Institute of Animal Breeding and Genetics Professorship for Animal Genetics and Pathogenetics Justus Liebig University Giessen Genetic factors of horn-related traits in small ruminants with special reference to the polled intersex syndrome (PIS) in the goat INAUGURAL-DISSERTATION submitted for the degree of Doctor of Agricultural Science (Dr. agr.) to the Faculty of Agricultural Sciences, Nutritional and Environmental Management zur Erlangung des Doktorgrades (Dr. agr.) im Fachbereich Agrarwissenschaften, Ökotrophologie und Umweltmanagement der Justus-Liebig-Universität Gießen submitted by M. Sc. Rebecca Simon from Leverkusen (North Rhine-Westphalia) Giessen, June 2024 page | II With permission of the Department of Agricultural Sciences, Ecotrophology and Environmental Management of the Justus Liebig University Giessen The examination committee: 1. Supervisor: Prof. Dr. Gesine Lühken Institute of Animal Breeding and Genetics Justus Liebig University Giessen, Germany 2. Supervisor: Prof. Dr. Cord Drögemüller Institute of Genetics University of Bern, Switzerland Examiner: Prof. Dr. Christine Wrenzycki Veterinary Clinic for Reproductive Medicine and Neonatology Justus Liebig University Giessen, Germany Examiner: Prof. Dr. Rod Snowdon Department of Plant Breeding Justus Liebig University Giessen, Germany Chair: Prof. Dr. Joachim Aurbacher Department of Business Administration of the Agricultural and Food Sector Justus Liebig University Giessen, Germany Date of disputation: 18.12.2024 page | III This work was supported by a doctoral scholarship from the H. Wilhelm Schaumann Foundation. page | IV Wenn wir wüssten, was wir tun, würde das nicht Forschung heißen, oder? ~Albert Einstein~ page | V Table of content List of tables ______________________________________________________________ VI List of figures ______________________________________________________________ VI List of abbreviations ________________________________________________________ VII 1. SUMMARY ___________________________________________________________ - 1 - 2. INTRODUCTION _______________________________________________________ - 1 - 2.1. Development, morphology and inheritance of horns (and scurs) _________________ - 2 - 2.1.1. Inheritance __________________________________________________________________ - 6 - 2.2. Special traits related with horn status ______________________________________ - 7 - 2.2.1. Scurs ________________________________________________________________________ - 7 - 2.2.2. Polyceraty ___________________________________________________________________ - 8 - 2.3. Differences in husbandry management for horned animals _____________________ - 9 - 2.4. Dehorning of small ruminants ____________________________________________ - 9 - 2.4.1. Methods, risks and regulation in Germany ________________________________________ - 10 - 2.5. Breeding for polledness ________________________________________________ - 11 - 2.5.1. Interaction of horn status and health-related characteristics __________________________ - 11 - 2.5.1.1. Polled Intersex Syndrome (PIS) _____________________________________________ - 11 - 2.5.1.1.1. Other causes for intersexuality in livestock ________________________________ - 12 - 2.5.1.2. Abnormalities of the brows and eyelids ______________________________________ - 12 - 2.5.1.3. Polled and Multisystemic Syndrome (PMS) ___________________________________ - 13 - 2.5.1.4. Abnormal skull shape, small body size and subfertility in Fleckvieh cattle ___________ - 13 - 2.5.1.5. Type 2 Scurs Syndrome ___________________________________________________ - 13 - 2.5.2. Genetic engineering __________________________________________________________ - 14 - 2.6. Aim of this study ______________________________________________________ - 14 - 3. ORIGINAL WORKS ____________________________________________________ - 15 - 3.1. First publication _______________________________________________________ - 15 - 3.2. Second publication ____________________________________________________ - 26 - 3.3. Third publication ______________________________________________________ - 43 - 4. DISCUSSION _________________________________________________________ - 54 - 4.1. Future studies – outlook ________________________________________________ - 58 - 4.2. Conclusion ___________________________________________________________ - 58 - References ______________________________________________________________ - 60 - Appendix _______________________________________________________________ - 72 - First publication _____________________________________________________________ - 72 - Second publication ___________________________________________________________ - 78 - Third publication ____________________________________________________________ - 80 - Acknowledgement _______________________________________________________ - 88 - Declaration _____________________________________________________________ - 89 - page | VI List of tables Table 1: Different horn forms and example breeds from different species displaying them. ................................................................................................................................................ - 5 - Table 2: Details on the variant segregating with polyceraty in sheep and goats. ................. - 8 - List of figures Figure 1: Varius horn shapes and sizes in non-domesticated representatives of small ruminants. .............................................................................................................................. - 3 - Figure 2: Variety of horn shapes in the cattle breed Angler Rotvieh..................................... - 6 - Figure 3: German Improved White buck with horn crusts. ................................................... - 6 - Figure 4: Polycerate icelandic ewe. ...................................................................................... - 58 - page | VII List of abbreviations ADM Anaesthesia delegation model AMH Anti-Mullerian Hormone gene ARHGAB15 Rho GTPase activating protein 15 gene bp basepair(s) cm centimeter CRISPR Clustered Regularly Interspaced Short Palindromic Repeats DHH Desert hedgehog gene DMRT1 Doublesex and mab-3 related transcription factor 1 gene DSD Disorder(s) in sexual development EMBL European Molecular Biology Laboratory e.g. exempli gratia ERG ETS transcription factor ERG gene FOXL2 Forkhead box L2 gene GATA4 GATA binding protein 4 gene GTDC1 Glycosyltransferase like domain containing 1 gene HOXD1 Homeobox D1 gene HSC hematopoietic stem cells ISAG International Society for Animal Genetics Kb kilobase(s) KCNJ15 potassium inwardly rectifying channel subfamily J member 15 gene Mb Megabase(s) MOWS or MWS Mowat-Wilson Syndrome NRXN1 Neurexin 1 gene PCR Polymerase chain reaction PIS Polled Intersex(uality) Syndrome PISRT1 PISRT1 lncRNA PMS Polled and Multisystemic Syndrome RXFP2 Relaxin family peptide receptor 2 gene SFRP4 Secreted frizzled related protein 4 gene SIM1 SIM bHLH transcription factor 1 gene SNP Single nucleotide polymorphism STIM1 Stromal interaction molecule 1 gene SUED split upper eyelid defect TALEN Transcription activator-like effector nuclease TWIST1 Twist family bHLH transcription factor 1 gene U.S. United States UTR Untranslated region WNT3 WNT family member 3 gene WT1 WT1 transcription factor gene ZEB2 Zinc finger E-box binding homeobox 2 gene page | - 1 - 1. SUMMARY Bovidae belong to the family of ruminants which unite a range of even-toed ungulates like cattle, sheep, goat and antelopes. The group of pecorans is very heterogeneous in their phenotypic appearance, but have a common feature, the horns, which appear in pairs. The characteristics associated with the presence or absence of horns are as diverse as the group itself. In livestock, especially cattle and goats, polledness is often seen as a desirable trait, as it can reduce the risk of injury to humans and flockmates. In order to achieve this, young horned