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Rapid advances in genetics, genomics and imaging have given insight into the molecular and cellular basis of behaviour in a variety of model organisms with unprecedented detail and scope. It is increasingly becoming routine to isolate behavioural mutants, clone and characterise mutant genes and discern the molecular and neural basis for a behavioural phenotype. Conversely, reverse genetic approaches have made it possible to straightforwardly identify genes of interest in whole-genome sequences and generate mutants that can be subjected to phenotypic analysis. In this latter approach, it is the phenol typing that presents the major bottleneck; when it comes to connecting phenotype to genotype in freely behaving animals, analysis of behaviour itself remains superficial and time-consuming. However, many proof-of-principle studies of automated behavioural analysis over the last decade have poised the field on the verge of exciting developments that promise to begin closing this gap.
In the broadest sense, our goal in this chapter is to explore what we can learn about the genes involved in neural function by carefully observing behaviour. This approach is rooted in model organism genetics but shares ideas with ethology and neuroscience, as well as computer vision and bioinformatics. After introducing Caenorhabditis elegans as a model, we will survey the research that has led to the current state of the art in worm behavioural phenol typing and present current research that is transforming our approach to behavioural genetics.
The worm as a model organism
Caenorhabditis elegans is a nematode worm that lives in bacteria-rich environments such as rotting fruit and has also been isolated from insects and snails which it is thought to use for longer-range transportation (Barriere & Felix 2005, Lee et al. 2011). In the laboratory, it is commonly cultured on the surface of agar plates seeded with a lawn of the bacterium Escherichia coli as a food source. On plates, worms lie on either their left or right side and crawl by propagating a sinuous dorso-ventral wave from head to tail.
Despite immense achievements in the past century in hygiene control, and the development of vaccines and antibiotics, infectious diseases continue to pose a tremendous threat to public health globally. There are still devastating infections for which there are no effective vaccines or antimicrobial therapies. Moreover, the problem of drug resistance in bacteria and viral populations and the increasing appreciation that pathologies resulting from infections are responsible for a number of chronic conditions, are creating an ever-growing need for novel preventive and therapeutic approaches. In line with this, a new host-targeted approach has been suggested for antimicrobial drug research that exploits the central role of the host cell during infection. Decades of research have taught us that infections are supported by host cell functions, and that infection pathology is frequently host dependent. Accordingly, the pharmacological targeting of host cell factors promises novel opportunities to prevent and treat infectious disease. Such an approach may be anticipated to expand the number of druggable targets, produce broad-spectrum compounds and impede the generation of resistance. The discovery of RNA interference (RNAi) has created opportunities to explore gene functions in cellular systems in a targeted manner. RNAi loss-of-function approaches have proved invaluable for the identification of host proteins important for pathogen viability. These approaches can be applied on a high-throughput scale, which demands sophisticated liquid handling and high-content image analysis. Here, we provide an overview of the current status of high-content screening (HCS) in loss-of-function analyses in infectious disease research and discuss how these powerful techniques can be applied to identify host factors with previously unknown roles in infection and its pathology.
The challenge of fighting infectious diseases
Infections by pathogenic species of bacteria, viruses, fungi and protozoa have had considerable impact on mankind throughout history. Advances in our understanding of the importance of hygiene control, and later, improvements in diagnostics and the development and successful employment of vaccines and antimicrobial drugs, have substantially benefited human health, and provided social and economic benefits.
Despite great strides in the development of anti-cancer strategies over the last 50 years, treatment regimens continue to cause significant toxicity and fail to fully eradicate disease. Enhancing the current state of therapy will require: (1) the expansion of available tumor selective and therapeutically tractable molecular targets, (2) the development of methods to provide a rational approach to identifying effective combinatorial drug cocktails, and (3) molecular markers that can accurately predict sensitive patient populations. To this end, efforts that reveal the molecular architecture supporting tumorigenic phenotypes are essential. RNA interference (RNAi)-mediated loss of function screens have emerged as a method for wholesale identification of tumor-specific dependencies that modulate chemo responsiveness. Here, we provide a broad overview of how genome-scale RNAi screening is being implemented.
