应用多重PCR技术检测性器官异常患者的性别决定基因

采用针对人SRY基因及X与Y染色体同源序列的两对引物进行多重聚合酶链反应技术检测204例新生儿脐血DNA,男性均显示590bp、 355bp及280bp 3条扩增带,女性只有590bp1条扩增带,性别鉴定准确率为100%。又检测47例性器官异常患,13例社会性别为女性者只出现590bp扩增带,与核型性别结果一致。 34例社会性别为男性者中,有2例SRY基因检测结果阴性,只出现590bp带,核型为46,XX,证明这两例患者社会性别不相符,经病理证实后应诊断为女性假两性畸形。 Abstract:This investigation adapted multiplex PCR technique with two pairs of primer to determine sex according to the human SRY gene and X,Y chromosome analogical sequence.We detected bellybutton blood DNA from 204 newborns.Male revealed three bands:590bp,355bp and 280bp,and females only had one band which was 590bp,the accuracy of sex determination was 100%.47 sexual abnormal patients were tested.The results showed that 13 cases,whose social sex were female,had one 590bp band which consistent with their chromosome type sex.In the rest 34 cases,whose social sex were male,2cases showed,on the contrary,only one 590bp band and their chromosome type were 46,XX.This proved that the two patients did not consistent with their social sex,and pathological analysis showed that they were female pseud-hermaphroditism.     

黑麂Y染色体的鉴别和Sry基因的克隆及定位

以流式细胞仪分离小麂(Muntiacus reevesi)Y染色体和黑麂(Muntiacus crinifrons)Y1,Y2,X+4和1号染色体,利用DOP-PCR技术富集了分离的各单条染色体。然后,将小麂的Y染色体的DOP-PCR产物经Cy3标记后直接作为涂染探针,应用染色体涂染技术与雌雄黑麂的核型标本进行杂交,确认了黑麂真正的Y染色体为Y2染色体。再以黑麂的Y1,Y2,X+4和1号染色体的DOP-PCR产物为模板,用人的特异性的SRY（sex determining region of the Y chromosome）基因引物对其进行扩增,结果表明黑麂只有Y2染色体出现了SRY扩增片段。然后扩增产物克隆和测序,比较它与人的同源性,初步把黑麂的Sry基因定位在Y2染色体上。最后提取雄性黑麂的基因组DNA,并用同一对引物对其进行扩增,亦得到Sry基因的片段,对此扩增片段进行克隆,测序,结果表明其与Y2染色体得到的Sry基因片段完全一样,与人SRY基因的同源性均为83%。 Abstract：The single Y chromosome of Muntiacus reevesi and Y1,Y2 ,X+4,1 chromosome of Muntiacus crinifrons were obtained by flow-sorting ,then they were amplified through DOP-PCR . After that, the metaphase karyotype of Muntiacus crinifrons were painted by using the product of the DOP-PCR of the Y chromosome of Muntiacus reevesi as a special probe and the result showed that Y2 chromosome was the real Y chromosome of Muntiacus crinifrons. Secondly the product of the DOP-PCR of Y1,Y2,X+4,1 chromosome of Muntiacus crinifrons were used as the templates of the next amplification using the special primer devised according to the human SRY gene .One band was obtained only from Y2 chromosome, then it was cloned to the T-vector and sequenced. The Sry gene sequence of Muntiacus crinifrons was acquired and the conclution was that there are 83% homology between the human and Muntiacus crinifrons. It was testified that in all mammal Sry gene is consertive. On the other side the Sry gene was located to the Y2 chromosome of the Muntiacus crinifrons.     

利用差减杂交分离具有进化保守性的睾丸表达的基因

The mechanisms of sexual development and regulation display extraordinary diversity between phyla. However, recent studies show that there must be underlying evolutionary conservatism between different mechanisms. In order to examine evolutionary conservatism in molecular mechanisms of sex determination, the natural sex reversal process of the rice field eel (Monopterus albus) was used to identify conserved functional genes involved in sex determination and differentiation. Firstly, a mini cDNA library was constructed from the cDNAs expressed specifically in the human testis. The cDNA library was screened with first strand cDNA probes from the intersex gonads of rice field eels. Secondly, likely positive clones were arrayed onto two nylon membranes, which were then hybridized with the first strand cDNAs probes from rice field eel intersex and female gonads respectively. Two positive clones were obtained, which showed obvious differential expression between intersex and female gonads. DNA sequencing analysis reveal that the insert fragments of these two clones were 895 bp and 596 bp long respectively. BLAST results indicate that the 895 bp fragment is the 3′ part of the No.AJ011779 gene, located on 6q21, encoding a homologue of yeast Sec63p protein. An interesting finding is that one member of the human Sec gene family, Sec61γ , is expressed in the testis and involved in the process of testis development. The 596 bp fragment was 97% identical with the No.BG723880 EST, located on clone RP11 12M5 of chromosome 1 This needs further study. We infer that these two genes should also play a role in sexual development.     