animals were or still are dehorned. In view of the ever-increasing controversy and tightening of the legal situation in this regard, for example in Germany, breeding and selection for genetically polled livestock offers a possible alternative. While knowledge about inheritance and underlying gene variants for horn-specific traits is extensive for cattle, there are still some gaps in knowledge for small ruminants (sheep and goats) - even for long-known phenomena. The polled intersex syndrome (PIS) in goats, which describes the fact that female, homozygous polled goats are infertile hermaphrodites, has been described since the 1940s. Due to the wide variability in phenotypic expression, affected animals are often difficult to identify. This restricts breeding for polledness in goats. Despite the published association with an 11.7-kb deletion on chromosome 1 that affects the transcription of the PISTR1 and the FOXL2 gene, it was not possible for a long time to develop a genetic test for the early detection of affected individuals that could be used in practice. The identification of a complex rearranging structural variant, consisting of the named deletion in combination with an inverse inserted duplication, associated with PIS was made possible through the use of long-read whole genome sequencing in the context of this work. This finding made it possible for the first time to develop an early applicable genetic test to identify all three possible genotypes, as well as the sex. Subsequent publications have confirmed this variant for all goat breeds examined worldwide. Also for the trait polledness in sheep a causal variant in form of an 1.78-kb sized insertion in the 3‘-UTR region of the RXPF2 gene on chromosome 10 has been published for some time. However, it has been shown several times that this does not segregate with the trait in all breeds. This applies in particular to breeds with variable or sex-specific horn status. This was mainly confirmed for the Icelandic sheep in this study. In sheep, the RXFP2 gene has been shown to be not only associated with the presence/absence of horns but also with other traits related to horns, such as size and shape. Horn size showed some association with a previously published RXFP2 variant in the Icelandic sheep as well. The inclusion of polycerate Icelandic sheep in the present work confirmed the segregation of a previously published 4-bp insertion in HOXD1 (ovine chromosome 2) with this trait, but also brought new findings. For the first time, this insertion was detected in polled individuals of polycerate origin. And a simultaneous observation of the HOXD1 and RXFP2 variants mentioned allows the assumption that polledness in Icelandic sheep of polycerate origin is not controlled by the RXFP2 insertion described. This work has helped to solve some unknowns, especially with regard to the PIS of the goat. However, insights were also gained that raise further questions, such as polledness in sheep of polycerate origin. These should be investigated more intensively in other breeds and larger samples in the future. The further development of molecular genetic methods can also help to clarify further horn-specific traits and verify initial indications, like those found in this study. page | - 2 - 2. INTRODUCTION Domestication of the livestock species cattle, sheep and goat in the Middle East (Fertile Crescent) laid the foundation for today's agriculture, but also for the emergence of the current diversity of breeds and traits of farm animals (Diamond, 2002). Through intensified husbandry of the mentioned species, humans have come into closer contact with them. The use of livestock was beneficial for mankind (Ahmad et al., 2020). But the close contact with the animals also revealed the risks for humans and flockmates. To minimize the increased risk of injury, but also payout losses for reduced meat quality due to bruising (Youngers et al., 2017; Mendonça et al., 2016) lower milk yield and quality due to udder injuries or bruising on valuable cuts, dehorning of young animals is used especially in cattle farming. Since this procedure is associated with stress and potentially also pain for the animals, it is partly regulated by law, and in the case of goats in Germany, for example, it is already prohibited completely (according to the German Animal Welfare Act §6 paragraph 1). Breeding for polledness can be seen as an animal friendly alternative, although this possibility is also associated with restrictions, especially in goats. In this species, polledness is associated with interferences in sexual development, known as polled intersex syndrome (PIS) (Asdell, 1944; Eaton, 1945). Prior to the start of this work, there is no genetic test available to help identify affected animals as early as possible. While in cattle the knowledge about the genetic factors underlying polledness is great, in small ruminants many pieces are still missing. In sheep, for example, an 1.78 kb-sized insertion on chromosome 10 associated with polledness has been known for several years, but this does not segregate with the trait in all breeds (Wiedemar and Drögemüller, 2015; Lühken et al., 2016). Other variants are not yet known. Consequently, information on the functional basis of such horn-related traits of interest for breeding and knowledge on possible targets for selective breeding is still required. 2.1. Development, morphology and inheritance of horns (and scurs) Horns are referred to as cranial appendages or headgear in bovine species, which have to be delimited from the antlers in cervids, ossicones in giraffids and pronghorns in antelopes – all belonging to the family of ruminants (Davis et al., 2011). Little is known about the evolution of this manifold trait (Davis et al., 2011). The occurrence of polledness in cattle can be traced back to ancient times. Depictions of polled cattle have been found on petroglyphs and in burial sites in ancient Egypt. It is estimated that those polled individuals had a very high value, as they were never depicted as working animals (Schafberg and Swalve, 2015). Depictions of hornless goats can also be found on ancient (approx. 3000 BC) Egyptian illustrations (reviewed in Amills et al., 2017). The horn morphology varies among species, which becomes evident when examining the non- domesticated relatives of small ruminants (Figure 1), as well as between breeds. The latter is particularly evident in sheep, but also known in cattle (Figure 2). Individuals can express a variety of horn forms (Table 1), ranging from small and slightly curved ones up to massive spiral ones. The diameters can be rather rounded or angular. Sometimes forms also differ between the sexes of one breed. Additionally there is a special semi-form known in sheep and cattle. The small, mis-shaped and flexible scurs, which are, in contrast to normal horns, not page | - 3 - attached to the skull (Gehrke et al., 2020b; White, W.T. and Ibsen, H.L., 1936; Long and Gregory, 1978). Their existence in goats is not proven, but breeders sometimes report hornlike structures for this species as well (personnel communication, Figure 3). Nevertheless, a common feature of all horns in different species is their origin in the cells of the neural crest (Guo et al., 2021). Figure 1: Various horn shapes and sizes in non-domesticated representatives of small ruminants. All pictures were taken in the Museum of Natural History in Vienna, Austria. 1. line (from left to right): Argali (dt.: Argali; Ovis ammon), Dall’s sheep (dt.: Dallschaf ; Ovis dalli), Mouflon (dt.: Mufflon; Ovis gmelini musimon), Siberian bighorn sheep (dt.: Schneeschaf; Ovis nivicola), Bighorn sheep (dt.: Dickhornschaf; Ovis Canadensis). 2. line (from left to right): Alpine ibex (dt.: Alpensteinbock; Capra ibex), Siberian ibex (dt.: Sibirischer Steinbock; Capra sibirica), Pyrenean ibex (dt.: Pyrenäensteinbock; Capra pyrenaica), Kuban tur (dt.: Westkaukasischer Steinbock; Capra caucasica), Daghestan tur (dt.: Ostkaukasischer Steinbock; Capra cyclindricornis), Agrimi (dt.: Kretaziege; Capra aegagrus cretica). 