Cytogenetic analysis allows detection of animals with chromosomal abnormalities, which may affect the productive and reproductive functions. This study aimed to evaluate tR1/29 abnormality incidence in Senepol cattle evaluated in this study. Blood samples were obtained from 94 females and 61 males from farms and AI centers (artificial insemination) at São Paulo State. 15 karyotypes were analyzed, 11 (74.2%) had normal chromosome complement (2n = 60), 58 autosomes acrocentric chromosomes morphology and two submetacentrics sex chromosomes (XX in females and XY in males). The remaining 40 (25.8%) had abnormalities of type Robertsonian 1/29 (tR1/29), 37 in heterozygous, and three in homozygous. Results confirm the importance of cytogenetic analysis. Negative effect on reproduction of tR1/29 anomaly is still controversial, but are particularly relevant to the industry of bulls destined to replacement or semen collection and embryo donors cows, since the identification and subsequent removal of animals carrying this anomaly may prevent further problems in herds.
One of the main objectives of cytogenetics is the detection of chromosomal anomalies; they can be either numerical or structural. In the specific case of cattle, the chromosomal complement is considered of great importance due to its relationship with fertility (Raudsepp & Chowdhary, 2016). Robertsonian translocations, especially 1/29, have been associated with decreased fertility and early embryonic mortality (Santos et al., 1989; Bonnet-Garnier et al., 2008). This is initially described as anomaly (Gustavsson, 1979) that is found spread throughout the world in different breeds of cattle, particularly in the species Bos taurus taurus, which are more often found in herds of beef cattle than in dairy systems (Popescu & Pech, 1991; Corredor & Jiménez,2005) . In Brazil, it is described in the races Blond d’Aquitaine, Charolesa, Chianina, Caracu, Marchigiana, Pardo Suiss Pitangueiras, Red Poll and Senepol (Pinheiro et al., 1994; Issa et al., 2008). Due to its productive characteristics and tolerance to tropical conditions, the Senepol breed has increased considerably in some regions of Brazil. Consequently, the demand for genetic material (semen and embryos) of this breed had a significant increase. In Santos et al. (1989) the Senepol obtained the second position in the production of sperm of the beef breeds, with approximately 240.591 doses (ASBIA, 2014). However, a restrictive factor for the propagation of the Senepol breed is the finding of chromosomal anomalies, in particular of the Robertsonian translocation 1/29, in a significant number of animals (approximately 28% in published results), probably due to their kinship with the Red Poll race, where it comes from.Animals carrying the Robertsonian translocation tR1/29 are not recognized by the phenotype. Meanwhile, when the parent is heterozygous, it produces unbalanced gametes, and therefore, unbalanced zygotes that will have a little chance of continuing their development, resulting in high rates of early embryonic mortality. In addition, 50% of the products will carry the reordering described above. If on the other hand, one of the parents is homozygous, all the offsprings will be the carriers of translocation in heterozygosis. Therefore, the evaluation of the cytogenetic status of the animals of this breed, especially of the bulls and matrices intended for large-scale reproduction (bulls in sperm collection and as embryo donors), must be carried out systematically to avoid the propagation of genetic material, which can put the rest of the population at risk.The objective of this study was to evaluate the incidence of chromosomal abnormalities in Senepol cattle breed in the São Paulo state.
