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Traditional breeding techniques, applied incrementally over thousands of years, have yielded huge benefits in the characteristics of agricultural animals. This is a result of significant, measurable changes to the genomes of those animal species and breeds. Genome editing techniques may now be applied to achieve targeted DNA sequence alterations, with the potential to affect traits of interest to production of agricultural animals in just one generation. New opportunities arise to improve characteristics difficult to achieve or not amenable to traditional breeding, including disease resistance, and traits that can improve animal welfare, reduce environmental impact, or mitigate impacts of climate change. Countries and supranational institutions are in the process of defining regulatory approaches for genome edited animals and can benefit from sharing approaches and experiences to institute progressive policies in which regulatory oversight is scaled to the particular level of risk involved. To facilitate information sharing and discussion on animal biotechnology, an international community of researchers, developers, breeders, regulators, and communicators recently held a series of seven virtual workshop sessions on applications of biotechnology for animal agriculture, food and environmental safety assessment, regulatory approaches, and market and consumer acceptance. In this report, we summarize the topics presented in the workshop sessions, as well as discussions coming out of the breakout sessions. This is framed within the context of past and recent scientific and regulatory developments. This is a pivotal moment for determination of regulatory approaches and establishment of trust across the innovation through-chain, from researchers, developers, regulators, breeders, farmers through to consumers.

Foreign Agricultural Service of the U.S. Department of Agriculture; U.S. Department of Agriculture Published version The work of author Eric M. Hallerman in this area is supported by a grant from the Foreign Agricultural Service of the U.S. Department of Agriculture. The work of author Diane Wray-Cahe is supported by the U.S. Department of Agriculture. Public domain – authored by a U.S. government employee

Genome editing in agriculture and food is leading to new, improved crops and other products. Depending on the regulatory approach taken in each country or region, commercialization of these crops and products may or may not require approval from the respective regulatory authorities. This paper describes the regulatory landscape governing genome edited agriculture and food products in a selection of countries and regions. Supplementary Information The online version contains supplementary material available at 10.1007/s11248-021-00257-8.

A study was conducted to elucidate hormonal control of ketogenesis and glycogen deposition in primary cultures of porcine hepatocytes. Hepatocytes were isolated from pigs (54-68 kg) by collagenase perfusion and seeded into collagen-coated T-25 flasks. Monolayers were established in medium containing fetal bovine serum for 1 day and switched to a serum-free medium for the remainder of the culture period. Hepatocytes were maintained in DMEM/M199 containing 1% DMSO, dexamethasone (10(-6) or 10(-7) M), linoleic acid (3.4 x 10(-5) M), and carnitine (10(-3) M) for 3 days. On the first day of serum-free culture, insulin was added at 1 or 100 ng/ml and glucagon was added at 0, 1, or 100 ng/ml. Recombinant human leptin (200 ng/ml) was added during the final 24 h; medium and all cells were harvested on the third day. Concentrations of acetoacetate and beta-hydroxybutyrate (ketone bodies) in media and glycogen deposition in the cellular compartment were determined. Ketogenesis was highly stimulated by glucagon (1 and 100 ng/ml) and inhibited by insulin. In contrast, glycogen deposition was stimulated by insulin and attenuated by glucagon; high insulin was also associated with a reduction in the ketone body ratio (acetoacetate:beta-hydroxybutyrate). High levels of dexamethasone stimulated ketogenesis, but inhibited glycogen deposition at low insulin. Culture of cells with leptin for 24 h, over the range of insulin, glucagon, and dexamethasone concentrations had no effect on either glycogen deposition or ketogenesis. These data suggest that while adult porcine hepatocytes are indeed sensitive to hormonal manipulation, leptin has no direct influence on hepatic energy metabolism in swine.

