*Article* **Prevalence of Sex-Related Chromosomal Abnormalities in a Large Cohort of Spanish Purebred Horses**

**Sebastián Demyda-Peyrás 1,2,\*, Nora Laseca 3, Gabriel Anaya 3, Barbara Kij-Mitka 4, Antonio Molina 3, Ayelén Karlau 1,2 and Mercedes Valera <sup>5</sup>**


**Simple Summary:** Horses are well known for the increased number of individuals carrying chromosomal abnormalities related to the sex pair, which have been identified as a major cause of idiopathic infertility. However, large-scale populational studies evaluating the occurrence of these chromosomal aberrations in commercial or wild populations are extremely scarce. We, therefore, performed a cytogenetic analysis on a large dataset of 25,237 individuals, gathered over a period of 24 months, using a two-step genomic-based diagnostic methodology. We first screened the entire population, analyzing the results of short tandem repeats parentage testing to determine individuals showing abnormal results. Thereafter, the positive samples, together with the individuals showing morphological abnormalities in the reproductive tract, were reanalyzed using a single nucleotide polymorphism (SNP)-based procedure to determine the occurrence of chromosomal abnormalities. Our results showed that the overall prevalence of individuals carrying chromosomal alterations was close to 0.05%, with blood chimerism and 64,XY sex-reversed mares the most common type of aberrations detected. In addition, one case of Turner and one of Klinefelter syndrome, as well as a small number of individuals carrying complex karyotypes, were also detected. However, these results should be taken with caution since the occurrence of X chromosome monosomy, a sex-related chromosomal aberration commonly reported in mares, cannot be screened using the methodology employed in this study. To our knowledge, this is the largest study performed aimed at determining the prevalence of the most important chromosomal abnormalities in the domestic horse.

**Abstract:** Chromosomal abnormalities are largely associated with fertility impairments in the domestic horse. To date, over 600 cases of individuals carrying abnormal chromosome complements have been reported, making the domestic horse the species with the highest prevalence. However, studies analyzing the prevalence of chromosomal diseases in whole populations are scarce. We, therefore, employed a two-step molecular tool to screen and diagnose chromosomal abnormalities in a large population of 25,237 Pura Raza Español horses. Individuals were first screened using short tandem repeats parentage testing results and phenotypic evaluations. Those animals showing results suggesting chromosomal abnormalities were re-tested using a single nucleotide polymorphism (SNP)-based diagnostic methodology to accurately determine the chromosomal complements. Thirteen individuals showed a positive screening, all of which were diagnosed as chromosomally abnormal, including five 64,XY mares with sex development disorders (DSD) and four cases of blood chimerism (two male/female and two female/female cases). In addition, we detected one Turner and one Klinefelter syndrome and two individuals carrying complex karyotypes. The overall prevalence in the entire population was ~0.05%, with the prevalence of 64,XY DSD and blood chimerism ~0.02% and ~0.016%, respectively. However, the overall results should be taken with caution since the individuals carrying Turner syndrome (in full (63,X) or mosaic (mos 63,X/64,XX) forms) cannot be

**Citation:** Demyda-Peyrás, S.; Laseca, N.; Anaya, G.; Kij-Mitka, B.; Molina, A.; Karlau, A.; Valera, M. Prevalence of Sex-Related Chromosomal Abnormalities in a Large Cohort of Spanish Purebred Horses. *Animals* **2023**, *13*, 539. https://doi.org/10.3390/ ani13030539

Academic Editors: Leopoldo Iannuzzi and Pietro Parma

Received: 2 January 2023 Revised: 27 January 2023 Accepted: 28 January 2023 Published: 3 February 2023

**Copyright:** © 2023 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

detected due to limitations in the methodology employed. Finally, the lack of agreement between populational studies performed using karyotyping or molecular methods is discussed. To our knowledge, this is the largest populational study performed evaluating the prevalence of the most common chromosomal abnormalities in the domestic horse.

**Keywords:** chromosomal abnormalities; horse; genomic; prevalence; SNP-array; cytogenetics

#### **1. Introduction**

Chromosomal abnormalities related to the sex pair are a common genetic disease in domestic horses, *Equus caballus*. This knowledge is not new [1] but was demonstrated 30 years ago by Power [2], who compiled the karyotyping results of nearly 400 cases showing chromosomal aberrations. More recently, Bugno-Poniewierska and Raudsepp [3] established that chromosomal disorders are among the most common non-infectious causes of subfertility, infertility, and congenital defects in the species, accounting for ~30% of horses with reproductive or developmental problems. However, cytogenetic analysis in such individuals is not a common practice and is far from being a systematic practice in large populations and/or commercial herds [4]. This lack of testing may make it difficult to determine the prevalence of chromosomal abnormalities in horses, especially since some individuals carrying chromosomal aberrations may be phenotypically normal, thus avoiding detection [5].

The most comprehensive and largest cytogenetic evaluation of a horse population existing to date was performed 15 years ago by Bugno, et al. [6], who reported a prevalence of chromosomal abnormalities related to the sex pair close to 2% by karyotyping 500 horses selected randomly. In contrast, Kakoi, et al. [7] conducted a large-scale analysis of 17,471 newborn light-breed foals in Japan using high-throughput molecular methods (instead of karyotyping) and found a much lower prevalence of chromosomal abnormalities, approximately 0.01%. Similarly, Anaya, et al. [8] used molecular methods to detect blood chimerism in 21,097 Pura Raza Español (PRE) horses, reporting a prevalence of 0.01%, which is 20 times lower than the previous existing reports. Given the discrepancy existing between results obtained using different methodologies, any additional data produced will help us to determine a more accurate rate of chromosomal abnormalities in the domestic horse.

Thirty years ago, Bowling, et al. [9] demonstrated the usefulness of short tandem repeats (STR) genotyping to detect chimerism in horses. More recently, this approach was further employed to detect the same type of chromosomal abnormality in an American Bashkir Curly [10] and several Pura Raza Español horses [8,11]. However, the standardized STR panel employed for parentage testing in the species includes only one ECAX marker and none located in the ECAY. Therefore, most of the aberrations associated with reproductive impairments, such as sex reversions (DSD) or ECAX monosomy [5], cannot be detected. To solve this issue, Kakoi, et al. [7] developed an extended STR panel with better coverage of sex chromosomes, detecting 17 individuals with abnormal complements in a large population of Japanese horses. However, more recently, our laboratory validated a novel, more accurate methodology for chromosome testing, based on the analysis of the information provided by single nucleotide polymorphism (SNP) genotyping arrays, in the PRE breed [12]. This method, which can detect almost any type of chromosomal abnormalities, was integrated as an auxiliary tool of the PRE breeding program in 2021.

The Pura Raza Español horse is one of the oldest and most important horse breeds bred in Europe [13], with more than 250,000 active individuals bred in over 60 countries in the present day [14]. Its studbook was created in 1912, and since then, it has been managed by the Real Asociación Nacional de Criadores de Caballos de Pura Raza Española (ANCCE), following a closed enrolment policy. For this reason, to be included in the PRE studbook, all individuals need to perform mandatory DNA testing to confirm the parentage assignation, as well as a phenotypic characterization to avoid the enrolment of individuals with morphological variations forbidden by the PRE breeding program bylaws. Since 2021, all PRE individuals showing any reproductive abnormality in the mandatory pre-enrolment phenotypic assessment or those whose STR-based parentage test results showed any abnormal or incongruent results (more than 2 alleles per loci or incompatibility between genotypes and phenotypic sex) are being flagged as presumptive carriers of chromosomal abnormalities, and submitted for further investigation using SNP genotyping. Two years later, almost 25,237 horses have now been screened in one of the largest cytogenetic studies conducted on the species.