3. line (from left to right): Japanese serow (dt.: Japanischer Serau; Capricornis crispus), Sumatran serow (dt.: Südlicher Serau ; Capricornis sumatrensis) 4. line (from left to right): Bharal (dt.: Blauschaf; Pseudois nayaur), Mountain gaot (dt.: Schneeziege; Oreamus americanus), Himalayan tahr (dt.: Himalaya-Tahr; Hemitragus jemlahicus). 5. line: Barbary sheep (dt.:Mähnenspringer ; Ammotragus lervia). page | - 4 - Horns are skin organs enclosed in a keratin sheath, which develop from the epithelial bud and the proliferation of the connective tissue underneath. The bony horn cone formed in this way is connected to the frontal sinus in the first year of life at the latest; in small ruminants this happens at a younger age than in cattle. The blood supply to the horn is provided by the terminal branches of the temporal artery and vein (A. and V. temporalis). Innervation is mainly through the zygomaticotemporal nerve (N. zygomaticotemporalis) (König and Liebich, 2019). page | - 5 - Table 1: Different horn forms and example breeds from different species displaying them. Please note, the list does not claim to be complete and the scheme of the sheep head is used for every form for simplification, information about horn forms was taken from Porter et al., 2016. Horn form Example breed (species) Scheme Spiral Skudde (Ovis aries), Grey Horned Heath (Ovis aries) Horizontal screwed Roux du Valais (Ovis aries), Valais Blacknose Sheep (Ovis aries) V-formed and twisted Racka sheep Zackelschafe (Ovis aries) Crescent curved (a: upwards) (b: backwards) Peacock goat (Capra hircus), Saanen goat (Capra hircus) Cameroon sheep (Ovis aries) a b Curved outwards Capra Sempione (Capra hircus) Lyre-shaped Scottish Highland Cattle (Bos taurus) Hungarian Grey (Bos Taurus) Short and strong, bent upwards Hérens Cattle (Bos Taurus) page | - 6 - Figure 2: Variety of horn shapes in the cattle breed Angler Rotvieh. Please note the most common shape being displayed in the upper row, while also shapes like bend inwards horns (second row, left) or horns extending far outward with horn tips pointing upwards (second row, middle and right) can be found l. All cows come from the same flock. Figure 3: German Improved White buck with horn crusts. According to the breeder, the animal had shed its approximately 5 cm horn stubs, considered as scur-like structures, a few days bevor the picture was taken. However, these would grow back, according to the statement. (Picture: G. Lühken) 2.1.1. Inheritance Clarification of the challenging question of the inheritance of polledness in ruminants has occupied many scientists since the 1900s. Nevertheless, it is now considered proven, that page | - 7 - horns represent the wild type in cattle and that this trait is recessive to polled (P) (Aldersey et al., 2020). The situation is similar for goats in which polledness (P) dominates horns (p) (Asdell und Crew 1925), but different for sheep, where differences between breeds might be obvious. For instance for some breeds, polledness is described as recessive trait (Clutton-Brock and Pemberton, 2004; Pickering et al., 2009). Whereas Johnston at al. (2011) observed a sex- dependent type of gene action in which the horned allele is dominant in male Soay sheep and the two alleles being additive in female Soay sheep. For the merino population polledness is described as dominant (Dolling, 1960). Due to the heterogeneity of the molecular causes of inherited polledness in the different species it is suggested that the underlying mutations occurred independently. While in cattle (Bos Taurus OMIA:00483-9913 and Bos indicus OMIA:00483-9915 (Nicholas and Tammen, 1995)) knowledge about the traits underlying gene variants is great (Medugorac et al., 2012; Medugorac et al., 2017; Gehrke et al., 2020b; Gehrke et al., 2020a; Lamb et al., 2020; Aldersey et al., 2023), it is still incomplete in sheep (OMIA: 000483-9940 (Nicholas and Tammen, 1995)). To date, all studies in various sheep breeds with different origins, using multiple, ever-evolving methods, point to a central role of the relaxin family peptide receptor 2 (RXFP2) gene in the inheritance of horn-related traits in this species (e.g. Dominik et al., 2012; Johnston et al., 2011). However, only the 1.78 kb-sized insertion into the 3’-UTR region or RXFP2 has been confirmed causal for polledness in breeds with sex-independent horn status (Wiedemar and Drögemüller, 2015; Lühken et al., 2016). Additional variants in or close to RXFP2 gene can only be referred to as markers, often just for specific breeds, for horn size and shape (Pan et al., 2018; Johnston et al., 2010) or the absence of horns (Duijvesteijn et al., 2018). Therefore, not just the existence of horns in sheep (Ovis aries) is associated with RXFP2, but also morphological horn attributes. For example a haplotype consisting of two SNPs was found to be associated with higher length and a spiral form in various Chinese breeds (Pan et al., 2018). However, not in Thinhorn sheep (Ovis dalli, Figure 1, first line) in which two loci one on ovine chromosome 2 and 3 each, where found to be associated with horn length (Sim and Coltman, 2019). The locus for polledness in goats was mapped to the distal end of chromosome one (Vaiman et al., 1997). The variant underlying polledness in goats is connected with intersexuality (for details refer to chapter 2.5.1.1.) and is described as an 11.7 kb-sized deletion affecting the transcription of two genes - polled intersex syndrome regulated transcript 1 (PISTR1) and forkhead box L2 (FOXL2) (Pailhoux et al., 2001). 2.2. Special traits related with horn status In addition to the occurrence of a solid pair of horns, additional types have been described. They refer to the development of horn-like structures or the occurrence of a large number of horn pairs. 2.2.1. Scurs In contrast to horns, scurs are defined as small bony structures that are not firmly fused with the skull. Sometimes they are also referred to as knobs. The scurs phenotype is variable in size (Dove, 1935). Its occurrence has been proven in both sheep (Ibsen, 1944) and cattle (Long and Gregory, 1978; Capitan et al., 2009); there is no confirmation for goats, but breeders report page | - 8 - the occurrence of small, unattached horns in this species as well (Figure 3). Although not fully elucidated yet, most of the evidence on this sex-influenced trait is available in cattle (OMIA: 000894-9913 (Nicholas and Tammen, 1995)). The long accepted mode of inheritance of scurs being a dominant trait in bulls, while two copies of allele Sc are required in females to express the trait (White, W.T. and Ibsen, H.L., 1936) has meanwhile been disproven (Gehrke et al., 2020b). However, it was confirmed that animals carrying scurs are heterozygous for one of the known polled variants (Gehrke et al., 2020b). The scurs locus was mapped to the bovine chromosome 19 (Asai et al., 2004) but recently a genome-wide linkage mapping showed significant loci on two chromosomes, giving rise to the hypothesis of scurs being a polygenic trait (Gehrke et al., 2020b; Mariasegaram et al., 2010). A phenotypically similar but still different trait, named type II scurs (OMIA: 001593-9913 (Nicholas and Tammen, 1995)), was exclusively found in a Charolais family. In contrast to type I scurs these map to bovine chromosome 4 and segregate with a frame-shift mutation (p.A56RfsX87) in twist family bHLH transcription factor 1 (TWIST1) gene (Capitan et al., 2011). No homozygous individuals could have been found, pointing towards a lethal factor, which is in accordance with embryonic lethality in TWIST1 knock-out mice (Chen and Behringer, 1995). 