SUMMARY Interspecies blastocyst complementation enables organ-specific enrichment of xenogenic pluripotent stem cell (PSC) derivatives. Here, we establish a versatile blastocyst complementation platform based on CRISPR-Cas9-mediated zygote genome editing and show enrichment of rat PSC-derivatives in several tissues of gene-edited organogenesis-disabled mice. Besides gaining insights into species evolution, embryogenesis, and human disease, interspecies blastocyst complementation might allow human organ generation in animals whose organ size, anatomy, and physiology are closer to humans. To date, however, whether human PSCs (hPSCs) can contribute to chimera formation in non-rodent species remains unknown. We systematically evaluate the chimeric competency of several types of hPSCs using a more diversified clade of mammals, the ungulates. We find that naïve hPSCs robustly engraft in both pig and cattle pre-implantation blastocysts but show limited contribution to post-implantation pig embryos. Instead, an intermediate hPSC type exhibits higher degree of chimerism and is able to generate differentiated progenies in post-implantation pig embryos.

Transgenesis provides a method of expressing novel proteins in milk to increase the functional benefits of milk consumption. Transgenic goats expressing human lysozyme (hLZ) at 67% of the concentration in human breast milk were produced, thereby enhancing the antimicrobial properties of goats' milk. The objective of this study was to investigate the impact of pasteurized milk containing hLZ on growth, the intestinal epithelium, and an enteropathogenic Escherichia coli (EPEC) infection in young weaned pigs. Pigs were placed into 4 groups and fed a diet of solid food and either control (nontransgenic) goats' milk or milk from hLZ-transgenic goats. Growth was assessed by weight gain. Nonchallenged pigs were necropsied after 6 wk, whereas the remaining pigs were necropsied at 7 wk following bacterial challenge. We determined the numbers of total coliforms and E. coli and examined small intestinal histology for all pigs. Complete blood counts were also determined pre- and postchallenge. Challenged pigs receiving hLZ milk had fewer total coliforms (P = 0.029) and E. coli (P = 0.030) in the ileum than controls. hLZ-fed pigs also had a greater duodenal villi width (P = 0.029) than controls. Additionally, nonchallenged hLZ-fed pigs had fewer intraepithelial lymphocytes per micron of villi height (P = 0.020) than nonchallenged controls. These results indicate that the consumption of pasteurized hLZ goats' milk has the potential to improve gastrointestinal health and is protective against an EPEC in young weaned pigs. These same benefits may occur in young children if they were to consume milk from hLZ-transgenic goats.

Avian gastrointestinal (GI) tracts are highly populated with a diverse array of microorganisms that share a symbiotic relationship with their hosts and contribute to the overall health and disease state of the intestinal tract. The microbiome of the young chick is easily prone to alteration in its composition by both exogenous and endogenous factors, especially during the early posthatch period. The genetic background of the host and exposure to pathogens can impact the diversity of the microbial profile that consequently contributes to the disease progression in the host. The objective of this study was to profile the composition and structure of the gut microbiota in young chickens from two genetically distinct highly inbred lines. Furthermore, the effect of the Salmonella Enteritidis infection on altering the composition makeup of the chicken microbiome was evaluated through the 16S rRNA gene sequencing analysis. One-day-old layer chicks were challenged with S. Enteritidis and the host cecal microbiota profile as well as the degree of susceptibility to Salmonella infection was examined at 2 and 7 days post infection. Our result indicated that host genotype had a limited effect on resistance to S. Enteritidis infection. Alpha diversity, beta diversity, and overall microbiota composition were analyzed for four factors: host genotype, age, treatment, and postinfection time points. S. Enteritidis infection in young chicks was found to significantly reduce the overall diversity of the microbiota population with expansion of Enterobacteriaceae family. These changes indicated that Salmonella colonization in the GI tract of the chickens has a direct effect on altering the natural development of the GI microbiota. The impact of S. Enteritidis infection on microbial communities was also more substantial in the late stage of infection. Significant inverse correlation between Enterobacteriaceae and Lachnospiraceae family in both non-infected and infected groups, suggested possible antagonistic interaction between members of these two taxa, which could potentially influences the overall microbial population in the gut. Our results also revealed that genetic difference between two lines had minimal effect on the establishment of microbiota population. Overall, this study provided preliminary insights into the contributing role of S. Enteritidis in influencing the overall makeup of chicken’s gut microbiota.

The potential for applying biotechnology to benefit animal agriculture and food production has long been speculated. The addition of human milk components with intrinsic antimicrobial activity and positive charge to livestock milk by genetic engineering has the potential to benefit animal health, as well as food safety and production. We generated one line of transgenic goats as a model for the dairy cow designed to express human lysozyme in the mammary gland. Here we report the characterization of the milk from 5 transgenic females of this line expressing human lysozyme in their milk at 270 microg/mL or 68% of the level found in human milk. Milk from transgenic animals had a lower somatic cell count, but the overall component composition of the milk and milk production were not different from controls. Milk from transgenic animals had a shorter rennet clotting time and increased curd strength. Milk of such nature may be of benefit to the producer by influencing udder health and milk processing.

ABSTRACTHuman milk contains antimicrobial factors such as lysozyme and lactoferrin that are thought to contribute to the development of an intestinal microbiota beneficial to host health. However, these factors are lacking in the milk of dairy animals. Here we report the establishment of an animal model to allow the dissection of the role of milk components in gut microbiota modulation and subsequent changes in overall and intestinal health. Using milk from transgenic goats expressing human lysozyme at 68%, the level found in human milk and young pigs as feeding subjects, the fecal microbiota was analyzed over time using 16S rRNA gene sequencing and the G2 Phylochip. The two methods yielded similar results, with the G2 Phylochip giving more comprehensive information by detecting more OTUs. Total community populations remained similar within the feeding groups, and community member diversity was changed significantly upon consumption of lysozyme milk. Levels ofFirmicutes(Clostridia) declined whereas those ofBacteroidetesincreased over time in response to the consumption of lysozyme-rich milk. The proportions of these major phyla were significantly different (P< 0.05) from the proportions seen with control-fed animals after 14 days of feeding. Within phyla, the abundance of bacteria associated with gut health (BifidobacteriaceaeandLactobacillaceae) increased and the abundance of those associated with disease (Mycobacteriaceae,Streptococcaceae,Campylobacterales) decreased with consumption of lysozyme milk. This study demonstrated that a single component of the diet with bioactivity changed the gut microbiome composition. Additionally, this model enabled the direct examination of the impact of lysozyme on beneficial microbe enrichment versus detrimental microbe reduction in the gut microbiome community.