A Novel Method for Sexing Day-Old Chicks Using Endoscope System
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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 chicks and 88.3% in RIR chicks, confirmed by observ- layer and broiler production. A novel method for sex- ing gonads after laparotomy or appearances at 80 d of Downloaded from https://academic.oup.com/ps/article-abstract/95/11/2685/2399384 by guest on 22 March 2019 ing day-old chicks was developed using an endoscope age. Accuracy of sexing male chicks (95.0%) was higher system. The probe of the endoscope was inserted from than that of female chicks (86.0%). The overall accu- the cloaca into the intestine of a chick, and the pres- racy of sexing was 90.2% in the present study, and ence of testes or ovary was observed through the wall the accuracy would be improved by continuous training of the intestine. The picture image was displayed on in the handling of the endoscope. The endoscope sys- the monitor. Sexing was performed in White Leghorn tem devised in this study requires no specific skills and (WL) and Rhode Island Red (RIR) chicks using this anyone can perform sexing of chicks after short-term new system. The accuracy of sexing was 91.1% in WL 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 Although this molecular sexing method is accurate, it is impossible to apply it to a large number of chicks in Sexing day-old chicks is very important for poultry practice due to time and cost. There is also a method production. Females are used exclusively for commer- for sexing using “Chick Tester” (Thozai Sangyo Boeki, cial layers, whereas males are preferable in commercial Tokyo, Japan). With this method, a monitor probe is broilers due to their fast growth. Several methods of inserted into the intestine of a chick, and the testes or chick sexing have been developed so far (Kaleta and ovary is observed directly through the wall of intestine. Redmann, 2008). The most popular method is vent sex- Although this method does not require specific skills, it ing (Masui et al., 1924; Masui and Ito, 1931), which is is difficult to use due to the indistinct, low-resolution performed just by looking for the presence or absence picture images it yields. Production of the “Chick of a rudimentary male sex organ after everting the vent Tester” has ceased since the 1970s. In the present area of chicks. Although this method is fast and accu- study, we modified an endoscope system for small ani- rate, it requires the observer to be well trained and to mals to make a novel endoscope system that can dete- have a great deal of practice. Another method is feather rmine the sex of day-old chicks quickly and accurately. sexing, which uses sex-linked feather characteristics + genes such as barring and non-barring (B/b ) genes, MATERIALS AND METHODS silver and gold (S/s+) genes, or slow and fast feather- + ing (K/k ) genes (Etches, 1996). Although this method Chickens and Animal Care is easy in practice, it can be applied only to limited chicken breeds and cross breeds. Additionally, a molec- Fertilized eggs of White Leghorn (WL) and Rhode ular sexing technique has been developed (Clinton, Island Red (RIR) were obtained by artificial insemina- 1994). Since male chickens have ZZ and females have tion. Chicks were hatched by incubating the fertilized ZW sex chromosomes, sex can be identified by ana- eggs. All animals received humane care as outlined in lyzing the presence or absence of the W chromosome. the Guide for the Care and Use of Experimental Ani- mals (NARO Institute of Livestock and Grassland Sci- ence, National Institute of Agrobiological Sciences, An- C 2016 Poultry Science Association Inc. Received March 24, 2016. imal Care Committees). Accepted April 26, 2016. 1Current address: Tsukuba Technical Support Center, NARO, 2 Ikenodai, Tsukuba, Ibaraki 305–0901, Japan Endoscope System for Chick Sexing 2Current address: Institute of Agrobiological Sciences, NARO, 2-1-2 Kannondai, Tsukuba, Ibaraki 305–8602, Japan The endoscope system for chick sexing involves an en- 3Corresponding author: [email protected] doscope camera and a camera control unit (TESALA 2685 2686 OTSUKA ET AL. Downloaded from https://academic.oup.com/ps/article-abstract/95/11/2685/2399384 by guest on 22 March 2019 Figure 1. Newly devised endoscope system. Figure 2. Endoscope camera unit. Figure 3. Sexing day-old chicks using endoscope system. Glass camera probe is inserted from cloaca into intestine of a chick. Thin