Here, we present the results of the screening for sex-related chromosomal alterations in a large population of PRE horses, with the aim of establishing a more accurate estimation of the prevalence of chromosomal abnormalities in the species.

#### **2. Materials and Methods**

#### *2.1. Animal Samples*

All the individuals analyzed in this study are included in the screening program for chromosomal abnormalities of the Real Asociación Nacional de Criadores de Caballo Pura Raza Español (ANCCE) studbook. All of them provided blood samples collected by the ANCCE official veterinary services, according to the breeding association standard protocol for parentage testing, before their enrollment in the studbook. During the last 24 months, 25,237 individuals were evaluated (12,569 in 2021 and 12,668 in 2022).

#### *2.2. Genotyping and Chromosomal Analysis*

DNA was obtained from biological samples (either whole blood or hair bulbs) using regulation extraction kits from Qiagen (Madrid, Spain). Thereafter, the samples were first genotyped using the 17 STR panel for determining parentage in horses proposed by the International Society for Animal Genetics (ISAG) in a multiplexed determination using a set of commercially-available fluorescent-labeled primers (StockMarks kit for horses, PE Applied Biosystems, Foster City, CA, USA). In the same reaction, we determined the presence or absence of ECAX and ECAY gene-specific *amelogenin* markers using a slight modification of the PCR reaction proposed by Hasegawa, et al. [15]. Finally, the genotyping and allele calling was performed by capillary electrophoresis using an Applied Biosystems 3130xl DNA sequencer (ANCCE, Spain).

Any individuals who showed abnormal genotyping results (more than 3 alleles in different loci or those showing a discrepancy between the phenotypic and genotypic sex in the ECAX specific marker (LEX003) or AMELX and AMELY fragments, according to Anaya-Calvo [16]) or which showed phenotypic abnormalities in the external reproductive organs were further genotyped using a medium density SNP array chip (Equine GGP 70K, Neogen Inc, Scotland, UK). Finally, the chromosomal complements of these animals were determined according to the methodology validated in the PRE by Pirosanto, et al. [12]. In addition, these individuals were reinspected phenotypically by an official ANCCE veterinarian in situ to confirm the phenotypic sex and to determine the presence or absence of phenotypic abnormalities in the reproductive tract.

#### **3. Results**

Thirteen individuals (0.051% of the total population analyzed) were submitted for chromosomal analysis during the 24-month period (Table 1). Among them, three were submitted due to the existence of morphological abnormalities in the external gonads, four showed three or more alleles in a single locus in the parentage testing STR panel, and six showed incongruences between phenotypic and genotypic sex. In all the cases, the presence of chromosomal abnormalities was confirmed by the SNP genotyping (Table 1).


**Table 1.** Individuals carrying chromosomal abnormalities during the 24-month period in the PRE breed.

A short tandem repeat (STR) based parentage test was performed according to Demyda-Peyras, et al. [11]. Single nucleotide polymorphism (SNP) genotyping analyses were performed according to Pirosanto, et al. [12]. N.d.: not detected.

The results show that 64,XY DSD sex reversal mare (see Figure 1) was the most common syndrome detected (five cases, 0.02%), followed by blood chimerism (four cases, 0.015%). Interestingly, two of them were male/female chimeras, whereas the remaining two were female/female chimeras (see Figure 2).

**Figure 1. Copy number alterations (CNA) analysis profile of a 64,XY DSD mare**. Analysis of the B allele frequency (BAF) and Log R ratio (LRR) values from a 64,XY DSD individual according to [10]. BAF values (upper part) depicting hemizygous markers (close to 0 or 1) in the non-pseudoautosomal region (XPAR, green). Conversely, the XPAR (in yellow) is mostly heterozygous (values close to 0.5). LRR values (lower part) are close to 0 in XPAR (in yellow), depicting diploidy. On the contrary, values in the non-XPAR region (in purple) are close to −0.5, depicting monosomy.

**Figure 2. Copy number alterations (CNA) analysis profile of 64,XY/64,XX and 64,XX/64,XX blood chimeras.** Analysis of the B allele frequency (BAF) and Log R ratio (LRR) values of the ECAX from 64,XY/64,XX (Sample 4, left part) and 64,XX/64,XX individuals (Sample 10, shown on the right of the figure) according to Pirosanto, et al. [12].

In addition, we detected a Klinefelter horse (65,XXY), a Turner's mare (63,X) and two mosaicisms (63,X/64,XX and 63,X/64,XY). Finally, the overall prevalence of individuals carrying chromosomal abnormalities in the PRE during the last 24 months was 0.051% (13/25,237). However, it should be mentioned that individuals carrying 63,X ECAX monosomy or low-level chimerisms could be screened using the diagnostic pipeline employed in this study, and therefore, the overall prevalence is most likely underestimated.

#### **4. Discussion**

It is well established that the domestic horse is the domestic species with the highest number of individuals carrying chromosomal alterations [17], most of which are associated with reproductive failures and abnormal phenotypes [5]. This knowledge is mostly built on the compelling number of cases reported to date, such as the ~400 horses compiled by Power [2] 30 years ago or the 214 horses analyzed by the TAMU cytogenetic lab over the last 20 years [3]. However, large-scale cytogenetic surveys analyzing whole populations are scarce. Here, we establish the incidence of the most important chromosomal abnormalities in a population of 25,237 Pura Raza Español horses.

Several reasons have been suggested for the lack of large cytogenetic surveys in horses, such as the inability of field practitioners to establish an association between infertility and chromosomal failures, but also the scarce availability of commercial laboratories providing karyotyping services in the species [4]. In addition, the classical cytogenetics techniques are slow, expensive, and require the collection and shipping of biological samples for cell culture. However, 20 years ago, Kakoi, et al. [7] proposed the viability of the use of STR parentage genotyping as a screening tool to detect chromosomal abnormalities in horses. Based on this idea, we established a relationship with the Pura Raza Español breeding association, which allowed us to detect several individuals carrying chromosomal abnormalities in the PRE breed, including chimerisms [11], DSD sex reversal horses [18] and complex karyotypes [19], such as normal and abnormal cell lines originating in two different individuals in the same horse. However, it was not until more recently, with the validation of an SNP-based methodology [12], that chromosomal screening was integrated into the breeding program. Since then, more than 25,000 horses have been screened, thus producing one of the most comprehensive datasets analyzed to date.

So far, two large-scale studies have been performed to analyze horse populations from a cytogenetic point of view. In 2005, Kakoi, et al. [7] analyzed 17,471 light-breed Japanese horses using a molecular approach based on STR parentage genotyping, similar to the one used in our study. The authors were able to detect 18 individuals with presumptive chromosomal abnormalities in the sex pair, establishing a prevalence of close to 1/1000. In the second step, they employed an extended ECAX/ECAY STR panel, thus confirming the existence of 13 Turner mares (63,X, 0.07%), 4 Klinefelter horses (65,XXY, 0.02%), and a 65,XXX mare (0.006%). However, only four of these cases could be confirmed by karyotyping due to the lack of availability of biological samples. Two years later, Bugno, et al. [6] karyotyped 500 young Polish horses from several breeds, reporting 10 individuals (2%) with chromosomal abnormalities. Among them, the authors reported a male/female chimera (64,XX/64,XY, 0.2%), one Turner syndrome (63,X, 0.2%), 7 ECAX mosaicisms (63,X/64,XX, 1.4%), and an individual with an autosomal trisomy in the ECA31. Our study agrees with the results provided by Kakoi, et al. [7], probably due to the fact that they were performed using a similar diagnostic methodology in which a large population was first screened, and then only the horses suspected of carrying chromosomal abnormalities were re-analyzed for confirmation. In contrast, the later study karyotyped all 500 horses, with the most common abnormality reported being 63,X/64,XX mosaicism, which was barely detected in our study (just one case). Since this later syndrome is difficult to screen using molecular methods [20], it may explain the differences observed between these different approaches. However, it is also worth mentioning that an overall incidence equal to that reported by [6] would be equivalent to detecting nearly 500 horses carrying chromosomal abnormalities in our PRE dataset, which is far higher than the figure we found. In this context, Shah, et al. [21] reported a 33% discordance rate between karyotyping and molecular methods in the analysis of human miscarriages, with the appearance of chromosomal artifacts due to prolonged culture being one of the potential causes. This lack of consensus was even more noticeable when the growth of cultured cells was stimulated with phytohaemagglutinin (PHA), which resulted in an increased number of abnormalities in comparison with the same samples analyzed from unstimulated cultures [22]. However, we previously compared the results of karyotyping and molecular methods in 30 PRE horses (10 carrying chromosomal abnormalities) with 100% of accuracy [23]. For this reason, we hypothesize that this could be an additional cause to explain the differences observed among studies, such as the breed effect, or maybe another, which was not analyzed in the present report.