Materials and Methods
Blood samples from Senepol cattle, located in three Artificial Insemination Centers (11 bulls in sperm collection) and four farms (50 adult females designated for replacement and 94 embryo donors), in the São Paulo state, were collected in the period from 2004 to 2014. The collection of blood (jugular vein) was performed by heparinized and sterile vacuum tubes (vacutainer). On the same day of the collection, the samples were sent to the cytogenetics laboratory of the Genetics and Animal Reproduction Research Center of the city of Nova Odessa, state of São Paulo for processing according to the technique (Moorhead et al., 1960), with some alterations. Succinctly, immediately after the arrival of the sample, 20 drops of blood were transferred to a culture flask previously prepared with 4 ml of MEM medium, 1 ml of fetal bovine serum and 0.1 ml of phytohemagglutinin and antibiotics. After an incubation period of 47 hours at 38 °C, 0.1 ml of 0.0016% colchicine solution was added, continuing the incubation for over 1 hour. Next, the material was centrifuged for 8 minutes at 800 rpm and the supernatant was discarded. Hypotization of the cells was achieved by the addition of 10 ml of KCl solution (0.075 M at 38 °C) and incubation for more than 8 minutes. After this period, fixation was performed, by the addition of a volume of 0.5 ml of fixative (three parts of methanol: one part of acetic acid). Next, the material was homogenized and centrifuged at 800 rpm for 8 minutes and the supernatant was discarded. Subsequently, 5 ml more of fixing solution was added and centrifuged again, discarding the supernatant (this step was carried out three times). Then, 1 ml of fixative was added and kept in the freezer for 2 hours. From this material, eight drops were removed and placed on cooled glass sheets. After 2 days, the preparations were stained for 8 minutes with 3% Giemsa solution. Chromosome analysis, in metaphase, was carried out with an optical microscope with a clear immersion lens and clear camera. After the design and accounting of the chromosomes, the best cells were photographed to mount the karyotype. For each animal, approximately 30 cells were analyzed. The comparison analysis of the incidence of translocation in males and females was made by the Chi-square test (Gomes, 2009).
Results and DiscussionThe results of the reading of the karyotype confirmed in 115 (74.2%) of the animals the typical pattern of the species Bos taurus taurus, this means, bovine 2n = 60, with constitution 60, XX for females and 60, XY for males, being 58 acrocentric autosomes and the submetacentric X and Y chromosomes (Fig. 1a), which were considered normal according to the detailed definition (Melander, 1959). In the remaining 40 (25.8%) animals, the presence of the Robertsonian translocation type 1/29 chromosomal anomaly was observed, being 37 in heterozygotes (Fig. 1b) and three in homozygotes (Fig. 1c) in Table 1.
Table 1: Frequency of chromosomal abnormalities in Senepol cattle from Sperma centers and farms at São Paulo state
|Breed Senepol||Group||Number of Bovine||Karyotype Normal (%)||tR1/29 Heterozygote (%)||tR1/29 Homozygote (%)||tR1/29 Total (%)|
|D||94||76 (80.8)||17 (18.1)||1 (1.1)||18 (19.2)|
|T1||11||8 (72.7)||2 (18.2)||1 (9.1)||3 (27.3)|
|T2||50||31 (62.0)||18 (36)||1 (2.0)||19 (38.0)|
|Total||155||115 (74.2)||37 (23.9)||3 (1.9)||40 (25.8)|
D = embryo donators, T1 = Bulls for sperma harvest, T2 = female adults for reposition, tR1/29 = Robertsonian translocation.
