Volume 14 | December
Research Article | 08 Dec 2021
Antioxidant activity and protective effect of propolis against carbon tetrachloride-induced liver and kidney injury by modulation of oxidative parameters
Redouan El-Haskoury, Noori Al-Waili, Zeineb Kamoun, Mohamed Makni, Ahmed Al-Waili, and Badiaa Lyoussi

Volume-14 | Issue-12 | Article-1 | https://doi.org/10.14202/vetworld.2021.3076-3083

Preview Abstract
Propolis has a protective effect against cellular damage caused by toxic agents such as drugs, metals, xenobiotics, and chemicals. The aim of this study was to investigate the antioxidant activity and the effect of ethanolic extract of propolis on carbon tetrachloride (CCl4)-induced oxidative stress on kidney and liver injury in rat. The study quantified phenol, flavone, and flavonol in propolis and assessed antioxidant activity using 2, 2-diphenyl-1-picrylhydrazyl, ferric reducing antioxidant power, and molybdate. The investigators used four groups of rats to study the effect of propolis on CCl4-induced toxicity. Propolis extract was given orally (500 mg/kg) for 12 days, and CCl4 (1 mL/kg) was administered intraperitoneally on day 5 of the experiment. Blood and tissue samples of the liver and kidney were collected on day 13 to measure biochemical and oxidative parameters. The parameters included malondialdehyde (MDA), protein carbonyl formation (PCO), advanced oxidation protein products (AOPP), catalase (CAT), glutathione peroxidase (GPx), reduced glutathione (GSH), and ascorbic acid (AA). Biochemical parameters included liver enzymes, blood urea (BU), creatinine, and uric acid (UA). CCl4 decreased antioxidant agents, including CAT, GPx, GSH, and AA in the liver and kidney tissues. The oxidative agents' levels, including MDA, PCO, and AOPP, increased by CCl4 compared to the control group. CCl4 increased liver enzymes, UA, BU, and creatinine in the blood samples. Propolis significantly alleviated liver and kidney function, improved antioxidant parameters, and decreased levels of oxidative agents. The data showed for the 1st time that Moroccan propolis has a protective effect against CCl4-induced kidney and liver toxicity by maintaining the activity of the antioxidant defense system, which was most likely due to its antioxidant activity. Keywords: antioxidant, carbon tetrachloride, kidney, liver, propolis, toxicity.

Volume-14 | Issue-12 | Article-2 | https://doi.org/10.14202/vetworld.2021.3084-3090

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Determination of trace amounts of vitamins in multi-component feed premix is a troublesome analytical procedure. In this study, a simple and rapid high-performance liquid chromatography (HPLC) method was developed and validated for the concurrent detection and quantitation of four water-soluble vitamins such as thiamine, riboflavin, pyridoxine, and cyanocobalamin in veterinary feed premixes. The chromatographic separation of the vitamins was carried out at 35°C temperature on a reversed-phase C18 column using a gradient pump mode. Mobile phase constituents were solvent (a): 25 mM Potassium dihydrogen phosphate and 5 mM sodium hexanesulfonate in deionized water having pH-4.0 and solvent and (b) 5 mM sodium hexanesulfonate in methanol. Detection was performed with HPLC ultraviolet/visible detection set at 278 and 361 nm wavelength in two different channels. The flow rate was 1.2 mL/min and the total run time was 25 min. The method was validated according to the International Conference on Harmonization and Food and Drug Administration guidelines and acceptance criteria for system suitability, precision, linearity, and recovery were met in all cases. The relative standard deviation for system suitability and precision was <2% for all vitamins. The linearity of the calibration curves was excellent (R2>0.999) at concentration of 5, 10, 15, 20, 25, and 30 μg/mL for all vitamins. The limits of detection values were 0.0125, 0.0017, 0.0064, and 0.0065 μg/mL for thiamine, riboflavin, pyridoxine, and cyanocobalamin, respectively, and the limits of quantification values were 0.0378, 0.0051, 0.0213, and 0.0198 μg/mL for thiamine, riboflavin, pyridoxine, and cyanocobalamin, respectively. The recovery percentages ranged from 88% to 115%. The overall parameters of the proposed method met the validation criteria and this method could be a highly desirable technique for routine analysis of water-soluble vitamins in veterinary feed premix. Keywords: method development, veterinary feed-premix, water-soluble vitamin.
Research Article | 09 Dec 2021
Improving the knowledge of high school students about zoonotic diseases from pets in Medellín-Colombia
Natalia Uribe Corrales, Karen Velásquez Giraldo, Catalina María Saldarriaga Garcés, and Angie Lorena Navarro Giraldo