2.2.2. Polyceraty Polyceraty describes the occurrence of more than two horns. Up to six horns are reported (Porter et al., 2016). This trait is mainly found in sheep (Dýrmundsson, 2005), where some breeds, like Jacob sheep (Porter et al., 2016), carry multiple horns as signature trait. Polycerate goats are described as well (Giovanoli, 1919; Herrera et al., 2007). In both species a link to eyelid deformity is described (Herrera et al., 2007; Henson, 1981; Gascoigne et al., 2017). How and why polyceraty evolved remains unknown, but recently two slightly different variants (Table 2), in the homeobox D1 (HOXD1) gene on ovine chromosome 2 and caprine chromosome 2 were found to segregate with the trait in sheep and goats, respectively (Allais- Bonnet et al., 2021) confirming first mapping trials (Greyvenstein et al., 2016; He et al., 2016; Kijas et al., 2016). No information is available about the occurrence of multihornedness in cattle. Table 2: Details on the variant segregating with polyceraty in sheep and goats.(modified after Allais-Bonnet et al., 2021; Nicholas and Tammen, 1995) Species Chromo- some Gene Variant Location Mode of inheritance Reference Sheep (ovis aries) 2 HOXD1 Deletion, 4 bp Oar_rambouillet _v1.0: NC_056055.1 (133949709..13 3947471) Autosomal co- dominant Allais- Bonnet et al. 2021 Goat (capra hircus) 2 HOXD1 Delins, 137 kb ARS1: NC_030809.1 (115593830..11 5596023) Autosomal dominant Allais- Bonnet et al. 2021 page | - 9 - 2.3. Differences in husbandry management for horned animals Reviewing recommendations for commercial goat housing and management Zobel et al. (2019) stated that the available information is limited and a great proportion is rather based on practical experiences than on science-based research (Zobel et al., 2019). Horns in goats are used in offense and defense (Geist, 1960) for various resources, including sexual partners and food (Shi and Dunbar, 2006; Shank, 1972; Geist, 1966; Lundrigan, 1996; Stankowich and Caro, 2009). Goats have a complex social structure and constant group formation of relatively small core groups (~ 12 individuals Stanley and Dunbar, 2013), with a strict hierarchy (Zobel et al., 2019). Groups are formed and structured by rank fights (agonistic behavior) and affiliative behavior (Zobel et al., 2019). It was shown that there is a higher tendency for injuries regarding the udder in horned compared to polled flocks (Waiblinger et al., 2010). Horns do not only bear a risk for flockmates but also for stockpersons to become physically injured (Knierim et al., 2015; Braun et al., 2016; Goldblum et al., 1999; Katsos et al., 2019; Tijjani et al., 2015). However, injuries caused by horn-induced bruises can also cause economic damage, for example by reducing milk yield or by lowering the meat quality of valuable carcass cuts (Mendonça et al., 2016; Youngers et al., 2017; Collins and Huey, 2014). In intensive-housing systems, animals cannot always avoid flockmates and maintain individual distance, potentially leading to social conflicts and horn use (Aschwanden et al., 2008). Therefore, the named risk factors need to be considered in housing and management of horned livestock and often require adaptations. To give more space for evasion measures like a lower stocking density, the offer of a raised level (Zobel et al., 2019) and additional feeding space (Loretz et al., 2004; Waiblinger et al., 2010) in combination with modified feeding rails (Aschwanden et al., 2009; Hillmann et al., 2014; Waiblinger et al., 2010) are advised. In addition, a stable herd/flock structure, e.g. by a longer service life, is recommended as a measure to avoid social stress and associated combative interactions (Waiblinger et al., 2010). Waiblinger et al. (2010) stated that named improvements of housing and general management increase animal welfare in goats in general, independent of the respective horn status. It has also been shown that management and the housing environment, in particular the availability of space, are decisive factors in the successful keeping of horned cows (Menke et al., 1999; Waiblinger et al., 2001). For sheep no recommendations are found in the literature. 2.4. Dehorning of small ruminants To account for the risk factors associated with horns described in chapter 2.3. disbudding is a routine management practice in cattle and goats husbandry in many countries worldwide (Boyd, 1988; Cozzi et al., 2015). Even though several countries and the European Union (EU) defined animal welfare principals (e.g. European Union, 1998, 2008)) which include the avoidance of pain induced by management procedures like for example disbudding it is often still seen as a routine measurement, due to legal exceptions. page | - 10 - In addition to legal restrictions that aim at avoiding pain caused by interventions on the animal, also functional properties of horns, e.g. in thermoregulation (Parés-Casanova and Caballero, 2014) are discussed as arguments against dehorning. Disbudding of sheep is not a common practice, as many commercially used breeds are bred genetically polled. 2.4.1. Methods, risks and regulation in Germany Three common methods of disbudding are cautery, cryosurgical and caustic paste (Bengtsson et al., 1996; Hempstead et al., 2018b; Vickers et al., 2005). According to a survey in European countries cautery method using an hot iron was preferred in disbudding calves (Gottardo et al., 2011; Staněk et al., 2018) while in the U.S. state Wisconsin caustic paste was the primary method (Saraceni et al., 2021) . All named methods cause pain to the treated animals (Heinrich et al., 2010; Allen et al., 2013; Stafford and Mellor, 2005; Waiblinger et al., 2010), whereby caustic paste and cryosurgical disbudding appeared to cause greater acute pain in goat kids when compared to the cautery method (Hempstead et al., 2018a). In addition to the animal welfare aspect, economic reasons can also be evoked for the use of analgesics. Investigations have shown that pain can lead to reduced feed intake and thus reduced daily weight gains (Bates et al., 2015; Borderas et al., 2009). Cortisol concentration, expressing pain and stress, was even elevated in animals in which dehorning was performed under local anesthesia, showing that a combination with analgesics is important in goat kids (Alvarez et al., 2009) and calves (Stock et al., 2013). This contradicts earlier results in calves, where a reduced pain sensitivity was observed after dehorning under local anesthesia (Graf and Senn, 1999). Nevertheless, especially in goats, disbudding-related injuries occur often. Especially cautery disbudding, if performed incorrectly, has a high potential of skull damage, leading to thermal injuries of the brain (Hempstead et al., 2018a; Sanford, 1989; Waiblinger et al., 2010), being still considered the most effective method compared to others (Still Brooks et al., 2021). A large proportion (> 50%) of the observed brain injuries in goats in a retrospective study could be diagnosed as suppurative inflammation due to, among other things, injuries from dehorning (Allen et al., 2013). This confirms findings from a New Zealand working group showing a high risk of brain injury from thermal disbudding of neonatal kids (Thompson et al., 2005). Another factor that enlarges the risk of injuries is that most veterinarians will only proceed disbudding of goat kids irregularly and therefore lacking on experience/practice (Clayton, 2013). To address this factor, in 2008 a system has been established in Switzerland where only certified farmers are allowed to perform the mandatory anesthesia and dehorning of their own kid goats independently – “anesthesia delegation model (ADM)” (Alsaaod et al., 2014; Wagmann et al., 2018). Due to Wagmann et al. (2018) the success of this program is questionable as in over one third of the analyzed cases the anesthesia was inadequate. Another reported issue with improper disbudding is the remaining of horns or parts of them, causing pain as well (Battini et al., 2014). In Germany, the legal situation differs significantly from other countries, even though the painful intervention has also been the subject of criticism elsewhere for a long time. Referring to paragraphs §5 and §6 of the German Animal Welfare Act, the prohibition of avoidable pain and suffering, the dehorning of sheep, goats and cattle is prohibited as a routine measure in page | - 11 - Germany (Deutscher Bundestag, 2006). However, there is currently an exception for the latter (§ 5 (3)2. and §6 (1)3.), allowing calves younger than six weeks to be dehorned even without the use of anesthesia (Deutscher Bundestag, 2006). 