Anticipatory Behavior for a Mealworm Reward in Laying Hens Is Reduced by Opioid Receptor Antagonism but Not Standard Feed Intake
It is widely accepted that the absence of suffering no longer defines animal welfare and that positive affective experiences are imperative. For example, laying hens may be housed in environments that do not cause chronic stress but may lack particular resources that promote positive affective experiences, such as conspecifics or effective enrichment. Despite a consensus of how important positive affect is for animal welfare, they are difficult to identify objectively. There is a need for valid and reliable indicators of positive affect. Pharmacological interventions can be an effective method to provide insight into affective states and can assist with the investigation of novel indicators such as associated biomarkers. We aimed to validate a pharmacological intervention that blocks the subjective hedonistic phase associated with reward in laying hens via the administration of the non-selective (μ, δ, and κ) opioid receptor antagonist, nalmafene. We hypothesized that nonfood deprived, hens that did not experience a positive affective state when presented with a mealworm food reward due to the administration of nalmefene, would show minimal anticipatory and consummatory behavior when the same food reward was later presented. Hens (n = 80) were allocated to treatment groups, receiving either nalmefene or vehicle (0.9% saline) once or twice daily, for four consecutive days. An anticipatory test (AT) was performed on all days 30 min post-drug administration. Behavioral responses during the appetitive and consummatory phase were assessed on days 1, 3 and 4. Anticipatory behavior did not differ between treatment groups the first time hens were provided with mealworm food rewards. However, antagonism of opioid receptors reduced anticipatory and consummatory behavior on days 3 and 4. Feed intake of standard layer mash was not impacted by treatment, thus nalmefene reduced non-homeostatic food consumption but not homeostatic consumption. Behavioral observations during the AT provided no evidence that nalmefene treated hens were fearful, sedated or nauseous. The results suggest that we successfully blocked the hedonistic subjective component of reward in laying hens and provide evidence that this method could be used to investigate how hens perceive their environment and identify associated novel indicators to assess hen welfare.
Smallholder farmer community-based breeding program of indigenous chickens key to in-situ genetic resource conservation and enhancing rural livelihoods in Zambia
Agriculture is the primary livelihood for many nations. Most rural communities in Sub-Saharan Africa (SSA) depend on agriculture for their livelihood but are also custodians of essential animal genetic resources in the region. Nearly 80% of smallholder farmers rear small livestock, including indigenous chickens (IC) (Gallus domesticus) for household income, food and nutritional security. However, populations of IC genetic resources over the past decade have experienced erosion and extinction and thus have the potential to affect future production and biodiversity negatively. Among the avian species, many chicken varieties are in danger with 62% of chicken strains being of unknown status, 33% at risk of extinction and 3.5% extinct. These negative changes can adversely affect rural communities in SSA. Therefore, genetic resource conservation focused on in-situ selection, development, multiplication and utilisation of Indigenous chickens in rural communities should be considered. In SSA, Zambia has also experienced poor performance, including loss of breeds of IC attributed to disease and weak markets dominated by intensive broiler and egg chicken production. Therefore, this research project will focus on a community-based breeding program in Zambia to formulate solutions for local smallholder farmers to mitigate the losses of IC genetic resources. The three main objectives are to; i) strengthen sustainable selection, multiplication, utilisation and conservation of IC, (ii), improve efficiency in production and disease control of IC (iii), and explore potential market value chains of IC. The methodology will involve undertaking a survey, focus group discussion, and interviews for 50 households to collect social-economic data throughout 2021. This data will include breeds reared, production systems used, common poultry diseases, and marketing systems practised. Identification of challenges faced by farmers involved in IC production will be through the engagements and consensus of the research team. A bio-economic model will then be used to evaluate the profitability and economic contributions of the essential biological traits concerning consumer-market demands (Okeno et al., 2012). The model will describe the production system used and the effects of biological characteristics on costs due to labour, veterinary, brooding, feeding requirements and revenue from the sale of eggs, growers, and culled breeding stock. This research aims to facilitate future benefits for both smallholder farmers through improved livelihoods, and consumers are having access to healthy IC eggs and meat from the market.
Pharmacological intervention of the reward system in the laying hen has an impact on anticipatory behaviour
Valid reliable indicators of positive affective state are a critical component of welfare assessments, however, these are currently lacking for laying hens. Pharmacological interventions that disrupt specific neural pathways have shown to be a useful tool when validating indicators of affective states. As such, we utilised and validated the μ-opioid receptor antagonist nalmefene to block the reward pathway in laying hens.
