Supplementary Material for: The Aachen Minipig: Phenotype, Genotype, Hematological and Biochemical Characterization, and Comparison to the Göttingen Minipig

<b><i>Background:</i></b> The pig is one of the most frequently used large animal models for biomedical research, especially in the field of translational research and surgical models. While standard livestock breeds are used in short-term and acute studies, minipig breeds are the preferred breeds in long-term and chronic studies due to their limited growth and body weight. <b><i>Objective:</i></b> In consideration of the 3R principle (refinement, reduction, replacement) and the increasing demand, the aim of this study was to generate a new, robust, non-specific-pathogen-free minipig breed, the Aachen minipig. <b><i>Methods:</i></b> Phenotype, genotype, and hematological as well as clinical chemistry parameters were characterized, and reference values of the Aachen minipig were generated and compared to the values in the commonly used Göttingen minipig. Organ weights of the heart, kidney, liver, lung, spleen, and brain were determined using a laboratory balance. Blood samples were collected for hematology and clinical chemistry. Assessment of genetic diversity was performed by microsatellite markers. Nasal swabs were collected from 11 individual minipigs representing 6 races for DNA extraction. DNA was quantified and the identity and origin of the Aachen minipigs at the genomic level was determined by microsatellites. <b><i>Results:</i></b> The Aachen minipig established here is based on the Mini-LEWE breed and consists of the Vietnamese potbelly pig, the Schwäbisch Hällisch Landpig, the German Landrace, and the Minnesota minipig. Relative organ weights (lung, heart, kidneys, brain), hematology (hemoglobin, hematocrit, platelet count, mean corpuscular hemoglobin concentration, segmented neutrophils, lymphocytes, eosinophils, basophils), and clinical chemistry parameters (sodium, calcium, chloride, alkaline phosphatase, aspartate aminotransferase, alanine aminotransferase, gamma-glutamyl transferase, lactate dehydrogenase, triglycerides, blood urea nitrogen, creatinine, total bilirubin, total protein, creatine kinase) of the Aachen minipigs and the Göttingen minipigs were not significantly different. Significant differences where only seen in relative organ weights (liver, spleen), hematology (red blood cell count, mean corpuscular volume, mean corpuscular hemoglobin, white blood cell count, banded neutrophils, monocytes), and clinical chemistry parameters (inorganic phosphorus, potassium, glucose, cholesterol, albumin, amylase). <b><i>Conclusion:</i></b> The Aachen minipig is a suitable model for research due to its similarity to other minipig breeds, especially the Göttingen minipig. The reference values established in this study may be used for the comparison of scientific data and encourage the use of the Aachen minipig as an animal model for biomedical research.**背景：** 猪是生物医学研究中最常用的大型动物模型之一，尤其在转化研究（translational research）领域及外科模型构建中。短期急性研究通常采用标准畜禽品种，而小型猪（minipig）品种因生长受限、体重可控，成为长期慢性研究的首选品种。 **目的：** 鉴于3R原则（优化、减少、替代，refinement, reduction, replacement）及日益增长的研究需求，本研究旨在培育一种全新、健壮的非无特定病原体（non-specific-pathogen-free）小型猪品种——亚琛小型猪（Aachen minipig）。 **方法：** 本研究对亚琛小型猪的表型、基因型、血液学及临床生化指标进行了系统表征，建立了其参考范围，并与常用的哥廷根小型猪（Göttingen minipig）的对应指标开展对比分析。使用实验室天平测定了心脏、肾脏、肝脏、肺脏、脾脏及大脑的器官重量；采集血液样本用于血液学及临床生化检测；采用微卫星标记（microsatellite markers）评估遗传多样性。从覆盖6个品种的11头小型猪个体中采集鼻拭子以提取基因组DNA，对DNA进行定量后，通过微卫星标记明确了亚琛小型猪在基因组水平的种属特征及起源。 **结果：** 本研究培育的亚琛小型猪以Mini-LEWE品种为基础，由越南大肚猪（Vietnamese potbelly pig）、施瓦本-哈尔施兰猪（Schwäbisch Hällisch Landpig）、德国长白猪（German Landrace）及明尼苏达小型猪（Minnesota minipig）杂交选育而来。亚琛小型猪与哥廷根小型猪的相对器官重量（肺、心脏、肾脏、大脑）、血液学指标[血红蛋白（hemoglobin）、血细胞比容（hematocrit）、血小板计数、平均红细胞血红蛋白浓度、分叶中性粒细胞、淋巴细胞、嗜酸性粒细胞、嗜碱性粒细胞]及临床生化指标[钠、钙、氯、碱性磷酸酶、天冬氨酸氨基转移酶、丙氨酸氨基转移酶、γ-谷氨酰转移酶、乳酸脱氢酶、甘油三酯、血尿素氮、肌酐、总胆红素、总蛋白、肌酸激酶]均无显著差异。仅在相对器官重量（肝脏、脾脏）、血液学指标[红细胞计数、平均红细胞体积、平均红细胞血红蛋白量、白细胞计数、杆状中性粒细胞、单核细胞]及临床生化指标[无机磷、钾、葡萄糖、胆固醇、白蛋白、淀粉酶]中存在显著差异。 **结论：** 亚琛小型猪与其他小型猪品种（尤其是哥廷根小型猪）具有高度相似性，是适用于生物医学研究的理想动物模型。本研究建立的参考值可用于科研数据的对比分析，可为亚琛小型猪作为动物模型的推广应用提供数据支撑。