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

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.

Cerebellar Abiotrophy Across Domestic Species.

Cerebellar abiotrophy (CA) is a neurodegenerative disorder affecting the cerebellum and occurs in multiple species. Although CA is well researched in humans and mice, domestic species such as the dog, cat, sheep, cow, and horse receive little recognition. This may be due to few studies addressing the mechanism of CA in these species. However, valuable information can still be extracted from these cases. A review of the clinicohistologic phenotype of CA in these species and determining the various etiologies of CA may aid in determining conserved and required pathways necessary for proper cerebellar development and function. This review outlines research approaches of studies of CA in domestic species, compared to the approaches used in mice, with the objective of comparing CA in domestic species while identifying areas for further research efforts.

Lysozyme-rich milk mitigates effects of malnutrition in a pig model of malnutrition and infection.

Malnutrition remains a leading contributor to the morbidity and mortality of children under the age of 5 years and can weaken the immune system and increase the severity of concurrent infections. Livestock milk with the protective properties of human milk is a potential therapeutic to modulate intestinal microbiota and improve outcomes. The aim of this study was to develop an infection model of childhood malnutrition in the pig to investigate the clinical, intestinal and microbiota changes associated with malnutrition and enterotoxigenic Escherichia coli (ETEC) infection and to test the ability of goat milk and milk from genetically engineered goats expressing the antimicrobial human lysozyme (hLZ) milk to mitigate these effects. Pigs were weaned onto a protein-energy-restricted diet and after 3 weeks were supplemented daily with goat, hLZ or no milk for a further 2 weeks and then challenged with ETEC. The restricted diet enriched faecal microbiota in Proteobacteria as seen in stunted children. Before infection, hLZ milk supplementation improved barrier function and villous height to a greater extent than goat milk. Both goat and hLZ milk enriched for taxa (Ruminococcaceae) associated with weight gain. Post-ETEC infection, pigs supplemented with hLZ milk weighed more, had improved Z-scores, longer villi and showed more stable bacterial populations during ETEC challenge than both the goat and no milk groups. This model of childhood disease was developed to test the confounding effects of malnutrition and infection and demonstrated the potential use of hLZ goat milk to mitigate the impacts of malnutrition and infection.

Young Pigs Consuming Lysozyme Transgenic Goat Milk Are Protected from Clinical Symptoms of Enterotoxigenic Escherichia coli Infection.

Background: Diarrheal diseases in infancy and childhood are responsible for substantial morbidity and mortality in developing nations. Lysozyme, an antimicrobial component of human milk, is thought to play a role in establishing a healthy intestinal microbiota and immune system. Consumption of breast milk has been shown to prevent intestinal infections and is a recommended treatment for infants with diarrhea.Objective: This study aimed to examine the ability of lysozyme-rich goat milk to prevent intestinal infection.Methods: Six-week-old Hampshire-Yorkshire pigs were assigned to treatment groups balanced for weight, sex, and litter and were fed milk from nontransgenic control goats (GM group) or human lysozyme transgenic goats (hLZM group) for 2 wk before they were challenged with porcine-specific enterotoxigenic Escherichia coli (ETEC). Fecal consistency, complete blood counts, intestinal histology, and microbial populations were evaluated.Results: Pigs in the hLZM group had less severe diarrhea than did GM pigs at 24 and 48 h after ETEC infection (P = 0.01 and 0.05, respectively), indicating a less severe clinical disease state. Relative to baseline, postmilk hLZM pigs had 19.9% and 137% enrichment in fecal Bacteroidetes (P = 0.028) and Paraprevotellaceae (P = 0.003), respectively, and a 93.8% reduction in Enterobacteriaceae (P = 0.007), whereas GM pigs had a 60.9% decrease in Lactobacillales (P = 0.003) and an 83.3% enrichment in Burkholderiales (P = 0.010). After ETEC infection, hLZM pigs tended to have lower amounts (68.7% less) of fecal Enterobacteriaceae than did GM pigs (P = 0.058). There were 83.1% fewer bacteria translocated into the mesenteric lymph nodes of hLZM pigs than into those of GM pigs (P = 0.039), and hLZM pigs had 34% lower mucin 1 and 61% higher tumor necrosis factor-α expression in the ileum than did GM pigs (P = 0.046 and 0.034, respectively).Conclusion: Results of this study indicate that human lysozyme milk consumption before and during ETEC infection has a positive effect on clinical disease, intestinal mucosa, and gut microbiota in young pigs.