2.5. Breeding for polledness Taking animal welfare concerns and the legislation into account the need for an alternative of dehorning becomes obvious. As previously described, polledness occurs naturally in sheep, goats and cattle and therefore the basics for breeding for polledness in those species are present. In some cases genetic testing for polledness is available to support breeders with early information about an individual’s genotype (Randhawa et al., 2020). Nevertheless, this approach has limitations, as described below. The first one to be mentioned is the risk of inbreeding, accompanied by the loss of genetic diversity, when trying to push the trait of polledness into a mainly horned population (Schafberg and Swalve, 2015; Windig et al., 2015; Scheper et al., 2016). Spurlock et al. (2014) described the risk of losing genetic merit which is associated with the selection for the polled trait in cattle (Spurlock et al., 2014). Another risk, which is especially important for goats, is that polledness can be linked to undesired traits or even defects. 2.5.1. Interaction of horn status and health-related characteristics Horn status, in peculiar polledness, is often thought to have a negative influence on performance parameters in livestock. Such adverse associations are difficult to prove scientifically, as performance is influenced multifactorially (Scheper et al., 2021; Cozzi et al., 2015; Goonewardene et al., 1999). Nevertheless, there are also known characteristics of varying impact on the affected animal, whose association with the horn status is easier to prove. Some of the described characteristics associated with polledness are also known to affect only individual families. 2.5.1.1. Polled Intersex Syndrome (PIS) PIS is the most commonly known constraint in farm animals that is related to the horn status. The phenomenon was first described in 1944 when an unusual sex ratio, increased number of animals with male habit, was observed in polled goat flocks (Asdell, 1944). It became clear that, in contrast to other horn-bearing species, there is a connection between the absence of horns and disorders in sex development in domestic goats. Particular effects are seen in females (XX), which are affected by phenotypically variable intersexuality when homozygous polled (Pannetier et al., 2012; Just et al., 1994; Vaiman et al., 1997). The phenotypic variability complicates the early detection of affected goats (Szatkowska et al., 2014). While polledness is known to be dominant inherited, the associated intersexuality is a recessive trait. Infertility or lower fertility in male PIS-affected goats may occur, but the exact circumstances and impacts have not yet been discovered (Pannetier et al., 2005). An association between PIS- affection and various growth traits could be ruled out, at least for Guanzhong dairy goats (Zhang et al., 2020). The locus for PIS was mapped to chromosome one (Vaiman et al., 1997) and narrowed down to a 11.7-kb sized deletion in the region of PIS-regulated transcript 1 page | - 12 - (PISRT1) and forkhead box L2 (FOXL2) gene, affecting both genes transcription (Pailhoux et al., 2001). The influence of FOXL2 on sex determination and regulation in horn bud differentiation was confirmed in further studies (Allais-Bonnet et al., 2013; Boulanger et al., 2014). For example, loss of function of FOXL2 has been shown to lead to female-to-male sex reversal (Boulanger et al., 2014). A genome wide association study confirmed the association of the region previously published with PIS. Furthermore, evidence was found for a single genetic, but more complex basis then suggested for PIS in European and non-European breeds (Kijas et al., 2013). The identification of affected animals, early in life was considered particularly important for the agricultural livestock production but so far there are only attempts realized that focus on sex determination in polled goats. For example via a simple PCR detection of X- and Y-specific variants in the amelogenin gene, focusing on the detection of XX-males (Fábián et al., 2017). 2.5.1.1.1. Other causes for intersexuality in livestock The already described phenomenon of PIS is not the only known cause of intersexuality in goats. And also for other farm animals the condition of intersexuality is not rare – various causes have been described. One well-known cause of intersexuality is freemartinism (Padula, 2005; Nicholas and Tammen, 1995), mainly occurring in mixed-gender twin gestation in cattle (OMIA 000393-9913), but cases in sheep (OMIA 000393-9940) and goats (Szatkowska et al., 2014) are reported as well. Due to anastomosis in early gestation, masculinizing hormones (anti-mullerian hormone and testosterone) and hematopoietic stem cells (HSC) are transferred from the male to the female embryo in heterosexual twins, causing the female to be born infertile (XX/XY chimerism) in most of the cases (Padula, 2005). This is possible because the male gonad differentiation begins earlier in gestation then in females and gets active with steroid production (Short and Bulaban, 1994). In addition, a number of gene variants are known to be associated with disorders of sexual development (DSD) in humans and mice (Larson et al., 2012; Eggers and Sinclair, 2012) which might also be involved in similar cases in livestock. Yang et al. (2021) showed that altered expression of some of those genes where detectable in intersex goats as well. Thereby they found that in the expression of WT1 transcription factor (WT1), doublesex and mab-3 related transcription factor 1 (DMRT1), GATA binding protein 4 (GATA4), Anti-Mullerian hormone (AMH), and desert hedgehog (DHH) genes differed between testicular (male habit) and ovarian (female habit) types of intersexes (Yang et al., 2021). 2.5.1.2. Abnormalities of the brows and eyelids In polled cattle, a link between polledness and a specific eyelash-and-eyelid phenotype is described. A supernumerary row of lashes on the inner part of the eyelid is reported in polled animals, as well as an eyelid hypertrichosis (extensive hair growth) (Allais-Bonnet et al., 2013). In sheep and goats, abnormalities of eyebrows and eyelids have also been described, not in connection with polledness, but in the presence of an increased number of horns (polyceraty) (Allais-Bonnet et al., 2021; Lühken and Drögemüller, 2021). The congenital split upper eyelid defect (SUED) for example is primarily found in polyceratous sheep and was first described in Jacob sheep (Henson, 1981). Little is known on the effects on animal welfare, for example due page | - 13 - to the severity of damage to the cornea surface, or performance parameters of affected individuals (Gascoigne et al., 2017). A case report on goat flocks in Extremadura, Spain, showed that the association also exists in multiple-horned goats (Herrera et al., 2007). It is not yet known whether the variants in the HOXD1 gene recently identified as causal for multihornedness in sheep and goats are also associated with the occurrence of these eyelid anomalies (Allais-Bonnet et al., 2021). 