Short Communication |
Corresponding author: Andréia Pires Amancio ( andreiaamancio5@gmail.com ) Academic editor: Nina Bulatova
© 2019 Andréia Pires Amancio, Sabrina Sara Moreira Duarte, Rafael Carneiro Silvafael, Alex Silva da Cruz, Danilo Conrado Silva, Claudio Carlos da Silva, Aparecido Divino da Cruz.
This is an open access article distributed under the terms of the Creative Commons Attribution License (CC BY 4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Citation:
Amancio AP, Duarte SSM, Silvafael RC, da Cruz AS, Silva DC, da Silva CC, da Cruz AD (2019) Banded karyotype of Nelore cattle (Bos taurus indicus Linnaeus, 1758). Comparative Cytogenetics 13(3): 265-275. https://doi.org/10.3897/CompCytogen.v13i3.36449
|
Chromosome banding techniques were applied and standardized to obtain karyotype characteristics for the first time in Brazil of Nelore cattle – Bos taurus indicus Linnaeus, 1758 – (bovine subspecies most prominent in Brazilian livestock). Blood samples were collected from the animals of the School of Agrarian and Biological Sciences of the Pontifical Catholic University of Goiás, two males and two females of pure breed. These samples were submitted to the cell culture method to study metaphase chromosomes. Chromosome banding techniques (C, G and NOR) revealed the karyotype architecture of Nelore cattle common with that of other breeds of zebu cattle formerly karyotyped. The diploid chromosome number was invariably normal, 2n = 60. C-banding revealed C-positive heterochromatin in centromeric regions almost in all chromosomes. G-banding presented the expected band pattern in the respective chromosome pairs in correspondence with the established chromosomal patterns for the species. Ag-staining for nucleolus organizer regions (AgNOR) was identified on the telomeric end of the long arm in 7 autosomal chromosomes. In this study we found more regions in chromosomes with staining than presented in the literature for the Bos indicus group (BIN). These NOR regions were repeated on the same chromosomes for the 4 animals studied.
AgNOR, Brazil breeds, Cytogenetics, Karyotype, Zebu
Nelore is an important bovine breed and well noted in Brazil for its meat production, body size and sturdiness. However, the meat industry has demanded products of higher quality. Thus, in the last 5 years, specifically with respect to meat production, the cross between Angus (taurine) and Nelore (zebu) breeds has been growing in Brazil. Indiscriminate crossing of Nelore cattle may result in a dilution of the breed and a decline in their number which may result in complete genetic extinction. Consequently, the conservation of the original breed is necessary (
Despite this trend in the market, Nelore still comprises up to 80% of the national cattle of bovine breeds raised for meat, mostly due to its combination of productivity and adaptability to the tropics (
Cytogenetic studies are highly useful for genetic characterization and for effective conservation of the species seriously at risk of extinction (
Despite the extensive genomic investigation in cattle, not so many novelties are reported about bovine chromosomes that could be an excellent and inexpensive tool to provide important pieces of information useful for animal characterization, herd management, and evolutionary studies of breeds (
For correct identification of individual chromosomes, several banding techniques were developed, broadly divided into two categories: those that produce bands along the entire chromosome (Q, G, and R) and those that mark specific regions of each chromosome (C, T, or NOR) (
We are presenting in this work the necessary characterization of Nelore’s chromosomes using G-, C-, and NOR-branding methodologies.