Cancer chemotherapy
Cytotoxic chemotherapy
Goodman and Gilman's 1946 discovery that lymphosarcomas respond to nitrogen mustard demonstrated that tumor cells may have an enhanced sensitivity to chemical poisons as compared to their normal counterparts. This finding revolutionized cancer treatment as it indicated that in addition to radiation and surgery, the only available modalities at the time, drugs could also be administered to reduce tumor burden (Goodman et al. 1946). Following on these initial observations, over the ensuing 50 years, an arsenal of cytotoxic agents were developed to treat a range of cancer types (Chabner & Roberts 2005, Strebhardt & Ullrich 2008).
The majority of these agents, as with the nitrogen mustard, share a common characteristic: they induce genomic damage. For example, agents such as cisplatin cause inter-and intrastrand DNA cross links. This DNA damage can lead to the inhibition of cell division by activating an arrest in the cell cycle to allow for DNA repair through the nucleotide excision repair (NER) pathway. This pathway is coupled to apoptotic programs that are activated if overwhelming damage is detected (Plunkett et al. 1995, Siddik 2003, Wang & Lippard 2005).
By
Xin Wang, Cancer Research UK Cambridge Institute,
Ke Yuan, Cancer Research UK Cambridge Institute,
Florian Markowetz, Cancer Research UK Cambridge Institute
How to link genotypes and phenotypes is a long-standing question in modern biology. Modern high-throughput approaches are key technologies at the forefront of genetic research. They enable the analysis of a biological response to thousands of experimental perturbations and require a tight collaboration between experimental and computational scientists. Perturbation studies and computational approaches have revolutionized research in functional genomics and genetics and promise to lay the foundation for personalized medicine. For modern high-throughput technologies, computation is as important as experimentation. Genome-wide image-based RNA interference (RNAi) screens, for example, are only feasible because of computational techniques. Computational skills to analyse the data have become as important as experimental skills to generate the data.
Design and analysis of phenol typing screens depend on the number of genes perturbed and the richness of the phenotype observed (Figure 6.1). At one extreme are high-throughput screens with single reporters, e.g. a genome-wide screen for new components of a pathway. At the other extreme are perturbations of individual genes with very rich phenotypes, e.g. assessing the effects of a single gene perturbation on several molecular levels over time. Between these two extremes lie a variety of possible screen designs. Two widely used scenarios are small-scale perturbations (<20 genes) of a single target pathway with rich readouts, e.g. a global transcriptional profile, and medium-scale perturbations (hundreds of genes) with multi-parametric readouts, e.g. cell morphology or growth in different media. In the following we will discuss statistical and computational methodologies for functional analysis in all four scenarios.
Scenario 1: Genome-wide screens with single reporters
RNAi screens have been frequently and successfully applied for functional profiling of genes on a large scale (Boutros & Ahringer 2008). The vast majority of these applications use a single phenotype (e.g. cell viability, growth rate, activity of reporter constructs) to characterize the function of genes in specific biological pathways.
The advent of sequencing technologies has revolutionized our understanding and approach to studying biological systems. Indeed, whole-genome sequencing projects have already targeted many different species, enabling the identification of most genes in those organisms. However, observed phenotypes cannot be explained by genes alone, but rather by the interactions that their products establish under some environmental conditions (Waddington 1957). Thus, it is through the analysis of these interaction net-works (e.g. regulatory, metabolic, molecular, or genetic) that we can better understand the genotype-to-phenotype relationship, the complexity and evolution of organisms, or the differences among individuals of the same species. The topology and dynamics of these biological networks can be unveiled by systematic perturbation of their nodes (i.e. genes). For instance, upon single-gene deletions in Saccharomyces cerevisiae under standard laboratory conditions, most genes (∼80%) were not found to be essential for cell viability (Giaever et al. 2002). Though many of these genes may be required for growth in other environments (Hillenmeyer et al. 2008), this result suggests extensive functional redundancy among genes. Such functional buffering confers robustness to biological networks and shields the cellular machinery from genetic perturbations (Hartman et al. 2001). Additionally, the small effect on phenotype that many gene deletions exhibit (see Figure 2.1) evidences that single perturbations alone cannot capture the complexity of the genotype-to-phenotype relationship. Therefore, a combinatorial approach to gene perturbations is best suited to elucidate biological systems and can enable a better characterization of genes and cellular functioning.
