TJVM Vol. 48 No. 4 December 2018

5586 Tawatsin A. et al. / Thai J Vet Med. 2018. 48(4): 551-558. บทคดั ย่อ อัตราการติดเชื้อสูงของไวรัสซิกาในยุงทเี่ ก็บจากแหล่งระบาดของไวรัสซกิ า ณ ภาคตะวันออกของประเทศไทย อภิวัฏ ธวชั สิน1 อัจฉรา ภมู 2ี ,5 อุษาวดี ถาวระ1 พชั ราวรรณ ศิริโสภา1 วรรณภา ฤทธสิ นธิ์3 กฤษณพงศ์ ธรรมโกศล4 เพราพิลาศ อินตะยศ4 ยทุ ธนา จอ้ ยจนิ ดา2 สภุ าภรณ์ วชั รพฤษาดี2 ธีระวัฒน์ เหมะจฑุ า2 เผดจ็ สริ ิยะเสถียร5* โรคไขซ้ กิ า โรคไขช้ ิคุนกนั ยา และโรคไข้เลือดออกเดงกีเป็นโรคทีเ่ กดิ จากไวรัสทน่ี า้ โดยแมลงที่เกิดการอุบัติใหมแ่ ละอุบตั ซิ ้า ซง่ึ ไวรัส เหลา่ นจี ะถกู สง่ ผา่ นไปยงั มนุษย์โดยผ่านการกดั ของยงุ ลาย ไมน่ านมานไี วรสั ซกิ าไดพ้ บอบุ ตั ใิ หมข่ ึนในประเทศไทยและพบอกี หลายๆประเทศ โดยเฉพาะประเทศในเขตร้อนและก่ึงเขตร้อน และเป็นทีท่ ราบกนั ดีวา่ โรคตดิ เชอื ดังกลา่ วเป็นโรคที่ไมม่ ีวัคซนี ป้องกนั และไมม่ ยี ารักษาที่เฉพาะ ดงั นนั การควบคมุ การเกิดโรคทม่ี ีประสทิ ธิภาพจึงเป็นเพียงการควบคุมแมลงพาหะ อกี ทังเพื่อเข้าใจวงจรการแพรก่ ระจายของไวรสั และยุง การศกึ ษานจี งึ ไดอ้ อกแบบเพื่อตรวจหาการตดิ เชอื ไวรสั ซิกา ไวรสั ชิคนุ กันยา และไวรัสไข้เลือดออกเดงกีในยุงตามธรรมชาติดว้ ยเทคนคิ ทางอณู ชีววทิ ยา โดยเก็บยงุ ตัวเตม็ วัยและลกู น้ายุงบริเวณรอบบา้ นคนไขท้ ีต่ ้าบลแกลง จังหวดั ระยอง ประเทศไทย ซงึ่ ไวรัสชิคนุ กนั ยา และไวรัส ไขเ้ ลอื ดออกเดงกีจะตรวจสอบด้วยเทคนคิ วนั สเตป็ มลั ติเพลก็ เรยี ลไทม์ อาร์ที-พีซีอาร์ สว่ นไวรัสซกิ าตรวจดว้ ยเทคนิคเนสเตด็ อาร์ที-พซี ีอาร์ จากผลการทดลองพบวา่ ยุงทเ่ี กบ็ จากธรรมชาติใหผ้ ลบวกตอ่ ไวรัสซิกาจา้ นวน 8 (10.3%) ตัวอย่าง โดยพบในยุงลายบา้ น (Aedes aegypti) เพศเมีย 5 (6.4%) ตวั อย่างและเพศผจู้ า้ นวน 2 (2.6%) ตัวอยา่ ง และทนี่ ่าสนใจพบวา่ พบในตวั อย่างยงุ แม่ไก่ (Armigeres subalbatus) เพศ เมียจา้ นวน 1 ตวั อยา่ ง อกี ทงั ยังพบการติดเชือไวรสั ชคิ นุ กนั ยาจ้านวน 5 (6.4%) ตัวอย่างในยงุ ลายบา้ นเพศเมยี 3 (3.8%) ตวั อย่างและในระยะ ลูกนา้ ของยุงลายบ้านจา้ นวน 2 (2.6%) ตวั อย่าง และไม่พบการตดิ เชอื ไวรสั ไข้เลอื ดออกเดงกีในทกุ ตวั อย่าง เม่อื นา้ ล้าดบั นวิ คลีโอไทดข์ อง ตัวอย่างท่ีให้ผลบวกตอ่ ไวรสั ซกิ ามาสรา้ งแผนภูมติ ้นไมว้ ิวฒั นาการพบวา่ อยใู่ นกลุ่มของเอเชยี (Asian lineage) การศึกษานจี ึงเปน็ การศกึ ษา เบืองตน้ ในการส้ารวจหาเชอื ในยุงท่พี บในแหล่งระบาดของโรคเพื่อศกึ ษาหาแมลงพาหะท่สี ามารถกอ่ โรคไขซ้ กิ า โรคไขช้ คิ ุนกันยา และโรค ไข้เลือดออกเดงกี ขอ้ มลู ท่ไี ด้จากการศกึ ษานีจะชว่ ยใหเ้ ขา้ ใจถงึ อัตราการติดเชอื ของไวรสั ซิกา ไวรัสชิคนุ กนั ยา และไวรัสไข้เลอื ดออกเดงกีในยงุ ทเี่ ก็บจากธรรมชาตไิ ด้ และอาจจะน้าไปสูก่ ารควบคุมยงุ พาหะให้มปี ระสิทธภิ าพในอนาคต คาสาคญั : ไวรัสซิกา ไวรัสชคิ นุ กนั ยา ไวรัสไข้เลือดออกเดงกี ยุง ประเทศไทย 1สถาบนั วจิ ัยวิทยาศาสตรส์ าธารณสขุ กรมวทิ ยาศาสตรก์ ารแพทย์ กระทรวงสาธารณสุข 2ศนู ย์วทิ ยาศาสตรส์ ุขภาพโรคอุบัตใิ หม่-ศูนย์ปฏบิ ตั กิ ารโรคทางสมอง โรงพยาบาลจุฬาลงกรณ์ สภากาชาดไทย และ คณะแพทยศาสตร์ จุฬาลงกรณม์ หาวทิ ยาลัย 3สาํ นักงานป้องกนั ควบคุมโรคท่ี 6 จงั หวัดชลบุรี กรมควบคุมโรค กระทรวงสาธารณสขุ 4หลกั สตู รวทิ ยาศาสตรก์ ารแพทย์ คณะแพทยศาสตร์ จฬุ าลงกรณม์ หาวทิ ยาลัย 5หน่วยปฏิบัตกิ ารวจิ ัยชวี วิทยาของแมลงพาหะนําโรคและโรคตดิ ต่อนําโดยแมลง ภาควิชาปรสติ วทิ ยา คณะแพทยศาสตร์ จฬุ าลงกรณ์ มหาวิทยาลยั *ผูร้ ับผิดชอบบทความ E-mail: [email protected]

Original Article Hemostatic efficacy of sheep-derived fibrin glue for liver biopsy in swine Siriluck Luckanahasaporn1 Theerawat Tharasanit2 Passakorn Briksawan1 Sumit Durongphongthron1* Abstract Fibrin glue has been widely used for controlling hemorrhage and sealing tissue in surgery. The purpose of this study was to evaluate the hemostatic efficacy of sheep-derived fibrin glue. Six Marino sheep were used to collect concentrate fibrinogen preparation by the ammonium sulfate precipitation method. Crossbred pigs (n=6) were used to evaluate hemostatic efficacy by monitoring whole blood clotting time by the glass slide method, bleeding time and bleeding quantity during liver biopsy. Mean whole blood clotting time on a glass slide started at 201.1±90.47 and 4.43±3.73 seconds, and was completed at 447.83±63.77 and 31.93±4.28 seconds in control and treatment groups respectively. Two-sited open liver biopsies were performed, each biopsy site was assigned as either control or treatment groups. One ml of sheep-derived fibrin glue on the bleeding surface was applied at the treatment sited group. Bleeding quantity was estimated by the increased weight of filter paper after blood straining. Mean bleeding quantity was 0.94±0.38 and 0.1±0.12 g, and mean bleeding time was 175.18±11.80 and 68.08±28.84 seconds in control and treatment sites, respectively. Whole blood clotting time, bleeding time, and bleeding quantity were significantly less in the treatment-sited group, compared to the control-sited group (p<0.01). In conclusion sheep-derived fibrin glue could be applied as an effective hemostatic agent to control hemorrhage after liver biopsy in swine. Keywords: fibrinogen, liver biopsy, sheep-derived fibrin glue, swine, thrombin 1Department of Veterinary Surgery, Faculty of Veterinary Science, Chulalongkorn University, Bangkok 10330. Thailand 2Department of Obstetrics, Gynaecology and Reproduction, Faculty of Veterinary Science, Chulalongkorn University, Bangkok 10330. Thailand *Correspondence: [email protected] Thai J Vet Med. 2018. 48(4): 559-565.

560 Luckanahasaporn S. et al. / Thai J Vet Med. 2018. 48(4): 559-565. Introduction to evaluate the hemostatic efficacy of sheep-derived fibrin glue. Whole blood clotting time by glass slide Liver biopsy is the gold standard for method, bleeding time and bleeding quantity during diagnosis and prognosis in liver disease (Watson and the liver biopsy in swine were selected to determine the Bunch, 2009). There are many indications for liver efficacy of sheep-derived fibrin glue. biopsy such as evaluation of abnormal liver function, alteration of liver structure, confirmation or staging of Materials and Methods neoplastic diseases, and assessment of disease progression (Vasanjee et al., 2006). Bleeding is one of Animals: This study was approved by the Institutional the most common and serious complications (0.32% - Animal Care and Use Committee (IACUC), 0.35%), the next complication is death after liver biopsy Chulalongkorn University (Approval No. 13310065) (0.11%) (Alkozai et al., 2009; Al-Ghamdi, 2011). Six healthy Marino sheep 30–50 kg., were Fibrin glue was first used as a topical used for sheep-derived fibrin glue preparation. hemostatic agent in 1960 and developed for Approximately 90 ml of blood was collected from the commercial products in late 1970 (Tabélé et al., 2012). jugular vein into a tube containing 10 ml of Two major components of fibrin glue are fibrinogen anticoagulant (3.7% w/v sodium citrate) and then and thrombin.After combining these two components, gently mixed immediately. Six healthy crossbred pigs fibrinogen will be converted into fibrin clot, which is 18–22 kg., were used to evaluate hemostatic efficacy by the final product of the coagulation cascade, by liver biopsy (n=6). thrombin in the presence of calcium ion (de Boer et al., 2012; Tabélé et al., 2012). In general, fibrin glue is a Experimental design natural product with great biocompatibility, non Experiment 1: a glass slide method was used to toxicity to tissues, does not stimulate inflammation nor evaluate whole blood clotting time. Forty microliters delay wound healing and degrades by the normal (µl) of blood were collected from each pig and then fibrinolytic pathway (Gibble and Ness, 1990; each sample was divided into control (n=6) and Radosevich et al., 1997; Morey et al., 2001; Morikawa, treatment (n=6) groups with 20 µl of blood per each 2001; Aksoy et al., 2009). Presently, fibrin glue has been sample. widely used in a variety of surgical procedures, including plastic surgery, cardiothoracic surgery, Experiment 2: Bleeding time and bleeding quantity vascular surgery, neurosurgery, gastrointestinal following by liver biopsy were used to evaluate surgery, and hepatic surgery (Morikawa, 2001). hemostatic efficacy.Two open liver biopsy sites were Several studies have shown that the application of performed using endoscopic cup biopsy forceps. The fibrin glue in liver surgery is effective in reducing the biopsy sites of each pig were divided into control (n=6) bleeding quantity and hemostatic time (Wheaton et al., and treatment (n=6) groups. 1994; Davidson et al., 2000; Paulson et al., 2000; Karpelowsky et al., 2006; Taha et al., 2006). Fibrin glue preparation: Sheep blood samples were centrifuged at 4°C 600gfor 20 minutes to obtain plasma The two basic categories of fibrin glue are and all plasma samples were pooled. Concentrated commercial fibrin glue and in-house fibrin glue. fibrinogen was prepared by the ammonium sulfate Methods for producing concentrated fibrinogen precipitation method. Saturated ammonium sulfate (80 involve cryoprecipitation and chemical precipitation gram in distilled water 100 ml) was added into pool methods such as precipitation by ethanol, polyethylene plasma at a ratio of 1:5 to plasma and then centrifuged glycol and ammonium sulfate (Silver et al., 1995a, at 4°C 1600g for 5 minutes. Supernatants were Wang et al., 1995). Several studies have indicated discarded and pellets were resuspended in distilled ammonium sulfate precipitation as yielding higher water at a ratio of 1:10 to plasma and then centrifuged fibrinogen concentration, and therefore higher at 4°C 1600g for 5 minutes. Soluble protein was bonding strength of fibrin glue (Siedentop et al., 1985; collected as fibrinogen-rich fraction protein (Silver et Silver et al., 1995a). The advantages of using in-house al., 1995b)(Fig 1). fibrin glue include reducing the risk of disease transmission, immune reaction, and cost of surgery Lyophilized commercial bovine thrombin, (Tabélé et al., 2012). However, the limitations of using 1000 International Unit (IU) per vial (EMD Millipore in-house fibrin glue are the low concentrations of Inc., SDG, USA), was used in this study. Thrombin fibrinogen due to blood collection from animals with solution was generated by adding 1 ml calcium low body weight, with coagulopathy, or with liver chloride at a concentration of 40 mmol/L in order to disease and especially for the use in urgent surgery make the final concentration of 1000 IU/ml of procedures (Gibble and Ness, 1990; Wheaton et al., thrombin. 1994). Until the present time, the use of commercial fibrin glue has not been a mainstay of common practice Concentration of fibrinogen: Fibrinogen concentration in veterinary surgery due to economic concern. was measured by the heat precipitation method (Millar et al., 1971). One hundred microliters of plasma were Therefore in this study, in-house fibrin glue heated for 3 minutes in a water bath (56 ± 1°C) and then was developed from concentrated sheep-derived centrifuged at room temperature at 2000 RPM for 3 fibrinogen by the ammonium sulfate precipitation minutes. The supernatant was evaluated for its protein method, which can be an alternative method to control concentration (g/dl) as fibrinogen-free fraction protein hemorrhage. Moreover, sheep-derived fibrin glue can (Protein 1) by a refractometer. Fibrinogen-rich fraction be produced from plasma as the byproduct of sheep protein (Protein 2), which is the final result of the blood agar production. The objective of this study was

Luckanahasaporn S. et al. / Thai J Vet Med. 2018. 48(4): 559-565. 561 ammonium sulfate precipitation method, was protein 1 multiply with 1000 (fibrinogen concentration measured for its concentration (g/dl) by (mg/dl) = (Protein 2 – Protein 1) x 1000) (Millar et al., refractrometer. Fibrinogen concentration (mg/dl) was 1971) calculated as the difference between the protein 2 and Figure 1 Concentrated fibrinogen preparation by the ammonium sulfate precipitation method (Modified from Silver et al., 1995b) Whole blood clotting time: Twenty microliters of blood Two biopsy sites were allocated as either control or were dropped onto a glass slide with either 20 µl of treatment groups. Approximately 1 ml of sheep 0.9% saline (w/v), or 20 µl of sheep-derive fibrin glue derived fibrin glue was applied onto the bleeding in the control and treatment groups respectively. The surface in the treatment group. Bleeding quantity was clotting time was determined as the time from the estimated by applying no.1 filter paper (Whatman®; blood dropping onto the glass slide until blood clotting GE Healthcare, Buckinghamshire United Kingdom) was first mentioned (second) (Gross et al., 1929). without pressure on the biopsy site until hemostasis was completed. The filter papers were weighed before Liver biopsy: Liver biopsies were performed under and after blood absorption to measure the amount of general anesthesia. The animals were premedicated blood loss in term of weight. The time that hemostasis and inducted with 2.2 mg/kg of ketamine (Calypsol®; accomplished was recorded as bleeding time (second). Gedene Richter Inc., Budapest, Hungary), 2.2 mg/kg Suturing of the biopsy site in the control group or of xylazine (Seton®; Calier Inc., Barcelona, Spain )and reapplying 1 ml of sheep-derived fibrin glue in the treatment group was considered if bleeding continued 4.4 mg/kg of zolazepam-tiletamine (Zoletil®; Virbac to occur for more than 3 minutes. After complete Inc., Texas, USA) intramuscularly, analgesic of hemostasis, the liver and peritoneal cavities were meperidine (Demerol®; Hospira, Illinois, USA) inspected for 30 minutes to assess if there were intramuscularly (Smith et al., 1997) and antibiotic rebleeding. Then, the animals were euthanized using prophylaxis of amoxicillin-clavulanic acid (Synulox®; pentobarbital sodium (Nembutal®; Ceva Santé Pfizer Inc., NY, USA) 8.75 mg/kg intramuscularly. Animale Inc., Libourne, France) 100 mg/kg Anesthesia was maintained with isoflurane and intravenously (Shaw and Reilly, 2001). oxygen delivery by endotracheal intubation. The heart rate (BPM), systolic blood pressure (mmHg) and Statistical analysis: Fibrinogen concentration was oxygen saturation (%) were monitored during the described as descriptive analysis. Paired t-test was period of anesthesia. Two sites of liver biopsy were used to evaluate the statistical significance of whole performed at the peripheral area using 5 mm. blood clotting time on a glass slide, bleeding time and endoscopic cup biopsy forceps at the left lateral lobe.

562 Luckanahasaporn S. et al. / Thai J Vet Med. 2018. 48(4): 559-565. bleeding quantity between control and treatment higher compared to the concentration before groups. precipitation. Results Experiment 1: There was statistically significant difference in whole blood clotting time between The fibrinogen concentration was 3000 mg/dl control and treatment groups (p<0.01)(Table1) (Fig (30mg/ml) after precipitation, which was 7.5 times 2A). Table 1 Whole blood clotting time (mean±SD) in control and treatment groups Groups Start clot (seconds) Complete clot (seconds) Control 201.1±90.47a 447.83±63.77b Treatment 4.43±3.73a 31.93±4.28b Same superscript letters in the same column represent statistically significant (p < 0.01) AB BB Figure 2 Complete blood clot on glass slide and fibrin clot on bleeding surface (Fig. 2a) whole blood clotting was performed after binding blood with fibrin glue on a glass slide in the treatment group (Fig. 2b) fibrin clot (arrow) was covered on bleeding surface after applied sheep-derive fibrin glue for controlling hemorrhage in the treatment group Experiment 2: After the liver biopsy, bleeding time and surface in all animals in treatment groups (Fig 2B). All bleeding quantity in the treatment group were animals in treatment groups a had lower bleeding time significantly lower compared with the control group and bleeding quantity compared with animals in the (p<0.01) (Table 2,3). In the control group, suturing was control group (100%). Rebleeding did not occur in all animals. required to achieve hemostasis in 5/6 animals (83.33%). Fibrin clot was presented on the bleeding Table 2 Bleeding time (mean±SD) after liver biopsy in control and treatment groups Groups Bleeding time (seconds) Control 175.18±11.8a Treatment 68.08±28.84a Same superscript letters in the same column represent statistically significant (p < 0.01) Table 3 Bleeding quantity (mean±SD) after liver biopsy in control and treatment groups Groups Bleeding quantity (ml) Control 0.94±0.38a Treatment 0.1±0.12a Same superscript letters in the same column represent statistically significant (p < 0.01) Discussion efficient in achieving fast and durable hemostasis as well as not causing adverse effects. In addition, it Liver biopsy is an important procedure for the should be cost efficient and user friendly (Moench et diagnosis, treatment options and prognosis of liver al., 2010). In general, fibrin glue has been widely used disease in patients. In normal animals, liver biopsy is in liver surgery to reduce hemostatic time and bleeding generally safe and can be accomplished with minimal quantity (de Boer et al., 2012). The bonding strength of blood loss (Vasnjee et al., 2006; Rothuizen and Twedt, fibrin glue and the patrient’s hemostatic time are 2009). However, patients with liver disease that may related to fibrinogen and thrombin concentrations require liver biopsy as a diasnostic procedure are at a respectively (Durham et al., 1987; Harris et al., 1988; higher risk of bleeding due to the coexistent of Wheaton et al., 1994; Kheirabadi et al., 2001; coagulopathy (Paulson et al., 2000). Several topical MacGillivray, 2003). Fibrinogen concentrations in haemostatic agents are used in liver surgery to reduce cryopreccipitated plasma are approximately 20 mg/ml hemorrhage. The ideal hemostatic agent should be (Dresdale et al., 1985) and in chemical precipitated

