Comparison of vent sexing and polymerase chain reaction for reliable sex determination in guinea fowls

K. M. J. AHIAGBE*, B. B. KAYANG, A. NAAZIE, P. K. BOTCHWAY & A. A. AGBOLOSU (K. M. J. A.: Animal Research Institute, Council for Scientific and Industrial Research, P.O. Box AH 20, Achimota, Accra, Ghana; B. B. K., P. K. B.: Department of Animal Science, University of Ghana, Legon, Ghana; A. N.: Livestock and Research Centre, University of Ghana, Legon,Ghana.; A. A. A.: Department of Animal Science, University for Developmental studies, Tamale, Ghana) *Corresponding author's current email address: [email protected]

ABSTRACT The guinea fowl is an important poultry species with great economic potential in Africa. It is a monomorphic bird with less conspicuous sexual dimorphism. Inability to sex birds accurately early in their life imposes multiple challenges on breeding, conservation and production of these birds. Several methods have been employed for sexing monomorphic birds each with specific advantages and disadvantages. In the present study, sexes of 215 guinea fowls were determined by Polymerase Chain Reaction (PCR), Vent Sexing and determination of gonads. PCR was used to amplify a sequence homologous to EcoR1 fragment of 0.6 kb (EE0.6) using Universal Sex Primer (USP)1 and USP3 and internal control primers. Vent sexing could only diagnose 48.7 % of males accurately while it was able to diagnose females with an accuracy of 81%. Results from PCR was in complete agreement with sex indicated by gonads. Differences in results between PCR and vent sexing were significant (p < 0.05). Vent Sexing alone is not reliable for sexing guinea fowls prior to breeding decisions. Molecular sexing using the method described is recommended for accurate sex determination for breeders and researchers while future research is necessary to develop farmer friendly guidelines for reliable sex determination of guinea fowls.

Original Scientific Paper. Received18 Jul 16; revised 20 Apr 17

Introduction Due to its West African origin the indigenous The helmeted guinea fowl (Numida meleagris) population of this species is expected to be rich is an important poultry species in West Africa. It in adaptive genes representing a high degree of is native to Africa and derives its name from genetic variation.Guinea fowls are hardy, Guinea Coast where it is believed to have disease resistant (Jacob & Pescatore, 2013) and evolved (Crowe, 1978). In Ghana guinea fowl is hence suitable for low input agriculture common widely reared in the three Northern regions in tropical countries. where small holder farmers make greater Demand for guinea fowl always financial gains compared to raring chicken exceeds the supply in Ghana (Karbo & Avornyo, (Teye & Gyawu, 2001; FAO, 2014). It is an 2006). Therefore, development of the guinea important source of income and protein and fowl industry plays a vital role in strategies to plays important socio-cultural, economic and ensure food and nutrition security particularly in nutritional roles for its keepers (Dei & Karbo, the Northern part of the country. However the 2004; Avornyo et al., 2013; Dei et al., 2014). development of the guinea fowl industry as a