Definition of genetic interaction
Genetic interactions reveal functional relations between genes that contribute to a pheno-typic trait. William Bateson first introduced the term, formerly known as epistasis (see Phillips [1998] for a description on the origin and evolution of the definition), to refer to an allele at one locus preventing a variant at another from manifesting its effect (Bateson 1909).
Death of calves around parturition is a matter of concern for dairy farmers. Relatively high stillbirth rates and unfavourable trends have been reported for Holstein heifers in the Netherlands and several other countries. In our study, we investigated herd differences, genetic parameters and genotype by environment interaction for heifer calf livability. A large dataset with data from calvings between 1993 and 2012 of Dutch dairy farms was used. There were considerable differences between herds in livability of calves from heifers, with averages ranging from 74% to 95%. Both herds with relatively high and low averages showed the same negative trend between 1993 and 2012, with largest declines in herds with the lowest averages. We found that heritability and genetic variation of first parity livability were substantially larger in herd environments where the likelihood of stillbirth was high v. environments where stillbirth was at a low level. The genetic correlations between herd environment levels were all very close to unity, indicating that ranking of sires was similar for all environments. However, for herds with a relatively high stillbirth incidence selecting sires with favourable breeding values is expected to be twice as profitable as in herds with a relatively low stillbirth incidence.
The effects of early life events on average daily weight gain from birth to day 21 (ADG) of suckling pigs kept at different room temperatures (15°C, 20°C and 25°C) from birth to weaning were investigated. Data were collected from litters born by 61 sows in a loose housing system. The ADG for piglets with low birth weight (estimated for birth weights below the 10% percentile) was estimated to be 20 to 30 g higher per day at room temperature 20°C to 25°C compared with 15°C. In contrast, the ADG during the lactation period decreased for larger piglets (estimated for birth weights above the 10% percentile) by 28 g/day at room temperature 25°C compared with 15°C. Thus, high ambient temperatures (20°C to 25°C) are favourable for the growth in smaller piglets during lactation. Neither latency to first suckle nor birth-induced hypoxia, measured as concentration of umbilical cord lactate, affected the growth rate of the piglets. Lowest rectal temperature during the first 24 h after birth had a long-term negative effect on ADG (P<0.05), so that piglets with a lowest rectal temperature of 32.8°C (10% percentile) had an ADG which was on average 19 g lower per day than piglets with a rectal temperature of 37.3°C (90% percentile). Our results showed that hypothermia at birth, low birth weight and high number of suckling piglets lead to reduced ADG during the suckling period. The results suggest that keeping the room temperature at 20°C during lactation to some extent could compensate for the otherwise negative effects of low birth weight on ADG in piglets without decreasing the ADG of high birth weight piglets. However, to avoid hypothermia in the smallest piglets it may be beneficial to increase the room temperature above 20°C during the farrowing period of loose housed sows.
BW of replacement heifers is rarely measured on commercial farms, making it difficult to evaluate the success of management practices related to calf growth. Our aims were to describe variability among commercial farms in Holstein heifer BW, determine how BW differences varied with management and propose a method of estimating calf growth based upon single measurement. Heart girth circumference was used to estimate BW of 576 heifers 48 to 70 weeks of age on 33 different farms (on average 11±6 heifers/farm) in British Columbia, Canada. Regression analysis showed a linear relationship of BW with age (BW (kg)=116+5×age (weeks)). Residuals from this regression were averaged across heifers within each farm to identify farms where heifers were heavier or lighter than would be predicted on the basis of their age; farm average residuals ranged from −54 to 72 kg. Farms with heifers showing the highest residual BW also had the highest rates of gain for pre-weaned calves. These results indicate that farms able to rear faster growing calves before weaning were also rearing faster growing heifers at breeding, and suggest that management of milk-fed calves is a particularly important component of replacement heifer management.
