Department of Veterinary Integrative Biosciences, Texas A&M University, College Station, TX, USA
Introduction
Cats are unique among mammals in that over forty genetic crosses between different wild cat species, and between wild cats and domestic cats, have been documented to produce viable hybrid offspring (Figure 1). Popular examples include the liger (male lion x female tiger) and tigon (male tiger x female liger). Among the nearly 70 domestic cat breeds are a number of interspecies hybrids. Remarkably, these crosses are most often accomplished by natural mating, despite millions of years separating the species from their most recent common ancestors. This ease of hybridization between different species is aided by the strong degree of similarity between feline genomes: all but seven cat species (i.e., the South American cat lineage including margays and ocelots) share the same chromosome number, with identical or nearly identical chromosome structure.16 Individual cat species or groups of species are distinguished by one or two morphologically distinct chromosomes. However, these differences do not correspond to large-scale changes in gene order.2 In addition, the DNA sequence itself is very similar between all cat species: > 99% genomic identity between species of the genus Felis (which includes the domestic cat) and > 97% genomic identity across the entire family.
| Figure 1. Phylogeny and timescale of the cat family Felidae | 
Arrows show some of the documented examples of successful interspecies hybridization. Red font indicates interspecific crosses that are the foundation of the three most common hybrid domestic breeds.9 |
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The most common interspecies breeds are the Bengal (domestic cat x Asian leopard cat [ALC], Prionailurus bengalensis), Savannah (Domestic cat x African serval, Profelis serval), and Chausie (Domestic cat x Jungle cat, Felis chaus). The Bengal is one of the most popular cat breeds in the world.8 Domestic cats will naturally interbreed with African servals (Profelis serval) in captivity to produce the Savannah breed, with over 10,500 registered members worldwide.15 The Chausie (Domestic cat x Jungle cat) is one of the more recently established breeds. All three of these "exotic" breeds are recognized by TICA, none by CFA, and only the Bengal by other registries. Other less common interspecies hybrids include the Safari cat (Domestic cat x Geoffroy's cat, Leopardus geoffroyi) and the Marguerite (Domestic cat x Sand cat, Felis margarita). These hybrids are currently restricted to F1 or early backcross generations.
Reproduction
One of the most significant health-related problems observed in exotic cat breeds is the high prevalence of infertility in early (and even later) generation hybrid males. Infertility may arise due to either poor sperm quality (ejaculates with a high frequency of abnormal sperm or teratospermia) or lack of sperm altogether (azoospermia). Teratospermia can impair the success of feline breeding programs and even in vitro fertilization efforts in wild cat breeding programs.14 Teratospermia is commonly found in domestic animal breeds, endangered species and is also found in infertile human patients. Many wild felid species/subspecies, including cheetahs, Asian lions, Florida panthers, and clouded leopards, consistently ejaculate > 75% structurally abnormal sperm, which confounds assisted reproduction efforts and wildlife breeding programs.14 Though the wild cat phenotypes are known to result from inbreeding, they mirror those found in sterile exotic breeds, suggesting common underlying genes/pathways are affected. Understanding the relative contribution of genetics to sperm defects could have measurable impacts on assisted breeding strategies for managing and conserving endangered wild cat species, where a direct correlation exists between proportion of normal sperm and in vitro fertilization success. Unfortunately, the genetic bases of feline teratospermia and infertility are both poorly understood.
The foundation lines for each exotic domestic breed almost exclusively originate through mating female domestic cats to males of the wild species. Fertilization may be accomplished by natural mating, or more rarely, via artificial insemination. The reproductive success of feline interspecies crosses is dictated by a genetic principle known as Haldane's rule, which states that in the F1 offspring of interspecies hybrid crosses it is the male sex (one carrying different sex chromosomes: X & Y) which is sterile or absent, whereas the female (carrying two like chromosomes: XX) is fertile.7 As a result of this sex bias, breeding lines are normally perpetuated via unidirectional crossing of fertile F1 females to male domestic cats.
