Abstract

   Congenital developmental abnormalities in piglets, such as intersex and
   aproctia, adversely affect survival rates, growth performance, and
   genetic breeding efficiency in pig populations. To elucidate their
   genetic basis, we performed a genome-wide association study (GWAS) on
   1030 Large White pigs. We combined 50 K SNP chip data with SWIM-based
   genotype imputation to enhance the resolution of genetic variation
   detection, followed by MLM analysis. Our results identified 53
   significant SNPs, with 52 associated with intersex and 1 with aproctia.
   Key candidate genes included MAD1L1, ID4, EFNA5, and PPP1R16B for
   intersex and ARNT2 for aproctia. Functional enrichment analysis
   highlighted pathways related to gonadal development (e.g.,
   progesterone-mediated oocyte maturation) and embryonic morphogenesis.
   Collectively, the identification of these SNPs and candidate genes
   advances our understanding of the genetic architecture of intersex and
   aproctia in piglets. These findings provide actionable insights for
   optimizing genetic breeding strategies and improving health management
   in Large White pig production, with potential implications for reducing
   economic losses caused by congenital disorders.

1. Introduction

   In pig genetic breeding programs, intersex and aproctia are critical
   congenital anomalies that adversely affect population health and
   productivity. Disorders of sex development (DSD), a group of congenital
   conditions characterized by chromosomal, gonadal, or anatomical sex
   abnormalities [[54]1], cause the abnormal development of reproductive
   organs, thus affecting the reproductive capacity of individuals.
   Intersex pigs, which exhibit a female chromosomal karyotype (38,XX) but
   lack the sex-determining region Y (SRY) gene, are classified as
   38,XX-DSD (SRY-negative) and represent a type of DSD. These disruptions
   impair reproductive organ development and individual fertility [[55]2].
   Affected individuals often display ambiguous genitalia, including a
   hypertrophied clitoris, vulvar malformations, and occasionally
   scrotal-like structures or penile enlargement, despite a predominantly
   female chromosomal profile [[56]3]. The offspring of carrier boars
   exhibit intersex incidence rates exceeding 4–5% [[57]3], resulting in
   infertility, behavioral aggression during fattening, and economic
   losses due to compromised carcass quality [[58]4]. Although
   intersexuality has been well studied in horses [[59]5] and cattle
   [[60]6], the genetic mechanisms underlying porcine DSD remain poorly
   characterized, with only a few candidate genes identified.

   Congenital aproctia, a rare gastrointestinal malformation affecting
   approximately 0.02–0.06% of the global population, exhibits a
   male-biased sex ratio and familial aggregation in 1–9% of cases.
   Although environmental influences cannot be ruled out, genetic factors
   are the primary determinants of aproctia susceptibility, as evidenced
   by heritability estimates ranging from 0.11 to 0.35 in pig populations
   [[61]7,[62]8]. Genome-wide studies in Pietrain × Landrace crosses have
   mapped aproctia-associated loci to sus scrofa chromosome1 (SSC1), SSC9,
   and SSC12, involving genes such as FMN, BMP4, HOXB5, and HOXB9,
   suggesting that the disease may be controlled by single-gene or
   multi-gene inheritance [[63]9]. In addition, other studies have also
   identified candidate pathogenic genes such as GLI2 [[64]10,[65]11] and
   IHH [[66]11] on SSC15 of Landrace pigs and Large White pigs. In
   addition, a survey of congenital malformations in Australia found that
   the overall incidence of the disease was 0.27% [[67]12]. This disease
   is particularly serious in boars because it usually causes death,
   usually within 1 to 3 days. Although surgical correction is the only
   treatment, the postoperative survival rate is low. For sick sows, some
   individuals will form rectovaginal fistulas, meaning that feces can be
   discharged from the vaginal opening, so some sows can survive, and
   their survival rate within 1 month of age is about 50% [[68]13]. A
   congenital imperforate anus has adverse effects on pig health, breeding
   efficiency, and animal welfare, including acute intestinal obstruction,
   secondary infection, increased mortality, and decreased reproductive
   efficiency.

   Despite the economic and welfare implications of intersex and aproctia
   in pig production, the genetic architecture of these anomalies remains
   incompletely characterized, hindering the development of molecular
   screening tools. This study aims to address this gap by conducting a
   genome-wide association study (GWAS) in a large cohort of 1030 Large
   White pigs, integrating high-density SNP data and imputation strategies
   to identify novel candidate genes and pathways underlying these
   developmental disorders. In addition, since pigs have certain
   similarities with humans in physiological and genetic characteristics,
   the results of this study can also provide valuable references for the