Selection of animals for improved feed efficiency can affect sustainability of animal production because the most efficient animals may face difficulties coping with challenges. The objective of this study was to determine the effects of an inflammatory challenge (using an intravenous injection of complete Freund’s adjuvant – CFA) in piglets from two lines of pigs divergently selected during the fattening period for a low (RFI−) or a high (RFI+) residual feed intake (RFI; difference between actual feed intake and theoretical feed requirements). Nitrogen and energy balances (including heat production – HP – and its components: activity-related HP – AHP, thermic effect of feeding, and resting HP) were measured individually in thirteen 20-kg BW castrated male piglets (six and seven from RFI+ and RFI− line, respectively) fed at the same level (1.72 MJ ME/kg BW0.60 per day) from 3 days before to 3 days after CFA injection. Dynamics of dietary U-13C-glucose oxidation were estimated from measurements of 13CO2 production on the day before and 3 days after the CFA injection. Oxidation of dietary nutrients and lipogenesis were calculated based on HP and O2 consumption and CO2 production. The data were analyzed as repeated measurements within piglets in a mixed model. Before CFA injection, RFI− piglets had a lower resting energy expenditure than RFI+ piglets, which tended to increase energy retention because of a higher energy retention as fat. The CFA injection did not affect feed intake from the day following CFA injection onwards but it increased energy retention (P=0.04). Time to recover 50% of 13C from dietary glucose as expired 13CO2 was higher in RFI+ piglets before inducing inflammation but decreased after to the level of RFI− piglets (P<0.01). Oxidation of U-13C-glucose tended to slightly increased in RFI− piglets and to decreased in RFI+ piglets (P=0.10) because of CFA. Additionally, RFI− piglets had a lower respiratory quotient during the 1st day following the CFA injection whereas RFI+ piglets tended to have a higher respiratory quotient. In conclusion, selection for RFI during the fattening period also affected the energy metabolism of pigs during earlier stages of growth. The effects of CFA injection were moderated in both lines but the most efficient animals (RFI−) exhibited a marked re-orientation of nutrients only during the 1st day after CFA, and seemed to recover thereafter, whereas the less efficient piglets expressed a more prolonged alteration of their metabolism.
Measuring and mitigating methane (CH4) emissions from livestock is of increasing importance for the environment and for policy making. Potentially, the most sustainable way of reducing enteric CH4 emission from ruminants is through the estimation of genomic breeding values to facilitate genetic selection. There is potential for adopting genetic selection and in the future genomic selection, for reduced CH4 emissions from ruminants. From this review it has been observed that both CH4 emissions and production (g/day) are a heritable and repeatable trait. CH4 emissions are strongly related to feed intake both in the short term (minutes to several hours) and over the medium term (days). When measured over the medium term, CH4 yield (MY, g CH4/kg dry matter intake) is a heritable and repeatable trait albeit with less genetic variation than for CH4 emissions. CH4 emissions of individual animals are moderately repeatable across diets, and across feeding levels, when measured in respiration chambers. Repeatability is lower when short term measurements are used, possibly due to variation in time and amount of feed ingested prior to the measurement. However, while repeated measurements add value; it is preferable the measures be separated by at least 3 to 14 days. This temporal separation of measurements needs to be investigated further. Given the above issue can be resolved, short term (over minutes to hours) measurements of CH4 emissions show promise, especially on systems where animals are fed ad libitum and frequency of meals is high. However, we believe that for short-term measurements to be useful for genetic evaluation, a number (between 3 and 20) of measurements will be required over an extended period of time (weeks to months). There are opportunities for using short-term measurements in standardised feeding situations such as breath ‘sniffers’ attached to milking parlours or total mixed ration feeding bins, to measure CH4. Genomic selection has the potential to reduce both CH4 emissions and MY, but measurements on thousands of individuals will be required. This includes the need for combined resources across countries in an international effort, emphasising the need to acknowledge the impact of animal and production systems on measurement of the CH4 trait during design of experiments.