Volume-14 | Issue-12 | Article-3 | https://doi.org/10.14202/vetworld.2021.3091-3098

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The "One Health" concept is a global strategy that recognizes that public health is related to animal health and the environment; however, the role of domestic animals and their involvement in the transmission of zoonoses is often underestimated. The aim of the study was to evaluate and improve the knowledge about zoonotic diseases of domestic animals in high school students from Medellín, Colombia. A quasi-experimental intra-subject study was carried out. This study was conducted with 11th-grade students from four schools in Medellín, Colombia. A structured multiple-choice questionnaire was used from March 2021 to May 2021. The research had two phases, first, "naive" knowledge and learning. Then, descriptive, association, and comparative analysis were carried out using absolute and relative frequencies, Pearson's Chi-square test, and MacNemar's test with a value of p<0.05 considered statistically significant. A research poll from 315 students of four private schools found that feeding their pets with raw food and leftovers cooked for human consumption were common practices; the results also show a lack of knowledge of their pets' immunization deworming status. It was understood that when the students were able to identify at least two symptoms of zoonoses, one route of its' transmission and two preventive measures, we found that only 12.49% of the polled students had proper knowledge of the disease in domestic animals. After conducting an educational strategy, the findings show a general increase in knowledge, leading us to accept that the academic approach was adequate to statistically increase the recognition of symptoms, routes of transmission and preventive measures (p=0.00). The use of the theoretical lecture is effective to improve the understanding of the concept of transmission of diseases from animals to humans; in addition, the results show an increase of knowledge in high school students of the related symptoms, transmission routes, and preventive measures of zoonoses diseases in the region. Keywords: cats, diseases, dogs, education, zoonoses.
Research Article | 13 Dec 2021
Antimicrobial resistance profile of extended-spectrum beta-lactamases, adenosine-monophosphate-cyclic, and carbapenemase-producing Gram-negative bacteria isolated from domestic animals
Jôiciglecia Pereira dos Santos, Valesca Ferreira Machado de Souza, Marcos Wilker da Conceição Santos, Juliany Nunes dos Santos, Natilene Silva dos Santos, Angélica Prado de Oliveira, Valquíria Tatiele da Silva Rodrigues, Ianei Oliveira Carneiro, and Layze Cilmara Alves da Silva Vieira

Volume-14 | Issue-12 | Article-4 | https://doi.org/10.14202/vetworld.2021.3099-3104