One of the key questions about the use of molecular methods for detecting chromosomal abnormalities is their accuracy in determining the presence of mosaicisms/chimerisms in which the percentage of abnormal cells is low [24]. This is particularly important since Power [2] reported that 15% of the 401 individuals carrying chromosomal abnormalities were 63,X/64,XX mares. These limitations were recently demonstrated in horses by Szczerbal, et al. [20] using a highly accurate technique (ddPCR) and by our group using an array-based SNP methodology [12]. However, this is also a well-known problem in humans, where the ability to detect low levels of mosaicism with less than 10% of abnormal cells is questionable [25]. Pienkowska-Schelling, et al. [26] recently reported a substantial increase in the rate of ECAX mosaicism (63,X/64,XX) in fertile mares by performing classical karyotyping. This finding agrees (although the incidence was different) with the reports demonstrating that some of these individuals can develop normally and produce offspring [6,27]. Nevertheless, most of the cases of 63,X/64,XX described are associated with subfertility [17]. Despite the fact that the use of molecular methods for this particular syndrome is still in its infancy, its importance may be limited in the whole population in comparison with the other chromosomal abnormalities reported to date [2,3].

One important limitation of this study worth mentioning is the inability to screen Turner's syndrome (63,X; ECAX monosomy) using parentage STR markers since the standardized test performed worldwide includes only one STR located in the ECAX. In this context, Bugno, et al. [6] reported a prevalence value of 0.2% (1 in 500) for this abnormality in the whole population (1/500). Similarly, several reports [2,17,28,29] have established that ECAX monosomy can account for approximately 30/40% of all the chromosomal abnormalities detected in horses. In contrast, Kakoi, et al. [7] reported a prevalence of this abnormality close to 0.075% (13/17,471), although they were only able to determine those individuals in which the foal's ECAX was of paternal origin. Since we are not able to estimate the incidence of such aberrations in PRE, we extrapolated the results obtained

in other breeds (30% of incidence among chromosomal abnormalities) to our dataset to obtain a hypothetical incidence for comparison purposes. This estimated value (0.016%) is ten times lower than that reported by Bugno, et al. [6] but more in line with that reported by Kakoi, et al. [7] as well as with the incidence of this syndrome in humans (0.04%, according to Bondy [30]), although a large-scale study would be needed to determine the real prevalence of this syndrome in horse populations. Here, SNP-based methodologies can accurately detect mares carrying 63,X complements [12]. Given the introduction of SNP arrays and the genotyping of horses in breeding programs [31–33]), we should expect to find an increase in the detection of this chromosomal abnormality in large populations of horses within the next few years.

Disorders in sex development constitute another common syndrome in horses [5]. Among these, the most common is the sex-reversal mare (64,XY DSD), in which the individual shows a mare phenotype instead of carrying male chromosomal complements [34], even in the PRE breed [18]. In our study, we used a combination of an ECAX STR marker (LEX003) together with determining two fragments of the sex-related *amelogenin* gene (AME), which reduces the possibility of misdiagnosis close to null. However, Martinez, et al. [35] reported a very low percentage of inconsistencies in the AMEY testing (8/100,000) in a large population of horses, suggesting that they might be the result of a translocation (in the paternal line) from the Y to another chromosome. In our case, all the individuals showing a positive AMEY amplification were confirmed as 64,XY DSD by SNP genotyping (6/6). In terms of prevalence, Power [2] stated that 28% of the individuals carrying chromosomal abnormalities showed a 64,XY karyotype, whereas Bowling, et al. [28] detected 22 individuals showing this chromosomal arrangement among 98 mares. In our case, the incidence of 64,XY DSD among individuals showing abnormal karyotypes was ~40%. However, 63,X was not included in this study, and therefore, results may, to some extent, be overestimated. Interestingly, we determined a prevalence of 0.02% 64,XY DSD in the whole population analyzed. This result is in full agreement with the largest screening programs performed for this syndrome in the species, which reported the same value [35]. Interestingly, neither [6] nor [7] reported any individual showing a DSD in their populational studies. However, the fact is that analysis of almost 235,000 individuals, together with the long history of cases reported during the last 40 years, suggest that 64,XY DSD is one of the major chromosomal abnormalities in the domestic horse.

Finally, we were able to detect four cases of blood chimerism in the whole population (4/25,237; 0.016%). These results agree with Anaya, et al. [8] (0.024%), who analyzed ~21,000 PRE foalings but were ten times lower than those reported by Bugno, et al. [6]. Since the electropherogram pattern obtained in STR parentage can only be caused either by blood chimerism or by cross-contamination of the sample, the use of an additional DNA sample is mandatory. In our case, the results obtained using DNA obtained from hair bulbs were normal (not chimeric). However, SNP genotyping also allowed us to detect 64,XX/64,XX blood chimeras, which cannot be detected using classical or molecular karyotyping. Since it is perfectly possible to misdiagnose chimeric samples as normal, we believe that the occurrence of one case of blood chimerism every 5000 foals is a reasonable and reliable estimation for the domestic horse.

#### **5. Conclusions**

Chromosomal abnormalities associated with the sex pair are a noticeable problem in horse fertility. In this study, we demonstrated that the use of a combined technique, including STR and SNP genotyping, can detect most of these genetic abnormalities in horses at an early age. We detected the existence of chromosomal abnormalities in 0.05% of the 25,237 PRE individuals analyzed over the 24-month period within the official breeding program. However, we were also able to estimate a reliable prevalence for specific chromosomal abnormalities, such as 64,XY DSD and blood chimerism, by analyzing one of the largest datasets to date. However, the overall prevalence could be underestimated because of our inability to screen 63,X individuals due to methodological limitations. Finally, we

suggest that the increasing use of SNP genotyping within breeding programs will allow us to detect most of the individuals carrying chromosomal abnormalities in the next few years in a reliable, systematic way.

**Author Contributions:** Conceptualization: S.D.-P., M.V. and A.M.; Methodology: N.L., G.A. and S.D.-P.; Formal analysis: G.A., N.L., B.K.-M. and A.K.; Data curation: G.A., N.L., B.K.-M. and A.K.; Investigation: G.A., N.L., B.K.-M. and A.K.; Resources: A.M., M.V. and S.D.-P. Writing—original draft preparation, B.K.-M., G.A. and N.L.; Writing—review and editing: B.K.-M., G.A., S.D.-P. and M.V.; Supervision: A.M. and S.D.-P.; Project management: S.D.-P. All authors have read and agreed to the published version of the manuscript.