Luckanahasaporn S. et al. / Thai J Vet Med. 2018. 48(4): 559-565. 563 plasma range from 13-57 mg/ml (Park and Cha 1993; In conclusion, sheep-derived fibrin glue can Kjaergard and Weis-Fogh 1994), similar to this study be used as an effective topical hemostatic agent for result, fibrinogen concentration was 30 mg/ml after controlling hemorrhage following liver biopsy in ammonium sulfate precipitation. Although the swine and could further be adapted for liver biopsy concentration was lower than commercial fibrin glue, procedure in clinically veterinarian practice. we believe that the bonding strength of the sheep- derived fibrin glue we prepared was adequate to be Acknowledgements used in liver biopsy protocols because there was no rebleeding occurring in any animal. In addition, This study was financially supported by the previous studies were accomplished using lower 90th year Chulalongkorn scholarship, Chulalongkorn fibrinogen concentration (22±0.7 mg/ml and 10.72 University, Bangkok, Thailand. We are grateful for the mg/ml) as hemostatic agent in liver biopsy models laboratory technique assistance of Dr. Narong (Wheaton et al., 1994; Davidson et al., 2000). The Tiptanavattana and staff of the Research Unit for advantages of chemical precipitation over Obstetrics and Reproduction in Animals, cryoprecipitation are its convenience and the rapid Chulalongkorn University, as well as graduate method to concentrate of the fibrinogen (Silver et al., students and staff of the surgery unit, Small animal 1995a). Thrombin concentration of in-house fibrin glue Teaching Hospital, Faculty of Veterinary Science, normally ranges from 500-1000 IU/ml (Wheaton et al., Chulalongkorn University for surgical assistance. 1994). Since the bleeding time is perhaps as important as bleeding quantity,we decided to use a high References concentration of thrombin (1000 mg/ml) to rapidly reduce the bleeding time. The mean bleeding time was Aksoy H, Rabus A, Uras F, Savci N, Alatlo FC, Erseven significantly shorter in the sheep-derived fibrin glue G and Emekli N 2009. Evaluation of the effect of treatment group than control group. Mean bleeding fibrin glue prepared from single-donor plasma on quantity in the treatment group was 0.1±0.12 g. This wound healing in rats. Hacet J Faculty Pharm. showed up as the same direction of results from the 29(2): 83-93. previous study, using commercial fibrin glue as a/the hemostatic agent after tru-cut liver biopsy in swine, in Alkozai EM, Lisman T and Porte RJ 2009.Bleeding in which mean bleeding quantity was 0.1 ml (Paulson et liver surgery: prevention and treatment.Clin al., 2000). However, bleeding quantity after using tru- Liver Dis. 13(1): 145-154. cut liver biopsy is slightly higher than using endoscopic cup biopsy forceps in normal animals Al-GhamdiASG 2011. Complications of Liver Biopsy. (Vasanjee et al., 2006). The conversion of fibrinogen to In: Liver biopsy. 1st ed. H Takahashi (ed). Rijeka: fibrin is caused by thrombin in the presence of calcium InTech. p. 363-370. ions. The concentration of calcium chloride in in-house fibrin glue varied from 20-160 mmol/L (Wang et al., de Boer MT, Boonstra EA, Lisman T and Porte RJ 2012. 1995). In this study, 40mmol/L calcium chloride was Role of fibrin sealant in liver surgery. Dig Surg. used due to the report of the previous study showing 29(1): 54-61. high bonding strength and rapid formation of fibrin glue after 20-40 mmol/L of calcium chloride was Davidson BR, Burnett S, Javed MS, Scifalian A, Moore added (Wang et al., 1995). Moreover, this concentration D and Doctor N 2000.Experimental study of a was similar to concentration in commercial fibrin glue novel fibrin sealant for achieving haemostasis (Tisseel®; Baxter, Illinois, USA). Unlike most following partial hepatectomy. Brit J Surg. 87(6): commercial fibrin glue, the antifibrinolytic agent such 790-795. as aprotinin was not added in this study due to several studies suggesting that aprotinin is not a necessary Dresdale A, Bowman FO Jr, Malm JR, Reemtsma K, component because fibrinolysis of glue before Smith CR, Spotnitz HM and Rose EA 1985. complete hemostasis has not been reported (Durham et Hemostatic effectiveness of fibrin glue derived al., 1987; Gibble and Ness 1990; Wheaton et al., 1995). from single-donor fresh frozen plasma. Ann Moreover, some present commercial fibrin glue Thorac Surg. 40(4): 385-387. products also does not contain aprotinin as its composition (Quixil®; OMRIX Biopharmaceuticals, Durham LH, Willatt DJ, Yung MW, Jones I, Stevenson Kiryat Ono, Israel). However, antifibrinolytic agent A and Ramadan MF 1987.A method for should be added when using fibrin glue at high levels preparation of fibrin glue. J Laryngol Otol. 101: fibrinolysin organs such as the lung, kidney, prostatic 1182-1186. gland, and uterus, because long-term bond strength is required (Gibble and Ness 1990) and tranexamic acid Gibble JW and Ness PM 1990. Fibrin glue: the perfect is commonly used as an antifibrinolytic agent in in- operative sealant?.Transfus. 30(8): 741-747. house fibrin glue (Radosevich et al., 1997). Gross P 1929.Duration of anticoagulant action of Further studies on the adverse effect by heparin in vivo in relation to dosage.ExpBiol immunogenicity reaction from using xenogenic Med. 26: 383-384. products, prevention of biliary leakage, and hemostatic efficacy in coagulopathy animals are required in order Harris DM, Siedentop KH and Sanchez B 1988. to use sheep-derived fibrin glue as the topical Autologous fibrin tissue adhesive: factors hemostatic agent in liver disease patients. influencing bonding power. Laryngoscope.98: 731-733. Karpelowsky JS, Numanoglu A, Bax NMA and Rode H 2006. Laparoscopically assisted liver biopsy with a hemostatic plug.SurgEndosc. 20: 1626–1628. Kheirabadi BS, Pearson R, Rudnicka K, Somwaru L, MacPhee M, Drohan W and Tuthill D 2001. Development of animal model for assessment of

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Luckanahasaporn S. et al. / Thai J Vet Med. 2018. 48(4): 559-565. 5653 บทคดั ย่อ ประสิทธิภาพในการห้ามเลอื ดของกาวไฟบรินแบบเตรียมเองจากเลอื ดแกะ ในการทาศัลยกรรมตดั ชิ้นเน้ือตับเพื่อตรวจในสกุ ร ศริ ลิ กั ษณ์ ลัคนหัสพร1 ธีรวฒั น์ ธาราศานิต2 ภาสกร พฤกษะวนั 1 สมุ ติ ร ดุรงค์พงษธ์ ร1* กาวไฟบรินถูกใช้อย่างกวา้ งขวางในการศลั ยกรรมเพือ่ ช่วยห้ามเลือด และประสานเนอื้ เย่อื การศึกษาคร้งั น้มี จี ดุ ประสงคเ์ พ่อื ประเมนิ ประสิทธิภาพในการห้ามเลอื ดของกาวไฟบรนิ จากเลอื ดแกะ จากระยะเวลาการแขง็ ตัวของเลือดบนแผ่นสไลด์ ระยะเวลาตกเลอื ด และ ปริมาตรการตกเลือดในระหว่างศลั ยกรรมตัดชิน้ เนื้อตับเพื่อตรวจในสุกร แกะจานวน 6 ตวั ถูกใช้ในการเตรยี มไฟบริโนเจนเขม้ ขน้ ดว้ ยวิธี ตกตะกอนด้วยแอมโมเนยี มซลั เฟต สุกรจานวน 6 ตวั ถกู ใชใ้ นการศึกษาประสทิ ธภิ าพกาวไฟบรนิ จากเลอื ดแกะ โดยค่าเฉล่ียระยะเวลาทเ่ี ลือด เร่มิ แข็งตวั บนแผน่ สไลด์เทา่ กบั 201.1±90.47 และ 4.43±3.73 วนิ าที และเลือดแข็งตวั โดยสมบูรณท์ ี่ 447.83±63.77 และ 31.93±4.28 วนิ าที ในกลมุ่ ควบคุมและกลุ่มทดลองตามลาดับ ทาการศลั ยกรรมตดั ชิน้ เนื้อตับเพือ่ ตรวจในสกุ รตัวละ 2 ตาแหน่ง โดยเนื้อตบั แตล่ ะตาแหน่ง จะถูกแบง่ เป็นกลุ่มควบคุมและกลมุ่ ทดลอง โดยกลมุ่ ทดลองได้รบั การหา้ มเลอื ดดว้ ยกาวไฟบรินจากเลือดแกะปรมิ าตร 1 มลิ ลิลติ ร ประเมินปริ มาตรการตกเลือดจากน้าหนักท่ีเพ่มิ ขึน้ ของกระดาษกรองจากการซับเลือด ซงึ่ พบวา่ พบวา่ มคี ่าเฉลยี่ เท่ากับ 0.94±0.38 กรมั และ 0.1±0.12 กรมั และระยะเวลาเสียเลอื ดมีคา่ เฉลี่ย 175.18±11.80 วินาที และ 68.08±28.84 วินาที ในกลุ่มควบคุมและกลมุ่ ทดลองตามลาดับ ผล การศึกษาพบวา่ ระยะเวลาทเ่ี ลือดแข็งตัวบนแผ่นสไลด์ ระยะเวลาตกเลือด และปริมาตรการตกเลือดในระหวา่ งศลั ยกรรมตัดช้นิ เน้อื ตบั เพือ่ ตรวจมีความแตกตา่ งอย่างมีนัยสาคัญระหวา่ งกลมุ่ ควบคุมและกลุ่มทดลอง (p<0.01) โดยสรปุ กาวไฟบรินจากเลือดแกะมปี ระสทิ ธิภาพดใี น การหา้ มเลือดจากการศัลยกรรมตดั ชิน้ เนอ้ื ตับเพอื่ ตรวจในสกุ ร คาสาคัญ: ไฟบริโนเจน ศัลยกรรมตัดช้ินเนื้อตบั เพื่อตรวจ กาวไฟบรนิ จากเลือดแกะ สุกร ทรอมบิน 1ภาควชิ าศลั ยศาสตร์ คณะสตั วแพทยศาสตร์ จฬุ าลงกรณม์ หาวทิ ยาลยั ปทุมวนั กรุงเทพ ฯ 10330 2ภาควิชาสูตศิ าสตร์ เธนเุ วชวิทยา และวิทยาการสบึ พนั ธ์ุ คณะสตั วแพทยศาสตร์ จุฬาลงกรณ์มหาวิทยาลยั ปทมุ วนั กรงุ เทพ ฯ 10330 *ผู้รับผดิ ชอบบทความ E-mail: [email protected]



Original Article Small dosages of alpha1 adrenoceptor antagonist on goat semen quality and seminal fluid volume Sakdichod Kimsakulvech1* Sookruetai Boonmasawai1 Dulyatud Gronsang1 Chowalit Nakthong2 Yupaporn Lanamtiang3 Abstract The frozen goat semen process must wash seminal fluid from goat semen. A small dosage of alpha1 adrenoceptor antagonist or tamsulosin may reduce seminal fluid in goat. A latin square 4x4 was applied to twelve male goats to receive either normal saline (NSS), tamsulosin 3, 6 and 9 g/kg intramuscular injection at one-week intervals. Semen collection by artificial vagina and libido scoring was performed within one-hour post injection. Semen quality and seminal fluid were evaluated after semen collection The results showed that none of the libido scores in the groups was different. Anejaculations occurred in 1 (8.3%), 3 (25%) and 4 (33.3%) using tamsulosin 3, 6 and 9 g/kg, respectively. Only, the volume of seminal fluid in tamsulosin 9 g/kg has significantly lower than the NSS group. Other semen quality did not show any significant difference. Although, in this experiment, the alpha1 adrenoceptor antagonist or tamsulosin could reduce the amount of seminal fluid in goats anejaculation was promoted in all dosages of tamsulosin one hour after injection. Therefore, this drug may not suitable in reducing seminal fluid when producing goat frozen semen. Keywords: alpha1 adrenoceptor antagonists, tamsulosin, seminal fluid, semen quality, goat 1Department of Preclinical and Applied Animal Sciences, Faculty of Veterinary Science, Mahidol University 2Department of Clinical science and Public health, Faculty of Veterinary Science, Mahidol University 3Veterinary Medical Center for Livestock and Wildlife Animal Hospital, Faculty of Veterinary Science, Mahidol University *Correspondence: [email protected] Thai J Vet Med. 2018. 48(4): 567-572.

568 Kimsakulvech S. et al. / Thai J Vet Med. 2018. 48(4): 567-572. Introduction 19). Twelve mixed breed male goats aged 1 to 3 years and weighing 30 to 45 kg were bought from the farmer. In many mammal species seminal fluid has an Male goats were kept in a single pen and separated important role for spermatozoa metabolism, function, from female goats at the Veterinary Medical Center for survival and transportation in the female reproductive Livestock and Wildlife Animal Hospital, Faculty of tract (Juyena and Stelletta, 2012). Nevertheless, seminal Veterinary Science, Mahidol University, fluid in goats may become toxic and decrease Kanchanaburi, Thailand. Before any testing, all goats spermatozoa survival in the frozen semen process were evaluated for their health and judged to be (Ferreira et al., 2014). The goat seminal fluid washing brucellosis disease free by the Rose-bengal test. They procedure can increase the spermatozoa survival rate were confirmed to have a good libido and a normal in the frozen semen process (Memon et al., 1985; Kucuk ejaculating ability using an artificial vagina (AV) et al., 2014). The seminal fluid washing procedure uses producing and normal semen quality. a centrifuge to pack spermatozoa down at the bottom of a centrifuge tube. A packed spermatozoa does not This experiment was designed as a 4 x 4 Latin facilitate the evaluation of viable spermatozoa. In square. Each male was administered normal saline addition, the dead spermatozoa are included in the (NSS, control), Tamsulosin 3 g/kg or 6.743 nmol/kg pack of spermatozoa packed pellets. These dead (TAM3), Tamsulosin 6 g/kg or 13.485 nmol/kg spermatozoa release a reactive oxygen species (ROS) (TAM6) and Tamsulosin 9 g/kg or 20.229 nmol/kg within the packed pellets which causes live (TAM9) intramuscularly at one-week intervals. spermatozoa membrane damage (Aitken and Clarkson, 1987). Moreover, the washing procedure Solution preparation: Tamsulosin was purchased from increases the time of the freezing process and loses Sigma-Aldrich Pte Ltd. TAM (50 mg) was dissolved in spermatozoa during spinning. Interestingly, if we can 1 ml dimethylsufoxide (DMSO) to generate a reduce seminal fluid in ejaculated goat semen, the concentrated stock solution which was then diluted washing procedure may not be essential in the frozen with additional DMSO (200 l) prior to administration. goat semen process which may lead to an increase in Normal saline (200 l) was administered during the the viability and number of spermatozoa. control trial. Alpha1 adrenoceptor antagonist drugs are Libido scoring and semen collection: Semen was used to treat benign prostatic hyperplasia (BPH) in collected using an AV and an estrous female to trigger humans (Welliver and Essa, 2016). Tamsulosin is a mating behavior at and after drug administration for drug of the alpha1 adrenoceptor antagonist type with 15 - 60 minutes. Libido was scored by a modified a high affinity for alpha1a and 1d adrenoceptors in the version of Frydrychova (Frydrychova et al., 2011), each human prostate gland. This drug induces smooth male had the chance to copulate once or twice. In the muscle relaxation in the human prostate (Kawachi, first attempt, if no semen was found in the AV 1998). In goats, a high dosage of tamsulosin between 60 collecting tube after thrusting, the male with - 120 g/kg can suppress ejaculation within 3 hours of ejaculatory suppression was allowed to have a second injection (Kimsakulvech et al., 2015a,b). Noticeably in chance of mounting within 10 mins. Subsequently, if the recovery period or 12 hours after tamsulosin 120 the goat did not copulate in 10 mins, the ejaculation of g/kg injection, all goats had normal ejaculation while this goat was classified as anejaculation. a higher spermatozoa concentration than in the control group was recorded. It is suggested that the increasing Ejaculatory scoring: Ejaculation was classified into spermatozoa concentration may result from the two groups: anejaculation and complete ejaculation. accessory sex gland smooth muscle remaining relaxed Anejaculation was defined as a lack of semen in the AV because of the small tamsulosin dosage remaining in collecting tube and complete ejaculation as semen the body. Decreasing the seminal fluid in goats by volume containing spermatozoa in the collecting tube. alpha1 adrenoceptor antagonist drug or tamsulosin may be possible with a small dosage. A lower dosage Semen quality assessment: The semen volume, mass of tamsulosin on semen quality had been shown in spermatozoa movement score, percentages of motile, humans (Hisasue et al., 2006). Tamsulosin 0.2 and 0.4 live spermatozoa, spermatozoa concentration, and mg/ person reduced semen volume and fructose in total spermatozoa per ejaculate were measured to male volunteers. The reduction of these parameters assess semen quality. Seminal fluid volume was receiving small dosage tamsuloin may have resulted evaluated and calculated according to the percentage from alpha1 adrenoceptor antagonist inhibiting of seminal fluid for assessed tamsulosin affecting accessory gland secretion (Mann, 1974; Marconi et al., accessory sex organ function. 2009). However, the effect of small dosage of tamsulosin on reducing seminal fluid in goats has not Semen volume was measured using a been revealed. The present study investigates small tuberculin syringe. Mass spermatozoa movement was dosages of tamsulosin on seminal fluid volume and scored from 0 (immotile) to 5 (high), and the semen quality in goats. percentage of motile spermatozoa was measured using light microscopy. Spermatozoa concentration was Materials and Methods estimated by hemocytometer. The percentage of live spermatozoa was based on the hypo-osmotic swelling The protocol was approved by the Animal test (Fonseca et al., 2005). The total number of Usage and Ethics Committee of the Veterinary Science spermatozoa was calculated from spermatozoa Faculty, Mahidol University (ID no. MUVS 2016-05- concentration and semen volume. After semen measurement, the semen of each

Kimsakulvech S. et al. / Thai J Vet Med. 2018. 48(4): 567-572. 569 goat was centrifuged at 5,000 rpm for 15 minute. with a difference of p < 0.05. All values were shown as Seminal fluid was separated from spermatozoa and the mean and standard error of the mean (SEM). was measured by tuberculin syringe. The percentage of seminal fluid was calculated by semen volume. Results Statistical analysis: The semen volume, percentages All goats copulated at the first attempt within of motile and live spermatozoa, spermatozoa 2 minutes of face to face contact. The libido score of concentration, total spermatozoa, seminal fluid each group is shown in table 1. Anejaculation is shown volume and the percentage of seminal fluid were in the tamsulosin injection group. Tamsulosin 3, 6 and analyzed using analysis of variance for Latin square designs. The libido score and mass spermatozoa 9 g/kg had 1, 3 and 4 anejaculation, respectively. The movement score were examined by the Chi-square test. semen quality of anejaculation goats was not include in Mean values were considered statistically significant the statistical calculation. Only seminal fluid and the percentage of seminal fluid in the tamsulosin 9 g/kg group was significantly lower than the control groups. Other semen parameters did not show any statistically significant difference as is shown in table 2. Table 1 Libido score (mean + SEM) between control (NSS), TAM3, TAM6 and TAM9 n NSS TAM3 TAM6 TAM9 Libido score 12 5.0 4.92±0.08 5.0 4.92±0.08 Table 2 Comparison of goat semen quality and seminal fluid volume (mean + SEM) between NSS, TAM3, TAM6 and TAM9 Parameters n NSS n TAM3 n TAM6 n TAM9 Semen volume (ml) 12 0.65±0.07 11 0.552±0.08 9 0.439±0.08 8 0.419±0.09 Seminal fluid volume (ml) 12 0.48±0.05a 11 0.36±0.05 9 0.291±0.05 8 0.261±0.06b Sediment semen (ml) 12 0.17±0.03 11 0.19±0.03 9 0.15±0.04 8 0.16±0.04 Percentage of seminal 12 75.18±3.5a 11 67.88±3.6 9 64.52±4.1 8 59.51±4.3b plasma per semen volume 12 4.75±0.17 11 4.73±0.18 9 4.56±0.20 8 4.50±0.21 Mass spermatozoa 12 91.7±4.3 11 80.91±4.5 9 85.56±5.0 8 81.25±5.3 movement score 12 4.31±0.5 11 3.97±0.6 9 3.65±0.6 8 3.50±0.7 Percentage of spermatozoa 12 49.21±4.4 11 40.23±4.6 9 40.50±5.1 8 35.5±5.4 motility 12 2.94±0.5 11 2.60±0.6 9 1.84±0.6 8 1.47±0.7 Spermatozoa concentration (x109 cells) Percentage of live spermatozoa Number of spermatozoa per ejaculate (x109 cells) ab Values with different superscripts in the same row are significant difference at p < 0.05 Discussion In addition to seminal fluid volume, semen quality parameters did not show a significant Tamsulosin, alpha1 adrenoceptor antagonist, difference between groups. Concentration of the 3 - 9 g/kg, IM caused anejaculation in goats within 60 minutes of injection. Tamsulosin 9 g/kg promoted a tamsulosin 6 - 9 g/kg tended to reduce the mean higher anejaculation than other groups. The number of semen volume, spermatozoa concentration and anejaculation in goats increased with an increase of the number of spermatozoa per ejaculate more than the tamsulosin dosage. In previous reports, tamsulosin 0.2 mg/person (about 3 g/kg) in humans caused tamsulosin 3 g/kg. Tamsulosin 9 g/kg tended to ejaculatory dysfunction. The tamsulosin decreased the effect semen quality similar to volunteers receiving capacity of seminal vesicle contraction leading to a tamsulosin 0.8 mg/person (Hellstrom and Sikka, reduction in semen volume (Hisasue et al., 2006). The 2006a; Hellstrom and Sikka, 2006b). In a previous higher dosage of tamsulosin 0.8 mg/person (about study, in the recovery period 12 hours after tamsulosin 11.42 g/kg) promoted 35 percent anejaculation (Hellstrom and Sikka, 2006b). In goats, tamsulosin 9 120 g/kg injection, all goats had normal ejaculation g/kg caused 66.6 percent of anejaculation. These with a higher spermatozoa concentration than control results suggest that the suppressed ejaculation with group (Kimsakulvech et al., 2015a). In the recovery tamsulosin seemed to effect goats more than humans. period, the remaining tamsulosin in the body that was However, the effect on anejaculation with tamsulosin received at a high dosage of tamsulosin could result in dosage in this study was dose dependent, consistent only the inhibition of the accessory sex glands function. with a previous study (Kimsakulvech et al., 2015a). However, the small dosage of the tamsulosin in this experiment did not demonstrate any spermatozoa concentration difference. The rate of drug delivery

