for lean (L) or fast (F) growth. Second and fourth generation pigs were used to determine effects on fibre type composition, fibre diameters and capillary density in the Longissimus Lumborum (LL). Significant differences in histochemical properties were found in the LL between L- and F-pigs, but only in the 4th generation. L-pigs had significantly less type I and more type IIB myofibres compared to F-pigs in both sexes. In both lines lower type I and higher type IIB percentages were determined in gilts compared with boars. Significant differences in meat quality properties between L- and F-pigs were found in the LL, but only in the 4th generation. The a* values were significantly higher in F- versus L-pigs and in boars versus gilts. L* values were not significantly different between lines. However, L* values were significantly lower in 4th versus 2nd generation pigs and in boars versus gilts. In conclusion, genetic selection on lean versus fast growth induces differences in fibre type composition of the LL. These differences become visible in the 4th generation, when a certain selection effect is achieved. Fibre type composition was influenced by the gender of the animal. Meat quality showed significant differences between lines in meat colour (a* value), but only in the 4th generation. Capillary density and fibre area between L- and F-lines showed minor differences, which could be explained by the differences in weight and age of the pigs of both lines.

Evaluation of invasive instruments for assessment of veal colour at time of classification.

In a random selection of 480 veal carcasses, invasive measurements were made on the longissimus lumborum (LL) and biceps femoris (BF) with the Colormet, Hennessy Grading Probe (HGP4; LL only) and Invasive Meat Colour Meter. Surface colour of the rectus abdominis (RA) was determined instrumentally with the Minolta Chromameter and Surface Meat Colour Meter and visually by two persons, using a colour standard. All measurements were made at 30-40 min post mortem. Of the measurements made on the RA, the Surface Meat Colour Meter was best related to both visual judgements (r = -0·79; -0·73). The correlation coefficient between both visual judgements was 0·77. Clear differences were found between the invasive instruments in their ability to determine veal colour. The correlation coefficients between the invasive measurements and both visual judgements varied for the Colormet L (∗)-value from -0·18 to -0·35, for the HGP4 from -0·40 to -0·49, and for the Invasive Meat Colour Meter from -0·69 to -0·74. Both for LL and BF the best relationships were found between the Invasive Meat Colour Meter and the Surface Meat Colour Meter (RA).

Causes for variation in pork quality.

Research was carried out on 260 pigs that were slaughtered in 12 batches in the slaughter facilities of ID-DLO at Zeist. The practical circumstances were highly standardized. The 'animals' meat quality was good with only little variation; 46 animals showed a 'slightly' aberrant quality and 6 'slight' DFD. Carcasses within the quality categories 'PSE' and 'slight' PSE did not occur. Statistically significant effects on meat quality could be shown for the duration of the resting period before slaughter and the muscular contractions occurring while stunning, shackling and exsanguination, despite the minor variation in pork quality under standardization. A longer resting period before slaughter induced significantly lower temperatures in the m.semimembranosus and the loin at 45 min post mortem and a slightly higher ultimate pH, accompanied by a somewhat darker colour (24hr post mortem). A connection between the animal's behaviour at the fattening station and ultimate pork quality could not be shown. The same counts for fighting in the resting pen of the slaughter house and the pig's behaviour in the stunning area. Muscular contractions during and after stunning had a negative effect on pork quality, causing a more rapid drop in pH, a faster development of rigor mortis and a reduced water holding capacity. An imperfect electrical stunning procedure caused an increase in muscular contractions.

Instrumental colour classification of veal carcasses.