**Funding:** This study was funded by UCO-FEDER 20 REF. 1380999-R (Junta de Andalucía, Spain), and AGL2017-84217-P (Mineco, Spain) grants (Antonio Molina, PI) and PICTA-2021-0063 grant (FONCyT, ANPCyT, Argentina), Sebastián Demyda-Peyrás (PI). Ayelén Karlau is a CONICET fellow.

**Institutional Review Board Statement:** Ethical review and approval were waived for this study since the data analyzed was produced by the ANCCE as a part of their official breeding program.

**Informed Consent Statement:** Not applicable.

**Data Availability Statement:** All the data analyzed in this study belong to the Pura Raza Española breeding program. The ANCCE allows its use for scientific purposes under a specific agreement of collaboration with the MERAGEM group. Therefore, the genetic and genomic data employed in this study is not publicly available. Access can be granted by request to the corresponding authors upon agreement with the ANCCE.

**Acknowledgments:** We would like to thank the ANCCE for providing STR data and samples.

**Conflicts of Interest:** The authors declare no conflict of interest.

#### **References**


**Disclaimer/Publisher's Note:** The statements, opinions and data contained in all publications are solely those of the individual author(s) and contributor(s) and not of MDPI and/or the editor(s). MDPI and/or the editor(s) disclaim responsibility for any injury to people or property resulting from any ideas, methods, instructions or products referred to in the content.

### *Review* **Molecular Cytogenetics in Domestic Bovids: A Review**

**Alessandra Iannuzzi 1, Leopoldo Iannuzzi <sup>1</sup> and Pietro Parma 2,\***


**Simple Summary:** Molecular cytogenetics, and particularly the use of fluorescence in situ hybridization (FISH), has allowed deeper investigation of the chromosomes of domestic animals in order to: (a) create physical maps of specific DNA sequences on chromosome regions; (b) use specific chromosome markers to confirm the identification of chromosomes or chromosome regions involved in chromosome abnormalities, especially when poor banding patterns are produced; (c) better anchor radiation hybrid and genetic maps to specific chromosome regions; (d) better compare related and unrelated species by comparative FISH mapping and/or Zoo-FISH techniques; (e) study meiotic segregation, especially by sperm-FISH, in some chromosome abnormalities; (f) better show conserved or lost DNA sequences in chromosome abnormalities; (g) use informatic and genomic reconstructions, in addition to CGH arrays in related species, to predict conserved or lost chromosome regions; and (h) study some chromosome abnormalities and genomic stability using PCR applications. This review summarizes the most important applications of molecular cytogenetics in domestic bovids, with an emphasis on FISH mapping applications.

**Abstract:** The discovery of the Robertsonian translocation (rob) involving cattle chromosomes 1 and 29 and the demonstration of its deleterious effects on fertility focused the interest of many scientific groups on using chromosome banding techniques to reveal chromosome abnormalities and verify their effects on fertility in domestic animals. At the same time, comparative banding studies among various species of domestic or wild animals were found useful for delineating chromosome evolution among species. The advent of molecular cytogenetics, particularly the use of fluorescence in situ hybridization (FISH), has allowed a deeper investigation of the chromosomes of domestic animals through: (a) the physical mapping of specific DNA sequences on chromosome regions; (b) the use of specific chromosome markers for the identification of the chromosomes or chromosome regions involved in chromosome abnormalities, especially when poor banding patterns are produced; (c) better anchoring of radiation hybrid and genetic maps to specific chromosome regions; (d) better comparisons of related and unrelated species by comparative FISH mapping and/or Zoo-FISH techniques; (e) the study of meiotic segregation, especially by sperm-FISH, in some chromosome abnormalities; (f) better demonstration of conserved or lost DNA sequences in chromosome abnormalities; (g) the use of informatic and genomic reconstructions, in addition to CGH arrays, to predict conserved or lost chromosome regions in related species; and (h) the study of some chromosome abnormalities and genomic stability using PCR applications. This review summarizes the most important applications of molecular cytogenetics in domestic bovids, with an emphasis on FISH mapping applications.

**Keywords:** animal cytogenetics; cattle; river buffalo; sheep; goat; FISH mapping; PCR

#### **1. Introduction**

The application of cytogenetics to domestic animals emerged about 60 years ago with the study of normal stained chromosome preparations from some cases of domestic animals with reproductive defects [1–3]. However, the discovery of the Robertsonian

**Citation:** Iannuzzi, A.; Iannuzzi, L.; Parma, P. Molecular Cytogenetics in Domestic Bovids: A Review. *Animals* **2023**, *13*, 944. https://doi.org/ 10.3390/ani13050944

Academic Editor: Emilia Bagnicka

Received: 31 January 2023 Revised: 28 February 2023 Accepted: 2 March 2023 Published: 6 March 2023

**Copyright:** © 2023 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

translocation (rob) involving cattle chromosomes 1 and 29 [4,5] and the demonstration of its deleterious effects on fertility [6,7] was what piqued the interest of many scientific groups and focused their attention on studying the chromosomes of domestic animals. This approach was particularly useful for selecting bulls to be used for artificial insemination, as it could avoid the transmission of chromosome abnormalities (i.e., rob1;29) from bull carriers to their progeny. Evolutionary studies also benefitted from advancements beyond normal chromosome staining. Among the various studies, the most important was the study of the Bovidae family by Wurster and Benirske [8], who looked at the diploid number and shape of chromosomes. They concluded that while the diploid number varies from 38 to 60 among all bovid species, the number of chromosome arms (Fundamental Number = NF) varies only between 58 and 62, with three exceptions; therefore, they hypothesized a high degree of autosome arm conservation among all bovid species. This hypothesis was later confirmed with the application of chromosome banding techniques [9], which ushered in a new era of chromosome studies in various domestic animal species, allowing (a) the establishment of standard karyotypes of the most important domestic species as a point of reference for various applications; (b) better characterization and identification of the chromosomes involved in chromosome abnormalities of domestic animals [10], particularly domestic bovids [11], pigs [12], horses [13], and dogs [14]; (c) the study of the chromosome homologies between related and unrelated species [15–17]; and (d) the study of chromosome fragility in animals exposed in vivo or in vitro to particular mutagens [18,19]. The molecular cytogenetics, particularly the introduction of fluorescence in situ hybridization (FISH), offered a deeper investigation of the chromosomes of domestic animals through: (a) the physical mapping of specific DNA sequences on chromosome regions; (b) the use of specific chromosome markers for the identification of chromosomes or chromosome regions involved in chromosome abnormalities, especially when poor banding patterns are produced; (c) better anchoring of radiation hybrid (RH) and genetic maps to specific chromosome regions; (d) better comparisons of related and unrelated species by comparative FISH mapping and/or Zoo-FISH techniques; (e) the study of meiotic segregation, especially by sperm-FISH, in some chromosome abnormalities or aneuploidies in both oocytes and embryos; (f) better demonstration of conserved or lost DNA sequences in chromosome abnormalities by CGH (comparative genomic hybridization) or SNP (singlenucleotide polymorphism) arrays; (g) the use of informatic and genomic reconstructions, in addition to CGH arrays, for the prediction of conserved or lost chromosome regions in related species; and (h) the study of chromosome abnormalities and genomic stability using PCR (polymerase chain reaction).

This review summarizes the most important applications of molecular cytogenetics in domestic bovids, with particular emphasis on FISH mapping applications.

