Publications by authors named "Alison L Van Eenennaam"

40 Publications

Electroporation-Mediated Genome Editing of Livestock Zygotes.

Front Genet 2021 13;12:648482. Epub 2021 Apr 13.

Department of Animal Science, University of California, Davis, Davis, CA, United States.

The introduction of genome editing reagents into mammalian zygotes has traditionally been accomplished by cytoplasmic or pronuclear microinjection. This time-consuming procedure requires expensive equipment and a high level of skill. Electroporation of zygotes offers a simplified and more streamlined approach to transfect mammalian zygotes. There are a number of studies examining the parameters used in electroporation of mouse and rat zygotes. Here, we review the electroporation conditions, timing, and success rates that have been reported for mice and rats, in addition to the few reports about livestock zygotes, specifically pigs and cattle. The introduction of editing reagents at, or soon after, fertilization can help reduce the rate of mosaicism, the presence of two of more genotypes in the cells of an individual; as can the introduction of nuclease proteins rather than mRNA encoding nucleases. Mosaicism is particularly problematic in large livestock species with long generation intervals as it can take years to obtain non-mosaic, homozygous offspring through breeding. Gene knockouts accomplished the non-homologous end joining pathway have been more widely reported and successfully accomplished using electroporation than have gene knock-ins. Delivering large DNA plasmids into the zygote is hindered by the zona pellucida (ZP), and the majority of gene knock-ins accomplished by electroporation have been using short single stranded DNA (ssDNA) repair templates, typically less than 1 kb. The most promising approach to deliver larger donor repair templates of up to 4.9 kb along with genome editing reagents into zygotes, without using cytoplasmic injection, is to use recombinant adeno-associated viruses (rAAVs) in combination with electroporation. However, similar to other methods used to deliver clustered regularly interspaced palindromic repeat (CRISPR) genome-editing reagents, this approach is also associated with high levels of mosaicism. Recent developments complementing germline ablated individuals with edited germline-competent cells offer an approach to avoid mosaicism in the germline of genome edited founder lines. Even with electroporation-mediated delivery of genome editing reagents to mammalian zygotes, there remain additional chokepoints in the genome editing pipeline that currently hinder the scalable production of non-mosaic genome edited livestock.
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http://dx.doi.org/10.3389/fgene.2021.648482DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8078910PMC
April 2021

Functional annotations of three domestic animal genomes provide vital resources for comparative and agricultural research.

Nat Commun 2021 03 23;12(1):1821. Epub 2021 Mar 23.

Department of Animal Science, University of California, Davis, Davis, CA, USA.

Gene regulatory elements are central drivers of phenotypic variation and thus of critical importance towards understanding the genetics of complex traits. The Functional Annotation of Animal Genomes consortium was formed to collaboratively annotate the functional elements in animal genomes, starting with domesticated animals. Here we present an expansive collection of datasets from eight diverse tissues in three important agricultural species: chicken (Gallus gallus), pig (Sus scrofa), and cattle (Bos taurus). Comparative analysis of these datasets and those from the human and mouse Encyclopedia of DNA Elements projects reveal that a core set of regulatory elements are functionally conserved independent of divergence between species, and that tissue-specific transcription factor occupancy at regulatory elements and their predicted target genes are also conserved. These datasets represent a unique opportunity for the emerging field of comparative epigenomics, as well as the agricultural research community, including species that are globally important food resources.
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http://dx.doi.org/10.1038/s41467-021-22100-8DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7988148PMC
March 2021

Comparison of Gene Editing Versus Conventional Breeding to Introgress the Allele Into the Tropically Adapted Australian Beef Cattle Population.

Front Genet 2021 11;12:593154. Epub 2021 Feb 11.

Department of Animal Science, University of California, Davis, Davis, CA, United States.

Dehorning is the process of physically removing horns to protect animals and humans from injury, but the process is costly, unpleasant, and faces increasing public scrutiny. Genetic selection for polled (hornless), which is genetically dominant to horned, is a long-term solution to eliminate the need for dehorning. However, due to the limited number of polled Australian Brahman bulls, the northern Australian beef cattle population remains predominantly horned. The potential to use gene editing to produce high-genetic-merit polled cattle was recently demonstrated. To further explore the concept, this study simulated introgression of the allele into a tropically adapted Australian beef cattle population via conventional breeding or gene editing (top 1% or 10% of seedstock bulls/year) for 3 polled mating schemes and compared results to baseline selection on genetic merit (Japan Ox selection index, $JapOx) alone, over the course of 20 years. The baseline scenario did not significantly decrease the 20-year allele frequency (80%), but resulted in one of the fastest rates of genetic gain ($8.00/year). Compared to the baseline, the conventional breeding scenarios where polled bulls were preferentially used for breeding, regardless of their genetic merit, significantly decreased the 20-year allele frequency (30%), but resulted in a significantly slower rate of genetic gain ($6.70/year, ≤ 0.05). The mating scheme that required the exclusive use of homozygous polled bulls, resulted in the lowest 20-year allele frequency (8%), but this conventional breeding scenario resulted in the slowest rate of genetic gain ($5.50/year). The addition of gene editing the top 1% or 10% of seedstock bull calves/year to each conventional breeding scenario resulted in significantly faster rates of genetic gain (up to $8.10/year, ≤ 0.05). Overall, our study demonstrates that, due to the limited number of polled Australian Brahman bulls, strong selection pressure on polled will be necessary to meaningfully increase the number of polled animals in this population. Moreover, these scenarios illustrate how gene editing could be a tool for accelerating the development of high-genetic-merit homozygous polled sires to mitigate the current trade-off of slower genetic gain associated with decreasing allele frequency in the Australian Brahman population.
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http://dx.doi.org/10.3389/fgene.2021.593154DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7905321PMC
February 2021

One-step generation of a targeted knock-in calf using the CRISPR-Cas9 system in bovine zygotes.

BMC Genomics 2021 Feb 12;22(1):118. Epub 2021 Feb 12.