2.5.1.3. Polled and Multisystemic Syndrome (PMS) The PMS was just described in the polled progeny of a single Charolais sire (Capitan et al., 2011). A number of other symptoms could be observed in the affected offspring. These included among others facial dysmorphism, variable neurological disorders, chronic diarrhea and anomalies in reproduction with low progesterone levels. The latter one described for nine out of 14 PMS-affected females. Furthermore the unusual gender distribution in the offspring population points towards a male-specific lethal factor, inherited dominantly (Capitan et al., 2011). A 3.7-Mb deletion on bovine chromosome two was found to be causative, for which the founder bull was a somatic mosaic. The variant affects two complete genes (zinc finger E-box binding homeobox 2 (ZEB2) and glycosyltransferase like domain containing 1 (GTDC1)) and a part of the Rho GTPase activating protein 15 (ARHGAB15) gene (Capitan et al., 2011). Loss of ZEB2 is also known in humans, affected individuals have Mowat-Wilson syndrome (MOWS or MWS), which has symptomatic similarities to PMS (Birkhoff et al., 2021; Mowat et al., 1998). 2.5.1.4. Abnormal skull shape, small body size and subfertility in Fleckvieh cattle Another condition, which was found in a polled Fleckvieh cattle bull and its progeny is as well segregating with a mutation in the second exon of ZEB2 (Gehrke et al., 2020a). The described polled condition is in association with a deformed skull, reduced height and subfertility, whereby also here a connection to the MOWS was drawn. A de novo 11-bp deletion in ZEB2 gene was found to be causative, and was first detectable in the affected polled Fleckvieh cattle bull, that was born to horned parents, which led to the exclusion from the classic dominant hornless variants (Gehrke et al., 2020a). 2.5.1.5. Type 2 Scurs Syndrome The autosomal dominant inherited type 2 scurs, just present in Charolais cattle, that segregate with a frameshift mutation in the TWIST1 gene (bovine chromosome 4), differ from the classical scurs (Capitan et al., 2011). What distinguishes type 2 scurs from classic ones is that in addition to horn abnormalities (loosely attached small or deformed appendices), skull interfrontal suture synostosis is also present. An elongated protrusion on the forehead is visible in affected animals, getting more prominent with age. It is considered that the condition has no severe consequences in heterozygous state. However, as no individual was observed that carries the underlying variant in a homozygous state, it is presumed to act as a lethal factor (Capitan et al., 2011). page | - 14 - 2.5.2. Genetic engineering A promising and relatively new approach to advance the breeding for polledness, especially in those species where polledness is associated with additional undesirable traits, is the use of genetic engineering. Public and research focus was drawn on these techniques when in 2012 the Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-Cas9 system, originally found in adaptive immunity in bacteria, was published, highlighting its potential for programmable genome editing (Jinek et al. 2012). Nevertheless, already before that attempts were made, editing of polled cattle using so called transcription activator-like effector nucleases (TALENs), was achieved (Tan et al., 2013). The result-oriented evaluation of genetic engineering by means of gene scissors led to a classification as classical genetic engineering by the European Court of Justice and was groundbreaking in the European Union (EuGH, 2018). Products produced in this way are therefore subject to the strict guidelines for genetically modified organisms, which regulate their release (European Parliament and the Council, 2001). The judgement tried to reflect the average public opinion. The public is highly skeptical of genetically modified foods, with country-specific differences in attitude (Frewer et al., 2013; Canavari and Nayga, 2009). Nevertheless, the methodology is applied, also in the field of farm animals. Especially polledness in cattle was already focused (Carlson et al., 2016), while it was not applied for polledness in small ruminants yet. 2.6. Aim of this study The aim of this work was to address genetic polledness in small ruminants, since the knowledge here is only rudimentary compared to the scientific knowledge for the same trait in cattle. This is despite the fact that the trait is associated with severe breeding impairments in goats due to the polled intersex syndrome (PIS). In this area, the aim of the work was to establish a practical genetic test for early detection of affected animals and thereby facilitate practical breeding for polledness in goats. Since the trait polledness in bovid species is highly diverse in many aspects, including the genetic basis, there is no review so far that compares this trait in the most important commercially used species cattle, sheep and goats. In order to provide such an up-to-date overview and comparison, the preparation of a review was another important goal of this work. The collection of current research results on the inheritance of horn-related traits shows that, especially in sheep, only little across-breed knowledge has been generated in recent years. However, many aspects showed a certain breed dependency. In order to lay the foundation for further research in this field, another aim of this work was to verify known variants and markers for traits related to horn status in the breed Icelandic sheep, as this breed is diverse, highly isolated and has never been analyzed for horn-related traits on the genetic basis in detail. page | - 15 - 3. ORIGINAL WORKS 3.1. First publication Simon, R.; Lischer, H. E. L.; Pieńkowska-Schelling, A.; Keller, I.; Häfliger, I. M.; Letko, A.; Schelling, C.; Lühken, G.; Drögemüller, C. (2020): New genomic features of the polled intersex syndrome variant in goats unraveled by long-read whole-genome sequencing. In: Animal Genetics 51 (3). DOI: 10.1111/age.12918. - Parts of this publication were presented as poster and talk at the 37th conference of the International Society for Animal Genetics (ISAG) 2019 and awarded a poster prize and a travel bursary. Additionally parts of this publication were presented as talk at the European Molecular Biology Laboratory (EMBL) Symposium: The Molecular Basis and Evaluation of Sexual Dimorphism (2020) and at the International Congress on the Breeding of Sheep and Goats (2020). - Contribution in: investigation, writing – original draft preparation, writing – review and editing, visualization page | - 16 - page | - 17 - page | - 18 - page | - 19 - page | - 20 - page | - 21 - page | - 22 - page | - 23 - page | - 24 - page | - 25 - page | - 26 - 3.2. Second publication Simon, R.; Drögemüller, C.; Lühken, G. (2022): The complex and diverse genetic architecture of the absence of horns (polledness) in domestic ruminants, including goats and sheep. In: Genes 13 (5). DOI: 10.3390/genes13050832. - Contribution in: conceptualization, formal analysis, writing—original draft preparation, writing—review and editing, visualization page | - 27 - page | - 28 - page | - 29 - page | - 30 - page | - 31 - page | - 32 - page | - 33 - page | - 34 - page | - 35 - page | - 36 - page | - 37 - page | - 38 - page | - 39 - page | - 40 - page | - 41 - page | - 42 - page | - 43 - 3.3. Third publication Simon, R; Elísabetardóttir, K.; Lühken, G. (2024): Analysis of genetic variants for different horn phenotypes and their inheritance in Icelandic sheep. In: Archieves Animal Breeding 67 (2). DOI: 10.5194/aab-67-237-2024 - Parts of this publication were presented as poster at the 39th conference of the International Society for Animal Genetics (ISAG) 2023. Contribution in: conceptualization, formal analysis, investigation, visualization, writing – original draft preparation, writing – review and editing page | - 44 - page | - 45 - page | - 46 - page | - 47 - page | - 48 - page | - 49 - page | - 50 - page | - 51 - page | - 52 - page | - 53 - page | - 54 - 4. DISCUSSION The genetic factors influencing various horn traits exhibit considerable variation across bovid species, but also within species. In the context of livestock keeping, polledness is a particularly