羊驼染色体核型及其G分带的研究

为了从选种、杂交改良、疾病诊断以及性别决定的遗传机制等方面为羊驼的繁育与推广提供更为有效的细胞遗传学资料,本试验采用外周血淋巴细胞培养法及胰酶-EDTA法分析了23只胡阿基亚型羊驼（Huacaya alpaca,雌20只,雄3只）的染色体核型及其G-分带,结果表明：羊驼二倍体染色体数目为2n=74,雄性羊驼核型为74,XY;雌性羊驼核型为74,XX。其中,1～20对常染色体为亚端着丝粒染色体,21～36对常染色体为亚中着丝粒染色体和中着丝粒染色体,X为中着丝粒染色体,Y为端着丝粒染色体。G-带分析表明,羊驼G带明暗相间,显现出不同的带纹,且羊驼每对染色体都有其独特的带纹特征,其带纹数目和精细程度随着染色体长度的增加而增加。Abstract: Blood samples from 23 Huacaya alpacas, 3 males and 20 females, were used to study chromosomes and karyotypes, so as to provide some effective cytogenetic bases for the selection, improvement by crossing, disease diagnosis of alpacas, and genetic mechanisms of sex determination. Peripheral blood lymphocyte culture was used to prepare chromosome. A method of trypase-EDTA was used for G-banding. The results showed as follows: The number of diploid chromosomes was 2n=74, with the karyotype 74, XY and 74, XX for males and females respectively. Thirty-six homologous pairs of chromosomes were autosomes, in which chromosomes pairs No.1 to No.20 were acrocentric-subterminal and No.21 to No.36 metacentric-submetacentric. And X chromosome was metacentric, Y chromosome telocentric. The analysis of G-bands showed that bright and dark bands appeared by turn. It showed different bands. And every pair of chromosomes had its distinct band, and the longer the chromosomes, the more the number of bands, and the more clear the bands.     

Variation in chromosome number in the seedling progeny of a somaclone of Paspalum dilatatum

The somaclone,C39,derived by tissue culture from the obligate apomict Paspalum dilatatum cv Raki(2n=50),had 50 chromosomes and a karyotype apparently identical to Raki.SC2 seedlings of C39 showed a high degree of phenotypic variation which was often associated with increased chromosome numbers,but some of the variant seedlings were karyotypically indistinguishable from Raki or C39.Plants with increased chromosome numbers exhibited a high degree of intraplant chromosome variation(aneusomaty).In one of the SC2 seedlings,the chromosome number of root tip cells varied from 58 to 82 and in several other seedlings the range was more than 10.The results suggested that the ability to form seed apomictically was much reduced in C39 and that this plant showed some capacity for sexual reproduction and the resulting seedlings,with a chromosome number of about 70,were genetically unstable.Of 11 SC2 seedlings examined cytologically,6 did not produce any viable seed.Seedlings grown from seed of the remaining 5 plants showed that aneusomaty persisted in the SC3 generation.SC3 seedlings which were phenotypically similar to their maternal parent showed a similar range of chromosome numbers to that parent.Some of the SC3 seedlings exhibited an even wider range of chromosome numbers(e.g.56-136),and these plants were all dwarfs.     

Sex differences in morphine-induced behavioral sensitization and social behaviors in ICR mice

Gender and genetic strain are two prominent variants that influence drug abuse. Although certain sexrelated behavioral responses have been previously characterized in ICR mice, little is known about the effects of sex on morphine-induced behavioral responses in this outbred strain. Therefore, in this study, we investigated the sex differences of morphine-induced locomotion, anxiety-like and social behaviors in ICR mice. After morphine or saline exposure for four consecutive days(twice daily), increased locomotion, more time spent in the central area, as well as attenuated rearing and self-grooming behaviors were found in morphine-treated females in an open field; no differences were found in locomotion and the time spent in the central area between male and female controls. When interacting with the samesex individuals, female controls were engaged in more social investigation, following, body contacting and self-grooming behaviors than controls; morphine exposure reduced contacting and self-grooming behaviors in females; in contrast, these effects were not found in males. These results indicate that female ICR mice are more prosocial and are more susceptible to morphine exposure than males.     