Department of Animal Science, University of California - Davis, Davis, CA, USA.

Background: The homologous recombination (HR) pathway is largely inactive in early embryos prior to the first cell division, making it difficult to achieve targeted gene knock-ins. The homology-mediated end joining (HMEJ)-based strategy has been shown to increase knock-in efficiency relative to HR, non-homologous end joining (NHEJ), and microhomology-mediated end joining (MMEJ) strategies in non-dividing cells.

Results: By introducing gRNA/Cas9 ribonucleoprotein complex and a HMEJ-based donor template with 1 kb homology arms flanked by the H11 safe harbor locus gRNA target site, knock-in rates of 40% of a 5.1 kb bovine sex-determining region Y (SRY)-green fluorescent protein (GFP) template were achieved in Bos taurus zygotes. Embryos that developed to the blastocyst stage were screened for GFP, and nine were transferred to recipient cows resulting in a live phenotypically normal bull calf. Genomic analyses revealed no wildtype sequence at the H11 target site, but rather a 26 bp insertion allele, and a complex 38 kb knock-in allele with seven copies of the SRY-GFP template and a single copy of the donor plasmid backbone. An additional minor 18 kb allele was detected that looks to be a derivative of the 38 kb allele resulting from the deletion of an inverted repeat of four copies of the SRY-GFP template.

Conclusion: The allelic heterogeneity in this biallelic knock-in calf appears to have resulted from a combination of homology directed repair, homology independent targeted insertion by blunt-end ligation, NHEJ, and rearrangement following editing of the gRNA target site in the donor template. This study illustrates the potential to produce targeted gene knock-in animals by direct cytoplasmic injection of bovine embryos with gRNA/Cas9, although further optimization is required to ensure a precise single-copy gene integration event.
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http://dx.doi.org/10.1186/s12864-021-07418-3DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7881600PMC
February 2021

Evaluation of mutation rates, mosaicism and off target mutations when injecting Cas9 mRNA or protein for genome editing of bovine embryos.

Sci Rep 2020 12 18;10(1):22309. Epub 2020 Dec 18.

Department of Animal Science, University of California - Davis, Davis, CA, USA.

The CRISPR/Cas9 genome editing tool has the potential to improve the livestock breeding industry by allowing for the introduction of desirable traits. Although an efficient and targeted tool, the CRISPR/Cas9 system can have some drawbacks, including off-target mutations and mosaicism, particularly when used in developing embryos. Here, we introduced genome editing reagents into single-cell bovine embryos to compare the effect of Cas9 mRNA and protein on the mutation efficiency, level of mosaicism, and evaluate potential off-target mutations utilizing next generation sequencing. We designed guide-RNAs targeting three loci (POLLED, H11, and ZFX) in the bovine genome and saw a significantly higher rate of mutation in embryos injected with Cas9 protein (84.2%) vs. Cas9 mRNA (68.5%). In addition, the level of mosaicism was higher in embryos injected with Cas9 mRNA (100%) compared to those injected with Cas9 protein (94.2%), with little to no unintended off-target mutations detected. This study demonstrated that the use of gRNA/Cas9 ribonucleoprotein complex resulted in a high editing efficiency at three different loci in bovine embryos and decreased levels of mosaicism relative to Cas9 mRNA. Additional optimization will be required to further reduce mosaicism to levels that make single-step embryo editing in cattle commercially feasible.
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http://dx.doi.org/10.1038/s41598-020-78264-8DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7749171PMC
December 2020

Genetic Engineering of Livestock: The Opportunity Cost of Regulatory Delay.

Annu Rev Anim Biosci 2021 02 13;9:453-478. Epub 2020 Nov 13.

Department of Agricultural and Resource Economics, University of California, Berkeley, California 94720, USA; email:

Genetically engineered (GE) livestock were first reported in 1985, and yet only a single GE food animal, the fast-growing AquAdvantage salmon, has been commercialized. There are myriad interconnected reasons for the slow progress in this once-promising field, including technical issues, the structure of livestock industries, lack of public research funding and investment, regulatory obstacles, and concern about public opinion. This review focuses on GE livestock that have been produced and documents the difficulties that researchers and developers have encountered en route. Additionally, the costs associated with delayed commercialization of GE livestock were modeled using three case studies: GE mastitis-resistant dairy cattle, genome-edited porcine reproductive and respiratory syndrome virus-resistant pigs, and the AquAdvantage salmon. Delays of 5 or 10 years in the commercialization of GE livestock beyond the normative 10-year GE product evaluation period were associated with billions of dollars in opportunity costs and reduced global food security.
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http://dx.doi.org/10.1146/annurev-animal-061220-023052DOI Listing
February 2021

Efficient One-Step Knockout by Electroporation of Ribonucleoproteins Into Zona-Intact Bovine Embryos.

Front Genet 2020 7;11:570069. Epub 2020 Sep 7.

Department of Animal Science, University of California, Davis, Davis, CA, United States.

Somatic cell nuclear transfer or cytoplasm microinjection have been used to generate genome-edited farm animals; however, these methods have several drawbacks that reduce their efficiency. This study aimed to develop electroporation conditions that allow delivery of CRISPR/Cas9 system to bovine zygotes for efficient gene knock-out. We optimized electroporation conditions to deliver Cas9:sgRNA ribonucleoproteins to bovine zygotes without compromising embryo development. Higher electroporation pulse voltage resulted in increased membrane permeability; however, voltages above 15 V/mm decreased embryo developmental potential. The zona pellucida of bovine embryos was not a barrier to efficient RNP electroporation. Using parameters optimized for maximal membrane permeability while maintaining developmental competence we achieved high rates of gene editing when targeting bovine OCT4, which resulted in absence of OCT4 protein in 100% of the evaluated embryos and the expected arrest of embryonic development at the morula stage. In conclusion, Cas9:sgRNA ribonucleoproteins can be delivered efficiently by electroporation to zona-intact bovine zygotes, resulting in efficient gene knockouts.
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http://dx.doi.org/10.3389/fgene.2020.570069DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7504904PMC
September 2020

A comparative analysis of chromatin accessibility in cattle, pig, and mouse tissues.