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The production of beta-lactamase enzymes, such as extended-spectrum beta-lactamase (ESBL), adenosine-monophosphate-cyclic (AmpC), and Klebsiella pneumoniae carbapenemase (KPC), is one of the most important mechanisms of bacterial resistance to antimicrobials. Gram-negative bacteria show significant resistance due to various intrinsic and acquired factors. These intrinsic factors include low permeability of the outer membrane, various efflux systems, and the production of beta-lactamases, while acquired factors include chromosomal mutation and acquisition of resistance genes by horizontal transfer. Mobile elements such as plasmids, integrative conjugative elements, mobilizable islands, or transposable elements are involved in horizontal transfer. At present, the Gram-negative pathogens of most concern are Acinetobacter baumannii, Pseudomonas aeruginosa, and those belonging to the Enterobacteriaceae family (e.g., Escherichia coli, K. pneumoniae, and Proteus mirabilis). This study aimed to evaluate the profile of antimicrobial resistance and the production of the enzymes ESBL, AmpC, and KPC, in 21 gram-negative bacteria isolated from domestic animals treated at the University Veterinary Hospital (HVU) of the Federal University of Western Bahia (UFOB). The biological samples (21) were inoculated to brain heart infusion broth, blood agar, and MacConkey agar and incubated for 24-72 h at 37°C. Gram staining and identification through biochemical tests and matrix-associated laser desorption/ionization time-of-flight mass spectrometry were conducted. To evaluate the antimicrobial resistance profile, the disk diffusion method was used, and 25 antibiotics were employed. For the detection of ESBL, the disk approximation method was applied using chromogenic agar. The presence of KPC was observed using chromogenic agar and the Hodge test. For AmpC evaluation, the disk approximation method was used. The most isolated agent was E. coli (66.66%, 14/21), followed by K. pneumoniae and P. mirabilis (both 14.29%, 3/21), and then Pasteurella spp. (4.76%, 1/21). The bacterial isolates showed high levels of resistance against clindamycin, penicillin, imipenem, polymyxin, cefoxitin, gentamycin, cefotaxime, ceftazidime, cephalothin, ceftriaxone, ciprofloxacin, trimethoprim/sulfamethoxazole, chloramphenicol, and tetracycline. The best effectiveness rates were observed for cefepime, streptomycin, amoxicillin-clavulanate, aztreonam, nalidixic acid, tobramycin, levofloxacin, amikacin, and meropenem. All biological isolates showed multiple resistance to at least three of the antibiotics tested (3/25), and some showed resistance to 24 of the antibiotics tested (24/25). Among the 21 pathogens analyzed, 8 were ESBL producers (38.09%); of these, 6 were identified as E. coli (28.57%), and 2 were identified as K. pneumoniae (9.52%). Two strains of K. pneumoniae produced both ESBL and KPC. None of the isolates were producers of AmpC. The results found in the present work raise concern about the level of antimicrobial resistance among pathogens isolated from domestic animals in Brazil. The results highlight the need for the development and implementation of anti-resistance strategies to avoid the dissemination of multiresistant pathogens, including the prudent use of antimicrobials and the implementation of bacterial culture, antimicrobial sensitivity, and phenotypic tests for the detection of beta-lactamase enzymes in bacteria isolated from animals. Keywords: antimicrobial resistance, extended-spectrum beta-lactamase, Klebsiella pneumoniae carbapenemase, phenotypic tests.

Volume-14 | Issue-12 | Article-5 | https://doi.org/10.14202/vetworld.2021.3105-3110

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A new set of primers (400 base pairs partial of VP2) was designed and used for the infectious bursal disease virus (IBDV) screening test. Using this new primer set, the enzymes MboI and BstNI were unable to differentiate the field and vaccine strains. As a result, a new simple, cheap, and appropriate tool for strain differentiation is required. The objective of this study was to develop the appropriate restriction fragment length polymorphism (RFLP) and multiplex reverse transcription-polymerase chain reaction (RT-PCR) for the differentiation of classic IBDV (cIBDV) strains and very virulent IBDV (vvIBDV) strains in Thailand. Ninety seven bursa of Fabricius from 16 farms were collected from farms in the eastern and central regions of Thailand. RT-PCR screening showed that 82 samples were positive for IBDV and 15 samples were negative. Then, selected samples were sequenced from each farm with a positive test. The sequencing results showed that samples from six of the farms were vvIBDV and samples from the other six farms were cIBDV. Although the whole genome sequencing was incomplete, both the sequencing results of segment A and segment B showed high similarity between cIBDV and vvIBDV. Restriction enzyme cutting site and primers for multiplex RT-PCR were hard to design. An RT-PCR-RFLP method was developed, but it failed to differentiate IBDV strains. However, the multiplex RT-PCR was able to differentiate cIBDV from vvIBDV. Four primers were used in the multiplex RT-PCR. These four primers were used together in one reaction at an annealing temperature of 45°C. Therefore, multiplex RT-PCR is a less complicated, cheaper, and less time-consuming method for the differentiation of cIBDV and vvIBDV strains. Keywords: infectious bursal disease virus, multiplex reverse transcription-polymerase chain reaction, restriction fragment length polymorphism, strain, Thailand.
Research Article | 14 Dec 2021
Sociodemographic, income, and environmental characteristics of individuals displaying animal and object hoarding behavior in a major city in South Brazil: A cross-sectional study
Graziela Ribeiro da Cunha, Camila Marinelli Martins, Maysa Pellizzaro, Christina Pettan-Brewer, and Alexander Welker Biondo