Genetically engineered livestock for agriculture: a generation after the first transgenic animal research conference.

At the time of the first Transgenic Animal Research Conference, the lack of knowledge about promoter, enhancer and coding regions of genes of interest greatly hampered our efforts to create transgenes that would express appropriately in livestock. Additionally, we were limited to gene insertion by pronuclear microinjection. As predicted then, widespread genome sequencing efforts and technological advancements have profoundly altered what we can do. There have been many developments in technology to create transgenic animals since we first met at Granlibakken in 1997, including the advent of somatic cell nuclear transfer-based cloning and gene editing. We can now create new transgenes that will express when and where we want and can target precisely in the genome where we want to make a change or insert a transgene. With the large number of sequenced genomes, we have unprecedented access to sequence information including, control regions, coding regions, and known allelic variants. These technological developments have ushered in new and renewed enthusiasm for the production of transgenic animals among scientists and animal agriculturalists around the world, both for the production of more relevant biomedical research models as well as for agricultural applications. However, even though great advancements have been made in our ability to control gene expression and target genetic changes in our animals, there still are no genetically engineered animal products on the market for food. World-wide there has been a failure of the regulatory processes to effectively move forward. Estimates suggest the world will need to increase our current food production 70 % by 2050; that is we will have to produce the total amount of food each year that has been consumed by mankind over the past 500 years. The combination of transgenic animal technology and gene editing will become increasingly more important tools to help feed the world. However, to date the practical benefits of these technologies have not yet reached consumers in any country and in the absence of predictable, science-based regulatory programs it is unlikely that the benefits will be realized in the short to medium term.

Production of human lactoferrin and lysozyme in the milk of transgenic dairy animals: past, present, and future.

Genetic engineering, which was first developed in the 1980s, allows for specific additions to animals' genomes that are not possible through conventional breeding. Using genetic engineering to improve agricultural animals was first suggested when the technology was in the early stages of development by Palmiter et al. (Nature 300:611-615, 1982). One of the first agricultural applications identified was generating transgenic dairy animals that could produce altered or novel proteins in their milk. Human milk contains high levels of antimicrobial proteins that are found in low concentrations in the milk of ruminants, including the antimicrobial proteins lactoferrin and lysozyme. Lactoferrin and lysozyme are both part of the innate immune system and are secreted in tears, mucus, and throughout the gastrointestinal (GI) tract. Due to their antimicrobial properties and abundance in human milk, multiple lines of transgenic dairy animals that produce either human lactoferrin or human lysozyme have been developed. The focus of this review is to catalogue the different lines of genetically engineered dairy animals that produce either recombinant lactoferrin or lysozyme that have been generated over the years as well as compare the wealth of research that has been done on the in vitro and in vivo effects of the milk they produce. While recent advances including the development of CRISPRs and TALENs have removed many of the technical barriers to predictable and efficient genetic engineering in agricultural species, there are still many political and regulatory hurdles before genetic engineering can be used in agriculture. It is important to consider the substantial amount of work that has been done thus far on well established lines of genetically engineered animals evaluating both the animals themselves and the products they yield to identify the most effective path forward for future research and acceptance of this technology.

Assessing unintended effects of a mammary-specific transgene at the whole animal level in host and non-target animals.