大鳞副泥鳅ZZ/ZW型性别决定的细胞遗传学证据

大鳞副泥鳅是鲤形目、鳅科的鱼类。其2n数为48,核型组成为12m+4sm+32 t(雄性),11m+5sm+32t(雌性)。根据银染带和C带特征分析,证实大鳞副泥鳅为ZZ/ZW型性别决定。Z染色体为中部着丝粒染色体,在其长臂端部有Ag -NOR存在。 W染色体为亚中部着丝粒染色体,在其长臂末端也有Ag-NOR存在,同时还有一深染的居间C带,这是W染色体独有的带纹特征。 Abstract:Paramisgurnus dabryanus belongs to Cypriniformes,Cobitidae.Its 2n is 48.The karyotype formula is 12m+4sm+32t(in male),11m+5sm+32t(in female).According to the Ag-NORs band and C-band patterns,we consider that its sex determination is of ZZ/ZW type.The Z chromosome is a metacentric one with Ag-NORs located on its arm end.The W chromosome is a submetacentric with Ag-NORs located on the terminal of its long arm.There is a darkly stained C-band on the long arm of W chromosome.This band is a characteric of the W chromosome.     

Reconsidering sex differences during place learning in tungara frogs

In tungara frogs, female mate choice requires remembering the location and/or calls of preferred males who advertise from fixed positions within a breeding pond. A previous study found that, when solving a place discrimination task in the laboratory, female tCingara frogs were able to learn a visual cue to solve the task, whereas males were not. In that task, male performance appeared to be inhibited, in part, by their attempt to use egocentric cues. We tested whether the sex difference in place learning previously reported would generalize to other training parameters with different cues available by eliminating the potential to use egocentric cues and increasing the number of trials per day. As before, frogs were given a choice between a red or yellow door, one of which led to shelters and return to their home cage. In the current testing conditions, we detected a preference for the red door;thus, we only considered frogs rewarded to the yellow door. Training was associated with an in crease in correct choices and an in creased preference for the yellow door. However, there was no evide nee for a sex d iff ere nee in learni ng. In summary, un der the curre nt training conditions, we fou nd that the appare nt female advantage in place learning was no Ion ger evide nt. Future studies that in vestigate sex d iff ere nces in cue pref ere nee and/or ability to switch among cues will further illuminate the conditions under which sex differences in learning are manifest in tungara frogs.     

Tumor suppressor gene PTEN affects anoikis via both PKB and FAK in human lung carcinoma cell lines

PTEN was identified in 1997 as a new tumor suppressor gene on human chromosome 10q23, deletions and mutations of this gene were associated with a variety of human cancers such as glioblastoma and prostate cancer. In this study Northern blot analysis was carried out and one major band at 2 kb region was observed in all 8 HLCC samples, consistent with previous reports. The result showed that the PTEN gene were expressed and its mRNA level similar in all cell lines tested. To determine whether the PTEN mRNA level reflects the parallel level of protein, the level of PTEN protein was examined by Western blot.PTEN protein level was high in H460 and detectable in A549, A4, A7, L1 cells, not detectable in 95C, 95D, A1     

