Comparative Evaluation Of Two Techniques Of Sex Determination In Lovebirds (Agapornis Spp.)

1 1 2 1 Maria-Carmen1Faculty of Veterinary TURCU Medicine,*, Lucia-Victoria University of BEL Agricultural, Tommaso Sciences COLLARILE and Veterinary and MedicineDana Liana Cluj-Napoca, PUSTA Romania 2Clinica per animali esotici, CVS, Roma, Italia *corresponding author: [email protected]

Bulletin UASVM Veterinary Medicine 77(2)/2020 Print ISSN 1843-5270; Electronic ISSN 1843-5378 doi:10.15835/buasvmcn-vm:2020.0030

Abstract Agapornis spp

Most lovebirds ( .) have no distinct sexual dimorphic traits. The objective of the paper was to compare the results of two sexing methods, surgical sexing by celioscopy and DNA sexing by PCR from blood samples, in order to evaluate their effectiveness. Materials used to carry out the studies were a 2.7 mm telescope and endoscopy unit, surgical instruments and DNA sampling kits provided by Exomed.cz laboratory. Forty-two lovebirds were included in this study. The endoscopic sexing procedure was performed by the method described by Divers. Blood sampling for DNA testing by PCR method was performed from the metatarsal vein. The results were different in the case of one lovebird, endoscopic sexing revealed it as male, compared to DNA testing, where it was identified as female. DNA sexing is a non-invasive method that might be more accurate than celioscopy in this species,Keywords: and bird Agapornis owners have spp., easier access to it.

celioscopy, DNA sexing, lovebird, PCR

Introduction Agapornis canus Agapornis spp Agapornis pullarius lovebird ( ). The red-faced lovebird Most lovebirds ( .) do not show ( ) is probably the least bred sexual dimorphism. Out of the nine species of species of love parrot. It is considered a small lovebirds, only three show sexual dimorphism lovebird, reaching a body weight of about 37 (Forshaw, 2010). Agapornis grams. The male has the inner face of the wings roseicollisThe lovebirds that do not showAgapornis sexual fischeri dimo­ colored black and theAgapornis head bright taranta red, brighter phism are the peach-colored lovebird ( than the female. The black-winged lovebird or the Agapornis), Fischer’s personatus lovebird ( ), Abyssinian lovebird ( ) is usually the masked lovebird or the yellow-collaredAgapornis love­ found at higher altitudes. It is considered a large swindernianusbird ( ), the black-collared size lovebird, and can reach a body weight of about lovebird Agapornisor the Swindern lilianae lovebird ( 50 grams. Males have black feathers on the inner Agapornis), the nigrigenisLiliana lovebird or the Nyasa faceAgapornis of their canus wings and a red forehead, and females lovebird ( ) and the black-cheeked are completely green. The Madagascar lovebird lovebird ( ) (Kuchinski, 1995). ( ) is the only lovebird found on the AgapornisThe three pullarius species of lovebirds that display island of Madagascar and is considered the most sexual dimorphism are the Agapornisred-faced tarantalovebird primitive of the genus Agapornis. It is considered ( ), the black-winged lovebird the smallest lovebird, weighting arround 30 or the Abyssinian lovebird ( ) grams. The male has gray feathers on his head and and the gray-faced lovebird or the Madagascar the female is completely green (Kuchinski, 1995). (Agapornis Spp.) 107