Volume-14 | Issue-12 | Article-6 | https://doi.org/10.14202/vetworld.2021.3111-3118

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Hoarding cases have not been researched in depth in developing countries, such as Brazil. This study aimed to describe the characteristics of people with hoarding behavior in Curitiba, Brazil. A cross-sectional study was conducted based on complaints about hoarding situations received by the City Hall. The data on sociodemographic, income, and environmental characteristics of individuals displaying animal and object hoarding behavior were obtained and analyzed using descriptive statistics and multiple correspondence analyses. Out of the 113 hoarding cases reported, 69 (61.06%) were fully assessed. Most of the participants (43; 62.32%) were women, and it was observed that most of the animal hoarding cases were women (p=0.02). The average age was 62.47 years old, and most of them (44; 63.76%) had studied up to the middle school level. People associated with object hoarding belonged to the lower income category (p=0.031). In most cases, the homes had an unpleasant odor (45; 65.21%), and this was prevalent in cases involving women (p=0.004) and animals (p=0.001). The risk of fire (24 [34.78%]) and landslip (9 [13.04%]) was more frequent in the case of object hoarding (p=0.018 and 0.021, respectively). The description of characteristics of individuals with hoarding behavior may assist in understanding the magnitude of this public health problem in Brazil and shed light on the need to develop studies on the health conditions of people and animals that live in these situations. Keywords: epidemiology, hoarding, population characteristics.
Research Article | 15 Dec 2021
Validation of single-step genomic predictions using the linear regression method for milk yield and heat tolerance in a Thai-Holstein population
Piriyaporn Sungkhapreecha, Ignacy Misztal, Jorge Hidalgo, Daniela Lourenco, Sayan Buaban, Vibuntita Chankitisakul, and Wuttigrai Boonkum

Volume-14 | Issue-12 | Article-7 | https://doi.org/10.14202/vetworld.2021.3119-3125