Three experiments, involving over 56 000 veal carcasses, were carried out to develop a method for instrumental colour classification of veal carcasses at 45 min post mortem with the Minolta CR300. The method should produce results similar to those of the 10-point colour-system, which is currently in use in The Netherlands. In the first experiment, the colour of veal carcasses was visually assessed by three observers and instrumentally measured with two Minolta CR300 devices. The average of the results of the three observers and the two Minolta devices were used for discriminant analysis to analyse yield classification rules, which allowed to assignment of the carcasses to classes of the 10-point colour-system. Identical classifications for Minolta and observer were obtained for 63.2% of the carcasses. In the second and third experiment, the classification rules with the Minolta CR300 were used to predict the colour class. For the new samples, identical classification results were obtained for 50-55% of the carcasses. In 41-44% of the carcasses the classification results differed by less than one class. The results show that the Minolta CR300 is under practical conditions able to predict the 10-point colour classes and can be used for on-line colour classification in a slaughterhouse.

Effects of housing conditions of slaughter pigs on some post mortem muscle metabolites and pork quality characteristics.

The results of two experiments studying the effects of housing conditions on post mortem muscle metabolism and meat quality are presented. In the first experiment slaughter pigs (n=71) from two different crossings, were raised at a density of 0.7 m(2)/animal (10 pigs/pen) either with (enriched) or without (conventional) a bedding of daily fresh straw. In the second experiment, animals (n=60) were reared under either conventional or free range housing (1.25 m(2)/animal, 30 pigs per pen) conditions. Main differences between conventional and enriched housed pigs were found in the biceps femoris (BF) muscle, and related to temperature, drip loss and colour. Association studies revealed that there were significant correlations between early (0-4 h) post mortem muscle pH and glycogen and lactate concentrations and between muscle pH measured at 1 h post mortem and muscle rigidity, expressed as the rigor value. There were significant differences with respect to meat quality and post mortem metabolism of the longissimus lumborum (LL), between conventional and free-range pigs. Early post mortem pH (0-4 h) was highest in the free-range pigs, due to low lactate concentrations. The relatively fast pH decline in the conventionally housed group resulted in increased amounts of water exuding from the meat, as measured by the filter paper method. The ultimate pH, was lowest in the free-range animals. The differences in ultimate pH, however, had no effect on drip or cooking losses. It can be concluded from these results that enrichment of the housing system has little effect on the course of the post mortem metabolism and meat quality. However, increasing the freedom of movement by applying lower stocking densities, affects the post mortem muscle metabolism, resulting in reduced water exudation. Correlations up to 0.7 between energetic parameters are not sufficiently high to be useful to predict meat quality of individual carcasses.

Selection of SNP from 50K and 777K arrays to predict breed of origin in cattle.

Reliable breed assignment can be performed with SNP. Currently, high density SNP chips are available with large numbers of SNP from which the most informative SNP can be selected for breed assignment. Several methods have been published to select the most informative SNP to distinguish among breeds. In this study, we evaluated Delta, Wright's FST, and Weir and Cockerham's FST, and extended these methods by adding a rule to avoid selection of sets of SNP in high linkage disequilibrium (LD) providing the same information. The SNP that had a r2 value>0.3 with any of the SNP already selected were discarded. The different selection methods were evaluated for both the 50K SNP and 777K Bovine BeadChip. Animals from 4 cattle breeds (989 Holstein Friesian, 97 Groningen White headed, 137 Meuse-Rhine-Yssel, and 64 Dutch Friesian) were genotyped. After editing 30,447 and 452,525 SNP were available for the 50K and 777K SNP chip, respectively. All selection methods showed that only a small set of SNP is needed to differentiate among the 4 Dutch cattle breeds, whereas comparison of the selection methods showed only small differences. In general, the 777K performed marginally better than the 50K BeadChip, especially at higher confidence thresholds. The rule to avoid selection of SNP in high LD reduced the required number of SNP to achieve correct breed assignment. The Global Weir and Cockerham's FST performed marginally better than other selection methods. There was little overlap in the SNP selected from the 2 BeadChips, whereas the number of SNP selected was about the same.

Contributions to an animal trait ontology.