Biological samples were collected from four animals (2 male, 2 female), products industrial breeding Nelore, belonging to the study station of the Faculty of Agrarian of Biological Sciences / Pontifical Catholic University of Goiás. The herd maintained at lots of 28 m² of pasture and fed with fodder twice a day. Both males were 28 months old, weighing about 430kg. Both females were 35 months old, weighing about 480 kg. Blood samples of about 3ml of peripheral blood from the external jugular vein of each animal were kept in vaccum tubes containing heparin to prevent blood clotting and cooled on ice until arriving at the laboratory. Conventional cytological techniques were applied adapted to local and laboratory conditions of peripheral blood culturing and chromosome preparation (
Cell culture was performed from 1ml of blood sample transferred into RPMI 1640 (Gibco RPMI 1640 Medium) (4ml), enriched with FBS (Fetal Bovine Serum, Gibco (1ml), PHA (Phytohemagglutinin, Gibco) and antibiotics (Penicillin G sodium salt, Sigma-Aldrich) (100U/μL). The cell suspension was stored in an incubator at 38 °C under 5% of carbon dioxide (CO2) for 71 hours. After this time, 75μl of colchicine (Colcemid, Gibco) was added and incubation continued for an additional 30 minutes. Subsequently, samples were transferred to a 15ml conical tube and centrifuged for 10 minutes at 1000rpm, and then the supernatant was discarded (leaving about 1ml of material in the tube). A total of 10ml of hypotonic solution (KCl at 0.075 M) was added into the tube and incubated for 35 minutes at 38 °C, 5% CO2. The cells were then fixed with Carnoy’s solution (3 parts of methanol to 1 part of acetic acid), fixation was performed for 10 minutes at room temperature and immediately centrifuged for 10 minutes at 1000rpm. The cell pellet was fixed by three successive washes with the fixative, until the material became clear. Fixed cells were maintained in a suspension with 5ml of fixative in the refrigerator until the time of chromosomal analysis.
The cell suspension was dropped on a microscope slide over a water-bath steaming at 60 °C. Slides were previously cleaned and degreased to guarantee adequate spreading of metaphases. Metaphase spreads were aged in the refrigerator for 2 days. Subsequently, the slides were soaked in 0.2N HCl solution for 10 min, rinsed in distilled water. DNA denaturation was carried out in a solution of 5% barium hydroxide for 15 min at 37 °C, slides were rinsed in distilled water at room temperature. After drying, the slides were stained with 10% Giemsa’s solution for 5 minutes (KaryoMAX Giemsa Stain Solution).
For the GTG banding, slides with the metaphase spreads were stored at room temperature for 7 days. After aging, slides were treated in 0.025% trypsin solution (Gibco) diluted in 4mL of PBS at 37 °C for 6–7 seconds. Afterwards, slides were stained in 5% Giemsa’s solution for 5 minutes (KaryoMAX Giemsa Stain Solution).
Ag-staining of NORs (Nucleolus Organizer Regions) was carried out after aging the slides for 2 days in a refrigerator. Subsequently, 2 drops of 50% silver nitrate (AgNO3, Sigma-Aldrich) and 2 drops of 2% gelatin diluted in 1% formic acid were added to the material and covered by a glass coverslip. The slide was then placed into a humid chamber at 65 °C protected from light for a time ranging from 3 to 5 minutes until the slide surface showed a copper-like color.
Metaphases were captured using white light microscopy with the aid of a karyotyping station consisting of a microscope Axioplan 2 Imaging (Carl Zeiss, Alemanha) with motorized platinum controlled by Metafer 3.4.0 software (Metasystems Corporation, Germany). Captured images were analyzed using IKAROS (Metasystems Corporation, Germany).
Twenty metaphases of each animal were analyzed. The lengths of chromosomes in micrometers were measured in mitotic metaphase of male and female cells. Karyotype symmetry/asymmetry index (S/AI), the mean length of short arm (Ls), length of long arm (Ll), total length of arm (LT), arm ratio (AR-long/short chromosome), centromeric index (CI) and type of chromosome and formula were estimated according to
All chromosomes measurements were translated by computation using software IKAROS (Metasystems Corporation, Germany), after pairing each pair of homologs in G- banded karyotype. Homologs were paired for all four animals, according to sex, and the final chromosome measurement corresponded to arithmetic mean of individual estimation for each chromosome.
The study of Brazilian Nelore cattle adds to the list of the zebu (B. t. indicus) breeds so far karylogically investigated. The diploid number in all 4 studied animals was found to be 60, consisting of 29 pairs of autosomes and one pair of sex chromosomes – the karyotype constitution, common to domestic cows of taurine/B. taurus and zeburine/B. indicus origin and established in all former reports (
The Brazilian Nelore line originated from Ongole, a predominant breed in India (
The measures for autosomes did not vary between male and female. Therefore, here we show the corresponding figures for the males in order to show all autosomal and both heteromorphic sex chromosome for the studied subspecies. All chromosomes measurements were represented in Table
The average measurements and arm ratio of the entire chromosome complement for male Bos taurus indicus Linnaeus, 1758, after homologs were paired up following GTG- banding.
