Sport horse breeding is focused on the improvement of the genetic ability of warmblood riding horses for performance in competitions. In open subpopulations, an extensive exchange of the breeding stock between breeding organizations has been going on for decades. Significant improvements in reproduction techniques (e.g. chilled and frozen semen) have further facilitated the simultaneous use of breeding stallions in multiple countries and a large exchange of genetic material as a consequence (Koenen et al., 2004). The liberalization of European animal breeding legislation and an increasing diversity of equestrian sports have led to a constant rise in the number of horse breeds and breed registries. In addition, there is a trend towards an international expansion of the bigger established sport horse breeds (Aurich and Aurich, 2006). In this way, the importation of foreign genetic material (from foreign sport horse breeds) is present in open populations and gives the breeders the opportunity to choose foreign stallions that best contribute to the improvement of their horses' performance, as regards their physical, physiological, and/or genealogical characteristics (Bartolomé et al., 2011).

Still, there are some sufficiently closed studbooks which aim to preserve genetic resources (Zechner et al., 2002; Pjontek et al., 2012; Cosenza et al., 2019). In closed populations, genetic progress is slower, and it is difficult to obtain high-performance offspring while maintaining the genetic structure and diversity within the breed. Considering sport horse breeds, the Trakehner (TRAK) studbook represents a predominantly closed breeding system based on purebreeding (Trakehner Verband, 2025), whereas most contemporary sport horse breeding programmes operate as open studbooks, facilitating continuous genetic exchange across populations (Doyle et al., 2022). The TRAK breed is one of the oldest riding horse breeds and has long been acknowledged as an original breed, even internationally. Genetically, the breed can be traced back directly to the foundation of the Main Stud in Trakehnen (East Prussia) by a royal decree of the Prussian King in 1732. TRAK are a riding horse breed bred in accordance with the principles of pure breeding, with a high genetic proportion of English and Arab thoroughbred, Shagya, and Anglo-Arab, in keeping with the selection criteria established for the breed (Trakehner Verband, 2022). In other sport horse breeding programmes, TRAK stallions are commonly used for crossbreeding mares (Schröder et al., 2010; Roos et al., 2015; Oldenburger Pferdezuchtverband, 2022). In Lithuania TRAK were used for cavalry and sport (Krikščiūnas and Skaisgiris, 1939). Nowadays, according to the data of the Lithuanian Farm Animal Register, the total population of TRAK in Lithuania decreased by 25 % from 2014 to 2021.

Historical records indicate that the foundation of the Trakehnen Stud involved horses from studs located in Lithuania Minor (East Prussia), such as the Sereitlaukis estate (Račkauskaitė et al., 2021). Therefore, it could be assumed that local Lithuanian horses, including the Zemaitukai (ZEM), could have contributed to the early formation of the TRAK breed. ZEM horses have been known since the 6th–7th centuries (Gleß, 1989). Until the middle of the 19th century, the breed was dominant in the country. However, more intensive agricultural development has led to crossbreeding and dangerous declines of the population. After World War II in the first Lithuanian state studbook (1948), barely 17 purebred ZEM were entered; in 1994, only 30 horses were left (Macijauskienė et al., 2009). To restore the population, the studbook was opened, and three stallions of historically related breeds (Lithuanian large type Zemaitukai, Estonian native, and Arab) were used for a very low number of mares (Macijauskienė, 2009). The population of ZEM horses has increased, but the breed is still classified as endangered.

The Arabian (ARAB) horse is another breed of closed populations and one of the world's oldest breeds of any domesticated animals (Cosgrove et al., 2020), but the history of the breed's modern populations, described in pedigree records, is no longer than 200 years (Głażewska, 2010). The first European studs in Poland, Germany, and Hungary were established in the early nineteenth century. In Lithuania, ARAB were already used for breeding in 1937 (Krikščiūnas and Skaisgiris, 1939). Lithuania became a member of the World Arab Horse Organization (WAHO) in 1994. In 2007, 65 ARAB horses were already kept in the breeding nucleus of the national stud (Barisevičius et al., 2007). The breed is listed as transboundary (FAO, 2022).

In 1999, the development of a promising horse population suitable for all classical equestrian disciplines was initiated in Lithuania. This population was maintained as an open breeding structure, allowing local sport horse mares and stallions from various foreign breeds to be included. By 2007, six genetically dominant breed groups were identified within the population based on genotype composition: Hanoverian, TRAK, Holsteiner, Thoroughbred, Budyonny, ARAB, and others (Šveistienė, 2007). According to the World Breeding Federation for Sport Horses (WBFSH), 10 domestic and 30 foreign-approved stallions were used in the breeding programme of the Lithuanian Riding Horse in 2020 (WBFSH, 2021). In 2024, the breed was officially renamed to Lithuanian Warmblood horse (LW) to reflect its composition and alignment with international sport horse classification.