To evaluate the effect of vascular occlusion on the size of radiofrequency (RF) ablation lesions and to evaluate embolization as an occlusion method.The kidneys of six swine were surgically exposed. Fifteen RF ablation lesions were created in nine kidneys by using a 2-cm-tip single-needle ablation probe in varying conditions: Seven lesions were created with normal blood flow and eight were created with blood flow obstructed by means of vascular clamping (n = 5) or renal artery embolization (n = 3). The temperature, applied voltage, current, and impedance were recorded during RF ablation. Tissue-cooling curves acquired for 2 minutes immediately after the ablation were compared by using regression analysis. Lesions were bisected, and their maximum diameters were measured and compared by using analysis of variance.The mean diameter of ablation lesions created when blood flow was obstructed was 60% greater than that of lesions created when blood flow was normal (1.38 cm +/- 0.05 [standard error of mean] vs 0.86 cm +/- 0.07, P.001). The two methods of flow obstruction yielded lesions of similar mean sizes: 1.40 cm +/- 0.06 with vascular clamping and 1.33 cm +/- 0.07 with embolization. The temperature at the probe tip when lesions were ablated with normal blood flow decreased more rapidly than did the temperature when lesions were ablated after flow obstruction (P.001), but no significant differences in tissue-cooling curves between the two flow obstruction methods were observed.Obstruction of renal blood flow before and during RF ablation resulted in larger thermal lesions with potentially less variation in size compared with the lesions created with normal nonobstructed blood flow. Selective arterial embolization of the kidney vessels may be a useful adjunct to RF ablation of kidney tumors.

The purpose of this study was to examine the effects of dietary betaine over a range of concentrations (between 0 and 0.5%) on growth and body composition in young feed-restricted pigs. Betaine is associated with decreased lipid deposition and altered protein utilization in finishing pigs, and it has been suggested that the positive effects of betaine on growth and carcass composition may be greater in energy-restricted pigs. Thirty-two barrows (36 kg, n = 8 pigs per group) were restrictively fed one of four corn-soybean meal-skim milk based diets (18.6% crude protein, 3.23 Mcal ME/kg) and supplemented with 0, 0.125, 0.25, or 0.5% betaine. Feed allotment was adjusted weekly according to BW, such that average feed intake was approximately 1.7 kg for all groups. At 64 kg, pigs were slaughtered and visceral tissue was removed and weighed. Carcasses were chilled for 24 h to obtain carcass measurements. Subsequently, one-half of each carcass and whole visceral tissue were ground for chemical analysis. Linear regression analysis indicated that, as betaine content of the diet was elevated from 0 to 0.5%, carcass fat concentration (P = 0.06), P3 fat depth (P = 0.14) and viscera weight (P = 0.129) were decreased, whereas total carcass protein (P = 0.124), protein deposition rate (P = 0.98), and lean gain efficiency (P = 0.115) were increased. The greatest differences over control pigs were observed in pigs consuming 0.5% betaine, where carcass fat concentration and P3 fat depth were decreased by 10 and 26%, respectively. Other fat depth measurements were not different (P > 0.15) from those of control pigs. In addition, pigs consuming the highest betaine level had a 19% increase in the carcass protein:fat ratio, 23% higher carcass protein deposition rate, and a 24% increase in lean gain efficiency compared with controls. Dietary betaine had no effects (P > 0.15) on growth performance, visceral tissue chemical composition, carcass fat deposition rate, visceral fat and protein deposition rates, or serum urea and ammonia concentrations. These data suggest that betaine alters nutrient partitioning such that carcass protein deposition is enhanced at the expense of carcass fat and in part, visceral tissue.