Hybrid sterility manifests similarly in each of the breeds, with F1 and early backcross generation males exhibiting azoospermia and severe seminiferous tubule degeneration.3 Testes of F1 males from both crosses show Leydig cell hyperplasia with seminiferous tubules characterized by Sertoli-cell-only phenotype and an absence of a defined lumen. First-generation backcross males generally show a similar, though often milder phenotype, with the occasional presence of spermatogonia and early spermatocytes. Infertile late-generation backcross hybrids show meiotic progression, but still may produce low amounts of sperm with a high proportion of abnormalities (Figure 2).
| Figure 2. Hybrid testis histopathology | 
(A) Testis cross-section from a fertile fourth-generation backcross Bengal possessing high sperm concentration consisting of spermatozoa with normal elongated heads. (B) Testis cross-section from a (teratospermic) sibling to the cat in (A), possessing very low sperm concentration and globozoospermia.3 |
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F1 hybrid females from both breeds are fertile, thus they are used to establish foundation lines of each breed by unidirectional backcross mating to fertile domestic cat males, until fertile backcross hybrid males are produced. The backcross generation in which each hybrid breed regains fertility varies with evolutionary distance between the pair of parent cat species (as much as 10 and 7 million years for the parents of the Savannah and Bengal, respectively). Within our study population some Bengals produced viable sperm as early as the second backcross generation, and Savannahs as early as the third backcross generation, although this is considered rare in the breeding communities, who regularly backcross early generation females to late generation, fertile hybrid males.3 Savannahs of the fourth backcross generation (or F5 by breeder terminology) are typically fertile, while litters with both infertile and fertile offspring are not uncommon in the third backcross generation. This narrow and predictable window of achieving fertility is similar to that observed in the Bengal breed. In the Chausie breed, male fertility is restored after fewer backcross generations (2–3) than the more divergent Bengal and Savannah interspecies hybrids, suggesting a potentially simpler genetic basis (i.e., fewer acquired genetic incompatibilities). However, because fertile later generation hybrid backcross males are often used to perpetuate breeding lines via intercrossing with fertile F1 or early generation backcross females, the incidence of infertility can be unpredictable.
Genetics
My lab has been involved in mapping and analyzing the genome of the domestic cat for nearly two decades,2,11,12 and now with a good quality genome sequence in hand, we are using tools from the feline genome project to identify genes influencing infertility and other breed-specific phenotypes in the most common exotic cat breeds. The goal of our research is to better understand the molecular and cellular processes that lead to infertility, with the added hope that these findings will translate into practical benefits for breeders, where wild cat genes strongly associated with hybrid sterility could be detected in DNA samples of potential breeders, and ultimately used to select against carriers of these genes that manifest as sterility in males. Additional benefits of studying exotic cat hybrids relate to increasing our general knowledge of genes that cause human infertility, as well as conservation-related applications.
The genetic basis of hybrid male sterility in cats is polygenic, involving genes with both large and small influence. Interspecies crosses are powerful resources for mapping complex traits and examining the mechanisms of speciation, requiring fewer individuals to map genes with moderate to large phenotypic effects.10 Our first study analyzed cohorts of sterile and fertile Bengals and Savannahs in independent genome-wide association studies (GWAS) to identify genetic markers associated with the sterility phenotype.3 We leveraged the Illumina domestic cat SNP array to detect genetic markers fixed in wild progenitor species but variable in the domestic cat. The results of our two GWAS produced biologically relevant genetic associations for five chromosomal regions in Savannahs and three in Bengals (Figure 3A–C).
In the Savannah (Figure 3A) and Bengal (Figure 3B) GWAS we identified five and three, respectively, statistically significant SNPs hybrid incompatibilities underlying genes (Figure 3C) in three general categories of biological and molecular processes:
1. Acrosomal development (GRM8, VPS53, SNAP25)
2. Blood-testis barrier compartmentalization (CADM1, AKAP9)
3. Transcriptional regulation (ZSCAN25, DNMT3L)
It is noteworthy that genes in the latter category represent the top GWAS hits in both Bengals and Savannahs. This finding is consistent with our observations of large amounts of transcriptional misregulation in the testes of sterile hybrids when compared to testis transcription measurements from fertile parent species or fertile backcross hybrids. The most notable amount of gene misregulation was observed on the X chromosome of both hybrids. This is consistent with previous observations in fruit fly and mouse interspecific hybrids, which display a disproportionately "Large X-Effect" on hybrid male sterility.5,6,13 More recent research into the genetic ancestry of these three hybrid breeds using next generation sequencing has shown that males who possess one or more wild cat (i.e., ALC, serval, jungle cat) X chromosome haplotype blocks on an otherwise largely domestic cat genetic background are generally infertile, whereas virtually all fertile hybrid males possess an X chromosome of domestic cat ancestry. These results thus also support a major role of the X chromosome, in combination with the autosomal genes we identified,3 underlying feline hybrid male sterility.
| Figure 3. Manhattan plots and marker details for GWAS in two hybrid feline breeds | 
A) Five markers (SAV1-5) associated with hybrid male sterility in the Savannah breed. B) Three markers (BEN1-3) associated with hybrid male sterility in the Bengal breed. C) Table showing associated Illumina SNP markers, association statistics, chromosomal location, and candidate gene implicated in the sterility phenotype. |
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