In 2000, the development of the second riding horse population was launched, and the studbook of the Baltic Hanoverian horse was established. The foundation of the Baltic Hanoverian population was a cross between Hanoverian and other sport horses. Hanoverians, TRAK, and Thoroughbreds were the main breeds of light-riding horses between World Wars I and II in Lithuania (Krikščiūnas and Skaisgiris, 1939). The national stud in Zagare became the basis for Hanoverian horse breeding for several decades. In 2022, the name of the studbook was changed to Baltic Warmblood (BW), and currently many other international breeds are used. The studbook registry of the breed is administered in accordance with the rules of WBFSH.

Characterization of animal genetic resources is essential for informed decision-making in conservation and breeding programmes (FAO, 2007; Kierkegaard et al., 2020). In horse populations, pedigree data remain a valuable tool for assessing genetic structure and variability, particularly when molecular information is limited or unavailable. Pedigree-based approaches have been widely applied to analyse genetic diversity, track founder contributions, and assess inbreeding trends, thereby providing insight into the effects of selection, breed formation, and population openness (Duru, 2017; Bokor et al., 2022; Próchniak et al., 2024). In Lithuania, pedigree analyses were complemented by immunogenetic tools, including blood group and protein polymorphism typing, which were introduced in 1985 and applied in breeding control and the characterization of local breeds (Krikščiūnas, 1990). Although these methods are no longer routinely used in many countries, the standardized and long-term dataset they produced allows for the evaluation of genetic relationships and intra- and inter-population diversity. Moreover, these data may serve as a valuable reference for future molecular studies, especially in native and regional horse populations where genomic data remain limited.

Genetic variability under tightly controlled breeding in a declining population is essential. This study aimed to characterize the genetic variability of the TRAK population and its relationships with other horse populations in Lithuania by applying pedigree and parentage verification data. The results provide important insights into the effects of breed formation history and breeding practices on genetic structure, supporting informed decisions in the conservation and sustainable use of equine genetic resources.

One of the fundamental aims in conservation genetics is to preserve genetic variability within populations, as it is generally assumed to be positively correlated with population viability. In the management of livestock populations, this entails minimizing the loss of genetic variation due to inbreeding and genetic drift while also preventing undesired gene flow between different breeds (Thirstrup et al., 2008; Frankham, 2010). For breeds maintained as small, closed populations, such as TRAK and ZEM, within-population diversity conservation is particularly relevant.

Our results revealed that estimates of Ne depend heavily on the method used and the population characteristics. In TRAK, Ne calculated via the number of reproducing animals was even lower than in the restored ZEM breed. However, Ne estimated via the rate of inbreeding was dramatically low in ZEM, reflecting strong inbreeding pressure. This outcome is likely due to the high proportion of inbred individuals in the ZEM population and supports earlier findings that Ne estimates are highly sensitive to pedigree completeness, mating patterns, and population structure (Leroy et al., 2013; Bokor et al., 2022). Similar patterns have been reported in endangered horse populations analysed using pedigree-based approaches, such as the Hucul and Cleveland Bay breeds, where Ne estimates based on inbreeding rates were substantially lower and revealed stronger inbreeding pressure compared to estimates based on the number of breeding animals (Somogyvári et al., 2018; Dell et al., 2020). This is further supported by studies combining pedigree and genomic approaches, which have shown that pedigree-based estimates may underestimate inbreeding and effective population size, particularly in long-established closed populations affected by bottlenecks and hidden founder contributions (Poyato-Bonilla et al., 2022). Open populations, such as BW and LW, had high Ne values based on breeding animal counts. However, these figures decreased significantly when estimated via ΔF, with reductions of over 60 %, highlighting unbalanced parentage and hidden relatedness. This underlines the importance of using complementary metrics, as emphasized by Martyniuk et al. (2010), to gain an accurate understanding of breed endangerment beyond FAO's basic Ne thresholds.

The relatively high Ne of TRAK (via breeding animals) and moderate inbreeding level (F= 0.0373) suggest that current breeding strategies are effective in maintaining genetic diversity despite a 25 % population decline. Conservation programmes for such populations should prioritize not only the number of reproducers but also balanced parental contributions, minimization of related matings, and elongated generation intervals to slow genetic erosion (Caballero, 2020; Zechner et al., 2002). An illustrative example of the benefits of coordinated breeding is the case of the endangered Cleveland Bay, where structured mate allocation using SPARKS software increased Ne from less than 50 to over 170 in under 2 decades (Dell et al., 2020).

The average generation interval of 12 years in TRAK supports a conservative breeding pattern, being comparable to other Lithuanian sport horse breeds and slightly higher than that of German TRAK (10 years; Teegen et al., 2009). Longer generation intervals are generally associated with slower rates of genetic change and reduced accumulation of inbreeding, which may contribute to maintaining genetic diversity in structured breeding populations (Caballero, 2020). This may help stabilize the genetic structure of the population and reduce generational drift.

The analysis of the genetic data provided additional insights into population differentiation. Similar approaches based on blood group markers have been successfully applied in endangered horse populations, such as the Hucul breed, where immunogenetic analyses revealed considerable allelic diversity and provided a basis for understanding population structure and guiding breeding strategies (Popadiuk, 2019). Importantly, the analysis included all available genotyped horses and a subset representing active breeding animals.