570 Kimsakulvech S. et al. / Thai J Vet Med. 2018. 48(4): 567-572. dependent on blood flow in each organ may have been Dziuk PJ and Norton HW 1962. Influence of drugs the cause of this difference (Kok-Yong and Lawrence, affecting the autonomic system on seminal 2015). The possibility is that accessory sex glands may ejaculation. J Reprod Fertil. 4: 47-50. have a lower blood flow than the epididymis or vas deferens. Also, in the recovery period of tamsulosin Ferreira VdS, Mello MRBd, Fonseca CEMd, Dias ÁCF, 120 g/kg, the tamsulosin might also affect only Cardoso JM, Silva RB and Martins Júnior WP accessory sex glands. Seminal fluid decreased from 2014. Effect of seminal plasma and egg yolk accessory sex glands dysfunction. In this experiment, concentration on freezability of goat semen. Rev one hour after injection, the duration period might not Bras Zootec. 43(10): 513-518. have been enough to diffuse tamsulosin from the vas deferens and epididymis, also it might have inhibited Fonseca JF, Torres CAA, Maffili VV, Borges AM, the contraction of these organs. Santos ADF, Rodrigues MT and Oliveira RFM 2005. The hypoosmotic swelling test in fresh goat Autonomic nervous system control of spermatozoa. Anim Reprod. 2(2): 139-144. seminal fluid secretion in goats is not well understood. Previous reports have explained that the autonomic Frydrychova S, Opletal L, Macakova K, Lustykova A, nervous system is the main control of seminal fluid Rozkot M and Lipensky J 2011. Effects of herbal secretion in rats and boars (Dziuk and Norton, 1962; preparation on libido and semen quality in boars. Dziuk and Mann, 1963; Kepper and Keast, 1997; Reprod Domestic Anim. 46(4): 573-578. Coolen et al., 2004). The importance of a sympathetic nervous system on seminal fluid secretion has been Hellstrom W and Sikka SC 2006a. Effect of (alpha)- shown in rats. Alpha adrenoceptor antagonists such as blockers on sperm parameters in healthy adult prazosin reduced the seminal vesicle pressure in rats men. Eur Urol Suppl. 5(2): 217-217. (Hsieh et al., 1998). This study reveals the inhibition of a sympathetic nervous system by alpha1 adrenoceptor Hellstrom WJG and Sikka SC 2006b. Effects of acute antagonist or tamsulosin 9 g/kg can significantly treatment with tamsulosin versus alfuzosin on reduce seminal fluid in the control group. A ejaculatory function in normal volunteers. J Urol. sympathetic nervous system could be one part of the 176(4): 1529-1533. seminal fluid secretion control in goat. The sympathetic control ejaculation in goats had an effect Hisasue S, Furuya R, Itoh N, Kobayashi K, Furuya S on spermatozoa ejection (Kimsakulvech et al., 2015b). and Tsukamoto T 2006. Ejaculatory disorder Interestingly, the small tamsulosin dosage in this study caused by alpha-1 adrenoceptor antagonists is still had an effect on spermatozoa ejection. One hour not retrograde ejaculation but a loss of seminal after tamsulosin injection at this dosage may not be emission. Int J Urol. 13(10): 1311-1316. enough to reduce the effect on vas deferens or epididymis on spermatozoa ejection. Hsieh JT, Chang HC, Law HS, Hsieh CH and Cheng JT 1998. In vivo evaluation of serotonergic agents The lowest dosage of alpha1 adrenoceptor and alpha-adrenergic blockers on premature antagonist in this study, tamsulosin 3 g/kg could ejaculation by inhibiting the seminal vesicle reduce seminal fluid to 75 percent of control group pressure response to electrical nerve stimulation. with other semen quality and was not distinctly Br J Urol. 82(2): 237-240. different from the control group. However, only one anejaculate occurred at this dosage and this cannot Juyena NS and Stelletta C 2012. Seminal plasma: an exclude the possibility of using tamsulosin for essential attribute to spermatozoa. Journal of reducing seminal fluid prior to production in the andrology. 33(4): 536-551. frozen goat semen process. Kawachi Y 1998. Effect of tamsulosin on urodynamics Acknowledgements in benign prostatic hypertrophy. Curr Ther Res. 59(3): 149-161. The grant was supported by the Faculty of Veterinary Science, Mahidol University. Kepper ME and Keast JR 1997. Location, immunohistochemical features, and spinal References connections of autonomic neurons innervating the rat seminal vesicles. Biol Reprod. 57(5): 1164- Aitken RJ and Clarkson JS 1987. Cellular basis of 1174. defective sperm function and its association with the genesis of reactive oxygen species by human Kimsakulvech S, Suttiyotin P and Pinyopummin A spermatozoa. J Reprod Fertil. 81(2): 459-469. 2015a. Dose-dependent effects of tamsulosin on the ejaculation and semen quality in bucks. Turk Coolen LM, Allard J, Truitt WA and McKenna KE 2004. J Vet Anim Sci. 39(4): 465-471. Central regulation of ejaculation. Physiol Behav. 83(2): 203-215. Kimsakulvech S, Suttiyotin P and Pinyopummin A 2015b. Effects of alpha1-adrenoceptor antagonist Dziuk PJ and Mann T 1963. Effect of atropine on the (tamsulosin) on incidence of ejaculation and composition of semen and secretory function of semen quality in the goat. Andrologia. 47(3): 354- male accessory organs in the boar. J Reprod Fertil. 359. 5(1): 101-108. Kok-Yong S and Lawrence L 2015. Drug Distribution and Drug Elimination. In: Basic pharmacokinetic concepts and some clinical applications. T A Ahmed. Rijeka, InTech, p. 99-116. Kucuk N, Aksoy M, Ucan U, Ahmad E, Naseer Z, Ceylan A and Serin I 2014. Comparison of two different cryopreservation protocols for freezing goat semen. Cryobiology. 68(3): 327-331. Mann T 1974. Secretory function of the prostate, seminal vesicle and other male accessory organs of reproduction. 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Kimsakulvech S. et al. / Thai J Vet Med. 2018. 48(4): 567-572. 571 Marconi M, Pilatz A, Wagenlehner F, Diemer T and Weidner W 2009. Impact of infection on the secretory capacity of the male accessory glands. International Braz J Urol. 35(3): 299-309. Memon MA, Bretzlaff KN and Ott RS 1985. Effect of washing on motility and acrosome morphology of frozen-thawed goat spermatozoa. Am J Vet Res. 46(2): 473-475. Welliver C and Essa A 2016. Sexual Side Effects of Medical and Surgical Benign Prostatic Hyperplasia Treatments. Urol Clin North Am. 43(3): 393-404.

5720 Kimsakulvech S. et al. / Thai J Vet Med. 2018. 48(4): 567-572. บทคัดย่อ ผลของ อัลฟ่าวัน อะดรีโนเซบ็ เตอร์ แอนตาโกนิส ปริมาณนอ้ ยต่อคุณภาพน้าเช้อื และปรมิ าณน้าเลย้ี งอสุจใิ นแพะ ศักดโิ ชต คมิ สกุลเวช1* สขุ ฤทัย บุญมาไสว1 ดุลยทรรศน์ กรณฑ์แสง1 เชาวลติ นาคทอง2 ยภุ าภรณ์ ลาน้าเทยี่ ง3 การผลติ น้าเช้ือแชแ่ ข็งในแพะจ้าเป็นต้องมีขบวนการลา้ งน้าเลย้ี งอสุจิ (seminal fluid) การลดขัน้ ตอนนี้โดยใช้สารกล่มุ อัลฟ่าวนั อะดรีโนเซบ็ เตอร์ แอนตาโกนสิ (alpha1 adrenoceptor antagonist) หรือ แทมซโู ลซนิ (tamsulosin) อาจชว่ ยลดปรมิ าณนา้ เล้ยี งอสจุ ิได้ โดยไม่ต้องมขี ั้นตอนการล้างน้าเล้ยี งอสุจิ การทดลองนใี้ ช้รูปแบบ 4x4 ลาตินสแคว์ โดยใชแ้ พะเพศผู้ 12 ตวั ฉีด น้ากล่นั หรือ สารละลาย tamsulosin ขนาด 3 6 และ 9 ไมโครกรัม ตอ่ กโิ ลกรมั เขา้ กล้ามเนอื้ สลบั กัน ฉดี หา่ งกัน 1 สปั ดาห์ จนแพะทกุ ตวั ไดร้ ับสารละลายครบทกุ ชนิด เก็บนา้ เช้อื ด้วยชอ่ งคลอดเทยี ม และประเมินความต้องการทางเพศหลงั ฉีดภายใน 1 ชว่ั โมง ประเมนิ คณุ ภาพน้าเช้อื และปริมาณน้าเล้ยี ง อสจุ ิหลงั ไดน้ ้าเชือ้ ความตอ้ งการทางเพศไม่มคี วามแตกต่างระหวา่ ง การฉีด tamsulosin ขนาด 3 6 และ 9 ไมโครกรมั ตอ่ กิโลกรมั ทา้ ใหไ้ ม่ เกิดการหลัง่ น้าเช้อื จ้านวน 1 (8.3 เปอร์เซ็นต์), 3 (25 เปอรเ์ ซ็นต)์ และ 4 (33.3 เปอร์เซ็นต์) ครั้งตามล้าดับ ปรมิ าณน้าเลย้ี งอสจุ ิในกลมุ่ ฉีด 9 ไมโครกรมั ตอ่ กิโลกรมั น้อยกวา่ กล่มุ ฉีดน้ากลั่นอยา่ งมนี ัยส้าคญั เทา่ นัน้ ส่วนคุณภาพน้าเชอ้ื อ่ืนไม่มีความแตกตา่ งกันอยา่ งมีนยั ส้าคญั การ ทดลองนี้สารกลมุ่ alpha1 adrenoceptor antagonist หรือ tamsulosin มีผลลดปริมาณน้าเลี้ยงอสุจใิ นแพะแต่มผี ลใหไ้ ม่มกี ารหลง่ั น้าเชอื้ ภายหลงั ฉดี 1 ช่วั โมง จึงอาจยงั ไมเ่ หมาะสมสา้ หรับการนา้ ไปใช้ลดปรมิ าณนา้ เลย้ี งอสจุ สิ า้ หรบั ผลติ นา้ เชอ้ื แชแ่ ข็งแพะ คา้ สา้ คัญ: แอลฟา่ วัน-อะดรโี นเซบ็ เตอร์-แอนตาโกนิส แทมซูโลซิน น้าเลย้ี งอสุจิ คุณภาพนา้ เชอ้ื แพะ 1ภาคปรคี ลินกิ และสตั วศาสตร์ประยกุ ต์ คณะสัตวแพทยศาสตร์ มหาวทิ ยาลยั มหิดล 2ภาคเวชศาสตรค์ ลนิ ิกและการสาธารณสขุ คณะสัตวแพทยศาสตร์ มหาวิทยาลยั มหดิ ล 3โรงพยาบาลสตั วป์ ศุสตั วแ์ ละสตั วป์ า่ ปศุปาลนั คณะสตั วแพทยศาสตร์ มหาวทิ ยาลยั มหดิ ล *ผู้รบั ผดิ ชอบบทความ E-mail: [email protected]

Original Article Computed tomographic contrast enhancement and tumor angiogenesis in canine oral tumors Urapa Klansnoh1 Wijit Banlunara2 Nan Choisunirachon1* Abstract The potential value of bolus tracking contrast enhanced computed tomography (CT) in the diagnosis of oral tumors in dogs was investigated by examining the relationship between contrast-enhanced CT image density and microvascularization. Among 20 dogs with oral tumors, aged 8 – 17 years, weight 3.6 – 40.0 kg, 13 dogs were suffering with melanoma (MM), 4 with squamous cell carcinoma (SCC), and 3 with fibrosarcoma. Based on contrast-enhanced CT scan, the time lag from the injection of contrast medium into the right cephalic vein until the contrast medium returned to the mid-cervical external jugular vein at the level of the 4th cervical vertebrae significantly correlated with body weight (p < 0.01). The mean of post-contrast-enhancement increase in intra-tumor image density, measured in Hounsfield Units (HU), was 127.09 ± 38.58%, with the highest values detected in SCC (166.88 ± 23.47%, p < 0.05), followed by MM (122.78 ± 37.90%) and fibrosarcoma (99.37 ± 31.58%). The mean of microvessel density (MVD, a measure of vascularization determined by vWF-immunohistochemistry) of all tumors was 36.7 ± 11.7 vessels/mm2, with the highest MVD values in SCC (47.5 ± 5.3 vessels/mm2, p < 0.05), followed by MM (35.3 ± 11.6 vessels/mm2) and fibrosarcoma (27.2 ± 6.5 vessel/mm2). The values for MVD and intra-tumor HU in post-contrasted-enhanced CT scans significantly correlated (p < 0.01). Keywords: angiogenesis, computed tomography, dog, oral, tumor 1Department of Veterinary Surgery, Faculty of Veterinary Science, Chulalongkorn University, Bangkok, Thailand, 10330 2Department of Pathology, Faculty of Veterinary Science, Chulalongkorn University, Bangkok, Thailand, 10330 *Correspondence: [email protected] Thai J Vet Med. 2018. 48(4): 573-581.

574 Klansnoh U. et al. / Thai J Vet Med. 2018. 48(4): 573-581. Introduction enhanced CT estimation technique for canine nasal tumors could be applied as the oxygenation parameter In canine patients, oral tumors can be found prior radiation procedure. However, the previous in areas such as the oral mucosa, gingiva, palate, technique has limitations due to the fact that the tongue and tonsils. Generally, the malignancy of an acquisition data was derived from only a small area of oral tumor is a function of the tumor cell origin. Oral selected tumor tissue. Since canine oral tumors are tumors can be benign, such as epulis or papilloma, but frequently presented in aging companion pets other most canine oral tumors are malignant. The majority of diagnostic procedure revealed less information for malignant tumors are malignant melanomas (MM) but prognosis and treatment manner. Therefore, the aims squamous cell carcinoma (SCC) and fibrosarcoma are of this study were (1) to develop a diagnostic imaging also reported frequently (Niemiec, 2008). Because these technique by means of bolus tracking contrast- types of tumors are characterized by high growth rate enhanced CT images for canine head and neck tumors and invasiveness, it is essential that precise diagnoses that can be applied as a prospective estimator of tumor and clinical staging be determined prior to the malignancy through the tumor vascularization in initiation of treatment. related different size of dog breeds, (2) to investigate the correlation between HU attenuation number of an In clinical practice, oral tumor location and oral tumor after contrast-enhanced CT and tumor invasion may ordinarily be assessed using basic angiogenesis as quantified by MVD, and (3) to diagnostic images such as skull radiographs. However, compare the attenuation numbers with the MVD for due to the superimposed bony tissue, precise various types of oral tumors, tumor location and tumor information is difficult to obtain in this way. Currently, staging. advanced diagnostic imaging techniques such as computed tomography (CT) are increasingly being Materials and Methods applied in veterinary medicine. CT has the advantage of providing multi-planar images from the volumetric Animals: 20 canine oral tumor patients presented at the data of the patient, which eliminates the conventional oral surgical unit, The Small Animal Teaching radiographic limitation, especially in the head and Hospital, Faculty of Veterinary Science, neck area. When analyzed on a computer, CT data can Chulalongkorn University between March 2014 and be displayed in several grey scales, enhancing the November 2015 were enrolled in this study. All dogs ability of a veterinarian to detect any anatomical were clinically examined and confirmed to be affected lesions (Ohlerth and Scharf, 2007). In addition to by oral neoplasia. If a patient had been treated by conventional CT scanning, intravenous iodinated surgical resection or chemotherapy, the dog was contrast medium can be used to reveal highly excluded from the study. All available information vascularized lesions (Miles et al., 2012), which is concerning the enrolled dogs, including history, helpful in investigating the affected area in disorders symptoms, physical examination results, oral tumor such as inflammatory diseases and tumors. evaluation and laboratory data, was recorded. In addition, the affected dogs in this study were clinical It is well known that tumor staged in accordance with the tumor size and the neovascularization plays an important role in tumor regional metastatic status from lymph node biopsy at growth and metastasis. Several researchers have the time of oral mass removal and the distant demonstrated that tumor angiogenesis, detected via metastatic status from whole body metastatic intra-tumoral microvessel density (MVD), can be used screening through CT images. as one of the malignant indicators (Weidner, 1995; Restucci et al., 2003; Wolfesberger, et al., 2008). This study was approved by the Institutional Although MVD has been shown to be a good indicator Animal Care and Use Committee (IACUC) in of tumor malignancy, it has had the disadvantage that accordance with university regulations and policies assessment requires invasive procedures such as tissue governing the care and use of laboratory animals, and biopsy or tumor resection for histopathology. Ouyang consent was obtained from each dog owner. The et al. (2017) have reported that the intensity of a animal use protocol number was 1431045. contrast-enhanced CT image, measured by Hounsfield Unit (HU) attenuation number, can provide an Computed tomography: Prior to CT examination, all estimator of MVD in renal cell carcinoma. Despite the dogs were examined to confirm that hematologic and disagreement of CT value observed through the serum biochemistry results were within normal limits attenuation number and density including the area of for anesthetic procedures. On the day of CT microvessels detecting on different renal cell examination, all dogs fasted for 8 hours and carcinoma (RCC) subtypes which were clear cell RCC intravenous fluid rehydration was given using a and papillary RCC, overall findings suggest that crystalloid solution (Acetar®, 10 ml/kg/hour) through contrast-enhanced CT images could be clinically the right cephalic vein. The dog was then sedated with advantageous for malignancy assessment. In diazepam (Diapine®, Atlantic laboratories, Thailand, companion animals, several studies have reported the 0.3 mg/kg) intravenously. Subsequently, general utilization of contrast-enhanced CT images for anesthesia was induced using propofol (Lipuro®1%, detecting the anatomical alteration from maladies of Melsungen, Germany, 6 - 10 mg/kg, IV). After the head and neck (Codner et al., 1993; Drees et al., endotracheal intubation, the dogs were maintained 2009; Ghirelli et al., 2013). Nevertheless, non-dynamic, under general anesthesia using 2 – 5% isoflurane contrast-enhanced CT images were less advantageous (Isofurane®, Bethlehem, U.S.A.). The dog was placed for malignancy estimation (Kafka et al., 2004). Camp et on the CT bed (64-slice helical CT unit; Optima al. (2000) has reported that the dynamic contrast-

Klansnoh U. et al. / Thai J Vet Med. 2018. 48(4): 573-581. 575 CT660®, GE, Thailand) with its head pointing into the HU at the mid-cervical external jugular vein, were gantry and all forelegs caudally positioned. The center recorded in Digital Imaging and Communications in of the skull was set at the isocenter. After the pre-scan Medicine (DICOM) files, and analyzed using Osirix® phase, the field of view (FOV) was set to cover the software (Osirix®, Geneva, Switzerland) at the whole area extending from the nostril to the 7th workstation. To determine the HU numbers cervical vertebra, and the pre-contrast scan was comparing pre- and post- contrast enhancement, CT acquired using the following parameters: slice images were viewed on a soft tissue window (WW = thickness = 0.625 mm, pitch = 0.531 mm per tube 75 HU and WL = 450 HU). Small ROIs were randomly rotation; 120 kVp; 100 mA. Before the post-contrast- drawn on the oral tumor in 5 areas and the HU number enhanced CT images were obtained, a region of interest at each area was recorded. In the case of mineralized (ROI) was designated at either the right or left external tumor tissue, mineralized areas were omitted. To jugular vein at the mid 4th cervical vertebra. The reproduce the same locations for comparisons of HU contrast medium, using iohexol (Omnipaque®, Cork, numbers between the pre- and post-contrast-enhanced Ireland, 600 mgI/kg), was administered through the CT series, the ROIs of the pre-contrast-enhanced series right cephalic vein by automatic injector (2 ml/sec). were saved and then imported into the post-contrast- Once the HU number in the specified ROI reached 100 enhanced CT. The percentage of post-contrast HU, post-contrast CT images were recorded (Fig. 1). enhancement was calculated using the formula (Post- All CT data, including the lag period from contrast contrast-enhanced HU – Pre-contrast-enhanced HU x medium injection until the enhanced HU reached 100 100 / Pre-contrast-enhanced HU). Figure 1 Dynamic contrast-enhanced computed tomographic (CT) image for canine head and neck area. The region of interest (ROI) on a mid-cervical CT image was drawn at the cervical jugular vein at the level of the 4th cervical vertebra. As soon as the contrast was circulated to the ROI such that the value reached 100 Hounsfield unit (HU), a post-contrast-enhanced CT scan was acquired. The time lag from injection of the contrast medium into the right cephalic vein until the 100 HU enhancement was detected at the ROI was noted and compared between dogs.

