TJVM Vol. 49 No. 3 September 2019

244 Chaisri W. et al. / Thai J Vet Med. 2019. 49(3): 243-248. Introduction was purchased from Sigma-Aldrich (St. Louis, USA) Analytical grade methanol, HPLC grade of methanol Curcumin, the main active component or rhizome and water were purchased from Labscan (Bangkok, of turmeric (Curcuma longa L.), exhibits an important Thailand). Trypticase Soy Broth (TSB), Trypticase Soy role in the wound healing process by promoting Agar (TSA) and Muller-Hinton agar (MHA) were antioxidant, antimicrobial, anti-inflammatory and skin purchased from Merck (Darmstadt, Germany). regeneration activities (Cheppudira et al., 2013; Hewlings and Kalman, 2017; Moghadamtousi et al., Turmeric extraction Turmeric powders (1000 g) were 2014; Vanden and Haegeman, 2010). Previous studies extracted with 15 L of methanol using maceration on wound healing properties have shown that technique for 20 h at room temperature. Subsequently, curcumin can improve the rate of epithelialization, the extracts were filtered using 0.45 micron filter enhance granulation tissue, collagen deposition and papers and concentrated using a vacuum rotary wound contact (Cheppudira et al., 2013; Sidhu et al., evaporator with a controlling temperature of 45oC 1998; Vanden and Haegeman, 2010). (Büchi Labortechnik AC, Flawil, Switzerland). The extract were dried in a conventional oven until Mastitis in dairy cows has been established as the reaching a constant weight and stored at 4oC until use. most costly disease in the dairy industry worldwide. The extract was re-dissolved in methanol to obtain the For healthy teats, pre-milking and post-milking teats final concentration of 5 µg/ml before being subjected dipped in appropriate antiseptics for eliminating to HPLC analysis. bacteria on the teat skin has been found to be one of the most effective procedures for reducing the incidence of Determination of curcumin in crude extract The new intramammary infection (Gleeson et al., 2018; percentage of curcumin in the turmeric crude extract Leslie et al., 2006). Various chemical antiseptics are was analyzed using High Performance Liquid available in commercial teat disinfectants such as Chromatography (HPLC). The separation was chlorine, iodine, iodophor, hydrogen peroxide, and implemented on Schimadzu CBM-20A controller, LC- chlorhexidine (Gleeson et al., 2018; Leslie et al., 2006; 20AD pump, SIL-20AHT auto injector, the fluorescence Rasmussen and Larsen, 1998). In many cases, however, detector SPD-20A (Shimadzu, Tokyo, Japan) and inappropriate milking procedures and/or the use of operating software of LC-solution. The milking machines without regularly maintenance chromatographic method was carried out isocratically causes damage to the teat orifice such as increased with a flow rate of 1 ml/min. The mobile phase callosity, hyperkeratosis, cracks, sores and erosion at consisted of methanol and water (85:15 v/v). the teat ends (Cerqueira et al., 2018; Neijenhuis et al., Separation was achieved using a Phenomenex® C18 (5 2001), and consequently increases risk of new μm; 4.6 mm x 250 mm, 5 µm) analytical column intramammary infections (Mulei, 1999; Neijenhuis et connected to an Inersil® C18 (50 mm x 4.6 mm, 5 µm) al., 2001). guard column. The column temperature was set at 35oC. The detection wavelengths were set at 426 nm Correction of milking procedures and milking and 539 nm for excitation and emission, respectively. machines will stop damage to the teat orifice, and the The injection volume was 20 μL and the running time subsequent natural wound healing process will cure was 8 mins. Calibration curves were prepared by the damaged teat end. To accelerate the wound healing dissolving an accurate weight of curcumin standard process, the use of turmeric extracts and/or in and further diluted in methanol to obtained combination with other antiseptics might be an ideal concentration ranges of 1-10 µg/ml. antiseptic teat dipping disinfectant for farms with damaged teat end problems. From our literature Bacterial preparation For antimicrobial activities of reviews, curcumin was found to be synergistic in the tested substances, 2 reference strains including combination with 8 different antibiotic groups against Staphylocococcus aureus ATCC25923 and Escherichia coli S. aureus (Teow and Ali, 2015), but there were no ATCC12228 were purchased and used. Field strain of studies for other incidental mastitis pathogens such as mastitis bacteria were obtained from a stock of stored S. agalactiae, other streptococci, other staphylococci or bacteria, Faculty of Veterinary Medicine, Chiang Mai those known as coagulase negative staphylococci University. Prior to study, all selected bacterial (CNS), and gram negative bacteria. In screening isolations had been confirmed for their species by antimicrobial testing for various plant extracts, the MALDI-TOF Mass Spectrometry (Bruker Daltonics) agar diffusion method has been the sufficient accepted according to the manufacturer’s recommendation. method to evaluate the antimicrobial activity (Valgas et Spectra were analyszed using Bruker Biotyper al., 2007; Sugathan et al., 2012). Therefore, the objectives softwareReal Time Classification 3.0 software as of this study were to determine the screening describe by (Barreiro et al., 2010). The isolates with antibacterial effects of turmeric extract and its species level identification scores ≥ 2.00 using the combination with various antiseptics against manufacture’s criteria, log scores ≥ 2.00 were used. In microorganisms causing dairy mastitis. In addition, a total, fifty field strains comprising five strains for each concentration of curcumin in turmeric extract was pathogen of 10 mastitis pathogens including determined. Streptococcus agalactiae, Streptococcus uberis, Streptococcus dysgalactiae, Staphylococcus aureus, Materials and Methods Staphylococcus epidermidis, Staphylococcus chromogenes, Staphylococcus hyicus, Staphylococcus xylosus, Animals: The study was conducted with ethics Staphylococcus haemalyticus, Staphylococcus simulant, Chemicals and reagents Turmeric powders were purchased from a supermarket in Chiang Mai, Thailand. Curcumin standard at 99.8% purification

Chaisri W. et al. / Thai J Vet Med. 2019. 49(3): 243-248. 245 Escherichia coli and Klebseilla pneumoniae were used. (CFU)/ml. To confirm the viability of bacteria, These microorganisms were maintained in 15-20% counting the number of bacteria was performed before glycerol and kept at -80 oC. All the bacterial strains experiment. The bacterial suspension was inoculated at were recovered by sub-culturing in Trypticase Soy the surface of Muller-Hinton agar using a sterile cotton Broth (TSB), incubation at 37 oC for 8-12 h and/or swab. Wells with a 6.0 mm diameter were made using growing on Trypticase Soy Agar (TSA) at 37 oC for 24 a croxi broiler. The wells were filled with fifty h when required. microliters of each compound and incubated for 24 h. The diameters of inhibition zones were measured in Antibacterial trial design study The turmeric extract, millimeters after incubation other antiseptics, turmeric extracts in combination with other antiseptics and a commercial 0.5% iodine teat Statistical analysis: The concentration of curcumin dipping antiseptic (Iodine teat dip) available in the from turmeric extract was described in percentage by local market were used to compare their antibacterial weight. All pathogens were separated into 5 groups activities. The turmeric extracts were dissolved in including 1) S. agalactiae, 2) other streptococci from DMSO to obtain the concentration of 5%w/v and used Streptococcus uberis, Streptococcus dysgalactiae, 3) S. as a control group (Klawitter et al., 2012). Other aureus, 4) coagulase negative staphylococci (CNS) from antiseptics used in this study included 5% povidone Staphylococcus epidermidis, Staphylococcus chromogenes, iodine (PVP), 0.5% v/v hydrogen peroxide (H2O2) Staphylococcus hyicus, Staphylococcus xylosus, (Rios-Castillo et al., 2017), 0.5% v/v chlorine (Cl2), and Staphylococcus haemalyticus, Staphylococcus simulant, 0.5% v/v chlorhexidine (Gleeson et al., 2009; Hicks et and 5) Gram negative bacteria from Escherichia coli and al., 1981), and were prepared using distilled water as Klebseilla pneumoniae. Antibacterial activities separated dissolved solutions. The combinations of turmeric for bacterial group of PVP teat dip, turmeric extract, extract with other antiseptic compounds were H2O2, Cl2, and chlorhexidine were determined by their prepared using turmeric extract, dissolved with DMSO diameters of inhibition zones and were analyzed using and Tween, and then mixed with each prepared analysis of variance (ANOVA). Pairwise comparisons antiseptic compound to obtain the final concentration among groups were performed using Duncan’s test of turmeric 5% and antiseptic 0.5% v/v (Gleeson et al., and the significant difference was defined at P < 0.05. 2009; Zaikin et al., 2013). Results The antibacterial activities of all prepared compounds were determined using the agar diffusion Curcumin extraction: The extraction yield of turmeric method according to The National Committee for powder in this study was 27.2%. The HPLC analysis of Clinical Laboratory Standard (NCCLS, 1997). Briefly, curcumin showed single peaks at retention times of prepared bacterial strains were separately inoculated 4.11 mins (Figure 1). The value of curcumin yield was in 0.9% normal saline, incubated at 37°C for 24 h and 1.73%. the turbidity adjusted with 0.5 McFarland to obtain a final concentration of 108 colony-forming units Figure 1 Chromatogram of curcumin in turmeric extract inhibition zones for all bacterial groups of turmeric extracts ranged from 6.46 mm for CNS to 11.7 mm for Antibacterial activities of turmeric extract and its other streptococci. The most effective antibacterial combination with other antiseptic compounds: compounds against mastitis pathogens were Antibacterial activities indicated by the inhibition zone chlorhexidine for S. agalactiae (25.2 ± 0.97mm) and of turmeric extracts and all antiseptic compounds with and without combinations with turmeric extracts, other streptococci (23.5 ± 0.37 mm), H2O2 for S. aureus separated for bacterial groups, are shown in Figure 2. (40.2 ± 0.2 mm) and CNS (35.9 ± 1.06 mm), and Cl2 for Averages and standard error of means (SEM) of the gram negative bacteria (28.6 ± 2.28 mm). On all inhibition zones of commercial iodine teat dips were 15 bacterial groups, turmeric extracts had significantly ± 0.55, 16 ± 0.70, 18.4 ± 0.81, 20.8 ± 0.95 and 20 ± 3.42 lower inhibition zones than those of the iodine teat dip. mm for S. agalactiae, other streptococci, S. aureus, CNS and Gram negative bacteria, respectively. The In comparison to the iodine teat dip, inhibition zones

246 Chaisri W. et al. / Thai J Vet Med. 2019. 49(3): 243-248. of PVP were not significantly lower in other were significantly decreased, excepting S. agalactiae for streptococci and gram negative bacteria, but were Cl2 and gram negative bacteria for chlorhexidine. In significantly lower in S. agalactiae, S. aureus and CNS. contrast, the inhibition zones of H2O2 against all pathogens were not decreased after combination with Among all combination compounds, combination turmeric extracts (p > 0.05). In addition, the inhibition between turmeric extracts and H2O2 was the only zone of H2O2 for other streptococci (11.8 ± 0.2 mm) was compound without shorter inhibition zones than the significantly increased after combination with turmeric commercial iodine teat dip against all bacterial groups. extract (16 ± 0.63 mm). After combination with turmeric extracts, most antibacterial activities of both Cl2 and chlorhexidine Figure 2 Antibacterial activities indicating by inhibition zone of turmeric extracts and all antiseptic compounds with and without combinations with turmeric extracts separated for bacterial groups. a,b,c,d,e,f different letters indicated significant difference at P < 0.05. Discussion sources of turmeric powder, extraction conditions such as solvent types, solvent to turmeric ratio, times, and The yield of curcumin found in this study at 1.73% temperature of storages (Paulucci et al., 2013; Sogi et al., was considered as a low yield level as obtained from 2010). In this study, 50 field strains used comprising 5 Paulucci and colleagues (2013) at 0.1 to 1.8 %. In contrast, Sogi et al. (2010) obtained a curcumin yield of strains for each pathogen from 10 mastitis pathogens 4.5-12.9% that was 3 to 7 times the yield from the represented the majority of mastitis pathogens present study. The low curcumin yields in this study worldwide including this area (Leelahapongsathon et might be due to several factors such as different al., 2016; Suriyasathaporn, 2011; Suriyasathaporn et al., 2012). The results from this study might be used for the

Chaisri W. et al. / Thai J Vet Med. 2019. 49(3): 243-248. 247 further development of antiseptic teat dipping for Acknowledgements mastitis. This research project is supported by Chiang Mai Efficacies of antiseptics and their combination with University. turmeric extracts determined by inhibition zones are shown in Figure 2. The control iodine compound, as References the commercial iodine teat dip, had high inhibition zones for all pathogens. Iodine has been commonly Barreiro JR, Ferreira CR, Sanvido GB, Kostrzewa M, used as a commercially effective disinfectant for teat Maier T, Wegemann B, Bottcher V, Eberlin MN and dipping around the world (Foret et al., 2005; Galton, dos Santos MV 2010. \"Short communication: 2004). The effectiveness of our prepared PVP against Identification of subclinical cow mastitis pathogens bacteria was lower than the control, and this might in milk by matrix-assisted laser have been caused by the different molecular weight desorption/ionization time-of-flight mass polymer of iodine (Zaikin et al., 2013). Excepting gram spectrometry. J Dairy Sci. 93(12): 5661-5667. negative bacteria, chlorhexidine had significantly better antibacterial efficiency than the control in all Cheppudira B, Fowler M, McGhee L, Greer A, Mares pathogen groups (Figure 2). For H2O2 and Cl2, both A, Petz L, Devore D, Loyd DR and Clifford JL 2013. compounds had significantly higher inhibition zones Curcumin: a novel therapeutic for burn pain and than the control in S. aureus, CNS and gram negative wound healing. Expert Opin Investig Drugs. bacteria, but significantly lower in both S. agalactiae 22(10): 1295-1303. and other streptococci for H2O2. Ebrahimi A, Hemati M, Habibian-Dehkordi S, Curcumin has been known for a long time for its Bahadoran S, Khoshnood S, Khubani S, Dokht Faraj antibacterial properties (Teow and Ali, 2015). In this M and Hakimi-Alni R 2014. Chlorhexidine study, turmeric extracts expressed low effective digluconate effects on planktonic growth and antibacterial activities, which might be caused by the biofilm formation in some field isolates of animal low concentration of curcumin in our extract. After bacterial pathogens. Jundishapur J Nat Pharm combination with turmeric extracts, significant Prod. 9:e14298. decreases of antibacterial activity were shown in combinations of turmeric and either Cl2 or Foret CJ, Corbellini C, Young S and Janowicz P 2005. chlorhexidine against almost all of the mastitis Efficacy of two iodine teat dips based on reduction pathogens. With limitation on knowledge of of naturally occurring new intramammary antibacterial efficacy of combinations of turmeric and infections. J Dairy Sci. 88(1): 426-432. other antiseptics, a report showed that curcumin reduced the antimicrobial activity of ciprofloxacin, an Galton DM 2004. Effects of an automatic postmilking antibiotic, against Salmonella thyphi and S.thyphimurium teat dipping system on new intramammary (Marathe et al., 2013). In contrast to Cl2 or infections and iodine in milk. J Dairy Sci. 87(1):225- chlorhexidine, H2O2 did not reduce its antimicrobial 231. efficacy after combination with turmeric extracts (Figure 2). Unlike any other antiseptics in this study, Gleeson D, O'Brien B, Flynn J, O'Callaghan E and Galli H2O2 acts as an oxidant by producing hydroxyl free F 2009. Effect of pre-milking teat preparation radicals (•OH) which attack essential cell components procedures on the microbial count on teats prior to of bacteria, causing disturbances in the structure and cluster application. Ir Vet J. 62(7): 461-467. permeability of the cell wall and the cytoplasmic membrane. These different antibacterial qualities of Gleeson D, Flynn J and O'Brien B 2018. Effect of pre- H2O2 might be related to the penetration of curcumin milking teat disinfection on new mastitis infection enhancing their antibacterial activities (McDonnell and rates of dairy cows. Ir Vet J. 71(11): 1-8. Russell, 2001). Hewlings SJ and Kalman DS 2017. Curcumin: A review In conclusion, H2O2 was the only antiseptic that can of its' effects on human health. Foods. 6(10): 1-11. combine with turmeric extract, for use in promoting the wound healing process, without any loss of Hicks WG and Kennedy TJ 1981. Evaluation of a teat antibacterial efficacy. Although chlorhexidine had the dip of chlorhexidine digluconate (.5%) with highest antibacterial efficacy, especially for glycerin (6%). J Dairy Sci. 64(11): 2266-2269. streptococci, it was previously reported that chlorhexidine could induce biofilm development of E. Klawitter M, Quero L, Klasen J, Gloess AN, coli, S. aureus and S.agalactiae (Ebrahimi et al., 2014). In Klopprogge B, Hausmann O, Boos N and Wuertz K contrast, H2O2 was able to destroy both 2012. Curcuma DMSO extracts and curcumin microorganisms and their biofilm matrix (Lineback et exhibit an anti-inflammatory and anti-catabolic al., 2018). This might support the advantages of effect on human intervertebral disc cells, possibly combining turmeric extract and H2O2 for further by influencing TLR2 expression and JNK activity. J development of future antiseptic teat dipping Inflamm. 9(1): 1-14. products. Due to the screening antimicrobial testing in this study, the establishment of synergistic and/or Leelahapongsathon K, Schukken YH, Pinyopummintr antagonistic properties of turmeric extract in various T and Suriyasathaporn W 2016. Comparison of antiseptics need further studies for example the transmission dynamics between Streptococcus uberis determination of minimal inhibitory concentration and Streptococcus agalactiae intramammary (MIC) to be confirmed. infections. J Dairy Sci. 99(2): 1418-1426. Leslie KE, Vermony E, Bashiri A and Dingwell RT 2006. Efficacy of two hydrogen peroxide teat disinfectants against Staphylococcus aureus and Streptococcus agalactiae. J Dairy Sci. 89(9): 3696-3701. Lineback CB, Nkemngong CA, Wu ST, Li X, Teska PJ and Oliver HF 2018. Hydrogen peroxide and sodium hypochlorite disinfectants are more effective against Staphylococcus aureus and