Fatty acid-free albumin has been the standard carrier for intravenous infusion of fatty acids to study in vivo lipid metabolism. However, subjects can have adverse reactions to infusion of albumin. We sought an alternative to albumin as a carrier for intravenous infusion of fatty acids, using the pig as a model. Cyclodextrins are naturally occurring water-soluble molecules that can serve as carriers for lipid-soluble compounds. 13C-palmitate was complexed to either 20% methyl-beta-cyclodextrin, 20% 2-hydroxypropyl-beta-cyclodextrin, or 5% porcine albumin (isotopic purity of infusates: 99.22+/-0.06%). 13C-palmitate-albumin was infused under fed conditions and 13C-palmitate-methyl-beta-cyclodextrin was infused under fasted and fed conditions in 50-kg pigs. Palmitate remained in solution at 4 degrees C in methyl-beta-cyclodextrin, but precipitated at 25-30 degrees C in 2-hydroxypropyl-beta-cyclodextrin. Pigs infused with 13C-palmitate-methyl-beta-cyclodextrin maintained normal body temperature and appetite; those infused with 13C-palmitate-albumin became anorexic and exhibited other negative side effects to albumin. Palmitate oxidation rates under fed conditions were similar using either 13C-palmitate-methyl-beta-cyclodextrin or 13C-palmitate-albumin complexes. Fasting increased 13C-palmitate-methyl-beta-cyclodextrin oxidation by approximately eight-fold. These data suggest that methyl-beta-cyclodextrin may be a suitable substitute for albumin in fatty acid metabolism studies in swine.

Plasma amino acid concentrations fall during insulin infusion. Amino acid concentrations can be maintained using an infusion of amino acids if their plasma concentration can be determined within a few minutes. We developed a spectrophometric assay which determines the total concentration of all three branched-chain amino acids in plasma within 1 min. The enzyme leucine dehydrogenase oxidatively deaminates leucine, isoleucine, and valine, with stoichiometric reduction of NAD that is measured using a spectrophotometer. The assay was developed in both a kinetic and end-point format. For the kinetic assay the buffer conditions were formulated to obtain equivalent rates with all three amino acids so that it could be used in samples containing unknown mixtures. For the end-point assay additional enzyme was added so that an end-point could be reached within 1 min. The application of the kinetic assay for "clamping" the branched-chain amino acids during hyperinsulinemic euglycemic clamps in humans is demonstrated.

Cytotoxicity testing of extracts from medical device materials is typically conducted at 37 degrees C. It may be more relevant to screen extracts from device materials for in vitro cytotoxicity at temperatures found in febrile patients. To address this, the cytotoxicity of selected chemicals, drugs, and medical device extracts was evaluated in vitro following incubation at normothermic (37 degrees C) and hyperthermic (39 degrees C) conditions. In L929 cells, the percentage of cell death increased from 2-fold to more than 4-fold after chemical exposure when cells were maintained at 39 degrees C. Extracts of some medical devices and materials showed a 10-fold increase in cytotoxicity when cells were maintained at 39 degrees C as compared to 37 degrees C. For many of the substances in this study, exogenous compounds that are toxic at normothermic conditions (37 degrees C) are more cytotoxic under hyperthermic conditions (39 degrees C). The toxicity of compounds was more readily discernable at the higher incubation temperature, even at lower concentrations. In vitro cytotoxicity testing of chemicals and extracts at febrile temperatures can provide more sensitive and relevant biocompatibility tests than under normothermic conditions alone.

To evaluate a surface conductive radiofrequency (RF) coagulation instrument (Tissuelink FB3.0) in laparoscopic and open partial nephrectomy (PN) in hereditary kidney cancer. The lesion depth and viability in the pathologic specimens from a surgical series and an acute porcine model were characterized under conditions of vascular perfusion and occlusion.A total of 19 patients underwent 20 laparoscopic and open procedures with the device. Data were acquired on tumor number, size, operative time, blood loss, length of stay, renal function, complications, pathologic diagnosis, and surgical-margin status. Renal lesions were created in pigs with the device, ultrasonic shears, and a standard electrocautery for specified time intervals and operative energy settings. These lesions were analyzed for depth, diameter, and tissue viability.In 20 separate (14 laparoscopic; 6 open) procedures in 19 patients, a total of 112 tumors were removed (range 1-31 tumors per procedure). The median operative time, blood loss, and length of stay were 310 minutes, 250 mL, and 4 days, respectively. There were no positive surgical margins. Median preoperative and postoperative creatinine concentrations were similar (1.0 v 1.0 mg/dL). The average treatment margin depth was 3 mm. In the porcine experiments, the treatment depth in the unclamped vascular model was significantly less in than the clamped model (4.0 +/- 1.7 mm v 7.0 +/- 1.6 mm; P0.05). Lesion depth and diameter increased with treatment time. Viability depth correlated well with the depth of the visible thermal lesions (Pearson correlation 0.989).This RF energy device can provided adequate and uniform hemostatic control without hilar clamping during laparoscopic and open PN for hereditary renal tumors. Gross measures of renal function after surgery appeared clinically unchanged. Coagulation depth is dependent on both tissue perfusion and time in the porcine model.