#### **2. The Fluorescence In Situ Hybridization (FISH) Technique**

The FISH mapping technique is based on two main principles: the target and the probe. The target can be a whole chromosome (or chromosome arms) or a specific chromosome region. The probe is prepared according to the size of the target and is typically: (a) cDNA (generally applied when the target gene is a multi-copy); (b) cosmids with DNA insert sizes of 20–40 kb; (c) bacterial artificial chromosomes (BACs) with DNA insert sizes of 100–300 kb; (d) yeast artificial chromosome (YAC) clones (these are actually not used because they have a low cloning efficiency and show a high level of chimerism); (e) chromosome painting probes (obtained by cell sorter or chromosome microdissection techniques) that can visualize parts of or entire chromosomes; and (f) CGH arrays to check for genomic gains or losses. The probes are labeled directly with fluorochromes or indirectly with molecules that bind to the probe via fluorochrome-conjugated antibodies. The probe is specific for the target, based on complementary DNA base pairing, which allows the fluorescence-labeled probes to hybridize and form specific fluorescent signals on specific chromosome regions.

The advent of the fluorescence in situ hybridization (FISH) technique, initially applied to human chromosomes [20,21], noticeably expanded cytogenetics research and investigations applied to domestic animals due to the possibility of revealing specific chromosome regions, entire chromosomes, or chromosome arms according to the choice of probe. One of the great advantages of the FISH technique is that it can be applied to interphase cell nuclei, meiotic preparations (sperm and oocytes), embryos, and elongated chromatin fibers, in addition to metaphase chromosomes, thereby allowing more complete cytogenetic investigations of animal cells. The following sections describe the main uses of FISH in domestic bovids.

#### *2.1. FISH and Chromosome Abnormalities*

The first study to apply FISH for the precise identification of the chromosomes involved in a chromosome abnormality was published by Gallagher et al. [22], who discovered an X-autosome translocation (X;23) using both Q-banding and a BoLA Class I cDNA probe. The probe shows hybridization signals to the normal chromosome 23 and to the translocated autosomal material present on the X chromosome, allowing a more precise localization of MHC (major histocompatibility complex) in cattle than was achieved earlier by genetic mapping. Several subsequent studies also applied FISH to obtain better confirmation of the chromosome(s) involved in abnormalities (especially when banding was poor) and identification of the break points, especially in reciprocal translocations. Table 1 shows the main studies that applied FISH mapping, either alone or in combination with other classical cytogenetic techniques (e.g., C-banding, G-banding, R-banding, and Ag-NORs), to study the chromosome abnormalities of domestic bovids in somatic cells at the metaphase (Figure 1) or interphase nuclei of germinal cells, such as sperm and oocytes, or embryos at different cell stages.

**Table 1.** FISH mapping approaches applied for the detection of chromosome abnormalities in domestic bovids. The type of chromosome abnormality, the techniques used (including FISH), the main results, and authors are reported.



**Table 1.** *Cont.*


**Table 1.** *Cont.*


**Table 1.** *Cont.*

A more complete classification of all chromosome abnormalities studied by classical cytogenetic techniques alone or (in some cases) with other molecular cytogenetic techniques is provided by Iannuzzi et al. [11].

Two examples of the importance of the use of FISH for the correct identification of the chromosomes involved in chromosome abnormalities of cattle were a case of autosome trisomy and two types of Robertsonian translocations. A case of autosome trisomy 28 in an abnormal calf, revealed by both R-banding and FISH mapping with a specific molecular marker [33], was identified, and the same abnormality was reported earlier as trisomy 22 using only the banding technique [86]. Two robs earlier reported as rob (4;8) [87] and rob (25;27) [88] in cattle were later corrected as rob (6;8) and rob (26;29), respectively, using C-, G-, and R-banding and FISH mapping with specific molecular markers and the use of HSA painting probes [28].

**Figure 1.** FISH mapping with a BAC clone mapping proximal to BTA29 (large arrow) and proximal to q-arms (BTA1) of rob (1;29) (small arrows). Indeed, a small chromosome region of 5,4 Mb translocated from proximal BTA29 to the proximal region of BTA1 (with an inversion), originating rob (1;29) [56]. Different colors indicate different BACs.

Table 1 shows that FISH mapping applications were used for the diagnosis of chromosome abnormalities in both metaphase (the majority) and interphase cells, the latter applied to lymphocyte nuclei (Figure 2), sperm (Figure 3), oocytes, and embryos.

**Figure 2.** FISH mapping in an interphase nucleus of a female river buffalo affected by X-trisomy. Note the three hybridization signals due to the X chromosome PGK marker.

**Figure 3.** Sperm-FISH in a river buffalo bull carrying a rob (1p;18) using BAC probes for BBU 1p (red), BBU 1q (green), and BBU 18q (yellow) chromosomes. Normal sperm nucleus with 1/1/1 fluorescent phenotype and separate signals on left. Unbalanced sperm nucleus with 1/0/1 fluorescent phenotype on right.

Concerning the studies on meiotic preparations, those performed on the synaptonemal complexes (SCs), especially in spermatocytes, were particularly important for establishing the regularity of the pairing processes during the pachytene substage of meiotic prophase in animals carrying chromosome abnormalities (reviewed in [89]). Recent analyses of meiotic preparations have been performed using immune fluorescence approaches and have provided more detailed information on SCs [90–92]. Other studies have addressed the fragile sites in the chromosomes of domestic animals (reviewed by [93]), and limited studies have used CGH and SNP arrays to establish possible genomic losses occurring during chromosome rearrangements (Table 1).

FISH mapping was also very important for the definitive establishment of the agreement between various chromosome nomenclatures due to some discrepancies found during the Reading conference [94] and the subsequent ISCNDA1989 [95] (the inverted position between BTA4 and BTA6, as well as the correct position of BTA25, BTA27, and BTA29). This aspect was vital for the clinical cytogenetics of domestic bovids, as it allowed a correct identification of the chromosomes involved in chromosome abnormalities. During the Texas conference [96], specific molecular markers (only type I loci) were selected for each bovine syntenic group and each cattle chromosome based on previous standard chromosome nomenclatures.

The next advance was the application of FISH mapping by two labs that used 31 selected BAC clones (from the Texas Conference) on RBG- and QBH-banded cattle preparations [97]. The chromosome-banding homologies among bovids (cattle, sheep, goats, and river buffalo) were then used to establish a definitive standard chromosome nomenclature for the main domestic bovid species [98]. Subsequent studies using FISH mapping and the same Texas markers on river buffalo, sheep, and goat R-banded chromosomes [99,100] definitively confirmed the chromosome homologies among domestic bovids, as established at the ISCNDB2000 [98].

#### *2.2. FISH in Physical Mapping*

The identification of the DNA structure [101] paved the way for the development of in situ hybridization technology. In the early stages of its development, this technology allowed the localization of genes using radioactive probes [102]. It was also used in studies of domestic animals [103,104], but the greatest diffusion of the physical mapping of genes awaited the development of fluorescent probes [105]. At that moment, we entered the golden years of gene mapping, and domestic animals were not excluded. One of the first examples was the localization of bovine alpha and beta interferon genes [106], and this localization was rapidly replicated in buffalos, goats, and sheep [107,108]. Subsequently, many other localizations were obtained using this technology (Figure 4).

**Figure 4.** FISH mapping of type II loci in river buffalo R-banded chromosomes. FITC signals (arrows) of the markers and RBH banding were separately acquired by two different microscope filter combinations. Then signals were precisely superimposed to R-banded chromosomes (*Drawn from Iannuzzi et al., Cytogenet Cell Genet. 102, 65–75, 2003, DOI: 1 0.1159/000075727, S. Karger AG, Basel* [109]).

Considering the practical impossibility of compiling a complete list of all gene localizations obtained using this technology, some significant examples are listed in Table 2.

**Table 2.** Gene mapping obtained with FISH in domestic bovids. Type I and type II markers are expressed with polymorphic (SSRs, microsatellite, STSs) sequences, respectively.



