Research Note. A novel method for sexing day-old chicks using endoscope system

Research Note A novel method for sexing day-old chicks using endoscope system Makoto Otsuka,,1 Osamu Miyashita,,1 Mitsuru Shibata,,1 Fujiyuki Sato,,1 and Mitsuru Naito,2,3 NARO Institute of Livestock and Grassland Science, 2 Ikenodai, Tsukuba, Ibaraki 305 0901, Japan; and National Institute of Agrobiological Sciences, 2-1-2 Kannondai, Tsukuba, Ibaraki 305 8602, Japan ABSTRACT Sexing day-old chicks is important for layer and broiler production. A novel method for sexing day-old chicks was developed using an endoscope system. The probe of the endoscope was inserted from the cloaca into the intestine of a chick, and the presence of testes or ovary was observed through the wall of the intestine. The picture image was displayed on the monitor. Sexing was performed in White Leghorn (WL) and Rhode Island Red (RIR) chicks using this new system. The accuracy of sexing was 91.1% in WL chicks and 88.3% in RIR chicks, confirmed by observing gonads after laparotomy or appearances at 80 d of age. Accuracy of sexing male chicks (95.0%) was higher than that of female chicks (86.0%). The overall accuracy of sexing was 90.2% in the present study, and the accuracy would be improved by continuous training in the handling of the endoscope. The endoscope system devised in this study requires no specific skills and anyone can perform sexing of chicks after short-term training. Key words: day-old chick, endoscope, gonad, PCR, sexing 2016 Poultry Science 95:2685 2689 http://dx.doi.org/10.3382/ps/pew211 INTRODUCTION Sexing day-old chicks is very important for poultry production. Females are used exclusively for commercial layers, whereas males are preferable in commercial broilers due to their fast growth. Several methods of chick sexing have been developed so far (Kaleta and Redmann, 2008). The most popular method is vent sexing (Masui et al., 1924; Masui and Ito, 1931), which is performed just by looking for the presence or absence of a rudimentary male sex organ after everting the vent area of chicks. Although this method is fast and accurate, it requires the observer to be well trained and to have a great deal of practice. Another method is feather sexing, which uses sex-linked feather characteristics genes such as barring and non-barring (B/b + ) genes, silver and gold (S/s + ) genes, or slow and fast feathering (K/k + ) genes (Etches, 1996). Although this method is easy in practice, it can be applied only to limited chicken breeds and cross breeds. Additionally, a molecular sexing technique has been developed (Clinton, 1994). Since male chickens have ZZ and females have ZW sex chromosomes, sex can be identified by analyzing the presence or absence of the W chromosome. C 2016 Poultry Science Association Inc. Received March 24, 2016. Accepted April 26, 2016. 1 Current address: Tsukuba Technical Support Center, NARO, 2 Ikenodai, Tsukuba, Ibaraki 305 0901, Japan 2 Current address: Institute of Agrobiological Sciences, NARO, 2-1-2 Kannondai, Tsukuba, Ibaraki 305 8602, Japan 3 Corresponding author: mnaito@affrc.go.jp 2685 Although this molecular sexing method is accurate, it is impossible to apply it to a large number of chicks in practice due to time and cost. There is also a method for sexing using Chick Tester (Thozai Sangyo Boeki, Tokyo, Japan). With this method, a monitor probe is inserted into the intestine of a chick, and the testes or ovary is observed directly through the wall of intestine. Although this method does not require specific skills, it is difficult to use due to the indistinct, low-resolution picture images it yields. Production of the Chick Tester has ceased since the 1970s. In the present study, we modified an endoscope system for small animals to make a novel endoscope system that can determine the sex of day-old chicks quickly and accurately. MATERIALS AND METHODS Chickens and Animal Care Fertilized eggs of White Leghorn (WL) and Rhode Island Red (RIR) were obtained by artificial insemination. Chicks were hatched by incubating the fertilized eggs. All animals received humane care as outlined in the Guide for the Care and Use of Experimental Animals (NARO Institute of Livestock and Grassland Science, National Institute of Agrobiological Sciences, Animal Care Committees). Endoscope System for Chick Sexing The endoscope system for chick sexing involves an endoscope camera and a camera control unit (TESALA