Risk assessment in transgenic plants is intrinsically different than that for transgenic animals; however both require the verification of proper transgene function and in conjunction, an estimate of any unintended effects caused by expression of the transgene. This work was designed to gather data regarding methodologies to detect pleiotropic effects at the whole animal level using a line of transgenic goats that produce the antimicrobial protein human lysozyme (hLZ) in their milk with the goal of using the milk to treat childhood diarrhea. Metabolomics was used to determine the serum metabolite profile of both the host (lactating does) and non-target organism (kid goats raised on control or hLZ milk) prior to weaning (60 days), at weaning (90 days) and 1 month post-weaning (120 days). In addition, intestinal histology of the kid goats was also carried out. Histological analysis of intestinal segments of the pre-weaning group revealed significantly wider duodenal villi (p = 0.014) and significantly longer villi (p = 0.028) and deeper crypts (p = 0.030) in the ileum of kid goats consuming hLZ milk. Serum metabolomics was capable of detecting differences over time but revealed no significant differences in metabolites between control and hLZ fed kids after correction for false discovery rate. Serum metabolomics of control or hLZ lactating does showed only one significant difference in an unknown metabolite (q = 0.0422). The results as a whole indicate that consumption of hLZ milk results in positive or insignificant intestinal morphology and metabolic changes. This work contributes to the establishment of the safety and durability of the hLZ mammary-specific transgene.

Consumption of transgenic milk containing the antimicrobials lactoferrin and lysozyme separately and in conjunction by 6-week-old pigs improves intestinal and systemic health.

Lactoferrin and lysozyme are antimicrobial and immunomodulatory proteins produced in high quantities in human milk that aid in gastrointestinal (GI) health and have beneficial effects when supplemented separately and in conjunction in human and animal diets. Ruminants produce low levels of lactoferrin and lysozyme; however, there are genetically engineered cattle and goats that respectively secrete recombinant human lactoferrin (rhLF-milk), and human lysozyme (hLZ-milk) in their milk. Effects of consumption of rhLF-milk, hLZ-milk and a combination of rhLF-and hLZ-milk were tested on young pigs as an animal model for the GI tract of children. Compared with control milk-fed pigs, pigs fed a combination of rhLF and hLZ (rhLF+hLZ) milk had a significantly deeper intestinal crypts and a thinner lamina propria layer. Pigs fed hLZ-milk, rhLF-milk and rhLF+hLZ had significantly reduced mean corpuscular volume (MCV) and red blood cells (RBCs) were significantly increased in pigs fed hLZ-milk and rhLF-milk and tended to be increased in rhLF+hLZ-fed pigs, indicating more mature RBCs. These results support previous research demonstrating that pigs fed milk containing rhLF or hLZ had decreased intestinal inflammation, and suggest that in some parameters the combination of lactoferrin and lysozyme have additive effects, in contrast to the synergistic effects reported when utilising in-vitro models.

Consumption of transgenic cows' milk containing human lactoferrin results in beneficial changes in the gastrointestinal tract and systemic health of young pigs.

Lactoferrin is an antimicrobial and immunomodulatory protein that is produced in high quantities in human milk and aids in the gastrointestinal (GI) maturation of infants. Beneficial health effects have been observed when supplementing human and animal diets with lactoferrin. A herd of genetically engineered cattle that secrete recombinant human lactoferrin in their milk (rhLF-milk) have been generated which provide an efficient production system and ideal medium for rhLF consumption. The effects of consumption of rhLF-milk were tested on young pigs as an animal model for the GI tract of children. When comparing rhLF-milk fed pigs to non-transgenic milk fed pigs (control), we observed that rhLF-milk fed pigs had beneficial changes in circulating leukocyte populations. There was a significant decrease in neutrophils (p = 0.0036) and increase in lymphocytes (p = 0.0017), leading to a decreased neutrophil to lymphocyte ratio (NLR) (p = 0.0153), which is an indicator of decreased systemic inflammation. We also observed changes in intestinal villi architecture. In the duodenum, rhLF-milk fed pigs tended to have taller villi (p = 0.0914) with significantly deeper crypts (p < 0.0001). In the ileum, pigs consuming rhLF-milk had villi that were significantly taller (p = 0.0002), with deeper crypts (p < 0.0001), and a thinner lamina propria (p = 0.0056). We observed no differences in cytokine expression between rhLF-milk and control-milk fed pigs, indicating that consumption of rhLF-milk did not change cytokine signaling in the intestines. Overall favorable changes in systemic health and GI villi architecture were observed; indicating that consumption of rhLF-milk has the potential to induce positive changes in the GI tract.