BMC Genomics 2020 Oct 7;21(1):698. Epub 2020 Oct 7.

Department of Animal Science, University of California Davis, Davis, CA, 95616, USA.

Background: Although considerable progress has been made towards annotating the noncoding portion of the human and mouse genomes, regulatory elements in other species, such as livestock, remain poorly characterized. This lack of functional annotation poses a substantial roadblock to agricultural research and diminishes the value of these species as model organisms. As active regulatory elements are typically characterized by chromatin accessibility, we implemented the Assay for Transposase Accessible Chromatin (ATAC-seq) to annotate and characterize regulatory elements in pigs and cattle, given a set of eight adult tissues.

Results: Overall, 306,304 and 273,594 active regulatory elements were identified in pig and cattle, respectively. 71,478 porcine and 47,454 bovine regulatory elements were highly tissue-specific and were correspondingly enriched for binding motifs of known tissue-specific transcription factors. However, in every tissue the most prevalent accessible motif corresponded to the insulator CTCF, suggesting pervasive involvement in 3-D chromatin organization. Taking advantage of a similar dataset in mouse, open chromatin in pig, cattle, and mice were compared, revealing that the conservation of regulatory elements, in terms of sequence identity and accessibility, was consistent with evolutionary distance; whereas pig and cattle shared about 20% of accessible sites, mice and ungulates only had about 10% of accessible sites in common. Furthermore, conservation of accessibility was more prevalent at promoters than at intergenic regions.

Conclusions: The lack of conserved accessibility at distal elements is consistent with rapid evolution of enhancers, and further emphasizes the need to annotate regulatory elements in individual species, rather than inferring elements based on homology. This atlas of chromatin accessibility in cattle and pig constitutes a substantial step towards annotating livestock genomes and dissecting the regulatory link between genome and phenome.
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http://dx.doi.org/10.1186/s12864-020-07078-9DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7541309PMC
October 2020

Harnessing endogenous repair mechanisms for targeted gene knock-in of bovine embryos.

Sci Rep 2020 09 29;10(1):16031. Epub 2020 Sep 29.

Department of Animal Science, University of CA - Davis, Davis, CA, USA.

Introducing useful traits into livestock breeding programs through gene knock-ins has proven challenging. Typically, targeted insertions have been performed in cell lines, followed by somatic cell nuclear transfer cloning, which can be inefficient. An alternative is to introduce genome editing reagents and a homologous recombination (HR) donor template into embryos to trigger homology directed repair (HDR). However, the HR pathway is primarily restricted to actively dividing cells (S/G2-phase) and its efficiency for the introduction of large DNA sequences in zygotes is low. The homology-mediated end joining (HMEJ) approach has been shown to improve knock-in efficiency in non-dividing cells and to harness HDR after direct injection of embryos. The knock-in efficiency for a 1.8 kb gene was contrasted when combining microinjection of a gRNA/Cas9 ribonucleoprotein complex with a traditional HR donor template or an HMEJ template in bovine zygotes. The HMEJ template resulted in a significantly higher rate of gene knock-in as compared to the HR template (37.0% and 13.8%; P < 0.05). Additionally, more than a third of the knock-in embryos (36.9%) were non-mosaic. This approach will facilitate the one-step introduction of gene constructs at a specific location of the bovine genome and contribute to the next generation of elite cattle.
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http://dx.doi.org/10.1038/s41598-020-72902-xDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7525238PMC
September 2020

Author Correction: Genomic and phenotypic analyses of six offspring of a genome-edited hornless bull.

Nat Biotechnol 2020 Feb;38(2):245

Department of Animal Science, University of California, Davis, CA, USA.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.
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http://dx.doi.org/10.1038/s41587-020-0423-5DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7007411PMC
February 2020

Genomic and phenotypic analyses of six offspring of a genome-edited hornless bull.

Nat Biotechnol 2020 02 7;38(2):225-232. Epub 2019 Oct 7.

Department of Animal Science, University of California, Davis, CA, USA.

Genome editing followed by reproductive cloning was previously used to produce two hornless dairy bulls. We crossed one genome-edited dairy bull, homozygous for the dominant P Celtic POLLED allele, with horned cows (pp) and obtained six heterozygous (Pp) polled calves. The calves had no horns and were otherwise healthy and phenotypically unremarkable. We conducted whole-genome sequencing of all animals using an Illumina HiSeq4000 to achieve ~20× coverage. Bioinformatics analyses revealed the bull was a compound heterozygote, carrying one naturally occurring P Celtic POLLED allele and an allele containing an additional introgression of the homology-directed repair donor plasmid along with the P Celtic allele. These alleles segregated in the offspring of this bull, and inheritance of either allele produced polled calves. No other unintended genomic alterations were observed. These data can be used to inform conversations in the scientific community, with regulatory authorities and with the public around 'intentional genomic alterations' and future regulatory actions regarding genome-edited animals.
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http://dx.doi.org/10.1038/s41587-019-0266-0DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7007412PMC
February 2020

Management of lethal recessive alleles in beef cattle through the use of mate selection software.

Genet Sel Evol 2019 Aug 6;51(1):36. Epub 2019 Aug 6.

Department of Animal Science, University of California, Davis, CA, 95616, USA.

Background: Recessive loss-of-function (LOF) alleles at genes which are essential for life, can result in early embryonic mortality. Cattle producers can use the LOF carrier status of individual animals to make selection and mate allocation decisions.