interesting characteristic. Horned livestock can pose an increased risk of injury to both flockmates and handlers (Braun et al., 2016; Menke et al., 1999). Injuries among the animals can lead to economic losses, for example due to reduced milk quality and quantity (Mendonça et al., 2016; Youngers et al., 2017). From a broad perspective, there are three different approaches to address this issue. Recommendations for adjustments in management and the husbandry environment (Aschwanden et al., 2009, 2008; Hillmann et al., 2014; Loretz et al., 2004), which cannot be implemented universally for various reasons. The practice of mechanical dehorning / disbudding, as a second approach, is increasingly under criticism in the context of the intensified animal welfare debate. For example, the dehorning of goat kids is already completely prohibited by law in Germany (Deutscher Bundestag, 2006). The approach of genetic polledness and the associated breeding and selection in this area is considered an animal-friendly alternative. What is practically and largely unproblematic in the cattle sector (Prayaga, 2007) – the genetic basis is known for various cattle breeds and no severe associations are known – leads to problems in goat breeding. According to Asdell’s 1944 description, the connection between the dominant trait of polledness and the recessive trait of intersexuality in goats results in homozygous polled female goats being intersexual (also known as “polled-intersexes”), expressed in an variable phenotypic extent (Asdell, 1944). This inhomogeneity of the phenotype leads to difficulties in the early detection of affected animals in practice. Subsequently this leads to economic losses and reduced breeding success (infertility of polled-intersexes). The development of genetic testing based on the already published 11.7 kb-sized insertion on chromosome 1 (Pailhoux et al., 2001; Zhang et al., 2020) failed for European breeds. Although Zhang et al. (2020) reported that differentiation of all three possible genotypes for the PIS mutation was possible in their study. Using long-read whole genome sequencing, a much more complex variant was found that is linked to the already known insertion (Simon et al., 2020). This now known to be smaller ~ 10 kb-sized insertion, combined with an inversely inserted ~ 480 kb-sized duplicated segment of a region downstream of chromosome 1 has been confirmed several times for other breeds since this first publication (E et al., 2020; Guo et al., 2022). With reference to the new findings, not only the two genes PISRT1 and FOXL2, but also potassium inwardly rectifying channel subfamily J member 15 (KCNJ15) and ETS transcription factor ERG (ERG) are potentially influenced by the observed variant. Copy number variants in KCNJ15 were just recently shown to significantly correlate with growth traits in four out of five analysed Chinese goat breeds (Zhao et al. 2024). Furthermore, it has been shown that the published complex variant leads to an interchromosomal rearrangement and the formation of loop structures of chromosome 1 in the region of the FOXL2 gene (E et al., 2020). The assumption that this structural change leads to an altered expression of FOLX2 or neighboring genes still needs to be verified (E et al., 2020). The role of FOXL2 in mammalian sex page | - 55 - determination is undisputable (Migale et al., 2021) and it has already been shown that its loss of function leads to female-to-male sex reversal in goats (Boulanger et al., 2014). A recent gene expression study indicates that various pathways and thereby various physiological systems are involved in the development of intersexual goats (Han et al., 2022). However, it is important to note that the cause of intersexuality in those analyzed Huai goats (Han et al., 2022) has not been fully explained; As such, any direct link to PIS should be viewed with caution. However, looking at the polled/horned trait in goats independently, a recent publication investigating more than 300 genes showed an association in the comparison of whole genome data from 31 polled individuals (three breeds) and 15 horned individuals (one breed). Stromal interaction molecule 1 (STIM1) on chromosome 15 and neurexin 1 (NRXN1) on chromosome 11 were stated as possible candidate genes for the horned phenotype in goats (Wan et al., 2023). Information found on individual breeds in which the association between polledness and intersexuality is not supposed to occur could neither be confirmed by literature research nor, in the case of Maltese goats, by carrying out the genetic test developed by Simon et al. (2020). In general, the described variant is present in all polled individuals examined, but not in horned individuals. It can therefore be assumed that all breeds worldwide are equally affected by PIS, caused by the same complex variant, and the associated challenges influence breeding for polledness in this species. In cattle, the Celtic polled (Medugorac et al., 2012) variant has already been successfully integrated/inserted into a horned bulls fibroblasts genome by genome editing, thus producing polled offspring (Schuster et al., 2018; Schuster et al., 2020). Already ahead, attempts were successful which used other endonucleases, e.g. TALEN (Tan et al., 2013). No information is available on whether attempts have been made to use one of the known polled variants in one of the species to edit in the presence or absence of horn in another species. For instance, it could be tested if inserting the Celtic polled variant from cattle, ~200 bp-sized, into the genome of a horned goat leads to the expression of the desired trait without side effects (intersexuality) in the target species, for example using the clustered regularly interspaced short palindromic repeats (CRISPR) system (Jinek et al., 2012). Another interesting aspect in addition to generating polled goats without associated intersexuality, would be to investigate whether only one of the two combined variants (Simon et al., 2020) leads to polledness, and if so which one. For cattle, a similar study was recently published (Hennig et al., 2022). It was shown that the mere 10 bp deletion, which is replaced by a 212 bp duplication of a DNA segment in the Celtic variant, does not alone lead to polledness. Only the combination of deletion and duplication leads to the lack of horn bud development (Hennig et al., 2022). However, it remains debatable whether this genome editing methods, if successful, would have any practical use for breeding polled goats. European legislation at least classifies CRISPR technology as genetic engineering (EuGH, 2018), which makes its use in food producing animals challenging. In addition, consumer concerns must be seen as a limiting factor (Canavari and Nayga, 2009), even if the potential of genome editing remains undisputed (Wang and Doudna, 2023; van Eenennaam, 2019). In the field of gene-edited plants, there has page | - 56 - been a recent push to change European regulations (Vanderschuren et al., 2023; Nature Plants E, 2023). The European Parliament voted to ease the regulation of gene-edited crops, applying to changes that could also have been achieved by conventional breeding (European Parliament, 2024). However, by the new findings the development of a genetic testing for PIS was finally possible and thereby already offers valuable implications for the management and breeding of polled goats (Simon et al., 2020). It is important to note that intersexuality in goats can occur independently of polledness as well. XX/XY chimerism (freemartinism), as recently described by Paredes et al. (2024) in one of two case reports, is one of the causes (Paredes et al., 2024). Cases like these cannot be detected using the mentioned genetic testing. Comparing the current state of knowledge on the genetic factors of polledness in cattle, goats, and sheep reveals two observations (Simon et al., 2022). First, the variety