576 Klansnoh U. et al. / Thai J Vet Med. 2018. 48(4): 573-581. Histopathology and microvessel density: As soon as (vessel/mm2), defined as the MVD expression for each the CT procedure had finished, the dogs were hot spot area, was then recorded at 100x magnification. subjected to oral mass resection. After surgical resection of the oral tumor, a 1 cm3 sample was excised Statistical analysis: All data were analyzed using from the tumor mass and fixed in 10% neutral buffered Prism7 (Graphpad software, California, USA). formalin for 24 h prior to tissue processing for Measurements were expressed as mean and standard histopathologic investigation. A series of 4 μm thick deviation. The correlation between canine body weight sections were then stained with hematoxylin-eosin and the time lag from injection of the contrast medium with special staining; Masson Fontana for melanin until the contrast medium showed up at the mid- pigment and Mason Trichome for fibrous tissue; or cervical jugular vein was analyzed using the Pearson immune-stained for endothelial cells using polyclonal correlation test. The MVD number and the percentage rabbit anti-human Von Willebrand factor (vWF; Dako, of post-contrast-enhanced HU among tumor groups Glostrup, Denmark). Briefly, after deparaffinization in were compared using a Kruskal-Wallis test. The xylene and rehydration in graded ethanol, section correlation between the MVD number and the slides were washed with distilled water and phosphate percentage of post-contrast-enhanced HU was buffer saline (PBS) solution. In cases of MM, samples analyzed using the Pearson correlation test. Tests with were demelanized by incubating in 0.25 % potassium p < 0.05 were defined as statistically significant. permanganate for 45 min followed by an oxalic acid solution for 3 min. Then, all samples were immune- Results stained using the chain polymer conjugated method. To retrieve protein antigens from samples, section Clinical demographic data: Among the 20 canine slides were incubated in a sodium citrate buffer, pH patients with oral tumors, there were 15 males (12 6.4, for 5 min using the heat-mediated, microwave intact and 3 castrated) and 5 females (2 intact and 3 method. Endogeneous peroxide was then inactivated neutered). The average age was 12.0 ± 2.3 years old (8 by incubating the section slides with 3% hydrogen – 17 years old). The average body weight was 15.5 ± peroxidase. After washing with PBS, non-specific 12.0 kg (3.6 – 40.0 kg). There were 6 Poodles, 4 mixed protein binding was saturated by normal blocking breed dogs, 2 Pugs, 2 Golden retrievers, 2 Thai dogs serum, using 8% skimmed milk in PBS at room and 1 Shitzu, Rottweiler, German shepherd and terrier. temperature for 30 min. Subsequently the section slides Histologically, 13 dogs were affected by MM, 4 by SCC, were incubated with vWF (1:200) at 4° C overnight. The and 3 by fibrosarcoma. samples were then washed and labeled with a chained polymer solution containing HRP-conjugated The most frequent location of oral tumors was antibodies against rabbit IgG (EnVisionTM-HRP the gingiva (13 dogs), followed by lip (5 dogs), buccal labeled polymer, DAKO) for 45 min. Tissue sections mucosa (1 dog), and hard palate (1 dog). After clinical were visualized using a liquid DAB/hydrogen staging in accordance with world health organization peroxidase solution (DAKO). Nuclear counter-staining criteria by means of tumor size and tumor invasion of each section was done by Mayer’s hematoxylin either of regional or distant area, 5 dogs were found to before dehydration by graded ethanol, clearing in be at clinical stage II (2 dogs with MM, 2 with SCC, and xylene and covering with a cover-glass. Renal tissue 1 with fibrosarcoma), 1 dog was at clinical stage III was applied as a positive control, while primary (MM), and 14 dogs were at clinical stage IV (10 dogs antibody replacement with PBS was used as a negative with MM, 2 with SCC, and 2 with fibrosarcoma). control. Computed tomographic data: The time lag from To evaluate the MVD expression of each injection of the contrast medium into the right cephalic sample by veterinary pathologists, 5 “hot spots” which vein until the HU reached 100 at the mid-cervical were the most condense vWF-immune-stained vessels external jugular vein ranged from 15.0 to 49.0 seconds on the tissue were primarily selected at 40x (mean 17.11 ± 12.26 seconds). The time lag strongly magnification, and the density of vWF-positive vessel correlated with the body weight (range 3.8 to 40.0 kg) of the canine patients (R = 0.87, p < 0.01; Fig. 2). Figure 2 Correlation between body weight of canine patients and time lag between injection of contrast medium into the right cephalic vein and circulation of the contrast medium to the mid-cervical jugular vein.

Klansnoh U. et al. / Thai J Vet Med. 2018. 48(4): 573-581. 577 The mean percentage increase in post- was 36.7 ± 11.7 vessels/mm2 (range 19.7 – 54.6 contrast-enhanced HU attenuation number at the soft vessels/mm2). Similar results of the MVD number tissue of the oral tumor areas compared to the pre- compared to the tumor locations and clinical stages contrast-enhanced CT images was 127.09 ± 38.58% were detected at the CT parameter and neither location (range 62.90 – 196.00%). From the results, locations and nor clinical stage effected the MVD in tumor tissue. clinical stages of tumors were not effected to the CT Considered by location, gingival masses revealed attenuation number. By location, between gingival and equal numbers of MVD (37.6 ± 12.4 vessels/mm2, 21.2 lip masses that expressed as the greatest population in – 54.6 vessels/mm2) to those of lip masses (37.6 ± 13.0 this study, post-contrast HU numbers of both locations vessels/mm2, 19.7 – 51.3 vessels/mm2; p = 0.9971). were not significantly different (125.60 ± 37.99 %, 71.80 Likewise, the lower clinical stage of oral tumors – 195.60 % and 134.80 ± 51.51 %, 62.90 – 196.00% for (clinical stage 1 -2) revealed a slightly higher post- gingival and lip masses, respectively; p = 0.7278). contrast HU number (38.2 ± 13.25 vessels/mm2, 19.7 – Similar results were detected at the clinical stages of 51.3 vessels/mm2) than those of the higher clinical oral tumors. The lower clinical stage of oral tumors stage patients (clinical stage 3 – 4; 36.4 ± 11.6 (clinical stage 1 -2) revealed a slightly lower post- vessels/mm2, 21.1 – 54.6 vessels/mm2) but a statistical contrast HU number (126.06 ± 36.25 %; 62.90 – 196.00 difference was not detected (p = 0.7973). %) than those of the higher clinical stage patients (clinical stage 3 – 4; 130.16 ± 49.52 %; 71.80 – 195.60 %) Comparing different types of tumors, SCC but a statistical difference was not detected (p = 0.8707). showed significantly higher MVD values (47.5 ± 5.3 vessels/mm2, 40.0 – 51.5 vessels/mm2) than those Interestingly, tumor types having an effect on found in MM (35.3 ± 11.6 vessels/mm2, 21.2 – 54.6 the CT value by means of SCC showed significantly vessels/mm2) and fibrosarcoma (27.2 ± 6.5 higher post-contrast HU numbers (161.88 ± 23.47 %, vessels/mm2, 19.7 – 31.9 vessels/mm2) (p < 0.05, Fig. 143.60 – 196.00%) than those for MM (122.78 ± 37.90%, 4). 71.80 – 195.60%) and fibrosarcoma (99.37 ± 31.58%, 62.90 – 117.90%) (p < 0.05, Fig. 3). A comparison between the post-contrast enhanced HU attenuation number and the intratumor Microvessel density: The mean MVD of the tumor MVD showed significant correlation (R = 0.52, p < 0.01, tissue after immunohistochemical staining with vWF Fig. 5). Figure 3 Post-contrast-enhanced computed tomographic (CT) Hounsfield Unit (HU) values for different types of canine oral tumor. Squamous cell carcinoma showed significantly HU than malignant melanoma or fibrosarcoma (P < 0.05). Figure 4 Intratumor microvessel density (MVD) values after immunohistochemical staining with vWF. The highest value of MVD was found in squamous cell carcinoma, followed by malignant melanoma and fibrosarcoma (P < 0.05).

578 Klansnoh U. et al. / Thai J Vet Med. 2018. 48(4): 573-581. Figure 5 Statistically significant correlation between post-contrast-enhanced Hounsfield Units in the ROIs of the computed tomographic images and intra-tumor microvessel density (P < 0.01). Discussion with the goal of reducing the artifact in carotid CT angiography caused by contrast medium CT image is an increasingly popular imaging modality administration, we chose to administer the contrast for veterinary clinical diagnoses, especially in the areas medium into the right cephalic vein rather than the of neurology (Iwamoto et al., 1993; Axlund and usual left forelimb injection site (Demirpolat et al., Hudson, 2003), orthopedics (Gemmill et al., 2005; 2011). Also, compared to the previous dynamic Holsworth et al., 2005), soft tissue surgery, and contrast-enhanced CT image for predicting the tumor veterinary oncology (Kafka et al., 2004). In addition to oxygenation in canine nasal tumor for which data were the plain or non-contrast enhanced CT image, it has obtained from a single location scanning at various been reported that an increase in post-contrast- time periods (Camp et al., 2000) so that the results were enhanced CT density, as measured by HU value, has sometimes hard to represent the whole neoplastic been correlated with an increased intratumoral MVD condition, our study was designed by whole area-CT (Ouyang et al., 2017), which itself is a useful indicator scanning followed by picking up 5 randomized areas of tumor malignancy. Therefore, the measurement of to represent the neovascularization through HU post-contrast enhanced HU in CT images has been attenuation number. Therefore, the present study suggested as a technique for the clinical investigation should be much more representative of the actual of tumor malignancy. Among locations, due to their tumor angiogenesis in each tumor type. complex structure, tumors involving the head and neck area are among the most difficult to examine pre- Among 20 canine oral tumor patients in this operatively, either by physical examination or using study, our results were consistent with previous conventional imaging modalities such as radiography clinical demographic data in that most of the canine or ultrasonography, further imaging diagnostic oral patients (Dorn and Priester, 1976; Vos and van der modality such as utilization of CT images that can Gaag, 1987; Niemiec, 2008; Dobson, 2013). provide all of the structural changes such as tumor location, invasion and malignancy parameter, for Although our study revealed that tumor example; the tumor angiogenesis in companion animal location and clinical staging did not count as a can be applied as the guideline to diagnosis, treatment malignant factor effecting either of neovascularization and prognosis in clinical practice. Thus, the purpose of or CT value in tumor tissue, those factors are currently this study was to explore and compare tumor debatable. It has been reported that the clinical stages angiogenesis of canine oral tumors, evaluated using of oral squamous cell carcinoma in human patients dynamic post-contrast-enhanced HU values and have been influenced to the MVD expression, microvessel density. especially in the early clinical stage (Wirsling et al, 2016). However, Rubin et al. (1999) reported that MVD In this study, to maximize the value of expression was not significantly related to stage in measuring intratumoral post-contrast HU among other tumor type. By location, Dhanuthai et al (2013) different sizes of canine patients, we designed the post- additionally revealed that tumor site was not contrast CT protocol by drawing the ROI at the same associated with MVD expression in salivary gland anatomical level among different sizes of canine tumors but the tumor type did. Since there has been patients, instead of using duration setting methods as less information about the clinic-pathological seen in other protocol such as the dynamic CT protocol parameters such as age gender, tumor stage and commonly used for the liver (Fukushima et al., 2012). location that could effect the intraoral tumoral MVD In our protocol, the mid-cervical jugular vein was expression and other tumor progressed parameters in selected to draw the ROI instead of the common companion animals such as dogs and cats, further carotid artery used in other CT protocols for the head study with a large group would unveil useful and neck area because we wanted to make sure that the information. contrast medium circulated to the vascular bed of the oral tumor prior obtaining the post-contrast CT data. After computed tomographic examination, The goal was to improve the comparability of post- tumors among MM, SCC and fibrosarcoma revealed contrast enhancement measurements of oral tumors slightly different characteristics. Most of the MM and among canine patient of different sizes. In addition, fibrosarcoma canine patients showed a heterogeneous texture, whereas all of the SCC showed a homogeneous

Klansnoh U. et al. / Thai J Vet Med. 2018. 48(4): 573-581. 579 pattern and almost all samples showed alike results as kinetics in canine nasal tumors. Vet Radiol the histopathology samples. The result of SCC in canine patient was different from an earlier study of CT Ultrasound. 41(5): 403 – 408. in feline SCC (Gendler et al., 2010). Although this study did not compare the MVD expression between normal Codner EC, Lurus AG, Miller JB, Gavin PR, Gallina A, and tumor tissues due to ethical issues, the HU in the tumor area was significantly higher than those of Barbee DD 1993. Comparison of computed opposite intraoral normal tissue. In this study, either of MVD or HU was measured only in soft tissues, to tomography with radiography as a noninvasive avoid distortion of HU by including mineralization, which was scantly found in all patients and also to diagnostic technique for chronic nasal disease in avoid at the necrotic tissue in all tumors. However, HU measurement in some areas might be done in the dogs. J Am Vet Med Assoc. 202(7): 1106 – 1110. intratumoral granulation tissue due to poor detection of granulation tissue on the CT image than that of the Demirpolat G, Yüksel M, Kavukçu G and Tuncel D MVD on histopathology. Although no previous study has examined neovascularization examined using 2011. Carotid CT angiography: Comparison of dynamic CT or MVD, it has been reported that tumor angiogenesis evaluated by vascular endothelium image quality for left versus right arm infections. growth factor (VEGF) expression was highest in SCC and lowest in fibrosarcoma (Sobszynkska-Rak et al., Diagn Interv Radiol. 17(3):195 – 198. 2014), which is consistent with our observations. SCC is not the most common type of oral tumor in canine Dhanuthai K, Sappayatosok K, Yodsanga S, patients, but it is among the most malignant. Tumor angiogenesis in canine SCC might be up-regulated by Rojanawatsirivej S, Pausch NC and Pitak-Arnnop several molecular pathways. One of the most notorious molecules important for tumor angiogenesis is P 2013. An analysis of microvessel density in cyclooxygenase II. It has been reported that SCC involves high COX II expression (Mohammed et al., salivary gland tumours: a single centre study. The 2004). Therefore, further investigation of tumor angiogenesis, including possible anti-angiogenesis Surgeon II. 147 – 152. treatment in SCC, might be helpful in clinical practice. Due to the small sample size of canine patients which Dobson J 2013. Breed-predispositions to cancer in is one of the limitation, and the fact that other malignancy parameters such as the proliferative index pedigree dogs. ISRN Vet Sci. 2013: 941275. (PI) and survival period of each canine patient were not quantified in this study, further investigation on a Dorn CR and Priester WA 1976. Epidemiologic larger scale would clearly be useful. analysis of oral and pharyngeal cancer in dogs, In conclusion, contrast enhanced CT HU on oral tumor imaging in dogs correlates to tumor cats, horses, and cattle. J Am Vet Med Assoc. microvascular density and may be useful as a less invasive indicator of neovascularization and 169(11): 1202 – 1206. malignancy. Drees R, Forrest LJ and Chappell R 2009. Comparison Conflict of interest: There is no conflict of interest on this study. of computed tomography and magnetic Acknowledgements resonance imaging for the evaluation of canine This research was supported by the intranasal neoplasia. J Small Anim Pract. 50(7): Scholarship from the Graduate School, Chulalongkorn University to Commemorate the 72nd Anniversary of 334 – 340. His Majesty King Bhumibol Aduladej and a Graduate Thesis Grant, Graduate School, Chulalongkorn Fukushima K, Kanemoto H, Ohno K, Takahashi M, University. 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J Vet Dent. 30(2): 72 – 76. Holsworth IG, Wisner ER, Scherrer WE, Filipowitz D, Kass PH, Pooya H, Larson RF, Schulz KS 2005. Accuracy of computed tomographic evaluation of canine radio-ulnar incongruence in vitro. Vet Surg. 34(2): 108 – 113. Iwamoto KS, Norman, A, Freshwater DB, Ingram M and Skillen RG 1993. Diagnosis and treatment of spontaneous canine brain tumors with a CT scanner. Radiother Oncol. 26(1): 76 – 78. Kafka UC, Carstens A, Steenkamp G and Symington H 2004. Diagnostic value of magnetic resonance imaging and computed tomography for oral masses in dog. J S Afr Vet Assoc. 75(4): 163 – 168. Miles KA, Lee TY, Goh V, Klotz E, Cuenod C, Bisdas S, Groves AM, Hayball MP, Alonzi R and Brunner T 2012. Current status and guidelines for the assessment of tumour vascular support with dynamic contrast-enhanced computed tomography. Eur Radiol. 22(7): 1430 – 1441.

580 Klansnoh U. et al. / Thai J Vet Med. 2018. 48(4): 573-581. Mohammed SI, Khan KN, Sellers RS, Hayek MG, DeNicola DB, Wu L, Bonney PL and Knapp DW 2004. Expression of cyclooxygenases-1 and 2 in naturally-occurring canine cancer. Prostaglandins Leukot Essent Fatty Acids. 70(5): 479 – 483. Niemiec BA 2008. Oral pathology. Top Companion Anim Med. 23(2): 59 – 71. Ohlerth S and Scharf G 2007. Computed tomography in small animals-basic principles and state of the art applications. Vet J. 173(2): 254 – 271. Ouyang AM, Wei ZL, Su XY, Li K, Zhao D, Yu DX and Ma XX 2017. Relative computed tomography (CT) enhancement value for the assessment of microvascular architecture in renal cell carcinoma. Med Sci Monit. 31: 3706 – 3714. Restucci B, Maiolino P, Paciello O, Martano M, De Vico G and Papparella S 2003. Evaluation of angiogenesis in canine seminoma by quantitative immunohistochemistry. J Comp Pathol. 128(4): 252 – 259. Rubin MA, Buyyounouski M, Bagiella E, Sharir S, Neugut A, Benson M, Taille A, Katz AE, Olsson CA and Ennis RD 1999. Microvessel density in prostate cancer: lack of correlation with tumor grade, pathologic state, and cilinical outcome. Urology. 53(3): 542 – 547. Sobczynkska-Rak A, Polkowska I and Silmanowicz P 2014. Elevated vascular endothelial growth factor (VEGF) levels in the blood serum of dogs with malignant neoplasm of the oral cavity. Acta Vet Hung. 62(3): 362 – 371. Vos JH and van der Gaag I 1987. Canine and feline oral- pharyngeal tumours. J Vet Med Series A. 34(6): 420 – 427. Weidner N 1995. Current pathologic methods for measuring intratumoral microvessel density within breast carcinoma and other solid tumors. Breast Cancer Res Treat. 36(2): 169 – 180. Wolfesberger B, Tonar Z, Witter K, Guija de Arespacohaga A, Skalicky M, Walter I, Thalhammer JG and Egger GF 2008. Microvessel density in normal lymph nodes and lymphomas of dogs and their correlation with vascular endothelial growth factor expression. Res Vet Sci. 85(1): 56 – 61. Wirsing AM, Rikardsen OG, Steigen SE, Uhlin-Hansen L and Hadler-Olsen E 2016. Present of tumour high-endothelial venules is an independent positive prognostic factor and stratifies patients with advanced-stage oral squamous cell carcinoma. Tumour Biol. 37(2): 2449 – 2259.

Klansnoh U. et al. / Thai J Vet Med. 2018. 48(4): 573-581. 58719 บทคัดย่อ การศกึ ษาการเปรยี บต่างของระดบั สารเพิ่มความชดั ภาพบนภาพรงั สสี ่วนตดั อาศัยคอมพวิ เตอร์ และการกาเนดิ หลอดเลอื ดใหม่ในเนอื้ งอกชอ่ งปากของสนุ ัข อุราภา กลั่นเสนาะ1 วจิ ติ ร บรรลุนารา2 แนน ชอ้ ยสุนริ ชร1* การศกึ ษาครั้งนีเ้ ป็นการศกึ ษาการเพม่ิ ข้นึ ของระดบั สารเพ่มิ ความชัดภาพบนภาพรงั สสี ว่ นตดั อาศัยคอมพวิ เตอร์เพ่อื ชว่ ยในการ วนิ ิจฉัยโรคเนอ้ื งอกภายในชอ่ งปากของสุนัข ผา่ นการศกึ ษาความสมั พันธ์ ระหวา่ งความหนาแน่นของการเพม่ิ ขน้ึ ของสารเปรยี บตา่ งบน ภาพรังสรี ่วมกบั การเพ่มิ ขนึ้ จานวนหลอดเลือดฝอยภายในเนื้องอกช่องปาก โดยทาการศึกษาในสุนขั จานวน 20 ตวั ที่มีเนื้องอกในช่องปาก สุนัข ดงั กลา่ วมอี ายุระหวา่ ง 8 - 17 ปี น้าหนกั ตัวระหวา่ ง 3.6 - 40.0 กโิ ลกรัม โดยสุนขั จานวน 13 ตัวป่วยดว้ ยมะเรง็ เมด็ สี สุนัขจานวน 4 ตัวป่วย ด้วยมะเร็งสแควร์มัสเซลล์ และสนุ ัขจานวน 3 ตัวป่วยดว้ ยมะเร็งไฟโบรซาโครมา ผลการศึกษาพบระยะเวลาระหวา่ งการฉดี สารเพมิ่ ความชดั ภาพบรเิ วณหลอดเลอื ดดาขาหนา้ จนกระทง่ั สารเพิม่ ความชัดภาพเดินทางถงึ บริเวณหลอดเลอื ดดาใหญบ่ รเิ วณกลางคอที่ตาแหน่งกระดูกสนั หลังส่วนคอช้ินที่ 4 มีความสัมพนั ธก์ บั นา้ หนกั ตัวของสตั วป์ ่วย (p < 0.01) ค่าเฉลย่ี การเพ่ิมขึน้ ของสารเพมิ่ การชัดภาพในในเน้อื เยอ่ื ชอ่ งปากมี คา่ เทา่ กับ 127.09 ± 38.58 % โดยพบค่าสงู ท่สี ุดในมะเร็งสแควรม์ ัสเซลล์ (166.88 ± 23.47%, p < 0.05) ตามมาด้วยมะเรง็ เมด็ สี (122.78 ± 37.90%) และมะเร็งไฟโบรซาโครมา (99.37 ± 31.58%) ตามลาดบั คา่ เฉลยี่ การเพมิ่ ขน้ึ ของจานวนความหนาแน่นหลอดเลือดขนาดเล็กใน เนื้องอกผ่านการตรวจด้วยวิธที างอิมมูโนฮสี โตเคมดี ว้ ยโปรตีนวอนวิลลแิ บรนด์มคี ่าเทา่ กับ 36.7 ± 11.7 หลอดเลอื ดตอ่ ตารางมลิ ลิเมตร โดยคา่ ดงั กล่าวมีคา่ สงู สดุ ในมะเร็งสแควรม์ สั เซลล์ (47.5 ± 5.3 หลอดเลอื ดตอ่ ตารางมลิ ลเิ มตร, p < 0.05) ตามมาดว้ ยมะเรง็ เมด็ สี (35.3 ± 11.6 หลอดเลอื ดต่อตารางมลิ ลิเมตร) และมะเรง็ ไฟโบรซาโครมา (27.2 ± 6.5 หลอดเลอื ดตอ่ ตารางมลิ ลเิ มตร) ตามลาดบั พบความสัมพนั ธ์กัน ระหวา่ งความหนาแนน่ ของหลอดเลอื ดขนาดเลก็ และการเพิม่ ขน้ึ ของระดบั สารเพ่ิมความชัดภาพบนภาพรงั สีสว่ นตัดอาศยั คอมพวิ เตอร์อยา่ งมี นยั สาคัญทางสถติ ิ (p < 0.01) คาสาคัญ: การกาเนิดหลอดเลอื ดใหม่ ภาพถา่ ยรงั สสี ่วนตัดอาศยั คอมพวิ เตอร์ สุนัข ชอ่ งปาก เนอื้ งอก 1ภาควิชาศลั ยศาสตร์ คณะสัตวแพทยศาสตร์ จฬุ าลงกรณม์ หาวทิ ยาลัย ปทมุ วัน กรงุ เทพ ฯ 10330 2ภาควิชาพยาธวิ ิทยา คณะสตั วแพทยศาสตร์ จฬุ าลงกรณ์มหาวิทยาลยั ปทุมวนั กรงุ เทพ ฯ 10330 *ผู้รบั ผิดชอบบทความ E-mail: [email protected]



Original Article The potential use of phytoestrogen containing the herb, Pueraria mirifica, for bone healing in osteoporotic monkeys Donlaporn Kittivanichkul1 Nan Choisunirachon2 Kumpanart Soontornvipart2 Suchinda Malaivijitnond1,3* Abstract Within the last decade, the positive effects of Pueraria mirifica on osteoporosis have been established in rats and monkeys. However, the effect of P. mirifica on bone healing has not been elucidated. In this study, post-menopausal osteoporotic monkeys were subjected to an iliac crest biopsy, divided into two groups (5 animals/group), and fed daily with standard monkey diet alone (PMP0 group) or mixed with 1,000 mg/kg body weight of P. mirifica powder (PMP1000 group) for 16 months. The progression of the bone healing was continuously assessed by X-ray radiography, and three individuals from each group were selected for a computed tomographic (CT) scan at 0, 8 and 16 months. The individual from each group that showed the greatest progression of bone healing, based upon the CT image, was selected for a second biopsy of the right ilium after 16 months of treatment and histological changes determined. The perimeter and area measured by the X-ray radiograph were significantly decreased earlier in the PMP1000 group compared to the PMP0 group, and the significant differences of the perimeter between the groups were detected in month 3, 6 and 8. The 3D-CT scan showed a progressive bone healing in one PMP1000 individual, while histological examination indicated a lower number of fibrocartilage cells and a higher amount of new bone formation compared to the PMP0 monkey. In conclusion, PMP treatment could accelerate the progression of bone fracture healing in naturally postmenopausal osteoporotic monkeys, in addition to the previously reported amelioration of bone loss. Keywords: Bone mineral contents, Estrogen deficiency, Macaca fascicularis, Osteoporosis, Pueraria mirifica, X-ray 1Department of Biology, Faculty of Science, Chulalongkorn University, Bangkok, Thailand 2Department of Veterinary Surgery, Faculty of Veterinary Science, Chulalongkorn University, Bangkok, Thailand 3National Primate Research Center of Thailand, Chulalongkorn University, Saraburi, Thailand *Correspondence: [email protected] Thai J Vet Med. 2018. 48(4): 583-593.