Salmonella enhances osteogenic differentiation in adipose-derived mesenchymal stem cells.

The potential of mesenchymal stem cells (MSCs) for tissue repair and regeneration has garnered great attention. While MSCs are likely to interact with microbes at sites of tissue damage and inflammation, like in the gastrointestinal system, the consequences of pathogenic association on MSC activities have yet to be elucidated. This study investigated the effects of pathogenic interaction on MSC trilineage differentiation paths and mechanisms using model intracellular pathogen Salmonella enterica ssp enterica serotype Typhimurium. The examination of key markers of differentiation, apoptosis, and immunomodulation demonstrated that Salmonella altered osteogenic and chondrogenic differentiation pathways in human and goat adipose-derived MSCs. Anti-apoptotic and pro-proliferative responses were also significantly upregulated (p < 0.05) in MSCs during Salmonella challenge. These results together indicate that Salmonella, and potentially other pathogenic bacteria, can induce pathways that influence both apoptotic response and functional differentiation trajectories in MSCs, highlighting that microbes have a potentially significant role as influencers of MSC physiology and immune activity.

Consumption of lysozyme-rich milk can alter microbial fecal populations.

Human 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 of Firmicutes (Clostridia) declined whereas those of Bacteroidetes increased 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 (Bifidobacteriaceae and Lactobacillaceae) 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.

Mapping of equine cerebellar abiotrophy to ECA2 and identification of a potential causative mutation affecting expression of MUTYH.

Equine Cerebellar Abiotrophy (CA) is a neurological disease found in Arabian horses. CA is characterized by post-natal degeneration of the Purkinje cells of the cerebellum. Signs of CA include ataxia, head tremors, and a lack of balance equilibrium. We have discovered a linkage of the CA phenotype to a microsatellite marker on ECA2 and identified a region of conserved homozygosity spanning approximately 142 kb. Complete sequencing of the four genes in this region identified one SNP found only in Arabian horses, located in exon 4 of TOE1 and approximately 1200 base pairs upstream of MUTYH, adjacent to a possible binding site for the transcription factor GATA2. qPCR analysis of cDNA from the cerebella of affected and unaffected horses suggested that MUTYH expression is down-regulated in affected horses. This SNP may therefore have a function effect on TOE1, or a regulatory effect on MUTYH by negatively affecting the binding affinity of GATA2.

MSC therapy in livestock models.

Mesenchymal stem cells (MSCs) have great value as therapeutic tools in a wide array of applications in regenerative medicine. The wide repertoire of cell functions regarding tissue regeneration, immunomodulation, and antimicrobial activity makes MSC-based therapy a strong candidate for treatment options in a variety of clinical conditions and should be studied to expand the current breadth of knowledge surrounding their physiological properties and therapeutic benefits. Livestock models are an appropriate resource for testing the efficacy of MSC therapies for their use in biomedical research and can be used to improve both human health and animal agriculture. Agricultural animal models such as pigs, cattle, sheep, and goats have grown in popularity for in vivo research relative to small animal models due to their overlapping similarities in structure and function that more closely mimic the human body. Cutaneous wound healing, bone regeneration, osteoarthritis, ischemic reperfusion injury, and mastitis recovery represent a few examples of the types of disease states that may be investigated in livestock using MSC-based therapy. Although the cost of agricultural animals is greater than small animal models, the information gained using livestock as a model holds great value for human applications, and in some cases, outcompetes the weight of information gained from rodent models. With emerging fields such as exosome-based therapy, proper in vivo models will be needed for testing efficacy and translational practice, i.e., livestock models should be strongly considered as candidates. The potential for capitalizing on areas that have crossover benefits for both agricultural economic gain and improved health of the animals while minimizing the gap between translational research and clinical practice are what make livestock great choices for experimental MSC models.

LincRNA#1 knockout alone does not affect polled phenotype in cattle heterozygous for the celtic POLLED allele.

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.

A deletion at the polled PC locus alone is not sufficient to cause a polled phenotype in cattle.

