Genetic Variability in Lithuanian Black and White Cattle with Different Proportions of Holstein Bloodline

Effective management of animal genetic diversity has become an important issue in cattle mating programs (Kukuckova et al., 2017). Modern livestock programs can be implemented to control inbreeding in the next generation and minimize genetic relationships between selected individuals (Weigel, 2001). Furthermore, correct information about cattle pedigree is fundamental for the appropriate development of breed and selection programs to improve productivity in farming.

The Holstein cattle breed is  characterized by a shorter reproduction time and produces a high amount of milk (Coffey et al., 2016; Pishchan et al., 2021). However, despite the obvious advantages, the Holstein cattle breed represents a significant threat to biological diversity worldwide due to its extensive crossbreeding with other usually local breeds (Lee and Shin, 2018). It has been established that interbreeding of Holstein cattle with other dairy cows can increase their economic efficiency due to gene variety expansion and a decrease in the cows’ stress and depression levels (Begley et al., 2010).

The Lithuanian Black and White is an old native dairy cattle breed that makes up 70% of all cattle in Lithuania (Prusevičius, 2014). This cattle breed is part of Lithuania’s cultural heritage and is exclusive to Lithuania. Like many other breeds, Lithuanian Black and White cattle have a close genetic relationship with the Holstein breed. Since 1973, Lithuanian Black and White cattle have been intensively improved using American and Canadian Holsteins, as well as Danish and German Black and White and British and Holstein Friesians (Baltrėnaitė et al., 2003).

In Lithuania, few studies have investigated genetic diversity and genetic relationships in cattle breeds (Peciulaitiene et al., 2007; Šveistienė and Jatkauskienė, 2008; Lapickis et al., 2021). Characterization of breeds is increasingly important in the context of assessments of genetic diversity and conservation of genetic resources (FAO, 2011). Thus, the objective of the present study was to determine the genetic variability of Lithuanian Black and White and Holstein cattle breeds by the percentage presence of bloodlines.

The present study was conducted during 2019-2020 at the Biology department of Vytautas Magnus university. Semen or ear tissue samples (n = 126) were taken from native cattle breeds in Lithuania (Marijampolė district): Lithuanian Black and White (BW, n = 121) plus Holsteins (HL, n = 5) included in the study for reference purposes. All the samples were collected from several agricultural companies and data about the level of Holstein bloodline were provided by the Lithuanian Black and White Cattle Improvers Association. Lithuanian BW cattle were divided into five groups by their level of Holstein bloodline: individuals with more than 93.75%, individuals with 83-90%, individuals with 71-78%, individuals with 62-70% and individuals with less than 50%. Plus, five pure-blooded HL individuals (names “BREWMASTER”, “DICKEY”, “EPIC”, “FLAX”, “KESSLER-ET”) and one pure-blooded Lithuanian BW cow (name “IKRAS”).

Genomic DNA from tissue samples was extracted using the Thermo Scientific Genomic DNA Purification Kit (Thermo Scientific, Lithuania), following the manufacturer’s instructions. Sixteen microsatellites were selected from the panel recommended by the Food and Agriculture Organization and the International Society for Animal Genetics for genetic characterization of cattle breeds (Table 1) (FAO, 2011).



Microsatellites were amplified in three multiplex PCRs (3 to 4 co-amplified loci). Each PCR was performed in a 15 μL reaction mixture containing 1 μL of each primer pair, 7.5 μL 2xPCRMix (Thermo Scientific, Lithuania) and 2 μL each of DNA and H₂O. Two separate reactions with different temperature requirements were used-PCR program I: denaturation at 95°C for 5 min, 35 passes of 95°C for 45 s, 50-60°C for 45 s, 72°C for 30 s and an extension 72°C for 10 min; PCR program II: 95°C for 15 min, 3 passes of 84°C for 2 min, 54°C for 20 s, 72°C for 10 s, then 35 passes of 94 °C for 15 s, 54°C for 20 s, 72°C for 10 s, followed by a final extension at 72°C for 30 min. Amplified DNA fragments were mixed with formamide and GeneScan™ 500 LIZ size standard (Applied Biosystems, USA) and then loaded in the ABI 3100 Genetic Analyzer (Applied Biosystems, USA) for capillary electrophoresis. Allele size was assigned using GeneMapper v4.0 software (Applied Biosystems, USA).

GeneAlEx 6.501 was used to estimate allele frequencies, the total number of alleles, the number of different private and dominant alleles. The principal coordinate analysis (PCoA) was performed to identify structure in the distribution of genetic variation. Nei’s genetic distances and FST values were used to identify genetic differentiation between different cattle breeds (Peakall and Smouse, 2012; Nei, 1973).

A total of 126 individuals’ samples were successfully genotyped at 13 microsatellite loci and altogether 251 alleles were detected (Table 2). Three loci (AGLA232, BM1824, RT29), either failed to amplify or the amplified alleles were difficult to score. The number of different alleles ranged from 8 (BOVIRP) to 29 (BM2830 and TGLA122). The BW cattle group with <50 % Holstein bloodline had the largest number of alleles with 49 (Table 2). The presence of dominant alleles was also abundant, with 36 different dominant alleles appearing in 13 microsatellite loci (Table 2). The number of different dominant alleles per locus ranged from 1 (BOVIRP) to 5 (BM2830). The BW cattle group with <50% Holstein bloodline had the highest number of dominant alleles (Table 2). No private alleles were identified in two loci (BOVFSH and TGLA53) in the HL group.