Chromosome pair | Total length (μm) | Long arm (μm) | Short arm (μm) | Arm ritio (long/short) | Centromeric index | Chromosome type |
---|---|---|---|---|---|---|
1 | 67,9 | 61,7 | 6,2 | 9,952 | 9,131 | A |
2* | 60,9 | 54,3 | 6,6 | 8,227 | 10,837 | A |
3* | 57,8 | 51,8 | 6 | 8,633 | 10,381 | A |
4* | 56,7 | 50,8 | 5,9 | 8,610 | 10,406 | A |
5 | 53,5 | 48,4 | 5,1 | 9,490 | 9,533 | A |
6 | 52,6 | 46,9 | 5,7 | 8,228 | 10,837 | A |
7 | 49,9 | 44,6 | 5,3 | 8,415 | 10,621 | A |
8 | 50,5 | 45,1 | 5 | 8,352 | 10,693 | A |
9 | 49,5 | 44,4 | 5,1 | 8,706 | 10,303 | A |
10 | 47,4 | 42,2 | 5,2 | 8,115 | 10,970 | A |
11* | 45,6 | 40,6 | 5,0 | 8,120 | 10,965 | A |
12 | 42,2 | 37 | 5,2 | 7,115 | 12,322 | A |
13 | 38,7 | 33,1 | 5,6 | 5,911 | 14,470 | A |
14 | 40,3 | 35 | 5,3 | 6,604 | 13,151 | A |
15 | 38,5 | 33,5 | 5,0 | 6,700 | 12,987 | A |
16 | 38,6 | 33 | 5,6 | 5,893 | 14,508 | A |
17 | 37,8 | 32,2 | 5,6 | 5,750 | 14,815 | A |
18 | 35,6 | 30,2 | 5,4 | 5,593 | 15,169 | A |
19 | 33,5 | 28 | 5,5 | 5,091 | 16,418 | A |
20 | 32,3 | 26,4 | 6 | 4,475 | 18,266 | A |
21 | 31,7 | 26,5 | 5,2 | 5,096 | 16,404 | A |
22 | 32,2 | 26,9 | 5,3 | 5,075 | 16,460 | A |
23 | 31 | 26,1 | 4,9 | 5,327 | 15,806 | A |
24 | 29,5 | 24,2 | 5,3 | 4,566 | 17,966 | A |
25* | 28,2 | 23 | 5,2 | 4,423 | 18,440 | A |
26 | 26,5 | 21,3 | 5,2 | 4,096 | 19,623 | A |
27 | 26,6 | 21,5 | 5,1 | 4,216 | 19,173 | A |
28* | 25,3 | 20,2 | 5,1 | 3,961 | 20,158 | A |
29 | 22,6 | 18 | 4,6 | 3,913 | 20,354 | A |
X | 66,6 | 44,2 | 22,4 | 1,973 | 33,634 | SM |
y | 29,4 | 23,7 | 5,7 | 4,2 | 19,388 | A |
The chromosome pairs indicate evidence of interchromosomal asymmetry. S/AI for Nelore karyotype was 2.97 and 2.98 for female and male animals, respectively, classified its karyotype between symmetric and asymmetric, most likely due to the presence of the X chromosomes. The karyotype formulae were also different for male and female Nelore cows, corresponding, respectively, to 1SM+59A and 2SM+58A. For additional discussion about the importance to know the values of the karyotype symmetry/asymmetry in higher animals, readers are strongly advised to read the work of
With respect to sex chromosomes in Nelore, in our results the ratio between X and Y chromosomes was 2.45 indicating a remarkable in level of allosomic heteromorphism, a common observation among animals harboring XY sex determination mechanism, leading to an evolutionary stronger reproductive isolation (
Chromosome X is relatively a few larger than chromosome 1, the largest acrocentric chromosome in the bovine karyotype. X/1 proportion is close to one (1,1µm). On the other hand, Y chromosome is close in size to autosomal chromosomes 24 (BIN), with an average size of 29,5µm then compared to the smallest acrocentric chromosome 29 (BIN), Y/29 proportion was found to be 1,3. Due to its acrocentric morphology and its small size, the Y chromosome of Nelore can easily be confused with several other small autosomal chromosomes that are also acrocentric. Here we report difficulty in the identification of Y(BIN) when relying only on Giemsa staining, just as reported by
However, C-, GTG-, and NOR-banding provided a better morphological characterization of all chromosomes, including Y chromosome in Nelore, facilitating the proper differentiation of autosomal and sexual chromosomes for the breed.
