Source: UNIV OF CALIFORNIA (VET-MED) submitted to
SLAUGHTER HORSE TRANSPORT STRESS: FUNCTIONAL GENOMIC STUDY
Sponsoring Institution
Cooperating Schools of Veterinary Medicine
Project Status
TERMINATED
Funding Source
Reporting Frequency
Annual
Accession No.
0202472
Grant No.
(N/A)
Project No.
CALV-APHIS03-9106-06
Proposal No.
(N/A)
Multistate No.
(N/A)
Program Code
(N/A)
Project Start Date
Apr 1, 2003
Project End Date
Mar 31, 2005
Grant Year
(N/A)
Project Director
Stull, C. L.
Recipient Organization
UNIV OF CALIFORNIA (VET-MED)
(N/A)
DAVIS,CA 95616
Performing Department
VETERINARY MEDICINE EXTENSION
Non Technical Summary
The public in the US has become more concerned with many different animal welfare issues in the last 20 years. Transportation conditions of horses to slaughter has become a visible issue in the media. This research will make available a functional genomics approach for assessing stress during transport of slaughter horses and to use this tool for collecting data necessary for drafting recommendations for regulations of slaughter horses which will benefit the welfare and health of horses. Science-based regulations will be beneficial in foreign trade discussions.
Animal Health Component
100%
Research Effort Categories
Basic
(N/A)
Applied
50%
Developmental
50%
Classification

Knowledge Area (KA)Subject of Investigation (SOI)Field of Science (FOS)Percent
3153810102050%
3153810103025%
3153810109025%
Goals / Objectives
Since many different factors are integrated with stress response to transport, there is a need to identify the cellular mechanisms that trigger a stress reaction and apply this data to objectively evaluating the welfare of slaughter horses undergoing different transport conditions.
Project Methods
To identify key genes and patterns of gene expression that are reliable indicators of transport stress of slaughter horses transported long distances (24 hours) under commercial conditions (similar stocking density, bedding materials, care and horse subjects) using a straight-deck trailer. To correlate these identified gene patterns with traditional stress indices including immunological, hormonal and pathological parameters collected in the field for the application of predicting the severity of transport stress and it's impact on welfare of slaughter horses.

Progress 04/01/03 to 03/31/05

Outputs
Thirty-eight mature light horses of various breeds were used in a two transport treatment in a cross-over experimental design. The two transport treatments consisted of either 24 h of continuous transport (24T) or two 12 h transit periods separated by offloading, resting, and feeding horses for 12 h (12/12T). A total of four trips were scheduled such that each group was studied over both transit treatments. Six horses were randomly selected from the reciprocal group to serve as control horses (CH) for the transported horses (TH). The initial proposal described using subtractive hybridization for the detection of differentially-expressed genes. In order to maximize the potential for identifying as many biologically relevant genes as possible we subsequently decided to expand the experimental design and approach the investigation from three directions, using three independent techniques, namely ACP technology, suppression subtractive hybridization and differential display. To optimize the ACP technology, cDNA was synthesized from peripheral blood mononuclear cell-derived RNA from the pre-transit and post-transit samples of a single horse in the 24 hour transport protocol. By using a dT-ACP oligonucleotide in the reverse transcription reaction, an ACP adaptor was added to the cDNA products during this step. A two stage PCR was then used to generate PCR products that would be present in an amount representative of the cDNA. The optimization of these reactions is vital because it affects the performance of the regulator sequence on the 5 and 3-ends of the ACP oligonucleotide, which determines the generation of specific differentially expressed gene products. The optimization of this step has taken many months, but we have now designed conditions suitable for use with a range of ACP oligonucleotides. The gel bands representing possible differentially-expressed genes have been excised from the gels, and are currently being purified for cloning and nucleotide sequencing. The nucleotide sequences will allow us to identify each isolated gene and a decision to pursue further assays will be made. We have optimized real-time quantitative-PCR assays for a number of equine cytokine genes.

Impacts
Objectives 1) Identify genes with transport-associated changes in peripheral blood leukocyte gene expression using subtractive hybridization 2) Develop quantitative real time RT-PCR assays for each of the differentially expressed genes 3) Compare levels of gene expression between

Publications

  • No publications reported this period


Progress 01/01/04 to 12/31/04

Outputs
Suppression subtractive hybridization is a recently developed technique that allows the comparison of two populations of expressed genes, and also facilitates the isolation of genes that are expressed in one individual but not the other. In this study SSH will be used to identify transport stress-associated expression of genes in the peripheral blood leukocytes of horses. It is hoped that the detection of such genes will help us determine some of the molecular mechanisms by which transit stress affects the equine immune system, and provide a basis for the development of techniques by which the expression of these genes can be accurately quantified. Such assays could provide a highly sensitive means of measuring subclinical stress in transported animals. Hypothesis 1) Transport induces a specific pattern of gene expression involving functionally important genes of the equine immune system. 2) Horses with transport associated immunological perturbations show both quantitative and qualitative differences in expression of these genes compared to normal horses.

Impacts
Objectives 1) Identify genes with transport-associated changes in peripheral blood leukocyte gene expression using subtractive hybridization 2) Develop quantitative real time RT-PCR assays for each of the differentially expressed genes 3) Compare levels of gene expression between normal horses and those with immunological perturbations

Publications

  • No publications reported this period