248 Chaisri W. et al. / Thai J Vet Med. 2019. 49(3): 243-248. Pseudomonas aeruginosa biofilms than quaternary Teow SY and Ali SA 2015. Synergistic antibacterial ammonium compounds. Antimicrob Resist Infect activity of curcumin with antibiotics against Control. 7(154): 1-7. Staphylococcus aureus. Pak J Pharm Sci. 28(6): Marathe SA, Kumar R, Ajitkumar P, Nagaraja V and 2109-2114. Chakravortty D 2013. Curcumin reduces the antimicrobial activity of ciprofloxacin against Valgas C, De Souza SM, Smânia EFA and Smânia A Salmonella typhimurium and Salmonella typhi. J 2007. Screening methods to determine antibacterial Antimicrob Chemother. 68(1): 139-152. activity of natural products. Braz J Microbiol. 38(2): McDonnell G and Russell AD 1999. Antiseptics and 369-380. disinfectants: activity, action, and resistance. Clin Microbiol Rev. 12(1): 147-179. Vanden Berghe W and Haegeman G 2010. Epigenetic Middleton JR, Saeman A, Fox LK, Lombard J, Hogan JS remedies by dietary phytochemicals against and Smith KL 2014. The national mastitis council: a inflammatory skin disorders: myth or reality?. Curr global organization for mastitis control and milk Drug Metab. 11(5): 436-450. quality, 50 years and beyond. J Mammary Gland Biol Neoplasia. 19(3-4): 241-251. Zaikin VG, Borisov RS, Polovkov NY, Zhilyaev DI, Moghadamtousi SZ, Kadir HA, Hassandarvish P, Tajik Vinogradov AA and Ivanyuk AV 2013. H, Abubakar S and Zandi K 2014. A review on Characterization of low-molecular weight iodine- antibacterial, antiviral, and antifungal activity of terminated polyethylenes by gas curcumin. BioMed Res Int. Article ID: 186864. chromatography/mass spectrometry and matrix- Mulei CM 1999. Teat lesions and their relationship to assisted laser desorption/ionization time-of-flight intramammary infections on small-scale dairy mass spectrometry with the use of derivatization. farms in Kiambu district in Kenya. J S Afr Vet Eur J Mass Spectrom. 19(3): 163-173. Assoc. 70(4): 156-157. Neijenhuis F, Barkema HW, Hogeveen H and Noordhuizen JPTM 2001. Relationship between teat-end callosity and occurrence of clinical mastitis. J Dairy Sci. 84(12): 2664-2672. Paulucci VP, Couto RO, Teixeira-Crustiane CC and Luis-Alexandre PF 2013. Optimization of the extraction of curcumin from Curcuma longa rhizomes. Rev Bras Farmacogn. 23(1): 94-100. Rasmussen MD and Larsen HD 1998. The effect of post milking teat dip and suckling on teat skin condition, bacterial colonisation, and udder health. Acta Vet Scand. 39(4): 443-452. Rios-Castillo AG, Gonzalez-Rivas F and Rodriguez- Jerez JJ 2017. Bactericidal efficacy of hydrogen peroxide-based disinfectants against gram-positive and gram-negative bacteria on stainless steel surfaces. J Food Sci. 82(10): 2351-2356. Sidhu GS, Singh AK, Thaloor D, Banaudha KK, Patnaik GK, Srimal RC and Maheshwari RK 1998. Enhancement of wound healing by curcumin in animals. Wound Repair Regen. 6(2): 167-177. Sogi DS, Sharma S, Oberoi DPS and Wani IA 2010. Effect of extraction parameters on curcumin yield from turmeric. J Food Sci Technol. 47(3): 300-304. Sugathan S, Manilal A, Selvin J, Idhayadhulla A, Kumar RS and Panikkar MVN 2012. Evaluating the antagonistic potential of seaweed-associated marine bacteria collected from the southwest coast of India. Asian J Anim Vet Adv. 7: 578-587. Suriyasathaporn W 2011. Epidemiology of subclinical mastitis and their antibacterial susceptibility in smallholder dairy farms, Chiang Mai province, Thailand. J Anim Vet Adv. 10(3): 316-321. Suriyasathaporn W, Chupia V, Sing-Lah T, Wongsawan K, Mektrirat R and Chaisri W 2012. Increases of antibiotic resistance in excessive use of antibiotics in smallholder dairy farms in northern Thailand. Asian-Australas J Anim Sci. 25(9): 1322- 1328.

Original Article Comparison of different methods for sperm vitality assessment in frozen-thawed Holstein bull semen Kakanang Buranaamnuay Abstract The present study evaluated the accuracy of four methods, i.e. eosin-nigrosin, trypan blue, hypo-osmotic swelling test (HOST) and Hoechst 33342 (H342)/propidium iodide (PI) for assessing bovine sperm vitality. Frozen-thawed semen (n = 30) of Holstein bulls was layered on 40%/80% percoll solutions to isolate viable spermatozoa. An aliquot of viable spermatozoa was kept at 37°C (live sample); the rest was submitted to cold shock (dead sample). The two aliquots were mixed in three proportions, corresponding to 0%, 50% and 100% of viable cells; the sperm vitality was analyzed. The percentages of viable spermatozoa evaluated with the four methods significantly correlated with the expected vitality (rs = 0.92 – 0.94, P<0.001). Comparing methods of evaluation, the sperm vitality assessed by eosin- nigrosin and trypan blue was comparable (P>0.05) when live and dead samples were used. For the live sample, eosin- nigrosin (82.37 ± 1.48%) and trypan blue (81.00 ± 1.67%) yielded significantly greater results than HOST (50.14 ± 1.68%) and H342/PI (56.69 ± 1.76%) (P<0.001). The percentage of viable spermatozoa in the dead sample (13.45 ± 0.86%) was highest when HOST was exploited (P<0.001); still, the sperm vitality acquired by this technique become the lowest when the live sample was evaluated (P<0.001 to P=0.03). In conclusion, while HOST and fluorescence stains H342/PI with the protocol used in this study are not trustworthy methods, eosin-nigrosin and trypan blue are accurate techniques for sperm vitality assessment in frozen-thawed Holstein bull semen. Keywords: cattle, spermatozoa, membrane integrity, staining techniques, freezing Molecular Agricultural Biosciences Cluster, Institute of Molecular Biosciences (MB), Mahidol University, Nakhon Pathom 73170, Thailand *Correspondence: [email protected] (K. Buranaamnuay) Thai J Vet Med. 2019. 49(3): 249-255.

250 Buranaamnuay K. / Thai J Vet Med. 2019. 49(3): 249-255. Introduction 1996), monkeys (Cai et al., 2005) and other mammals (Garner and Johnson, 1995). Using H342/PI for rhesus Vitality of spermatozoa is requisite for successful monkey ejaculates, the correlation between fertilization. Therefore, irrespective of animal species, percentages of expected vitality and viable sperm vitality is always included in semen analysis spermatozoa stained only with H342 was found to be (Vasan, 2011; Morado et al., 2015; Sutovsky, 2015). very strong both when evaluated by fluorescence Currently, there are several methods for assessing the microscopy and flow cytometry (Cai et al., 2005). vitality of spermatozoa. Dye exclusion assays, Besides vitality evaluation, H342 has been utilized in traditional vitality tests, such as eosin, eosin-nigrosin computer assisted sperm analysis (CASA) (Farrell et al., and trypan blue are of the most common methods 1996; Oliveira et al., 2013) and flow cytometric sorting (Brito et al., 2003), and eosin-nigrosin is recommended of spermatozoa (Garner, 2009). For CASA, H342 helps as a vitality test by the World Health Organization distinguish spermatozoa from other particulate (WHO) for use in clinical Andrology laboratories material such as somatic cells in semen and egg yolks (WHO, 2010). The dye exclusion tests are grounded in or whole milk in freezing media while in the process of the permeability of the plasma membrane. The plasma flow sorting, H342 is used to measure DNA mass in X- membrane of viable spermatozoa is a barrier to dye and Y-chromosome-bearing mammalian spermatozoa. penetration; therefore, after incubation with the dyes, According to previous results, it has been proved that viable spermatozoa remain colorless. On the other exposure of spermatozoa to H342 did not affect sperm hand, damaged or dead spermatozoa lack membrane DNA, the fertilizing capacity of the viable spermatozoa structural integrity and, as a result, allow the entry of or the developmental competence of the resultant the dyes (Eliasson and Treichl, 1971). It has been embryos (Morrell and Dresser, 1989; Seidel and reported that trypan blue contained in the triple Garner, 2002; Cran, 2007). staining technique and eosin-nigrosin have been effective for vitality evaluation in bovine fresh To date, there are no comparative studies on the spermatozoa but in frozen-thawed semen the eosin- effectiveness of using eosin-nigrosin, trypan blue, nigrosin technique overestimated the number of viable H342/PI as well as HOST for assessing sperm vitality. cells that were able to fertilize oocytes (Felipe-Perez et The present study was therefore designed to compare al., 2008). the accuracy of these methods for evaluating the sperm vitality of frozen-thawed Holstein bull semen. The In addition to the permeability of the plasma results obtained will be useful for selecting the membrane, sperm vitality can also be evaluated by appropriate effective method for sperm vitality determining the ability of the sperm membrane to assessment. maintain equilibrium between the intracellular-fluid and extracellular-fluid compartments using the hypo- Materials and Methods osmotic swelling test (HOST) (WHO, 2010). During the HOST procedure, spermatozoa are exposed to Chemicals: All reagents, unless otherwise other hypotonic solutions; influx of the solutions results in specified, were from Sigma-Aldrich (St. Louis, MO, the expansion of the cytoplasmic space especially in the USA). tail. Coiling of sperm tails can be easily observed with a phase contrast microscope (Jeyendran et al., 1992; Ethics Statement: No live animals were included in Hossain et al., 1998). A higher percentage of swollen any of the experiments described in this article. Frozen spermatozoa indicate the presence of viable bovine semen used for sperm vitality evaluations was spermatozoa having a functional plasma membrane supplied from a commercial dairy farm named Farm (Ramu and Jeyendran, 2013). HOST is considered to Chokchai®, which is situated in Nakhon Ratchasima be a useful method for clinical and practical province in the northeastern region of Thailand. applications apart from its diagnostic value in selecting viable but immotile spermatozoa for intracytoplasmic Specimens: Frozen semen in mini-(0.25 mL) plastic sperm injection (ICSI) although, in humans, lower straws (Minitub GmbH, Tiefenbach, Germany) was fertilization rates have been obtained from this sperm produced from 30 ejaculates of Holstein bulls, whose population as compared with cases where motile and their fertility had been proved, for daily use in artificial viable spermatozoa were used (Tsai et al., 1997). In insemination on the farm. The protocol for freezing boars, it has been shown that HOST test correlated semen according to a disclosure by a member of staff is significantly with in vivo fertility and indicated briefly described here. At room temperature (27 ± 2°C), possible sperm damage due to cold shock (Perez-Llano raw semen with the progressive motility of 65% or et al., 2001). Contrarily, HOST test is not sufficiently more was extended in Tris-citric acid-egg yolk-fructose sensitive to discriminate between bovine semen diluent (Buranaamnuay et al., 2015) to attain 120x106 samples of intermediate fertility (Rota et al., 2000). spermatozoa/mL in final sperm concentration. The diluted semen was placed at 4°C for 4 h, loaded into The assessment of sperm vitality has been the straws and frozen by a rapid freezing method in a enhanced by the availability of new fluorescent stains styrofoam box. targeting sperm DNA. The combination of these nucleic acid stains, one stain to identify viable Sperm preparation: In order to assess the vitality of the spermatozoa e.g. Hoechst 33342 (H342) and SYBR-14 spermatozoa, 2 ‒ 3 frozen semen straws from the same and another to stain only dead spermatozoa e.g. ejaculates and the same bulls were thawed (37°C for 30 Hoechst 33258 (H258), propidium iodide (PI) and s) and three defined percentages of the sperm vitality ethidium bromide, have been effective methods for (i.e. 0%, 50% and 100%) were made by the following determining sperm vitality in boars (Garner et al.,

Buranaamnuay K. et al. / Thai J Vet Med. 2019. 49(3): 249-255. 251 procedure. Immediately after thawing, the total movement of spermatozoa during evaluation, 10 µL of motility of spermatozoa was evaluated visually under formol saline [0.9% (w/v) NaCl and 0.1% (v/v) 40% a light microscope (400x; Helmut Hund GmbH, formaldehyde in distilled water] (Dott and Foster, Wetzlar-Nauborn, Germany) to check whether the 1975) was added to the sample. Microscopic slides post-thaw spermatozoa were viable. The thawed were prepared by placing a drop of the stained sample semen that contained viable spermatozoa was then on the slide and covering with a cover slip. Slides were placed on the topmost layer of the 40% and 80% Percoll examined under the 1000 x magnification, with gradient (semen: 40% Percoll: 80% Percoll = 1:3:3, v/v) immersion oil, of a bright-field microscope. A total of and centrifuged at 400 x g for 15 mins. Percoll solutions 200 sperm heads was counted, and the percentage of were prepared beforehand by the dilution of Percoll® viable (unstained) spermatozoa was calculated and (GE Healthcare, Little Chalfont, UK) in sperm-TALP noted. (10X and 1X) (Buranaamnuay, 2013). The sperm pellet was washed in 2 mL sperm-TALP by single Hypo-osmotic swelling test (HOST): The HOST is an centrifugation (200 x g, 5 min); and then the washed alternative to the dye exclusion test for the assessment pellet was resuspended in sperm-TALP to acquire of sperm vitality (WHO, 2010). It estimates approximately 80 x 106 spermatozoa/mL. This sperm physiological functionality not structural integrity as portion was assumed to be 100% viable sample, since evaluated by the dye exclusion method, of the sperm the total sperm motility checked at this stage was not plasma membrane (Lin et al., 1998). A swelling solution less than 75%. (75 mOsm) had been prepared by combining Na-citrate dihydrate with D-fructose in distilled water. A total of To provide quantifiable proportions of viable and 50 µL sample was mixed with 300 µL of the swelling dead spermatozoa, the 100% viable sample was solution. After incubation for 20 min at 37 °C, a small divided into two parts. The first part was placed in a drop of the mixture was placed on a microscope slide water bath at 37°C to maintain sperm vitality. The overlaid with a coverslip and 200 spermatozoa were other part was submitted to cold shock to induce analyzed by phase-contrast microscopy (400x; Nikon, cellular damage by two cycles of suddenly placing it in Melville, NY, USA). Only swollen cells of different a -80°C freezer (model 720, capacity 0.566 m3; Thermo types were considered positive and reported as a Fisher Scientific Inc., Waltham, MA) for 7 min and percentage of all spermatozoa analyzed. For an thawing at 37°C. At this stage, motile spermatozoa accurate assessment, sperm samples without the were not found according to the sperm motility swelling solution were also examined for spontaneous evaluation. This sperm portion was therefore deemed swelling or spermatozoa with coil tails. The number of to be a 0% viable sample. Then, a sample with 50% swollen cells in the unincubated samples was deducted viable spermatozoa was made by mixing aliquots of from the HOST score in order to obtain the percentage 100% and 0% viable samples at a ratio of 1:1. In each of viable spermatozoa. semen sample, three defined percentages of sperm vitality (i.e. 0%, 50% and 100%) were prepared in total H342/PI staining: The H342/PI staining procedure and used for sperm vitality evaluations. used in the present study was modified from Garner et al. (1996). A 22 µM working solution of H342 was Evaluations of sperm vitality: In this study, the vitality prepared from a stock solution (22 mM) by dilution in of spermatozoa was appraised by four techniques, as distilled water. The working solution of PI was 1 mM described below. in phosphate buffered saline (PBS). An aliquot (75 µL) of sample was stained with 4 µL of H342 and 4 µL of PI One-step eosin-nigrosin staining: The eosin-nigrosin working solutions. The sample was incubated in test is a dye exclusion method recommended by WHO darkness for 30 min at 37°C. Immediately prior to to be used as a sperm vitality test. For each sample, a assessment, a drop of formol saline was added to 20 μl aliquot was placed on a pre-warmed glass slide; prevent sperm movement. Fluorescent staining was the same amount of eosin-nigrosin dyes [0.6% (w/v) monitored using a fluorescence microscope (400x; eosin Y and 5% (w/v) nigrosin dissolved in distilled Axioskop, Carl Zeiss Jena GmbH, Jena, Germany). The water] was dropped on. After mixing for 30 secs, 10 μL viable, dead and moribund spermatozoa emit bright of the mixture was taken to spread on another clean blue, pinkish red and both blue and red, respectively glass slide and dried on a 37°C warm plate. The dried when the H342/PI-stained samples are excited with smear is then observed for the vitality of spermatozoa the ultraviolet (UV) light. The percentage of sperm using bright-field microscopy (1000x). The viable vitality was determined from a total of 200 spermatozoa with intact plasma membrane are not spermatozoa counted. stained by the eosin dye. On the other hand, the dye penetrates the membrane-damaged spermatozoa; dark Statistical analysis: All statistical analyses were pink or red sperm heads are seen. In each sample, the performed using the SPSS software (version 17.0 for percentage of sperm vitality was calculated based on Windows, SPSS Inc., Chicago, IL, USA). An assessment 200 total spermatozoa counted. of the normality of data was undertaken by the Kolmogorov-Smirnov test. Correlation coefficients Trypan blue staining: The vitality of spermatozoa was between expected (i.e. 0%, 50% and 100% vitality) and also evaluated by the vital stain Trypan blue, which measured (i.e. % vitality obtained through four tests) penetrates into the post acrosomal region of dead cells values were made using linear regression analysis. (Felipe-Pérez et al., 2008). The sperm sample was Correlations among pairs of measured variables were diluted with 0.4% trypan blue solution in 1:1.2 (v/v) calculated using a Spearman correlation. One-way ratio and then incubated at 37°C for 15 min. To stop the