泰和乌骨鸡的核型与带型研究

采用外周血淋巴细胞培养--空气干燥法,对泰和乌骨鸡染色体核型和带型进行了研究。结果表明：泰和乌骨鸡体细胞染色体数目2n=78,染色体基本臂数AF=90,1、9号染色体及Z、W性染色体为中央着丝粒（m）染色体,2、4、7号染色体为亚中央着丝粒（sm）染色体,3、6、8、10号染色体为端着丝粒（t）染色体。G带研究表明：前10对大型染色体可分为29个区,190条带。C带处理发现,所有母鸡分裂相中W性染色体都出现C带并整条深染。Ag-NORs研究发现：Ag-NORs常定位于1、2号常染色体短臂和Z性染色体短臂端部;Ag-NORs数目分布范围为1～6;平均每个细胞的Ag-NORs数在雌、雄鸡中分别为2.94和2.96。 Abstract:This study made the chromosome slides of Taihe Silkies by the peripheral blood lymphocyte culture-drying method,and analyzed Taihe Silkies karyotype and band pattern.The results are as follow:The diploid chromosome number of Taihe Silkies was 2n=78,the basic number of chromosome arms was AF=90 and the sex chromosome type was ZZ(♂)/ZW(♀).According to the measured relative length,arm ratio and centromeric index,the first 10 pairs of macro-chromosomes are described as follows:No.1 ,9 and Z,W chromosomes were metacentrics,No.2,4,7 were submetacentrics,and No.3,6,8,10 were telocentrics.Studies on Taihe Silkies′ G-band showed that the first 10 pairs of macro-chromosomes can be divided into 29 zones and 190 bands.Being treated by C-banding technique,a totally dark-stained and easily indentified W-chromosome always showed up in the female metaphase configurations.Ag-NORs were located in the short arms′ telomere of No.1,2 euchromosomes and Z sexchromosome,the Ag-NORs number varied from 1-6. -NORs     

蛋鸽早期性别DNA分子鉴定

蛋鸽早期性别的鉴定一直是制约蛋鸽业发展的瓶颈问题。在本研究中,我们采用PCR扩增和琼脂糖凝胶电泳检测的方法,对20日龄期的100只蛋鸽的性别连锁基因染色体解旋酶DNA结合基因CHD(chromo-helicase DNA binding)进行跨内含子扩增检测。蛋鸽早期DNA分子电泳检测结果与成年后的真实性别核对结果表明:所有雌性蛋鸽扩增结果含有两条带,其大小分别为280bp和250bp,雄性蛋鸽仅有一条280bp的条带;泊松统计分析发现,蛋鸽早期DNA分子判定结果达到统计学上显著水平,可以作为蛋鸽早期性别鉴定的一种方法。本实验实现了以DNA分子为基础的早期蛋鸽性别鉴定,将对蛋鸽业早期的饲养成本降低和早期雄性蛋鸽的淘汰起到准确的指导作用。     

Identification of a RAPD marker linked to sex determination in Pistacia vera using bulked segregant analysis

The Random Amplified Polymorphic DNA (RAPD) technique was used to amplify DNA segments, with the objective of finding markers linked to sex determination in the dioecious species, Pistacia vera. Progenies from two female parents pollinated by a common male parent were studied. Two bulks of DNA were made in each cross, one from males and one from females, by pooling an equal weight of fresh leaves from each individual contributing to the bulk prior to DNA extraction. DNA was extracted from each bulked sample and from each of the contributing individuals. DNA was also extracted from 14 cultivars of P. vera and from 94 open-pollinated, fewweeks-old P. vera seedlings of unknown sex. Seven hundred different decamer oligonucleotide primers were used to perform DNA amplification, with 1 of these (OPO08) producing a 945 bp amplification band that was present only in the bulked female samples and absent in the bulked male samples of the two crosses. The relationship between band presence and female sex expression was conserved in every individual obtained from the two crosses and in the 14 cultivars unrelated to the crosses. We propose that this band is tightly linked to the gene(s) that control sex determination in pistachio. The OPO08₉₄₅ RAPD marker could be used in a breeding program to screen the gender of pistachio plants long before they reach reproductive maturity, resulting in considerable savings of time and economic resources. In order to verify that assumption we screened 94 additional seedlings with the OPO08 primer and obtained results consistent with a 11 male:female ratio.     

Identification of sex chromosomes in lake trout (Salvelinus namaycush)

In the male trout there is a difference in the quinacrine banding and C-banding patterns between the two homologs of the second largest chromosome pair. This chromosome is the only large submetacentric in the karyotype, making it easy to identify and suggesting that the sex chromosomes have become differentiated since the time of tetraploidization. In males one homolog has a medium-to-large quinacrine bright heterochromatic band on the end of the short arm, while the other lacks it completely. In females both homologs have medium-to-large quinacrine bright heterochromatic bands. Approximately half the progeny from every lake trout cross studied and half the eggs from every lake trout population examined were heteromorphic for a difference in this chromosome band. Results from sexed fish, reciprocal F1 hybrids between brook trout and lake trout, and gynogenetic haploids are all consistent with the interpretation that chromosome 2 is the sex chromosome. These results suggest that the addition of heterochromatin to the X can be the first step in the inhibition of crossing over between the X and Y chromosomes required for sex chromosome differentiation.     