584 Kittivanichkul D. et al. / Thai J Vet Med. 2018. 48(4): 583-593. Introduction non-human primate models. Recently, the therapeutic effects of P. mirifica have been confirmed in macaques Bone fractures are a common orthopedic (Kittivanichkul et al., 2016a), where P. mirifica could problem in which the continuity of the respective bone ameliorate the loss of cortical bone mass in naturally is completely or partially disrupted. Indeed, almost postmenopausal cynomolgus monkeys, especially at half of all fractures are related to osteoporosis, which is the diaphysis site. Apart from the effect on bone mass, characterized by a low bone mass and deterioration of P. mirifica also improved the bone structure by the bone microarchitecture (Kanis et al., 2013). It has decreasing the endosteal circumferences and been reported that osteoporotic fractures cause a increasing the cortical thickness at the diaphysis site of higher mortality and morbidity than cancers (except both the radius and tibia. lung cancer), particularly hip fractures that cause 10– 20% increased mortality in age-matched women With respect to the positive effect of P. mirifica (Pisani et al., 2016). Currently, osteoporotic fractures on osteoporosis in rodents and in non-human are an increasing public health and socioeconomic primates, its efficacy on bone fracture healing has been problem and cost about $20 billion per year in the raised. Although the Food and Drug Administration United States (Cummings and Melton, 2002) and €30.7 (USFDA) has approved many anti-osteoporotic drugs billion in Europe (Johnell and Kanis, 2006). The for osteoporotic patients, they have undesirable side incidence of osteoporotic fractures is growing effects on bone healing. For example, alendronate, an significantly according to the global demographic anti-bone resorptive agent that has been widely used trend. The number of osteoporotic hip fractures for osteoporosis since it can increase bone density, has reached 1.7 million in 1990 and has been projected to been reported to have a negative impact on bone reach 21 million in 2050 (Cummings and Melton, 2002). healing (Kates and Ackert-Bicknell, 2016). Moreover, in osteoporotic or osteopenia patients who had It is well known that estrogen deficiency is a spontaneous nonspinal fractures, alendronate therapy major risk factor of osteoporosis (Ettinger, 2003). Thus, resulted in the delay or prevention of bone fracture estrogen or estrogen-like compounds are widely used healing due to the severe suppression of bone turnover for the prevention and therapeutics of osteoporosis in (Odvina et al., 2005). Although osteoporosis can be postmenopausal women (Beil et al., 2010). Estrogen induced in many animal species, spontaneous deficiency or supplementation also directly affects the fractures with no adequate trauma can only be seen in bone healing process (Tella and Gallagher, 2014). For non-human primates (Egermann et al., 2005). Besides, example, estrogen deficient mice showed an impaired osteoporosis naturally and consequently occurs only in periosteal callus formation, diminished chondrocytes, macaques and humans (Egermann et al., 2005). Thus, less mineralization, and a thinner and more porous this study aimed to test whether P. mirifica, which had bone cortex compared to control mice. However, when positive effects on osteoporosis, could accelerate the they were treated with estrogen, the fracture healing bone fracture healing process in osteoporotic monkeys. was progressed by increasing the chondrocyte area, stimulating mineralization and giving a thicker cortex Materials and Methods (Tella and Gallagher, 2014). This is supported by a Animals: In an attempt to mimic the bone fracture study in humans that noted that the lack of 17- healing process in osteoporotic animals, naturally estradiol in menopausal women promoted the postmenopausal monkeys that showed bone loss were development of fat tissue from bone marrow cells selected for this study. Ten female cynomolgus rather than the osteogenic lineage (Justesen et al., 2001). monkeys (Macaca fascicularis) which were (i) more than Although estrogen seems to be beneficial for the 20 years old, (ii) amenorrhea for at least 1 year and (iii) treatment of both bone healing and osteoporosis, it also had total bone mineral content (BMCs) at the radius has adverse side effects, such as the promotion of and tibia of between -0.5 to -1 standard deviation (SD) estrogen-dependent cancers and diseases compared to 33 premenopausal monkeys (Fig. 1), were (Gambacciani et al., 2003). selected for this experiment. The premenopausal monkeys that were selected as a reference of normal Pueraria mirifica is a leguminous Thai plant BMCs in this study were fully mature females with at least three consecutive regular menstrual cycles. They belonging to the Family Leguminoceae. Its tuberous were housed in a group cage (4–8 animals/cage), roots contain at least 17 phytoestrogenic substances which allowed them to move freely, at the Krabok-Koo including genistin, genistein, daidzin, daidzein, Wildlife Breeding Center, Chachoengsao Province, puerarin and miroestrol (Malaivijitnond, 2012), and Thailand. The post-menopausal monkeys were housed have been used in Thai folklore medicine for the in individual cages at the Primate Research Unit, rejuvenating qualities in aged women and men for a Chulalongkorn University, Thailand. The monkeys century. In the past decade, its anti-osteoporotic effects were exposed to a 12:12 h light: dark cycle at ambient have also been widely tested (Urasopon et al., 2007; temperature. Monkeys were fed daily with monkey Urasopon et al., 2008; Malaivijitnond, 2012; chow (Perfect Companion Group. Co., Ltd. Samut Tiyasatkulkovit et al., 2012; Tiyasatkulkovit et al., 2014; Pakran, Thailand) in the morning (09:00–10:00 am) and Kittivanichkul et al., 2016a; Suthon et al., 2016a). P. fresh fruit supplemented in the afternoon (01:00–02:00 mirifica and its phytoestrogen contents stimulated bone pm) and water ad libitum. All animal procedures were formation and suppressed bone resorption both in vitro approved by Faculty of Science, Chulalongkorn (Tiyasatkulkovit et al., 2012) and in vivo (Urasopon et University Animal Care and Use Committee (CU- ACUC: Protocol Review no. 1123015, August 8, 2011). al., 2007; Urasopon et al., 2008; Suthon et al., 2016a) in rodent models. Because rats are lacking in the Haversian system in the cortical bone, the anti- osteoporotic effects of P. mirifica were tested further in

Kittivanichkul D. et al. / Thai J Vet Med. 2018. 48(4): 583-593. 585 Animal health for each animal was checked daily by phytoestrogen in the Pueraria plant) and miroestrol (a veterinary staff and animal caretakers. species-specific phytoestrogen in P. mirifica which Plant materials: The dried Pueraria mirifica Airy Shaw exhibits the highest estrogenic activity in the plant), & Suvatabandhu powder (PMP) (lot no. 141023) was before being accepted into the manufacture process. kindly provided by Dr. I. Sandford Schwartz (Smith Quality control of the manufactured PMP was done by Naturals Co., Ltd., Bangkok, Thailand). The tuberous the high-performance liquid chromatography (Bio- roots of P. mirifica were collected from Chiang Mai, Botanica Inc., Hauppauge, NY, USA). After receiving northern Thailand and a voucher specimen has been the PMP, the phytoestrogen contents in the PMP were kept at the Professor Kasin Suvatabhandhu Herbarium confirmed again by liquid chromatography-tandem Thailand (voucher specimen number BCU011045), mass spectrometry (LC-MS/MS) as reported Department of Botany, Faculty of Science, previously (Kittivanichkul et al., 2016a). The puerarin Chulalongkorn University, Thailand. The materials and miroestrol contents were 18.6 mg/100 g PMP (lot were examined for at least two species-specific no. 141023) and 233 µg/100 g PMP (lot no. 141023), phytoestrogens; puerarin (a species-specific respectively. Figure 1 Bone mineral content (BMC) of the (A, C) radius and (B, D) tibia at the (A, B) metaphysis site and (C, D) diaphysis site of pre-menopausal monkeys (n= 33) and post-menopausal monkeys (n = 10) that were selected for the bone healing study. Data are shown as the mean ± S.E.M., and * represents p< 0.05 compared with the pre-menopausal monkeys. Experimental design: Prior to the experiment, all (Kittivanichkul et al., 2016a). The dose of PMP treatment was adjusted with the monkey’s BW every 10 postmenopausal monkeys were subject to an iliac two months. crest biopsy. Two bone fracture defects were created at Progression of bone healing was accessed by radiography (X-ray) and the determination was the right ilium wing (Fig. 2). Three days after the performed immediately after the biopsy procedure (month 0) and then at 1, 2, 3, 4, 6, 8, 12 and 16 months surgical procedure, the monkeys were randomly afterwards. Three individuals from each PMP0 and PMP1000 group were randomly selected for computed divided into two groups (n = 5 per group) and fed with tomography (3D-CT) scans at month 0, 8 and 16. As the bones in aged postmenopausal monkeys are highly standard monkey diet either alone (PMP0) or mixed porous and fragile, biopsy is difficult to perform and only an individual from each group that showed the with PMP at a dose of 1,000 mg/kg body weight greatest progression of bone healing based on the 3D reconstructed CT images was selected for a second (BW)/day (PMP1000) at 08:00–09:00 am for 16 months. biopsy at the right ilium after 16 months of the PMP0 The age (29.6 ± 1.8 y and 26.0 ± 2.3 y, respectively) and menopause period (5.8 ± 1.4 y and 7.8 ± 1.2 y, respectively) were not significantly different between the PMP0 and PMP1000 groups. The dose of PMP used in this study was based on its efficacy in retaining cortical bone loss in naturally postmenopausal cynomolgus monkeys reported previously

586 Kittivanichkul D. et al. / Thai J Vet Med. 2018. 48(4): 583-593. or PMP1000 treatment and examined for histological carprofen (Zoetis, Pasippany, New Jersey, USA) was changes. subcutaneously injected as an analgesic. The operation was performed under strict aseptic techniques to avoid Bone biopsy procedure: The monkeys were any complications. The ilium wing was palpated and subcutaneously premedicated with tramadol (4 mg/kg the biopsied position was marked. Each bone defect BW; T.P. Laboratories (1969), Bangkok, Thailand) and was created by a 1 x 1 cm cut from the lateral ilium cefazolin (25 mg/kg BW; Biolab. Co., Ltd., wing (Fig. 2). The monkeys were intramuscularly Samutprakarn, Thailand). After that monkeys were injected with tramadol (3 mg/kg BW) and cefazolin (20 anesthetized by intramuscular injection of mg/kg BW) immediately after surgery and this tiletamine/zolazepam (3 mg/kg BW; Virbac, continued twice a day for 4 days. The monkey’s health Nonthaburi, Thailand) and medetomidine was checked twice daily for any sign of sickness or hydrochloride (40 g/kg BW; Vetcare, Joneboro, AR, complications. USA). During the surgical procedure, 4 mg/kg BW Figure 2 Bone defects were created by a 1 x 1 cm cut from the lateral ilium wing in each monkey. In the magnified insert image, the dashed line and shaded grey area indicate the perimeter and area measurement of the radiography, respectively. Assessment of bone repair by radiography: Monkeys in a supine position on the radiolucent V-pad were anesthetized by intramuscular injection with a positioning device. After the pre-scan phase, the region mixture of 3 mg/kg BW tiletamine/zolazepam and 40 of interest was set to cover the whole area of the pelvic µg/kg BW medetomidine hydrochloride. They were girdle and helical CT images were acquired at 120 kVp, placed ventrodorsally and the ilium wing was 250 mAs, pitch speed of 0.531 and a slice thickness of palpated and aligned parallel to the cassette. A 2-cm 1.25 mm. Subsequently, the CT images were analyzed calibration scale was also placed on the cassette at in the digital imaging and communication in medicine every exposure. A portable X-ray machine (model (DICOM) format using the open source workstation PXP-60HF, Poskom, Goyang, Korea) was used with a viewer (Osirix Lite Version V.7.0.2, Bernex, constant source to image distance of 70 cm while the Switzeland). To measure the bone structure kilovoltage peak (kVp) and milliampere seconds parameters of both two post-operative ilium defects at (mAs) were adjusted to acquire a proper image. After different time points, the CT images were subjected to exposure, the X-ray cassette was developed with a 3D multiplanar reconstruction and were revealed digital X-ray reader model FCR Capsula, Fujifilm and under the bone window (WL = 300, WW = 1500). For the resulting of each bone defect was used to measure each ilium defect, the depth was repeatedly measured the perimeter and area of the damage by a specialist at the top (0%), center (50%) and lower (100%) position using the Osirix Lite (Version V.7.0.2, Bernex, of the lateral height of the defect (see Fig. 4). Moreover, Switzeland), as a blind assay, and the average value of the perimeter and area of each bone defect were also two bone defects in each monkey was used for further measured. analysis. Bone histology: The second biopsy of the post- Assessment of bone repair by 3D-CT scans: The scan treatment right ilium was performed at the end of the was performed to unveil the anatomical lesion of the treatment period (month 16) as explained above. The ilium at 0, 8 and 16 months after the biopsy using a 64 bone specimens were trimmed, washed in sterile slice multidetector CT scanner (model Optima CT660, normal saline solution and fixed in 10% neutral buffer GE, Bangkok, Thailand). All monkeys were formalin at room temperature. The specimens were immobilized by intramuscular injection of a mixture of decalcified, dehydrated, paraffin embedded, sectioned 3 mg/kg BW tiletamine/zolazepam and 40 µg/kg BW and stained with hematoxylin and eosin (H&E) by the medetomidine hydrochloride and then were scanned Vet & Vitro Central Lab, Bangkok, Thailand.

Kittivanichkul D. et al. / Thai J Vet Med. 2018. 48(4): 583-593. 587 Figure 3 The percent change from baseline of (A) perimeter and (B) area of bone defect, as determined by radiography, during the 16-month experimental period. Open square and closed circle indicate the PMP0 and PMP1000 groups, respectively. Data are shown as the mean ± S.E.M., derived from five monkeys in each group. a, b and a', b' represent p < 0.05 and p < 0.01 compared between the baseline (month 0) and other time points of the PMP0 and PMP1000 groups, respectively. * represents p < 0.05 compared between the PMP0 and PMP1000 groups. Statistical analysis: The radiography and CT data are PMP1000 groups (perimeter = 3.590.28 cm for PM0 presented as the mean ± standard error of the mean (S.E.M). As there was no significant interaction group and 3.740.21 cm for PM1000 group; area = between the time and treatment when tested by two- way repeated measures ANOVA, the effects of time on 0.630.10 cm2 for PM0 group and 0.690.07 cm2 for bone healing in each monkey group, comparing values PM1000 group), the values are thus adjusted to percent between month 0 and other time points, were analyzed changes from the baseline (month 0) values. Compared using a paired t-test. Moreover, the effects of P. mirifica to month 0, the perimeter of the bone defect in the treatment compared between the PMP0 and PMP1000 PMP0 group, assessed by X-ray radiography, groups at the same time point were analyzed using an significantly decreased starting at month 2 of the unpaired t-test. A level of p< 0.05 was accepted as treatment, while a significant decrease was detected significantly different. Statistical analyses were earlier (since month 1) in the PMP1000 group (Fig. 3A). performed using the IBM SPSS Statistics for windows, In concordance with the perimeter, the area of the bone version 20.0 software. defect was significantly decreased starting at month 1 in both groups (Fig. 3B). Comparing the PMP0 and Results PMP1000 groups, the perimeter of the bone defect of the PMP0 group was significantly higher than the Clinical and physical examinations: All monkeys PMP1000 group at month 3, 6 and 8. Although it recovered rapidly after surgery and bone biopsy, tended to be higher, no significant differences between without any sign of infection or complication. The the PMP0 and PMP1000 groups were detected in the routine health check by veterinarians found that the area throughout the 16-month post-biopsy study monkeys regularly consumed food and drank water. period. Moreover, the animals remained in good health throughout the 16-month post-biopsy study period. The area (Fig. 4A), perimeter (Fig. 4B) and depth (Fig. 4C) of the 3D reconstructed CT images of Decreasing size of the bone defect under PMP the bone defect in the PMP0 and PMP1000 groups were treatment: Since the perimeter and area of the bone not significantly different, considering either the defects are different when comparing the PMP0 and comparison between month 0, 8 and 16 of both monkey groups or between the PMP0 and PMP1000 groups at the same time point. However, the CT image clearly

588 Kittivanichkul D. et al. / Thai J Vet Med. 2018. 48(4): 583-593. revealed the progression of bone healing in one were evidently shallower at month 8 and 16 compared monkey of the PMP1000 group where the bone defects to those in the PMP0 group (Fig. 5). Figure 4 The (A) area, (B) perimeter and (C) depth of the bone defect, as determined by 3D-CT scans at month 0, 8 and 16. Open square at the iliac crest indicates the area of magnification. White and grey boxes in the graphs indicate the PMP0 and PMP1000 groups, respectively. Box plot indicates the median and 25th and 75th percentiles. Figure 5 Reconstructed 3D-CT scans at month 0, 8 and 16 of the representative PMP0 (upper panel) and PMP1000 (lower panel) monkeys.