The aim of this study was to determine the genetic background of longitudinal residual feed intake (RFI) and BW gain in farmed mink using random regression methods considering heterogeneous residual variances. The individual BW was measured every 3 weeks from 63 to 210 days of age for 2139 male+female pairs of juvenile mink during the growing-furring period. Cumulative feed intake was calculated six times with 3-week intervals based on daily feed consumption between weighing’s from 105 to 210 days of age. Genetic parameters for RFI and BW gain in males and females were obtained using univariate random regression with Legendre polynomials containing an animal genetic effect and permanent environmental effect of litter along with heterogeneous residual variances. Heritability estimates for RFI increased with age from 0.18 (0.03, posterior standard deviation (PSD)) at 105 days of age to 0.49 (0.03, PSD) and 0.46 (0.03, PSD) at 210 days of age in male and female mink, respectively. The heritability estimates for BW gain increased with age and had moderate to high range for males (0.33 (0.02, PSD) to 0.84 (0.02, PSD)) and females (0.35 (0.03, PSD) to 0.85 (0.02, PSD)). RFI estimates during the growing period (105 to 126 days of age) showed high positive genetic correlations with the pelting RFI (210 days of age) in male (0.86 to 0.97) and female (0.92 to 0.98). However, phenotypic correlations were lower from 0.47 to 0.76 in males and 0.61 to 0.75 in females. Furthermore, BW records in the growing period (63 to 126 days of age) had moderate (male: 0.39, female: 0.53) to high (male: 0.87, female: 0.94) genetic correlations with pelting BW (210 days of age). The result of current study showed that RFI and BW in mink are highly heritable, especially at the late furring period, suggesting potential for large genetic gains for these traits. The genetic correlations suggested that substantial genetic gain can be obtained by only considering the RFI estimate and BW at pelting, however, lower genetic correlations than unity indicate that extra genetic gain can be obtained by including estimates of these traits during the growing period. This study suggests random regression methods are suitable for analysing feed efficiency and BW gain; and genetic selection for RFI in mink is promising.
The objectives of this study were to determine: (1) the effect of wheat dried distillers grain with solubles (DDGS) inclusion, and (2) dietary feed enzyme (FE; Econase XT) supplementation in a finishing diet containing wheat DDGS on fatty acid profile of the pars costalis diaphragmatis muscle of beef cattle. A total of 160 crossbred yearling steers with initial BW of 495±38 kg were blocked by BW and randomized into 16 pens (10 head/pen). The pens were randomly assigned to one of the four treatments: (1) control (CON; 10% barley silage and 90% barley grain-based concentrate, dry matter (DM) basis); (2) diet containing 30% wheat DDGS in place of barley grain without FE (WDG); (3) WDG diet supplemented with low FE (WDGL; 1 ml FE/kg DM); and (4) WDG diet supplemented with high FE (2 ml FE/kg DM). The pars costalis diaphragmatis muscle samples were collected from cattle at slaughter at the end of the finishing period (120 days) with a targeted live weight of 650 kg. No differences in organic matter intake, final BW and average daily gain were observed among treatments. However, steers fed WDG had greater (P<0.01) feed conversion ratio than those fed CON, and increasing FE application in wheat DDGS-based diets tended (P<0.10) to linearly decrease feed conversion ratio. In assessing the effects of including WDG diets without FE, concentration of total polyunsaturated fatty acids (PUFA) in muscle tended to be greater (P<0.10) for steers fed WDG than steers fed CON. In addition, inclusion of wheat DDGS into the diet increased (P<0.05) concentration of CLA and vaccenic acid (VA) in muscle and also resulted in a higher (P<0.05) ratio of n-6/n-3 PUFA compared with that from steers fed CON diet. Increasing FE application in wheat DDGS-based diets did not modify the concentrations of individual or total fatty acids. These results suggest that inclusion of wheat DDGS in finishing diets may improve fatty acid profile of beef muscle which could benefit human health.