Methods: Two beef cattle breeding strategies i.e. (1) selection against LOF carriers as parents and (2) simultaneous selection and mate allocation to avoid the occurrence of homozygous offspring in three scenarios, which differed in number and frequency of LOF alleles were evaluated using the mate selection program, MateSel. Scenarios included (a) seven loci with high-frequency LOF alleles, (b) 76 loci with low-frequency LOF alleles, and (c) 50 loci with random high- and low-frequency LOF alleles. In addition, any savings resulting from the information obtained by varying the percentage (0-100%) of the herd genotyped, together with segregation analysis to cover ungenotyped animals, were calculated to determine (1) which percentage optimized net profit for a fixed cost of genotyping ($30/test), and (2) the breakeven cost for genotyping.

Results: With full knowledge of the LOF alleles carried by selection candidates, the most profitable breeding strategy was always simultaneous selection and mate allocation to avoid homozygous affected offspring (aa) as compared to indiscriminate selection against carrier parents (Aa). The breakeven value of genotyping depended on the number of loci modeled, the LOF allele frequencies, and the mating/selection strategies used. Genotyping was most valuable when it was used to avoid otherwise high levels of embryonic mortalities. As the number of essential loci with LOF alleles increased, especially when some were present at relatively high minor allele frequencies, embryonic losses increased, and profit was maximized by genotyping 10 to 20% of a herd and using that information to reduce these losses.

Conclusions: Genotyping 100% of the herd was never the most profitable outcome in any scenario; however, genotyping some proportion of the herd, together with segregation analysis to cover ungenotyped animals, maximized overall profit in scenarios with large numbers of loci with LOF alleles. As more LOF alleles are identified, such a mate selection software will likely be required to optimally select and allocate matings to balance the rate of genetic gain, embryonic losses, and inbreeding.
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http://dx.doi.org/10.1186/s12711-019-0477-3DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6683482PMC
August 2019

Application of genome editing in farm animals: cattle.

Transgenic Res 2019 08;28(Suppl 2):93-100

University of California, Davis, USA.

Milk and meat from cattle and buffaloes contribute 45% of the global animal protein supply, followed by chickens (31%), and pigs (20%). In 2016, the global cattle population of 1.0 billion head produced 6.5 billion tons of cows' milk, and 66 million tons of beef. In the past century, cattle breeding programs have greatly increased the yield per animal with a resultant decrease in the emissions intensity per unit of milk or beef, but this has not been true in all regions. Genome editing research in cattle to date has focused on disease resistance (e.g. tuberculosis), production (e.g. myostatin knockout; production of all-male offspring), elimination of allergens (e.g. beta-lactoglobulin knockout) and welfare (e.g. polled or hornlessness) traits. Modeling has revealed how the use of genome editing to introduce beneficial alleles into cattle breeds could maintain or even accelerate the rate of genetic gain accomplished by conventional breeding programs, and is a superior approach to the lengthy process of introgressing those same alleles from distant breeds. Genome editing could be used to precisely introduce useful alleles (e.g. heat tolerance, disease resistance) and haplotypes into native locally-adapted cattle breeds, thereby helping to improve their productivity. As with earlier genetic engineering approaches, whether breeders will be able to employ genome editing in cattle genetic improvement programs will very much depend upon global decisions around the regulatory framework and governance of genome editing for food animals.
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http://dx.doi.org/10.1007/s11248-019-00141-6DOI Listing
August 2019

Proposed U.S. regulation of gene-edited food animals is not fit for purpose.

NPJ Sci Food 2019 20;3. Epub 2019 Mar 20.

1Department of Animal Science, University of California, Davis, CA USA.

Dietary DNA is generally regarded as safe to consume, and is a routine ingredient of food obtained from any living organism. Millions of naturally-occurring DNA variations are observed when comparing the genomic sequence of any two healthy individuals of a given species. Breeders routinely select desired traits resulting from this DNA variation to develop new cultivars and varieties of food plants and animals. Regulatory agencies do not evaluate these new varieties prior to commercial release. Gene editing tools now allow plant and animal breeders to precisely introduce useful genetic variation into agricultural breeding programs. The U.S. Department of Agriculture (USDA) announced that it has no plans to place additional regulations on gene-edited plants that could otherwise have been developed through traditional breeding prior to commercialization. However, the U.S. Food and Drug Administration (FDA) has proposed mandatory premarket new animal drug regulatory evaluation for all food animals whose genomes have been intentionally altered using modern molecular technologies including gene editing technologies. This runs counter to U.S. biotechnology policy that regulatory oversight should be triggered by unreasonable risk, and not by the fact that an organism has been modified by a particular process or technique. Breeder intention is not associated with product risk. Harmonizing the regulations associated with gene editing in food species is imperative to allow both plant and animal breeders access to gene editing tools to introduce useful sustainability traits like disease resistance, climate adaptability, and food quality attributes into U.S. agricultural breeding programs.
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http://dx.doi.org/10.1038/s41538-019-0035-yDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6550240PMC
March 2019

The Importance of a Novel Product Risk-Based Trigger for Gene-Editing Regulation in Food Animal Species.

CRISPR J 2018 04 9;1:101-106. Epub 2018 Apr 9.

Department of Animal Science, University of California , Davis, California.

A 2017 draft Food and Drug Administration guidance proposes mandatory, multigenerational premarket new animal drug evaluation of all "intentional genomic alterations" induced by site-directed nucleases such as CRISPR in food animal genomes, irrespective of the novelty of the alteration or the existence of any hazards in the resulting product. Such a regulatory approach will effectively introduce additional layers of regulatory scrutiny on products produced using gene editing that are no different from those that could have been obtained using conventional breeding. Although this guidance may not directly impact the basic research community, due to Food and Drug Administration regulatory discretion around genetic modifications in model species, the potential opportunity cost of placing high regulatory costs on gene-edited food animals based solely on the use of modern molecular methods to introduce genomic alterations has potential global implications in terms of sustainability of food animal production.
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http://dx.doi.org/10.1089/crispr.2017.0023DOI Listing
April 2018

Association of plasma haptoglobin concentration and other biomarkers with bovine respiratory disease status in pre-weaned dairy calves.