2520 Buranaamnuay K. / Thai J Vet Med. 2019. 49(3): 249-255. A1N9 OVA and Tukey’s HSD post-hoc test were used to determine the difference in the percentages of sperm nigrosin and trypan blue were commensurate (P>0.05) vitality among tests. Data are represented as means ± when the ratios of viable spermatozoa in the sample SEM; the level of significance was set at P<0.05. were 0% and 100% (Table 2). However, when the proportion of viable spermatozoa was 50%, trypan Results blue gave significantly, but marginally, higher in the results than eosin-nigrosin staining (P=0.04). The In the present study, the mean percentages of results in the 50% and 100% viable samples evaluated viable spermatozoa assessed by eosin-nigrosin and with eosin-nigrosin and trypan blue were significantly trypan blue, for ratios of 0:100, 50:50 and 100:0 of the greater compared with the vitality assessed by HOST live and dead mixtures were 6.50%, 46.63% and 82.37% and H342/PI (P<0.001). The percentage of viable and 6.73%, 50.77% and 81.00%, respectively. The spermatozoa in dead samples (13.45 ± 0.86%) was the correlations between the vitality evaluated by these highest when the HOST method was used (P<0.001 for two techniques with the expected percentages were rs all). On the other hand, the sperm vitality acquired by = 0.94 (P<0.001) (Table 1 and Fig 1 A and B). Similarly, this technique (50.14 ± 1.68%) became the lowest when very high positive correlations (rs = 0.92 ‒ 0.94, the samples containing 100% of living cells were P<0.001) were found between the expected values and evaluated (P<0.001 to P=0.03). Regardless of the the viable population detected by HOST and H 342/PI mixture ratio, the percentages of viable spermatozoa (Table 1 and Fig 1 C and D). were positively and significantly associated among the four evaluation methods; the correlation coefficients Comparing the methods of vitality evaluation, the were between 0.84 and 0.95 (P<0.001, Table 1). percentages of viable spermatozoa assessed by eosin- Table 1 Coefficients of correlation (rs) and significance level (P value) between the percentages of sperm vitality, evaluated by four different methods and expected values Expected values Eosin-nigrosin Trypan blue HOST1 H342/PI2 Eosin-nigrosin Trypan blue 0.94 0.94 0.92 0.94 HOST (P<0.001) (P<0.001) (P<0.001) (P<0.001) 0.95 0.86 0.92 (P<0.001) (P<0.001) (P<0.001) 0.84 0.93 (P<0.001) (P<0.001) 0.86 (P<0.001) 1The sperm vitality detected by the hypo-osmotic swelling test 2The sperm vitality evaluated by staining with Hoechst 33342 and propidium iodide (PI) Table 2 The mean percentages (± SEM) of viable frozen-thawed bovine spermatozoa assessed by four different methods, for ratios of 100:0, 50:50 and 0:100 of the living and killed mixtures Expected percentage Method of sperm vitality evaluation of sperm vitality Eosin-nigrosin Trypan blue HOST1 H342/PI2 0% 6.50 ± 1.19 b 6.73 ± 1.19 b 13.45 ± 0.86 a 0.73 ± 0.31 c 31.47 ± 1.16 c 50% 46.63 ± 0.96 b 50.77 ± 1.00 a 32.07 ± 1.21 c 56.69 ± 1.76 b 100% 82.37 ± 1.48 a 81.00 ± 1.67 a 50.14 ± 1.68 c Values with different letters (a, b, c) indicate significant difference within row 1The sperm vitality detected by the hypo-osmotic swelling test 2The sperm vitality evaluated by staining with Hoechst 33342 and propidium iodide (PI) Discussion humans (Eliasson and Treichl, 1971; Bjorndahl et al., In the present study, the accuracy of 2003), monkeys (Cai et al., 2005), horses (Kutvolgyi et techniques for sperm vitality evaluation was analyzed al., 2006), pigs (Zhou et al., 2004) and mice (Al-Saleh in frozen-thawed bovine semen. This has yet to be and Al-Otaiby, 2014). carried out in dairy cattle, to the author knowledge. The results showed that all four techniques studied Besides high correlations with the expected vitality, (three assessed with vital stains and one evaluated by the percentages of viable spermatozoa obtained with measurement of osmoregulatory capacity) correlated significantly with the expected vitality percentages. the four methods correlated significantly with one This indicates that both measurements of structural another especially between the results obtained with integrity using eosin-nigrosin, trypan blue and vital stains, for which correlation coefficients of more H342/PI and functional integrity using HOST of sperm than 0.90 were shown. The present finding is in plasma membrane are usable for sperm vitality agreement with a previous report where vital stains assessment in Holstein bulls. In accordance with the present results, previous studies have demonstrated eosin-nigrosin, trypan blue and PI in combination the usefulness of such tests in the evaluation of sperm with carboxyfluoresceindiacetate (CFDA) or SYBR-14 vitality in several mammalian species including were tested in beef bull semen and high correlations between the results were investigated (Brito et al., 2003). This could suggest that these vital stains provide measures of the same sperm trait, i.e. plasma membrane integrity. The mechanism of action of these

Buranaamnuay K. et al. / Thai J Vet Med. 2019. 49(3): 249-255. 253 dyes depends on the properties of the cell membrane (Felipe-Pérez et al., 2008). Comparing single- (trypan which separates the inside of the cell from the external blue) and dual- (eosin-nigrosin) staining methods, environment. Eosin and trypan blue, that in an differentiation of viable from dead cells using bright aqueous solution at physiological pH are anionic, do field microscopy is easier for dual- than single-staining not penetrate the intact plasma membrane of viable methods. This could be a possible reason explaining cells due to the negative charge of the cell membrane; why in the 50% viable samples of the present study the in contrast, these dyes penetrate dead or damaged sperm vitality evaluated with eosin-nigrosin was cells, staining the cytoplasm (Kwolek-Mirek and different from that assessed with trypan blue; Zadrag-Tecza, 2014). To interpret the results, for eosin nonetheless, such difference was marginally staining, viable cells are colorless or faint pink and significant (P=0.04). Disregarding a trivial difficulty dead cells show red or dark pink. In cases of combining during assessment, both eosin and trypan blue staining with eosin, nigrosin helps provide a dark background techniques require neither of expensive material nor for increased contrast and yields reliable evaluations highly sophisticated equipment (Bjorndahl et al., 2003; using ordinary light microscope optics (WHO, 2010). Felipe-Pérez et al., 2008); it is therefore highly possible For trypan blue staining, colorless cells are scored as for a small laboratory and even in the field to adopt viable cells while, in spermatozoa, cells with blue color these methods in semen analysis. at the post acrosomal region are classified as dead cells Figure 1 Regression plots for correlations between the percentages of viable frozen-thawed bovine spermatozoa evaluated by eosin-nigrosin staining (A), trypan blue staining (B), hypo-osmotic swelling test (C) and Hoechst 33342 and propidium iodide staining (D) and the expected percentages. Apart from eosin-nigrosin and trypan blue, the bovine spermatozoa, it has been speculated that, other vitality of frozen-thawed bovine spermatozoa was also than staining conditions, spermatozoa from different evaluated with nucleic acid specific-stains H342/PI. bulls and even some spermatozoa within an ejaculate The proportions of membrane-intact spermatozoa may differ in dye permeability; this results in identified by H342/PI were significantly inferior to variations in staining efficiency among cells (Garner, those detected with eosin-nigrosin or trypan blue 2009). Nonetheless, it is worth noting that results of the stains. The difference in the proportions of viable cells present study were in accord with those observed in acquired by these staining methods is potentially due beef bull semen, for which CFDA/PI or SYBR-14/PI to the fact that these dyes bind to different cell fluorescent stains instead of H342/PI were used and components, i.e. eosin and trypan blue stain the yielded significantly lower sperm vitality compared cytoplasm while H342/PI stain the nucleus. with eosin-nigrosin and trypan blue (Brito et al., 2003). Furthermore, for H342 staining, cell types and staining As stated above, the HOST and vital stains measure conditions such as concentrations of the dye, acidity and alkalinity of media as well as incubation different aspects of the sperm plasma membrane, i.e. temperature and time are important factors in dye the HOST evaluates functional activity while vital stains assess structural integrity (Lin et al., 1998). This uptake by the cells (Lalande et al., 1981; Weisenfeld, 2007) and, hence, the percentage of cell vitality. For fact may be used to explain differences in the sperm vitality percentages between the HOST and vital stains

254 Buranaamnuay K. / Thai J Vet Med. 2019. 49(3): 249-255. in the present study. The HOST yielded significantly Kulnasan Saikhun and Ms. Kornkanok Promthep for greater results in the dead (0% viable) sample but providing the laboratory facilities. provided significantly lower results in the 100% viable sample. The present finding, in the dead sample, Funding: This research did not receive any specific agreed with Munuce et al. (2000), who reported higher grant from funding agencies in the public, commercial vitality values in the HOST than in the eosin-nigrosin or not-for-profit sectors. when freeze-killed human spermatozoa were evaluated. The authors suggested that when References spermatozoa are killed by freezing, structural integrity of the sperm plasma membrane is lost before the Al-Saleh AA and Al-Otaiby AM 2014. Evaluation effect capacity to regulate osmotic equilibrium (Munuce et of dacarbazine on mice testis tissues and sperm al., 2000). Similarly, in humans, it has been revealed parameters. Int Res J Pharm App Sci. 4: 9‒13. that poor quality spermatozoa with borderline membrane integrity stained red with eosin can exhibit Bjorndahl L, Soderlund I and Kvist U 2003. Evaluation swelling in HOST (Chan et al., 1991). In addition, the of the one-step eosin-nigrosin staining technique results of the present study found that the 100% viable for human sperm vitality assessment. Hum sample was in concurrence with previous findings Reprod. 18: 813‒816. which indicated that the response of spermatozoa to hypo-osmotic stress does not depend only on the Brito FC, Barth AD, Bilodeau-Goeseels S, Panich PL morphological integrity of the sperm plasma and Kastelic P 2003. Comparison of methods to membrane (Brito et al., 2003) and that during the HOST evaluate the plasmalemma of bovine sperm and procedure hypo-osmotic shock on its own induces their relationship with in vitro fertilization rate. membrane damage and therefore decreases the Theriogenology 60: 1539‒1551. percentages of viable spermatozoa (Ramirez et al., 1992). Buranaamnuay K 2013. Sperm-TALP: an alternative extender for retrieving and diluting epididymal Although the proportions of viable spermatozoa sperm in the domestic cat. Reprod Dom Anim. 48: acquired by means of H342/PI staining and HOST 912‒917. were more likely to deviate from the expected values, in comparison to eosin-nigrosin and trypan blue, Buranaamnuay K, Sangsuwan P, Changsangfa C, vitality assessment by these two techniques offers Faisaikarm T and Kaeoket K 2015. Influence of some advantages over staining with eosin-nigrosin or discontinuous PureSperm® and OptiPrepTM trypan blue. The most prominent one is that viable gradient centrifugations on bovine sperm quality spermatozoa detected with these techniques can be and the sex ratio of in vitro produced embryos. further used for therapeutic procedures such as ICSI Chiang Mai J Sci. 42: 637‒649. (Tsai et al., 1997), and the resultant embryos and offspring are normal (Cran et al., 1995; Garner et al., Cai K, Yang J, Guan M, Ji W, Li Y and Rens W 2005. 1996; McNutt and Johnson, 1996). Moreover, using Single UV excitation of Hoechst 33342 and HOST as a vitality assessment method, proportions of propidium iodide for viability assessment of rhesus HOST positive spermatozoa were significant monkey spermatozoa using flow cytometry. Arch predictors of sperm fertility in beef bulls (Brito et al., Androl. 51: 371‒383. 2003) and boars (Perez-Llano et al., 2001). Therefore, to confirm the usefulness of sperm vitality assessment Chan PJ, Tredway DR, Corselli J, Pang SC and Su BC techniques for Holstein bulls, the relationships 1991. Combined supravital staining and between the percentage of sperm vitality evaluated hypoosmotic swelling test. Hum Reprod. 6: 1115‒ with these four methods and fertility in vitro and in vivo 1118. should also be determined. Cran DG 2007. XY sperm separation and use in In conclusion, while the fluorescence stains artificial insemination and other ARTs. Soc. H342/PI with the protocol used in this study are not Reprod. Fertil. (Suppl) 65: 470–491. trustworthy stains to assay sperm vitality, eosin- nigrosin and trypan blue staining techniques can be Cran DG, Johnson LA and Polge C 1995. Sex taken as effective diagnostic tests for the preselection in cattle: a field trial. Vet Rec. 136: 495‒ discrimination of viable from nonviable spermatozoa 496. in Holstein bulls. Nevertheless, in vitro and/or in vivo fertility of frozen-thawed spermatozoa should also be Dott HM and Foster GC 1975. Preservation of investigated to confirm the benefit of these vitality differential staining of spermatozoa by formol assessment techniques for this animal. citrate. J Reprod Fertil. 45: 57‒60. Conflicts of interest: There are no conflicts of interest Eliasson R and Treichl L 1971. Supravital staining of regarding the publication of this article. human spermatozoa. Fertil Steril. 22: 134–137. Acknowledgements Farrell PB, Foote RH and Zinaman MJ 1996. Motility and other characteristics of human sperm can be Frozen Holstein bull semen was kindly supplied by measured by computer-assisted sperm analysis of Farm Chokchai®. The author is grateful to Assist. Prof. samples stained with Hoechst 33342. Fertil Steril. 66: 446‒453. Felipe-Perez YE, Juarez-Mosqueda ML, Hernandez- Gonzalez EO and Valencia JJ 2008. Viability of fresh and frozen bull sperm compared by two staining techniques. Acta Veterinaria Brasilica 2: 123‒130. Garner DL 2009. Hoechst 33342: the dye that enabled differentiation of living X-and Y-chromosome bearing mammalian sperm. Theriogenology 71: 11‒ 21.

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Original Article A feasibility of ultrasonographic assessment for femoral trochlear depth and articular cartilage thickness in canine cadavers Phuangporn Boonchaikitanan Nan Choisunirachon Kumpanart Soontornvipart* Abstract Trochleoplasty or trochlear groove deepening is one of surgical correction techniques for patellar luxation (PL) that caused cartilage alterations of the distal femur and this technique may not be always necessary. To evaluate trochlear groove for PL correction preoperative planning, several diagnostic image techniques have been applied. In this study, ultrasonography (USG) was introduced in the trochlear evaluation as pre-operative planning because it’s user-friendly and radiationless. USG imaging of 22 distal femurs from small breed of canine cadavers were evaluated for trochlear groove depth (TGD) and trochlear cartilage thicknesses (CT) at medial condyle (MCCT), femoral groove (FGCT) and lateral condyle (LCCT), and compared to those of other distal femoral evaluations such as conventional radiography and/or anatomical appearance observed through stereomicroscope (STR). The results showed that TGD on radiograph was significantly deeper than those on USG and STR (P = 0.0099 for USG and P = 0.0021 for STR) but TGD on USG and STR was not significant difference (P > 0.9999). MCCT, FGCT and LCCT were evaluated and compared only between USG and STR and the results showed that only FGCT between techniques were not significant difference (P = 0.0646). At the condyles, MCCT and LCCT on USG were significantly thicker than those on STR (P = 0.006 for MCCT and P = 0.0004 for LCCT). In conclusion, USG was a reliable technique for TGD and FGCT evaluations. However, to evaluate the MCCT and LCCT by USG, CT may be slightly exaggerating. This diagnostic imaging technique could be applied in clinical practice and further evaluations of bone appearance between the normal and PL dogs should be done. Keywords: cartilage thickness, dog, femoral trochlea, stifle, ultrasonography Department of Veterinary Surgery, Faculty of Veterinary Science, Chulalongkorn University, Bangkok, Thailand, 10330 *Correspondence: [email protected] (K. Soontornvipart) Thai J Vet Med. 2019. 49(3): 257-264.

258 Boonchaikitanan P. et al. / Thai J Vet Med. 2019. 49(3): 257-264. Introduction computed tomography can provide three-dimensional information, several issues should be concerned. For Patellar luxation (PL) is one of the most frequent example; high radiation exposure comparing to orthopedic diseases found in small breed dogs in radiography, requirement of chemical immobilization several countries (Vidoni et al., 2006; Soontornvipart et such as deep sedation or anesthesia and also al., 2013). The etiology of this diseases is not only requirement of strict positioning (Marino and Loughin, developmental disease, but can also be counted as an 2010). acquired orthopedic disease caused by trauma or orthopedics surgery (Hayes et al., 1994). Common In addition to radiography and computed features of PL include quadriceps displacement, tomography, ultrasonography (USG) that is a low cost, shallowed femoral trochlear groove, torsion of tibial non-invasive and radiation less, is comfortably tuberosity and patellar alta (Hans et al., 2016). technique for animals. Althogh the skyline view of the Furthermore, malposition of the quadriceps muscles stifle, which the patellar is vertically direction to the X- can induce angulation and rotation between distal ray beam from cranioproximal to craniodistal direction femur and proximal tibia. This phenomenon (Meier et al., 2001), is a routinely procedure to observe subsequently increases stress to articular cartilage and distal femoral trochlear. This study selected the gross cranial cruciate ligament. Therefore, long term of stifle appearance of bone structure on STR as the gold joint instability due to the progression of PL may lead standard in stead of skyline view due to stifle to stifle osteoarthritis (OA). radiograph could not provide information of the trochlear cartilage (Wolski et al., 2011). USG can be In general, surgical correction by rearrangement of done in conscious dogs or under only light sedation. the quadriceps and stabilization of patella to be fitted Besides, USG can be useful for clinical evaluation of in femoral trochlear groove is one of the treatments of almost all intra-articular structures such as articular choice (Kowaleski et al., 2012; van der Zee, 2015). cartilage, intra-articular effusion and articular tendon Surgical corrections including both of soft tissue and (Marino and Loughin, 2010). Laasanen and colleagues bone reconstruction were reported to be recommended (2006) reported that USG could be applied as for PL in dogs. (Slocum, 1993; Hayes and Boudreiau et quantitative assessment of the impaired structural al., 1994; Arthers and SJ, 2006). Nevertheless, bone integrity of cartilage and subchondral bone in porcine reconstruction with trochleoplasty; femoral trochlear model. In dogs, it has been reported that USG was used deepening, was reported to cause the damage of to estimate structure of femoral joint (Kramer et al., hyaline cartilage and finally were permanently 1997; Hansen et al., 2017). Since the PL is one of the replaced by fibrocartilagenous tissue (Thompson, 1975; most frequent orthopedic diseases found in small dogs Hunziker, 2002; van der Zee, 2015). Fibrocartilage and USG is one of the imaging modalities that current patch creation from trochleoplasty does not have the broadly available, USG observing the trochlear depth same properties as that of hyaline cartilage (O’Driscoll, and adjacent structures at the stifle would be one of the 1998). In addition to fibrocartilageneous replacement attractive procedures for preoperative surgical following the trochleoplasty, the unfit between patella and planning for PL. Therefore, the aim of this study was new outline of the sulcus caused abnormal pressure at the stifle to assess distal femoral structures, all of trochlear depth and articular cartilage thicknesses (CT) at the joint and led to cartilage degradation (Daems et al. 2009). distal femur, through USG comparing to the stifle Interestingly, Linney and colleagues (2011) reported radiographs and gross anatomy in canine cadavers. that surgical treatment for grade II, III and IV of PL by only tibial tuberosity transposition (TTT) and lateral Materials and Methods retinaculum imbrication in dogs without trochlear groove deepening showed satisfying results. These Animals: Twenty two stifle joints acquired from 11 dogs showed only 19.8% of patellar reluxation and small breed dogs after death at the surgical unit of osteoarthritis were not found during eight weeks post- Small Animal Teaching Hospital, Faculty of Veterinary operatively. Therefore, to treat PL with surgical Science, Chulalongkorn University between April 2018 correction, trochlear groove deepening may not always and October 2019 under permission of owners. All be necessary, and preoperative planning by evaluating cadavers were mature without history of stifle fracture of distal femoral structures by means of trochlear or luxation, metabolic disease, and stifle surgery. If depth and surrounding cartilage condition should be stifle deformity was detected and there were history of done prior trochleoplasty selection. PL, the cadaver will be excluded from the study. To achieve the evaluation of distal femoral structures, Prior the study procedure, all samples were several imaging modalities such as conventional radiography marked at the deepest level of the trochlear groove and computed tomography have been reported. On using two Kirschner wires (K-wire) before studied as a radiograph, noticeable transformation of the joint marker of each techniques (Fig. 1). To achieve the could not be promptly observed in early osteoarthritis location, skin at the lateral and medial sites of distal (Innes et al., 2004; Marino and Loughin, 2010; Alam et femur were stabbed to locate the long digital extensor al., 2011). In contrast, advanced technique such as tendon. Guided pins were then parallel drilled parallel computed tomography images of trochlear drilled at the cranial and caudal to origin of the long measurement can provided the three-sided depths of digital extensor tendon from the lateral site to the the trochlear groove; proximal aspect gauged at the medial site by using a battery power drill. The distance level of the proximal point of the fabellae, distal aspect between two pins marks was 3 mm. The drilling gauged at proximal to the intercondylar notch and the procedure was slowly done to ensuring that pins did center aspect gauged halfway between the distal and not bend and interrupt when flex and extend of stifles proximal points (Towle et al., 2005). Although,