In TRAK, both datasets showed high allelic richness (Na= 3.833 and 3.417, respectively), and observed heterozygosity increased in the reference group (Ho from 0.460 to 0.488). This pattern indicates successful retention – or even improvement – of genetic diversity among active reproducers, despite the constraints of a partly closed studbook. Similar trends were observed in ZEM, where Ho and He also improved compared to earlier reports (Juras and Cothran, 2005), suggesting that the implementation of structured conservation breeding is having a positive effect.

BW and LW populations exhibited the highest heterozygosity across both datasets, in line with their open breeding structure and frequent introduction of imported stallions. However, the slight decline in Ae in reference groups hints at potential erosion of rare alleles under stabilized selection pressure. Petersen et al. (2013) similarly observed that open studbook breeds generally display higher heterozygosity but may undergo narrowing of genetic bases over time due to directional selection.

In contrast, ARAB horses showed consistently low heterozygosity and a narrow genetic base, with minimal differences between full and reference datasets. Although the total number of alleles was similar to other populations, ARAB had the highest proportion of rare alleles and lacked some variants observed in other breeds. These findings align with previous research on genetically uniform, closed populations and highlight the importance of long-term breeding coordination (Zechner et al., 2002).

Nei's genetic distances and Fst values, calculated for both datasets, revealed stable patterns of interpopulation relationships. TRAK showed the closest proximity to BW and LW, reflecting its historical influence on Lithuanian sport horse breeding. Meanwhile, the largest genetic distance was observed between TRAK and ZEM, as expected for historically and functionally distinct populations. The moderate but increasing differentiation between TRAK and ARAB (Fst 0.015 to 0.021) in the reference dataset suggests that structured breeding in recent years is strengthening internal breed cohesion.

The assignment test further confirmed these patterns: 57.9 % of all TRAK horses were correctly assigned to their population, while this figure increased to 62.1 % in the reference group, indicating greater genetic consistency among actively bred individuals. This pattern was even more pronounced in ARAB and ZEM, where 83 % and 89 % of individuals, respectively, were assigned correctly in both datasets, consistent with their closed genetic histories and minimal admixture.

Multivariate analyses (PCA and clustering) supported the relationships described above. In the full dataset, TRAK, BW, and LW overlapped substantially, while in the reference group, population boundaries became more distinct. TRAK formed a more compact and centralized cluster, indicating increasing within-population cohesion. Heatmap analysis placed the reference populations of BW and LW as the closest relatives, with TRAK closely associated within the same cluster – consistent with historical data indicating that TRAK horses formed a significant genetic base for the Lithuanian Warmblood studbook (Šveistienė, 2007). This clustering pattern was particularly evident in the reference groups, supporting the idea that recent breeding practices have maintained historical genetic links.

The maintenance of genetic diversity within TRAK, despite its closed structure, is likely supported by the permitted inclusion of Thoroughbred and Arabian ancestry, as allowed by breeding standards (Nolte et al., 2019). This partly explains the moderate genetic proximity observed between TRAK and ARAB in our study. In contrast, native breeds such as ZEM do not benefit from this kind of gene flow, which places greater responsibility on internal diversity management.

Breeding systems and studbook structures have a profound impact on genetic diversity, influencing both within-breed variability and between-breed differentiation (Curik et al., 2017). Partly closed breeding systems, such as the TRAK studbook, are effective in maintaining breed identity but may contribute to genetic erosion over time (Leroy et al., 2013). Conversely, open systems, like those of LW and BW, enable greater genetic input from diverse sources, yet they pose challenges related to managing inbreeding and maintaining structured selection schemes (Pjontek et al., 2012). These results highlight the importance of regular genetic monitoring and tailored breeding strategies to ensure sustainable population management. The integration of genomic tools is essential for more accurate characterization of genetic architecture, controlling inbreeding and optimizing selection decisions (Yaro et al., 2017). Therefore, combining pedigree information with molecular data can support both conservation and performance goals in national horse breeding programmes.

Despite the limited population size, the Trakehner horse population in Lithuania demonstrated a favourable level of genetic diversity and heterozygosity. The genetic relationships observed between Trakehner and other horse populations (particularly BW and LW) are consistent with historical patterns of breed development and reflect recent gene flow. However, the observed population decline highlights the need for well-planned genetic management to prevent further erosion of genetic variation and to safeguard biodiversity.

This study underscores the importance of continued monitoring and strategic breeding efforts. While traditional selection practices have contributed to the current population structure, they may also pose risks to genetic diversity in the long term. Incorporating genomic selection tools could help to mitigate these effects by maintaining or even enhancing genetic variability. Strategic genetic management, including the use of genomic tools, should be considered an important component in efforts to preserve genetic variation and ensure the long-term sustainability of horse breeds in Lithuania.

Importantly, from 2025, national support has been introduced in Lithuania for genetic testing and parentage verification of breeding horses. This initiative is expected to facilitate the wider application of molecular tools, including microsatellite- and SNP-based analyses, in breeding programmes. The increasing availability of such data will provide a valuable foundation for more precise genetic evaluation, improved pedigree verification, and more effective long-term management of genetic diversity in Lithuanian horse populations.