J Vet Diagn Invest 2019 Jan 17;31(1):40-46. Epub 2018 Oct 17.

Department of Animal Sciences, Auburn University, Auburn, AL.

We conducted a nested, case-control study of pre-weaned dairy calves ( n = 477; 4 California dairy farms) to assess the association between bovine respiratory disease (BRD) and hematologic biomarkers, including plasma haptoglobin (Hp) and plasma bactericide (PB). At each location, heifer or bull dairy calves were observed 2-4 times per week until confirmed as BRD-positive using parallel interpretation of thoracic ultrasound examination and auscultation. In addition, control calves were enrolled after being confirmed as BRD-negative using ultrasound and auscultation. Complete blood counts (CBC), PB, and Hp concentrations were measured. Hp values were higher in calves with confirmed BRD than in controls ( p < 0.01). The area under the curve (AUC) for the various biomarkers was obtained from the corresponding receiver operating characteristic curves. The AUC for Hp was 0.68, a value greater than those for PB or the remaining CBC parameters, indicating that Hp may be the most useful biomarker of BRD in pre-weaned dairy calves. The cutoff value for Hp was 0.195 g/L.
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http://dx.doi.org/10.1177/1040638718807242DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6505765PMC
January 2019

Simulation of introgression of the allele into the Jersey breed via conventional breeding vs. gene editing.

Transl Anim Sci 2018 Sep 27;2(Suppl 1):S57-S60. Epub 2018 Sep 27.

Department of Animal Science, University of California, Davis, CA.

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http://dx.doi.org/10.1093/tas/txy054DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7200810PMC
September 2018

Genome-wide identification of tissue-specific long non-coding RNA in three farm animal species.

BMC Genomics 2018 Sep 18;19(1):684. Epub 2018 Sep 18.

Department of Animal Science, University of California, Davis, Davis, CA, USA.

Background: Numerous long non-coding RNAs (lncRNAs) have been identified and their roles in gene regulation in humans, mice, and other model organisms studied; however, far less research has been focused on lncRNAs in farm animal species. While previous studies in chickens, cattle, and pigs identified lncRNAs in specific developmental stages or differentially expressed under specific conditions in a limited number of tissues, more comprehensive identification of lncRNAs in these species is needed. The goal of the FAANG Consortium (Functional Annotation of Animal Genomes) is to functionally annotate animal genomes, including the annotation of lncRNAs. As one of the FAANG pilot projects, lncRNAs were identified across eight tissues in two adult male biological replicates from chickens, cattle, and pigs.

Results: Comprehensive lncRNA annotations for the chicken, cattle, and pig genomes were generated by utilizing RNA-seq from eight tissue types from two biological replicates per species at the adult developmental stage. A total of 9393 lncRNAs in chickens, 7235 lncRNAs in cattle, and 14,429 lncRNAs in pigs were identified. Including novel isoforms and lncRNAs from novel loci, 5288 novel lncRNAs were identified in chickens, 3732 in cattle, and 4870 in pigs. These transcripts match previously known patterns of lncRNAs, such as generally lower expression levels than mRNAs and higher tissue specificity. An analysis of lncRNA conservation across species identified a set of conserved lncRNAs with potential functions associated with chromatin structure and gene regulation. Tissue-specific lncRNAs were identified. Genes proximal to tissue-specific lncRNAs were enriched for GO terms associated with the tissue of origin, such as leukocyte activation in spleen.

Conclusions: LncRNAs were identified in three important farm animal species using eight tissues from adult individuals. About half of the identified lncRNAs were not previously reported in the NCBI annotations for these species. While lncRNAs are less conserved than protein-coding genes, a set of positionally conserved lncRNAs were identified among chickens, cattle, and pigs with potential functions related to chromatin structure and gene regulation. Tissue-specific lncRNAs have potential regulatory functions on genes enriched for tissue-specific GO terms. Future work will include epigenetic data from ChIP-seq experiments to further refine these annotations.
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http://dx.doi.org/10.1186/s12864-018-5037-7DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6145346PMC
September 2018

Tissue Tropism in Host Transcriptional Response to Members of the Bovine Respiratory Disease Complex.

Sci Rep 2017 12 20;7(1):17938. Epub 2017 Dec 20.

Division of Animal Sciences, University of Missouri, Columbia, MO, United States of America.

Bovine respiratory disease (BRD) is the most common infectious disease of beef and dairy cattle and is characterized by a complex infectious etiology that includes a variety of viral and bacterial pathogens. We examined the global changes in mRNA abundance in healthy lung and lung lesions and in the lymphoid tissues bronchial lymph node, retropharyngeal lymph node, nasopharyngeal lymph node and pharyngeal tonsil collected at the peak of clinical disease from beef cattle experimentally challenged with either bovine respiratory syncytial virus, infectious bovine rhinotracheitis, bovine viral diarrhea virus, Mannheimia haemolytica or Mycoplasma bovis. We identified signatures of tissue-specific transcriptional responses indicative of tropism in the coordination of host's immune tissue responses to infection by viral or bacterial infections. Furthermore, our study shows that this tissue tropism in host transcriptional response to BRD pathogens results in the activation of different networks of response genes. The differential crosstalk among genes expressed in lymphoid tissues was predicted to be orchestrated by specific immune genes that act as 'key players' within expression networks. The results of this study serve as a basis for the development of innovative therapeutic strategies and for the selection of cattle with enhanced resistance to BRD.
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http://dx.doi.org/10.1038/s41598-017-18205-0DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5738336PMC
December 2017

Whole-Genome Sequencing and Concordance Between Antimicrobial Susceptibility Genotypes and Phenotypes of Bacterial Isolates Associated with Bovine Respiratory Disease.

G3 (Bethesda) 2017 09 7;7(9):3059-3071. Epub 2017 Sep 7.