Boonchaikitanan P. et al. / Thai J Vet Med. 2019. 49(3): 257-264. 259 during observation. K-wires (0.1 mm in diameter) were positioning was achieved, skyline stifle radiograph (X- then replaced in the guided holes. ray) was taken. The trochlear groove depth (TGD) was measured at the most depth of groove comparing to Radiography of the distal femur: All radiographs of the stifle joints were done with sternal recumbency and the adjacent condyles (Fig. 2A and 2B). TGD was done by pelvic limbs were maximal flexion (Towle et al., 2005). measuring in two directions; horizontal and vertical The primary X-ray beam was vertically centered to the lines. At first, the horizontal line was drawn from the femoral trochlear groove. Calibrate ball was placed at highest point from medial to lateral trochlea and the level of femoral trochlear for calibration of all vertical line was then measured at the most depth of radiographic parameters. As soon as the best the trochlear groove until the horizontal line and reported as TGD (Fig. 2A). Figure 1 AB The anatomical marks at the distal femur before the experiment to validate the similar location among imaging procedures. A: a lateral view of distal femur and B: the cross section view of femoral trochlea site. TGD A MCC LCC D T T < B < FGCT < ^ < C < < Figure 2 Distal femoral trochlear on different imaging procedures. A: an illustration of distal femur represented the measurement methods for trochlear groove depth (TGD), all dashed lines were presented cartilage thickness (CT) including of medial femoral condyle cartilage thickness (MCCT), trochlear groove cartilage thickness (FGCT) and lateral femoral condyle cartilage thickness (LCCT); B: a skyline stifle radiograph; C: the transverse ultrasonographic image (USG) of distal femur; D: the transverse view of distal femur observed through stereomicroscopic image (STR). Pins sites were presented with arrow heads.

260 Boonchaikitanan P. et al. / Thai J Vet Med. 2019. 49(3): 257-264. Ultrasonography of distal femur: All stifles were addition, to compare the significant difference of two examined by USG scanner (LOGIQ P6®, GE, Seoul, data set such as parameters between left and right limb Korea) using a 10 MHz, linear transducer on transverse or parameter between USG and STR, a pair t-test and view at maximum flexion of the stifle. The USG beam Wilcoxon test were used for normal distributed data was vertically centered over the trochlea. After the best and non-parametric data set respectively. Besides, the view of femoral trochlear with the evidence of double correlation between the CT and body weight was test K-wires was detected on the cine loop, USG image was using linear regression. All statistical analyses were captured, Subsequently, all parameters such as TGD significant if the P value < 0.05. and adjacent cartilage thichnesses; medial condyle cartilage thickness (MCCT), femoral groove cartilage Results thickness (FGCT) and lateral condyle cartilage thickness (LCCT) were measured using digital caliper Clinical demographic data: Twenty-two stifles (Fig. 2A and 2C). To measure the CT, only the samples were collected from eleven canine cadavers. thicknesses of hypoechoic line at the highest point of There were pomeranian (n= 4), shih-tzu (n=3), and one medial and lateral condyles, and trochlear groove of each following breed: chihuahua, Lhasa apso, above the hyperechoic bone cortex were collected (Fig. miniature Pinscher and mongrel. There were six male 2C). and five female cadavers. The mean body weight was 4.94 ± 1.49 kg (2.5 - 7.0 kg). The mean age was 11.09 ± Stereomicroscopic examination for distal femur: All 2.91 years (5 - 13 years). parameters such as femoral trochlear depth and various sites of cartilage thickesses of distal trochlear The trochlear groove depth (TGD): The TGD among were grossly observed by stereomicroscope (STR) as investigation techniques were reported as mean ± SD, the gold standard and compared all parameters to 95% confidence interval and range (Table. 1) FDG on those of the radiograph and USG images. As soon as the skyline stifle radiograph was significantly deeper the radiographic and USG examinations were done, than those on the USG (P = 0.0099) and STR (P = 0.0021) both K-wire were removed. Then, distal femurs were whereas TGD on the USG and STR were not cut using oscillating saw (Synthes®, Johnson and significantly difference (P > 0.9999). Johnson, Germany) for anatomical observation using stereomicroscope (STR). Bones was cut at the K-wire The femoral trochlear cartilage thickness (CT): CT of marking sites with the 0.5 mm of sample thickness. different areas at the femoral trochlear were collected Subsequently, bone samples were reserved in a 5% and compared only on USG and STR (Table. 2). CT at formaldehyde solution before observing of TGD, the FGCT was comparable between USG and STR (P = MCCT, FGCT, LCCT as USG technique by STR 0.0646). In contrast, the CT at lateral sites of the image, (Olympus®, Olympus Corporation, Tokyo, Japan) both of MCCT and LCCT were significant difference. within twenty-four hours (Fig. 2A and 2D). The MCCT and LCCT on the USG were significantly thicker than those of those on the STR (P = 0.006 for Statistical analysis: All clinical demographic data MCCT and P = 0.0004 for LCCT, respectively; (Fig. 4). including image parameters consisting of TGD, MCCT, FGCT and LCCT were presented as means with Relationship of femoral groove CT and body weight: standard deviations. All data were tested using Considering with body weight of canine cadavers, the GraphPad Prism software version 8.2.0 (GraphPad FGCT seemed to be correlated with the body weight, Software, California, USA). At first, the normality test however, statistical significance differences were not was done by the Shapiro-Wilk test. To compare the detected on both of USG and STR (Fig. 5). TGD among method, Friedmann test was applied. In Table 1 The femoral groove depth (FGD) among observation technique; skyline stifle radiograph (Xray), ultrasonographic image (USG) and stereomicroscope (STR) Imaging modality Mean ± SD 95% Confidence interval Range Xray 1.31 ± 0.16 0.341043 0.28 – 3.45a,b 0.83 ± 0.07 0.154087 0.29 – 1.6a USG 0.78 ± 0.11 0.237899 0.20 – 2.38b STR a; P = 0.0099 b; P = 0.0021 Table 2 The femoral trochlear cartilage thicknesses obtained by ultrasound image (USG) and stereomicroscope (STR) Imaging modality Mean ± SD 95% Confidence interval Range USG 0.45 ± 0.04 0.07381 0.28 – 0.90a STR 0.42 ± 0.04 0.07487 0.25 – 1.00a a; P = 0.06

Boonchaikitanan P. et al. / Thai J Vet Med. 2019. 49(3): 257-264. 261 Figure 3 Trochlear groove depth (TGD) on skyline stifle radiography, ultrasonographic image (USG) and stereomicroscopic image (STR). The trochlear groove depth (TGD) on radiograph was significantly deeper than those of the USG (P = 0.0099) and STR (P = 0.0021). The trochlear groove depth (TGD) on between ultrasonographic image and stereomicroscopic image were not significantly significant difference (P > 0.9999). AB C Figure 4 Femoral trochlear cartilage thicknesses (CT) at medial femoral condyle cartilage thickness (MCCT) (A), trochlear groove cartilage thickness (FGCT) (B) and lateral femoral condyle cartilage thickness (LCCT) (C) on ultrasonographic image (USG) and stereomicroscopic image (STR). There was no significantly difference between two methods (P = 0.0646). However, There were significantly difference between MCCT (Pa = 0.006) and LCCT (Pb = 0.0004). Discussion USG for stifle in small animals such as fox. However, the USG results compared to those of radiograph and Despite radiography is the most common imaging modality for musculoskeletal diagnosis in dogs gross anatomy of distal femoral trochlear without (Kramer et al., 1997), USG was an interesting modality calibrating marker may affect the accuracy of the that was applied to investigate the intra-articular results (Miles et al., 2014). structures of stifle joint due to the scanner availability and radiation less technique compared to those of In this study, canine cadaver was selected to be the other modalities (Karim et al., 2004; Soler et al., 2007). sample of the study instead of alive canine patients due USG can provide the information of femoral trochlear appearance. Besides, trochlear cartilage can be to the availability of the femoral trochlea for gross observed in human stifle (Kazam et al., 2011). In measurement without any invasion. All selected addition to human study, there was a study of using of cadavers was donated and processed for the study soon right after death or within 24 hours with preserving the cadavers at 0-4 degree Celsius. Previous

262 Boonchaikitanan P. et al. / Thai J Vet Med. 2019. 49(3): 257-264. study reported that human chondrocytes remained All parameters such as femoral trochlear depth and viable at post mortem period (Alibegović et al., 2014). various sites of cartilage thickesses of distal trochlear The viability of chondrocytes was not significantly were grossly observed by STR as the gold standard and different at 9 days at post-mortem when keeping the compared all parameters to those of the radiograph sample at below than 35 degree Celcius and USG images. In this study, histological sample of (Alibegović et al., 2014). Therefore, the period and distal femoral trochlear under the microscope did not condition of stifle in this study could not effect to the prefer for evaluation since the routine chemical quality and quantity of femoral trochlear articular fixation solution for bone and cartilage such as cartilage. aldehyde based medium can create the disruption of Before the study, all distal femurs of canine the chondrocyte surfaces and caused the condrocyte retraction and shrinkage (Hunziker, 2002). Therefore, cadavers were marked with two parallel pins to observing of CT of distal femoral trochlear on confirm the comparable location among measurement histologic samples comparing to those on USG images methods. The location to insert marking pins was or gross appearance might not be accurate. selected to be the deepest point of the femoral trochlear or at the insertion of the long digital extensor tendon. Figure 5 A correlation between body weight and femoral groove cartilage thickness (FGCT) on ultrasound image (USG) and stereomicroscopic image (STR). Besides, to reach the hyperflexion of the stifle for cartilage thickess only USG and STR. The result skyline view by the sternal recumbency observation for suggested that the cartilage thickess only at trochlear appearance and depth of trochlear would be interfered groove was comparable between methods of USG and by the volume of the tight muscle. The more STR but not the condylar cartilage. To perform USG in hyperflexion of the thinner tight muscle could be more this study, linear transducer that produced linear array achieved than that of the thicker tight muscle. and image formation showed as rectangular image Therefore, on the sternal recumbency, hyperflexion of format was used (Von Ramm and Smith, 1983). Linear stifle in different muscle volumes of the tight would transducer was focusing of the ultrasound beam that effect to the angle of stifle and location (Miles et al., can be achieved by a concave lens with only in one 2014) of distal trochlea that need to be perpendicular to direction (Aldrich, 2007) Ultrasound beam reflection the primary x-ray beam. The different location of the firstly arrived at the center of the array. A few trochlear depth in different dog would be effect. Such microseconds later, the ultrasound beam was reflected that, the radiographic assessment is inconclusive and at the outer edges. In according to the delay of the further advanced imaging techniques are necessary ultrasound reflection at the side or the border of the (Marino and Loughin, 2010). image, the less tightly focused beam of ultrasound can be generated (Powers and Kremkau, 2011). Therefore, To observed on the STR, distal femur was cut at the the difference of CT at the medial and lateral condyles same direction of the double marking pin. At first, the observed between the USG and STR could be caused trochlear groove depths among modalities of by the location of the structure on ultrasonogram. The radiograph, USG and STR were compared. The more centralized location is preferred to enhance the trochlear groove depth on radiograph showed the accuracy of the observation. In addition to the location deepest value compared to those on USG and STR. The of the structure on ultrasonogram, frequency or sounds deepest of the trochlear groove on radiograph may be speed seems to impact on the CT too. In the difference due to radiograph provided information of only the of the structure such as tissue type, species and cortical bone without the CT (Towle et al., 2005; Hansen pathology of the tissue could effect on the sound speed et al., 2017). (Myers et al., 1995). To clarify the effect of sound on the cartilage among differences of sample such as breed In addition to trochelar groove, in this study, we investigated the differences of femoral trochlear

Boonchaikitanan P. et al. / Thai J Vet Med. 2019. 49(3): 257-264. 263 and body weight including the variation of bone time and temperature of in vitro culture conditions. pathology and the evaluation of enhancing sound J Forensic Sci. 59(2): 522-528. transmission instrument (Shen et al., 2014), further Arthurs GI and SJ Langley-Hobbs 2006. Complications investigation should be done. associated with corrective surgery for patellar In addition to differences of trochlear structure luxation in 109 dogs. Vet Surg. 35(6): 559-566. among imaging modalities, the CT seemed to have a Daems R, Janssens LA and Beosier YM. 2009. Grossly correlation to the bodyweight of cadavers. However, apparent cartilage erosion of the patellar articular due to the small number of the sample size in this surface in dogs with congenital medial patellar study, statistical significance was not detected. Therefore, further study with increasing number of the luxation. Vet Comp Orthop Traumatol. 22(3): 222- population of dogs would provide more information. 224. Moreover, this study was not compare the feasibility of Hans EC, Kerwin SC, Elliott AC, Butler R, Saunders the pre-operative USG to the gross appearance of WB and Hulse DA 2016. Outcome following femoral trochlear of patella luxation patient by means surgical correction of grade 4 medial patellar of the sensitivity and specificity of USG to detect the cartilage lesion. Therefore, further study should be luxation in dogs: 47 stifles (2001–2012). J Am Anim done. Hosp Assoc. 52(3): 162-169. Hansen JS, Lindeblad K, Buelund L and Miles J 2017. In conclusion, the USG provides more precise Predicting the need for trochleoplasty in canine information of the distal femur such as trochlear patellar luxation using pre- and intra-operative groove depth and CT than that of the stifle radiograph. Although, the location and direction to evaluate the CT assessments of trochlear depth. Vet Comp Orthop of distal femoral trochlear by USG must be performed Traumatol. 30(2): 131-136. when the sound beam is only centralized to the Hayes A, Boudrieau R and Hungerford L 1994. interested anatomical structure, this technique can be Frequency and distribution of medial and lateral clinically applied in veterinary practice both of patellar luxation in dogs: 124 cases (1982-1992). J preoperative diagnosis and proper treatment planning for patella luxation and also postoperative Am Vet Med Assoc. 205(5): 716-720. osteoarthritis detection. Hunziker EB 2002. Articular cartilage repair: basic The limitation of this study was that included science and clinical progress. A review of the samples in this present study were senior dogs. In current status and prospects. Osteoarthritis addition, postmortem changes, as well as, the Cartilage. 10(6): 432-463. preparing cadaver procedure (freezing, thrawing arthrotomy) may influence on cartilage properties. 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Original Article Effect of oxidized soybean oil on the immune response to porcine reproductive and respiratory syndrome modified live virus vaccine in nursery pigs Alongkot Boonsoongnern1,4,5 Pichai Jirawattanapong1 Win Surachetpong 2 Prapassorn Boonsoongnern3* Pariwat Poolperm1* Abstract This study evaluated the effect of oxidized soybean oil on the immune response to porcine reproductive and respiratory syndrome modified live vaccine (PRRS-MLV), and the oxidative stress status in nursery pigs. Seventy castrated weaned pigs, from a free PRRS virus infected herd, were divided into two groups: treatment group (n=63) and control group (n=7). The treatment pigs were vaccinated with the PRRS-MLV and then divided into three groups based on different diet types containing different soybean oils: (A) 5% fresh oil, (B) 5% heated oil for 43 h, and (C) 5% heated oil for 38 h. The control pigs were not vaccinated and consumed (A) diet. Blood samples were collected for PRRS immune response and for measuring oxidative status. No significant differences were observed among groups in malondialdehyde (MDA), superoxide dismutase-1 (SOD-1), ELISA titer, and SN antibodies against PRRS virus. Notably, the percentage of interferon-gamma (IFN-) producing cell increased significantly in group A in comparison to that in the other groups on D28 and D56. In contrast, the percentage of interleukin-10 (IL-10) producing cell was highest in group C, followed by group B and group A, respectively. Moreover, group C exhibited a higher number of IL-10 producing cells compared to group A on D56 (p=0.082). Oxidized soybean oil exerts a negative effect on the cell- mediated immune response to PRRS-MLV, especially in pigs fed oil containing high peroxide value and high numbers of total polar compounds. These findings suggest that pig farmers should be more concerned with the quality of oil used in the diet for nursery pigs. Keywords: Immune Response, Oxidative stress, Oxidized Soybean Oil, Nursery Pigs, PRRSV 1Department of Farm Resources and Production Medicine, Faculty of Veterinary Medicine, Kasetsart University, Kamphaeng Saen campus, Nakhon Pathom 73140 Thailand 2Department of Veterinary Microbiology and Immunology, Kasetsart University, Bangkok 10900 Thailand 3Department of Anatomy, Faculty of Veterinary Medicine, Kasetsart University, Bangkok 10900 Thailand 4Center for Agricultural Biotechnology, Kasetsart University, Kamphaeng Saen campus, Nakhon Pathom 73140 Thailand 5Center of Excellence on Agricultural Biotechnology: (AG-BIO/PERDO-CHE), Bangkok 10900 Thailand *Correspondence: [email protected], [email protected] (P. Poolperm, P. Boonsoongnern) Thai J Vet Med. 2019. 49(3): 265-271.