The Robertsonian translocation is formed by the rupture followed by the fusion of two acrocentric chromosomes, in the region of the centromeres, constituting a new metacentric or submetacentric chromosome (depending on the size of the chromosomes involved) and reducing the diploid number from one in the heterozygotes and two in the homozygotes. In tR1/29, the chromosome formed is a submetacentric, corresponding to one of the largest in the set, formed by chromosomes 1 and 29 (Fig. 1b and c). It is important to mention that in most cases, the animals that exhibit tR1/29 are not recognized by the phenotype. However, it is possible to affirm that 50% of the products of a heterozygous genitor will be the carriers of the rearrangement of the type found in the parent. If, on the other hand, one of the parents is homozygous, all the offsprings will be the carriers of translocation in heterozygosis.Regarding the incidence of this anomaly, described since the 1960s, it is found scattered throughout the world, in bullfighting breeds, constituting the most common chromosomal abnormality in bovines. In Brazil, this abnormality has been recorded in several European or synthetic bull breeds with European blood, such as Blond d’Aquitania, Charolais, Su, Caracu, Marchigiana, Pardo Suiza, Pitangueiras, Red Poll, and Senepol (Corredor & Jiménez, 2005; Ducos et al., 2008).The karyotype of the ancestors is very important in the formation of a “synthetic” or a “composite” race, because the Robertsonian translocation carrying reproducers transmit this anomaly to the offspring. As a result, the new breed may present a high incidence of the anomaly, as suggested by the results of some authors (ASBIA, 2014; Moorhead et al., 1960) in the animals of the Pitangueiras breed (5/8 Red Poll and 3/8 Cebu). These authors observed an incidence of approximately 30% of the carriers with tR1/29. Other authors (Pires et al., 2006) found an incidence of 27.3% in bulls of the Senepol breed (N’Dama × Red Poll) arranged in semen collection centers. The probable reason for this high incidence in both the Pitangueiras and Senepol breeds is the fact of having the breed Red Poll which was the carrier of tR1/29 (Halnan et al., 1980; Ducos et al., 2008) in its formation. Frebling et al. (1987) analyzed cytogenetically animals of the breed N’Dama, the other ancestral of the Senepol breed and no chromosomal rearrangement was detected. Meanwhile, crossings of Zebu × N’Dama and N’Dama × “Baoule” if they presented the referred chromosomal rearrangement (Frebling et al., 1987), with these results, it was not possible to detect if the rearrangement originated in the Cebu or in the race “Baoule” or in the N’Dama. If it happened in the last race, it is an ancestor of the Senepol, and for that reason could have transmitted the tR1/29.The implications of the presence of tR1/29 in reproduction are contradictory. While some studies have not found any negative effect (Lui et al., 1997), others suggest an association with low fertility. Thus, Hatayde et al., reported problems of subfertility in Pitangueiras and Marchigiana animals paired with animals carrying tR1/29. This negative effect on reproduction would be associated with the quality of the oocytes or the embryo in the early stages of development, as evidenced Bonnet-Garnier et al., 2008; Ducos et al., 2008). These authors observed an increase in metaphase II with cytogenetic abnormalities in oocytes from cows with tR1/29. In addition, other authors (Gustavsoon, 1979) reported that the female offspring of sires carrying tR1/29 in heterozygosis present a reduction in fertilization, probably due to early embryonic mortality. For others (Schumutz et al., 1991), it would be the embryonic loss and not the failures in fertilization are the cause responsible for the decrease in fertility. The authors also observed that the rate of embryos with unbalanced karyotype was higher when the female was a carrier of tR1/29 compared to the carrier bull.
Therefore, due to the potential risk of dissemination of large-scale chromosomal abnormalities capable of adversely affecting the productive or reproductive performance of animals, some countries such as France has established standards for the systematic monitoring of the karyotype of breed stock that are found in centers of Artificial Insemination. In that country, Ducos et al. (2000) reported the elimination of 10.9% of the bulls (Blond d’Aquitane) destined to the collection of sperm due to the fact of being carriers of chromosomal anomalies, mainly of tR1/29. Another way to reduce the incidence of chromosomal abnormalities in a livestock population would be the systematic discarding of carrier animals, as previously suggested (Pinheiro et al., 1995). Based on the cytogenetic tests in a herd of Pitangueiras cattle, the authors observed a reduction in the incidence of tR1/29 from 30.5% to 8.5%, after 7 years of selection, which simultaneously resulted in the reduction in the number of services per conception (from 2.2 to 1.8) and the interval between deliveries (from 443.2 to 412.4 days). Chi-square analysis, comparing the incidence of translocation in males and females, showed significance (2%) for males with a higher frequency of RR (Gomes, 2009; Raudsepp & Chowdhary, 2016) females, which are subject to a greater selection of subfertility, can be discarded when the carriers of the anomaly appear.
Senepol cattle show a high incidence of chromosomal abnormalities tR1/29. For this reason and in order to avoid or reduce the spread of genetic anomalies, it is recommended the systematic monitoring of carrier animals, in particular, of the bulls that are in sperm collection centers, replacement bulls for natural mating and female embryo donors due to their capacity to multiply unwanted genetics in the rest of the population.