2686 OTSUKA ET AL. Figure 1. Newly devised endoscope system. Figure 2. Endoscope camera unit. Thin Endoscope for Small Animals and Laboratory Animals, AE-C1, AVS, Tokyo, Japan), and a computer as shown in Figure 1. The endoscope camera has a probe (length: 110 mm, diameter: 2.7 mm; AE-E27110, AVS, Tokyo, Japan) with a thin and tube-like camera lens (length: 27 mm; diameter: 3.4 mm; OZ-AE27110- R, OZU Shokai, Tsukuba, Japan) made by processing a glass tube (Figure 2). The tip of the glass camera lens curves slightly (about 20 degrees), simplifying gonadal observation through the wall of intestine. The endoscope camera is fixed with a grip adaptor (adaptor length: 190 mm (aluminum), grip diameter: 35 mm, grip length: 70 mm (resin); Camera Unit Grip, Ozu Shokai, Tsukuba, Japan). The gonad s picture image is displayed on a PC monitor and recorded to memory using the video capture and attached software (GV- USB2/HQ, I-O Data, Kanazawa, Japan). Direct connection to the monitor is possible through S-video port. Figure 3. Sexing day-old chicks using endoscope system. Glass camera probe is inserted from cloaca into intestine of a chick. Observation of Chick Gonads by Endoscope System and Confirming Accuracy of Sexing Observation of gonads by the endoscope system was performed using day-old WL and RIR chicks. First, the chick is held, the fecal material is expelled, and the probe is inserted from the cloaca into the intestine (Figure 3). Testes or ovary observed through the wall of intestine were displayed on the monitor by adjusting the depth and angle of the probe, and sex was identified. Testes or ovary from 3 male chicks and 3 female chicks sampled from each breed were then observed under a dissection microscope (M205 FA, Leica Microsystems, Tokyo, Japan) after laparotomy to confirm the sex of the chick. Furthermore, the gonads were isolated from the chick, DNA was extracted, and molecular sexing was performed by PCR.

NOVEL METHOD FOR SEXING DAY-OLD CHICKS 2687 Chick Sexing in Practice by Endoscope System Sexing was performed on day-old WL and RIR chicks using the endoscope system. Time required for sexing 100 chicks was measured for both WL and RIR. Chicks were then sexed by observing gonads after laparotomy or by other appearances (comb size, feather characteristics, etc.) at 80 d of age. Blood was collected from 5 male chickens and 5 female chickens sampled from each breed at 101 d of age, and DNA was extracted. Molecular sexing was performed in each hen regardless of whether the sex determined by appearances was identical to the sex determined by the endoscope. Chick Sexing by PCR DNA was extracted from the isolated gonads or blood using a DNA extraction kit (SepaGene, EIDIA, Tokyo, Japan) according to the manufacturer s instructions. PCR analysis was then performed to detect the presence or absence of the W chromosome-specific repeating sequences using a programmable thermal controller (Model 9700; Perkin Elmer, Waltham, MA). PCR reaction mixture was prepared using Takara Ex Taq kit (PR001A, Takara Bio, Shiga, Japan), in 25 μl total volume containing 50 ng genomic DNA, 0.5 μm primers, 0.2 mm dntps, and 0.6 U DNA polymerase. The sequences of the primers for detecting W chromosomespecific repeating sequences were: 5 -CCC AAA TAT AAC ACG CTT CAC T-3,5 -GAA ATG AAT TAT TTT CTG GCG AC-3 (Clinton et al., 2001). Control PCR reactions were conducted with the same sample (a single tube assay) to ensure the presence of genomic DNA using primers: 5 -AGC TCT TTC TCG ATT CCG TG-3,5 -GGG TAG ACA CAA GCT GAG CC-3, which were designed to amplify the chicken 18S ribosomal gene (Clinton et al., 2001). After an initial denaturation step of 94 C for 2 min, 25 cycles of amplification were performed; DNA was denatured at 94 C for 30 s, annealed at 56 C for 30 s, and extended at 72 C for 30 s. Samples were then incubated at 72 Cfor5min. After amplification, the reaction product was separated on a 2% agarose gel, and visualized under UV irradiation after ethidium bromide staining. A band of 415 bp was detected in females but not in males, and a band of 256 bp was detected in each male and female reactions. RESULTS Chick Sexing by Endoscope System Testes and ovary could be observed by the endoscope system in WL and RIR chicks as shown in Figures 4A and 4B. In male chicks, both left and right testes were observed across the spine. In female chicks, only the left ovary could be observed clearly; the degenerated right ovary was hard to observe. To evaluate the accuracy of sexing by the endoscope system, testes or ovary were observed directly under a dissection microscope after laparotomy of the chicks. The results confirmed that sex judged by the endoscope system was identical to the sex judged by direct observation of gonadal morphology under a dissection microscope (Figure 5). Furthermore, molecular