Kittivanichkul D. et al. / Thai J Vet Med. 2018. 48(4): 583-593. 589 Bone histology: Since the measurements of area, PMP1000 group showed a lower number of perimeter and depth of bone defect did not indicate the fibrocartilage cells but a region of new bone formation, bone healing process, the bone histology was as indicated by the presence of chondrocyte cells, was examined. As expected, the PMP0 group showed a clearly observed (Fig. 6). thick layer of fibrocartilage cells. In contrast, the Figure 6 Longitudinal plane histology section (stained with H&E) of the ilium biopsy at month 16 of the (A, C, E) PMP0 and (B, D, F) PMP1000 treated monkeys at (A, B) 25X, (C, D) 100X and (E, F) 200X magnification. In Figs A and B, the host bone area is separated from the healing bone area by the black and thick line and the open squares indicate the area of magnification shown in Figs C and D, accordingly. In Fig C, the thick layer is a fibrous tissue. In Fig D, an area between the thick and dashed lines is a new bone forming area. The open squares in Figs C and D indicate the area of magnification shown in Figs E and F, respectively. The arrow head and arrow in Figs E and F indicate fibrocartilage and chondrocyte cells, respectively. The scale bars are 200 µm, 100 µm and 50 µm for 25X, 100X and 200X magnification. Discussion require extensive surgery (Malluche et al., 2007). From 99 iliac crest biopsies of osteoporotic patients, It has been reported that osteoporosis can complications were observed in only eight (8.1%) cases cause irregular bone healing and affect different stages (Hodgson et al., 1986). Furthermore, the progression of in the bone healing process depending on the species the iliac crest biopsies correlated with the pattern seen of animals used. In rats, osteoporosis affects callus for bone repair in the vertebrae, tibiae and femur formation and mineralization in the early and late (Meunier et al., 1973; Dorr et al., 1990; Dorr et al., 1993). stages of bone healing, respectively (Egermann et al., Another reason for performing an iliac crest biopsy in 2005). In osteoporotic sheep, a delay in the tibiae bone this study rather than the usual transiliac crest biopsy healing at callus formation and mineralization is was that the transiliac crest biopsy might lead to observed as well as altered mechanical properties (Lill immobility in the aged osteoporotic monkeys which et al., 2003). No study of bone fracture healing in were used this study. Therefore, the bone defect was osteoporotic monkeys has been conducted yet, this is created by cutting the lateral site of iliac crest, where because it is not easy to recruit naturally osteoporotic the weight bearing or tension is minimal (Malluche et monkeys. al., 2007). The iliac crest was selected for bone biopsy in In this study, PMP at a dose of 1,000 mg/kg this study because it was easily accessible and did not BW, which could alleviate cortical bone loss in

590 Kittivanichkul D. et al. / Thai J Vet Med. 2018. 48(4): 583-593. naturally post-menopausal cynomolgus monkeys of the remodeling process (bone resorption followed (Kittivanichkul et al., 2016a), potentially stimulated the by its formation) at the site of the iliac crest biopsy bone healing process as seen in the significant occurring from the bottom before moving to the edge decreases in the perimeter of bone defect in the which has a high bone surface area. PMP1000 group compared to the PMP0 group. Our results are in agreement with previous reports on the With respect to the bone histology, PMP0 effect of the phytoestrogens, puerarin, genistein and monkeys showed a thick layer of fibrocartilage cells daidzein, on bone defects in rabbits (Wong and Rabie, while the PMP1000 monkeys had fewer fibrocartilage 2007; 2009; 2010). These rabbit bone defects were cells but a higher level of new bone formation. This grafted with collagen matrix alone (control) or the indicates that bone healing in the PMP0 individuals same mixed with phytoestrogens. By the early healing was at the early stage of fibrocartilaginous callus stage (day 14) the presence of puerarin, genistein or formation because the fibrocartilage cells were not yet daidzein in the collagen matrix had significantly differentiated into a soft callus, while in the PMP1000 increased new bone regeneration by 554%, 520% or monkeys it was in the late stage of fibrocartilaginous 620%, respectively. formation up to the early stage of bony callus formation since chondrocytes or osteocytes could be Previously, it was reported that genistein and observed together with the existence of woven bone. daidzein enhanced osteogenesis while depressing This leads to the conclusion that bone healing can adipogenic differentiation of mesenchymal progenitor occur, in general, in the non-treated PMP0 monkeys, cells (Schilling et al., 2014). Likewise, it was shown that but the PMP1000 treatment accelerated the bone genistein and daidzein enhanced the osteogenic healing process. activity in mesenchymal and adipocyte stem cells (Strong et al., 2014). Moreover, in vitro studies on the Even though the progression of bone healing effects of P. mirifica on osteoblast and osteoclast cells gradually occurred throughout 16 months of the experiment, it should be noted that the bone defect of support the results of this study on bone repair in both the PMP1000 and PMP0 groups was still non- osteoporotic monkeys (Tiyasatkulkovit et al., 2012; union at month 16 of the study period. Based on the Suthon et al., 2016b). The administration of P. mirifica fact that the age of monkeys in this study was over 20 years old, cellular senescence and alteration of the extract or puerarin increased the mRNA expression microenvironment surrounding the bone cells might level of alkaline phosphatase (Alp) and osteoprotegerin be the cause of the incomplete bone healing. It has been (Opg) and decreased the receptor activator of nuclear reported that aging affects the proliferation and factor kappa-B ligand (Rankl)/Opg mRNA ratio in rat functional ability of osteal macrophages (Gibon et al., 2016), which are crucial in bone repair for coordinating osteoblast-like UMR 106 cells (Tiyasatkulkovit et al., the crosstalk between osteoclasts and osteoblasts (Cho, 2012) and primary baboon osteoblasts (Tiyasatkulkovit 2015). Furthermore, the bone remodeling cycle by et al., 2014). In primary baboon osteoblasts, P. mirifica osteoblasts and osteoclasts also became more lacking with age (Szulc and Seeman, 2009), potentially due to extract also increased collagen Type I mRNA a reduction in osteoblast precursors, mesenchymal expression (Tiyasatkulkovit et al., 2014). The stem cells and osteoblast life span (Boskey and mechanism of action was shown to initially pass Coleman, 2010). A study in non-human primates also through the estrogen receptor, since the upregulation showed a similar trend in that the osteoclast precursor, of Alp mRNA levels was abolished by pre-treatment hematopoietic cells, declining with age (Lee et al., 2005). with the estrogen receptor antagonist, ICI182780 (Tiyasatkulkovit et al., 2012). However, the subsequent In conclusion, PMP treatment could underlying mechanisms of actions of P. mirifica extract accelerate the progression of bone fracture healing in naturally postmenopausal osteoporotic monkeys, in or other phytoestrogens on bone healing in monkeys addition to the previously reported amelioration of are still unknown and need to be elucidated in the bone loss (Kittivanichkul et al., 2016b). Therefore, PMP future. has a high potential to be developed as an anti- osteoporotic agent and also for bone fracture healing One serious limitation of this study was the for use in humans or pet animals (e.g., dogs) whose number of animals used, especially for the CT scan. It cortical bone possesses a Haversian system and whose is difficult to acquire postmenopausal monkeys and it bone remodeling processes are similar to those in is much more difficult to recruit sufficient osteoporotic monkeys (Egermann et al., 2005). postmenopausal animals at the same time. So far, naturally osteoporotic monkeys are the most suitable Acknowledgements animal models to represent naturally post-menopausal osteoporotic women. Unfortunately, the progress of We thank Mr. Taratorn Kemthong, DVM and bone healing has a high variation between individuals, Miss Nusjira Boonwong, DVM and the animal care the 3D-CT images of only three monkeys per group takers at the National Primate Research Center of could not reach a statistically significant difference. Thailand, Chulalongkorn University for the animal However, the qualitative data from one monkey in the procedures; Associate Professor Dr. Wijit Banlunara, PMP1000 group evidently showed a greater DVM and Dr. Jirarach Kitana for their valuable advice progression of bone healing compared to the PMP0 on bone histology; Assistant Professor Dr. Phisit monkeys. Close examination of the 3D-CT image of Khemawoot for the LC-MS/MS analysis of the PMP; this monkey revealed that during the reduction in J.F. Advance Med Co., Ltd., Bangkok, Thailand for use depth and area of the bone defect, the perimeter actually increased. Combining the CT data and CT image together, we hypothesize that the shallower depth and wider perimeter of the bone defect at month 16 of the PMP1000 treatment was due to the initiation

Kittivanichkul D. et al. / Thai J Vet Med. 2018. 48(4): 583-593. 591 of the X-ray machine and digital x-ray reader; and Mr. Scientific Advisors of the International Osteoporosis F 2013. European guidance for the Ronnakrit Rojyindeelert for the illustration of bone diagnosis and management of osteoporosis in postmenopausal women. Osteoporos Int. 24(1): biopsy. Financial support by International Research 23-57. Kates SL and Ackert-Bicknell CL 2016. How do Integration: Chula Research Scholar, bisphosphonates affect fracture healing? Injury. 47 Suppl 1: S65-68. Ratchadaphiseksomphot Endowment Fund Kittivanichkul D, Charoenphandhu N, Khemawoot P and Malaivijitnond S 2016. Pueraria mirifica (GCURS_58_06_23_02 to S. Malaivijitnond); the alleviates cortical bone loss in naturally Thailand Research Fund through the Royal Golden menopausal monkeys. J Endocrinol. 231(2): 121- 133. Jubilee Ph.D. Program (PHD/0097/2557 to D. Kittivanichkul D, Watanabe G, Nagaoka K and Malaivijitnond S 2016. 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Kittivanichkul D. et al. / Thai J Vet Med. 2018. 48(4): 583-593. 55931 2 บทคดั ย่อ ศกั ยภาพของกวาวเครือขาวซ่งึ เปน็ พืชสมุนไพรทม่ี ีสารไฟโตรเอสโตรเจน ในการประสานเน้ือกระดกู ในลิงท่อี ยู่ในภาวะกระดูกพรนุ ดลพร กติ ติวนิชยก์ ลุ 1 แนน ชอ้ ยสุนิรชร2 กัมปนาท สุนทรวภิ าต2 สุจนิ ดา มาลัยวจิ ิตรนนท์1,3* จากท่ีมีรายงานผลของกวาวเครือขาวตอ่ โรคกระดกู พรนุ ในหนูและลิง แตไ่ มม่ ผี ลการศึกษาตอ่ การประสานของกระดกู ทห่ี ัก จงึ ได้ คดั เลอื กลิงแสมวัยหมดประจาเดอื นและกระดูกพรุนมาตัดชน้ิ เนอ้ื กระดูกเชงิ กรานออก แบง่ ลงิ ออกเป็นสองกลุ่ม ๆ ละ 5 ตวั คอื กลมุ่ ทไ่ี ด้รับ อาหารปกติ (PMP0) และกลุ่มท่ไี ดร้ ับอาหารผสมผงกวาวเครอื ขาว 1000 มิลลกิ รมั /กโิ ลกรมั /วนั (PMP1000) นาน 16 เดือน ติดตามการ ประสานของกระดูกจากภาพถา่ ยเอกซเรย์ (X-ray) แลว้ ส่มุ เลอื กลิง 3 ตวั /กลุ่ม มาถา่ ยภาพเอกซเรยค์ อมพิวเตอร์ (3D-CT) ในเดอื นท่ี 0 8 และ 16 จากนน้ั เลอื กลงิ ท่ีกระดกู ประสานดีที่สุดมาตดั ชิน้ เน้ือกระดูกเชิงกรานออกอกี คร้งั ในเดือนที 16 เพอ่ื นาไปวเิ คราะหท์ างมญิ ชวทิ ยา จาก ภาพ X-ray พบวา่ เส้นรอบวงและพ้นื ท่ที ่ตี ดั ชนิ้ เนอื้ กระดกู ออกในลิงกลุ่ม PMP1000 ลดลงเร็วกว่าลงิ กลมุ่ PMP0 โดยพบความแตกต่างของเส้น รอบวงระหวา่ งลิงท้งั สองกลุ่มในเดอื นท่ี 3, 6 และ 8 จากผล 3D-CT พบวา่ ลิงกล่มุ PMP1000 มีการประสานของกระดูกดีกว่าลิงกลมุ่ PMP0 อยา่ งสอดคล้องกับผลทางมิญชวทิ ยาท่พี บเน้ือกระดูกใหม่เพม่ิ ขึ้นมากกวา่ แต่มเี ซลล์กระดกู ออ่ นเส้นใยน้อยกวา่ การทดลองนี้แสดงให้เห็นว่า นอกจากผลต่อโรคกระดกู พรนุ แล้ว กวาวเครือขาวยงั ช่วยกระต้นุ การประสานของกระดูกท่ีหกั ด้วย คาสาคญั : มวลกระดกู ภาวะพรอ่ งฮอร์โมนเอสโทรเจน ลงิ แสม กระดกู พรนุ กวาวเครือขาว เอกซเรย์ 1ภาควชิ าชวี วิทยา คณะวทิ ยาศาสตร์ จฬุ าลงกรณม์ หาวิทยาลยั ปทุมวัน กรุงเทพฯ ประเทศไทย 2ภาควิชาศลั ยศาสตร์ คณะสตั วแพทยศาสตร์ จฬุ าลงกรณม์ หาวิทยาลยั ปทมุ วนั กรงุ เทพ ฯ ประเทศไทย 3ศูนย์วิจัยไพรเมทแห่งชาติ จุฬาลงกรณม์ หาวิทยาลัย สระบุรี ประเทศไทย *ผรู้ บั ผิดชอบบทความ E-mail: [email protected]



Original Article Comparison of sperm freezability and fertility among Hmong Black-Bone, Barred Plymouth Rock and Thai Native (Pradu Hang Dam) chickens Saennamphung Somtaw1 Pornjit Sonseeda3 Nalinee Tubtimtong1 Yupin Phasuk1,2 Thevin Vongpralub1,2* Abstract Semen cryopreservation is the most useful method for the long-term storage of chicken genetic resources. The objective of this study was to compare post- thaw semen quality of Hmong Black- Bone to Barred Plymouth Rock and Thai native ( Pradu Hang Dam) chickens in terms of sperm motility, viability and fertility. A total of 9 males of each breed were used; semen collected from each cock was subsequently evaluated under a microscope. Semen samples were diluted with Schramm diluent, cooled down from 20– 25oC to 5oC and diluent plus DMF ( dimethylformamide) was added ( 6% DMF of final volume when the sperm concentration was 1,200 × 106/ mL) . Semen was loaded into 0. 5 mL straws, frozen at -35oC for 12 mins and at -135oC for 5 min in nitrogen vapor, and then plunged into liquid nitrogen. After thawing the straws in cold water (5oC), sperm motility characteristics were analyzed by computer-assisted sperm analysis and viability was assessed by staining with SYBR-14 and propidium iodide and observing under a fluorescent microscope ( 400×) . Fertility tests of frozen semen were carried out by artificial insemination of layer hens. There was no significant difference in motility and viability between breeds. The fertility rates for frozen semen of Hmong Black- Bone, Barred Plymouth Rock and Thai native (Pradu Hang Dam) chickens were 74.96, 69.75 and 80.59%, respectively, which showed no significant difference between breeds ( P>0. 05) . The results show that semen cryopreservation in Hmong Black-Bone chickens achieved acceptable freezing ability and fertility for use in ex situ conservation. Keywords: fertility, Hmong Black-Bone chicken, semen cryopreservation 1Department of Animal Science, Faculty of Agriculture, Khon Kaen University, Khon Kaen, 40002, Thailand 2Research and Development Network Center for Animal Breeding (Native Chicken) Faculty of Agriculture, Khon Kaen University, Khon Kaen, 40002, Thailand 3Department of Animal Science, Faculty Agriculture and Technology, Nakhon Phanom University, Nakhon Phanom, 48000, Thailand *Correspondence: [email protected] Thai J Vet Med. 2018. 48(4): 595-602.

596 Somtaw S. et al. / Thai J Vet Med. 2018. 48(4): 595-602. Introduction 160728014, Batagro Agro Industry Co Ltd Thailand) 120–130 g/day and water was provided ad libitum. At present, more than 13% of avian species are threatened with extinction ( IUCN, 2015) . For For fertility testing, 162 commercial layer domestic chickens, 35% of breeds are currently hens (Romann Brown, Batagro Agro Industry Co Ltd considered to be at risk of extinction ( Pym, 2013) for Thailand) were used when they were 40 weeks old and various reasons, mainly related to modern agriculture, hen day production was approximately 85% at the commercial needs and exposure to health risks. beginning of the experiment. The animals were reared under natural environmental conditions throughout Indigenous chickens have long played an the experimental period, July to September 2016. This important role in smallholder farms and among local experiment was carried out at the Livestock Research people. Black- Bone chicken, a type of indigenous Farm, Faculty of Agriculture, Khon Kaen University, chicken, reportedly has dietary health benefits that located at 16o26’N latitude and 102o50’E longitude, include reducing fatigue, and anxiety, stimulating the Thailand. The experiment was approved by the metabolism; controlling blood sugar and blood Animal Ethics Committee ( Approval No. 33/ 2015) of pressure, and helping to strengthen the immune Khon Kaen University, Thailand. system ( Li et al., 2012) . Hmong Black- Bone chicken is one of the varieties of Black- Bone chicken. These Semen collection: Semen was collected two times a indigenous chickens have been raised for food and week throughout the study period using the dorso- used in traditional medicine by the Hmong people, an abdominal massage technique ( Burrows and Quinn ethnic group living in the mountainous regions of 1937) . Care was taken to avoid contamination with South China, Vietnam, Laos and Thailand, and, hence, cloaca products. Semen samples were collected into a are an economically important animal for these hill 1.5 mL micro tube containing 0.1 mL Schramm diluent tribe communities. Bouahom and colleagues ( 2007) ( Schramm, 1991) , composed of 0. 07 g magnesium reported that populations of this local high land acetate, 2.85 g sodium glutamate, 0.5 g glucose, 0.25 g chicken breed in Laos face the risk of extinction. inositol and 0. 25 g potassium acetate dissolved in 100 mL double-distilled water. To maximize semen quality In relation to biodiversity, indigenous and quantity, semen collection was always performed chickens are a reservoir of genes which may be by the same person. Semen samples were transported important for future use. Currently, frozen semen to the laboratory for microscopic evaluation of the preservation methods are the most practical way to mass motility score, ranging from 0– 5 ( 0 = no sperm maintain long-term storage of poultry semen (Blesbois movement; 5 = very rapid waves and whirlwinds et al., 2007); however, the tolerance of sperm to visible, more than 90% of sperm showing forward cryopreservation varies among chicken breeds (Long, movement) . Semen samples having good motility ( at 2006; Siudzińska and Lukaszewicz, 2008b). Many least a score of 4) were used in this study. studies have shown satisfactory fertility rates of cryopreserved chicken semen (Tselutin et al., 1999; Freezing Procedures: Semen collected from each cock Woelders et al., 2006; Thananurak et al., 2016). To our was subsequently evaluated under a microscope. knowledge, there is limited information available on the cryopreservation of semen from Hmong Black- Semen samples were diluted with Schramm diluent Bone chicken. The objective of the present study was to (Schramm, 1991) cooled down from 20–25oC to 5oC and determine the freezability and fertility of Hmong Black-Bone frozen semen compared to Barred diluent plus DMF (dimethylformamide) was added (6% Plymouth Rock and Thai native (Pradu Hang Dam) chicken frozen semen cryopreserved using a simple DMF of final volume when the sperm concentration vapor method. The result of this study allow for the was 1,200 × 106/ mL) . Semen was loaded into 0. 5 mL optimization of semen cryopreservation of highland indigenous chicken genetic resource. straws, frozen at -35oC for 12 min and at -135oC for 5 min in nitrogen vapor, and then plunged into liquid Materials and Methods nitrogen. (Vongpralub et al., 2011) Before frozen semen Animals and Their Management: Two- year- old males assessment, straws were thawed in an iced water bath of three breeds of domestic fowl ( Hmong Black- Bone, at 2– 5°C for 5 mins. Three frozen semen straws from Barred Plymouth Rock and Thai Native ( Pradu Hang each cock were used for post- thaw motility Dam) chickens) were used in this study. The Hmong characteristics evaluation. Sperm motility (total motile Black- Bone roosters were purchased from farmers in and progressive motile) was determined using a the high lands, 700 m above sea level, in Kek- noi sub- computer- assisted semen analyzer ( CASA) ( IVOS district, Phetchabun province, Thailand, one year 12.3D; Hamilton Thorne, Beverly, MA, USA). Briefly, 5 before starting the experiment. Barred Plymouth Rock µL samples of post- thaw semen and diluent at a ratio is an exotic breed introduced into Thailand more than of 1: 15 were loaded on close cover dual slides for 40 years ago, while Pradu Hang Dam is a Thai native sperm analysis. The temperature of the sample chicken breed kept by small farm holders for both meat influences sperm motility (Iguer-ouada and Verstegen, and also as fighting cocks. The Pradu Hang Dam has 2001) since spermatozoa move more slowly at lower black feathers, beak and leg, and white skin. Each temperature. The optimal temperature for analysis of breed was represented by 9 healthy cocks. The animals sperm motion is body temperature, 37– 38°C were housed in individual pens at the Animal Farm, ( Verstegen et al., 2002) . A customized CASA system Faculty of Agriculture, Khon Kaen University. Cocks was assembled, with a microscope fitted with a were offered commercial feed (Balance 924, Lot No. I warming stage, negative phase- contrast optics and video camera interfaced with a computer to digitize