Ghana Jnl Agric. Sci. 52, 17-23 18 K. M. J. Ahiagbe et al., (2018) Ghana Jnl agric. Sci. 52, 17-23 sustainable means of livelihood is limited by character such as colour or rate of several challenges including inability to feathering has been introduced into the breeds accurately sex guinea fowls (Teye et al., 2000). (Kalina et al., 2012). Although this has been Moreki & Radikara (2013), described inability achieved in chicken, so far no such breed of to sex guinea fowls as a challenge for guinea fowls with genetically incorporated commercialization of guinea fowl production in autosex characters in which feather sexing can Africa. be performed accurately has been developed. The guinea fowl is a monomorphic bird Vent sexing on the other hand is a method with no sexually dimorphic features expressed popularised by a Japanese Professor, Kioshi in early stages (Awotwi, 1975; Teye et al.,2000). Maui and is the most widely used method for Inability to determine sex of monomorphic chick sexing in chicken (Cerit & Avanus, birds results in multiple challenges in breeding, 2007b). Teye et al., (2000), proposed the use of breed improvement and several other points in vent sexing for sexing guinea fowls from the the guinea fowl value chain. Unless the sex of sixth week of age. parents is known, guinea fowl keepers and Sex in birds is genetically determined. The breeders cannot maintain a good breeding stock female is heterogametic (ZW) while the male is for selection. Teye et al., (2000), reported that homogametic (ZZ). Several assays based on the inability to sex the birds early often leaves a Polymerase Chain Reaction (PCR) have been flock with none or few females after sexing described to determine the genetic sex of following methods used by farmers. It becomes monomorphic birds (Griffiths & Tiwari, 1993; difficult for breeders and farmers to introduce Griffiths et al., 1998; Itoh et al., 2001; Huang et the correct sex ratio to ensure high fertility and al., 2003; Jensen et al., 2003; Horng et al., 2006; hatchability. Cerit & Avanus (2007b), reported Cerit & Avanus, 2007a). These assays are based that difference in care cost of male and female on variations in alleles or sequences present in Z and time spent for reproduction causes consi- and W chromosomes including Chromohelicase derable financial losses in raring monomorphic DNA binding protein (CHD) gene (Griffiths et birds. Thus, it is important to develop reliable al., 1998) and EcoR1 fragment of 0.6 bp methods for early sexing that can be easily sequence (EE0.6) described first in the long arm adopted by farmers. of Chicken W chromosome (Ogawa et al., Several methods have been used to sex 1997). Itoh et al., (2001) cloned EE0.6 monomorphic birds with varying degrees of sequences from 12 different avian species and accuracy including examining the cloacal duct, proposed four forward and three reverse primers endoscopy, hormone determination, and cell that can be usefull in determination of sex in 36 gene analysis (Jones et al., 1984; Cerit & different species of Carinatae family using PCR Avanus, 2007b). Laparoscopy yields the most with a suitable forward and reverse primer pair. reliable results although it can be harmful or Differential banding patterns were observed in lethal to the birds (Saino et al., 1999; Cerit & male and female guinea fowls for PCR with Avanus, 2007a). Faecal steroid sexing is Universal Sex Primer (USP)1 and USP3 as sex expensive, largely depends on the method used specific primers together with internal control while yielding accurate results only in breeding primers that amplify a common sequence season with mature birds (Cerit & Avanus, present on both W and Z chromosomes. Ahiagbe 2007b). et al., (2012) successfully applied these primers Feather sexing and vent sexing are among for non-invasive sex determination of guinea the most used methods by breeders for chick fowls including keets. sexing. Feather sexing can only be performed in In this study we determined genetic sex of well-established breeds in which an autosex young guinea fowls using primers described by Comparison of vent sexing and polymerase chain reaction 19

Itoh et al., (2001) by using a modified protocol PCR for sex determination with DNA extracted from and Primers used were Universal Sex Primer 1 compared the genetic sex with sex suggested by (USP1) vent sexing, using gender determined by the (5'CTATGCCTACCACMTTCCCTATTGC3'), presence of respective gonads as the standard. U n i v e r s a l S e x P r i m e r 3 ( U S P 3 ) (5'AGCTGGAYTTCAGWSCATCTTCT3') Materials and methods together with internal control primers namely Animals F o r w a r d C o n t r o l P r i m e r ( C P E 1 5 F ) Two hundred and fifteen 16 weeks old guinea (AAGCATAGAAACAATGTGGGAC), fowls from Ghanaian local breeds whose sex R e v e r s e C o n t r o l P r i m e r ( C P E 1 5 R ) could not be differentiated externally were used (5'AACTCTGTCTGGAAGGACTT3') (Itoh et for the study. The birds were randomly selected al., 2001). PCR was performed in a final volume from a grower phase flock maintained at the of 20 µl containing 0.2 mM each of dNTPs, 2 Livestock and Poultry Research Centre µM of each target and control primers, 20 ng of (LIPREC) of University of Ghana. extracted feather DNA, 0.25 U Taq Polymerase, 1 x Green Go Taq flexi buffer and 1.5 mM Vent sexing and samples Magnesium Chloride (GoTaq® PCR Core Sexes of each of the 215 birds were determined System I, Promega Corporation, Madison, by both vent sexing and PCR at 16 weeks. Each USA) by modifying the protocol described by bird was tagged and vent sexed by observing Itoh et al., (2001). The PCR conditions used for presence of phallus in the cloaca by a trained the thermal cycler (Bio-Rad C1000 TM Thermal animal breeder according to the procedure cycler, Bio-Rad Laboratories, Inc. USA) were as described by Teye et al., (2000). Feather follows: Initial denaturation at 95°C for 10 samples were collected into sterile polythene minutes, followed by 35 cycles at 95°C for 30 bags and transported for laboratory analysis. seconds, annealing at 60°C for 30 seconds, After sampling birds were maintained up to elongation at 72°C for 60 seconds and finally maturity. At 24 weeks all surviving birds were 72oC for 5 minutes for final elongation (Ahiagbe euthanized to confirm the sex by presence of et al., 2012). PCR was performed at Molecular ovaries and testes. Genetics Laboratory, Department of Animal The results from vent sexing were compared Science, University of Ghana, Legon, Ghana. with the genetic sex determined by PCR and the PCR products were separated on 1.5% aga- presence of respective gonads. rose gel at 100 V for 30 min by electrophoresis in TBE buffer (1 M Tris base, 1M Boric acid, 0.02 DNA extraction from feathers M EDTA), stained with gel red and visualized About 4 mm length of the proximal follicular under a UV transilluminator. Size of bands were end of the calamus was cut into pieces with determined relative to 100 bp size marker sterilized scissors. DNA was extracted from (Promega Corporation, Madison, USA). Appea- feather follicles using DNeasy Blood and Tissue rance of sex specific bands was studied to deter- Kit (Qiagen Inc., Valencia, CA, USA) from all mine genetic sex. PCR was performed in dupli- 215 birds at Biotechnology laboratory of cates to veryfy the reproducibility. Animal Research Institute, Accra, Ghana. Results from vent sexing were later evalua- Extracted DNA was stored at -200C until use. ted based on genetic sex as the standard due to its 20 K. M. J. Ahiagbe et al., (2018) Ghana Jnl agric. Sci. 52, 17-23