266 Boonsoongnern A. et al. / Thai J Vet Med. 2019. 49(3) 265-271. Introduction production by the peripheral blood mononuclear cells (PBMCs) (Couper et al., 2008; Diaz et al., 2005; Suradhat Inefficiency and inconsistency in pig rearing are and Thanawongnuwech, 2003). In the case of a chronic common problems encountered in pig farms PRRSV infection, the virus has been detected in several throughout the world, including Thailand. There are organs, especially in the lymph nodes, lungs, and several factors influencing this problem, such as swine tonsils, which can be evidence of low serum diseases, especially Porcine Reproductive and neutralization (SN) antibody titers and decreased Respiratory Syndrome (PRRS), farm management, interferon-gamma (IFN-) producing PBMCs (Diaz et numbers and quality of farm laborers, the market price al., 2005; Lopez and Osorio, 2004; Suradhat et al., 2015). of pork along with the price and quality of the raw In this context, the present study aimed to determine materials; all of these factors affect production the effects of oxidized soybean oil on the immune efficiency in the pig industry. This is especially true response to PRRS modified live vaccine and to evaluate during a decline in the market price of the finished pigs the oxidative stress parameters in nursery pigs. and rising feed prices, which are the reasons for the use of lower-grade raw materials in order to reduce the Materials and Methods costs of production since the feed cost is approximately 65%–70% of the total cost for pig production. Oxidized Animals and experimental design: The procedure for oil is frequently substituted for the high-grade oil in animal use employed in the present study was pig diet. Even though it is known that the oxidized oil approved by the Institutional Animal Care and Use may have an impact on the production efficiency and Committee, Kasetsart University. The harvest was the health of the pigs, it is nonetheless used widely in conducted under the Thai Research Institution No. Thai commercial pig farms and feed mills ACKU 04260. A completely randomized design with (Boonsoongnern et al., 2017). sub-sampling was employed for the study. The experiments were performed in the Veterinary Oxidative stress is a condition of imbalance Demonstration Farm Unit, Kasetsart University, between free radical generation and antioxidant levels Kampheang Saen Campus, Nakhon Pathom province, in the body. A good oil is altered in its physical and Thailand. The nursery building, with each pen of size chemical composition when exposed to high 2.0 × 2.0 meters (wide × length), was provided for the temperatures and moisture. In addition, oxidized oils weaning pigs. Each pen had one water nipple and one contain several toxic compounds, including 1,4- mechanical feeder. All the pigs were labeled with dioxane, benzene, toluene and hexyl-benzene. Several individual identity numbers. The duration of the study studies conducted on rats and mice have indicated that was eight weeks. oxidized oil is able to induce oxidative stress and exert a negative impact on growth performances (Olivero Seventy 25-day-old castrated pigs (Landrace × David et al., 2010; Olivero et al., 2011; Varady et al., 2011; Large White; BW 8.66 ±1.42 kg) were obtained from a Yen et al., 2010). free PRRSV-infected herd. The pigs were divided into two groups: the treatment group (n = 63) and the Oxidized oil or lipid peroxidation is a complex and negative control group (n = 7). The pigs in the negative dynamic process that degrades and produces control group were housed in a different barn to numerous peroxidation compounds that can attack an separate them from the pigs in the treatment group. oxygen molecule in unsaturated fatty acids (Labuza The negative control group pigs did not receive and Dugan, 1971). The rate of oxygen uptake by a fatty vaccination with the PRRSV modified live vaccine acid correlates with the degree of unsaturation and an (Ingelvac PRRS® MLV, Boehringer Ingelheim, increasing of the carbon chain length (Naudi et al., Germany) and consumed a diet containing 5% fresh 2011). Soybean oil contains a high number of soybean oil. The treatment group pigs received unsaturated fatty acids, so it is easily induced to vaccination with the PRRSV modified live vaccine, become oxidized oil (Perkins, 1995). following which they were sub-divided into three sub- groups (n = 21 for each sub-group): (A) pigs that were PRRS virus (PRRSV) affects the reproductive and fed on a diet containing fresh soybean oil, (B) pigs fed respiratory systems, resulting in late-term abortion, on a diet containing soybean oil heated for 43 h, and mummification, stillbirth, coughing, abdominal (C) pigs fed on a diet containing soybean oil heated for breathing, cyanosis, and death. It has been reported 38 h (Table 1). There were seven pens in each sub- that the virus suppresses the immune response in group, i.e., three pigs per pen. infected pigs, as evidenced by the outcomes of significantly increased interleukin 10 (IL-10) Table 1 Experimental design Groups (pig) PV (mEq/kg of oil) TPC (%) PRRS vaccination Negative control (7) 0.88 4.50 No Yes A (21) 0.88 4.50 Yes Yes B (21) 98.00 > 40.00 C (21) 168.00 37.00 PV, peroxide value; TPC, total polar compounds; PRRS, porcine reproductive and respiratory syndrome Preparation of oxidized soybean oil and diets: The oxygen at a flow rate of 10 L/min for different total oxidized soybean oil was prepared by heating fresh time durations (38 h and 43 h). The peroxide value (PV) soybean oil to 110–120 °C, while continuously injecting and the percentage total polar compound (%TPC) of

Boonsoongnern A. et al. / Thai J Vet Med. 2019. 49(3) 265-271. 267 the prepared oils were measured following the respectively). The oils were stored at 4 °C until use. In methods described in previous studies order to prepare the diets, broken rice-soybean-based (Boonsoongnern et al., 2017). The fresh soybean oil was diet was mixed with 5% of oil, generating the final observed to contain 0.88 mEq/kg of PV and 4.5% TPC. experimental diets. Feed formulations were isocaloric The PV for the oxidized soybean oils were 168 and diets, including the phase 1 diet (Day 0–28) and phase 2 diet (Day 29–56), as presented in Table 2. 98 mEq/kg of PV, and the %TPC in the oxidized soybean oils were 37% and >40% (for 38 h and 43 h, Table 2 Experimental feed composition and determined parameters Items Day 0-28 Day 29-56 Ingredients,% - Broken rice 43.33 52.42 - Soybean 9.62 19.06 - Soy protein concentrate (Milpro®200) 9.62 4.76 - Full fat soy 14.44 14.29 - Milk replacer (Agrolac®20/40) 14.44 - - Fresh or oxidized soybean oil 5.00 5.00 - Mono-dicalcium phosphate P21 1.44 2.29 - Lame stone 0.96 0.95 - Salt 0.19 0.48 - L-lysine HCl 0.39 0.35 - DL-methionine 0.29 0.21 - L-threonine 0.19 0.17 - Vitamin and mineral premix1 0.24 0.24 Calculated analysis - ME, kcal/kg 3,746.02 3,495.24 - CP,% 21.44 20.72 - Crude fiber,% 2.33 2.81 - Crude fat,% 11.06 8.21 - Calcium,% 0.91 0.85 - Available P,% 0.40 0.45 - Total lysine,% 1.51 1.38 - Met+Cyst:Lys 60.91 60.87 - Thr:Lys 65.56 66.67 - Try:Lys 18.54 19.57 1 One kilogram of vitamin and mineral premix contains vitamin A, 4,000,000 IU; vitamin D3, 400,000 IU; vitamin E, 13,000 g; vitamin K3, 0.6 g; vitamin B1, 0.8 g; vitamin B3, 1.3 g; vitamin B6, 0.8 g; vitamin B12, 0.8 g; niacin, 9,000 g; pantothenic acid, 6,000 g; biotin, 40 g; folic acid, 0.3 g; choline, 35,000 g; Cu, 32,000 g; Fe, 36,000 g; Mn, 10,000 g; Zn, 30,000 g; Co, 0.05 g; Se, 0.06 g and I, 0.4 g Sample collection: Blood samples of the animals were SN test was performed for all the sera obtained on Day obtained by extracting blood from the external jugular 0, 28, and 56. The serum samples were subjected to heat vein of the pigs at Day 0, 14, 28, and 56. The serum and plasma samples were stored at −20 °C until to be used treatment at 56 °C for 30 min, followed by two-fold for analysis. The sera were used for evaluating the dilution in DMEM (Merck KGaA, Darmstadt, antibodies against PRRSV using enzyme-linked Germany) supplemented with 10% FBS (Merck KGaA) immunosorbent assay (ELISA) and serum neutralizing and 50 µg gentamicin (Thermo Fisher Scientific, USA). (SN) techniques. Moreover, the heparinized blood at The dilution of the serum sample was performed in a Day 0, 28, and 56 was used for evaluating the PRRS- specific IFN- and the IL-10 producing PBMCs, and for 96-well plate, and an equal volume of the virus analyzing the enzymatic antioxidant activity and lipid solution was added to each well with a titer of 102 peroxidation. TCID50/mL. The microplate was then incubated at 37 °C for 60 min. The serum-virus mixtures were Laboratory analysis transferred to a fresh 96-well plate containing a Analysis of antibody response against PRRSV using ELISA technique: All the serum samples obtained at monolayer of MARC-145 cells. The SN titer was Day 0, 14, 28, and 56 were analyzed for antibodies defined as the highest dilution of the serum that against PRRSV using the ELISA IDEXX PRRS 3X Ab neutralized the cytopathic effects (CPE) on Day 3 post- Test Kit (IDEXX Laboratories, Inc., USA). The analysis incubation. Back titration of the virus was performed protocol was performed in accordance with the each time in order to confirm the correctness of the manufacturer’s instructions. S/P ratio greater than 0.4 was considered a positive detection of the antibodies viral titration. Each sample was run in duplicate and against PRRSV. the mean of the titers was calculated as the final value (Yoon et al., 1994). Analysis of antibody response against PRRSV using Serum neutralizing technique: PRRSV was isolated Flow cytometry: PRRS-specific IFN- and IL-10- from the Thai pig farm in 2004 (accession number MK774669) and propagated in MARC-145 cells. The producing PBMCs were analyzed using the flow cytometry technique. The PBMCs at Day 0, 28, and 56 were isolated using Ficoll®-Plaque Premium density gradient media (Merck KGaA, Darmstadt, Germany), following the method described in a previous study (Wongyanin et al., 2010). In order to conduct the in vitro stimulation, 1 × 106 PBMCs were cultured in a 96-

268 Boonsoongnern A. et al. / Thai J Vet Med. 2019. 49(3) 265-271. well-plate, with 0.05 multiplicity of infection (m.o.i.) of TBARS were evaluated by measuring the OD at 450 nm the PRRSV or mock, for 48 h. At 42 h of the incubation and 530 nm, respectively. period, a protein transport inhibitor (GolgiStopTM; BD Biosciences, San Jose, CA, USA) was added to the Statistical analysis: All the results were analyzed culture for accumulating IFN- and IL-10 inside the using one-way analysis of variance (ANOVA) with cells. At the end of the incubation period, the repeated measures and compared using Tukey’s stimulated cells were transferred to a U-shape-96-well multiple comparison test, utilizing the R program plate and washed twice using 1X phosphate-buffered version 3.1.3, R Core Team (2014), R: A language and saline (PBS) [pH 7.4]. In order to perform PBMC-IFN- environment for statistical computing, R Foundation staining, the PBMCs were stained intracellularly in for Statistical Computing, Vienna, Austria (URL several steps, for the detection of cytokines present in http://www.R-project.org/). The comparison these cells. In the first step, the cells were between the groups or the data were considered to be permeabilized and fixed with 100 L of BD Fix and statistically significant when p was < 0.05. PermTM (BD Biosciences) at 4 °C for 20 mins in the dark. In the second step, the cells were washed twice with Results 250 L BD Perm/WashTM buffer (BD Biosciences) and Immune response: S/P ratios of the antibodies against stained with 1:100 of anti-IFN--Alexa 647 (BD PRRSV exhibited an increasing trend from D0 to D28, Biosciences). In the third step, after two washes, the followed by a slight decrease at D56. All pigs exhibited stained cells were resuspended in 200 L of 2% positive ELISA results (cut-off S/P ratio ≥ 0.4) at two paraformaldehyde prepared in PBS in order to fix and weeks after the PRRS vaccination. No significant preserve the cells until used for the analysis of the differences were observed in the antibody response percentage of IFN--producing cells using a flow against PRRSV among the different groups (Fig. 1A). cytometer (BD AccuriTM C6 Cytometer, BD All the pigs vaccinated with PRRS modified live Biosciences). vaccine exhibited continuously increasing S/P ratios of the antibodies against PRRSV from D0 until D28, and In order to perform the PBMC-IL-10 staining, the a slight decrease in the ratio at D56. According to the results of the SN titer, continuously increasing titers cells were stained with 0.5 g/mL of biotinylated anti- were observed until D56 post-vaccination, although IL-10 (Invitrogen, USA) at 4 °C for 30 min in the dark, there were no significant differences between the followed by washing of the cells twice with 250 L BD groups. However, the SN titer exhibited the lowest Perm/WashTM buffer. Subsequently, the cells were value at D28 and D56 in group C (Fig. 1B). The SN secondarily stained with 1:100 Streptavidin-PE (BD antibody against PRRSV demonstrated results similar Biosciences) at 4 °C for 30 min in the dark, followed by to those obtained in ELISA. Although the pigs vaccinated with the PRRS vaccine were fed on diets two washes with 250 L BD Perm/WashTM buffer. containing the highest PV (group C), they exhibited Lastly, the stained cells were resuspended in 200 L of low levels of SN titer against PRRSV compared to the 2% paraformaldehyde prepared in PBS in order to fix other groups (groups A and B). and preserve the cells until they were used for the analysis of the IL-10-producing cells using the flow The IFN- was produced by the cells at D28 post- cytometer. vaccination, and its levels were increased sharply from D0 to D56, especially in group A. Notably, the pigs The background cut-off and flow cytometer belonging to group A exhibited a significantly higher validation were conducted by performing IgG1-PE number of IFN--producing cells at D28 and D56, isotype control staining using six and eight peak bead compared to those in group B and C (Fig. 1C). On the runs (BD Biosciences), respectively. Acquisition of other hand, the highest numbers of IL-10-producing 30,000 events and analyses were performed using the cells at D28 and 56 were obtained for group C, followed Accuri C6 flow cytometer (BD Bioscience). The data by B and A, respectively. As expected, the percentage provided the mean percentage (±standard deviation; of the IL-10-producing cells at D56 obtained for group SD) of the cytokine-producing cells, which were C was higher (p = 0.082) than that obtained for group calculated from the following expression: [(percentage A (Fig. 1D). The pigs fed on the oxidized oils of cytokine-producing cells obtained from the PRRSV) containing the highest level of PV exhibited a high – (cultured PBMC − percentage of cytokine-producing percentage of IL-10-producing cells, especially at D56, cells obtained from the mock-cultured PBMC)]. compared to the other groups. Oxidative stress parameters: Cu/Zn-superoxide Oxidant and antioxidant: The results of the present dismutase (SOD-1) and thiobarbituric acid reactive study demonstrated that the levels of MDA and SOD- substances (TBARS) are commonly used as markers of 1 exhibited an increase from D0 to D56 in all the oxidative stress. TBARS, which are among the most groups, although no significant differences were crucial chemical substances of lipid peroxidation, were observed between the groups. However, the quantified for measuring the malondialdehyde (MDA) concentration of plasma MDA at D56 was observed to levels. The plasma samples (Day 0, 28, and 56) were be the highest in group C, followed by group B and analyzed for the levels of enzymatic antioxidant group A, respectively (Table 3) activity using the ELISA assay kits (Cayman Chemical Company, USA). The end product of SOD-1 and

Boonsoongnern A. et al. / Thai J Vet Med. 2019. 49(3) 265-271. 269 Figure 1 The PRRS immune response of nursery pigs fed on the different level of oxidized soybean oils. (A) ELISA response, (B) serum neutralizing antibody against PRRSV, (C) PRRS-specific IFN- produced by the PBMCs, (C) PRRS-specific IL-10 produced by the PBMCs. Asterisks and different letters indicate significant differences among the groups at the same time point (mean ±SD). Statistical analysis was performed using the one-way ANOVA with repeated measures and Tukey’s correction for multiple comparisons, * p < 0.05. Table 3 The levels of lipid peroxidant and antioxidant in the blood plasma of nursery pigs Group (n) MDA (mM/L) SOD-1 activity (U/L) D 0 D 28 D 0 D 28 D 56 D 56 A (14) 2.46 ±0.17c 8.03 ±0.95b 36.74 ±7.18a 1.43 ±1.0c 15 ±97 ±3.78b 84.94 ±5.92a B (13) 2.23 ±0.15c 8.57 ±1.52b 58 ±93 ±9.92a 1.40 ±1.1c 13.95 ±2.81b 87.67 ±10.63a C (14) 2.07 ±0.14c 7.15 ±0.86b 68.99 ±10.23a 7.42 ±3.00c 20.83 ±4.07b 85.87 ±7.30a ns ns Significant ns ns ns ns Results are presented as mean ±SD MDA, malondialdehyde; SOD, superoxide dismutase ns = the mean values do not differ significantly (p > 0.05) a, b, c mean values in a row with different superscript letters were significantly different (p < 0.05) Discussion the virus was eliminated using the cellular immunity This study showed similar results to the previous provided by the PRRSV-specific IFN--producing cells reports that the ELISA antibody response against (Charerntantanakul et al., 2006; Loving et al., 2015; PRRSV could be detected beginning from the period Martelli et al., 2013). It is noteworthy that the oxidized between Day 9 and 12 after the PRRSV infection or oils exerted a negative impact on immunomodulatory vaccination with the attenuated PRRSV live vaccines activity (Brody, 2016). (Lopez and Osorio, 2004). The study finding was in agreement with those in previous studies that reported IL-10 is a well-known anti-inflammatory cytokine gradual and low-level responses of SN titer after that inhibits the synthesis of pro-inflammatory vaccination with modified live PRRS vaccine (Lopez cytokines. IL-10 is known to impede the activity of the and Osorio, 2004; Osorio et al., 1998). Th1 cells, NK cells, and macrophages, for the optimal clearance of pathogen, which may lead to tissue The number of IFN--producing cells correlated damage (Couper et al., 2008). In addition, IL-10 may act with the PRRSV protective immunity through which directly on the CD4+ T cells in inhibiting the proliferation and production of IL-2, IFN-, IL-4, IL-5,