Somtaw S. et al. / Thai J Vet Med. 2018. 48(4): 595-602. 597 and analyze the images ( Lange- Consiglio et al., 2013) . percentage of sperm viability of thawed sperm from For each sample, multiple microscope fields were Hmong Black- Bone, Barred Plymouth Rock and Thai analyzed. Some parameters were measured directly on native ( Pradu Hang Dam) chickens. Significant the digital images while others were calculated from (P<0.05) differences between individual males in each measurements of the percentage of motile and breed were found for all post- thawed sperm progressive motile cells. parameters. The percentage of sperm viability was assessed Fertility of Cryopreserved Semen: The effect of breed by staining with SYBR- 14 and propidium iodide ( PI) . on fertility of frozen semen is shown in Table 2. There Since SYBR- 14, a green- fluorescent cell membrane were no statistically significant differences (P>0.05) counterstain, does not penetrate dead cells, it is among breeds in fertility. Significant (P<0.05) commonly used to identify live cells in a population; differences were found within breeds regarding while PI, a red- fluorescent nuclear and chromosome fertility. counterstain, does not penetrate live cells and is commonly used to identify dead cells in a population. Discussion The protocol of two types of fluorescent staining was as follows: 2 µL SYBR- 14 was added to 150 µL post- The present study was undertaken to thaw semen and incubated for 10 min; next, 2 µL PI was investigate the sperm freezing ability and fertility of added and incubated for 5 min; then samples were Hmong Black- Bone chickens compared to Barred evaluated under a fluorescent microscope ( 400×) Plymouth Rock and Thai native ( Pradu Hang Dam) ( Olympus 1X71, Tokyo, Japan) , as described by chickens, under a suitable protocol for chicken semen Partyka et al. ( 2010) . For sperm viability assessment, cryopreservation which could pave the way for ex situ three frozen semen straws from each male were used. conservation of this highland chicken. Artificial Insemination: Assessment of the fertilizing It is generally accepted that the ability of ability of frozen semen was carried out by single intra- spermatozoa to survive under cryopreservation is vaginal insemination of layer hens. A total of 162 hens different in different avian species ( Blanco et al. , 2008; were divided into 27 groups of 6 hens, and each group Blanco et al. , 2012) and in different breeds or strains was assigned to be inseminated once by frozen semen ( Hammerstedt, 1995; Siudzińska and Lukaszewicz, pooled from six ejaculates of each cock. Artificial 2008a; Makhafola et al. , 2009; Purdy et al., 2009; Long insemination was performed using a tuberculin et al., 2010); intra-species variations in freezability have syringe containing 0. 4 mL of frozen semen ( the also been reported ( Blesbois et al. , 2007; Kowalczyk insemination dose was about 480 million and Lukaszewicz, 2015). spermatozoa/ artificial insemination) inserted into the vagina ( about 4 cm) . The fertilizing ability of sperm Massip and colleagues ( 2004) reported that cells was estimated from eggs collected on day 2– 8 the quality of frozen semen of chickens and ganders post- insemination. Fertility rates ( fertilized eggs / was superior to most other poultry. A study by incubated eggs × 100) were determined by candling Blesbois and colleagues ( 2005) also found that post- eggs on day 7 of incubation. thawed fowl semen showed significantly higher percentages of viable and morphologically normal Statistical Analysis: The percentages of sperm spermatozoa compared to the semen of turkeys and motility, viability and fertility were examined by guinea fowl. Numerous studies have reported on the randomized complete block design; statistical analysis effect of breed on the freezability of domestic fowl of data used the generalized linear model procedure of semen. In relation to freezability, Siudzińska and SAS ( 1996) . The means of the treatment effects were Lukaszewicz ( 2008a) found that post- thawed semen compared using Duncan’s multiple range test (P<0.05). quality was significantly different among four fancy fowl breeds; this was in agreement with a previous Results study by Tselutin and colleagues ( 1995) , in which the cryopreserved semen of four fancy fowl breeds was Fresh Semen Parameters: Fresh semen quality was assessed. Peters and colleagues ( 2008) compared the determined from the pooled ejaculates of each breed, quality of frozen semen of 7 chicken breeds and found six times per breed, during the collection period. Mean differences in post-thawed semen quality, although the semen volume, motility score ( 0– 5) and sperm same processing method was used. Similarly, Blesbois concentration for Hmong Back-Bone, Barred Plymouth and colleagues (2007) and Wishart (2009) reported that Rock and Thai native ( Pradu Hang Dam) chickens, the quality of frozen semen depends on differences respectively, were: 0.311.11 ± 24.84 mL, 4.68 ± 0.05 and between breeds. Long and colleagues ( 2010) also 3,054.63 ± 108.34 million cells/mL; 0.366.67 ± 25.15 mL, reported that there was significant variability in the 4.73 ± 0.02 and 3,112.96 ± 143.44 million cells/mL; and frozen semen quality of eight poultry lines, which 0. 320. 37 ± 22. 86 mL, 4. 70 ± 0. 03 and 3,199. 07 ± 171. 92 supported the results of previous studies. million cells/mL (data not shown in tables). The present experiment indicated that there Post-Thaw Sperm Characteristics: Mean total motility was similar post- thawed semen quality of Hmong and progressive motility parameters of thawed Black- Bone chickens and the two other breeds spermatozoa are shown in Table 1. There were no mentioned above. This is in accordance with previous differences among breeds (P>0.05). There also was no reports: Mosenene ( 2009) showed that there were no statistically significant difference ( P>0 . 0 5 ) in the significant differences between breeds in the frozen semen quality of Rhode Island Red, Potchefstroom Koekoek, New Hampshire and White Leghorn

5968 Somtaw S. et al. / Thai J Vet Med. 2018. 48(4): 595-602. ch2ickens; and Makhafola and colleagues ( 2009) reported that there was no difference in sperm different metabolic factors, including ATP survivability immediately after thawing between concentration in spermatozoa post- cryopreservation Potchefstroom Koekoek and Ovambo breeds. Recently, ( Wishart and Palmer, 1986) . The determination of Sonseeda (2013) studied three Thai native chicken lines sperm membrane permeability in various avian ( Leung Hang Khao, Pradu Hang Dam and Chee) to species under different osmotic conditions indicates determine differences in cryopreserved semen quality; that spermatozoa fluidity differs between species of the results showed that the line of Thai native cocks poultry (Blesbois et al., 2005). These differences partly had no effect on post-thaw semen quality. reflect differences in cholesterol content and may be related to species differences in semen freezability. In In the present study, a significant variation of chickens, intra- species variations in intra- breed post- thawed semen quality was obtained cholesterol/phospholipid content were also found to be ( Table 1) , even though good raw semen samples were related to semen freezability ( Ansah and Buckland, used. Many biological and biophysical factors may 1982). The intra-breed differences in freezability in the affect the ability of sperm cells to prevent damage present study may depend on variations in the caused by the freezing/thawing procedure (Holt, 2000). cholesterol/phospholipid content of sperm membranes These factors include membrane permeability, lipid in semen from individual cocks. composition and fluidity ( which affect motility) , and Table 1 Sperm freezability of Hmong Black-Bone, Barred Plymouth Rock and Thai native (Pradu Hang Dam) chickens Motility parameters (%) Breed Male # Viability Hmong 1 Motile Progressive VCL VSL VAP LIN ALH 44.27ab Black- 64.00a 16.67bc 135.07ab 57.57abc 77.10abc 43.00ab 5.87a 41.46b 2 55.67bc 118.77c 46.83cd 66.47cd 40.67d 5.30ab 44.86ab Bone 3 14.33bcd 130.53abc 54.00abcd 70.67bc 42.00bc 5.63ab 45.26ab 4 40.67bc 14.00bcd 135.37ab 59.27ab 78.87ab 44.00ab 5.63ab 47.92a 5 62.33a 14.67bcd 141.07a 64.33a 83.93a 45.67a 5.93a 43.41ab 6 59.33ab 19.33ab 134.27ab 61.00ab 78.10abc 45.33a 5.97a 46.22ab 7 50.67a 13.33bcd 124.93bc 50.63bcd 69.87bc 41.33cd 5.67ab 45.99ab 8 10.00d 106.63d 43.33d 56.67d 42.33bc 4.67b 47.81a 32.67c 23.00a 41.33cd 5.63ab 60.67ab 5.59±0.13 45.24±0.69 42.85±0.60 9 51.67ab 10.67cd 135.77ab 54.43abcd 71.57bc 5.77bc 42.69bc 44.00a 6.13ab 42.89bc Mean ± 53.07±3.51 15.11±1.36 129.16±3.57 54.60±2.77 72.58±2.69 42.67a 5.20c 45.29b SE 45.00a 6.67a 40.58c 1 54.67abc 10.33cd 127.83bc 55.83ab 74.00ab 43.33a 5.50c 50.57a 67.67a 19.67a 143.77a 60.43a 80.53a 45.67a 6.47a 45.42b 2 64.33ab 16.67ab 132.10ab 59.87a 75.87ab 43.00a 5.47c 45.98b 45.33a 6.43a 44.27bc 3 38.33b 5.23c 51.78a 43.00a 5.87±0.19 Barred 4 50.67abc 9.67b 137.60ab 60.00a 79.23a 43.37±0.73 45.50±1.21 Plymouth 5 42.33bc 10.00b 113.00d 49.60b 63.70c 5.83ab Rock 6 46.00abc 138.67ab 58.93a 77.57ab 49.33a 5.93a 45.35bc 7 40.67c 11.33b 116.93cd 52.90ab 69.13bc 42.00b 6.10a 44.27c 16.00ab 47.00ab 6.07a 40.42d 44.33ab 5.63ab 46.17bc 8 67.33a 10.33b 138.23ab 51.40ab 72.00abc 44.00ab 5.50ab 39.91d 42.33b 5.03b 48.57ab 9 59.33abc 15.00ab 126.07bc 52.90ab 68.80bc 46.67ab 5.63ab 50.19a 46.33ab 5.30ab 48.94ab Mean ± 54.78±3.50 22.00±1.22 130.47±3.48 55.76±1.39 73.43±1.84 44.67ab 5.67±0.12 51.83a SE 59.00ab 20.67a 135.83ab 66.70ab 84.07ab 45.18±0.79 46.18±1.39 1 2 66.00a 15.67ab 142.23ab 58.20abc 80.30ab 3 71.33a 21.67a 143.97a 67.70a 86.50a Pradu 4 64.33a 18.33a 144.00a 63.73abc 83.83ab 5 48.00b 14.00ab 127.93ab 55.73bc 72.93b Hang 6 58.67ab 15.33ab 136.70ab 58.17abc 75.93ab Dam 7 57.67ab 19.33a 126.50b 58.07abc 72.87b 8 65.67a 22.00a 132.50ab 60.97abc 77.40ab 9 30.00c 14.33b 126.87b 54.13c 72.30b 78.46±1.81 Mean ± 57.85±4.13 17.37±1.40 135.17±2.39 60.38±1.58 SE Different superscripts within a column indicate significant differences (P<0.05) The results of this study indicated that the of inbred chickens was different among groups (Bacon fertility ranges were 58. 54– 92. 31% for Hmong Black- et al., 1986; Kirby et al., 1998). Blesbois and colleagues Bone, 46. 34– 87. 5% for Barred Plymouth Rock and ( 2005) also reported that post- thawed native chicken 41. 18– 100% for Thai native ( Pradu Hang Dam) semen had lower fertility compared with commercial chickens, respectively; high intra- breed variation and breed chickens. In poultry, genetic selection for sperm significant differences ( P<0. 05) among cocks within freezability has the ability to improve the frozen semen breed were obtained. These results are contradictory to quality of chickens ( Siudzińska and Lukaszewicz, previous findings, where the fertility of frozen semen 2008; Long et al., 2010), geese (Lukaszewicz, 2010) and

Somtaw S. et al. / Thai J Vet Med. 2018. 48(4): 595-602. 599 turkeys (Iaffaldano et al., 2008; Long et al., 2014). This ( Ushiyama et al., 2016) , that affect individual suggests that random breeding conserved biodiversity differences in semen freezability. and resulted in a variation of fertility rates of cryopreserved semen. We propose that in the present In summary, fertility rates were not study the high individual within- breed variation significantly different among Hmong Black-Bone, caused non- significant differences in fertility among Barred Plymouth Rock and Thai native (Pradu Hang breeds. This result is in accordance with a previous Dam) chicken breeds. The present study showed high report by Hammerstedt ( 1995) , who showed that the individual intra-breed variation in post-thawed semen range of frozen semen fertility in chickens was 0– 90% quality and fertility. The fertility rates, obtained after ( mean fertility 60% ) , and also the results of Blesbois one-time artificial insemination with frozen semen, of and colleagues ( 2008) , which showed that the fertility all breeds were superior to the fertility standard (60%) rate of chicken frozen semen was from 9 to 94% , a of chicken cryopreserved semen. The fertility results of wider range compared with fresh semen (34–94%). The 74.96% for Hmong Black-Bone chicken cryopreserved high variation in cryopreserved semen fertility of semen, achieved using a simple vapor freezing individual chickens may be related to many biological procedure, suggest the feasibility of establishing a and biophysical factors, including a loss of sterols from sperm bank for the conservation of Hmong Black-Bone membranes and a depletion of membrane rafts genetic resources. Table 2 Fertility rates of frozen semen of Hmong Black-Bone, Barred Plymouth Rock and Thai native (Pradu Hang Dam) chickens Breed Male # Number of eggs Fertility (%) Hmong Black-Bone 1 39 84.62ab Barred Plymouth Rock 2 42 73.81bc 3 40 65.00cd 4 35 68.57cd 5 38 92.11a 6 39 92.31a 7 41 58.54d 8 42 71.43bc 9 41 65.85cd Mean ± SE 74.96±4.03 1 38 41 52.63bc 2 37 75.61ab 3 42 72.97ab 4 40 83.33a 5 41 65.00bc 6 40 46.34d 7 42 87.50a 8 38 73.81ab 9 71.05ab Mean ± SE 69.75±4.48 1 34 41.18d 2 40 100.00a 3 39 64.10bc Pradu Hang Dam 4 42 95.23a 5 41 85.37ab 6 40 100.00a 7 42 85.71ab 8 37 81.08ab 9 41 68.29bc Mean ± SE 80.59±6.44 Different superscripts within a column indicate significant differences (P<0.05) Acknowledgements References The authors would like to thank the Research Ansah GA and Buckland RB 1982. Genetic variation in and Development Network Center for Animal fowl semen cholesterol and phospholipid levels Breeding (Native Chicken), Faculty of Agriculture, and the relationships of these lipids with fertility Khon Kaen University for financial support. This work of frozen–thawed and fresh semen. Poult Sci. 61 was also supported by The Thailand Research Fund (4): 623–637. (TRF) contract grant IRG5980010. Bacon LD, Salter DW, Motta JV, Crittenden LB and Ogasawara FX 1986. Cryopreservation of chicken semen of inbred or specialized strains. Poult Sci. 65 (10): 1965–1971.

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6020 Somtaw S. et al. / Thai J Vet Med. 2018. 48(4): 595-602. 2 บทคัดย่อ การศกึ ษาเปรยี บเทยี บความทนได้ในการแช่แข็งและอัตราการผสมตดิ ในไกก่ ระดกู ดา สายพนั ธุม์ ง้ ไกบ่ าร์พลมี ัทรอ็ ค และไกพ่ ้นื เมืองไทย (ประดหู่ างดา) แสนนำ้ ผ้งึ สมทำ้ ว1 พรจติ สอนสีดำ3 นลนิ ี ทบั ทิมทอง1 ยพุ นิ ผำสขุ 1,2 เทวนิ ทร์ วงษ์พระลบั 1,2* การเกบ็ รักษาน้าเช้อื ดว้ ยวิธกี ารแชแ่ ขง็ เป็นวธิ ีการท่เี ปน็ ประโยชนท์ ส่ี ุดในการเก็บรกั ษาทรัพยากรพันธุกรรมของไก่ การศกึ ษานม้ี ี วัตถุประสงคเ์ พ่อื เปรียบเทยี บคณุ ภาพนา้ เชอ้ื ภายหลังการทา้ ละลายของไก่กระดกู ดา้ สายพันธ์ุม้ง กับไก่บาร์พลีมัทรอ็ ค และไก่พื้นเมืองไทย (ประดูห่ างดา้ ) ในด้านอตั ราการเคล่อื นทข่ี องอสจุ ิ อัตราการรอดชวี ิต และอตั ราการผสมติด การศึกษาคร้งั น้ีใชพ้ อ่ พันธสุ์ ายพนั ธล์ุ ะ 9 ตัว ท้าการ รดี เก็บนา้ เชอ้ื จากพอ่ พันธุไ์ ก่ ประเมนิ คณุ ภาพของน้าเชอ้ื พอ่ พนั ธุ์แต่ละตวั ภายใตก้ ล้องจลุ ทรรศน์ เจอื จางตวั อย่างน้าเชอ้ื ด้วยน้ายาเจอื จางสตู ร Schramm ลดอุณหภูมจิ าก 20-25 องศาเซลเซยี ส ไปที่ 5 องศาเซลเซยี ส และเจอื จางนา้ เช้อื ดว้ ยน้ายาเจือจางสตู รทมี่ ี DMF (dimethyl formamide) ความเข้มขน้ สดุ ท้าย 6 % โดยมีอสุจทิ ่ีความเขม้ ข้น 1,200 ล้านตวั / มลิ ลิลติ ร บรรจุน้าเช้อื ลงในหลอดฟางขนาด 0.5 มลิ ลลิ ติ ร แช่แข็งท่อี ณุ หภูมิ -35 องศาเซลเซยี ส เปน็ เวลา 12 นาที และท่ี -135 องศาเซลเซยี ส เป็นเวลา 5 นาที ในไอไนโตรเจนเหลว จากน้นั บรรจไุ วใ้ น ถังไนโตรเจนเหลว ทา้ ละลายน้าเช้อื ในนา้ เย็น (5 องศาเซลเซียส) ทา้ การตรวจวเิ คราะหอ์ ตั ราการเคลือ่ นทีข่ องอสุจิโดยเครอ่ื ง computer- assisted sperm analysis (CASA) และตรวจวเิ คราะหอ์ ตั ราการรอดชวี ติ โดยการยอ้ มสี SYBR-14 and propidium iodide ตรวจประเมนิ ภายใต้กลอ้ งกา้ ลงั ขยาย 400 เทา่ ทดสอบอตั ราการผสมตดิ โดยการผสมเทียมในแม่พันธุไ์ ก่ไข่ ผลการศึกษาพบวา่ อตั ราการเคล่อื นท่ีและอัตรา การรอดชีวติ ของอสจุ ิระหว่างไกแ่ ต่ละสายพนั ธ์ุ ไมม่ ีความแตกต่างอย่างมีนยั ส้าคญั ทางสถิติ อัตราการผสมตดิ ของนา้ เช้อื แบบแชแ่ ข็งของไก่ กระดูกด้าสายพนั ธุม์ ง้ ไก่บารพ์ ลมี ทั ร็อค และไกพ่ น้ื เมือง (ประดหู่ างด้า) มีคา่ เทา่ กบั 74.96, 69.75 และ 80.59 เปอรเ์ ซ็นต์ ตามลา้ ดับ ซึ่งไมม่ ี ความแตกตา่ งกนั อย่างมนี ัยส้าคัญทางสถิตริ ะหวา่ งสายพันธุ์ (P>0.05) ผลการศกึ ษาชใ้ี หเ้ หน็ วา่ น้าเช้ือแบบแชแ่ ข็งของไก่กระดูกด้าสายพนั ธม์ุ ้ง มคี วามทนได้ตอ่ การแชแ่ ข็ง และอตั ราการผสมตดิ สงู ในระดบั มาตรฐาน น้าเชื้อแบบแชแ่ ข็ง จงึ สามารถใช้เป็นเครอื่ งมือในการอนุรักษ์ พันธกุ รรมของไกก่ ระดูกด้าสายพันธุม์ ้งได้ คาสาคัญ: อตั ราการผสมติด ไกก่ ระดูกดา้ สายพนั ธ์ุม้ง น้าเชือ้ แช่แขง็ 1ภาควชิ าสัตวศาสตร์ คณะเกษตรศาสตร์ มหาวทิ ยาลยั ขอนแก่น ขอนแก่น ประเทศไทย 40002 2ศนู ยเ์ ครอื ข่ายวจิ ัยและพัฒนาด้านการปรบั ปรุงพันธุส์ ัตว์ (ไกพ่ ้ืนเมือง) คณะเกษตรศาสตร์ มหาวิทยาลัยขอนแก่น ขอนแก่น ประเทศไทย 40002 3สาขาวชิ าสัตวศาสตร์ คณะเกษตรและเทคโนโลยี มหาวิทยาลยั นครพนม นครพนม ประเทศไทย 48000 *ผูร้ ับผดิ ชอบบทควำม E-mail: [email protected]

Original Article Efficacy of different vaccination programs of recombinant HVT-NDV vaccine against genotype VII NDV Challenge in broiler chickens Nataya Charoenvisal1 Bubpa Supannamoke2 Rik Koopman3 Jiroj Sasipreeyajan1* Abstract This study determined the efficacy of recombinant HVT- NDV ( rHVT- NDV) vaccine simultaneously vaccinated with live vaccine at 1 day old and a booster vaccination with live vaccine at 10 days old against Asiatic, Genotype VII NDV in broiler chickens. The chickens were divided into 5 groups. The chickens in groups 1, 2 and 3 were vaccinated with rHVT- NDV vaccine and different live vaccines at 1 day old, and then finally received a booster vaccination at 10 days old. The chickens in groups 4 and 5 served as the positive and negative control groups, respectively. The chickens in groups 1, 2, 3 and 4 were challenged with NDV at 14, 21, 28 and 35 days old, 20 chickens of each group at a time. Clinical signs and mortality were observed for 14 days after each time of challenge. Blood samples were collected at 1 and 10 days old, on the virus inoculation day and 14 days post- inoculation, to determine NDV antibodies. Swab samples were collected from the chickens challenged with the virus at 35 days old to detect NDV RNA by real- time PCR. The results reveal that the protection rate of the vaccinated chickens, which were challenged at 14 days old was 70- 95% , while the protection rate of all vaccinated groups increased to 90- 100% when they were challenged at 21, 28 or 35 days old. The vaccinated groups had a lower rate of virus shedding when compared to the challenged control group. It is concluded that all of the vaccination programs of rHVT-NDV vaccine in this study could be used to reduce economic loss due to infection by Genotype VII NDV. Keywords: chicken, recombinant HVT-NDV vaccine, efficacy, Newcastle disease virus 1Avian Health Research Unit, Department of Veterinary Medicine, Faculty of Veterinary Science, Chulalongkorn University, Henri-Dunant Street, Bangkok 10330, Thailand 2Intervet (Thailand) Ltd., Bangkok 10121, Thailand 3MSD Animal Health, Wim de Körverstraat 35, 5831 AN, Boxmeer, The Netherlands *Correspondence: [email protected] Thai J Vet Med. 2018. 48(4): 603-611.