**Table 2.** *Cont.*

Localization sometimes involved a single gene [124,129] or a family of genes [132]. Other reports, however, mapped many genomic markers [100,141]. A point to remember is that FISH technology has significantly benefited from the availability of BAC genomic libraries—elements that represent the ideal source for the construction of the probes. Among these, the INRA library [144] and the CHORI-240 have played relevant roles. The publication of genomes [145–148] has since inevitably diminished interest in using this technology for mapping genetic factors, although genetic factor mapping continued for species whose genomes were sequenced later, such as the water buffalo [149]. However, this technology has proved useful in several aspects, including: a) the identification of errors in genomic assembly [150]; b) the refinement of genome assembly [151]; and c) the mapping of sequences not included in genomic assemblages [152]. Clearly, the interest today is very limited in locating a genetic factor in a species whose genomic sequence is available, but this does not mean that FISH technology is no longer indispensable for solving other problems related to the organization of genomes.

The mapping of genomic elements by FISH has also been used successfully for the physical mapping of data obtained by other technologies. The first examples concerned the physical anchoring of a genetic map to a chromosome [153–155] and the mapping of a synteny group to a specific chromosome [114]. Subsequent examples of the combined use of FISH and genetic maps followed [127,156].

#### *2.3. Comparative FISH Mapping*

Two main methods have been applied thus far to obtain a FISH mapping comparison between related and unrelated species: Zoo-FISH, which uses chromosome painting probes, and FISH mapping, which uses specific molecular markers of both type I and type II. ZooFISH is a molecular technique that provides an easier comparison between related and unrelated species from a macro point of view. The term was first reported by [157], based on earlier studies that used genomic chromosome painting probes, obtained by cell sorter chromosomes, to compare related species [158–160].

Zoo-FISH was first applied in domestic animals when human chromosome painting probes became commercially available. This approach demonstrated the conservation of several human chromosome segments in both domestic bovids (Table 3) and other domestic species (reviewed in [161]).

**Table 3.** Comparative FISH mapping in domestic bovids with related and unrelated species.


#### **Table 3.** *Cont.*



**Table 3.** *Cont.*

The use of human-chromosome painting probes allowed the identification of a substantial number of human chromosome segments (around 50) in bovid chromosomes [175,176,217–219]. Zoo-FISH has also been applied to correctly identify some chromosomes involved in the chromosome abnormalities shown in Table 1. The availability of specific painting probes obtained by both cell sorting and/or by the microdissection of specific chromosomes (or chromosome arms) from domestic animals extended these studies to investigations between related species (Table 3). For example, in cattle, Zoo-FISH was applied to study X-Y aneuploidy in sperm [55] and in oocytes [58] (Table 1). An interesting approach was demonstrated in two studies characterizing two cases of goat/sheep [220] and donkey/zebra [221] hybrids using multicolor FISH (M-FISH), starting from painting probes obtained from microdissected river buffalo chromosomes (or chromosome arms) and from flow-sorted donkey chromosomes, respectively.

Chromosome painting probes allow the delineation of large, conserved chromosome regions between related and unrelated species, as reported above. The use of comparative FISH mapping using several chromosome markers to map a single type I or type II locus along the chromosomes allows a more accurate establishment of the gene order within chromosome regions, thereby confirming that chromosome rearrangements occurred to differentiate related or unrelated species in key evolutionary studies (Table 3). These detailed comparisons have confirmed a high degree of autosome (or chromosome arm) conservation among all bovid species. The main autosome difference found thus far in bovids was a chromosome translocation of a proximal chromosome region from *Bovinae* chromosome 9 to *Caprinae* chromosome 14, as demonstrated by both chromosome banding and, in particular, by a molecular marker (COL9A1) mapping proximal to *Bovinae* chromosome 9 and proximal to *Caprinae* chromosome 14 (reviewed in [9]). This translocation involved a genome region of about 13 MB and was followed by an inversion in *Caprinae* chromosome 14, as demonstrated earlier [213]. This chromosome event was common to all remaining *Bovidae* subfamilies, leading to the conclusion that the *Bovinae* subfamily is an ancestor to the remaining *Bovidae* subfamilies (reviewed in [9]).

In contrast to autosomes, sex chromosomes are differentiated by more complex chromosome rearrangements. Indeed, the *Caprinae* X chromosome (as for all remaining X chromosomes of the other *Bovidae* subfamilies) is differentiated from the ancestor *Bovinae* X (very probably a large acrocentric chromosome, such as that of the water buffalo) by at least three chromosome transpositions and one inversion (reviewed in [9]). Detailed FISH mapping data are also useful for better anchoring of both genetic and RH maps [203,222–224]. The availability of detailed cytogenetic maps in bovid species allowed a better comparison of the bovid and human chromosomes, especially using type I loci. These comparisons facilitated the translation of genomic information from the human genome to the genomes of domestic animals, especially in those with no genome sequencing available. These comparisons also revealed a very high number of chromosome rearrangements that differentiate bovid species from humans. Indeed, the conservation of entire chromosomes or large regions of them between bovid and human chromosomes, as revealed by Zoo-FISH, was

the result of complex chromosome rearrangements that differentiated human and bovid species according to their gene order. An example is presented in Figure 5 which illustrates the comparison of FISH mapping between HSA2q and BTA2. As seen, when utilizing the Zoo-FISH technique with the HSA2q painting probe, almost all BTA2 is painted [217], indicating a high degree of chromosome conservation between the chromosomes of the two species. By conducting the same comparison using comparative FISH mapping and examining the gene order along the chromosomes of the two species, we observe a distinct gene order between the two species, thus revealing complex chromosome rearrangements that differentiated the chromosomes of the two species during their evolution.

**Figure 5.** Comparative FISH mapping between HSA2q and BTA2. Note the different gene order between the two chromosomes due to complex chromosome rearrangements occurred during the chromosome evolution of the two species (*Drawn from Di Meo et al., Animal Genetics 37, 299–300, 2006, Wiley Online Library* [140]).

#### *2.4. Fiber-FISH*

The various FISH mapping techniques developed for human cytogenetics (reviewed by [225]) include SKY-FISH (spectral karyotyping FISH), Q-FISH (quantitative FISH), M-FISH (multicolor FISH), heterochromatin-M-FISH, COBRA-FISH (combined binary ratio labeling FISH), cenM-FISH (centromere-specific M-FISH), and fiber-FISH. Among these techniques, only fiber-FISH and M-FISH have been applied to domestic bovids. The use of fiber-FISH yields high-resolution maps of chromosomal regions and related genes on a single DNA fiber. This approach establishes the physical location of DNA probes with a resolution of 1000 bp. It is particularly useful for detecting gene duplications, gaps, and variations in the nuclear genome. The DNA fibers are obtained from nucleated cells by releasing the DNA fibers from the nucleus, stretching them mechanically, and then fixing them on slides [226] (Figure 6). Table 4 summarizes the studies that have used this technique in domestic bovids.

**Figure 6.** Details of the fiber-FISH performed on a lymphocyte nucleus of cattle affected by arthrogryposis using a BAC clone containing the survival of motor neuron gene (SMN). The presence of two groups of linear hybridization signals (arrows) supports the hypothesis that SMN was at least duplicated [135].