Lameness in dairy herds is traditionally detected by visual inspection, which is time-consuming and subjective. Compared with healthy cows, lame cows often spend longer time lying down, walk less and change behaviour around feeding time. Accelerometers measuring cow leg activity may assist farmers in detecting lame cows. On four commercial farms, accelerometer data were derived from hind leg-mounted accelerometers on 348 Holstein cows, 53 of them during two lactations. The cows were milked twice daily and had no access to pasture. During a lactation, locomotion score (LS) was assessed on average 2.4 times (s.d. 1.3). Based on daily lying duration, standing duration, walking duration, total number of steps, step frequency, motion index (MI, i.e. total acceleration) for lying, standing and walking, eight accelerometer means and their corresponding coefficient of variation (CV) were calculated for each week immediately before an LS. A principal component analysis was performed to evaluate the relationship between the variables. The effects of LS and farm on the principal components (PC) and on the variables were analysed in a mixed model. The first four PC accounted for 27%, 18%, 12% and 10% of the total variation, respectively. PC1 corresponded to Activity variability due to heavy loading by five CV variables related to standing and walking. PC2 corresponded to Activity level due to heavy loading by MI walking, MI standing and walking duration. PC3 corresponded to Recumbency due to heavy loading by four variables related to lying. PC4 corresponded mainly to Stepping due to heavy loading by step frequency. Activity variability at LS4 was significantly higher than at the lower LS levels. Activity level was significantly higher at LS1 than at LS2, which was significantly higher than at LS4. Recumbency was unaffected by LS. Stepping at LS1 and LS2 was significantly higher than at LS3 and LS4. Activity level was significantly lower on farm 3 compared with farms 1 and 2. Stepping was significantly lower on farms 1 and 3 compared with farms 2 and 4. MI standing indicated increased restlessness while standing when cows increased from LS3 to LS4. Lying duration was only increased in lame cows. In conclusion, Activity level differed already between LS1 and LS2, thus detecting early signs of lameness, particularly through contributions from walking duration and MI walking. Lameness detection models including walking duration, MI walking and MI standing seem worthy of further investigation.
Pharmacogenetic/pharmacogenomic (PGx) testing is currently available for a wide range of health problems including cardiovascular disease, cancer, diabetes, autoimmune disorders, mental health disorders and infectious diseases. PGx contributes important information to the field of precision medicine by clarifying appropriate treatments for specific disease subtypes. Tangible benefits to patients including improved outcomes and reduced total health care costs have been observed. However, PGx-guided therapy faces many barriers to full integration into clinical practice and acceptance by stakeholders, whether practitioner, patient or payer. Each stakeholder has a unique perspective on the role of PGx testing, although all are similarly challenged with demonstrating or appraising its cost-to-benefit value. Coverage by insurers is a critical step in achieving widespread adoption of PGx testing. The acceleration of adoption of precision medicine in general and for PGx testing in particular will be determined by how quickly robust evidence can be accumulated that shows a return on investment for payers in terms of real dollars, for clinicians in terms of patient clinical responses, and for patients in terms of economic, health and quality of life outcomes. Trends in PGx testing utilization and uptake by payers in real-world practice are discussed; the role of pharmacoeconomics in assessing cost-effectiveness is highlighted using a case study in psychiatric care, and several issues that will affect adoption of PGx testing in the United States (US) over the next few years are reviewed.
The in situ degradation of the washout fraction of starch in six feed ingredients (i.e. barley, faba beans, maize, oats, peas and wheat) was studied by using a modified in situ protocol and in vitro measurements. In comparison with the washing machine method, the modified protocol comprises a milder rinsing method to reduce particulate loss during rinsing. The modified method markedly reduced the average washout fraction of starch in these products from 0.333 to 0.042 g/g. Applying the modified rinsing method, the fractional degradation rate (kd) of starch in barley, oats and wheat decreased from on average 0.327 to 0.144 h−1 whereas for faba beans, peas and maize no differences in kd were observed compared with the traditional washing machine rinsing. For barley, maize and wheat, the difference in non-fermented starch in the residue between both rinsing methods during the first 4 h of incubation increased, which indicates secondary particle loss. The average effective degradation of starch decreased from 0.761 to 0.572 g/g when using the new rinsing method and to 0.494 g/g when applying a correction for particulate matter loss during incubation. The in vitro kd of starch in the non-washout fraction did not differ from that in the total product. The calculated ratio between the kd of starch in the washout and non-washout fraction was on average 1.59 and varied between 0.96 for oats and 2.39 for maize. The fractional rate of gas production was significantly different between the total product and the non-washout fraction. For all products, except oats, this rate of gas production was larger for the total product compared with the non-washout fraction whereas for oats the opposite was observed. The rate of increase in gas production was, especially for grains, strongly correlated with the in vitro kd of starch. The results of the present study do not support the assumption used in several feed evaluation systems that the degradation of the washout fraction of starch in the rumen is much faster than that of the non-washout fraction.
