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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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Sistemas CRISPR-Cas , Zigoto , Animais , Bovinos/genética , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas , Reparo do DNA por Junção de Extremidades , Feminino , Edição de Genes , Técnicas de Introdução de Genes , MasculinoRESUMO
A live, infectious vaccine candidate for epizootic bovine abortion, designated EBAA Vaccine, USDA-APHIS Product code #1544.00, has been reported to be both safe and effective. Previous studies established that a single dose of EBAA vaccine administered to cows at potencies of either 2000 or 500 live P. abortibovis-infected murine spleen cells (P.a.-LIC) induced protective immunity for a minimum of 5 months. The current study employed 19 pregnant cows that were challenged with P. abortibovis in their 2nd trimester of gestation; 9 were vaccinated 17.2-months earlier as 1-year-olds with 2000 P.a.-LIC and 10 served as negative controls. Eighty-nine percent of the vaccinates gave birth to healthy calves as compared to 10% of challenge controls. Vaccine efficacy was significant when analyzed by prevented fractions (87.7%; 95% CI=0.4945-0.9781). Serologic data supports previous findings that pregnant cows with detectable P. abortibovis antibodies are immune to P. abortibovis challenge as demonstrated by the birth of healthy calves.
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Aborto Animal , Animais , Bovinos , Feminino , Gravidez , Aborto Animal/imunologia , Aborto Animal/prevenção & controle , Doenças dos Bovinos/imunologia , Doenças dos Bovinos/prevenção & controle , Estações do Ano , Vacinas Atenuadas/imunologia , Vacinas Atenuadas/administração & dosagem , Vacinas Bacterianas/imunologia , Vacinas Bacterianas/administração & dosagemRESUMO
A single locus on the X chromosome codes for androgen receptor (AR) although this gene is subject to alternative splicing. AR is expressed in multiple tissues in males and females and is essential for reproductive success in the male. Since male and female mice are viable following naturally occurring and engineered loss of function with male mice infertile as anticipated, functional deletion of AR in pigs was hypothesized to provide a genetic containment strategy for males with edited genomes. In addition, deletion of AR might be a method to manage boar taint, hence contributing to a perceived improvement in animal welfare. The CRISPR/Cas9 technology was used to edit either exon 2 or exon 5 of the pig AR gene. Although pregnancies were established following embryo transfer of edited embryos, they were not maintained beyond day 25. Furthermore, normal M:F sex ratios were present in edited blastocysts and 19-day fetuses, but all fetuses recovered on day 21 or later were female. The pig AR gene differs from the mouse in having a U2 spliceosome component encoded in the intronic region. Hence, the absence of fetal survival beyond day 25 may be due to interference with the U2 component rather than AR.
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Receptores Androgênicos , Spliceossomos , Masculino , Feminino , Gravidez , Suínos , Animais , Camundongos , Spliceossomos/genética , Receptores Androgênicos/genética , Feto , Íntrons , Éxons/genéticaRESUMO
NANOS3 is expressed in migrating primordial germ cells (PGCs) to protect them from apoptosis, and it is known to be a critical factor for germline development of both sexes in several organisms. However, to date, live NANOS3 knockout (KO) cattle have not been reported, and the specific role of NANOS3 in male cattle, or bulls, remains unexplored. This study generated NANOS3 KO cattle via cytoplasmic microinjection of the CRISPR/Cas9 system in vitro produced bovine zygotes and evaluated the effect of NANOS3 elimination on bovine germline development, from fetal development through reproductive age. The co-injection of two selected guide RNA (gRNA)/Cas9 ribonucleoprotein complexes (i.e., dual gRNA approach) at 6 h post fertilization achieved a high NANOS3 KO rate in developing embryos. Subsequent embryo transfers resulted in a 31% (n = 8/26) pregnancy rate. A 75% (n = 6/8) total KO rate (i.e., 100% of alleles present contained complete loss-of-function mutations) was achieved with the dual gRNA editing approach. In NANOS3 KO fetal testes, PGCs were found to be completely eliminated by 41-day of fetal age. Importantly, despite the absence of germ cells, seminiferous tubule development was not impaired in NANOS3 KO bovine testes during fetal, perinatal, and adult stages. Moreover, a live, NANOS3 KO, germline-ablated bull was produced and at sexual maturity he exhibited normal libido, an anatomically normal reproductive tract, and intact somatic gonadal development and structure. Additionally, a live, NANOS3 KO, germline-ablated heifer was produced. However, it was evident that the absence of germ cells in NANOS3 KO cattle compromised the normalcy of ovarian development to a greater extent than it did testes development. The meat composition of NANOS3 KO cattle was unremarkable. Overall, this study demonstrated that the absence of NANOS3 in cattle leads to the specific deficiency of both male and female germ cells, suggesting the potential of NANOS3 KO cattle to act as hosts for donor-derived exogenous germ cell production in both sexes. These findings contribute to the understanding of NANOS3 function in cattle and have valuable implications for the development of novel breeding technologies using germline complementation in NANOS3 KO germline-ablated hosts.