Department of Animal Science, University of California, Davis, California 95616

Extended laboratory culture and antimicrobial susceptibility testing timelines hinder rapid species identification and susceptibility profiling of bacterial pathogens associated with bovine respiratory disease, the most prevalent cause of cattle mortality in the United States. Whole-genome sequencing offers a culture-independent alternative to current bacterial identification methods, but requires a library of bacterial reference genomes for comparison. To contribute new bacterial genome assemblies and evaluate genetic diversity and variation in antimicrobial resistance genotypes, whole-genome sequencing was performed on bovine respiratory disease-associated bacterial isolates (, , , and ) from dairy and beef cattle. One hundred genomically distinct assemblies were added to the NCBI database, doubling the available genomic sequences for these four species. Computer-based methods identified 11 predicted antimicrobial resistance genes in three species, with none being detected in While computer-based analysis can identify antibiotic resistance genes within whole-genome sequences (genotype), it may not predict the actual antimicrobial resistance observed in a living organism (phenotype). Antimicrobial susceptibility testing on 64 , , and isolates had an overall concordance rate between genotype and phenotypic resistance to the associated class of antimicrobials of 72.7% ( < 0.001), showing substantial discordance. Concordance rates varied greatly among different antimicrobial, antibiotic resistance gene, and bacterial species combinations. This suggests that antimicrobial susceptibility phenotypes are needed to complement genomically predicted antibiotic resistance gene genotypes to better understand how the presence of antibiotic resistance genes within a given bacterial species could potentially impact optimal bovine respiratory disease treatment and morbidity/mortality outcomes.
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http://dx.doi.org/10.1534/g3.117.1137DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5592931PMC
September 2017

Genome Report: Identification and Validation of Antigenic Proteins from Using Genome Sequence Assembly and Reverse Vaccinology.

G3 (Bethesda) 2017 02 9;7(2):321-331. Epub 2017 Feb 9.

Department of Animal Science, University of California, Davis, California 95616

Epizootic bovine abortion (EBA), or "foothill abortion," is the leading cause of beef cattle abortion in California and has also been reported in Nevada and Oregon. In the 1970s, the soft-shelled tick , or "pajaroello tick," was confirmed as the disease-transmitting vector. In 2005, a novel Deltaproteobacterium was discovered as the etiologic agent of EBA (aoEBA), recently named This organism cannot be grown in culture using traditional microbiological techniques; it can only be grown in experimentally-infected severe combined immunodeficient (SCID) mice. The objectives of this study were to perform a genome assembly for and identify and validate potential antigenic proteins as candidates for future recombinant vaccine development. DNA and RNA were extracted from spleen tissue collected from experimentally-infected SCID mice following exposure to This combination of mouse and bacterial DNA was sequenced and aligned to the mouse genome. Mouse sequences were subtracted from the sequence pool and the remaining sequences were assembled at 50x coverage into a 1.82 Mbp complete closed circular Deltaproteobacterial genome containing 2250 putative protein-coding sequences. Phylogenetic analysis of predicts that this bacterium is most closely related to the organisms of the order Myxococcales, referred to as Myxobacteria. prediction of vaccine candidates was performed using a reverse vaccinology approach resulting in the identification and ranking of the top 10 candidate proteins that are likely to be antigenic. Immunologic testing of these candidate proteins confirmed antigenicity of seven of the nine expressed protein candidates using serum from immunized mice.
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http://dx.doi.org/10.1534/g3.116.036673DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5295582PMC
February 2017

Regulate genome-edited products, not genome editing itself.

Nat Biotechnol 2016 05;34(5):477-9

Department of Genetics, Cell Biology, and Development, University of Minnesota, Minneapolis, Minnesota, USA.

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http://dx.doi.org/10.1038/nbt.3566DOI Listing
May 2016

Identification of Gene Networks for Residual Feed Intake in Angus Cattle Using Genomic Prediction and RNA-seq.

PLoS One 2016 28;11(3):e0152274. Epub 2016 Mar 28.

VMRD Genetics R&D, Zoetis Inc., Kalamazoo, MI, United States of America.

Improvement in feed conversion efficiency can improve the sustainability of beef cattle production, but genomic selection for feed efficiency affects many underlying molecular networks and physiological traits. This study describes the differences between steer progeny of two influential Angus bulls with divergent genomic predictions for residual feed intake (RFI). Eight steer progeny of each sire were phenotyped for growth and feed intake from 8 mo. of age (average BW 254 kg, with a mean difference between sire groups of 4.8 kg) until slaughter at 14-16 mo. of age (average BW 534 kg, sire group difference of 28.8 kg). Terminal samples from pituitary gland, skeletal muscle, liver, adipose, and duodenum were collected from each steer for transcriptome sequencing. Gene expression networks were derived using partial correlation and information theory (PCIT), including differentially expressed (DE) genes, tissue specific (TS) genes, transcription factors (TF), and genes associated with RFI from a genome-wide association study (GWAS). Relative to progeny of the high RFI sire, progeny of the low RFI sire had -0.56 kg/d finishing period RFI (P = 0.05), -1.08 finishing period feed conversion ratio (P = 0.01), +3.3 kg^0.75 finishing period metabolic mid-weight (MMW; P = 0.04), +28.8 kg final body weight (P = 0.01), -12.9 feed bunk visits per day (P = 0.02) with +0.60 min/visit duration (P = 0.01), and +0.0045 carcass specific gravity (weight in air/weight in air-weight in water, a predictor of carcass fat content; P = 0.03). RNA-seq identified 633 DE genes between sire groups among 17,016 expressed genes. PCIT analysis identified >115,000 significant co-expression correlations between genes and 25 TF hubs, i.e. controllers of clusters of DE, TS, and GWAS SNP genes. Pathway analysis suggests low RFI bull progeny possess heightened gut inflammation and reduced fat deposition. This multi-omics analysis shows how differences in RFI genomic breeding values can impact other traits and gene co-expression networks.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0152274PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4809598PMC
August 2016

Sensitivity and specificity of on-farm scoring systems and nasal culture to detect bovine respiratory disease complex in preweaned dairy calves.

