Examination of the effect of weaning stress on the physiological and molecular regulation of immune function in circulating bovine leukocytes

Weaning is a multifaceted stressor and may involve numerous husbandry practices, including the abrupt separation of the calf from its dam, a nutritional adjustment to a non-milk diet and social reorganisation and, additionally, is often associated with other husbandry practices, such as housing and transport. Stress-induced disease susceptibility in livestock animals has documented economic and societal consequences and the sense of urgency to understand and intervene in these is becoming increasingly important. Available published research has examined the physiological and behavioural responses to weaning stress in calves, but no study has yet characterised the molecular response. This Ph.D. project aimed to obtain a greater understanding of the impacts of weaning on both the molecular and physiological responses in order to elucidate the immune mechanisms responsible for stress dependent immune suppression in calves post weaning. A series of studies were conducted to investigate i) the effect of weaning stress as an accumulative stressor on haematological and immunological variables to assess whether combining weaning with housing elicits a greater response than housing alone, ii) to (a) characterise, in male and female calves habituated to housing, the immune response to weaning stress at the physiological and molecular levels, and (b) assess the difference between calves weaned and housed in the presence of the dam and those weaned and housed away from the dam, and iii) the effect of accumulative stressors on the global transcriptomic response in blood leukocytes to weaning stress. The results of this thesis demonstrate that stress can have accumulative effects and by combining housing with weaning, an increase in neutrophil number and decrease in lymphocyte number was found. Concerning management at weaning, it is evident that calves, particularly intact male calves, may benefit from a weaning strategy where the calves are allowed contact with the dam but prevented from suckling for a number of days before total separation occurs. Weaning calves away from the dam also resulted in a much stronger stress response, resulting in an increase in the expression of inflammatory cytokines, the glucocorticoid receptor, TLR4, CD62L and Fas, than weaning calves next to the dam. Additionally, a far more potent inflammatory response, at the transcriptomic level, was reported to occur in weaned and housed calves compared with housed calves, indicating that stress activates the innate immune system to increase surveillance. An important finding of this thesis was that simultaneously weaning and housing of calves produces a perturbation to the homeostasis of the leukocyte transcriptome which was still present 7 days following weaning. Thus the identification of novel biomarkers and regulatory gene networks that are stress activated provides a mechanistic framework to characterise the multifaceted nature of weaning stress adaptation in beef calves

Biomarker responses to weaning stress in beef calves

The study objective was to investigate the physiological effects of weaning on beef calves and identify a panel of blood-based welfare biomarkers. On the day (d) of weaning (d 0), 16 spring-born, singlesuckled, beef bull calves that previously grazed with their dams at pasture, were assigned to one of two treatments: (1) control (n = 8), calves were loose-housed with their dam, (2) weaned (n = 8), calves were abruptly separated from their dam and loose-housed. Jugular blood was collected on d −4, 0, 1, 2, 3, 7, and 14 relative to weaning (d 0) and assayed for inflammatory and steroid variables. Total leukocyte counts were measured in whole blood. It is concluded that neutrophil number is a robust biomarker of stress and that plasma CXCL8 is a sensitive indicator of stress in weaned and control calves. In future studies, these two biomarkers should be central to the characterisation of stress responses

Examination of the effect of weaning stress on the physiological and molecular regulation of immune function in circulating bovine leukocytes

Weaning is a multifaceted stressor and may involve numerous husbandry practices, including the abrupt separation of the calf from its dam, a nutritional adjustment to a non-milk diet and social reorganisation and, additionally, is often associated with other husbandry practices, such as housing and transport. Stress-induced disease susceptibility in livestock animals has documented economic and societal consequences and the sense of urgency to understand and intervene in these is becoming increasingly important. Available published research has examined the physiological and behavioural responses to weaning stress in calves, but no study has yet characterised the molecular response. This Ph.D. project aimed to obtain a greater understanding of the impacts of weaning on both the molecular and physiological responses in order to elucidate the immune mechanisms responsible for stress dependent immune suppression in calves post weaning. A series of studies were conducted to investigate i) the effect of weaning stress as an accumulative stressor on haematological and immunological variables to assess whether combining weaning with housing elicits a greater response than housing alone, ii) to (a) characterise, in male and female calves habituated to housing, the immune response to weaning stress at the physiological and molecular levels, and (b) assess the difference between calves weaned and housed in the presence of the dam and those weaned and housed away from the dam, and iii) the effect of accumulative stressors on the global transcriptomic response in blood leukocytes to weaning stress. The results of this thesis demonstrate that stress can have accumulative effects and by combining housing with weaning, an increase in neutrophil number and decrease in lymphocyte number was found. Concerning management at weaning, it is evident that calves, particularly intact male calves, may benefit from a weaning strategy where the calves are allowed contact with the dam but prevented from suckling for a number of days before total separation occurs. Weaning calves away from the dam also resulted in a much stronger stress response, resulting in an increase in the expression of inflammatory cytokines, the glucocorticoid receptor, TLR4, CD62L and Fas, than weaning calves next to the dam. Additionally, a far more potent inflammatory response, at the transcriptomic level, was reported to occur in weaned and housed calves compared with housed calves, indicating that stress activates the innate immune system to increase surveillance. An important finding of this thesis was that simultaneously weaning and housing of calves produces a perturbation to the homeostasis of the leukocyte transcriptome which was still present 7 days following weaning. Thus the identification of novel biomarkers and regulatory gene networks that are stress activated provides a mechanistic framework to characterise the multifaceted nature of weaning stress adaptation in beef calves