Comparative Evaluation Of Two Techniques Of Sex Determination In Lovebirds

The determination of sex in parrots can be Forty-two lovebirds were included in this obtained by several methods, some being more study. The lovebirds were approximately 1 year accurate than others. These are represented by old and were raised by the same breeder. traditional methods, surgical methods, genetic The methods of choice for sexing the 42 love­ methods, hormonal methods for determining birds were surgical sexing by celioscopy and DNA steroid hormones and ultrasound methods of the sexing by PCR from blood sample. The objective genital tract (O’Malley, 2005; Stanford, 2010). of the paper was to compare the results of the Traditional methods of sexing birds involve two Endoscopicsexing methods method in of order bird sexing: to evaluate their observing the secondary sexual characteristics effectiveness. of the species, cloacal inspection, vent sexing (determining the width of the pubic arch), the Endoscopic sexing of the 42 lovebirds was size of the bird, the shape of the bird’s head and performed using the technique of celioscopy behavior studies (O’Malley, 2005). through the left lateral approach with the limb Genetic methods used for sexing birds involve, pulled cranially, as previously described by Divers in on the one hand, cytogenetic tests to highlight 2015. A pre-anesthetic consultation was necessary chromosomes by karyotyping (Fridolfsson and in order to declare the birds clinically healthy Ellegren, 1999), and on the other hand,et al., modern and not egg binding. The parrots were fasted for molecular genetic tests that determine the sex 1-2 hours prior to the procedure. Patients were of birds by DNA testing (Vučićević 2012). inhalatory anesthetized using a semi-closed non- Another method of early genetic sexing of birds is rebreathing circuit. Induction was made on mask autosexing of day-old chicks (Pusta, 2013). with 5% isoflurane, and maintenance with 2% Surgical methods of sexing birds involve vi­ isoflurane. The anesthetized patient was placed in sua­lizing the gonads following a laparotomy or right lateral recumbency with the wings extended laparoscopy under general anesthesia (O’Malley, dorsally. The left limb was pulled cranially, while 2005). the right limb was maintained in a normal position. Determining the level of steroid hormones in The area of interest (Fig. 1) was represented by a bird feces is a method of sexing birds that is based triangular shape delimited cranially by the last on the levels of estrogen or testosterone found in rib, caudally by the medial crural flexor muscle the feces of birds. Fresh faecal samples are required and ventral by the parallel that passes at the level to make this determination. Female feces have a of the sternum. Subsequently, the left flank area higher estrogen/testosterone ratio than maleset al. was prepared by plucking and antisepsis with and vice versa, males have a higher estrogen/ chlorhexidine. An incision of the skin of about 2-3 testosterone ratio than females (Bercovitz , mm was made caudal to the last rib and ventral 1978; Cerit and Avanus, 2007). of the medial crural flexor muscle. With the help Genital ultrasound is a method of sexing birds of a curved Pean forceps, the abdominal muscles that involves the identification of oviducts. Sex were punctured in order to penetrate into the determination by ultrasound is performed using caudal thoracic air sac. Through the orifice an ultrasoundet probeal placed at the cloacal level thus created, a 2.7 mm telescope was inserted of unanesthetized birds to identify the oviduct perpendicularly, penetrating the caudal thoracic (HildebrandtMaterials and., 1995). methods air sac. Penetration into the caudal thoracic air sac is confirmed by identifying the lung (central), cranial thoracic air sac (left), abdominal air sac Materials used to carry out the studies were (right), liver and proventriculus (ventral), ribs and Karl Storz Veterinary Endoscopy America Inc. intercostal muscles (dorsal). Subsequently, the laparoscopic equipment, light source, laparoscopy telescope is oriented caudally. Penetration into the camera, display, 2.7 mm telescope, surgical ins­ left abdominal air sac is achieved by perforating truments (15 blade, Pean forceps, scissors, sterile the membrane using the tip of the telescope. At gauze and 5-0 polyfilament absorbable suture this level, the kidney-gonad-adrenal gland triad ma­terial) and DNA sampling kits provided by is identified. The gonads are located at the cranial Exomed.cz laboratory. pole of the kidney. Sometimes the adrenal gland is covered by the sexuallyBulletin UASVM active Veterinary testicle Medicine enlarged 77 (2) / 2020 in 108 et al.

TURCU volume (Divers, 2015). Lastly, the skin was sutured chromosome is done by amplifying the CHD gene with polyfilament absorbable suture material. (chromo-helicase-DNA binding protein) found on Postoperatively, the lovebirds recovered from the sex chromosomes of birds, using the technique anesthesia within minutes. Postoperative anal­ of PCR (Polymerase Chain Reaction) followed gesia after endoscopy is recommended (Divers, by agarose gel electrophoresis (Fridolfsson and 2015).DNA Nonsteroidal testing by PCR anti-inflammatorymethod of bird sexing: drugs, Ellegren, 1999). The agarose gel is examined using such as meloxicam, were used in this case. an UV trans illuminator (Fridolfsson and Ellegren, 1999; Prathipa and Karthickeyan, 2017). Corres­ Birds have different sex chromosomes than ponding to the genes of the sex chromosomes, the mammals. Females are heterogametic and have ZW electrophoresis gel shows single bands in males sex chromosomes, while males are homogametic (ZZ), whereas in females double bands (ZW) and have ZZ sex chromosomes. DNA testing for (Prathipa and Karthickeyan, 2017). bird sexing involves identifying the W (female) DNA sexing in birds can be performed from chromosome. The identification of the female blood samples, feathers, eggshells or mouth swabs

Figure 1

. Patient positioning for endoscopic sexing

Figure 2. Agapornis spp.