Preview Abstract
Genomic selection improves accuracy and decreases the generation interval, increasing the selection response. This study was conducted to assess the benefits of using single-step genomic best linear unbiased prediction (ssGBLUP) for genomic evaluations of milk yield and heat tolerance in Thai-Holstein cows and to test the value of old phenotypic data to maintain the accuracy of predictions. The dataset included 104,150 milk yield records collected from 1999 to 2018 from 15,380 cows. The pedigree contained 33,799 animals born between 1944 and 2016, of which 882 were genotyped. Analyses were performed with and without genomic information using ssGBLUP and BLUP, respectively. Statistics for bias, dispersion, the ratio of accuracies, and the accuracy of estimated breeding values were calculated using the linear regression (LR) method. A partial dataset excluded the phenotypes of the last generation, and 66 bulls were identified as validation individuals. Bias was considerable for BLUP (0.44) but negligible (–0.04) for ssGBLUP; dispersion was similar for both techniques (0.84 vs. 1.06 for BLUP and ssGBLUP, respectively). The ratio of accuracies was 0.33 for BLUP and 0.97 for ssGBLUP, indicating more stable predictions for ssGBLUP. The accuracy of predictions was 0.18 for BLUP and 0.36 for ssGBLUP. Excluding the first 10 years of phenotypic data (i.e., 1999-2008) decreased the accuracy to 0.09 for BLUP and 0.32 for ssGBLUP. Genomic information doubled the accuracy and increased the persistence of genomic estimated breeding values when old phenotypes were removed. The LR method is useful for estimating accuracies and bias in complex models. When the population size is small, old data are useful, and even a small amount of genomic information can substantially improve the accuracy. The effect of heat stress on first parity milk yield is small. Keywords: accuracy, genomic selection, heat stress, linear regression method, ssGBLUP.
Research Article | 19 Dec 2021
Detection of invA virulence gene of multidrug-resistant Salmonella species isolated from the cloacal swab of broiler chickens in Blitar district, East Java, Indonesia
Freshindy Marissa Wibisono, Hayyun Durrotul Faridah, Freshinta Jellia Wibisono, Wiwiek Tyasningsih, Mustofa Helmi Effendi, Adiana Mutamsari Witaningrum, and Emmanuel Nnabuike Ugbo

Volume-14 | Issue-12 | Article-8 | https://doi.org/10.14202/vetworld.2021.3126-3131

Preview Abstract
The increasing number of multidrug-resistant (MDR) Salmonella species on poultry farms in Indonesia has caused concern regarding human health. This study was conducted to determine the presence of the virulence gene invA in MDR Salmonella species isolated from the cloacal swab of broiler chickens in Blitar district, East Java Province, Indonesia. Cloacal swab samples were collected by purposive sampling from 15 farms in four districts. Isolation and identification of bacteria were performed using standard microbiological techniques. Confirmation of MDR isolates was done using five different classes of antibiotics, including the beta-lactam, aminoglycoside, fluoroquinolone, phenicol, and monobactam groups. An antibiotic susceptibility test was conducted using the Kirby–Bauer disk diffusion method, and a polymerase chain reaction method was used to screen for the presence of invA. It was observed that 32.26% (50/155) of the samples were positive for Salmonella species. Of these 50 Salmonella isolates, 7 (14%) were identified as MDR strains. An important finding was the detection of invA in all the seven MDR Salmonella strains (100%) isolated from the cloacal swab of broiler chickens in Blitar district, East Java Province. Veterinarians have an extremely important role in monitoring the use of antibiotics in farm animals to mitigate the rapid spread of MDR organisms in our environment, which can otherwise cause serious economic losses and also public health issues. Keywords: broiler chicken, invA gene, multidrug-resistant, public health, Salmonella.
Research Article | 20 Dec 2021
Apigenin induces oxidative stress in mouse Sertoli TM4 cells
Sathaporn Jittapalapong, Thapanee Poompoung, and Samak Sutjarit

Volume-14 | Issue-12 | Article-9 | https://doi.org/10.14202/vetworld.2021.3132-3137

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Apigenin (API) is an estrogenic compound found in many plants. Sertoli cells reside in the testis and are a key target of environmental toxicants. This study aimed to examine the cytotoxicity, especially oxidative stress of API in mouse Sertoli TM4 cells. Mouse Sertoli TM4 cells were treated with 50 and 100 μM API for 48 h. Cell viability, lactate dehydrogenase (LDH) activities, glutathione reductase (GR) activities, production of reactive oxygen species (ROS), and malondialdehyde (MDA) levels were evaluated using various assays. Treatment with API at both 50 and 100 μM decreased viability and GR activity but increased LDH activity, ROS production, and MDA levels in mouse Sertoli TM4 cells. Exposure to API induced oxidative stress in mouse Sertoli TM4 cells. Keywords: apigenin, malondialdehyde, oxidative stress, reactive oxygen species, TM4 cells.
Research Article | 20 Dec 2021
Detection of microbial contamination in chicken meat from local markets in Surabaya, East Java, Indonesia
Dhandy Koesoemo Wardhana, Ajeng Erika Prihastuti Haskito, Muhammad Thohawi Elziyad Purnama, Devi Ayu Safitri, and Suwaibatul Annisa