Two DNA sequences specific for the canine Y chromosome

Data are presented on the characterization of two nucleotide sequences found exclusively in the DNA of male dogs. In polymerase chain reactions (PCRs) of canine genomic DNA with a decanucleotide primer of arbitrary sequence (OP-W17), two nucleotide segments (650 and 990 bp) were amplified only from male samples, whereas a number of other fragments between 400 and 2500 bp in size were amplified from both male and female samples. The two male-specific segments were cloned and sequenced, and terminal 24mer oligonucleotide primer pairs were synthesized. PCR with these specific primer pairs resulted in amplification of the two male-specific sequences only from DNA samples of 34 male dogs; no product was amplified from 42 samples of females. A segment of the SRY gene previously localized on the Y chromosome could be amplified in DNA samples that had the two new sequences. Eco RI digested genomic male DNA when hybridized with the 650 bp or the 990 bp sequences, resulted in a single band for each on Southern analysis; DNA from females did not yield any bands. Comparisons between the two new sequences and the SRY gene segment revealed no homologies. We concluded that the two new sequences are specific for the canine Y chromosome and do not contain the short characterized segment of the SRY gene.     

Loss of heterozygosity on chromosome 5 in Iranian esophageal cancer patients

There is a high incidence of esophageal squamous cell carcinoma (ESCC) in Iran. Non-functionality of some tumor suppressor genes has been reported in esophageal cancer. Loss of heterozygosity on chromosome 5 has also been reported in esophageal carcinomas. We assessed loss of heterozygosity along a region of the long arm of chromosome 5 (5q), from 5q23.1 to 5q23.2, by PCR amplifying DNA fragments of tumor tissues from patients with ESCC and their corresponding normal samples. The PCR products were electrophoresed on 6% non-denaturing polyacrylamide gels, and band intensity was shown by silver staining. Of 40 patients with ESCC, 27, 25 and 36% of informative cases showed allelic losses at microsatellite markers D5S1384, D5S1478 and D5S1505, respectively. Two of the 40 patients studied had microsatellite instability at marker D5S1384. Based on the fact that loss of heterozygosity with more than 22% incidence for a specific marker cannot be regarded as a random event, we add support to previous reports concerning the presence of tumor suppressor genes in this chromosome region and that they affect esophageal cancer development. According to the data in NCBI UniSTS, the PCR product size of human DNA with primers of the D5S1505 marker ranges from 243 to 275 bp, containing about 20 repeats of the TAGA tetranucleotide, while the amplicon size of one allele of one of our cases was 207 bp, with about 10 repeats of the TAGA tetranucleotide, which would be the shortest sequence reported so far.     

CHARACTERIZATION OF GENETIC MARKERS LINKED TO SEX DETERMINATION IN THE HAPLOID‐DIPLOID RED ALGA GRACILARIA CHILENSIS1

Bulk segregant analysis, random amplified polymorphic DNA (RAPD), and sequence characterized amplified region (SCAR) methods were used to identify sex‐linked molecular markers in the haploid‐diploid rhodophyte Gracilaria chilensis C. J. Bird, McLachlan et E. C. Oliveira. One hundred and eighty 10 bp primers were tested on three bulks of DNA: haploid males, haploid females, and diploid tetrasporophytes. Three RAPD primers (OPD15, OPG16, and OPN20) produced male‐specific bands; and one RAPD primer (OPD12), a female‐specific band. The sequences of the cloned putative sex‐specific PCR fragments were used to design specific primers for the female marker SCAR‐D12‐386 and the male marker SCAR‐G16‐486. Both SCAR markers gave unequivocal band patterns that allowed sex and phase to be determined in G. chilensis. Thus, all the females presented only the female band, and all the males only the male band, while all the tetrasporophytes amplified both male and female bands. Despite this sex‐specific association, we were able to amplify SCAR‐D12‐386 and SCAR‐G16‐486 in both sexes at low melting temperature. The differences between male and female sequences were of 8%–9% nucleotide divergence for SCAR‐D12‐386 and SCAR‐G16‐486, respectively. SCAR‐D12‐386 and SCAR‐G16‐486 could represent degenerated or diverged sequences located in the nonrecombining region of incipient sex chromosomes or heteromorphic sex chromosomes with sequence differences at the DNA level such that PCR primers amplify only one allele and not the other in highly specific PCR conditions. Seven gametic progenies composed of 19 males, 19 females, and the seven parental tetrasporophytes were analyzed. In all of them, the two SCAR markers segregated perfectly with sexual phenotypes.     