Improved understanding of the biology of traits of livestock species necessitates the use and combination of information that is stored in a variety of different sources such as databases and literature. The ability to effectively combine information from different sources, however, depends on a high level of standardization within and between various resources, at least with respect to the used terminology. Ontologies represent a set of concepts that facilitate standardization of terminology within specific domains of interest. The biological mechanisms underlying quantitative traits of farm animal species related to reproduction and host pathogen interactions are complex and not well understood. This knowledge could be improved through the availability of domain-specific ontologies that provide enhanced possibilities for data annotation, data retrieval, data integration, data exchange, data analysis, and ontology-based searches. Here we describe a framework for domain-specific ontologies and the development of 2 first-generation ontologies: Reproductive Trait and Phenotype Ontology (REPO) and Host Pathogen Interactions Ontology . In these first-generation ontologies, we focused on "female fertility in cattle" and "interactions between pigs and Salmonella". Through this, we contribute to the global initiative toward the development of an Animal Trait Ontology for livestock species. To demonstrate its usefulness, we show how REPO can be used to select candidate genes for fertility.

Longissimus muscle transcriptome profiles related to carcass and meat quality traits in fresh meat Pietrain carcasses.

High quality pork is consumed as fresh meat, whereas other carcasses are used in the processing industry. Meat quality is determined measuring technical muscle variables. The objective of this research was to investigate the molecular regulatory mechanisms underlying meat quality differences of pork originating from genetically different Piétrain boars. Piétrain boars were approved for high meat quality using a DNA marker panel. Other Piétrain boars were indicated as average. Both groups produced litters in similar Piétrain sows. The LM were sampled from 9 carcasses produced by approved boars and 8 carcasses of average boars. Total RNA was isolated, and an equal portion of each sample was pooled to make a reference sample representing the mean of all samples. Each sample was hybridized on microarrays against the reference in duplicate using a dye swaps design. After normalization and subtraction of 2 times the background, only genes expressed in at least 5 carcasses were analyzed. For all analyses the mean of the M-values relative to the reference (i.e., fold change), were used. Sixteen genes showed significant linear or quadratic associations between gene expression and meat color (Minolta a* value, Minolta L* value, reflection, pH 24 h) after Bonferroni correction. All these genes had expression levels similar to the reference in all carcasses. Studying association between gene expression levels and meat quality using only genes with expression statistically differing from the reference in at least 5 carcasses revealed 29 more genes associating with the technological meat quality variables, again with meat color as a main trait. These associations were not significant after Bonferroni correction and explained less of the phenotypic variation in the traits. Bioinformatics analyses with The Database for Annotation, Visualization and Integrated Discovery (DAVID) using the list of genes with more than 2-fold changed expression level revealed that these genes were mainly found in muscle-specific processes, protein complexes, and oxygen transport, and located to muscle-specific cellular localizations. Pathway analysis using the Kyoto Encyclopedia of Genes and Genomes (KEGG) database revealed pathways related to protein metabolism, cellular proliferation, signaling, and adipose development differing between the 2 groups of carcasses. Approved meat carcasses showed less variation in gene expression. The results highlight biological molecular mechanisms underlying the differences between the high meat quality approved and average boars.

A pathway analysis tool for analyzing microarray data of species with low physiological information.

Pathway information provides insight into the biological processes underlying microarray data. Pathway information is widely available for humans and laboratory animals in databases through the internet, but less for other species, for example, livestock. Many software packages use species-specific gene IDs that cannot handle genomics data from other species. We developed a species-independent method to search pathways databases to analyse microarray data. Three PERL scripts were developed that use the names of the genes on the microarray. (1) Add synonyms of gene names by searching the Gene Ontology (GO) database. (2) Search the Kyoto Encyclopaedia of Genes and Genomes (KEGG) database for pathway information using this GO-enriched gene list. (3) Combine the pathway data with the microarray data and visualize the results using color codes indicating regulation. To demonstrate the power of the method, we used a previously reported chicken microarray experiment investigating line-specific reactions to Salmonella infection as an example.