Bulletin UASVM Veterinary Medicine 77 (2) / 2020 Blood sample taken for DNA sexing in (Agapornis Spp.) 109

Comparative Evaluation Of Two Techniques Of Sex Determination In Lovebirds et al (Prathipa and Karthickeyan, 2017, Mataragka using the UV trans illuminator (Fridolfsson and ., 2018). Feathers and oral swabs are easier to Ellegren, 1999; Prathipa and Karthickeyan, 2017). collect. Moreover, oral swabs are easier to processet al., Regarding lovebirds, electrophoresis shows in than feathers, whereas the easiestAgapornis results to sppread males single bands, whereas in females double are obtained from blood samples (Mataragka bands, corresponding to the genes of the sex 2018). DNA sexing in lovebirds ( .) chromosomesResults and (Prathipa discussion and Karthickeyan, 2017). can be performed from blood samples, feathers or mouth swabs (Prathipa and Karthickeyan, 2017). In our case, during anesthesia for endoscopy, blood Endoscopic sexing of the 42 lovebirds included samples were taken from the metatarsal vein of in this study identified 26 males and 16 females. the 42 lovebirds (Fig. 2). PCR was performed using CelioscopyAgapornis through thespp left lateral approach made DNA testing kits provided by Exomed.cz. visualization of the gonads possible. The testicles DNA sexing in lovebirds involves the use of in male . have an oval shape, are PCR technique followed by polyacrylamide gel smooth and show obvious vascularity on their electrophoresis (Prathipa and Karthickeyan, surface (Fig. 3). They are found at the cranial pole 2017). In the case of lovebirds, DNA isolation by of the kidney, while identifying the kidney-gonad- PCR and amplification of the GHD gene is performed adrenal gland triad. The normal mature testicle using the P2 (5’-TCTGCATCGCTAAATCCTTT-3 ‘) of birds has an oval shape, is smooth and has and P8 (5’-CTCCCAAGGATGAGRAAYTG-3’) primer obvious vascularity on the surface. Sometimes the sets (Prathipa and Karthickeyan, 2017). The congenital testicle on the right side can be observed PCR reaction aims to amplify the DNA sequence through the transparency of the abdominal air containing the CHD1W gene using P2-P8 primers. sac In juvenile birds, the gonads are smaller and Initially, the DNA is denatured at 94° C for 2 minutes, less differentiated. In general, in juvenile birds, followed by a series of thermal reactions that the normal immature testis is smooth and has a gradually reduce the temperature by 1° C per cycle, sausage shape. The vas deferens with a sinuous starting at 60° C and reaching the temperature “zig-zag” appearanceAgapornis can be traced spp from the gonad of 50° C. Subsequently, another 25-35 additional to the cloacal level (Divers, 2015). cycles are run maintaining the temperature of 50° The ovaries of . females have C. After the last cycle, DNA extension is performed follicles at various stages of development forming at 72° C for 5 minutes (Fridolfsson and Ellegren, the shape of grapes (Fig. 4). The normal mature 1999; Prathipa and Karthickeyan, 2017). Thus, ovary of birds has the shape of grapes with the product obtained after PCR is separated by follicles during various stages of development. In polyacrylamide gel electrophoresis and stained some species, the gonads may have partial or total with ethidium bromide. The gel is examined physiological melanosis. Sexing by endoscopy

Figure 3. Agapornis spp

Mature testicle in . Bulletin UASVM Veterinary Medicine 77 (2) / 2020 110 et al.