270 Boonsoongnern A. et al. / Thai J Vet Med. 2019. 49(3) 265-271. and TNF- (Couper et al., 2008; Joss et al., 2000; Moore Commission, Ministry of Education. (AG- et al., 2001; Schandené et al., 1994). Moreover, Suradhat BIO/PERDO-CHE). We also thank the Thai Vegetable and Thanawongnuwech (2003) observed that the Oil Public Company Limited (TVO), Nakhon Pathom genotypes of both the PRRSV strains, namely the North province, Thailand for soybean oil provision and oil American (NA) and European (EU) strains, quality analysis. Moreover, we thank the Faculty of significantly induced IL-10 production through Veterinary Medicine, Kasetsart University, PBMCs. The findings of the present study, therefore, Kamphaeng Saen campus, Nakhon Pathom province, indicate that the oxidized oils impaired the immune Thailand for supporting facilities. responses. The results of the present study are in agreement with the findings of previous studies References conducted on poultry and swine (Calder, 2008; Dibner et al., 1996; Leff, 2001; Liang et al., 2015a). Importantly, Boonsoongnern A, Boonsoongnern P, Jirawattanapong MDA is produced during the lipid peroxidation P, Rattanavanichroj N and Poolperm P 2017. A process. It is more stable and changes many cell survey of quality of oils used in Thai pig farms. membranes especially immune cells (Van der Paal et Thai J Vet Med. 47: 475-480. al., 2015). Low levels of MDA and MDA adducts can be immune complement inducers. In the opposite way, Brody T 2016. Chapter 29 - Mechanisms of action-part high levels of them showed an adverse effect on IV (Infections), In: Clinical Trial 2nd ed. From T. immune cells (Willis et al., 2004). Brody ed. Academic Press, Boston, 635-662. Oxidative stress is induced by the common Calder PC 2008. The relationship between the fatty acid stressors in nursery pigs, such as housing composition of immune cells and their function. management, dietary factors and disease susceptibility Prostaglandins Leukot Essent Fatty Acids. 79: 101- on pig farms (Forbes, 2007; Jones et al., 2001; Melin et 108. al., 2004; Moeser et al., 2007). Oxidative stress may be evaluated through multiple markers associated with Charerntantanakul W, Platt R, Johnson W, Roof M, the overproduction of the reactive oxygen species Vaughn E and Roth JA 2006. Immune responses (ROS). Lipid peroxidation is commonly used as an and protection by vaccine and various vaccine indicator of the ROS-mediated damage to cell adjuvant candidates to virulent porcine membranes. MDA is useful as lipid oxidative stress reproductive and respiratory syndrome virus. Vet marker, while SOD-1 represents an antioxidant Immunol Immunopathol. 109: 99-115. enzyme. The MDA and SOD-1 levels exhibited a sharp increase from the beginning to the end of the study. Couper KN, Blount DG and Riley EM 2008. IL-10: the Meanwhile, the levels of lipid peroxidants and master regulator of immunity to infection. J antioxidants did not differ between the sub-groups Immunol. 180: 5771-5777. when measured on the same day (D0, D28, and D56). Rosero and colleagues (Rosero et al., 2015) also Diaz I, Darwich L, Pappaterra G, Pujols J and Mateu E reported that nursery pigs fed on oxidized soybean oil 2005. Immune responses of pigs after experimental exhibited high levels of MDA concentration in jejunal infection with a European strain of porcine mucosa. Some researchers found that oxidized reproductive and respiratory syndrome virus. 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Original Article The use of B-scan coupling with A-scan ultrasonography to characterize ocular biometry in canine absolute glaucoma Napasorn Ngamrojanavanit Natthaporn Tanthasathien Nirachon Srisamai Suchaya Taotongnuntasin Panrawee Phoomvuthisarn Nalinee Tuntivanich* Abstract This study was to investigate intraocular structures of canine absolute glaucomatous eyes using real time B-scan ultrasonography in accordance with the amplitude mode. Ten normal eyes and twenty absolute glaucomatous eyes were included in this study. Ocular biometry via closed-eye technique was performed using the Ultrascan® Imaging System (Alcon Surgical Laboratory, Fort Worth, USA), with a 10 MHz mechanical sector probe. Changes of intraocular structures were descriptively analyzed. Ocular biometry was measured, statistically analyzed and compared between both groups. While mean lens thickness was comparable between normotensive and glaucomatous eyes, mean lens equatorial length was higher in the latter group. Increased hyperechogenicity of lens was ultrasonographically observed in dogs with absolute glaucoma. Axial globe length was significantly greater in the experimental group than the control group (P<0.05). In relation to the appearance of the cone-shaped posterior wall, positive correlation between intraocular pressure and axial globe length with statistical significance was noted (r=0.5, P<0.05). Retinal detachment and lens displacement were also observed in absolute glaucomatous eyes. Not only does ultrasonography, with the 10 MHz frequency transducer, provides ocular biometry to specifically investigate changes of lens and the posterior segment of the canine absolute glaucomatous eyes, it can lead to further selection of appropriate treatment and awareness of post-treatment complications. Keywords: dogs, glaucoma, ocular biometry, ultrasonography Department of Veterinary Surgery, Faculty of Veterinary Science, Chulalongkorn University, 39 Henry Dunant Road, Phayathai, Pathumwan, Bangkok, 10330 Thailand *Correspondence: [email protected] (N. Tuntivanich) Thai J Vet Med. 2019. 49(3): 273-281.

274 Ngamrojanavanit N. et al. / Thai J Vet Med. 2019. 49(3): 273-281. Introduction mode or A-scan) and brightness mode (B-mode or B- scan) are applicable in animals. A-scan Glaucoma is one of the most important ocular ultrasonography offers a one-dimensional display of diseases that lead to permanent loss of vision. A echoes in a spike pattern. Time delay is required for prevalence of dogs suffering from primary breed sound waves to reach given ocular tissue interfaces. related glaucoma was reported by Gelatt in 2004 and The height of spikes describes strength of echoes. The Storm in 2009. Even though the prevalence of canine indication of an A-scan ultrasonogram is therefore to glaucoma has not been scientifically reported in measure distance between ocular interfaces. B-scan Thailand, there has been an increase in the number of ultrasonography represents two-dimensional image canine glaucoma at the Ophthalmology Unit, Animal receiving from an oscillating wave that passes through Teaching Hospital, Faculty of Veterinary Science, a cross-section of tissues. The great advantage of B- Chulalongkorn University, Thailand (personal mode ultrasonography is to observe the intraocular communication). Glaucoma occurs as a result of contents of the globe, obscured by ocular media aqueous humor accumulation; leading to an increase in opacity. Intraocular neoplasm, intraocular intraocular pressure (IOP). Disease progression inflammation, retinal detachment and vitreous consists of 3 stages (Gelatt, 2004). The first stage is the hemorrhage are good suggestion for ultrasonographic early stage with IOPs of 20-30 mmHg. There may be no investigation in cases with opaque ocular media symptomatic signs or mild changes of pupil, retina and (Gallhoefer et al., 2013). optic disc. If proper treatment has not received, vision will rapidly be threatened. Second is the mild stage According to the fact that the ocular media of dogs with IOPs of 30-40 mmHg. This stage is usually with absolute glaucoma is obscured from examination, noticeable by suffers from several clinical signs such as ultrasonography is therefore considered an applicable episcleral congestion, variable degrees of corneal diagnostic tool to evaluate anatomical changes within edema and slight buphthalmos. When IOPs is 40-50 glaucomatous eyes (Aldavood et al., 2012). Our mmHg, the disease has progressed to the advanced objective was to characterize ocular biometry in canine stage. Persistent changes associated include corneal absolute glaucoma with the use of B-scan coupling edema, mydriasis, optic disc cupping and retinal with A-scan ultrasonography. degeneration. Materials and Methods Ophthalmic signs of glaucoma may be defined as acute and chronic stages, majorly categorized by the Samples: A total of thirty eye balls from thirty duration and progression of the disease. Rapid small-breed dogs weighing not more than 10 kilograms elevation of IOP is usually observed in the acute stage, were included into the study. These dogs were which is accompanied by severe pain. The cornea has presented at the Ophthalmology Unit, Small Animal generalized edema associated with deep Teaching Hospital, Faculty of Veterinary Science, vascularization. The pupil is generally constricted. Chulalongkorn University. All procedures were Episcleral congestion is evident. Vision can rapidly be performed under informed consent provided by the lost within a few hours after IOP elevation. On the owners of all dogs. All procedures had been approved other hand, dogs presented with chronic signs of by the Chulalongkorn University Animal Care and Use glaucoma can tolerate ocular pain quite well. However, Committee with approved Animal Use Protocol changes of the anatomical structure of the eye in number 1731024, and care was taken to comply with chronic glaucoma are also noteworthy. They include the 3R concept. buphthalmos, permanent corneal opacity, lens opacity, intraocular hemorrhage, optic disc cupping and Eyes were divided into 2 groups; controls and atrophy, generalized retinal degeneration and phthisis experiments. There were 10 randomized control eyes bulbi (Renwick, 2002). The atrocious outcome of this (from 10 dogs) with IOP ranging from 5-20 mmHg in a whole developing process of glaucoma is an control group. Ophthalmic examinations revealed no irreversible blindness due to optic neuropathy (Miller intraocular inflammation, corneal disorders of and Bently, 2015). Absolute glaucoma will soon occur systemic diseases that affect intraocular pressure. when the disease has progressed toward the advanced There were twenty randomized eyes in the stage. The presence of buphthalmic eyeball will be in experimental group. They were from twenty dogs accordance with lagophthalmos, keratoconjunctivitis diagnosed with advanced/absolute glaucoma. They sicca, exposure keratopathy and equatorial had a history of IOP more than 35 mmHg. The disease staphyloma. had been progressing for not less than 3 months duration. All dogs were on ocular hypotensive Various diagnostic tools for human glaucoma medications. detection are recommended (Sharma et al., 2008). Though ocular ultrasonography is not one of those, it Procedures: All procedures had been approved by the is widely used to interpret changes within the eyeballs Chulalongkorn University Animal Care and Use (Dudea, 2011) when direct visualization is inaccessible. Committee with approved Animal Use Protocol It is a technique using high-frequency sound waves number 1731024, and care was taken to comply with travelling through tissue interfaces generating echoes. the 3R concept. Each image describes various acoustic characteristic of each tissue. In ophthalmic ultrasonography, a high- To categorize dogs into control and experimental frequency transducer within the range of 8-50 MHz is groups, they underwent general ophthalmic typically used (Millier and Bentley, 2015). Among examinations. Examinations included (1) Schirmer tear several types of ultrasonography, amplitude mode (A- test I, (2) neuro ophthalmic reflexes: menace response, dazzle reflex and pupillary light response, (3)

Ngamrojanavanit N. et al. / Thai J Vet Med. 2019. 49(3): 273-281. 275 fluorescein staining test (4) rebound tonometry; a accordance with the amplitude spike. measurement of intraocular pressure using the 2.1 Anterior chamber depth (ACD): ACD was Tonovet® (Tiolat Oy, Helsinki, Finland), (5) dark/light room direct eye examination, and (6) assessment of the measured from the corneal epithelium to the anterior anterior chamber using a slit lamp biomicroscope lens capsule. (Kowa SL-15; Kowa company, Ltd, Japan). 2.2 Lens thickness (LT): LT was measured Ocular ultrasonography was performed through from the axial point of the anterior to the posterior lens the eyelid in all awakened dogs by an experienced capsule. veterinarian. Interobserver variability was then minimized. Intra-observer reliability was still 2.3 lens equatorial length (LEL): LEL was maintained by using an automated optical axis measured from equator to equator of the lens. together with visual axis as an indicator of all measurements. Ultrasonographic images were 2.4 Vitreous chamber depth (VCD): VCD was obtained from a B-mode ultrasonogram coupling with measured from the axial point of the posterior lens amplitude spikes (single A&B mode), with the use of capsule to the fundus. the Ultrascan® Imaging System (Alcon Surgical Laboratory, Fort Worth, USA). The transducer probe 2.5 Axial globe length (AXL): AXL was was 10 MHz mechanical sector. A topical 0.5% measured from the corneal epithelium to the fundus. tetracaine hydrochloride was applied to the cornea at a Means (± SD) of each measurement from both groups dose of 2 drops within 3-minute interval times. All were calculated. Comparisons of means between dogs were gently handled with minimal restraint and control and experimental groups, using independent t- the transducer probe with acoustic coupling gel was test (SPSS program version 22.0, IBM Corporation, NY, perpendicular to the center of the cornea. The probe USA) at a significance level of p<0.05. Pearson’s position marker was always placed in the rostral to correlation test (SPSS program version 22.0, IBM caudal direction. Once parallel plane to visual axis was Corporation, NY, USA) was applied to evaluate the achieved, an oblique plan was performed to observe correlation between intraocular pressure and five intraocular architectures. Immediately after the parameters. Statistical significance was considered procedure was finished, the acoustic coupling gel was when p<0.05. flushed off to ensure the safety of the ocular surface. Ultrasonographic images were recorded for further Results analysis. Clinical demographic data of all patients is in Table Data collection and data analysis: There were two 1. Experimental eyes had IOP ranging from 31 to 63 major parts of data interpretation. mmHg. 1. Structural abnormalities detected by B- In the control group (Fig 1), the cornea was mode ultrasonograms were categorized and identified as a curved hyperechoic interface descriptively analyzed. immediately parallel to the eyelid. The anterior chamber was indicated as anechoic cavity between two 2. Measurement of length/depth of hyperechoic structures; corneal endothelium and intraocular structures from ultrasonographic images: anterior lens capsule. The lens appeared as an elliptical A measurements were performed by Adobe anechoic structure with hyperechoic anterior and Photoshop CC 2017 (©Adobe System, Inc., San Jose, posterior lens capsule. The vitreous chamber was in a USA). Each location from B-mode image was in spherical shape. It was occupied with anechoic materials. The posterior wall was revealed as a hyperechoic curvilinear structure at the back of the eyeball. Figure 1 Representative ultrasonographic image of a normal eye, along with measurements; anterior chamber depth (ACD), lens thickness (LT), lens equatorial length (LEL), vitreous chamber depth (VCD), and axial globe length (AXL).

276 Ngamrojanavanit N. et al. / Thai J Vet Med. 2019. 49(3): 273-281. Various ocular abnormalities were identified in five out of twenty eyes (25%). Most of the echoic lens glaucomatous eyes (Table 2). Partial hyperechoic were luxated to the ventral/posterior of the eyeballs homogenous material was evident in the anterior (Fig 2C). Mild to moderate intensities of echoic chamber (8/20; 40%) (Fig 2A). Fine dispersed opacity homogeneous materials were observed in the vitreous was ultrasonographically noticed in the anterior chamber of the glaucomatous eyes (5/20; 20%) (Fig 2D). chamber of two out of ten control eyes (20%). Ten Retinal detachment appeared as the v-y structure of a experimental eyes were hyperechoic (50%) (Fig 2B). linear echoic membrane projecting in front of the Increased echogenicity was noticed in lens capsules posterior wall. Of 20 eyes, three (15%) retinal which had apparent hyperechoic lens materials. In detachments were detected (Fig 2E). Obvious cone- controls, lens materials were hyperechoic in three out shaped posterior wall was identified in eight out of 20 of ten (30%) eyes. Displacement of lens was evident in (40%) in glaucomatous eyes (Fig 2F). Table 1 Clinical demographic data of all patients involved in this study. Dog Gender Age Breed Side of eye Averaged IOP (years) (mmHg) Chihuahua OS 1 Female 3 Mixed OS 13 Mixed OD 11 2 Male 10 OS 11 Shih Tzu OS 8 3 Female 8 Mixed OD 10 OS 4 Male 10 Shih Tzu OD 8 Mixed OD 12 5 Male 9 Mixed OS 9 OS 9 6 Male 10 Chihuahua OS 9 Shih Tzu OD 32 7 Female 8 OD 33 Mixed OS 44 8 Female 2 Mixed OS 31 Mixed OD 63 9 Female 8 Poodle OD 60 Shih Tzu OS 10 Female 13 Mixed OS 37 Shih Tzu OS 49 11 Female 7 Poodle OS 45 Shih Tzu 41 12 Male 10 Shih Tzu 40 Mixed 60 13 Male 11 Shih Tzu 14 Male 10 15 Female 11 16 Female 2 17 Female 13 18 Female 12 19 Female 13 20 Female 10 21 Male 13 22 Female 9 *Dog 1-10 = control group, Dog 11-30 = experimental group Table 2 Ocular abnormalities identified by ultrasonography. Ocular abnormality Number of eyes Hypoechogenic homogenous materials filling in the anterior chamber (total of 20) Hyperechoic lens materials 8 Displaced echoic lens in the vitreous chamber 10 5 Multiple point-liked echoic foci in the vitreous chamber 5 Hyperechoic membrane pointing from the posterior wall into vitreous chamber 3 8 Hyperechoic cone-shaped posterior wall of the eye Comparison of the two chambers between the Mean axial globe length in glaucomatous eyes was experimental group and controls revealed different significantly longer (20.36±3.01 mm), compared to that mean depth (Fig 3). Both mean anterior and vitreous of control eyes (16.49±0.82 mm) (Fig 5). It had a chamber depths of glaucomatous eyes were higher moderate positive correlation to IOP with statistical than those of the controls. Statistically, significant difference was noted in the vitreous chamber depth. significance (R=0.68; p0.05) (Fig 6). There was no There was a low degree of positive correlation between vitreous chamber depth and IOP without statistical statistical correlation between IOP and anterior significance (r=0.461; p>0.05). Slight changes of lens chamber depth (r=-0.135; p>0.05) as well as lens equatorial length (r=0.049; p>0.05). thickness and lens equatorial length is shown in figure 4. As compared to the controls, the mean lens equatorial length of the glaucoma group was longer, whereas the mean lens thickness was shorter (6.26±0.57 mm). A low degree of positive correlation was noted between lens thickness and IOP without statistical significance (r=0.308; p>0.05).

Ngamrojanavanit N. et al. / Thai J Vet Med. 2019. 49(3): 273-281. 277 Figure 2 Ultrasonographic images from absolute glaucomatous eyes demonstrated (2A) hypoechoic homogenous material filling the anterior chamber, (2B) hyperechoic lens materials, (2C) displaced spherical echoic lens into vitreous chamber, (2D) multiple point-liked echoic foci in the vitreous chamber, (2E) hyperechoic membrane pointing from the posterior wall into the vitreous chamber and (2F) hyperechoic cone-shaped posterior wall. Figure 3 Mean (±SD) of the anterior chamber depth (ACD) and vitreous chamber depth (VCD) of the control and experimental groups. Star (*) indicates statistical significance at a level of p < 0.05.

278 Ngamrojanavanit N. et al. / Thai J Vet Med. 2019. 49(3): 273-281. Figure 4 Mean (±SD) of the lens equatorial length (LEL) and lens thickness (LT) of the control and experimental groups. Figure 5 Mean (±SD) of the axial globe length of the control and experimental group. Star (*) indicates statistical significance at a level of p < 0.05.