Androstenone is a steroid pheromone occurring in the pubertal Leydig cells. Breeding against androstenone can decrease pheromone odour in swine meat but appears to cause unwanted side effects such as delayed onset of puberty. To study causality, global gene expression in developing boar testes at 12, 16, 20 and 27 weeks was investigated using a porcine cDNA microarray. The morphological status and androgenic levels of the same individuals have been described in a previous publication. In the present paper, expression of genes and pathways has been analysed with reference to these findings. Nine clusters of genes with significant differential expression over time and 49 functional charts were found in the analysed testis samples. Prominent pathways in the prepubertal testis were associated with tissue renewal, cell respiration and increased endocytocis. E-cadherines may be associated with the onset of pubertal development. With elevated steroidogenesis (weeks 16 to 27), there was an increase in the expression of genes in the MAPK pathway, STAR and its analogue STARD6. A pubertal shift in genes coding for cellular cholesterol transport was observed. Increased expression of meiotic pathways coincided with the morphological onset of puberty. Puberty-related change in Ca(2+) pathway transcripts, neurosteroids, neuronal changes and signalling in redox pathways suggested a developmental-specific period of neuromorphogenesis. Several growth factors were found to increase differentially over time as the testis matured. There may be interactions between MAPK, STAR and growth factors during specific periods. In conclusion, pathways for neurogenesis, morphological pathways and several transcripts for growth factors, which have known modulating effects on steroidogenesis and gonadotropins in humans and rodents, act at specific ages and developmental stages in the boar testis. The age dependency and complexity shown for development-specific testis transcripts must be considered when selecting phenotypic parameters for genetic selection for low androstenone. The results of selection based on measurement of phenotypic maturation and androstenone (or other steroid) levels at one specific age may differ depending on the age used. More research is necessary to find the optimal phenotype to use in order to reduce the unwanted side effects.
In vitro gas production studies are routinely used to assess the metabolic capacity of intestinal microbiota to ferment dietary fibre sources. The faecal inocula used during the in vitro gas production procedure are most often obtained from animals adapted to a certain diet. The present study was designed to assess whether 19 days of adaptation to a diet are sufficient for faecal inocula of pigs to reach a stable microbial composition and activity as determined by in vitro gas production. Eighteen multiparous sows were allotted to one of two treatments for three weeks: a diet high in fibre (H) or a diet low in fibre (L). After this 3-week period, the H group was transferred to the low fibre diet (HL-treatment) while the L group was transferred to the diet high in fibre (LH-treatment). Faecal samples were collected from each sow at 1, 4, 7, 10, 13, 16 and 19 days after the diet change and prepared as inoculum used for incubation with three contrasting fermentable substrates: oligofructose, soya pectin and cellulose. In addition, inocula were characterised using a phylogenetic microarray targeting the pig gastrointestinal tract microbiota. Time after diet change had an effect (P<0.05) on total gas production for the medium–fast fermentable substrates; soya pectin and oligofructose. For the more slowly fermentable cellulose, all measured fermentation parameters were consistently higher (P<0.05) for animals in the HL-treatment. Diet changes led to significant changes in relative abundance of specific bacteria, especially for members of the Bacteroidetes and Bacilli, which, respectively, increased or decreased for the LH-treatment, while changes were opposite for the HL-treatment. Changing the diet of sows led to changes in fermentation activity of the faecal microbiota and in composition of the microbiota over time. Adaptation of the microbiota as assessed by gas production occurred faster for LH-animals for fast fermentable substrates compared with HL-animals. Overall, adaptation of the large intestinal microbiota of sows as a result of ingestion of low and high fibre diets seems to take longer than 19 days, especially for the ability to ferment slowly fermentable substrates.