TURCU does not present difficulties in adult female birds. and Schmutz in 1987 was that surgical sexing In juveniles, however, the ovary is smaller and less with an otoscope was preferred over sexing by differentiated. In general, in juvenile birds, the examining chromosomes in lymphocyteet culture al. in normal immature ovary has an irregular shape all categories exceptAgapornis risk. spp with grooves on its surface and poorly highlighted Subsequently, in 2001, Sae-Heng sexed primary follicles. The twisted-looking oviduct 21 lovebirds ( .) ThatAgapornis already spphad can be traced from the gonad to the cloacal level sex, 12 males and 9 females. The study authors (Divers, 2015). compared two methods of sexing . The DNA sexing of the 42 lovebirds identified The methods compared were the traditional one, 25 males and 17 females by emphasizing the vent sexing, and DNA testing by PCR. Following sex chromosomes, specifically the W (female) traditional sexing, 11 maleset al.and 10 females were chromosome. One lovebird obtained different identified, whereas by sexing by PCR, 12 males DNA results compared to endoscopic sexing. The and 9 females. Sae-Heng (2001) concluded results of DNA tests wereet al. released by the Exomed. that the accuracy of sexing by the PCR method cz laboratory (Fig. 5). was 100% compared to 76.19% for the method of In 1984, Jones performed 1056 endo­ determining the vent sexing in lovebirds. Cerit and scopes on 144 non-sexually dimorphic bird species Avanus performed poultry sex determinations in of 15 orders. The purpose was to establish sex by 2007. They stated that the identification of sex in direct inspection of the gonads. Anesthesia for poultry can be done by vent sexing, laparoscopy, endoscopy was consideredet al. dangerous in elderly determining steroid hormones and DNA testing. and obese birds, where a mortality rate of up to The preference for one of these methods also 2% was found. Jones (1984) stated at the depends on the equipment of the laboratory and time that celioscopy, although it involves surgery, the experience of the examiners. The authors is generally more practical and accurate than stated that vent sexing requires well-trained examining lymphocyte chromosomes or analyzing experts, and sometimes even professionals can steroids in the feces. misidentify the sex of birds. The same authors In 1987, Prus and Schmutz compared sur­ argued that laparoscopy is performed under gical sexing using an otoscope with sexing by general anesthesia and is a dangerous method analyzing lymphocyte culture chromosomes on that can be fatal to birds. In the 2000s, DNA sexing 22 psittacines belonging to eight different spe­ procedures required large amounts of blood cies. The comparison of the two techniques and a few weeks to perform. Cerit and Avanus involved the following categories: success rate, (2017) concluded that DNA-based techniques are quality of determination, cost, efficiency and risk. more reliable than vent sexing, laparoscopy or The conclusion of the study conducted by Prus determining steroid hormones.

Figure 4. Agapornis spp

Mature ovary in . Bulletin UASVM Veterinary Medicine 77 (2) / 2020 (Agapornis Spp.) 111

Comparative Evaluation Of Two Techniques Of Sex Determination In Lovebirds et al Spilornis et al. cheelaIn 2009,hoya Hsu . compared endoscopic sexing sexing method. The results of the study by Wang with DNA sexing from blood samples of 20 (2013) showed that molecular methods have eagles. Initially Hsu et al sexed these a great potential in determining the sex of owls, eagles by endoscopy, and later the eagles were although their accuracy remains to be tested on sexed by DNA. The study by Hsu et al concluded larger cohorts. Wang et al claim that DNA testing that the DNA test result in an eagle did not match is a safe alternative to reduceet al. the misdiagnosis of the endoscopy result, but thiset al. was later proven to conventional sex by laparoscopy of birds. be caused by incorrect sex determination by the In 2013, Morinha studied DNA sexing endoscopy technique. Hsu (2009) concluded by analyzing high-resolution melting (HRM), an that determining the sex of eagles by DNA sexing advanced post-PCR method. The researchers method was as reliable as by laparoscopy method,et al applied this molecular sexing technique to a noting that the latter might be influencedOtus bakkamoena by age, number of 14 species of birds, belonging to five season and body size of birds. In 2013, Wang . orders and nine families. The samples collected sexed 8 males and 8 females of were feathers, muscle tissue, blood and mouth by two methods. The sex of these owls was initially swabs. For comparison, the sex of living individuals determined by laparoscopy, and the data were then was identified by endoscopy, while the sex of dead compared with the results obtained by the DNA birds was determined during the autopsy. Morinha

Figure 5.

DNA test result Bulletin UASVM Veterinary Medicine 77 (2) / 2020 112 et al.

TURCU et al.