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A long intergenic non-coding RNA (lincRNA#1) is overexpressed in the horn bud region of polled (hornless) bovine fetuses, suggesting a potential role in horn bud suppression. Genome editing was used to test whether the absence of this sequence was associated with the horned phenotype. Two gRNAs with high mutation efficiencies targeting the 5' and the 3' regions flanking the lincRNA#1 sequence were co-injected with Cas9 as ribonucleoprotein complexes into bovine zygotes (n = 121) 6 h post insemination. Of the resulting blastocysts (n = 31), 84% had the expected 3.7 kb deletion; of these embryos with the 3.7 kb deletions, 88% were biallelic knockouts. Thirty-nine presumptive edited 7-day blastocysts were transferred to 13 synchronized recipient cows resulting in ten pregnancies, five with embryos heterozygous for the dominant PC POLLED allele at the POLLED locus, and five with the recessive pp genotype. Eight (80%) of the resulting fetuses were biallelic lincRNA#1 knockouts, with the remaining two being mosaic. RT-qPCR analysis was used to confirm the absence of lincRNA#1 expression in knockout fetuses. Phenotypic and histological analysis of the genotypically (PCp) POLLED, lincRNA#1 knockout fetuses revealed similar morphology to non-edited, control polled fetuses, indicating the absence of lincRNA#1 alone does not result in a horned phenotype.
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Cornos , RNA Longo não Codificante , Alelos , Animais , Bovinos , Feminino , Heterozigoto , Fenótipo , Gravidez , RNA Longo não Codificante/genéticaRESUMO
Dehorning is a common practice in the dairy industry, but raises animal welfare concerns. A naturally occurring genetic mutation (PC allele) comprised of a 212 bp duplicated DNA sequence replacing a 10-bp sequence at the polled locus is associated with the hornless phenotype (polled) in cattle. To test the hypothesis that the 10 bp deletion alone is sufficient to result in polled, a CRISPR-Cas9 dual guide RNA approach was optimized to delete a 133 bp region including the 10 bp sequence. Timing of ribonucleoprotein complex injections at various hours post insemination (hpi) (6, 8, and 18 hpi) as well as in vitro transcribed (IVT) vs synthetic gRNAs were compared. Embryos injected 6 hpi had a significantly higher deletion rate (53%) compared to those injected 8 (12%) and 18 hpi (7%), and synthetic gRNAs had a significantly higher deletion rate (84%) compared to IVT gRNAs (53%). Embryo transfers were performed, and bovine fetuses were harvested between 3 and 5 months of gestation. All fetuses had mutations at the target site, with two of the seven having biallelic deletions, and yet they displayed horn bud development indicating that the 10 bp deletion alone is not sufficient to result in the polled phenotype.
Assuntos
Indústria de Laticínios/métodos , Feto/anatomia & histologia , Cornos/crescimento & desenvolvimento , Deleção de Sequência/genética , Animais , Sistemas CRISPR-Cas , Bovinos , Transferência Embrionária/métodos , Feto/embriologia , Genótipo , Fenótipo , RNA Guia de Cinetoplastídeos/genéticaRESUMO
Epizootic bovine abortion (EBA) is an arthropod-borne bacterial disease that causes significant economic loss for cattle producers in the western United States. The etiologic agent, Pajaroellobacter abortibovis, is an intracellular pathogen that has yet to be cultivated in vitro, thereby requiring novel methodologies for vaccine development. A vaccine candidate, using live P. abortibovis-infected cells (P.a-LIC) harvested from mouse spleens, was tested in beef cattle. Over the course of two safety studies and four efficacy trials, safety risks were evaluated, and dosage and potencies refined. No incidence of anaphylaxis, recognized health issues or significant impact upon conception rates were noted. Vaccination did result in subclinical skin reactions. Early fetal losses were noted in two trials and were significant when the vaccine was administered within 21 days prior to conception. Administration of the EBA agent (EBAA) vaccine as a single dose, at a potency of 500 P.a-LIC, 56 days prior to breeding, provided 100% protection with no early fetal losses. Seroconversion occurred in all animals following EBAA vaccination and corresponded well with protection of the fetus from epizootic bovine abortion.