**Table 4.** Studies using the fiber-FISH on domestic bovids.


#### *2.5. CGH Arrays*

The CGH array technology, an evolution of in situ comparative genomic hybridization (CGH), is a method of cytogenetic investigation that emerged in the 1990s to overcome the limitations of common banding cytogenetic analyses, especially those involving the presence of genomic imbalances, such as duplications or deletions [231,232]. In situ CGH technology has many similarities to FISH: the support used is the same, i.e., denatured metaphases fixed on slides and the approaches to label the probes are identical. However, in this case, the probes are produced using complete genomic DNA deriving from two subjects: typically, one healthy and one relating to the subject being investigated. The two DNAs are labeled with two different fluorochromes and then hybridized simultaneously on the slide. In the hybridization phase, a competition is therefore created between the probes, and in the presence of a normal chromosomal segment, an intermediate color is obtained, while in the presence of chromosomal alterations, a fluorescence closer to one of the two colors used is obtained. Although this technology has been widely used and has provided important results, its major limitation lies in the resolution. CGH array technology follows the same principle, but the support is no longer represented by slides but by synthetic DNA fixed on slides. Initially, the chips for CGH array analyses contained DNA extracted from BAC to provide as uniform a representation of the genome as possible [233]. Current CGH array analyses are performed using devices containing oligonucleotides chosen that uniformly cover the whole genome and achieve resolutions of 5–10 kb [234,235]. More information about this technology and its use is provided by [236]. In species of zootechnical interest, CGH array analyses (Figure 7) became common following the appearance of the first commercial arrays, and these analyses are conducted essentially for two purposes: the identification of copy number variation (CNV) polymorphisms and the characterization of chromosome anomalies. CNVs are polymorphic variations present very frequently in the genomes of higher organisms [237–239]. In humans, approximately 4.8–9.7% of the genome contains CNVs [240]. The introduction of commercial arrays has allowed the use of this technology to obtain a great amount of information about the distribution of CNVs

in species differences and how these variations are related to phenotypic traits. The transfer of this technology to the animal field and the availability of commercial arrays has led to the publication of several reports (Table 5).


**Figure 7.** Identification of the PAR region present on BTAX and BTAY. The PAR region (yellow box) is identified by comparing DNA obtained from a male subject and that obtained from a female subject using a SurePrint G3 Bovine CGH Microarray 180 k (Agilent Technologies, Santa Clara, CA, USA). *Parma P. Personal communication*.

#### **3. Combined Informatic and Genomic Information**

The publication of animal genomes [145–149,250] has made available a very large series of data that required the development of sophisticated analysis techniques and often required the use of computers with large processing capacities. The first bio-informatic analyses were used to assemble thousands of short genomic sequences, produced by modern high-throughput sequencing technologies, into genomes. Today, most of these programs are available free of charge through web pages that function as interfaces between the user and calculation tools [251]. Currently, dozens of bio-informatics programs are available to analyze the data contained in genomic assemblies, and many of these are accessible through various web platforms. Making a complete list is very complicated, in part because this is a rapidly evolving discipline that introduces, almost daily, new analytical tools.

#### *3.1. Visualization of Genomes*

The genomic sequences produced by the various assemblies can be visualized using one of the available websites available, including Genome Data viewer [252], UCSC Genome Browser [253], and Ensembl [254]. Currently, these websites provide the ability to view and process data relating to several genome assemblies (Table 6).

**Table 6.** Independent genomic assemblies that can be analyzed through the main genomic visualization sites.


<sup>1</sup> BTA = cattle; OAR = sheep; CHI = goat; BBU = water buffalo and BIN = Zebu. <sup>2</sup> Only genomic assemblages at the chromosomal level were considered and not those limited to scaffolds. <sup>3</sup> Genome data viewer. <sup>4</sup> USCS genome browser. <sup>5</sup> Ensembl genome browser.

These genome viewers are constantly evolving and contain several tools within them that allow the user to obtain highly relevant genetic data and information. This includes, but is not limited to, the possibility of: (a) identifying the structure of genetic factors (in terms of exon–intron boundaries); (b) identifying SNP polymorphisms in a particular region of the genome; (c) identifying the position of BACs by mapping the BES (Bac Ends Sequences, particularly useful when the user wants to choose the BACs to use in FISH analysis); (d) observing the genomic regions expressed in particular types of tissues; (e) analyzing the relationships between different assemblies of the same species; (f) visualizing the relationships between similar regions in different species (comparative genomics); and (g) viewing the repeating regions. In this review, we do not specify a best genome viewer, as this will often depend on personal needs and experience. However, as each genome

viewer has its own specific analysis tools, sometimes the best solution is to use all three to obtain more complete information.

#### *3.2. Use of Genomic Assemblies*

The availability of genomic assemblages has, on the one hand, limited the interest in the physical mapping of genomic elements, but has, on the other hand, allowed the evolution of a very large number of genetic and genomic analyses. Probably one of the most common uses (even if not directly related to cytogenetics) is to design primers for use in PCR amplifications. This operation can be performed using different software, both available for free and for a fee. Among those available free of charge, the most frequently used is Primer3 [255]. The availability of genomic assemblages also makes rapid evolutionary investigation possible (i.e., visualizing, in a simple and rapid way, the similarities that exist between the various genomic regions of different species). The publication of genomes has certainly had a great impact on cytogenetics (both negatively and positively). If the golden era of gene mapping has ended, the possibility of rapidly identifying BACs for use as probes in FISH experiments has certainly provided great benefits to cytogenetics, as it avoids long and tedious testing of BAC libraries. This aspect has allowed the rapid characterization of some chromosomal anomalies, such as a centromere repositioning event in cattle [66], a reciprocal translocation, also in cattle [62], and cryptic evolutionary rearrangements between cattle and sheep [213]. Finally, the rapid localization of BACs on genomes has allowed the development of complex approaches for the identification of chromosomal abnormalities, which are also difficult to identify [71]. Obviously, these are not all the possible uses of genomic assemblies, but they represent the best examples in relation to cytogenetics. Each genomic assembly contains substantial information that can be used for very specific purposes and avoids the need for probes that would be complex to synthesize. The continuous evolution of these data analysis tools creates difficulty in any attempt to compile their possible uses.

#### *3.3. Tools for Genomic Data Analyses*

Simultaneously with the publication of the genomes, bio-informatics tools were developed for the analysis of the vast amount of data generated—data that are characterized by both their great variety and their large quantity. One of the main repositories of tools for analyzing genomic data is Galaxy [251]. This repository provides access to bio-informatic analysis tools, which are constantly updated. SNP variations represent the major source of variation in genomes, and the genomes of the species covered in this review are no exception. Currently, identifying these sources of variation is quite simple (through modern high-throughput sequencing techniques at ever-lower cost), but this does not characterize the effect that these variations can cause. For this scenario, the variant effect predictor (VEP available on the Ensembl website) software is helpful [256].

Without a doubt, BACs represent one of the most useful tools for molecular cytogenetics, and, as previously mentioned, their identification in genomes is currently greatly facilitated. However, the current situation would not be possible without the existence of two important institutions that have dedicated part of their activities to the construction, maintenance, and distribution of BAC libraries: the BACPAC Resources Center (BPRC, https: //bacpacresources.org/ (accessed on 2 March 2023)) and INRA (http://abridge.inra.fr/ index.php?option=com\_flexicontent&view=item&cid=17&id=61&Itemid=202&lang=fr (accessed on 2 March 2023)). Through these two institutes, BACs belonging to different libraries can be obtained.

#### *3.4. Whole-Genome Sequencing*

In recent years, the decreasing costs of sequencing have made it possible to analyze many subjects. The purposes of these sequencings are different; in many cases, the aim is the identification of signatures of selection [257–259], but other purposes are represented, such as: (a) the identification of genetic variants in specific genes [260]; (b) the verification of data obtained regarding the identification of SNPs with chip arrays [261]; (c) the identification of the run of homozygosity in breeds intended for different productions [262]; (d) prediction and QTL mapping [263]; and (e) the identification of copy number variants [264] and transcriptome characterization [265]. Similar analyses were performed on sheep [266,267] and goats [268,269]. Additionally, in this case, the water buffalo seems to be slightly behind, as there are very few papers available on it [265].