604 Charoenvisal N. et al. / Thai J Vet Med. 2018. 48(4): 603-611. Introduction regulations of the European Union have decreed that it must have an intracerebral pathogenicity index (ICPI) Newcastle disease ( ND) is one of the major of not more than 0.4 (Suarez, 2013). The most common diseases affecting the poultry industry worldwide, and seed viruses for live vaccine production are LaSota, both broilers and layers of all ages are susceptible. This Clone 30, B1 and Ulster 2C strains. Live vaccine is disease is caused by the Newcastle disease virus efficacious in inducing IgA and local immunity with ( NDV) , also known as avian paramyxovirus type 1. It high levels of IgA, IgY and IgM (Takada and Hida, is a member of genus Avulavirus, family 1996; Seal et al., 2000; Suarez, 2013). However, Paramyxoviridae. NDV is a non- enveloped, non- maternally-derived antibody (MDA) is able to segmented, negative- sense RNA virus, which consists neutralize live vaccine (Suarez, 2013). Killed vaccines of 6 genes, which encode 6 structural proteins, are also produced from those lentogenic ND viruses including Nucleoprotein ( NP) , Phosphoprotein ( P) , and some of the mesogenic NDV. The killed vaccines Matrix protein (M), Fusion protein (F), Hemagglutinin- induce high and long-lasting neutralizing antibodies in neuraminidase ( HN) and Large protein ( L) . the circulation but induce low local immunity (Takada Importantly, F and HN are the surface glycoproteins, and Hida, 1996; Kapczynski et al., 2013; Suarez, 2013). which bind to the host cell and initiate infection, and Recombinant vaccines are generally available also induce host neutralizing antibodies. Moreover, the nowadays. The F or HN glycoprotein genes of NDV F protein cleavage site determines the virulence of were inserted into the virus vector of Poxvirus or NDV (Dortmans et al., 2011; Suarez, 2013; Ganar et al., serotype 3 Marek’s disease virus (Herpesvirus of 2014). turkey or HVT). The chicken’s immune system is able to produce neutralizing antibodies against F and HN NDV has been roughly classified into 4 glycoprotein of NDV and Poxvirus or Marek’s disease pathotypes depending on the virulence of the virus. virus (Seal et al., 2000). The recombinant HVT - NDV The first pathotype is avirulent NDV, which causes no (rHVT-NDV) vaccine used in this study is HVT virus disease. Secondly, is the lentogenic NDV, which has inserted with the F gene of genotype II NDV. The low virulence and causes low mortality, but may affect objective of this study was to determine the efficacy of egg production. Thirdly, there is the mesogenic NDV, rHVT-NDV vaccine simultaneously vaccinated with which causes moderate virulence, and may raise live NDV vaccine at 1 day old with a booster mortality up to 50% . The last pathotype is velogenic vaccination with live vaccine at 10 days old against NDV, which has high virulence and causes severe challenge with Asiatic, Genotype VII NDV in broiler disease with high mortality. The latter pathotype is chickens. further subdivided into neurotrophic velogenic and viscerotropic velogenic NDV ( OIE, 2009) . The strains Materials and Methods of NDV have been divided into 2 classes according to the nucleotide sequence of F and L genes (Umali et al., Animal: Four hundred, female, broiler chickens ( Ross 2013). Class I has low virulence and is typically isolated 308) were brought from the commercial hatchery to the from wild birds and Class II, which has been further experimental animal facility at the Livestock Hospital, divided into 16 genotypes ( Kapczynski et al. , 2013) . Faculty of Veterinary Science, Chulalongkorn Genotype I is low virulence, except for one velogenic University, Nakhon Pathom, Thailand. Commercial NDV that caused an outbreak in Australia. Genotype feed for broiler chickens and water were provided ad II is a low virulent NDV, which has been used as a seed libitum. The guidelines and legislative regulations on virus for vaccine production, such as B1, LaSota, Clone the use of animals for scientific purposes of 30 and VG/ GA strains. Genotypes III - IV are mostly Chulalongkorn University were followed as certified mesogenic NDVs, while, genotype V - IX and XI - XVI in the permission No. 16310113. are velogenic NDVs and genotype X is the low virulence virus ( Diel et al. , 2012b; Kapczynski et al. , Vaccines and virus: Vaccines used in this study include 2013; Suarez, 2013) . Moreover, from the year 2000 recombinant HVT- NDV ( rHVT- NDV) vaccine onward, genotype VII NDV has been causing big ( Innofusion® ND- SB, Intervet, Inc., USA) and 5 live outbreaks in many countries in Asia including attenuated vaccines, which were C2, NDV vaccine countries in East Asia, China, Taiwan, Korea, Japan, ( Nobilis® ND C2, Intervet International B.V., The Vietnam and Cambodia (Jeon et al., 2008; Miller et al., Netherlands) , Clone 30, NDV vaccine ( Nobilis® Clone 2010; Choi et al. , 2013; Umali et al. , 2013; Choi et al. , 30, Intervet International B.V., The Netherlands) , 2014; Ganar et al., 2014). The virus has been spreading combined MA5, IBV + Clone 30, NDV vaccine to Africa, some countries in Europe, North America ( Nobilis® MA5 + Clone 30, Intervet International B.V., (where it is called exotic NDV) and South America has The Netherlands), MA5, IBV vaccine (Nobilis® IB MA5, also been reported ( Kapczynski and King, 2005; Susta Intervet International B.V., The Netherlands) and 4-91, et al., 2011; Diel et al., 2012a). IBV vaccine ( Nobilis® IB 4- 91, Intervet International B.V., The Netherlands) . One dose of the recombinant Although, many strains of NDV circulate in vaccine contained at least 1,810 plaque forming units each part of the world, all of them are one serotype (PFU) of rHVT-NDV. One dose of live C2 and Clone 30 (Kapczynski et al., 2013). As a result, vaccination is a NDV vaccines contained at least 105.5 50% embryo common way of protecting chickens from NDV. infective dose ( EID50) and 106.0 EID50 of virus, Various types of NDV vaccine are commercially respectively. One dose of live Ma5 and 4-91 IBV available such as live attenuated, inactivated or killed, vaccines contained at least 103.0 EID50 and 103.6 EID50 of recombinant Pox-NDV and the recombinant herpes virus, respectively. One dose of a combined virus of turkey-NDV (rHVT-NDV) vaccines. Live vaccine mostly contains lentogenic NDV because

Charoenvisal N. et al. / Thai J Vet Med. 2018. 48(4): 603-611. 605 MA5+Clone 30 vaccine contained at least 103.0 EID50 of vaccination day. Firstly, water was taken out of the IBV and 106.0 EID50 of NDV, respectively. All the cage for 2 hours. Then, 1 liter of water was placed in vaccines were provided by Intervet (Thailand) Ltd. The the cage for 45 minutes. After 1 hour, the average water challenge virus was Asiatic, Genotype VII NDV (CU-2 intake per chicken was calculated by subtracting the strain, ICPI=1.86), which contained approximately 105 remaining water from 2 liters and dividing by the EID50/0.1 ml. number of chicken in one group (80 chickens). On the vaccination day, water was taken out from the cage for Experimental design: As soon as the chickens arrived 2 hours as on the previous day. Doses of the vaccine at the experimental site, they were randomly divided were added into a specific volume of water which was into 5 groups with 80 chickens in each group. Chickens determined from the previous day. Then, the chickens in groups 1- 3 were subcutaneously vaccinated with were allowed to consume water containing vaccine for rHVT-NDV vaccine at 1 day old, 1 dose or 0.2 ml/bird. around 45 minutes. Water intake had been Then, the chickens in group 1 were vaccinated intra- continuously monitored in order to ensure that each ocularly with C2 NDV vaccine, group 2 were chicken consumed the exact volume of water which vaccinated intra-ocularly with combined MA5, IBV + contained one dose of the vaccine. The chickens in Clone 30, NDV vaccine, and group 3 were vaccinated groups 4 and 5 were the positive and negative control intra-ocularly with MA5 strain and 4- 91 strain IBV groups, respectively. Next, each chicken in groups 1, 2, vaccines. At 10 days old, the chickens in groups 1 and 3 and 4 was orally challenged at 14, 21, 28 and 35 days 2 received the same booster vaccination with combined old, 20 chickens/group at a time, 0.1 ml/bird (Table 1). MA5, IBV + Clone 30, NDV vaccine by the drinking Clinical signs and mortality were observed for 14 days water route. The chickens in group 3 received a booster after each time of challenge. Dead chickens were vaccination with Clone 30 NDV vaccine by the necropsied and any gross lesions were observed. Body drinking water route. To perform a booster vaccination weights were measured on the inoculation day and 14 by the drinking water route, the volume of water days post inoculation (DPI). intake per chicken was measured 1 day before the Table 1 Vaccination program and age of challenge Group Vaccination program Number of chickens challenged with NDV 1 day old Booster at 10 days old 14 D 21 D 28 D 35 D 1 C2 (I/O) MA5+Clone 30 20 20 20 20 rHVT-NDV (S/Q) (DW) 2 MA5+Clone 30 (I/O) MA5+Clone 30 20 20 20 20 rHVT-NDV (S/Q) (DW) 3 MA5 (I/O) & 4/91 (I/O) Clone 30 20 20 20 20 rHVT-NDV (S/Q) (DW) 4 Non-vaccinated control - 20 20 20 20 5 Non-vaccinated control - - -- - NDV = Newcastle disease virus, D = day old C2 = C2 NDV vaccine, rHVT-NDV = recombinant Herpes virus of turkey-Newcastle disease virus vaccine, MA5+Clone 30 = combined MA5 IBV and Clone 30 NDV vaccine, MA5 & 4/91 = MA5 IBV and 4/91 IBV vaccines S/Q = subcutaneous injection, I/O = intra-ocular route, DW = drinking water route Serological test and real- time polymerase chain in copies number/ microliter. The R2 value of the tests were > 0. 95 and the threshold was set above the no reaction ( PCR) : Five blood samples per group were template control sample (NTC) (around 0.029). randomly collected at 1 day old. After that, blood samples were collected at 10 days old, on the Statistical analysis: Body weights and antibody titers inoculation day (0 DPI) and 14 DPI. Sera were collected were analyzed and compared between groups using and tested for NDV antibodies by the ANOVA and least significant difference (LSD) test haemagglutination-inhibition (HI) test. using SPSS software version 22. The percentage of mortality was calculated using Chi-square values. The virus shedding of the 35-day-old Differences between groups were considered inoculated chickens was determined by collecting 10 significant at p<0.05. The normal distribution was oropharyngeal swabs of 10 chickens and 10 cloacal tested using SPSS software version 22. swabs of another 10 chickens from each treatment group at 2, 4, 7, 10 and 14 DPI. The RNA from each Results swab sample in viral transporting medium was extracted using QIAamp® Viral RNA Mini kit (Qiagen, Mortality rate: The mortality of chickens in group 1 USA) , followed by converting the RNA samples to was 25 and 10 percent, when they received the cDNA using Thermo Scientific Revert Aid First Strand challenged virus at 14 and 21 days old, respectively. cDNA Synthesis Kit ( Thermo Fisher Scientific, USA) . But there was no mortality when they received the Real- time PCR was performed using Kapa Probe fast virus at 28 and 35 days old. The mortality of chickens qPCR kit ( Kapa Biosystems, USA) and Rotor- GeneTM in group 2 was 30, 10, 5 and 10 percent, when they Q real-time PCR machine (Qiagen, USA). Primers and received the challenge virus at 14, 21, 28 and 35 days fluorescence probe were specific to NDV’ s Matrix old, respectively. The mortality of chickens in group 3 gene. The real- time PCR machine revealed results in graphs, Ct value, and calculation of the concentration

606 Charoenvisal N. et al. / Thai J Vet Med. 2018. 48(4): 603-611. was 5 and 10 percent, when they received the challenge 95, 95, 100 and 100 percent, when they received the virus at 14 and 28 days old, respectively. But there was challenge virus at 14, 21, 28 and 35 days old, no mortality when they received the virus at 21 and 35 respectively. There was no mortality of chickens in days old. The mortality of chickens in group 4, which group 5, which was the non- vaccinated and was the non-vaccinated challenged control group, was unchallenged control group (Table 2). Table 2 Mortality rate after NDV inoculation Group NDV challenge at 14 days old 21 days old 28 days old 35 days old NumberA Percent NumberA Percent NumberA Percent NumberA Percent 1 5/20 25a,b 2/20 10a 0/20 0a 0/20 0a 2 6/20 30a 2/20 10a 1/20 5a 2/20 10a 3 1/20 5b,c 0/20 0a 2/20 10a 0/20 0a 4 19/20 95d 19/20 95b 20/20 100b 20/20 100b 5 0/20 0c 0/20 0a 0/20 0a 0/20 0a DPI = day post inoculation, D = day old A Number of dead chickens / Total chickens in the group a,b,c,d Different superscript (a, b, c, d) in each group of the same column means statistically significant difference (p<0.05). Body weight: The body weight was measured before 1.681 ± 291.65 at 14 DPI). The mean body weight of 28 challenge (0 DPI) and at 14 PDI. The mean body weight days old inoculated chickens was also no different of each measuring day was similar between each between each group (ranging from 1,201 ± 55.02 to treatment group. Except in group 4 of the 14-day-old 1,206 ± 56.51 at 0 DPI and from 2,312 ± 73.86 to 2,371 ± inoculated date where there was only one chicken left weighing 575 grams, while the mean body weight of 369.10 at 14 DPI). The mean body weight of 35 days old the chickens in the other groups at 28 days old (14 DPI) was between 1,204 ± 202.47 to 1,290 ± 230.50 grams. The inoculated chickens was also similar between each mean body weight of 21 days old inoculated chickens group (ranging from 1,638 ± 101.87 to 1,643 ± 128.66 at 0 was not different between each group (ranging from 810 ± 32.48 to 812 ± 31.48 at 0 DPI and from 1,505 to DPI and from 2,392 ± 525.00 to 2,572 ± 225.93 at 14 DPI) (Table 3). Table 3 Mean body weight on the challenge day (0 DPI) and 14 DPI Group NDV challenge at 1 14 days old 21 days old 28 days old 35 days old 0 DPI 14 DPI 0 DPI 14 DPI 0 DPI 14 DPI 0 DPI 14 DPI (14 D) (28 D) (21 D) (35 D) (28 D) (42 D) (35 D) (49 D) 456 ± 1,204 ± 811 ± 49.63 1,681 ± 1,206 ± 2,339 ± 1,641 ± 2,392 ± 27.62A (20)B 202.47 (15) (20) 291.65 (18) 56.51 (20) 238.50 (20) 104.94 (20) 525.00 (20) 2 456 ± 1,290 ± 811 ± 30.50 1,640 ± 1,204 ± 2,328 ± 1,643 ± 2,572 ± 26.39 (20) 230.50 (14) (20) 301.51 (18) 88.55 (20) 178.00 (19) 128.66 (20) 225.93 (18) 3 456 ± 1,271 ± 811 ± 577.94 1,660 ± 1,205 ± 2,371 ± 1,638 ± 2,466 ± 29.55 (20) 136.52 (19) (20) 228.47 (20) 78.25 (20) 369.10 (18) 101.87 (20) 261.71 (20) 4 456 ± 575 810 ± 32.81 1,505 1,205 ± -C 1,641 ± -C 30.01 (20) (1) (20) (1) 68.58 (20) 105.18 (20) 5 456 ± 1,230 ± 812 ± 31.48 1,632 ± 1,201 ± 2,312 ± 73.86 1,643 ± 2,421 ± 23.67 (20) 73.77 (20) (20) 135.98 (20) 55.02 (20) (20) 112.77 (20) 161.82 (20) DPI = day post inoculation, D = day old A All data of body weight in this table is presented as mean body weight (gm/bird) + standard deviation (SD). B Number in parentheses under each body weight means number of chickens in the group. C All chickens died due to ND before 14 DPI. Serological results: Chickens had high maternally- at 21, 28 and 35 days old. In all challenged chickens (groups 1-4), antibody titers at 14 DPI highly increased, derived antibodies ( MDA) at 1 day old ( 7. 00+ 1. 00 to compared to 0 DPI. In contrast, an increase of antibody 7.80+0.84 log2), but this slowly declined to 3.90+0.88 to titers in the negative control group ( group 5) was not 4.50+0.71 log2, when the chickens were 10 days old, and detected (Table 4). continuously declined to 2. 40+ 0. 50 to 3. 00+ 0. 97 log2 when the chickens were 14 days old. In chickens which Viral detection by real-time PCR: Virus shedding was determined by collecting 10 oropharyngeal swabs from had not received the vaccine ( groups 4 and 5) , the 10 chickens and 10 cloacal swabs from another 10 chickens. The swabs were collected from 35-day-old antibody titers slowly declined to 1. 45+ 0. 69 to NDV inoculated chickens (groups 1, 2, 3 and 4) at 2, 4, 1. 55+ 0. 83 log2 at 21 days old. The antibody titers of chickens in groups 1, 2 and 3 before virus inoculation were significantly higher than those of groups 4 and 5

Charoenvisal N. et al. / Thai J Vet Med. 2018. 48(4): 603-611. 607 7, 10 and 14 DPI. The results reveal that at 2 DPI, group (36 percent). The total swab samples of group 2 at 2, 4, 1 had the highest number of chickens (11 birds) shed 7, 10 and 14 DPI were 94 samples, of which 19 were the virus, groups 2 and 4 had 8 positive birds, and positive (20.21 percent). Forty-seven samples were group 3 had 5 positive birds. At 4 DPI, the number of oropharyngeal swabs, of which 5 were positive (10.64 birds that shed the virus was 5 birds, which was percent) and another 47 samples were cloacal swabs, of equaled in groups 1, 2 and 3, while group 4 had 14 which 14 were positive (29.79 percent). The total swab birds shed the virus. At 7 DPI, 6, 4 and 2 birds of groups samples of group 3 at 2, 4, 7, 10 and 14 DPI were 100 1, 2 and 3 shed the virus, respectively. All chickens in samples, of which 13 were positive (13 percent). Fifty group 4 were dead before 7 DPI. At 10 DPI, 6, 1, and 1 samples were oropharyngeal swabs, of which 8 were bird of groups 1, 2 and 3 shed the virus. At 14 DPI, 4 positive (16 percent) and another 50 samples were birds in group 1 and 1 bird in group 2 shed the virus, cloacal swabs, of which 5 were positive (10 percent). while there was no shedding found in group 3 birds. Total swab samples of group 4 at 2 and 4 DPI were 40 Total swab samples of group 1 at 2, 4, 7, 10 and 14 DPI samples, of which 22 were positive (55 percent). were 100 samples, of which 32 were positive (32 Twenty samples were oropharyngeal swabs, of which percent). Fifty samples were oropharyngeal swabs, of 11 were positive (55 percent) and another 40 samples which 14 were positive (28 percent) and another 50 were cloacal swabs, of which 11 were positive (55 samples were cloacal swabs, of which 18 were positive percent) (Table 5). Table 4 Antibody titers against NDV measured by the HI test Group ND-HI titer (log2)A 1 Maternally-derived NDV challenge NDV challenge at 21 NDV challenge at 28 NDV challenge at 35 2 antibodies at 14 days old days old days old days old 3 4 0 D 10 D 0 DPI 14 DPI 0 DPI 14 DPI 0 DPI 14 DPI 0 DPI 14 DPI (14 D) (28 D) (21 D) (35 D) (28 D) (42 D) (35 D) (49 D) 7.80 ± 3.90 ± 2.65 ± 7.53 ± 2.35 ± 10.78 ± 2.90 ± 10 ± 3.60 ± 11.20 ± 0.84 (5)B 0.88 (10) 0.99a,b 2.00b (15) 0.81b (20) 1.40a (18) 1.83a (20) 2.20b (20) 0.68a (20) 1.40a,b (20) 7.80 ± 4.50 ± (20) 2.05 ± 9.59 ± 0.45 (5) 0.71 (10) 0.76b,d 1.87b (18) 3.00 ± 8.21 ± (20) 2.10 ± 11.63 ± 3.85 ± 10.50 ± 7.20 ± 4.20 ± 0.97a (20) 2.22a,b (14) 8.45 ± 2.06c 1.21b (20) 1.80a (19) 0.88a (20) 1.47b (18) 1.10 (5) 0.92 (10) 4.85 ± (20) 2.40 ± 6.05 ± 1.65c 1.23c (20) 3.95 ± 11.83 ± 4.55 ± 11.30 ± 7.00 ± 4.20 ± 0.50b (20) (19) 8.00 1.61c (20) 1.10a (18) 0.89b (20) 1.13a,b (20) 1.00 (5) 1.03 (10) 1.45 ± (1) 2.60 ± 9.00 0.69a (20) 1.00 ± -C 1.00 ± -C 0.88a,b (1) 0.00d (20) 0.00c (20) (20) 5 7.20 ± 4.20 ± 2.60 ± 1.10 ± 1.55 ± 1.00 ± 1.00 ± 1.00 ± 0.00c 1.00 ± 1.00 ± 0.00c 0.45 (5) 0.79 (10) 0.99a,b 0.31d (20) 0.83a (20) 0.00d (20) 0.00d (20) (20) 0.00c (20) (20) (20) DPI = day post inoculation, D = day old A All data of HI titers in this table is presented as mean + standard deviation (SD). B Number in parentheses under each HI titer means number of samples tested. C All chickens died due to ND before 14 DPI. a,b,c,d Different superscript (a, b, c, d) in each group of the same column means statistically significant difference (p<0.05). Table 5 Number of NDV shedding chickens via oral and cloacal routes at 2, 4, 7, 10 and 14 DPI of 35 days old inoculated chickens that were positive for real-time PCR specific for NDV M gene Group Number of chickens that were NDV positive by real-time PCRA 2 DPI 4 DPI 7 DPI 10 DPI 14 DPI C O CTOC TOCTOC T O 0 T 4 (10) 4 1 4 7 11 2 3 5 (20) 2 4 6 (20) 2 4 6 (20) (10) 0 (20) (10 B) (10) (20) (10) (10) (10) (10) (10) (10) 1 (9) 1 (18) (9) 0 2 1 7 8 (20) 2 3 5 (20) 1 3 4 (18) 0 1 1 (18) 0 (10) 0 (20) (10) (10) (10) (10) (9) (9) (9) (9) (10) (-)C (-)C 3 4 1 5 (20) 1 4 5 (20) 2 0 2 (20) 1 0 1 (20) (-)C (10) (10) (10) (10) (10) (10) (10) (10) 4 3 5 8 (20) 8 6 14 (-)C (-)C (-)C (-)C (-)C (-)C (10) (10) (10) (10) (20) DPI = day post inoculation, O = oropharyngeal swab, C = cloacal swab, T = total oropharyngeal and cloacal swabs A All data in this table is number of samples that were NDV positive. B Number in parentheses means number of chickens examined. C All group 4 chickens died before 7 DPI.