Regional arterial hemodynamics correlates with distinct endothelial phenotypes that may be modified by risk factors to influence focal and regional susceptibility to atherosclerosis. We compared endothelial transcript profiles from hemodynamically distinct arterial regions in 15 mature pigs: males and females fed a normal diet, and males fed a high-fat diet (15% lard, 1.5% cholesterol) for two weeks. Hierarchical clustering analysis showed preferential grouping of arrays by region over risk factor. A set of differentially expressed genes was identified which clearly distinguished regions of disturbed flow from undisturbed flow; however, few differences were observed within the same region based on gender or diet. Consistent with previous results in the absence of risk factors, the balance in gene expression was not inherently pathological at this early time-point. The results implicate regional hemodynamics as a predominant epigenetic determinant of endothelial phenotypic heterogeneity underlying atherosusceptibility in vivo.

Dietary betaine may reduce carcass fat in growing pigs. We explored the effects of betaine on short-term growth and in vivo and in vitro fatty acid oxidation. Pigs were housed in metabolism crates and fed diets containing either 0% (control), 0.125% or 0.5% betaine at 80% of ad libitum energy intake. Fatty acid oxidation was measured during intravenous infusions of 1- C-palmitate and in hepatocytes incubated in the presence or absence of betaine and carnitine. 13 CO and palmitate isotopic enrichments were determined by mass spectrometry. Pigs consuming 0.125% and 0.5% 2 betaine for at least 9 days had growth rates that were 38% and 12% greater than controls, respectively. Feed efficiency was also improved with betaine. Fasting increased palmitate oxidation rates 7-8-fold (P-0.01), but betaine had no effect in either the fed or fasted state (P)0.1). For hepatocytes, carnitine but not betaine enhanced palmitate oxidation. This response suggests that previously observed reduction in adipose accretion must be via a mechanism other than oxidation. Betaine had no effect on plasma non-esterified fatty acids or urea nitrogen. Under the confinement conditions in this study, dietary betaine improved animal growth responses, but it had no apparent effect on either whole body or hepatic fatty acid oxidation.

We recently demonstrated in neonatal pigs that, with amino acids and glucose maintained at fasting levels, the stimulation of protein synthesis in longissimus dorsi muscle with feeding can be reproduced by a physiological rise in insulin alone. In the current report, we determine whether the response of protein synthesis to insulin in the neonatal pig is 1) present in muscles of different fiber types, 2) proportional in myofibrillar and sarcoplasmic proteins, 3) associated with increased translational efficiency and ribosome number, and 4) present in other peripheral tissues and in viscera. Hyperinsulinemic-euglycemic-amino acid clamps were performed in 7- and 26-day-old pigs infused with 0, 30, 100, or 1,000 ng · kg−0.66· min−1of insulin to reproduce insulin levels present in fasted, fed, refed, and supraphysiological conditions, respectively. Tissue protein synthesis was measured using a flooding dose ofl-[4-3H]phenylalanine. Insulin increased protein synthesis in gastrocnemius muscle and, to a lesser degree, masseter muscle. The degree of stimulation of protein synthesis by insulin was similar in myofibrillar and sarcoplasmic proteins. Insulin increased translational efficiency but had no effect on ribosome number in muscle. All of these insulin-induced changes in muscle protein synthesis decreased with age. Insulin also stimulated protein synthesis in cardiac muscle and skin but not in liver, intestine, spleen, pancreas, or kidney. The results support the hypothesis that insulin mediates the feeding-induced stimulation of myofibrillar and sarcoplasmic protein synthesis in muscles of different fiber types in the neonate by increasing the efficiency of translation. However, insulin does not appear to be involved in the feeding-induced stimulation of protein synthesis in visceral tissues. Thus different mechanisms regulate the growth of peripheral and visceral tissues in the neonate.