Ngamrojanavanit N. et al. / Thai J Vet Med. 2019. 49(3): 273-281. 279 Figure 6 Correlation between intraocular pressure (IOP) and axial globe length (AXL) of the control and experimental groups. Star (*) indicates statistical significance at a level of p < 0.05. Discussion glaucoma. It is presented as globe elongation that could lead to lens displacement. A significant increase B-mode ultrasonography provides good of mean vitreous chamber depth (He et al., 2012) and investigation through opaque ocular media in absolute mean axial globe length is related to axial globe glaucomatous eyes. Good animal restraint is required elongation in glaucomatous eyes (Cho, 2008). Positive to perform ocular ultrasonography. Rotation of correlation between mean axial globe length and IOP eyeballs away from the visual axis may result in in glaucoma was not only found in our study but also incorrect assessment (Squarzoni et al.,2010); reported in normotensive marmoset (Nickla et al., particularly length or depth measurements. In our 2002) and chicken (Schmid et al., 2003). Their mean study, we attempted to collect only ultrasonograms axial globe length increases at night when diurnal IOP where the optical axis is perpendicular among the is the highest. With time of glaucoma progression to cornea, lens surface and the posterior wall. Amplitude advanced stage, cupped disc due to loss of myelin and of spikes coupling with B scan was used (Cottrill et al., prominent lamina cribrosa atrophy may be part of the 1989; Hamidzada, 1999) as guidance for all reason for the longer depth of the mean vitreous measurements of ocular biometry. To ensure proper chamber and axial globe length. Appearance of data interpretation, one investigator was assigned to scattered hyperechoic materials in the vitreous measure ultrasonographic images presented via chamber, likely indicates vitreous degeneration or commercialized imaging software program. inflammatory cell dispersion, rather than vitreous hemorrhage. A cone-shaped posterior wall is a substantial consequence of long-term pressure in chronic

280 Ngamrojanavanit N. et al. / Thai J Vet Med. 2019. 49(3): 273-281. Loss of lens transparency, due to the increase of Chulalongkorn University. We would like to lens protein insolubility, ultrasonographically reflects acknowledge the staff of the Ophthalmology Unit, hyperechoic materials within the lens cortex and Small Animal Teaching Hospital, Faculty of Veterinary nucleus, clinically described as cataract formation Science, Chulalongkorn University for their assistance (Michael and Bron, 2011). Though aging can cause and support. degenerative changes of lens fiber observed in some of our control dogs mostly at middle age, prominent References interlenticular density was found in the glaucomatous group a the median age of 10 years. At the stage of Aldavood SJ, Zahedi R, Sohrabi Haghdoost I, Rezaie liquefactive lens degeneration, while there was a Kanooy M and Veshkini A 2012. Ultrasonographic slightly lower mean lens thickness due to lens findings of experimental glaucoma in rabbits. resorption in advanced glaucoma long-term extension Comp Clin Path. 22(4): 585-589. of zonular fibers from globe enlargement caused longer mean lens equatorial length. Our finding is Bentley E, Miller PE and Diehl KA 2003. Use of high- contrary to a study in patients with occludable open- resolution ultrasound as a diagnostic tool in angled glaucoma (George et al., 2003) where lens veterinary ophthalmology. J Am Vet Med Asso. thickness increased. Their patients were in middle age, 223(11): 1617-1622. when the water imbibition mechanism is supposely active during an early stage of cataract formation. Cho HJ, Woo JM and Yang KJ 2002. Ultrasound biomicroscopic dimensions of the anterior chamber Regardless of a clear anterior chamber in angle-closure glaucoma patients. Korean J ophthalmoscopically examined in controls, our study Ophthalmol. 16: 20-25. reveals hypoechoic appearance within the anterior chamber in both groups. We speculate that this artifact Cho YK 2008. Early intraocular pressure and anterior is because of insufficient resolution (Guhu et al., 2006) chamber depth changes after phacoemulsification due to low ultrasonic frequency of 10 MHz. In fact, and intraocular lens implantation in ultrasound biometry is known for better interpretation nonglaucomatous eyes. Comparison of groups of the posterior segment of the eye. With the use of 10- stratified by axial length. J Cataract Refract Surg. 15 MHz to assess anterior ocular segment, enough 34(7): 1104-1109. coupling gel (MacKay and Mattoon, 2015) or a standoff (Gonzalez et al., 2001) is recommended via a closed-eye Cottrill NB, Banks WJ, Pechman RD 1989. technique to avoid ultrasonographic artifacts. Ultrasonographic and bio- metric evaluation of the Nevertheless, the presence of hypoechoic material eye and orbit of dogs. Am J Vet Res. 50(6): 898–903. within the anterior chamber of glaucomatous eyes may indicate real pathological incidence related to the Dudea SM 2011. Ultrasonography of the eye and orbit. replacement of hyaluronic acid by fibronectin and Medical Ultrasonography. 13(2): 171-174. thrombospondin in glaucoma patients (Gabelt and Kaufman, 2005). In terms of depth, it is likely to find a Gabelt BT and Kaufman PL 2005. Changes in aqueous decrease of the anterior chamber depth, as a result of humor dynamics with age and glaucoma. Prog anterior lens displacement (Cho et al., 2002). The Retin Eye Res. 24(5): 612-637. increase in corneal thickness (Muir et al., 2004) as well as positive correlation between IOP and central corneal Gallhoefer NS, Bentley E, Ruetten M, Grest P, Haessig thickness in dogs (Park et al., 2011) may cause M, Kircher PR, Dubielzig RR, Spiess BN and Pot SA insignificant comparison of the anterior chamber depth 2013. Comparison of ultrasonography and in our study. For better interpretation of the anterior histologic examination for identification of ocular segment, ultrasound biomicroscopy providing high diseases of animals: 113 cases (2000-2010). J Am Vet frequency of 35-50 MHz is recommended (Bently et al., Med Assoc. 243(3): 376-388. 2003). Gelatt KN and MacKay EO 2004. Prevalence of the In conclusion, ocular biometry of canine absolute breed related glaucoma in pure-bred dogs in North glaucoma could be characterized with the use of B-scan America. Vet Ophthalmol. 7(2): 97-111. coupling with A-scan ultrasonography. With additional apparent peaks of A scan, determination of George R, Paul PG, Baskaran M, Ramesh SV, Raju P, ocular organs could be more precise and accurate. Arvind H, McCarty C and Vijaya L 2003. Ocular Several pathological changes were apparently biometry in occludable angles and angle closure associated with chronic increased intraocular pressure. glaucoma: a population-based survey. Br J Assessment of intraocular structures could therefore be Ophthalmol. 87: 399-402. beneficial for further surgical therapy, as well as long- term treatment of the disease complications. Gonzalez EM, Rodriguez A and Garcia I 2001. Review of ocular ultrasonography. Vet Radiol Ultrasoun. Conflict of interest: The authors declare no conflict of 42(6): 485-495. interest in the study. Guhu S, Bhende M, Baskaran M and Sharma T 2006. Acknowledgements Role of ultrasound biomicroscopy in the detection and localisation of anterior segment foreign body. The study was funded by the senior project grant Ann Acad Med Singafore. 35: 536-540. for veterinary students, Faculty of Veterinary Science, Hamidzada WA 1999. Agreeement between A-mode and B-mode ultralsonography in the measurement of ocular distances. Radiology and Ultrasound. 40(4): 502-507. He L, Wendt M and Glasser A 2012. Manipulation of intraocular pressure for studying the effects on accommodation. Exp Eye Res. 102 : 76-84. MacKay CS and Mattoon JS 2015. Eye. In: Small Animal Diagnostic Ultrasound. 3 ed. John S Mattoon and

Ngamrojanavanit N. et al. / Thai J Vet Med. 2019. 49(3): 273-281. 281 Thomas G Nyland (eds). Missouri: Elsevier Saunders. 128-154. Michael R and Bron AJ 2011. The aging lens and cataract: a model of normal and pathological ageing. Philos Trans R Soc Lond B Biol Sci. 366(1568) : 1278-1292. Miller PE and Bentley E 2015. Clinical Signs and Diagnosis of the Canine Primary Glaucomas. Vet Clin North Am Small Anim Pract. 45(6): 1183-1212. Muir KW, Jin J and Freedman SF 2004. Central corneal thickness and its relationship to intraocular pressure in children. Ophthalmology. 111(12): 2220-2223. Nickla DL, Wildsoet CF and Troilo D 2002. Diurnal Rhythms in Intraocular Pressure, Axial Length, and Choroidal Thickness in a Primate Model of Eye Growth, the Common Marmoset. Invest Opthalmol Vis Sci. 48(8): 2519-2528. Park Y-W, Jeoung M-B, Kim T-H, Ahn J-S, Ahn J-T, Park S-A, Kim S-E and Seo K 2011. Effect of central corneal thickness on intraocular pressure with therebound tonometer and the applanation tonometer in normal dogs. Vet Ophthalmol. 14(3): 169-173. Renwick P 2002. Glaucoma. In: BSAVA Manual of Small Animal Ophthalmology. 2nd ed. Gloucester: BSAVA 185-203. Schmid KL, Hills T, Abbott M, Humphries A, Pyne K and Wildsoet CF 2003. Relationship between intraocular pressure and eye growth in chick. Ophthal Physiol Opt. 23: 25-33. Sharma P, Sample PA, Zangwill LM, and Schuman JS 2008. Diagnostic Tools for Glaucoma Detection and Management. Surv Ophthalmol. 53 (SUPPL1): 17- 32. Squarzoni R, Perlmann E, Antunes A, Milanelo L and Barros PSdM 2010. Ultrasonographic aspect and biometry of Stripped owl's eyes (Rhinoptynx clamator). Vet Ophthalmol. 13(1): 86-90.



Original Article The expression of serum lactate dehydrogenase in canine oral tumors Nan Choisunirachon1* Urapa Klansnoh1 Panrawee Phoomvuthisarn1 Sirinun Pisamai1 Chutimon Thanaboonnipat1 Anudep Rungsipipat2 Abstract Lactate dehydrogenase (LDH) has been reported to be a prognostic indicator of human malignant melanoma (MM), lymphoma, renal cell carcinoma and prostate cancer. In veterinary medicine, elevation of serum LDH has been reported in canine lymphoma and mammary gland tumors. However, the expression of the LDH in oral tumor-bearing dogs has not been elucidated. In this study, serum LDH levels were evaluated and compared between healthy dogs (control; n = 20) and oral tumor-bearing dogs (n = 34). Half of the tumor-bearing dogs (17dogs) had oral MM, followed by 6 dogs with oral squamous cell carcinoma (SCC), 4 dogs with fibrosarcoma (FS), 3 dogs with benign epithelial tumors (BET), and 1 dog each with osteosarcoma, lymphoma, histiocytoma and acanthomatous ameloblastoma. Serum LDH was significantly higher in oral tumor-bearing dogs (P = 0.0006). Besides, serum LDH expression of MM, SCC and FS was significantly higher than that of the control group (P = 0.0078, 0.0030 and 0.0022, respectively). The expression of serum LDH did not significantly correlate with the location of the primary tumor or the clinical stage of the disease, the clinical stage II revealed the highest expression of the LDH though. The results indicate that serum LDH might be useful as an indicator for oral tumors but further study examining a larger number of patients with each tumor type would be advantageous. Keywords: dog, lactate dehydrogenase, oral tumor, serum 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] (N. Choisunirachon) Thai J Vet Med. 2019. 49(3): 283-288.

284 Choisunirachon N. et al. / Thai J Vet Med. 2019. 49(3): 283-288. Introduction provide relatively simple methods for evaluating prognosis. Although previous reports have indicated In normal metabolism, cells obtain energy through that LDH could be used as a predictor of recurrence in oxidative phosphorylation, in which pyruvate enters canine lymphoma (Marconato et al., 2010) and the mitochondrial tricarboxylic acid cycle prior to metastatic mammary gland tumors (Campos et al. complete oxidation. In situations of reduced oxygen 2012), the evaluation of LDH expression in oral cancer- availability, however, aerobic glycolysis is replaced to bearing dogs has not been attempted. Therefore, the some degree by anaerobic glycolysis, in which glucose purpose of this study was to preliminary compare the is converted into lactate (Gao and Chen, 2015). To make serum LDH levels among canine patients with oral the lactate usable as an energy source, lactate tumors, and those of healthy dogs. dehydrogenase (LDH) catalyses the conversion of lactate to pyruvate concomitant with the NAD+ to Materials and Methods NADH interconversion. In addition to hypoxic conditions, this LDH-catalysed conversion also occurs This study was approved by the Institutional at elevated levels in several diseases. Serum LDH has Animal Care and Use Committee (IACUC), been found to be a sensitive indicator of diseases Chulalongkorn University, in accordance with involving the heart, liver, lungs, and muscles (Draoui university regulations and policies governing the care and Feron, 2011). and use of laboratory animals, and consent was obtained from the dog owners. The animal use Due to their high energy needs (to fuel growth, protocol number was 1531017. including local invasion and metastasis), tumor cells are often modified to use anaerobic glycolysis rather The canine patients used in this study were divided than mitochondrial oxidative phosphorylation into 2 groups. The healthy control group (control) (Doherty and Cleveland, 2013). Even in a normoxic consisted of dogs that presented for an annual health condition, high-metabolism cells such as those in check-up, or for castration or ovariohysterectomy. The tumors obtain energy through glycolysis, especially in primary subjects consisted of a group of oral tumor- deeper tumors with less neovascularization (Sonveaux bearing dogs. In both groups, the patients were et al., 2008). This phenomenon has been named the examined using a full set of standard tests, such as a “Warburg effect”. LDH, therefore, plays a crucial role physical examination, complete blood count, serum in tumor metabolism, by interconversion of lactate and biochemistry, thoracic and abdominal radiography pyruvate, after which the lactate may be transported (for the controls), and radiography with abdominal into the tumor cells for use as a source of energy ultrasonography or whole body computed (Draoui and Feron, 2011). The expression of LDH is tomography (for the tumor-bearing group). Patients thus a signature of the high metabolism of a malignant that had any disease involving the liver, heart, or tumor, making LDH an interesting candidate for a muscles, or any other disease that could interfere with prognostic indicator. In human medicine, LDH has the serum LDH level, were excluded from this study. been applied as a biomarker for several types of tumors, including lymphomas, prostrate cancer, renal In the tumor group, the dogs were examined and cell carcinomas, and malignant melanomas (MM) all relevant information about their oral masses was (Miao et al., 2013). recorded, including location; size; shape; invasion of adjacent tissue; metastatic status; observations made In veterinary medicine, among canine tumors, oral through diagnostic imaging such as radiography, masses account for 5–7% (Kafka et al., 2004), which is a ultrasonography or computed tomography; and high incidence compared to other species (Dorn and histopathological results after excisional biopsy. The Priester, 1976). Oral neoplasms can be found at sites clinical stage for each dog was classified according to such as the mucosa, gingiva, palate, or tongue (Vos and the tumor size as follows: clinical stage I if the primary Gaag, 1987). The primary location of an oral tumor is tumor was less than 2 cm in diameter, clinical stage II strongly related to its clinical signs. Primary tumors if the primary tumor size was between 2 and 4 cm, and can cause facial deformity, gagging, difficulty in clinical stage III if the primary tumor size was greater mastication, or drooling. In advanced cases, primary than 4 cm. If any patient had an oral tumor of any size oral masses can invade adjacent areas such as the with bone invasion or metastasis, either at a regional retrobulbar region, causing exophthalmos, or the nasal lymph node or a distant location, the dog was classified cavity, causing epistaxis (Liptak and Withrow, 2007). as clinical stage IV (Liptak and Withrow, 2007). To enable proper treatment planning, including the To investigate the serum LDH level in each patient, prognosis of each tumor-bearing patient, the type of blood samples were collected from the cephalic vein oral neoplasm should be clarified. In general, oral with an awareness of hemolysis, placed in a plain tube neoplasms can be divided into two groups, benign and left at room temperature for 30 min before tumors such as epulis, and malignancies. The separating the serum by centrifugation. For the tumor malignant oral tumors seen in canine patients include group, the blood was collected prior to anaesthesia on MM, squamous cell carcinoma (SCC), fibrosarcoma the day of the excisional surgery and some at one day (FS), osteosarcoma, mast cell tumors, post-operatively. All serum samples were kept at -20 haemangiosarcoma, lymphoma, plasma cell tumors, °C and submitted within 36 hours to the laboratory for and multilobular tumors of bone (Todoroff and LDH level measurement by the ultraviolet kinetic Broodey, 1979). In addition to the cytological or method with Daisy solution (Daisy, Germany) using a histopathological results, other methods, such as blood chemistry analyzer (Sapphire 400, Durai, China). examination of abnormal cancer-related biological The survival period for each dog was obtained via a substances found in body fluids such as blood or urine, telephone interview.