accuracy reported elsewhere (Huang et al., Population structure according to PCR-based 2003; Horng et al., 2006; Cerit & Avanus 2007a; sexing and vent sexing Kalina et al., 2012) and confirmed during the Fig. 2 represents the population structure current study by the presence of respective according to genetic sex determined by PCR (a) gonads. and vent sexing (b). Out of the total of 215 birds sampled PCR detected 115 males (53.5%) and Statistical analysis 100 females (47.5%) while vent sexing Conformity or deviation of vent sex to/from identified 75 males (35%) and 140 females genetic sex was evaluated by performing Chi- (65%). squared test with Yates correction for continuity. Dichotomous Nominal correlation coefficient was also calculated to evaluate the correlation between genetic sex and gonadal sex and genetic sex and vent sex (Naiman et al., 1983). Percent error rate (number of birds wrongly assigned/total number of birds x 100%) was calculated for both PCR and vent sexing.

Results Fig. 2. Flock structure by PCR-based sexing (a) Genetic sex determined by PCR and rent sexing (b) When target sex specific genes were amplified using USP1 and USP3 in a single reaction with internal control primers CPE15F, Comparison of genetic sex with gonadal sex CPE15R with DNA extracted from feathers as All birds (115/215) identified as males by PCR template, samples of female origin produced 2 were characterized by the presence of testes bands of sizes 250 bp and 370 bp upon gel while the remainder (100/215) identified as electropho-resis while samples from males females contained ovaries when dissected after resulted only in a single band of 250 bp (Fig. 1). being euthanized. Seventy four of the females Results for genetic sex for all 215 birds were (74/100) showed developing follicles while the consistent in all duplicates. remaining twenty six (26/100) contained small non ovulating ovaries. Due to complete agreement of results from PCR with gonadal sex the genetic sex was directly compared with the results from vent sex.

Comparison of genetic sex (gonadal sex) with vent sexing Table 1 represents the frequencies of observa- tions based on genetic sex (gonadal sex) and 250bp vent sex determinations in the guinea fowl population studied. 370bp Comparison of vent sexing and polymerase chain reaction 21

TABLE 1 conformity of methods are compared statisti- Observed frequencies for sex determined by cally the flock structures determined and vent sexing for genetic male and female represented in Fig. 2 suggests a considerable guinea fowls confirmed by gonads deviation of the two methods. Majority of birds were males accor-ding to genetic sex while the Genetic sex/ Vent sex Fr equency % Frequency majority of birds were females according to vent Gonadal Sex sexing. There is no data available from previous Male (115) Male 56 26.05 work that describes population structure of Female 59 27.44 unselected populations in confinement or in wild Female (100) Female 81 37.67 popula-tions of guinea fowls for comparisons. Male 19 8.84 Confirmation of genetic sex with gonadal sex Total (215) 215 100 The results from PCR were completely in agreement with the sex identified by presence of Of the 115 males detected by PCR (and their respective gonads. Presence of gonads is confirmed by gonadal sexing), vent sexing the ultimate indicator of sex. This suggests that correctly identified only 56 (48.7%) and results from PCR are accurate and hence can be wrongly assigned 59 (51.3%) as females. reliably used to identify the sex of guinea fowls. Similarly, out of the 100 females detected by Use of PCR as a diagnostic tool for accurate sex PCR, sexing by the vent method accurately determination has been recommended by identified 81 (81%) and wrongly designated 19 previous studies on several species (Huang et (19%) as females. Vent sexing therefore had an al., 2003; Horng et al., 2006; Cerit & Avanus, overall error rate of 78/215 or 36.3% in this 2007; Kalina et al., 2012). Thus in this study study. there on we directly compared genetic sex and vent sexing in guinea fowls. Statistical Analysis Chi-squared calculated was 29.86 with Yates Comparison of genetic sex and vent sex correction. This suggests that results from vent In the current study vent sexing identified sexing deviate from results from genetic sex females with a greater accuracy (81%) than significantly. Dichotomous nominal correlation males (48.7%).This may be due to anatomical coefficient between genetic sex and vent sex differences between male and female guinea was 0.27 and was statistically significant (P < fowls. The male guinea fowl is characterized by 0.05).The correlation coefficient between the presence of phallus in the cloaca. However genetic sex and gonadal sex was +1. depending on the location, the phallus may not be visible during vent sexing. This may be one of Discussion the reasons for observed error rate in males using When target sex specific genes were ampli- vent sexing. Females do not possess phallus and fied using USP1 and USP3 in a single reaction this probably makes it easier to be identified by with internal control primers CPE15F and vent sexing. However further investigations into CPE15R from DNA extracted from feathers as anatomical differences of the cloacal duct of template, differential banding patterns observed guinea fowls is necessary to better understand for males and females were similar to earlier the reasons for this differences. Guidelines for reports by Itoh et al., (2001). Ahiagbe et al., vent sexing guinea fowls should be then (2012) reported similar results in their attempts developed while evaluating accuracy with to apply PCR for non-invasive sex determina- determination of gonads or PCR prior to tion of guinea fowl keets.Even before the disseminating the guidelines to breeders. 22 K. M. J. Ahiagbe et al., (2018) Ghana Jnl agric. Sci. 52, 17-23

