Global regulatory policies for animal biotechnology: overview, opportunities and challenges.

Genome editing (GnEd) has the potential to provide many benefits to animal agriculture, offering a means for achieving rapid growth, disease resistance, and novel phenotypes. The technology has the potential to be useful for rapidly incorporating traits into existing selectively bred animals without the need for crossbreeding and backcrossing. Yet only four products from animals created via biotechnology, all growth-enhanced fishes, have reached commercialization and only on a limited scale. The past failure of genetically engineered (or GM) products to reach conventional producers can largely be attributed to the high cost of meeting GMO regulatory requirements. We review the history of GMO regulations internationally, noting the influence of Codex Alimentarius on the development of many existing regulatory frameworks. We highlight new regulatory approaches for GnEd organisms, first developed by Argentina, and the adoption of similar approaches by other countries. Such new regulatory approaches allow GnEd organisms that could have been developed by conventional means to be regulated under the same rules as conventional organisms and in the future is likely to enhance the opportunity for biotech animals to enter production. Treating certain GnEd products as conventional has had a large impact on the variety of biotechnological innovations successfully navigating regulatory processes. We suggest that for the full potential of GnEd technologies to be realized, enabling public policies are needed to facilitate use of GnEd as a breeding tool to incorporate new traits within existing animal breeding programs, rather than only a tool to create distinct new products.

Towards progressive regulatory approaches for agricultural applications of animal biotechnology.

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.

Regulatory approaches for genome edited agricultural plants in select countries and jurisdictions around the world.

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.

Hyperthermia increases the cytotoxicity of many exogenous compounds.

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.

Radiofrequency-assisted laparoscopic partial nephrectomy: clinical and histologic results.

PURPOSE: 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. MATERIALS AND METHODS: 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. RESULTS: 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; P < 0.05). Lesion depth and diameter increased with treatment time. Viability depth correlated well with the depth of the visible thermal lesions (Pearson correlation 0.989). CONCLUSIONS: 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 determinants of arterial endothelial phenotype dominate the impact of gender or short-term exposure to a high-fat diet.

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.

Effects of perfusion on radiofrequency ablation in swine kidneys.

PURPOSE: To evaluate the effect of vascular occlusion on the size of radiofrequency (RF) ablation lesions and to evaluate embolization as an occlusion method. MATERIALS AND METHODS: 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. RESULTS: 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. CONCLUSION: 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.

Radiofrequency cauterization with biopsy introducer needle.

PURPOSE: The principal risks of needle biopsy are hemorrhage and implantation of tumor cells in the needle tract. This study compared hemorrhage after liver and kidney biopsy with and without radiofrequency (RF) ablation of the needle tract. MATERIALS AND METHODS: Biopsies of liver and kidney were performed in swine through introducer needles modified to allow RF ablation with the distal 2 cm of the needle. After each biopsy, randomization determined whether the site was to undergo RF ablation during withdrawal of the introducer needle. Temperature was measured with a thermistor stylet near the needle tip, with a target temperature of 70 degrees C-100 degrees C with RF ablation. Blood loss was measured as grams of blood absorbed in gauze at the puncture site for 2 minutes after needle withdrawal. Selected specimens were cut for gross examination. RESULTS: RF ablation reduced bleeding compared with absence of RF ablation in liver and kidney (P <.01), with mean blood loss reduced 63% and 97%, respectively. Mean amounts of blood loss (+/-SD) in the liver in the RF and no-RF groups were 2.03 g +/- 4.03 (CI, 0.53-3.54 g) and 5.50 g +/- 5.58 (CI, 3.33-7.66 g), respectively. Mean amounts of blood loss in the kidney in the RF and no-RF groups were 0.26 g +/- 0.32 (CI, -0.01 to 0.53 g) and 8.79 g +/- 7.72 (CI, 2.34-15.24 g), respectively. With RF ablation, thermal coagulation of the tissue surrounding the needle tract was observed. CONCLUSION: RF ablation of needle biopsy tracts reduced hemorrhage after biopsy in the liver and kidney and may reduce complications of hemorrhage as well as implantation of tumor cells in the tract.

Betaine improves growth, but does not induce whole body or hepatic palmitate oxidation in swine (Sus scrofa domestica).

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-(13)C-palmitate and in hepatocytes incubated in the presence or absence of betaine and carnitine. CO2 and palmitate isotopic enrichments were determined by mass spectrometry. Pigs consuming 0.125% and 0.5% 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.