Hairy cell leukemia: an analysis of the chromosomes of 26 patients.

We studied the chromosomes from 26 patients with hairy cell leukemia (HCL) to ascertain the frequency and types of consistent chromosomal abnormalities. Samples from 21 patients were obtained from peripheral blood cultures grown 24 and 48 h without phytohemagglutinin, or from bone marrow samples. Two male patients had similar, consistent abnormalities; one patient's karyotype was 46, X, +12; that of the second was 46, X, +C marker. In the latter case, the distal long arm of the C marker most closely resembled chromosome No. 12 from band q14 to q terminal, but the short arm and proximal long arm were of undetermined origin. Both karyotypes lacked the Y chromosome. Nine of the 21 patients had abnormalities in single cells. One patient had, in one sample, a single abnormal cell with an extra No. 3 and an extra No. 12 (48, XY, +3, +12), and in a later sample, a second cell of poor morphology which also could have been trisomic for No. 12. Another patient had one cell with an unusually bright short arm, as well as two cells, with different abnormalities, both involving the short arm of chromosome No. 1. The two patients with consistent chromosome abnormalities had rapidly progressive disease in spite of splenectomy, and their clinical course from the time of diagnosis was relatively short (5 and 7 months, respectively).     

Xp-duplications with and without sex reversal

Duplications in Xp including the DSS (dosage sensitive sex reversal) region cause male to female sex reversal. We investigated two patients from families with Xp duplications. The first case was one of two sisters with karyotype 46,XY, der(22), t(X;22)(p11.3;p11)mat and unambiguous female genitalia. The living sister was developmentally retarded, and showed multiple dysmorphic features and an acrocallosal syndrome. The second case was a boy with a maternally inherited direct duplication of Xp21.3-pter with the breakpoint close to the DSS locus. He had multiple abnormalities and micropenis, but otherwise unambiguous male genitalia. We performed quantitative Southern blot analysis with probes from Xp22.13 to p21.2 to define the duplicated region. Clinical, cytogenetic, and molecular data from both patients were compared with those of previously reported related cases. A comparison of the extragenital symptoms revealed no differences between patients with or without sex reversal. In both cases, the symptoms were non-specific. Among 22 patients with a duplication in Xp, nine had unambiguous female genitalia and a well-documented duplication of the DSS region. Two patients with duplication of DSS showed ambiguous external genitalia. From these data, we conclude that induction of testicular tissue may start in these patients, but that the type of genitalia depends on the degree of subsequent degeneration by a gene in DSS.     

DNA sexing in Humboldt Penguins (Spheniscus humboldti) from feather samples

Humboldt Penguins (Spheniscus humboldti) show little sexual dimorphism, and although males are usually heavier and larger than females, sexing by direct observation may be difficult, especially in young subjects. In this paper we evaluate the utility of the molecular approach, for sexing impuberal Humboldt Penguins from feathers. Firstly, a PCR test was used employing primers that amplify the homologous region of the CHD-W gene, unique in female, and the CHD-Z gene, occurring in the two sexes. The analysis of the PCR products showed a band of 370 bp in males and two bands of 370 and 380 bp in females. Additionally, to confirm these results, the PCR products were digested with HaeIII and Asp700 for RFLP analysis. Male PCR products showed two bands (310 and 60 bp) after digestion with HaeIII, and a unique band (370 bp) using Asp700, while all fragments obtained from females resolved into three bands using both HaeIII (380, 310 and 60 bp) and Asp700 (370, 270 and 110 bp), confirming the previous PCR sex determination. Results from these two different DNA-based tests were in accordance, in all cases, with sexes checked by preliminary cloacoscopy. Thus, it was found that the PCR method from feather samples alone is sufficient, reliable and without any risks for a rapid sexing in Humboldt Penguin. This non-invasive sexing technique can be useful at any age to verify the sex ratio in field populations and for gender identification in ex situ conservation programs.