Several of the loci used in this work have been analyzed in previous studies with different breeds of European cattle breeds bred in Lithuania: Danish Red, Swedish Red, German Black and White and German Red (Makstutienė et al., 2013). In Australia, HL cattle were investigated and found that they had an impact on other breeds over the years (Zenger et al., 2007). The study states that the level of individual breeding is increasing but does not yet have a significant effect on genetic differentiation. However, the level of estimated inbreeding is increasing and becoming a serious problem. In addition, it may change due to environmental impacts, local selection, genetic drifting and isolation. Similar accounts about the genetic variability of different cattle breeds have been reported in Senegal, Pakistan and Korea (Suh et al., 2014; Ndiaye et al., 2015; Hussain et al., 2017).

Private alleles were found in all the cattle groups except for the BW cattle group with 83-90% Holstein bloodline (Table 3). The HL had the highest number of private alleles at 10, followed by BW with >93.75% Holstein bloodline at 8. Special consideration should be given to 140 bp (RT9) and 150 bp (BM723) alleles, which only occurred in one BW cattle group (<50% Holstein bloodline) with a high frequency of 1.00 and 0.50 respectively, while the other private alleles occurred with a low frequency of less than 0.10-0.25. These results highlight the relevance of these markers for characterizing cattle breeds and potentially improving efficiencies in breeding programs.



Principal coordinates analysis (PCoA) revealed that the HL group was most distinct from the other groups as well as the individual of pure-blooded Lithuanian BW (Fig 1). As expected, the PCoA in the present study revealed that the HL cattle breed is genetically distant from the other cattle breed groups. There was a significant difference, which was clearly visually detectable. This is in complete agreement with the factorial correspondence analysis of individual cattle microsatellite genotypes calculated using GENETIX in the study by Suh et al., (2014) where the HL cattle breed is undoubtedly also separate.



The greatest Nei’s genetic distance (0.359) was observed between pure-blooded HL group and BW cattle group with less than 93.75% Holstein blood, nevertheless the lowest (0.132) was between BW groups with less than 50% Holstein blood and 71-78% Holstein blood (Table 4). The fixation index (FST) values ranged from 0.002 to 0.257 among different groups of cattle, indicating mixed levels (from low to high) of genetic differentiation (Table 4). The highest value was between the pure HL group and the BW cattle group with less than a 93.75% Holstein bloodline.



Many of the loci used in this study have been used for different breeds, such as native Korean breeds, Senegal’s Gobra zebu, Pakistan’s zebu, Indian cattle and native China breeds where investigations used the HL breed as comparative genetic material (Suh et al., 2014; Ndiaye et al., 2015; Gupta et al., 2016; Hussain et al., 2017; Ni et al., 2018). The present findings would seem to show that Nei’s statistic ranged from 0.132 to 0.359, which is very similar to that of Suh et al., (2014) who evaluated a range of 0.129 to 0.316. Further analysis showed FST values from 0 to 0.257 that correlate favorably with Hussain et al., (2017) (0.02445-0.22009) and Svishcheva et al., (2020) (0.0608-0.0955). Interestingly, the observation originated from the data comparison in many cattle breed microsatellites, repeating the tendency of high allelic richness and high accuracy of dominant and rare alleles (Makstutienė et al., 2013; Suh et al., 2014; Ndiaye et al., 2015; Hussain et al., 2017; Svishcheva et al., 2020). This may occur due to the high polymorphism level per locus (Radko, 2008). Generally, rare alleles in large populations occur because of random mutations and are distributed in the individuals in a heterogenic form. In small populations, rare alleles are distributed by possible mating between related individuals or crossbreeding (Hale et al., 2012).

Another reason for the appearance of random alleles is the decrease in genetic diversity that over time leads to the loss of specific genes that define the breed or are found in a very low frequency. This phenomenon is noticeable when the effective population size falls significantly. In addition, scientists have observed that the offspring kept in one herd are phenotypically similar, but genetically different (Lithuanian Black and White Cattle Improvers, 2011). The high number of rare alleles detected could include genetic drift or migration from population to population. The finding of the unique alleles in the individuals allows the assumption that these alleles survive as adaptive genes that could be significant in future usage in animal husbandry. DNA sequencing polymorphism studies provide tools to help decide which livestock populations should be targeted and which should be used to identify the DNA regions of unusual productivity and signs of wellness. This information will be used directly in future genetic improvement programs for cattle.

The results of this study showed that the genetic distance between Lithuania’s Black and White cattle individuals and the pure-blooded Holstein individuals was high. The Lithuanian Black and White cattle breed is part of Lithuania’s cultural heritage and is exclusive to Lithuania. Maintaining genetic diversity should be one of the major objectives of breeding programs, especially in native cattle breeds. Genetic marker information is an excellent alternative to pedigree data and could be used to reduce the chance of inbreeding and conserve genetic diversity in populations.

The authors would like to thank the Lithuanian Black and White Cattle Improvers Association for providing semen and ear tissue samples. This research was funded by the National Paying Agency under the Ministry of Agriculture of the Republic of Lithuania via the Genetic Analysis of Lithuanian Black and White, Holsteins, Lithuanian Red and Lithuanian Red and White cattle breeds project, grant number MT-19-16.

All authors declared that there is no conflict of interest.