Beside these observed differences in error and female guinea fowls in order to develop rates, Chi-square calculated was 29.86 with guidelines for vent sexing guinea fowls to Yates correction factor was significant. Thus increase accuracy of vent sexing. there is significant deviation of results expected from gonadal sex and from observed results Acknowledgement according to vent sexing, while results from The research was partly supported by Canadian PCR were in complete agreement with gonadal International Development Agency through sex. Dichotomous nominal correlation coeffi- Food and Agricultural Budgetory support cient between gonadal sex and vent sex was 0.27 Scheme of Council for Scientific and Indistrial representing a weaker positive correlation that Research, and Ministry of food and Agriculture is statistically significant (P<0.005), while a of Ghana. Authors are greatful for their financial correlation coefficient of +1 between gonadal support. Authors also ackowledge Managemnt sex and PCR results indicates that results from and staff of Livestock Poultry Resesearch two methods are perfectly correlated. Centre of University of Ghana and Animal This indicates that although theres a posi- Research Institute for their encouragement and tive correlation between genetic sex and vent contributions. sex, vent sexing is not a reliable technique for accurate sexing in the grower phase. As indica- REFERENCES ted by the results, genetic sex determined by Ahiagbe, K. M. J., Kayang, B. B., Avornyo, F. PCR is a reliable method for sex determination & Botchway, P. K., (2012) Development of in guinea fowls and can be used for sex determi- molecular Assay for rapid reliable and non- nation for breeding in any situation that needs invasive sex determination of Guinea fowl accurate information of the gender of parents. keets (Numida meleagris).CSIR-Animal As sex is determined by the genetic blue print of Research Institute 2012 Annual Report, an animal, molecular sexing provides a better CSIR-ARI, Accra, Ghana, 40-42. alternative for sexing monomorphic birds Avornyo, F. K., Salifu, S., Moomen, A. & including guinea fowls. Agbolosu, A. A. (2013) Effect of dietary protein on the performance of local guinea Conclusions and recommendations keets in the Northern Region of Ghana. Vent sexing alone is not reliable for accurate sex Greener Journal of Agricultural Sciences, determination of guinea fowls during the 3(7), 585-591. grower phase. Until further research identifies Awotwi, E. K. (1975) Some aspects of the sexually dimorphic features, if any, based on reproductive physiology of male and female scientific evidence verified by gonadal sex or guinea fowls. Unpublished MSc. Thesis. genetic sex, determination of genetic sex by Faculty of Agriculture, Univ. of Ghana, PCR with sex-specific primers is recommended Legon. for accurate sex determination for breeders and Cerit, H. & Avanus, K., (2007a) Sex researchers. However, PCR cannot be directly determination by CHDW and CHDZ genes adopted by farmers at the farm level. Therefore of avian sex chromosomes in Nymphicus the method described here can be applied to hollandicus, Turkish Journal of Veterinary validate indigenous knowledge of farmers on and Animal Sciences, 31(5), 371–374. sexing guinea fowls and to develop farmer Cerit, H. & Avanus, K., (2007b) Sex friendly guidelines for sex determination in identification in avian species using DNA guinea fowls. Further research is also necessary typing methods. World's Poultry Science to study anatomical differences between male Journal, 63, 91–100. Comparison of vent sexing and polymerase chain reaction 23

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