In our case, Y is acrocentric, as in the first descriptions of the zebu karyotype (
C-banding demonstrated dark bands (C-positive) on all centromeric region of autosomes, analyzed in the bovine material which showed well-defined heterochromatin blocks.
The GTG banding provides alternated light and dark bands on the chromosomes, the distribution of these bands is different for each chromosome, facilitating the identification of the homologous pairs.
Thus, in the GTG banding analysis the haploid set of Nelore cattle consists of 29 autosomes and 1 sexual pair including X and Y chromosome. The pair composition presented in Figure
GTG-banding profile for the pairing of the chromosomes of the Nelore karyotype. A female (XX) B male (XY).
The nucleolus organizer regions on the long arm of the pairs of autosomal chromosomes 2, 3, 4, 11, 22, 25, and 28 by NOR-banding technique in female and male respectively. A female (XX) B male (XY).
In spite of the diverse qualities that the GTG- banding provides, it requires an extended time of 7 days for preparation of the slides, along with the obtaining of metaphases in good condition for the analysis of the chromosomes.
The NOR technique, initially described by
The seven nucleolus organizing regions (NORs) were located on the autosomal chromosomes of cattle Nelore. The four animals that made up the sample group in this study presented the NORs in the same chromosomal pairs, which are the autosomal pairs 2, 3, 4, 11, 22, 25 and 28 shown in figure 3. In contrast,
Jantarat and colleagues (2009) performed the banding in C, G and NOR Thai’s native cattle (Bos taurus indicus) and the results were compared to our study. There was a difference in the result of the NOR technique, the Thai’s native cattle presented NOR in three pairs of autosomal chromosomes whereas for the studied Nelore breed, seven pairs of chromosomes presented the silver placement in the telomeric region.
About 80% of the Brazilian herd is composed of zebu breeds (Bos t. indicus), animals with more rusticity and easy adaptation to the predominant environment in the country (
There was no cytogenetic characterization by banding techniques (C-, GTG- and NOR) for the Nelore Brazilian breed. For the animals of this study, the C banding made possible an exact identification of the acrocentric chromosomes. The technique GTG-banding provided the correct characterization of the pairs homologues, especially the autosomal chromosomes of cattle that are all acrocentric. In particular, the in this study it was possible to identify nucleolus organizing regions in other chromosomes, different from what was already known for subspecies Bos t. indicus.
The variation in the composition of the chromosomes that make up the national herds, especially those in this study, can be explained by the many preceding intersections and inbreeding. This management practice is commonly used to increase the herd of animals with favorable traits. Therefore, our observation can be in correspondence to the work of
In addition to the banding techniques excellent for studies of morphology and chromosome classification, instead of new cytogenetic methodologies, such as Fluorescent In Situ Hybridization (FISH) and High Resolution Banding, can be used to understand chromosomal rearrangements and to clarify phenomena that may be related to the integrity of bovine genetic material (
The authors are grateful to the School of Agrarian and Biological Sciences of the Pontifical Catholic University of Goiás (PUC-Goiás) for authorizing the collection of bovine samples. We are also grateful for both Replicon Research Group of Pontifical Catholic University of Goias and Human Cytogenetics and Molecular Genetics Laboratory of Health Secretary of Goias State for logistical support for the execution of this study. The study was funded by the Coordenadoria de Aperfeiçoamento de Ensino Superior (CAPES) and from the Fundação de Amparo à Pesquisa do Estato de Goiás (FAPEG).