Benchmarking differential expression analysis tools for RNA-Seq: normalization-based vs. log-ratio transformation-based methods
Background: Count data generated by next-generation sequencing assays do not measure absolute transcript abundances. Instead, the data are constrained to an arbitrary "library size" by the sequencing depth of the assay, and typically must be normalized prior to statistical analysis. The constrained nature of these data means one could alternatively use a log-ratio transformation in lieu of normalization, as often done when testing for differential abundance (DA) of operational taxonomic units (OTUs) in 16S rRNA data. Therefore, we benchmark how well the ALDEx2 package, a transformation-based DA tool, detects differential expression in high-throughput RNA-sequencing data (RNA-Seq), compared to conventional RNA-Seq methods such as edgeR and DESeq2.
Results: To evaluate the performance of log-ratio transformation-based tools, we apply the ALDEx2 package to two simulated, and two real, RNA-Seq data sets. One of the latter was previously used to benchmark dozens of conventional RNA-Seq differential expression methods, enabling us to directly compare transformation-based approaches. We show that ALDEx2, widely used in meta-genomics research, identifies differentially expressed genes (and transcripts) from RNA-Seq data with high precision and, given sufficient sample sizes, high recall too (regardless of the alignment and quantification procedure used). Although we show that the choice in log-ratio transformation can affect performance, ALDEx2 has high precision (i.e., few false positives) across all transformations. Finally, we present a novel, iterative log-ratio transformation (now implemented in ALDEx2) that further improves performance in simulations.
Conclusions: Our results suggest that log-ratio transformation-based methods can work to measure differential expression from RNA-Seq data, provided that certain assumptions are met. Moreover, these methods have very high precision (i.e., few false positives) in simulations and perform well on real data too. With previously demonstrated applicability to 16S rRNA data, ALDEx2 can thus serve as a single tool for data from multiple sequencing modalities.
Alternatives to formulate laying hen diets beyond the traditional least-cost model
Owing to the high cost of feed for poultry, there is continuous pressure to formulate 'least-cost' diets that meet nutritional requirements. However, the main aim of any commercial enterprise is usually to maximize profits with the resources or inputs available, and the conventional or historic tool of least-cost ration formulation has limitations in a more demanding economic environment. The layer industry may experience particularly volatile changes in egg price, principally owing to changes in supply rather than demand, and in the past, it has been reported to possess less production and financial data reporting than other industries. Thus, increased flexibility during these uncertain times may give the layer industry greater opportunity and capacity to cope with market fluctuations. A practical example of how a laying hen operation may benefit from these approaches demonstrates that the maximum-profit solution does not always match the least-cost solution and that stochastic feed formulation may be used to accurately assign safety margins and define the level of certainty this safety margin will provide. Finally, as producers better understand how their hens respond to different dietary specifications, the opportunity arises to choose the set of specifications that result in maximum profits for their unique situations, rather than relying on least-cost diets formulated to nutrient requirements alone.
The Status of Indigenous Chicken Genetic Resources: An Analysis of Farmers’ Perspectives and Implications for Breed Conservation Priorities in Zambia
(1) Background: Indigenous chickens (Gallus domesticus) (ICs) are an essential component of agriculture and rural livelihood among 80% of small-scale farmers (SSFs) in Sub-Saharan Africa (SSA). However, in the past decade, significant losses of Indigenous Chicken Animal Genetic Resources (ICAnGR) resulting from poultry diseases, imported exotic breeds, poor market access, and uncontrolled crossbreeding have been reported. This study aimed to investigate the status of IC-AnGR and assess the major challenges affecting the production of ICs. (2) Methods: We surveyed 358 households in eastern, central, and southern livelihood zones, comprising 81.6% males and 17.6% females. (3) Results: Our study shows that respondents owned 16,112 ICs, 3026 goats, and 5183 herds of cattle. Overall, 77.4% of chicken breeds were ICs and 22.6% were exotic. Across the three zones, 18–44% reported the introduction or adoption of exotic breeds in the past decade, with most households sourcing breeding stock from local communities and family and friends at 45% and 28.6%, respectively. Farmers gave various reasons for adopting new chicken breeds, including fast growth (21.7%), larger mature sizes (21.66%), and resistance to diseases at 15.2%. Overall, 92.5% of farmers agreed or strongly agreed that some IC breeds disappeared in the past decade and nearly 90% were concerned. Some attributed the loss to poultry diseases. (4) Conclusion: Deliberate policies to promote the sustainable use and conservation of ICs are critical in Zambia.