(2013) concluded that the accuracy of DNA include patients at high risk of anesthesia, with sexing was 100%. They stated that simplicity, intercurrent conditions such as obesity or various speed, low cost and high sensitivity/specificity large, tumor-like masses that can compress or make this technique an excellent method for obliterate the air sacs and thus narrow the visual routine and large-scale sexing of birds. field of the laparoscopic camera. In birds with Celioscopy is a minimally invasive surgical ascites or intra-abdominal hemorrhage, the left technique that has many advantages such as speed or right lateral approach is not recommended and accuracy in determining the sex of birds, because fluids will inevitably enter the air sacs. In lower risks than laparotomy, reduced surgical such cases, the ventral approach on the midline stress, respiratory function is less affected and is recommended, if it is absolutely necessary to postoperative anesthesia and hospitalization perform an endoscopy. Other contraindications time are significantly reduced in comparison to are represented by females during egg laying celiotomy (Hernandez-Divers, 2005). Neverthe­ and major subcutaneous emphysema caused by less, endoscopy also has disadvantages such as perforation of the air sacs (Hernandez-Divers and surgical risk, which, although minor, is still pre­ Hernandez-Divers, 2004; Chai and Roman, 2005; sent (Greenacre and Gerhardt, 2016). A major Korbel, 2012). disadvantage of celioscopy is the high cost of the Recent studies show that DNA sexing is pre­ equipment (Hernandez-Divers and Hernandez- ferred over celioscopy because DNA testing is a non- Divers, 2004). Complications of celioscopy in birds invasive method that has many advantages such are rare, as most birds undergoing endoscopic as accuracy and precision in sexing, an economic sexing are young and clinically healthy (Divers, advantage, given the low cost of laboratory analysis 2015). Celioscopy is recommended only for of samples, it is considered safer because samples clinically healthy patients, as it is not an emergency do not endanger the lives of birds and do not put surgical procedure. This should be performed them at risk of infection, moreover feathers can by an experienced veterinarian to minimize the be collected from the nest thus avoiding the stress risk of complications (Chavez and Echols, 2007). of handling birds. Another advantage iset thatal., birds The most common complications of celioscopy of all ages can be safely sexed, especiallyet al juveniles are minor hemorrhages that occur most often (Griffiths, 2000; Bermúdez-Humarán 2002; due to lack of experience of the examiner. In Cerit and Avanus, 2007; Vučićević ., 2012). rare cases, subcutaneous emphysema may occur The main disadvantages of DNA sexing are the postoperatively due to the strictly cutaneous suture, equipment needed in order to perform it and the but it heals by itself within a few days if the bird is time it takes to issue the results, in comparison not subjected to effort. One method of preventing with endoscopic sexing, where the result is the occurrence of postoperative subcutaneous obtained on the spot (Griffiths, 2000). Due to its emphysema is to a suture both the skin and the many advantages, the most important being its abdominal muscles using absorbable sututre accuracy of almost 100%, genetic sex has many material. Massive intra-abdominal haemorrhages applications in behavioral medicine, conservative are considered major complications of celioscopy medicine, wild bird management, breeding of that can endanger the life of the animal, but which different species of birds, improving the breeding are very rare if the procedure is performed by programs of birds in captivity, in the analysiset alof an experienced veterinarian (Hernandez-Divers poultry reproduction strategies, in evolutionary and Hernandez-Divers, 2004; Divers, 2015). studies or in forensic medicine (Morinha ., Complications of celioscopy in birds are rare, 2012).Conclusions as most birds undergoing sexing are young and Agapornis spp clinically healthy (Divers, 2015). Celioscopy is recommended only for clinically healthy patients, Lovebirds ( .) present 9 breeds, as it is not an emergency surgical procedure. but only three of them show sexual dimorphism. This should be performed by an experienced Moreover, crossbreeding makes it even more veterinarian to minimize the risk of complications di­fficult­ to sex lovebirds by their phenotypic (Chavez and Echols, 2007).There are few situations characters. Bulletinin which UASVM celioscopy Veterinary Medicine has 77 contraindications. (2) / 2020 These (Agapornis Spp.) 113