Examination of the effect of weaning stress on the physiological and molecular regulation of immune function in circulating bovine leukocytes

Weaning is a multifaceted stressor and may involve numerous husbandry practices, including the abrupt separation of the calf from its dam, a nutritional adjustment to a non-milk diet and social reorganisation and, additionally, is often associated with other husbandry practices, such as housing and transport. Stress-induced disease susceptibility in livestock animals has documented economic and societal consequences and the sense of urgency to understand and intervene in these is becoming increasingly important. Available published research has examined the physiological and behavioural responses to weaning stress in calves, but no study has yet characterised the molecular response. This Ph.D. project aimed to obtain a greater understanding of the impacts of weaning on both the molecular and physiological responses in order to elucidate the immune mechanisms responsible for stress dependent immune suppression in calves post weaning. A series of studies were conducted to investigate i) the effect of weaning stress as an accumulative stressor on haematological and immunological variables to assess whether combining weaning with housing elicits a greater response than housing alone, ii) to (a) characterise, in male and female calves habituated to housing, the immune response to weaning stress at the physiological and molecular levels, and (b) assess the difference between calves weaned and housed in the presence of the dam and those weaned and housed away from the dam, and iii) the effect of accumulative stressors on the global transcriptomic response in blood leukocytes to weaning stress. The results of this thesis demonstrate that stress can have accumulative effects and by combining housing with weaning, an increase in neutrophil number and decrease in lymphocyte number was found. Concerning management at weaning, it is evident that calves, particularly intact male calves, may benefit from a weaning strategy where the calves are allowed contact with the dam but prevented from suckling for a number of days before total separation occurs. Weaning calves away from the dam also resulted in a much stronger stress response, resulting in an increase in the expression of inflammatory cytokines, the glucocorticoid receptor, TLR4, CD62L and Fas, than weaning calves next to the dam. Additionally, a far more potent inflammatory response, at the transcriptomic level, was reported to occur in weaned and housed calves compared with housed calves, indicating that stress activates the innate immune system to increase surveillance. An important finding of this thesis was that simultaneously weaning and housing of calves produces a perturbation to the homeostasis of the leukocyte transcriptome which was still present 7 days following weaning. Thus the identification of novel biomarkers and regulatory gene networks that are stress activated provides a mechanistic framework to characterise the multifaceted nature of weaning stress adaptation in beef calves

Transcriptomic analysis of the stress response to weaning at housing in bovine leukocytes using RNA-seq technology

peer-reviewedBackground: Weaning of beef calves is a necessary husbandry practice and involves separating the calf from its mother, resulting in numerous stressful events including dietary change, social reorganisation and the cessation of the maternal-offspring bond and is often accompanied by housing. While much recent research has focused on the physiological response of the bovine immune system to stress in recent years, little is known about the molecular mechanisms modulating the immune response. Therefore, the objective of this study was to provide new insights into the molecular mechanisms underlying the physiological response to weaning at housing in beef calves using Illumina RNA-seq.Results: The leukocyte transcriptome was significantly altered for at least 7 days following either housing or weaning at housing. Analysis of differentially expressed genes revealed that four main pathways, cytokine signalling, transmembrane transport, haemostasis and G-protein-coupled receptor (GPRC) signalling were differentially regulated between control and weaned calves and underwent significant transcriptomic alterations in response to weaning stress on day 1, 2 and 7. Of particular note, chemokines, cytokines and integrins were consistently found to be up-regulated on each day following weaning. Evidence for alternative splicing of genes was also detected, indicating a number of genes involved in the innate and adaptive immune response may be alternatively transcribed, including those responsible for toll receptor cascades and T cell receptor signalling.Conclusions: This study represents the first application of RNA-Seq technology for genomic studies in bovine leukocytes in response to weaning stress. Weaning stress induces the activation of a number of cytokine, chemokine and integrin transcripts and may alter the immune system whereby the ability of a number of cells of the innate and adaptive immune system to locate and destroy pathogens is transcriptionally enhanced. Stress alters the homeostasis of the transcriptomic environment of leukocytes for at least 7 days following weaning, indicating long term effects of stress exposure in the bovine. The identification of gene signature networks that are stress activated provides a mechanistic framework to characterise the multifaceted nature of weaning stress adaptation in beef calves. Thus, capturing subtle transcriptomic changes provides insight into the molecular mechanisms that underlie the physiological response to weaning stress

Examination of the bovine leukocyte environment using immunogenetic biomarkers to assess immunocompetence following exposure to weaning stress

The molecular mechanisms by which stress induces the development of pathologies remains unclear, although it is recognised that one of the major factors affecting health as a consequence of stress is the involvement of the neuroendocrine system. In cattle, a number of necessary husbandry practices have been shown to activate the stress response, yet very little is known about the impact these have at the molecular level. The objectives of the study were to characterise, in male and female beef calves, the immune response to weaning stress in bovine leukocytes at the physiological and molecular levels and to assess the difference between calves weaned in the presence of the dam and those weaned and penned away from the dam