Endoscope for Small Animals and Laboratory An- Observation of Chick Gonads by imals, AE-C1, AVS, Tokyo, Japan), and a computer Endoscope System and Confirming as shown in Figure 1. The endoscope camera has a Accuracy of Sexing probe (length: 110 mm, diameter: 2.7 mm; AE-E27110, AVS, Tokyo, Japan) with a thin and tube-like camera Observation of gonads by the endoscope system was lens (length: 27 mm; diameter: 3.4 mm; OZ-AE27110- performed using day-old WL and RIR chicks. First, R, OZU Shokai, Tsukuba, Japan) made by processing the chick is held, the fecal material is expelled, and a glass tube (Figure 2). The tip of the glass camera the probe is inserted from the cloaca into the intestine lens curves slightly (about 20 degrees), simplifying go- (Figure 3). Testes or ovary observed through the wall nadal observation through the wall of intestine. The of intestine were displayed on the monitor by adjusting endoscope camera is fixed with a grip adaptor (adap- the depth and angle of the probe, and sex was identified. tor length: 190 mm (aluminum), grip diameter: 35 mm, Testes or ovary from 3 male chicks and 3 female chicks grip length: 70 mm (resin); Camera Unit Grip, Ozu sampled from each breed were then observed under a Shokai, Tsukuba, Japan). The gonad’s picture image dissection microscope (M205 FA, Leica Microsystems, is displayed on a PC monitor and recorded to memory Tokyo, Japan) after laparotomy to confirm the sex of using the video capture and attached software (GV- the chick. Furthermore, the gonads were isolated from USB2/HQ, I-O Data, Kanazawa, Japan). Direct con- the chick, DNA was extracted, and molecular sexing nection to the monitor is possible through S-video port. was performed by PCR. NOVEL METHOD FOR SEXING DAY-OLD CHICKS 2687 Chick Sexing in Practice on a 2% agarose gel, and visualized under UV irradia- by Endoscope System tion after ethidium bromide staining. A band of 415 bp was detected in females but not in males, and a band of Sexing was performed on day-old WL and RIR chicks 256 bp was detected in each male and female reactions. 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 RESULTS or by other appearances (comb size, feather character- istics, etc.) at 80 d of age. Blood was collected from Chick Sexing by Endoscope System 5 male chickens and 5 female chickens sampled from Testes and ovary could be observed by the endoscope each breed at 101 d of age, and DNA was extracted. system in WL and RIR chicks as shown in Figures 4A Molecular sexing was performed in each hen regard- and 4B. In male chicks, both left and right testes were Downloaded from https://academic.oup.com/ps/article-abstract/95/11/2685/2399384 by guest on 22 March 2019 less of whether the sex determined by appearances was observed across the spine. In female chicks, only the identical to the sex determined by the endoscope. left ovary could be observed clearly; the degenerated right ovary was hard to observe. To evaluate the accu- Chick Sexing by PCR racy of sexing by the endoscope system, testes or ovary were observed directly under a dissection microscope DNA was extracted from the isolated gonads or blood after laparotomy of the chicks. The results confirmed using a DNA extraction kit (SepaGene, EIDIA, Tokyo, that sex judged by the endoscope system was identi- Japan) according to the manufacturer’s instructions. cal to the sex judged by direct observation of gonadal PCR analysis was then performed to detect the pres- morphology under a dissection microscope (Figure 5). ence or absence of the W chromosome-specific repeat- Furthermore, molecular sexing by PCR was performed ing sequences using a programmable thermal controller to evaluate the accuracy of sex identified by the mor- (Model 9700; Perkin Elmer, Waltham, MA). PCR re- phology of gonads (Figure 6). As a result, sex as judged action mixture was prepared using Takara Ex Taq kit by gonadal morphology and PCR analysis was identical (PR001A, Takara Bio, Shiga, Japan), in 25 μl total vol- in both breeds. ume containing 50 ng genomic DNA, 0.5 μM primers, 0.2 mM dNTPs, and 0.6 U DNA polymerase. The se- quences of the primers for detecting W chromosome- Chick Sexing by Endoscope System specific repeating sequences were: 5 -CCC AAA TAT and Evaluation of its Accuracy AAC ACG CTT CAC T-3,5-GAA ATG AAT TAT TTT CTG GCG AC-3 (Clinton et al., 2001). Con- Sexing was performed on 202 WL chicks and 103 trol PCR reactions were conducted with the same sam- RIR chicks by the endoscope system.