The elevated rate of muscle protein deposition in the neonate is largely due to an enhanced stimulation of skeletal muscle protein synthesis by feeding. To examine the role of insulin in this response, hyperinsulinemic-euglycemic-amino acid clamps were performed in 7- and 26-day-old pigs. Pigs were infused with 0, 30, 100, or 1,000 ng ⋅ kg−0.66 ⋅ min−1of insulin to mimic the plasma insulin levels observed under fasted, fed, refed, and supraphysiological conditions, respectively. Whole body amino acid disposal was determined from the rate of infusion of an amino acid mixture necessary to maintain plasma essential amino acid concentrations near their basal fasting levels. A flooding dose ofl-[4-3H]phenylalanine was used to measure skeletal muscle protein synthesis. Whole body amino acid disposal increased progressively as the insulin infusion rate increased, and this response was greater in 7- than in 26-day-old pigs. Skeletal muscle protein synthesis was stimulated by insulin, and this response was maximal at a low insulin infusion rate (30 ng ⋅ kg−0.66 ⋅ min−1). The stimulation of muscle protein synthesis by insulin was also greater in 7- than in 26- day-old pigs. These data suggest that muscle protein synthesis is more sensitive to insulin than whole body amino acid disposal. The results further suggest that insulin is a central regulatory factor in the elevated rate of muscle protein deposition and the increased response of skeletal muscle protein synthesis to feeding in the neonate.

Neonatal animals utilize their dietary amino acids for protein accretion with high efficiency, and this efficiency declines during early life. The factors responsible for this developmental change are unknown. Our objectives were to determine whether amino acid (AA) utilization is stimulated by insulin in the neonate and whether this response changes during the suckling period. Two hyperinsulinemic-euglycemic clamp infusion studies, using 10-2,000 ng insulin.kg-0.66.min-1, were performed in 7- and 26-day-old pigs. In study I, no AA were provided during the infusion, and the resultant decline in plasma AA levels was defined. In study II, plasma AA were clamped at near-fasting levels, and whole body utilization of exogenous AA was determined by measuring the rate of infusion of an AA mixture necessary to maintain basal plasma lysine concentrations. In study I, the half-maximal effective dose (ED50) for the fall in AA concentrations with increasing plasma insulin concentration was lower in 7- than in 26-day-old pigs, and the nadir in AA concentration was achieved by only 20 microU/ml insulin. In study II, the utilization of exogenous AA during hyperinsulinemic-euglycemic AA clamps exhibited a higher maximum response (Rmax) (49 vs. 26 mumol AAtotal.min-1.kg-1) and a lower ED50 (18 vs. 45 microU insulin/ml) in 7- than in 26-day-old pigs. Plasma urea nitrogen concentrations did not rise with increasing insulin and AA infusion rates. These results indicate that insulin stimulates the utilization of exogenous AA in neonatal pigs and that both the insulin sensitivity and responsiveness of AA utilization decline over the suckling period. The infused AA were likely utilized for protein accretion.

The enzymic determination of lysine, using saccharopine dehydrogenase, has been modified to determine plasma lysine concentrations in approximately 5 min. The correlation between lysine concentration determined by using HPLC and this enzymic assay was high (r2= 0.92). The main interfering compound was leucine which, at 250 µmol l–1, inhibited the rate measured with lysine by 17%. One application of this method allows plasma lysine to be ‘clamped’ under hyperinsulinemic euglycemic conditions. The technique may be applicable to the bedside investigation of the roles of hormones and nutrition in the regulation of protein metabolism.