sexing by PCR was performed to evaluate the accuracy of sex identified by the morphology of gonads (Figure 6). As a result, sex as judged by gonadal morphology and PCR analysis was identical in both breeds. Chick Sexing by Endoscope System and Evaluation of its Accuracy Sexing was performed on 202 WL chicks and 103 RIR chicks by the endoscope system. They were then sexed by observing gonads after laparotomy, or raised for more than 80 d and had their sex confirmed by their appearance and PCR analysis. Sex judged by appearance and PCR analysis was identical for 5 male and 5 female chicks analyzed from 5 males and 5 females each breed (Figure 7). Table 1 shows the results of sexing WL and RIR chicks. The majority of them were sexed correctly by the endoscope system with an accuracy of 91.1% (184/202) in WL chicks and 88.3% (91/103) in RIR chicks. The accuracy of sexing was Figure 4. Picture image of right testis (A) and left ovary (B) displayed on the monitor.

2688 OTSUKA ET AL. Figure 5. Confirmation of gonads by direct observation after sexing with the endoscope system. Testes (A and C) and left ovary (B and D) were observed under dissection microscope after laparotomy. A and B: WL; C and D: RIR. 95.0% (134/141) in male chicks and 86.0% (141/164) in female chicks, and the overall accuracy of sexing was 90.2% (275/305). Time required for sexing 100 chicks by the endoscope system was 28 min 44 s in WL and 44 min 5 s in RIR. DISCUSSION Currently, about 90% of commercial chicks among layers and broilers are sexed by feather characteristics, but vent sexing is still needed for most pure lines. However, the number of persons acquiring vent-sexing skills is decreasing rapidly, and furthermore, few schools teach those skills. Under these circumstances, development of a novel method for chick sexing has been widely desired. The endoscope system developed in this study will surely become invaluable for sexing day-old chicks. Concerning the sexual differentiation of male and female chicken embryos, gonads are bipotential and morphologically identical in males and females at the initial stage of embryonic development. Morphological differentiation of male and female gonads is first detectable at d 6.5 of incubation (Smith et al., 2007). In the male embryo, both left and right gonads develop and become functional. In the female embryo, the left gonad develops normally and becomes functional, but development of right ovary is less extensive and gradually regresses during embryonic development (Naito et al., 2007). At the time of hatching, male chicks have both left and right functional testes, and female chicks have a functional left ovary and a rudimentary right ovary. In the present study, a novel sexing method for dayold chicks has been developed using a newly devised endoscope system. With this method, a probe camera is inserted into the intestine of a chick and observes the testes or ovary through the wall of intestine. The gonadal image can be easily observed by displaying it on the monitor. By identifying the position of a chick s gonads correctly, accurate sexing becomes possible. Since this method is easily and quickly performed, it can be practically applied for chick sexing. Observing testes is easier than observing ovary, because a male chick has both left and right testes and the shape is unique compared to other organs. As a result, the accuracy of sexing was higher for male chicks than for female chicks in both WL and RIR. On the other hand, the accuracy of sexing was higher for WL chicks than for RIR chicks. From our experience, sexing of colored chicks is somewhat difficult compared with sexing WL chicks, probably due to the difference Figure 6. Sexing by PCR. Gonads from 3 male and 3 female day-old chicks (judged by the endoscope system) in WL and RIR were isolated, and DNA was extracted. PCR analysis was performed to confirm the accuracy of sexing by the endoscope system. Lane 1: size marker, Lane 2: male control, Lane 3: female control, Lane 4: negative control (DW), Lanes 5 7: male chicks (WL), Lanes 8 10: female chicks (WL), Lanes 11 13: male chicks (RIR), Lanes 14 16: female chicks (RIR). Figure 7. Sexing by PCR. Blood from 5 male chickens and 5 female chickens (judged by the endoscope system) at 101 d old in WL and RIR were collected and DNA was extracted. PCR analysis was performed to confirm the accuracy of sexing by the endoscope system. Lane 1: size marker, Lane 2: male control, Lane 3: female control, Lane 4: negative control (DW), Lanes 5 9: male chickens (WL), Lanes 10 14: female chickens (WL), Lanes 15 19: male chickens (RIR), Lanes 20 24: female chickens (RIR).