of genetic factors involved for a trait (polledness) that hardly differs between the species phenotypically. Interestingly studies found that the two genes FOXL2 and RXFP2 seem to be the only ones, to a different extent, which are involved in polledness in all three species (Simon et al., 2022). The second conspicuous feature is the fact that there are still major gaps in knowledge and this applies to sheep in particular. After it was shown that the ~1.8 kb-sized RXFP2 variant published nearly a decade ago (Wiedemar and Drögemüller, 2015) does not segregate with polledness in several breeds with variable horn status (Lühken et al., 2016), no further variants or candidate genes could be found in sheep (Nicholas and Tammen, 1995). The influence of RXFP2 on the horn bud development in sheep was confirmed by a recent study, that furthermore identified few other genes, such as SFRP4 and WNT3, which are involved as well (Luan et al., 2023). However, not only the genetic factors of polledness in sheep still raise questions, but also the inheritance of this trait has not been clarified across the species (N.K. Pickering, P.L. Johnson, B. Auvray, K.G. Dodds, J.C. McEwan, 2009; Clutton-Brock and Pemberton, 2004; Johnston et al., 2011). Other horn associated traits such as size, shape or number are highly variable in sheep as well. Associated variants or markers are often only described for individual breeds (Johnston et al., 2010; Pan et al., 2018; Duijvesteijn et al., 2018). To account such observed or expected breed differences the scientific consideration of new breeds can be helpful (Marshall, 1994; Salonen et al., 2019; Aldersey et al., 2020). One breed in which both polledness and variations in horn size and shape, as well as the characteristics of scurs and polyceraty occur are Icelandic sheep (Dýrmundsson and Niżnikowski, 2010). Detailed records about phenotypes and relationship, as the basis for animal breeding and genetic research (Seidel et al., 2020), are available through the breeders. These facts and the intensive isolation of the breed due to import restrictions caused by eradication programs in recent decades and the isolated location of Iceland make it an interesting breed to verify the findings published so far. The recent study of Simon et al. (2024) therefore fulfilled the first step to include the Icelandic sheep breed into the research field of horn associated traits (Simon et al., 2024). The results reflect the diversity of the characteristics associated with horns in sheep and the variability of previous publications and results (in the sense of not being generally valid). The 1.78 kb-sized RXFP2 variant associated with polledness was found in the population, but, as expected, there page | - 57 - was no perfect segregation with the horn status (absence / presence of horns) (Simon et al., 2024). It is possible that there is a similarity to cattle (Medugorac et al., 2012; Medugorac et al., 2017), in which different variants are known to be independently associated with the same trait polledness. Which breeds could cluster and on which basis, however, remains unclear in sheep and offers potential for further research. The same might apply for scurs in sheep, in which the association with the described RXFP2 variant was week (Simon et al., 2024). Observed tendencies of an influence of RXFP2 on horn size (Pan et al., 2018), which was also visible in Icelandic Sheep (Simon et al., 2024) once again indicates the great influence of this gene on horn-associated traits in sheep. In addition, just recently it was published that a RXFP2 haplotype, associated with spiral horn form in the wild species, was passed on from Iranian mouflons into different sheep breeds worldwide and contributed to their morphological differentiation (Cheng et al., 2023). A link between horn shape and another, already published marker in the RXFP2 gene (Pan et al., 2018) could not be confirmed in the analyzed Icelandic sheep (Simon et al., 2024). However, it is difficult to compare horn shapes of different breeds based on pictures. The standardized measurement of specific characteristics, such as the perimeter of the horn at the base of the horn, taking into account the age of the animal, can help to increase comparability, but at the same time complicate data collection in the field. Two completely new aspects that are connected with polyceraty and the associated 4 bp deletion in HOXD1 (Allais-Bonnet et al., 2021; Zhang et al., 2023) were also observed in the Icelandic sheep (Simon et al., 2024). The described variant was, in contrast to all analysed breeds until now, also present in phenotypically polled individuals deriving from polycerate families. In addition, it is apparent that polledness in these individuals is not controlled by the RXFP2 insertion (Simon et al., 2024). It would be important to know whether this applies to a larger sample set of Icelandic sheep, as well as other polycerate breeds. A closer look at these phenotypically polled animals with polycerate origin could also be a new starting point for further identification of additional variants associated with polledness in sheep. Literature review revealed that this has not been focused yet. There was no difference in the occurrence of HOXD1 variant between four- and six-horned sheep. Whether a further, possibly linked but yet unknown variant codes for the final number of horns is still unknown. However, it seems certain that the "supernumerous" horns also always occur in pairs. The breeders or owners were able to confirm that the horns had grown together in all cases where an uneven number of horns was initially observed (Figure 4). Taken together, the results once again confirm the assumption of a strong breed influence and the important role of the RXFP2 gene with regard to the investigated horn-associated traits in sheep, excluding polyceraty. page | - 58 - Figure 4: Polycerate icelandic ewe. Please note that the two left horns fused (red arrow) while the right ones are clearly separated in two horns (picture: María Fríðgerður Bjarnadóttir). 4.1. Future studies – outlook As still a lot of aspects of the topic horns / absence of horns remain unknown, this leaves space for further research particularly in sheep and goats. In the light of new techniques or their advancements there is a chance to solve long persisting problems, like shown in the current work for PIS. Even if PIS is already known since the 1940`s and it has been widely reported that the resulting phenotype is highly variable, the effects on the hormonal status of affected individuals, the exact phenotypic expressions in female homozygous hornless animals, and the influence of PIS on fertility in male goats remain largely unexplored. Necessary studies should aim to answer long unanswered questions, primarily whether female homozygous polled goats are sterile in every case and if so, if there is a way to solve that problem. 4.2. Conclusion With the findings of this work it was possible to proof previous publications and extend the knowledge in the field of genetic factors in horn status trait. With this, the findings especially on the polled intersex syndrome and the variant for polyceraty in Icelandic sheep highlighted new aspects and laid the groundwork for subsequent investigations to unravel the underlying genetic mechanisms of horn traits in small ruminants. Once again the findings proved the multifaceted nature of the genetic architecture governing these traits, especially in sheep. Updated information were summarized about polledness in sheep and goats including cattle, which before was often seen apart from each other. The work thereby offers valuable implications for a better understanding of genetic factors influencing horn status traits in bovidae and contributes an up to date summary of the body of knowledge in the field of genetic polledness. 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