Probiotic Bacillus subtilis 29,784 improved weight gain and enhanced gut health status of broilers under necrotic enteritis condition
The study investigated the benefit of a Bacillus subtilis probiotic (Bs 29,784) in necrotic enteritis (NE)-challenged broilers. Four treatments were performed with 312 male day-old Ross 308 reared in floor pens from day 0 to day 35: 2 groups fed control diet without or with NE challenge (CtrlNC and CtrlNE); 2 groups fed probiotic and antibiotic supplements in the control diet with NE challenge (ProNE and AntNE). Necrotic enteritis challenge procedures commenced with inoculation of Eimeria spp 1 mL/bird per os at day 9 and Clostridium perfringens EHE-NE18 (approximately 108 cfu/mL) 1 mL/bird per os at day 14 and day 15. Performance parameters were measured on day 16 and day 35. Lesion, cecal microbiota, and jejunal gene expression were analyzed on day 16. Necrotic enteritis challenge significantly suppressed the performance parameters compared with CtrlNC: 27% weight gain reduction, 11 points feed conversion ratio (FCR) increase at day 16, and 12% weight gain reduction, 5-point FCR increase at day 35. By day 35, ProNE and AntNE treatments enabled significantly higher weight gain (4 and 9%, respectively) than CtrlNE. Compared with CtlrNE and contrary to AntNE, ProNE treatment exhibited upregulation of genes coding for tight junctions proteins (CLDN1, JAM2, TJP1), cytokines (IL12, interferon gamma, TGFβ), and Toll-like receptors (TLR5, TLR21) suggesting enhanced immunity and intestinal integrity. 16S NGS analysis of cecal microbiota at day 16 showed a decreased alpha diversity in challenged groups. Principal component analysis of operational taxonomic unit (OTU) abundance revealed that ProNE and AntNE grouped closely while both distantly from CtrlNC and CtrlNE, which were separately grouped, indicating the similar effects of ProNE and AntNE on the OTU diversity that were however different from both CtrlNC and CtrlNE. Microbiota analysis revealed an increase of genera Faecalibacterium, Oscillospira, and Butyricicoccus; and a decrease of genera Ruminococcus, Lactobacillus, and Bacteroides; and an increase of the Firmicutes-to-Bacteroidetes ratio in ProNE and AntNE groups compared with the CtlrNE group. It is concluded that Bs 29,784 may enable improved health of broiler chickens under NE conditions thus performance implications.
Appraisal of matrix values for exogenous phytase alone or in combination with other enzymes in diets for broiler chickens
A trial was conducted to determine if full matrix values for phytase alone or in combination with xylanase and beta-glucanase (XB) were appropriate across multiple inclusion levels and combinations. It may be concluded that these matrix values are appropriate, with birds offered diets containing either phytase alone or in combination with XB overall generating a similar or improved performance than those offered control diets.
A field guide for the compositional analysis of any-omics data
Background: Next-generation sequencing (NGS) has made it possible to determine the sequence and relative abundance of all nucleotides in a biological or environmental sample. A cornerstone of NGS is the quantification of RNA or DNA presence as counts. However, these counts are not counts per se: their magnitude is determined arbitrarily by the sequencing depth, not by the input material. Consequently, counts must undergo normalization prior to use. Conventional normalization methods require a set of assumptions: they assume that the majority of features are unchanged and that all environments under study have the same carrying capacity for nucleotide synthesis. These assumptions are often untestable and may not hold when heterogeneous samples are compared. Results: Methods developed within the field of compositional data analysis offer a general solution that is assumption-free and valid for all data. Herein, we synthesize the extant literature to provide a concise guide on how to apply compositional data analysis to NGS count data. Conclusions: In highlighting the limitations of total library size, effective library size, and spike-in normalizations, we propose the log-ratio transformation as a general solution to answer the question, "Relative to some important activity of the cell, what is changing?"
Acute exposure to high temperature affects expression of heat shock proteins in altricial avian embryos
As the world warms, understanding the fundamental mechanisms available to organisms to protect themselves from thermal stress is becoming ever more important. Heat shock proteins are highly conserved molecular chaperones which serve to maintain cellular processes during stress, including thermal extremes. Developing animals may be particularly vulnerable to elevated temperatures, but the relevance of heat shock proteins for developing altricial birds exposed to a thermal stressor has never been investigated. Here, we sought to test whether three stress-induced genes – HSPD1, HSPA2, HSP90AA1 – and two constitutively expressed genes – HSPA8, HSP90B1 – are upregulated in response to acute thermal shock in zebra finch (Taeniopygia guttata) embryos half-way through incubation. Tested on a gradient from 37.5 ◦C (control) to 45 ◦C, we found that all genes, except HSPD1, were upregulated. However, not all genes initiated upregulation at the same temperature. For all genes, the best fitting model included a correlate of developmental stage that, although it was never significant after multiple-test correction, hints that heat shock protein upregulation might increase through embryonic development. Together, these results show that altricial avian embryos are capable of upregulating a known protective mechanism against thermal stress, and suggest that these highly conserved cellular mechanisms may be a vital component of early developmental protection under climate change.