NOVEL METHOD FOR SEXING DAY-OLD CHICKS 2689 Table 1. Results of sexing day-old chicks by endoscope system. Number of chicks Accuracy of sexing Breed Sex Sexed by endoscope Sexed correctly Sexed incorrectly by endoscope (%) WL (S line) Male 40 40 0 100.0 Female 65 63 2 96.9 Total 105 103 2 98.1 WL (D line) Male 48 44 4 91.7 Female 49 37 13 75.5 Total 97 81 17 83.5 RIR Male 53 50 3 94.3 Female 50 41 9 82.0 Total 103 91 12 88.3 Total Male 141 134 7 95.0 Female 164 141 24 86.0 Total 305 275 31 90.2 Correct sex was confirmed by observing gonads after laparotomy or appearance at 80 d of age. in intestinal wall thickness. The intestinal wall of colored chicks such as RIR seems to be a little thicker compared to WL chicks. As a result, picture image is not so clearly seen in RIR chicks than WL chicks and the time required for sexing is longer for RIR than for WL. Although the overall accuracy of sexing by the endoscope system (90.2% in the present study) is a little lower than vent sexing (usually more than 99%), the accuracy would be improved by continuous training in how to handle the endoscope. The endoscope system requires no specific skills, and anyone can perform it for the sexing of chicks after a short-term training. Poultry research stations or poultry breeding stations maintain so many pure lines of chicken breeds that feather sexing cannot be applied, and they also have difficulty in finding specialists for vent sexing. Since the population size of these pure lines is not so large, the endoscope system developed in this study could be used for sexing day-old chicks. ACKNOWLEDGMENTS The authors would like to thank Minoru Fukuura and Masanobu Kuramochi, OZU Shokai, for their help in devising the endoscope system, and the staff of the Poultry Management Section of NARO Institute of Livestock and Grassland Science for taking care of the birds, providing fertilized eggs, and assisting with chick sexing. REFERENCES Clinton, M. 1994. A rapid protocol for sexing chick embryos (Gallus g. domesticus). Anim. Genet. 25:361 362. Clinton, M., L. Haines, B. Belloir, and D. McBride. 2001. Sexing chick embryos: a rapid and simple protocol. Br. Poult. Sci. 42:134 138. Etches, R. J. 1996. Growth and sexual maturation. Pages 74 105 in Reproduction in Poultry. CAB International, Wallingford, UK. Kaleta, E. F., and T. Redmann. 2008. Approaches to determine the sex prior to and after incubation of chicken eggs and of day-old chicks. World s Poult. Sci. J. 64:391 399. Masui, K., J. Hashimoto, and I. Ono. 1924. The rudimental copulatory organ of the male domestic fowl with reference to the sexual differentiation of chickens. Jpn. J. Zootech. Sci. 1:153 164. Cited from pages 3 15. 1967 in Sex Determination and Sexual Differentiation in the Fowl. K. Masui, ed. Iowa State University Press, Ames, Iowa, USA. Masui, K., and S. Ito. 1931. Morphological and statistical studies on the genital process of day-old chicks (White leghorn) with special reference to sex discrimination in day-old chicks. Jpn. J. Zootec. Sci. 5:139 162. Cited from pages 16 38. 1967 in Sex Determination and Sexual Differentiation in the Fowl. K. Masui, ed. Iowa State University Press, Ames, Iowa, USA. Naito, M., T. Minematsu, T. Harumi, and T. Kuwana. 2007. Testicular and ovarian gonocytes from 20-day incubated chicken embryos contribute to germline lineage after transfer into bloodstream of recipient embryos. Reproduction. 134:577 584. Smith, C. A., K. N. Roeszler, Q. J. Hudson, and A. H. Sinclair. 2007. Avian sex determination: what, when and where? Cytogenet. Genome Res. 117:165 173.