#### **4. PCR-Based Methods and Molecular Cytogenetics**

The polymerase chain reaction (PCR) [270] is a method largely used to make millions of copies of a specific DNA sample in a fast and economical way for the detection, quantification, and typing of infectious diseases and genetic changes. Current PCR-based methods are distinguished as: (a) first-generation PCR, (b) second-generation quantitative PCR (qPCR), and (c) third-generation droplet-based digital PCR (dPCR). PCR detects endpoint, qualitative, or semi-quantitative assays by gel electrophoresis, separating DNA fragments according to size. The qPCR measures DNA/RNA in real time using PCR methods, fluorescent dyes, and fluorometry for relative quantification and quantitative assays with standard curves. The dPCR splits a PCR sample labeled with fluorescent dye into millions of microsamples to digitize the pool of DNA molecules with a single or no copy in each droplet. It quantifies the DNA/RNA copy number faster than qPCR based on standard curves [271].

In recent years, PCR-based methods have replaced the classic cytogenetic techniques for detecting chromosome abnormalities and aneuploidy due to greater precision, lower cost, and faster data than are possible with cytogenetic methods, because of the small quantities of DNA (30 ng) required from any stored or fresh biological samples. PCR-based approaches are most commonly used in bovid studies to examine sex chromosomes in early-sex-determination assays to detect aberrations (Table 7).


**Table 7.** PCR-based approaches on bovids for the detection of chromosomal aberrations.

Telomere assessment is another critical goal of cytogenetics research due to the central roles of telomeres in chromosome stability, aging, cancer development, apoptosis, and senescence. The telomeres consist of thousands of noncoding repetitive sequences of DNA composed of six nucleotide motifs (TTAGGG)n localized at the ends of chromosomes and are responsible for maintaining DNA integrity during each cell division. They are associated with several proteins, with the most abundant being the shelterin complex, which is made up of six different polypeptides. Telomeres also contain other genomic structures, such as T-loops, D-loops, G-quadruplexes (G4), R-loops, and long noncoding RNA (TERRA) [286].

In farm animals, telomere length (TL) did not receive much interest initially due to the difficulty in determining the natural limits of their lifespans. However, a recent study related TL to health, genome stability, and aging in cattle aged between 2 and 13 years and transformed TL into a sensitive biomarker for longevity and wellness (critical traits of selective breeding), responding to the "One Health" approach (improving animal welfare) [287]. TL is not often used as a unique marker of aging in humans because of its poor predictive accuracy due to increased telomere shortening in elderly humans as a consequence of age-related diseases (e.g., cancer, atherosclerosis, autoimmune disorders, obesity, chronic obstructive pulmonary disease, diabetes, hematological disorders, and neurodegenerative diseases) [288]. By contrast, TL proved to be a relevant biomarker of the general state of farm animals due to their lack of age-related pathologies [289,290].

Approaches for measuring TL include: (a) telomere restriction fragment (TRF) length [291]; (b) length analysis by Southern blotting; (c) fluorescent in situ hybridization (FISH) by flow cytometry (flow-FISH) or in metaphase cells (Q-FISH) [292,293]; and (d) PCR-based methods. Most of these methods have several limitations. For example, TRF and flow-FISH are labor-intensive and expensive; Southern blot analysis requires large amounts of genomic DNA, and Q-FISH works only on chromosomes (metaphase stage). Of the available methods, the PCR-based ones are the fastest, most recent, and least costly and require only small quantities of DNA (30 ng) from stored or fresh biological samples [294]. The qPCR method amplifies telomere repeats relative to a singlecopy gene (reference gene) according to a method described by Cawthon et al. [295] and follows the MIQE guidelines [296]. One limitation of qPCR is the inconsistent repeatability and reproducibility of different TL measurement methods, producing a high variation in results [297]. Several studies on humans and animals indicated that the DNA extraction method might affect TL measurements using q-PCR, as DNA yields were higher using the non-silica membrane kit (salting-out method), and DNA integrity on electrophoresis gels varied [298,299]. A recent study showed comparable results for DNA quality and purity (tested using a NanoDrop instrument and electrophoresis gels) in cattle blood and milk samples using two different extraction kits (a saltingout kit for blood and a silica membrane kit for milk samples) due to the difficulty of extracting DNA from milk matrices. The DNA quality results were similar in both matrices, demonstrating a synchronous trend between them for the first time [287].

#### **5. Current Developments and Knowledge Gaps**

Molecular cytogenetics is approaching its first 30 years of history and during this period, it performed important functions that evolved over time. It therefore seems normal that in the coming years, we will witness further developments; however, some approaches will always be current and irreplaceable. The FISH technology represents, and will represent, the main methodology for the verification of chromosomal anomalies eventually identified with other approaches, just as the CGH array technology that will be increasingly used for the identification of genomic variants linked to a particular phenotype. Molecular cytogenetics could be very useful for the study of those species which have not yet benefited from the genomic revolution, or which are still in its early stages: in this sense, the water buffalo (Bubalus bubalis) is the main example. Despite possessing a great economic importance, its genome has been decrypted and made available only recently, and the application of other technologies is very late. A further gap that can be filled is the development of a technological approach that can allow the identification of all chromosomal types identifiable by cytogenetic analyses. A similar approach has already been published [71], but only the transfer of SKY-FISH technologies [300] from humans to

bovids will bridge this gap. Finally, the certain decrease in costs will mean that even the species considered in this review will be able to benefit from long-read genomic sequencing, such as PacBio [301] and Oxford Nanopore [302].

#### **6. Conclusions**

The study of the chromosomes of domestic bovids is about to enter its seventh decade, and, as expected, it has undergone a notable evolution along the way. This evolutionary process for this discipline is mainly a result of the appearance of technologies that have significantly increased the potential of applied cytogenetics. Banding techniques, FISH, CGH arrays, and PCR have radically changed animal cytogenetics, making them irreplaceable tools for understanding the genetics of bred animals. Therefore, considering the history of cytogenetics, a quite easy prediction is that even the next evolutions will be dictated by technological advances. Predicting the next technological leap is difficult, but if we were to make a prediction, it would be that long-read genomic sequencing technologies will have important impacts on cytogenetics. Cytogenetics will likely retain its functionality, particularly in the confirmation of genomic results and the characterization of cytogenetic anomalies, as well as in evolutionary studies. This is because the most significant genetic mutations have accumulated at the chromosome level during the evolution of species. Finally, the implication and progresses from animal cytogenetics can be summarized as follows:


**Author Contributions:** Conceptualization, P.P, L.I. and A.I.; writing—original draft preparation, P.P, L.I. and A.I.; writing—review and editing, P.P. and L.I. All authors have read and agreed to the published version of the manuscript.

**Funding:** The study was supported by the project PON1\_486 GENOBU.

**Institutional Review Board Statement:** An Institutional Review Board statement was not required.

**Data Availability Statement:** Data sharing is not applicable to this article as no new data were created or analyzed in this study.

**Conflicts of Interest:** The authors declare no conflict of interest.

#### **Abbreviations**



#### **References**


**Disclaimer/Publisher's Note:** The statements, opinions and data contained in all publications are solely those of the individual author(s) and contributor(s) and not of MDPI and/or the editor(s). MDPI and/or the editor(s) disclaim responsibility for any injury to people or property resulting from any ideas, methods, instructions or products referred to in the content.

MDPI St. Alban-Anlage 66 4052 Basel Switzerland Tel. +41 61 683 77 34 Fax +41 61 302 89 18 www.mdpi.com

*Animals* Editorial Office E-mail: animals@mdpi.com www.mdpi.com/journal/animals

Academic Open Access Publishing

www.mdpi.com ISBN 978-3-0365-7588-9