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Observations of glacier mass changes are key to understanding the response of glaciers to climate change and related impacts, such as regional runoff, ecosystem changes, and global sea level rise. Spaceborne optical and radar sensors make it possible to quantify glacier elevation changes, and thus multi-annual mass changes, on a regional and global scale. However, estimates from a growing number of studies show a wide range of results with differences often beyond uncertainty bounds. Here, we present the outcome of a community-based inter-comparison experiment using spaceborne optical stereo (ASTER) and synthetic aperture radar interferometry (TanDEM-X) data to estimate elevation changes for defined glaciers and target periods that pose different assessment challenges. Using provided or self-processed digital elevation models (DEMs) for five test sites, 12 research groups provided a total of 97 spaceborne elevation-change datasets using various processing approaches. Validation with airborne data showed that using an ensemble estimate is promising to reduce random errors from different instruments and processing methods but still requires a more comprehensive investigation and correction of systematic errors. We found that scene selection, DEM processing, and co-registration have the biggest impact on the results. Other processing steps, such as treating spatial data voids, differences in survey periods, or radar penetration, can still be important for individual cases. Future research should focus on testing different implementations of individual processing steps (e.g. co-registration) and addressing issues related to temporal corrections, radar penetration, glacier area changes, and density conversion. Finally, there is a clear need for our community to develop best practices, use open, reproducible software, and assess overall uncertainty to enhance inter-comparison and empower physical process insights across glacier elevation-change studies.

Glacier mass balance has been estimated on individual glacier and regional scales using repeat digital elevation models (DEMs). DEMs often have gaps in coverage (“voids”), the properties of which depend on the nature of the sensor used and the surface being measured. The way that these voids are accounted for has a direct impact on the estimate of geodetic glacier mass balance, though a systematic comparison of different proposed methods has been heretofore lacking. In this study, we determine the impact and sensitivity of void interpolation methods on estimates of volume change. Using two spatially complete, high-resolution DEMs over southeast Alaska, USA, we artificially generate voids in one of the DEMs using correlation values derived from photogrammetric processing of Advanced Spaceborne Thermal Emission and Reflection Radiometer (ASTER) scenes. We then compare 11 different void interpolation methods on a glacier-by-glacier and regional basis. We find that a few methods introduce biases of up to 20 % in the regional results, while other methods give results very close ( % difference) to the true, non-voided volume change estimates. By comparing results from a few of the best-performing methods, an estimate of the uncertainty introduced by interpolating voids can be obtained. Finally, by increasing the number of voids, we show that with these best-performing methods, reliable estimates of glacier-wide volume change can be obtained, even with sparse DEM coverage.

Knowledge of the ice thickness distribution of glaciers and ice caps is an important prerequisite for many glaciological and hydrological investigations. A wealth of approaches has recently been presented for inferring ice thickness from characteristics of the surface. With the Ice Thickness Models Intercomparison eXperiment (ITMIX) we performed the first coordinated assessment quantifying individual model performance. A set of 17 different models showed that individual ice thickness estimates can differ considerably – locally by a spread comparable to the observed thickness. Averaging the results of multiple models, however, significantly improved the results: on average over the 21 considered test cases, comparison against direct ice thickness measurements revealed deviations on the order of 10 ± 24 % of the mean ice thickness (1σ estimate). Models relying on multiple data sets – such as surface ice velocity fields, surface mass balance, or rates of ice thickness change – showed high sensitivity to input data quality. Together with the requirement of being able to handle large regions in an automated fashion, the capacity of better accounting for uncertainties in the input data will be a key for an improved next generation of ice thickness estimation approaches.

Abstract 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.

Abstract Campylobacter jejuni is a very common cause of gastroenteritis, and is frequently transmitted to humans through contaminated food products or water. Importantly, C. jejuni infections have a range of short- and long-term sequelae such as irritable bowel syndrome and Guillain Barre syndrome. C. jejuni triggers disease by employing a range of molecular strategies which enable it to colonise the gut, invade the epithelium, persist intracellularly and avoid detection by the host immune response. The objective of this review is to explore and summarise recent advances in the understanding of the C. jejuni molecular factors involved in colonisation, invasion of cells, collective quorum sensing-mediated behaviours and persistence. Understanding the mechanisms that underpin the pathogenicity of C. jejuni will enable future development of effective preventative approaches and vaccines against this pathogen.

Abstract 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.

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.

Abstract 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.

Abstract 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.

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.