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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 PC Celtic POLLED allele, with horned cows (pp) and obtained six heterozygous (PCp) 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 PC Celtic POLLED allele and an allele containing an additional introgression of the homology-directed repair donor plasmid along with the PC 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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Bovinos/genética , Edição de Genes , Genoma , Alelos , Animais , Sequência de Bases , Cruzamento , Quimerismo , Feminino , Feto/fisiologia , Loci Gênicos , Genótipo , Cornos , Masculino , Fenótipo , Filogenia , Plasmídeos/genética , Polimorfismo de Nucleotídeo Único/genéticaRESUMO
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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Edição de Genes/métodos , Técnicas de Introdução de Genes/métodos , Engenharia Genética/métodos , Animais , Proteína 9 Associada à CRISPR/genética , Sistemas CRISPR-Cas/genética , Bovinos , Reparo do DNA por Junção de Extremidades/fisiologia , Reparo do DNA/genética , Genoma/genética , Recombinação Homóloga/genética , Microinjeções/métodos , RNA Guia de Cinetoplastídeos/genética , Reparo de DNA por Recombinação/genética , Zigoto/fisiologiaRESUMO
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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To compare the use of controlled intravaginal drug release (CIDR, EAZI-BREED™ CIDR® Sheep, USA, Zoetis) with the ram effect, a combination of CIDR and ram effect and the addition of P.G.600® to each of these scenarios on their contribution to inducing the onset of cyclicity and their ability to improve reproductive efficiency in a commercial sheep flock, a randomized clinical trial was conducted. Multiparous ewes (nâ¯=â¯283) were randomly assigned to one of six treatments prior to intact male introduction on day 0: only CIDR devices (day -5 to day 0) (nâ¯=â¯40), exposure to two vasectomized rams (d-14 to d0) (nâ¯=â¯55), CIDR (d-5 to d0) and an intramuscular injection of P.G.600® (d0) (nâ¯=â¯40), exposure to two vasectomized rams (d-14 to d0) and treated with CIDR devices (day -5 to day 0) (nâ¯=â¯40). exposure to vasectomized rams (d-14 to d0), CIDR (d-5 to d0) and an intramuscular injection ofP.G.600®, (d0) (nâ¯=â¯38), and a control group, without exposure to vasectomized rams, CIDRs or P.G.600® (nâ¯=â¯70). Ewes were exposed to intact males on day 0. Days to lambing from the earliest expected lambing date at 150 days post introduction of rams was compared between groups with survival analysis. The mean survival time was significantly shorter in all groups that used CIDR compared to the control group (Pâ¯<â¯0.003, adjusted for multiple comparisons). The addition of a teaser ram or a P.G.600® injection did not change survival time. In a Cox proportional hazard model for time to lambing, the use of CIDR had a hazard ratio of 1.62 (95% CI: 1.07, 2.45, Pâ¯=â¯0.23), while other variables (teaser ram, P.G.600®) were not significant. In a Poisson regression for the number of lambs born to each ewe, P.G.600® was not a significant variable, even when adjusted for age of ewe (Pâ¯=â¯0.74). The combination of the ram effect and CIDRs did not have an additive effect. The use of CIDR shortened time to cyclicity in study ewes and may provide an attractive option to advance lambing season. Further studies on the economics of this method are warranted.
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Progesterona/uso terapêutico , Ovinos/fisiologia , Administração Intravaginal , Animais , Cruzamento/métodos , Ciclo Estral , Fotoperíodo , Distribuição de Poisson , Progesterona/administração & dosagem , Modelos de Riscos Proporcionais , Análise de Regressão , Estações do AnoRESUMO
Optimal mineral nutrition is required for cattle reproduction, immune function, and structural development. Formal evaluation of the current mineral status of California beef cattle is currently lacking. In 2017, a survey was initiated that evaluated a panel of 10 different minerals in 14 counties across California. Samples were collected from 555 cattle at 50 different ranches. Region of the state significantly affected herd mineral status. Herd use of supplements was also significant, and increased most blood levels of the mineral(s) targeted for supplementation. Forage source was idiosyncratic on its effect of mineral status. Previous blood survey data showed selenium to be widely deficient in California cattle in the 1970s and 1980s, but in this case, it was generally adequate in all areas of California. This indicates a good producer understanding of where supplementation is needed. Copper deficiency was more widespread in the southern region when compared with further north. Zinc deficiency was seen ubiquitously statewide, with 36% of animals being deficient. Manganese has been largely ignored in California. This study is the first known documentation of manganese levels in the state. Sampling found 92% of cattle fell below critical manganese levels. However, further research to better define manganese critical levels is probably warranted. The status of other minerals is presented.
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CASE DESCRIPTION A 2-year-old primiparous miniature Scottish Highland cow with an unknown breeding date was evaluated for suspected hydrops. CLINICAL FINDINGS Transabdominal and transrectal ultrasonographic examination identified a large amount of hypoechoic fluid within an enlarged uterus; the fetus could not be identified. Presence of a severely distended uterus and concerns regarding associated health risks to the cow led to the decision to induce labor. Although fluids were expelled, parturition did not progress further over the following 48 hours. Vaginal examination revealed a partially dilated cervix and an abnormally shaped fetus that was too large to pass vaginally. TREATMENT AND OUTCOME Supportive care was provided to the cow, and a stillborn bull calf was delivered by cesarean section. Grossly evident chondrodystrophic dwarfism with hydrocephalus, compatible with so-called bulldog calf malformations, was confirmed by diagnostic imaging and histopathologic evaluation. The cow recovered from surgery uneventfully and was discharged from the hospital the following day. Genetic analysis of DNA from hair roots collected from the sire and dam confirmed both were carriers of an aggrecan-1 gene mutation (bulldog dwarfism1) previously associated with dwarfism and bulldog calf malformations in Dexter cattle. CLINICAL RELEVANCE To our knowledge, this is the first reported case of bulldog calf malformations associated with an aggrecan-1 gene mutation in miniature Scottish Highland cattle, confirming that at least 1 genetic mutation associated with this condition is found in cattle breeds other than Dexter. The findings highlighted the clinical importance of testing for known genetic diseases in breeding cattle, particularly among miniature breeds.