Comparative Evaluation Of Two Techniques Of Sex Determination In Lovebirds

Traditional methods of sexing birds have low mixing of the blood samples for DNA sexing could accuracy and are considered rather empirical. The not be excluded. only traditional method with 100% accuracy is In conclusion, DNA sexing is a non-invasive egg binding in females, but it has the disadvantage method that might be more accurate than of being belated. celioscopyAcknowledgments. in this species. In determining the steroids in bird droppings, erroneous results may occur due to seasonal va­ This research did not re­ riations and the age of the birds, especially outside ceive any specific grant from funding agencies in the mating season. The most reliable results the public, commercial, or not-for-profit sectors. are obtained from adult birds during the mating References season. Measuring steroids is a time consuming procedure. Because steroid levels vary, sex cannot be accurately identified by this technique. 1. Bercovitz AB, Czekala NM, Lasley BL (1978). A new Ultrasound of the genital tract has the advan­ method of sex‐ determination in monomorphic‐ birds.‐ The journal of Zoo Animal medicine, 9(4):114-124. tage of being atraumatic and the disadvantage of ‐ ‐ ‐de‐ 2. Bermúdez‐ Humarán LG, García García A, Leal Garza requiring an experienced examiner. This technique CH, Riojas Valdes VM, Jaramillo Rangel G, Montes is not an applicable technique for sexing parrots Oca Luna R (2002). Molecular sexing of monomorphic due to their small size. endangered Ara birds. Journal of Experimental Zoology, Laparoscopic sexing requires special equip­ 292(7):677-680. ment and experience from the examiner. Lapa­ 3. Cerit H, Avanus K (2007). Sex identification in avian roscopic sexing can be influenced by season, species using DNA typing methods. World’s Poultry Agapornis spp Science Journal, 63(1):91-100. age and body size of birds. The testicles in male 4. Chai N, Roman Y (2005). L’endoscopie invasive et Agapornis. have an sppoval shape, are smooth noninvasive. Le Point Vétérinaire, 253(3):2-9. and show obvious vascularity on their surface. 5. Chavez W, Echols MS (2007) Bandaging, endoscopy, and The ovary of . females has follicles surgery in the emergency avian patient. Veterinary Clinics at various stages of development forming the of North America: Exotic Animal Practice, 10(2):419-436. shape of grapes. No complications were observed 6. Divers SJ (2015). Endoscopic sex identification in after performing the endoscopic sexing of the 42 chelonians and birds (Psittacines, Passerines, and lovebirds. Raptors). Veterinary Clinics: Exotic Animal Practice, 18(3):541-554. Molecular DNA sexing methods allows re­ 7. Forshaw JM (2010). Parrots of the World (Vol. 70). liable, rapid and economical determination of sex Princeton University Press., pg. 154-159. in most bird species. Moreover, bird owners have 8. Fridolfsson AK, Ellegren H (1999). A simple and universal easier access to it. method for molecular sexing of non-ratite birds. Journal In the past surgical sexing of birds was pre­ of avian biology, 116-121. ferred. Nowadays, after genetic sexing techniques 9. Greenacre CB, Gerhardt L (2016). Psittacine and Passerine developed, DNA sexing is considered to have Birds. Exotic Animal Medicine for the Veterinary 100% accuracy, simplicity, speed, low cost, Technician, pg. 90. high sensitivity/specificity and is considered a 10. Griffiths R. (2000). Sex identification in birds. In Seminars in avian and exotic pet medicine (Vol. 9, No. 1, pp. 14-26). safe alternative to reduce the misdiagnosis of WB Saunders. laparoscopic sexing in birds. 11. Hernandez-Divers SJ (2005). Minimally invasive In the present study one lovebird obtained endoscopic surgery of birds. Journal of Avian Medicine different results at endoscopic sexing, where and Surgery, 19(2):107-121. it was identified as male, compared to DNA 12. Hernandez-Divers SJ, Hernandez-Divers SM (2004). testing, where it was identified as female. This is Avian diagnostic endoscopy. Compendium on continuing considered to be a consequence of human error. education for the practicing veterinarian. Given that the lovebirds had not yet reached sexual 13. Hildebrandt T, Pitra C, Sömmer P, Pinkowski M (1995). Sex identification in birds of prey by ultrasonography. Journal maturity and their ovaries were in various stages of Zoo and Wildlife Medicine, 367-376. of development, there is a possibility that the 14. Hsu HA, Wang PH, Chao MC, Chan FT, Wang LM, Lin PI, Ding examiner may have confused the immature ovary ST (2009). Use of random amplified polymorphic DNA with the testicle during endoscopy. Moreover, to identify several novel markers for sex identification Bulletin UASVM Veterinary Medicine 77 (2) / 2020 114 et al.

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