THE JOURNAL OF EXPERIMENTAL ZOOLOGY 280:217–219 (1998)

© 1998 WILEY-LISS, INC.

Sex Determination of Parrots Amazona a. aestivaand Amazona amazonica Using the PolymeraseChain Reaction

KEIKO KUSAMURA DE MATTOS,* SÍLVIA NASSIF DEL LAMA,ANA RITA CHERMAN SALLES, MERCIVAL ROBERTO FRANCISCO,ADRIANA MEDAGLIA, ALESSANDRA MOREIRA TOMASULO,FAUSTO MOREIRA SILVA CARMO, JAIRO MARQUES CAMPOS PEREIRA,AND MARCOS ANTÔNIO DE OLIVEIRADepartment of Genetics and Evolution, São Carlos Federal University, SãoCarlos, São Paulo, CEP-13565905, Brazil

ABSTRACT A method was developed for sex determination in two parrot species (Amazona a.aestiva and Amazona amazonica). A protocol is described for direct PCR amplification from parrotwhole blood using primers designed for Xho I repetitive regions of chicken W-chromosome. A W-specific fragment (approximate size of 124 bp) was amplified only in six DNA-female samples. Noamplification was observed in 19 male samples. Complete correspondence was observed betweenour sex identification results and those obtained with endoscopy, karyotype analyses, and behav-ioral observations. J. Exp. Zool. 280:217�219, 1998. © 1998 Wiley-Liss, Inc.

Grant sponsor: PPG-GEV-CAPES-CNPQ.*Correspondence to: Keiko Kusamura de Mattos, Departamento

de Genética e Evolução, Rodovia Washington Luís, Km 235, CEP-13565-905, Brazil. E-mail: dsdl@power.ufscar.br

Received 26 August 1997; Accepted 28 January 1998

External morphology does not permit sex de-termination in most Psittacidae species. Amongparrot species, Amazona albifrons is a rare ex-ception because its sexual dimorphism is evidentin its plumage pattern (Sick, ’97). Sex identifica-tion is useful for breeding in captivity, and theearlier the application of the procedure, thegreater the chances for reproduction.

Several methods have been described for sexdetermination in birds. Some are based on mor-phological analyses of ovaries and testicles by en-doscopy (Kummerfeld, ’90) or by ultrasonography(Hildebrandt et al., ’95). Because females are het-erogametic (WZ) and males homogametic (ZZ),bird sexing can be determined by karyotype analy-ses (Fillon and Seguela, ’95) or by flow cytometry(Tiersch and Mumme, ’93). DNA W-specific crosshybridization (Miyaki et al., ’92), and PCR testbased on primers specific to the CHD gene havealso been used for sex identification (Ellegren, ’96;Griffiths et al., ’96).

Methodology using amplification of a W-specificsequence by polymerase chain reaction was de-scribed for embryo sex diagnosis in domestic fowl(Gallus g. domesticus) by Petitte and Kegelmeyer(’95). The amplified sequence is included in theXho I family of repetitive elements which com-prise about 60% of the W chromosome in this spe-cies (Kodama et al., ’87; Saitoh et al., ’91).

We report here a method of sex identification

in two species of parrots based on amplificationof a W-specific sequence using primers designedfor chicken repetitive Xho I region. The procedureis simple and quick and because it requires onlyminute blood samples, it is suitable for use inyoung animals.

EDTA blood samples were collected from 19 Ama-zona a. aestiva and from six Amazona amazonica.Thirteen animals were sex-identified previouslythrough endoscopy (two males and one female),karyotyping (nine males and four females), and re-productive behavior (eggs posture, two females).

DNA was prepared according to a method de-scribed by Khatib and Gruenbaum (’96). Bloodsamples were diluted in NaOH 50 mM in the pro-portion 1:4 (v/v). Lysed cells were boiled for 10min, neutralized with Tris-HCl 1M pH 8.0 in theproportion 1:1 (v/v), and were diluted in sterilewater in the proportion 1:3 (v/v). Amplification wasperformed in 25 µl of final volume. This reactioncontains 5 µl of diluted blood (0.5–1.5 µg of DNA),2.5 mM of each primer (Xho I AACTACCACTT-TTCTCACGG and Xho II TTCAGAGTGATA-ACGCATGG Kodama et al., ’87), 1.25 mM of each

218 K.K. DE MATTOS ET AL.

one of the dNTPs, 1 Taq polymerase unit andbuffer with MgCl2 2.5 mM, KCl 50 mM, Tris-HClpH 8.3 10 mM, and 0.1 mg/ml of bovine serumalbumin. Cycling was done in a Perkin Elmerthermocycler with five cycles of 94°C (30 sec), 47°C(30 sec), 72°C (30 sec), followed by 35 cycles of94°C (20 sec), 47°C (20 sec), and 72°C (20 sec).The total volume of PCR reaction was loaded di-rectly into wells of 2% agarose gel stained withethidium bromide. After electrophoresis, bandscorresponding to amplified fragments were re-vealed under ultraviolet light.