J Vet Diagn Invest 2016 Mar 21;28(2):119-28. Epub 2016 Jan 21.

Veterinary Medicine Teaching and Research Center, Tulare, CA (Love, Lehenbauer, Aly)Departments of Population Health and Reproduction (Lehenbauer, Kass, Farver, Aly), University of California, Davis, CAAnimal Science (Van Eenennaam), University of California, Davis, CAStatistics (Drake), University of California, Davis, CA

The California (CA) and Wisconsin (WI) clinical scoring systems have been proposed for bovine respiratory disease complex (BRDC) detection in preweaned dairy calves. The screening sensitivity (SSe), for estimating BRDC prevalence in a cohort of calves, diagnostic sensitivity (DSe), for confirming BRDC in ill calves, and specificity (Sp) were estimated for each of the scoring systems, as well as for nasal swab cultures for aerobic bacteria and mycoplasma species. Thoracic ultrasound and auscultation were used as the reference standard tests interpreted in parallel. A total of 536 calves (221 with BRDC and 315 healthy) were sampled from 5 premises in California. The SSe of 46.8%, DSe of 72.6%, and Sp of 87.4% was determined for the CA system. The SSe of 46.0%, DSe of 71.1%, and Sp of 91.2% was determined for the WI system. For aerobic culture, the SSe was 43.4%, DSe was 52.6%, and Sp was 71.3%; for Mycoplasma spp. culture, the SSe was 57.5%, DSe was 68.9%, and Sp was 59.7%. The screening and diagnostic sensitivities of the scoring systems were not significantly different but the Sp of the WI system was greater by 3.8%. Scoring systems can serve as rapid on-farm tools to determine the burden of BRDC in preweaned dairy calves. However, users may expect the SSe to be less than the DSe when confirming BRDC in an ill calf.
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http://dx.doi.org/10.1177/1040638715626204DOI Listing
March 2016

Single Pathogen Challenge with Agents of the Bovine Respiratory Disease Complex.

PLoS One 2015 16;10(11):e0142479. Epub 2015 Nov 16.

Department of Veterinary Pathobiology, Texas A & M University, College Station, Texas, 77843-4467, United States of America.

Bovine respiratory disease complex (BRDC) is an important cause of mortality and morbidity in cattle; costing the dairy and beef industries millions of dollars annually, despite the use of vaccines and antibiotics. BRDC is caused by one or more of several viruses (bovine respiratory syncytial virus, bovine herpes type 1 also known as infectious bovine rhinotracheitis, and bovine viral diarrhea virus), which predispose animals to infection with one or more bacteria. These include: Pasteurella multocida, Mannheimia haemolytica, Mycoplasma bovis, and Histophilus somni. Some cattle appear to be more resistant to BRDC than others. We hypothesize that appropriate immune responses to these pathogens are subject to genetic control. To determine which genes are involved in the immune response to each of these pathogens it was first necessary to experimentally induce infection separately with each pathogen to document clinical and pathological responses in animals from which tissues were harvested for subsequent RNA sequencing. Herein these infections and animal responses are described.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0142479PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4646450PMC
June 2016

Immunological Response to Single Pathogen Challenge with Agents of the Bovine Respiratory Disease Complex: An RNA-Sequence Analysis of the Bronchial Lymph Node Transcriptome.

PLoS One 2015 29;10(6):e0131459. Epub 2015 Jun 29.

Division of Animal Sciences, University of Missouri, Columbia, Missouri, United States of America.

Susceptibility to bovine respiratory disease (BRD) is multi-factorial and is influenced by stress in conjunction with infection by both bacterial and viral pathogens. While vaccination is broadly used in an effort to prevent BRD, it is far from being fully protective and cases diagnosed from a combination of observed clinical signs without any attempt at identifying the causal pathogens are usually treated with antibiotics. Dairy and beef cattle losses from BRD are profound worldwide and genetic studies have now been initiated to elucidate host loci which underlie susceptibility with the objective of enabling molecular breeding to reduce disease prevalence. In this study, we employed RNA sequencing to examine the bronchial lymph node transcriptomes of controls and beef cattle which had individually been experimentally challenged with bovine respiratory syncytial virus, infectious bovine rhinotracheitis, bovine viral diarrhea virus, Pasteurella multocida, Mannheimia haemolytica or Mycoplasma bovis to identify the genes that are involved in the bovine immune response to infection. We found that 142 differentially expressed genes were located in previously described quantitative trait locus regions associated with risk of BRD. Mutations affecting the expression or amino acid composition of these genes may affect disease susceptibility and could be incorporated into molecular breeding programs. Genes involved in innate immunity were generally found to be differentially expressed between the control and pathogen-challenged animals suggesting that variation in these genes may lead to a heritability of susceptibility that is pathogen independent. However, we also found pathogen-specific expression profiles which suggest that host genetic variation for BRD susceptibility is pathogen dependent.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0131459PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4484807PMC
March 2016

Applied animal genomics: results from the field.

Annu Rev Anim Biosci 2014 Feb 2;2:105-39. Epub 2013 Dec 2.

Department of Animal Science, University of California, Davis, California 95616; email: ,

Genomic selection (GS) is the use of statistical methods to estimate the genetic merit of a genotyped animal based on prediction equations derived from large ancestral populations with both phenotypes and genotypes. It has revolutionized the dairy cattle breeding industry and has been implemented with varying degrees of success in other animal breeding programs, including swine, poultry, and beef cattle. The findings of empirical field studies applying GS to the breeding sectors of these main animal protein industries are reviewed. Several translational considerations must be addressed before implementing GS in genetic improvement programs. These include determining and obtaining economically relevant phenotypes and determining the optimal size of the training population, cost-effective genotyping strategies, the practicality of field implementation, and the relative costs versus the benefits of the realized rate of genetic gain. GS may additionally change the optimal breeding scheme design, and studies that address this consideration are also reviewed briefly.
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http://dx.doi.org/10.1146/annurev-animal-022513-114119DOI Listing
February 2014

Development of a novel clinical scoring system for on-farm diagnosis of bovine respiratory disease in pre-weaned dairy calves.

