In 2022, a groundbreaking medical milestone was achieved when a genetically modified pig heart was transplanted into a 57-year-old man, marking the world’s first successful porcine-to-human heart transplantation. Following that historic surgery, the second transplantation was just completed last month at the University of Maryland Medical Center (UMMC). Every year, over 3,000 heart transplant procedures are performed worldwide, and xenotransplantation can be a revolutionary solution for patients with end-stage heart diseases. Pigs not only provide food to humans, but also serve as ideal animal models for translational research due to their stunning similarities to humans, in particular the anatomy, genetics, physiology, and metabolism. Moreover, miniature pigs (minipigs) are especially suitable for porcine-to-human xenotransplantation because of the similar size, shape, and function of different tissues and organs. To establish a stable minipig line with homogenous genetic backgrounds for biomedical and clinical research, inbreeding strategies have been widely used. Since 1925, scientists around the world, including the United States, Britain, and Germany, have made multiple attempts to establish viable inbred pig lines, but most endeavors have eventually failed due to severe inbreeding depression and high mortality rates amongst the inbred offspring.
Around 1980, Professor Yangzhi Zeng, a breeder at the Yunnan Agricultural University in China, discovered a pig colony in Bangnuodong (21.60110° N, 100.43361° E), a remote minority Lahu village located in Xishuangbanna, Southwest China. This pig colony consisted of roughly 100 animals, laying the foundation for developing an inbred line since the village was highly isolated, without any other pig breeds being introduced. Moreover, only one boar was selected to mate with all sows in the village every year, setting the colony as an ideal candidate to establish a genetically inbred population. To set out the inbreeding schema, a sow and its own male offspring were selected to build an inbred line. Since then, scientists have been employing a strict full-sibling or parent-offspring mating strategy in every generation, along with careful artificial selection.
However, during the first four generations of inbreeding, a significant number of offspring, averaging more than 90% in each generation, perished due to severe inbreeding depression. The initial 10 generations of inbreeding are known to be exceptionally challenging. Fortunately, each generation had a few sows and boars survived, allowing the process to persist. A serendipity occurred when the mini-boar No. 521 was found in the fifth generation. Among all the pigs, No. 521 had the smallest body size with a weight of about merely 30 kg at 18 months of age. Professor Zeng hypothesized that a recessive ‘miniaturization gene’ was expressed in No. 521, and half of its offspring might also be minipigs during backcrossing. Indeed, subsequent experiments confirmed his hypothesis. From then on, No. 521 became the founding ancestor of the new minipig inbred line, later known as the Banna minipig inbred line (BMI), named after its hometown, Xishuangbanna.
Professor Jinlong Huo has been engaged in the cultivation and research of BMI since 2000, serving as the leading participant in BMI development. After more than 20 generations of inbreeding, BMI has achieved a significantly high level of homozygosity in their genome. However, Professor Huo also found that the BMI pigs suffered from male infertility, restricting BMI inbreeding progression and hindering its utilization.
Genetic mutations and abnormal gene expressions are the key drivers affecting male fertility. Especially, changes in the transcriptome, including mRNA expression, non-coding RNA expression, and alternative splicing (AS), influence diverse aspects of the RNA regulatory network during male reproduction. So far, high-throughput sequencing methods have been widely used to identify genes related to spermatogenic disorders, but Illumina short-read sequencing lacks the power to uncover the full landscape of alternative isoforms. To complement that, long-read sequencing significantly improves the resolution and accuracy of transcriptome assembly.
Professor Jinlong Huo was a visiting scholar at the University of Rochester, USA, from January 2019 to January 2021. He designed the recently published study with the help of Dr. Yu H. Sun during the COVID-19 pandemic. In this study, five main organs of BMI and non-inbred Diannan small-ear pigs (DSE) reproductive system, testicle, epididymis, vesicular gland, prostate gland, and bulbourethral gland, were sequenced using Illumina RNA-seq, PacBio Iso-seq, and Illumina small RNA-seq. Following that, the team constructed full-length transcriptomes across five glands and accurately quantified the AS events using junction reads from RNA-seq data. Furthermore, the study revealed that after >30 years of inbreeding, the number of single nucleotide polymorphisms (SNPs) in BMI tissues was significantly lower than that in non-inbred pigs, underscoring the impact of inbreeding on elevating homozygosity. The data obtained in this study not only serve as a rich resource for studying the reproduction of inbred pigs and non-inbred controls, but will also illuminate mechanistic discoveries that guide modern pig molecular breeding and improve the reproductive performance of Chinese indigenous pig breeds.
We, along with our collaborators, are expanding our work to include nine other local pig breeds in Yunnan (Saba pig, Baoshan pig, Lijiang pig, Dahe pig, Zhaotong pig, Gaoligongshan pig, Diannan small-ear pig, Mingguang small-ear pig, Diqing Tibetan pig) and even other provinces in China. Our main research interest remains to be the reproductive traits of boars. As the saying goes, "Good sows, good litters; good boars, good herds," highlighting the importance of boar reproduction on pig population. Our research will focus on understanding swine germplasm characteristics and addressing practical challenges in cultivating indigenous pig breeds in China. Our ambitions include a comprehensive and systematic exploration of gene regulatory networks and signaling pathways related to reproduction, verification of key functional genes, and providing new insights into the reproductive performance of indigenous pig breeds.