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.

Lysozyme transgenic goats' milk positively impacts intestinal cytokine expression and morphology.

In addition to its well-recognized antimicrobial properties, lysozyme can also modulate the inflammatory response. This ability may be particularly important in the gastrointestinal tract where inappropriate inflammatory reactions can damage the intestinal epithelium, leading to significant health problems. The consumption of milk from transgenic goats producing human lysozyme (hLZ) in their milk therefore has the potential to positively impact intestinal health. In order to investigate the effect of hLZ-containing milk on the inflammatory response, young pigs were fed pasteurized milk from hLZ or non-transgenic control goats and quantitative real-time PCR was performed to assess local expression of TNF-α, IL-8, and TGF-ß1 in the small intestine. Histological changes were also investigated, specifically looking at villi width, length, crypt depth, and lamina propria thickness along with cell counts for intraepithelial lymphocytes and goblet cells. Significantly higher expression of anti-inflammatory cytokine TGF-ß1 was seen in the ileum of pigs fed pasteurized milk containing hLZ (P = 0.0478), along with an increase in intraepithelial lymphocytes (P = 0.0255), and decrease in lamina propria thickness in the duodenum (P = 0.0001). Based on these results we conclude that consuming pasteurized milk containing hLZ does not induce an inflammatory response and improves the health of the small intestine in pigs.

Gene silencing in bovine zygotes: siRNA transfection versus microinjection.

The aim of this study was to compare gene silencing in bovine zygotes when small interfering RNAs (siRNAs) were introduced into bovine zygotes by microinjection or lipid-based transfection. In Experiment 1, E-cadherin siRNA was injected at 100 or 375 µM and compared with PBS-injected and non-injected controls. Embryos were then cultured in vitro for 7 days and periodically assessed for development. For transfection, zona-free zygotes were incubated in transfection medium with siRNA for 1h at 39°C and then cultured to Day 7. Injection of PBS or 375 µM E-cadherin siRNA resulted in a decrease in the number of embryos reaching the 8-cell stage (51.5% and 45.5%) or the blastocyst stage (39.0 and 32.5%) compared with non-injected controls (62.9 and 45.0%, respectively; P<0.05). Messenger RNA abundance was suppressed by 36 and 46% when siRNA targeting E-cadherin was injected at 100 and 375 µM, respectively, compared with controls (P<0.05). Transfection with 100 nM E-cadherin siRNA decreased development to the 8-cell stage (20.3 versus 53.0%) and blastocyst stage (7.2 versus 18.2%) compared with controls (P<0.05). Messenger RNA relative abundance was not different between controls (non-transfected or transfected with GAPDH or scrambled siRNA). However, transfection of zygotes with 100 and 200 nM E-cadherin siRNA led to a 72 and 38% reduction, respectively, in E-cadherin mRNA relative abundance in Day 7 blastocysts compared with controls (P<0.05).

Consumption of pasteurized human lysozyme transgenic goats' milk alters serum metabolite profile in young pigs.

Nutrition, bacterial composition of the gastrointestinal tract, and general health status can all influence the metabolic profile of an organism. We previously demonstrated that feeding pasteurized transgenic goats' milk expressing human lysozyme (hLZ) can positively impact intestinal morphology and modulate intestinal microbiota composition in young pigs. The objective of this study was to further examine the effect of consuming hLZ-containing milk on young pigs by profiling serum metabolites. Pigs were placed into two groups and fed a diet of solid food and either control (non-transgenic) goats' milk or milk from hLZ-transgenic goats for 6 weeks. Serum samples were collected at the end of the feeding period and global metabolite profiling was performed. For a total of 225 metabolites (160 known, 65 unknown) semi-quantitative data was obtained. Levels of 18 known and 4 unknown metabolites differed significantly between the two groups with the direction of change in 13 of the 18 known metabolites being almost entirely congruent with improved health status, particularly in terms of the gastrointestinal tract health and immune response, with the effects of the other five being neutral or unknown. These results further support our hypothesis that consumption of hLZ-containing milk is beneficial to health.