PeerJ 2014 2;2:e238. Epub 2014 Jan 2.

Veterinary Medicine Teaching and Research Center, School of Veterinary Medicine, University of California - Davis , Tulare, CA , USA ; Department of Population Health and Reproduction, School of Veterinary Medicine, University of California - Davis , Davis, CA , USA.

Several clinical scoring systems for diagnosis of bovine respiratory disease (BRD) in calves have been proposed. However, such systems were based on subjective judgment, rather than statistical methods, to weight scores. Data from a pair-matched case-control study on a California calf raising facility was used to develop three novel scoring systems to diagnose BRD in preweaned dairy calves. Disease status was assigned using both clinical signs and diagnostic test results for BRD-associated pathogens. Regression coefficients were used to weight score values. The systems presented use nasal and ocular discharge, rectal temperature, ear and head carriage, coughing, and respiratory quality as predictors. The systems developed in this research utilize fewer severity categories of clinical signs, require less calf handling, and had excellent agreement (Kappa > 0.8) when compared to an earlier scoring system. The first scoring system dichotomized all clinical predictors but required inducing a cough. The second scoring system removed induced cough as a clinical abnormality but required distinguishing between three levels of nasal discharge severity. The third system removed induced cough and forced a dichotomized variable for nasal discharge. The first system presented in this study used the following predictors and assigned values: coughing (induced or spontaneous coughing, 2 points), nasal discharge (any discharge, 3 points), ocular discharge (any discharge, 2 points), ear and head carriage (ear droop or head tilt, 5 points), fever (≥39.2°C or 102.5°F, 2 points), and respiratory quality (abnormal respiration, 2 points). Calves were categorized "BRD positive" if their total score was ≥4. This system correctly classified 95.4% cases and 88.6% controls. The second presented system categorized the predictors and assigned weights as follows: coughing (spontaneous only, 2 points), mild nasal discharge (unilateral, serous, or watery discharge, 3 points), moderate to severe nasal discharge (bilateral, cloudy, mucoid, mucopurlent, or copious discharge, 5 points), ocular discharge (any discharge, 1 point), ear and head carriage (ear droop or head tilt, 5 points), fever (≥39.2°C, 2 points), and respiratory quality (abnormal respiration, 2 points). Calves were categorized "BRD positive" if their total score was ≥4. This system correctly classified 89.3% cases and 92.8% controls. The third presented system used the following predictors and scores: coughing (spontaneous only, 2 points), nasal discharge (any, 4 points), ocular discharge (any, 2 points), ear and head carriage (ear droop or head tilt, 5 points), fever (≥39.2°C, 2 points), and respiratory quality (abnormal respiration, 2 points). Calves were categorized "BRD positive" if their total score was ≥5. This system correctly classified 89.4% cases and 90.8% controls. Each of the proposed systems offer few levels of clinical signs and data-based weights for on-farm diagnosis of BRD in dairy calves.
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http://dx.doi.org/10.7717/peerj.238DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3898311PMC
January 2014

Considerations related to breed or biological type.

Vet Clin North Am Food Anim Pract 2013 Nov 4;29(3):493-516. Epub 2013 Sep 4.

Department of Animal Science, University of California, One Shields Avenue, Davis, CA 95616, USA. Electronic address:

This article reviews the literature on breed, biological type, and breeding system and their impact on female fertility, especially as they relate to heifer development. The attributes of different breeding systems and their appropriate use is discussed. In addition, the extant and emerging selection tools that are available for replacement heifer selection are reviewed.
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http://dx.doi.org/10.1016/j.cvfa.2013.07.012DOI Listing
November 2013

GMOs in animal agriculture: time to consider both costs and benefits in regulatory evaluations.

J Anim Sci Biotechnol 2013 Sep 25;4(1):37. Epub 2013 Sep 25.

Department of Animal Science, 2113 Meyer Hall, University of California, One Shields Avenue, Davis, CA 95616, USA.

In 2012, genetically engineered (GE) crops were grown by 17.3 million farmers on over 170 million hectares. Over 70% of harvested GE biomass is fed to food producing animals, making them the major consumers of GE crops for the past 15 plus years. Prior to commercialization, GE crops go through an extensive regulatory evaluation. Over one hundred regulatory submissions have shown compositional equivalence, and comparable levels of safety, between GE crops and their conventional counterparts. One component of regulatory compliance is whole GE food/feed animal feeding studies. Both regulatory studies and independent peer-reviewed studies have shown that GE crops can be safely used in animal feed, and rDNA fragments have never been detected in products (e.g. milk, meat, eggs) derived from animals that consumed GE feed. Despite the fact that the scientific weight of evidence from these hundreds of studies have not revealed unique risks associated with GE feed, some groups are calling for more animal feeding studies, including long-term rodent studies and studies in target livestock species for the approval of GE crops. It is an opportune time to review the results of such studies as have been done to date to evaluate the value of the additional information obtained. Requiring long-term and target animal feeding studies would sharply increase regulatory compliance costs and prolong the regulatory process associated with the commercialization of GE crops. Such costs may impede the development of feed crops with enhanced nutritional characteristics and durability, particularly in the local varieties in small and poor developing countries. More generally it is time for regulatory evaluations to more explicitly consider both the reasonable and unique risks and benefits associated with the use of both GE plants and animals in agricultural systems, and weigh them against those associated with existing systems, and those of regulatory inaction. This would represent a shift away from a GE evaluation process that currently focuses only on risk assessment and identifying ever diminishing marginal hazards, to a regulatory approach that more objectively evaluates and communicates the likely impact of approving a new GE plant or animal on agricultural production systems.
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http://dx.doi.org/10.1186/2049-1891-4-37DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4015968PMC
September 2013