Figure 1 shows the electrophoretic pattern ofPCR-products obtained after amplification of par-rot DNA with primers for chicken Xho I repetitiveelement. An amplified fragment of approximately124 bp was observed in samples of previously sex-identified females in the two species. No amplifica-tion of the 124-bp fragment was observed in malesamples. By comparing sex-specific occurrence andintensity of the 124-bp fragment we concludedthat it should be a W-specific fragment similar tothe 169-bp fragment detected in the female chickensample. Beyond the sex-specific product, a range ofnonsex-specific amplification products appearing asa smear was observed in female and male samples.There was a difference in the amplification of thesex-specific and nonsex-specific sequences. In themale samples, the nonspecific fragments weremore intense, whereas in female samples, they

were less intense because of the Xho I simulta-neous amplification.

Efficient amplification of Xho I sequences withprimers designed for chicken DNA in parrot spe-cies and similarities found between amplified frag-ments suggest that the W-region is apparentlyconserved in the studied genera. The Xho I DNArepetitive family in chickens consists of 0.7-kb and1.1-kb fragments, produced by enzymatic restric-tion with Xho I, repeated 14,000 and 6,000 times,respectively. The 0.7-kb internal repeat unit con-sists of tandem repetitions of a 21-bp sequence, ap-proximately. This region presents several peculiarcharacteristics, such as molecular configuration, latereplication, and high affinity with nuclear proteins(Suka et al., ’93), showing its relationship with Wchromosome and heterochromatinization. Similarrepetitive units have been described in turkey andpheasant W chromosome (Saitoh et al., ’89).

The proposed method showed accurate sex iden-tification in both parrot species studied. Completeagreement was observed between our results, ob-tained using PCR methodology, and sex diagnosisrealized by other methods (13 samples). The greatchromosome conservation observed among the vari-ous species of the Amazona genus (Duarte andCaparroz, ’95) supports the application of thepresent technique in other species of this genus.This methodology is less traumatic and more ad-equate for small birds than endoscopy. It requires

Fig. 1. PCR products seen through a 2% agarose gel us-ing crude DNA from blood samples following amplificationwith specific primers for Xho I repetitive element. Lane 1,ladder. Lanes 2–4, Amazona aestiva male, A. amazonica

male, A. aestiva male. Lanes 5–7, A. aestiva female, A.aestiva female, A. amazonica female. Lane 8, Gallus g.domesticus female.

SEX DETERMINATION IN PARROTS 219

less blood and is quicker to perform thanks to PCRutilization and the elimination of a conventionalDNA extraction stage. We believe that these char-acteristics make this technique more advantageousthan karyotyping and W-specific hybridization. Itis simple and requires inexpensive equipment andreagents, and thus is particularly well-suited foruse in developing countries, where most endangeredspecies of birds are found.

Until now, sex identification in Brazilian par-rots has been performed mainly by karyotypeanalyses (Duarte and Caparroz, ’95). In some spe-cies of the genus Aratinga, sex has been deter-mined by cross-hybridization with a minisatelliteprobe that was designed originally for humans andis W-chromosome specific (Miyaki et al., ’92). Thesex of the last wild Spix’s macaw was identifiedby PCR test using a feather as DNA-containingmaterial (Griffiths and Tiwari, ’95). The introduc-tion of our methodology will be useful in facilitat-ing studies on natural populations and in optimizingreproduction in captivity.

In the sample studied, the number of females wasless than the number of males (6 females/19 males).Excluding the hypothesis that this bias results fromsmall sample size, it can be supposed that the re-sults either reflect sexual proportion in nature orare the consequence of selective pressures in cap-tivity. Differences in sexual proportion betweennatural populations and those in captivity may beclarified through future research.

ACKNOWLEDGMENTSThe authors thank Fernando Siqueira Magnani

and Francisco Rogério Paschoal of Ecologic Park ofSão Carlos/SP for the supply of blood samples.

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