Application of genetically engineered (GE) large animals carrying multi-allelic modifications has been hampered by low efficiency in production and extended gestation period compared to rodents. Here, we rapidly generated RAG2/IL2RG double knockout pigs using direct injection of CRISPR/Cas9 system into developing embryos. RAG2/IL2RG deficient pigs were immunodeficient, characterized by depletion of lymphocytes and either absence of or structurally abnormal immune organs. Pigs were maintained in gnotobiotic facility and evaluated for human norovirus (HuNoV) infection. HuNoV shedding lasted for 16 days in wild type pigs, compared to 27 days (until the end of trials) in RAG2/IL2RG deficient pigs. Additionally, higher HuNoV titers were detected in intestinal tissues and contents and in blood, indicating increased and prolonged HuNoV infection in RAG2/IL2RG deficient pigs and the importance of lymphocytes in HuNoV clearance. These results suggest that GE immunodeficient gnotobiotic pigs serve as a novel model for biomedical research and will facilitate HuNoV studies.

Pigs are an important resource in agriculture and serve as a model for human diseases. Due to their physiological and anatomical similarities with humans, pigs can recapitulate symptoms of human diseases, making them a useful model in biomedicine. However, in the past pig models have not been widely used partially because of the difficulty in genetic modification. The lack of true embryonic stem cells in pigs forced researchers to utilize genetic modification in somatic cells and somatic cell nuclear transfer (SCNT) to generate genetically engineered (GE) pigs carrying site-specific modifications. Although possible, this approach is extremely inefficient and GE pigs born through this method often presented developmental defects associated with the cloning process. Advancement in the gene-editing systems such as Zinc-Finger Nucleases (ZFNs), Transcription activator-like effector nucleases (TALENs), and the Clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated 9 (Cas9) system have dramatically increased the efficiency of producing GE pigs. These gene-editing systems, specifically engineered endonucleases, are based on inducing double-stranded breaks (DSBs) at a specific location, and then site-specific modifications can be introduced through one of the two DNA repair pathways: non-homologous end joining (NHEJ) or homology direct repair (HDR). Random insertions or deletions (indels) can be introduced through NHEJ and specific nucleotide sequences can be introduced through HDR, if donor DNA is provided. Use of these engineered endonucleases provides a higher success in genetic modifications, multiallelic modification of the genome, and an opportunity to introduce site-specific modifications during embryogenesis, thus bypassing the need of SCNT in GE pig production. This review will provide a historical prospective of GE pig production and examples of how the gene-editing system, led by engineered endonucleases, have improved GE pig production. We will also present some of our current progress related to the optimal use of CRISPR/Cas9 system during embryogenesis.

Average daily gain is an indicator of the growth rate, feed efficiency, and current health status of livestock species including pigs. Continuous monitoring of daily gain in pigs aids producers to optimize their growth performance while ensuring animal welfare and sustainability, such as reducing stress reactions and feed waste. Computer vision has been used to predict live body weight from video images without direct handling of the pig. In most studies, videos were taken while pigs were immobilized at a weighing station or feeding area to facilitate data collection. An alternative approach is to capture videos while pigs are allowed to move freely within their own housing environment, which can be easily applied to the production system as no special imaging station needs to be established. The objective of this study was to establish a computer vision system by collecting RGB-D videos to capture top-view red, green, and blue (RGB) and depth images of nonrestrained, growing pigs to predict their body weight over time. Over a period of 38 d, eight growers were video recorded for approximately 3 min/d, at the rate of six frames per second, and manually weighed using an electronic scale. An image-processing pipeline in Python using OpenCV was developed to process the images. Specifically, each pig within the RGB frame was segmented by a thresholding algorithm, and the contour of the pig was identified to extract its length and width. The height of a pig was estimated from the depth images captured by the infrared depth sensor. Quality control included removing pigs that were touching the fence and sitting, as well as those showing extremely distorted shape or motion blur owing to their frequent movement. Fitting all of the morphological image descriptors simultaneously in linear mixed models yielded prediction coefficients of determination of 0.72–0.98, 0.65–0.95, 0.51–0.94, and 0.49–0.93 for 1-, 2-, 3-, and 4-d ahead forecasting, respectively, of body weight in time series cross-validation. Based on the results, we conclude that our RGB-D sensor-based imaging system coupled with the Python image-processing pipeline could potentially provide an effective approach to predict the live body weight of nonrestrained pigs from images.

Generating porcine embryos in vitro is a critical process for creating genetically modified pigs as agricultural and biomedical models; however, these embryo technologies have been scarcely applied by the swine industry. Currently, the primary issue with in vitro-produced porcine embryos is low pregnancy rate after transfer and small litter size, which may be exasperated by micromanipulation procedures. Thus, in this review, we discuss improvements that have been made to the in vitro porcine embryo production system to increase the number of live piglets per pregnancy as well as abnormalities in the embryos and piglets that may arise from in vitro culture and manipulation techniques. Furthermore, we examine areas related to embryo production and transfer where improvements are warranted that will have direct applications for increasing pregnancy rate after transfer and the number of live born piglets per litter.