Choisunirachon N. et al. / Thai J Vet Med. 2019. 49(3): 283-288. 285 The clinical demographic data including serum epithelial tumors (BET), and 1 each with osteosarcoma, LDH expression was presented as a descriptive lymphoma, histiocytoma and acanthomatous analysis including the mean and SEM. The difference ameloblastoma. The locations of the tumors were of serum LDH levels between the control group and predominantly in the gingival area (25/34 dogs), the tumor group, or between the control group and followed by the lip (5/34 dogs), buccal mucosa (2/34), each tumor type was analysed using the Mann- and each of the tonsils and hard palate (1/30 dogs). The Whitney U test. Besides, the difference of serum LDH clinical stages were stage I for 9 dogs, stage II for 7 among tumor group in location and clinical stage were dogs, stage III for 13 dogs, and stage IV for 5 dogs. analysed using the Kruskal-Wallis test. All statistical Among the 5 dogs in clinical stage IV, 3 dogs were analysis was performed using GraphPad Prism finally diagnosed with MM with splenic (1 dog) or Version 7.0 (GraphPad Software, Sandiego, CA, USA). pulmonary (2 dog) metastases, a dogs affected with FS P values less than 0.05 were considered statistically with pulmonary metastasis and the last dog had significant. osteosarcoma with pulmonary metastasis. Results Serum LDH expression: The average serum LDH for the control group was 102.43 ± 16.89 U/l, whereas the Clinical demographic data: Among the 20 dogs in the average for the tumor group was 378.35 ± 84.24 U/l, control group, 10 were male (all neutered) and 10 were which was significantly higher (P = 0.0006, Fig. 1). female (8 intact and 2 spayed). The average age was 3.5 ± 0.1 years old. The average body weight was 9.4 ± 1.7 Consideration with out individual patient, SCC kg. There were 7 mixed breed dogs, 2 poodles, 2 Shih revealed the highest serum LDH expression (478.43 ± Tzu, 1 German Shepherd, 1 Chow Chow, 1 Thai, 1 240.43 U/l) following by MM (397.29 ± 136.37 U/l), FS Beagle, 1 Boston terrier, 1 Pekinese, 1 Terrier, 1 (265.85 ± 45.83 U/l) and BET (94.35 ± 64.35 U/l) which Pomeranian, and 1 Chihuahua. Of the 34 dogs in the SCC, MM and FS had significantly higher serum LDH tumor group, 23 were male (16 intact and 7 castrated) and 11 were female (8 intact and 3 spayed). The expression than that of control group (P = 0.0030, average age was 9.4 ± 7.1 years old. The average body 0.0078 and 0.0022, respectively; table.1 and Fig. 2). weight was 14.5 ± 1.89 kg. There were 14 mixed breed dogs, 7 Shih tzu, 5 Poodles, 3 Golden Retriever, 1 In addition, the expression of serum LDH did not German Shepherd, 1 Rottweiler, 1 Siberian husky, 1 significantly correlate with the location of the primary Bangkaew, 1 Pomeranion and 1 Pug. tumor or the clinical stage of the disease (Fig. 3). The clinical information for the tumor group is as Clinical stage II affected dogs (733.38 ± 341.75 U/l) had follows: Among 34 patients, there were 17 dogs with a higher expression of serum LDH followed by clinical MM, 6 with SCC, 4 with FS, 3 dogs with benign stage III (329.26 ± 72.76 U/l), clinical stage IV (315.10 ± 212.06 U/l) and clinical stage I (208.26 ± 43.89 U/l), respectively (P = 0.4239; Table. 2). Figure 1 The serum lactate dehydrogenase (LDH) expression between the control dog (control) and the oral tumor-bearing dogs (oral tumors). The serum LDH was significantly higher in the oral tumor gr. than in that of the control gr. (P = 0.0006). Figure 2 The serum lactate dehydrogenase (LDH) expression in control dogs (control) compared to those of oral malignant melanoma (MM), squamous cell carcinoma (SCC), fibrosarcoma (FS) and benign epithelial tumor (BET). The serum LDH expressions of MM, SCC and FS were significantly higher than that of the normal control dogs (P = 0.0078, 0.0030 and 0.0022, respectively).

286 Choisunirachon N. et al. / Thai J Vet Med. 2019. 49(3): 283-288. Figure 3 The serum lactate dehydrogenase (LDH) expression among 34 canine oral tumors considerated by each group of clinical stages. No statistically significant difference of serum LDH expression among groups. Table 1 The serum lactate dehydrogenase level (mean ± SEM) among normal control dogs and various canine oral tumor affected dogs Classified group Number Serum LDH level (U/l) Normal control dogs 20 102.43 ± 16.89 Oral tumor affected dogs 34 378.35 ± 84.24b Malignant melanoma 10 397.29 ± 136.37a Squamous cell carcinoma 3 478.43 ± 240.43a Fibrosarcoma 4 265.85 ± 45.83a Benign epithelia tumor 3 94.35 ± 64.35 Osteosarcoma 1 1156.00 Lymphoma 1 39.50 Histiocytoma 1 205.00 Acanthomatous ameloblastoma 1 491.90 a and b Significant difference at p < 0.01 and 0.001 compared to the normal control group, respectively. Table 2 The serum lactate dehydrogenase level (mean ± SEM) among various clinical stages of canine oral tumor affected dogs. Classified group Number Serum LDH level (U/l) Oral tumor affected dogs 34 378.35 ± 84.24 Stage I 9 208.26 ± 43.89 Stage II 7 733.38 ± 341.75 Stage III 13 329.26 ± 72.76 Stage IV 5 315.10 ± 212.06 Discussion The use of serum LDH for canine oral tumors has been reported for a small number of canine patients, To treat tumors, several processes involving tumor which included only 3 dogs with tonsillar carcinoma, 2 pathophysiology and tumor metabolism have been investigated, both in human and in veterinary with oral FS, and 1 dog each with oral SCC and oral medicine. Among these processes, the production of MM (Marconato et al., 2009). Therefore, our study lactate dehydrogenases (LDH), a family of NAD+- aimed to preliminarily compare serum LDH levels in NADH conversion enzymes that contribute to the canine oral-tumor bearing patients and in healthy cancer-related increase in glycolysis known as the dogs. “Warburg effect”, are considered to be reliable indicators of several types of human cancer, including In this study, the ages of the healthy dogs and the malignant melanoma, lymphoma, renal cell carcinoma, oral tumor bearing dogs were different because a and prostate cancer (Miao et al., 2013). The increased higher incidence of the oral neoplastia was found in LDH is probably a consequence of the fact that tumors senile patients. In addition to neoplastic conditions, have high-energy requirements for utilization of geriatric patients are prone to several aging maladies lactate, to fuel their rapid growth (Gao and Chen, 2015). that can interfere with the serum LDH level such as heart or liver diseases (Draoui and Feron, 2011). In canine cancer patients, LDH levels have been Evaluation of serum LDH as the neoplastic indicator is reported to be higher than in healthy dogs and dogs of more benefit in the young to middle age patients without tumors. Among malignancies, LDH since their health statuses are not concurrent with other expression was highest in lymphoma, histiocystic sarcoma, sarcoma, carcinoma, and mastocytoma, in LDH interference conditions. Nevertheless, the patient that order (Marconato et al., 2009). As a result, which selection in this study attempted to eliminate the matches the findings in human medicine, LDH is an interference LDH from other sources through all of the attractive candidate for a predictor of tumor recurrence blood work, imaging diagnosis and sample collection in canine lymphoma patients (Marconato et al., 2010). procedure. Therefore, the significantly higher serum In addition, LDH analysis can be applied to evaluate the metastatic status of canine malignant mammary LDH level in oral tumor affected dogs would come gland tumors (Campos et al., 2012). from the intra-oral tumor origin. The serum LDH was significantly elevated in the oral tumor group compared to those of the control group, however the distribution of serum LDH level in the oral tumor group revealed a wide range. The

Choisunirachon N. et al. / Thai J Vet Med. 2019. 49(3): 283-288. 287 variation of the LDH level in the oral tumor group Rachadaphiseksomphot Endowment Fund, and The might be due to the composition of serum LDH from Research Assistant Scholarships, Graduate School, both of benign and malignant oral tumor. Although 34 Chulalongkorn University. Authors would like to canine oral tumor patients were enrolled, the thank all supports from Suwannachard Animal distribution of various tumor types, including the Hospital for the research technique. primary location and clinical stage, resulted in a low number of dogs in each subcategory, which made it References difficult to observe statistical differences in LDH levels in some rare types of oral tumor. When comparing the Bartlett EK and Karakousis GC 2015. Current staging serum LDH level of each tumor to that of the control and prognostic factor in melanoma. 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Short Communication Detection of rabies virus specific antibody in stray dogs in Palestine Adnan Fayyad1* Nasr Jalboush1 Ibrahim Alzuheir1 Belal Abu Helal1 Mohammad Manasrah2 Abstract Stray dogs are considered as a major public health risk in rabies-endemic countries such as Palestine. The objective of the present study was to assess the presence of antibody against rabies in stray dogs in order to estimate the immune status of stray dogs for the purpose of risk management. The present study reports the first investigation for the detection of specific antibodies to rabies virus (RABV) in the serum of stray dogs in Palestine. The serum samples were collected randomly from 92 stray dogs from different seven Palestinian districts and were tested for the presence of antirabies antibodies by ELISA. Only 11.95% of stray dogs (n=11) had protective immune status against rabies with antirabies antibody titer (>0.5 IU) according to World Health Organization criteria. This result suggest that there is a high potential for reintroduction of canine rabies into stray dogs, leading to rabies transmission to human. Therefore, a new strategy to enable a broader vaccination coverage in stray dogs in conjunction with control breeding of these dogs must be launched to reduce the risk for transmitting rabies to human. Keywords: Antirabies antibody, ELISA, Rabies, Stray dogs 1Department of Veterinary Medicine, Faculty of Agriculture and Veterinary Medicine, An-Najah National University, Nablus – Palestine 2Independent Researcher in Animal Health and Zoonotic Diseases, Bethlehem - Palestine *Correspondence: [email protected] (A. Fayyad) Thai J Vet Med. 2019. 49(3): 289-293.

290 Fayyad A. et al. / Thai J Vet Med. 2019. 49(3): 289-293. Introduction wild animals (Yakobson et al., 2017). Stray dogs have a direct contact with wild animals such as red foxes and Rabies is a rapidly neuroinvasive infectious viral golden jackals thus representing a major risk for disease affecting humans and mammals (Jackson, transmitting the virus to other domestic animals and 2003). The disease is caused by an RNA virus belongs humans (Cleaveland and Hampson, 2017: Bannazadeh to the genus Lyssavirus of the family Rhabdoviridae et al., 2018). Therefore, it is important to assess the level (Yousaf et al., 2012). The virus has a high tropism to of antirabies antibodies in animals to verify the nervous system causing acute, fatal encephalitis effectiveness of rabies vaccination campaigns. (Yousaf et al., 2012). Foxes, coyotes, skunks and dogs However, to the best of our knowledge, no are considered to be the most important reservoirs for epidemiological studies have yet been conducted to rabies virus worldwide (Rupprecht et al., 2004), human assess antirabies antibodies in stray dogs in Palestine. cases transmitted from rabid dogs constitute most of In addition, the effectiveness of vaccination through human reported cases (Fooks et al., 2014). measuring the antirabies antibodies titer has not been evaluated before. The present study aimed for the first The disease is considered as fatal disease once the time to test for the presence of rabies virus (RABV) clinical signs of disease occur except for rare cases sepcificantibody in serum samples from stray dogs (Jackson, 2003). Despite continued attempts to treat from different Palestinian districts. The results of this human rabies, medical treatment are unsuccessful and study will be necessary for increasing awareness the disease remains with the highest case fatality ratio against rabies and developing a national strategy (Hemachudha et al., 2002). It’s estimated that 7 million program for controlling rabies in Palestine. people are exposed to rabies annually, most of them in developing countries resulting in 55,000 death cases Materials and Methods reported in Africa and Asia annually (Hampson et al., 2011). Study design: A cross-sectional study on RABV specific antibody in stray dogs was conducted in the northern Rabies was first reported in Palestine since during and southern Palestinian districts. Blood samples were the 4thCentury (king et al., 2004). In addition, rabies is randomly collected from 92 stray dogs in Nablus (n = practically endemic in most of Palestine and Israel 15), Jenin (n = 8), Tulkarm (n = 44), Qalqīlyaḧ (n = 5), (Shimshony, 1997). There is a little information Ramallah (n = 1), Bethlehem (n = 5) and Hebron (n = available for Palestine (king et al., 2004). Red foxes and 14) Palestinian districts in West Bank (Fig 1). golden jackals were the main vectors of rabies in Israel and Palestine. However, since 2009, canine rabies has re-emerged in the country and were considered as the main animal reservoir highlighting the risk of virus transmission between human and both domestic and Figure 1 Map of Palestinian districts in West Bank where samples were taken. Number of dogs with protective antirabies antibodies above the protective level recommended by WHO (≥0.5 IU). First number indicates positive results and the second number represents the total number of sampled dogs.

Fayyad A. et al. / Thai J Vet Med. 2019. 49(3): 289-293. 291 Animals: The study was conducted for a period of one (>0.5 IU) was considered as protective level according year from April 2018 to April 2019. Samples were to World Health Organization (WHO) criteria. collected from 92 stray dogs, whose history of vaccination against rabies was unknown. These dogs Statistical analysis: Data were analyzed with were captured by using an appropriate cage traps. The statistical software SPSS version 20th (SPSS Inc, USA). capturing method used in this study was humane and Chi-square test was used to determine the association gentle to minimize stress on animals. For sedation, between neutralizing antirabies antibodies and the dogs were injected with Xylazine hydrochloride location, sex and age of dogs. Differences were (Eurovet Animal Health, Bladel, Netherland) at a considered statistically significant at P ≤0.05. dosage rate of (1 mg/kg) body weight using intramuscular injection before blood collection (Cassu Results and Discussion et al., 2014). A trained veterinarian conducted sample collection after obtaining ethical approval from Serum samples were obtained from 92 stray dogs Palestinian Animal League Association (Palestine). with unknown history of rabies vaccination collected Dogs were released after applying ear tags for from seven Palestinian districts. Of these 43 dogs were identification. female (46.7%), 49 dogs were male (53.2%). Estimated ages of dogs ranged from 6 months to 4 years. Blood samples: Blood were obtained in plain tubes from the cephalic vein of each dog after appropriate The antibody titers were calculated by comparison preparation of the venipuncture site using (70% with antirabies antibody titer of (>0.5 IU) according to Ethanol). The samples were left to clot at room WHO reference serum (WHO, 2013). The antirabies temperature for 2 hours. The clot was removed by antibody titers above (0.5 IU) threshold were centrifuging at 1,000-2,000 x g for 10 minutes. The considered protective (WHO, 2013). The seropositive serum was then transferred to a new (2 ml) Eppendorf rate was calculated as the percentage of dogs tube and stored at -20°C until analysis. demonstrating antirabies antibody titers (>0.5 IU). Out of 92 stray dogs tested; only 11.95% (11/92) had Detection of antirabies antibody: For determination of protective antirabies antibody titers (>0.5 IU) antirabies immunoglobulin G antibody (IgG) in stray according to WHO (WHO, 2013). Since these dogs dogs, a commercially available rabies ELISA kit were stray dogs, its vaccination status was unknown. (Demeditec Diagnostics GmbH, Germany) was used From the 11 dogs harboring protective antibodies according to manufacturer's instructions. Positive and levels; 5 (number samples = 44) were from Tulkarm negative sera were provided in the kit. A serum titer of district, 4 (15) from Nablus district, 1 (14) from Hebron and 1 (8) from Jenin district Fig 1. Table 1 shows antirabies antibody titers in street dogs from seven different Palestinian districts. Table 1 RABV specific antiraby titer among stray dogs from seven Palestinian districts according to WHO criteria. Number of dogs RABV specific antibody titer (<0.125 IU) (0.125-0.250 IU) (0.250-0.500 IU) (≥0.5 IU) 11 (11.95%) 92 2 (2.17%) 51 (55.43%) 28 (30.43%) WHO = World Health Organization; IU = international unit; IgG = immunoglobulin G Statistical analysis of antirabies antibody titer Stray dogs have uncontrolled rising population, showed no significant differences between different representing a challenge in Palestine. Dogs were found Palestinian districts, sex and age of dogs (P>0.05). to be the main rabies reservoir and transmitter in Israeli Rabies is an important zoonotic, fatal and and Palestinian territories (Yakobson et al., 2017). progressive neurological disease affecting all warm- Moreover, almost 99% of human rabies cases are blooded animals (Singh et al., 2017). The disease resulted from dog bites (David and Yakobson, 2011). remains as an important public health issue because of These infected dogs move across the country borders its prevalence through the world (Burgos-Caceres, carrying the infection to wild and domestic animals 2011). Rabies is endemic in Israel and Palestine (king et including domestic and stray dogs because of the high al., 2004). Despite this, there has been to date sparse proximity between the populated urban and rural information available about the disease in Palestine. areas and thereafter transmitting the disease into According to the Palestinian Ministry of Health official humans (Gdalevich et al., 2000). Different preventive data, only during the year 2018, approximately, 826 measures have been applied to control rabies in cases of dog bites were presented to the local Palestine; these include vaccination of house hold governmental health centers and hospitals in Palestine dogs, small-scale vaccination campaigns and the for post-exposure prophylaxis measures (Ministry of national oral rabies vaccination (ORV) campaign health, personal communication, data not published). which are being performed since 2005 and covers all The estimated governmental cost of these measures is territories in Israel and Palestine (Yakobson et al., $ 15.000 annually. In addition, 30 animal cases of rabies 2006). However, monitoring of RABV specific were confirmed, of which 12 were reported in dogs antibodies in stray dogs to evaluate the previous during the year 2016 in Israeli and Palestinian control measures has never been conducted before in territories (Israeli Ministry of Agriculture, data not Palestine. published).

292 Fayyad A. et al. / Thai J Vet Med. 2019. 49(3): 289-293. In our study, we have used an ELISA test for the Gdalevich M, Mimouni D, Ashkenazi I, Shemer J 2000. detection of RABV specific antibody titer in the serum collected from stray dogs, this test provide high Rabies in Israel: decades of prevention and a sensitivity, specificity as well as easy utilization for screening of the antirabies antibody titers in animal’s human case. Public Health. 114: 484-7. serum (Piza et al., 1999). In this study, the incidence of protective immune status in stray dogs in Palestine Hampson K, Cleaveland S, Briggs D 2011. Evaluation was very low (11.95%) in comparable to other countries. For example, a higher level (49%-86%) of of cost-effective strategies for rabies post-exposure protective antirabies antibody in stray dogs with unknown history of rabies vaccination was reported in vaccination in low-income countries. PLoS Negl Thailand (Kasempimolporn et al., 2007). In addition, Ogawa (2009) reported that the protective Trop Dis. 5: e982. seroprevalence on stray dogs in Japan was 27.7% (Ogawa et al., 2009). These results indicate that the Hemachudha T, Laothamatas J, Rupprecht CE 2002. current rabies vaccination programs including the (ORV) campaign are limited. The vaccine delivery Human rabies: a disease of complex problem observed in stray dogs with ORV campaign is perhaps because these stray dogs are often live in neuropathogenetic mechanisms and diagnostic packs, whereby one dominant dog may consume several oral vaccine baits leaving the rest of the group challenges. Lancet Neurol. 1: 101-9. unvaccinated, therefore this vaccine could be a better choice for vaccinating wild animals rather than stray Israeli Ministry of Agriculture 2017. “World Rabies dogs and parenteral vaccination could be the method of choice for stray dogs (Yakobson et al., 2008). The Day – September 28, 2017.” results of this study necessitate the importance for turning into a more effective vaccination strategy and [Online].Available:http://www.moag.gov.il/en/ the urgent need to control stray dogs by collaboration with both Public Health and the Veterinary Services, Ministrys%20Units/Veterinary_Services/Rabies/ periodic surveys to test the immunization status in stray dogs, increasing the awareness about rabies and Pages/World_Rabies_Day.aspxhttps://www.moa compulsory vaccination in household dogs. g.gov.il/en/Ministrys%20Units/Veterinary_Servi Conflict of Interest: The authors declare no conflicts of interest. ces/Rabies/Pages/World_Rabies_Day-.aspx. Acknowledgements Accessed: April. 28, 2019. The authors would like to acknowledge An-Najah Jackson AC 2003. Rabies virus infection: an update. 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