Volume-14 | Issue-12 | Article-10 | https://doi.org/10.14202/vetworld.2021.3138-3143

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Chicken meat can be contaminated by microorganisms anywhere in the supply chain, from farm to market, and these microorganisms can be transmitted to humans through direct contact, contact with the environment, and food consumption. The microbial contamination has a serious impact on public health. This study aimed to analyze the microbial contamination of chicken meat sampled from local markets in Surabaya, East Java, Indonesia. A total of 60 samples of fresh chicken meat obtained from 10 traditional markets (six samples per market) were examined for the presence of bacteria. Staphylococcus aureus, Salmonella spp., and Escherichia coli were identified using Gram staining, culturing, and biochemical tests. The most probable number (MPN) method was used to identify E. coli. Most chicken meat samples were positive for S. aureus (58.3%), Salmonella spp. (48.3%), and E. coli (40%). The samples were considered positive for E. coli if the MPN value was higher than 1×101 CFU/g. High microbial contamination was found in all the chicken meat sampled from local markets in Surabaya. Such contamination can lead to foodborne diseases so, proper hygiene and sanitation standards should be followed from slaughterhouses to the end-users. Keywords: chicken meat, local markets, microbial contamination, public health, Surabaya.
Research Article | 24 Dec 2021
The first report of the seroprevalence of antibodies against Bartonella spp. in water buffaloes (Bubalus bubalis) from South Thailand
Sumalee Boonmar, Phirabhat Saengsawang, Watcharapong Mitsuwan, Decha Panjai, Kamchai Kidsin, Chalutwan Sansamur, and Ittidet Wichianrat

Volume-14 | Issue-12 | Article-11 | https://doi.org/10.14202/vetworld.2021.3144-3148

Preview Abstract
Bartonellosis is an emerging worldwide zoonosis caused by bacteria belonging to the genus Bartonella. Several studies have been conducted on the prevalence of Bartonella infections from animals and humans, including reports from wild and domestic ruminants. However, there has been only one report of Bartonella infection in water buffaloes from the northeastern part of Thailand. Moreover, the seroprevalence of Bartonella spp. in water buffaloes still remains unknown. This study was conducted to explore the prevalence of Bartonella spp. among water buffaloes from South Thailand using molecular and serological techniques. A total of 312 samples (156 blood and 156 sera) of 156 water buffaloes from 29 farms in Phatthalung Province, South Thailand, were collected from January to March 2021. All samples were screened for Bartonella spp. using polymerase chain reaction and indirect immunofluorescence assay. The seroprevalence of antibodies against three Bartonella spp. was 16.03% (25/156, 95% confidence interval: 10.65-22.74%), and among 25 water buffaloes with seroprevalence, 56%, 20%, and 24% were positive for antibodies against Bartonella henselae, Bartonella vinsonii subspp. berkhoffii, and Bartonella tamiae, respectively. No significant difference was detected among seroprevalence, gender, age, and ectoparasite infestation. This is the first report of the seroprevalence of antibodies against B. henselae, B. vinsonii subspp. berkhoffii, and B. tamiae in water buffaloes from South Thailand. Further studies are required on the epidemiology of Bartonella infection among water buffaloes, related personnel, and ectoparasites. Keywords: Bartonella henselae, Bartonella tamiae, Bartonella vinsonii subspp. berkhoffii, immunofluorescence assay, seroprevalence, water buffaloes.
Research Article | 25 Dec 2021
Diversity of gastrointestinal parasites in sympatric mammals in Moukalaba-Doudou National Park, Gabon
Serge-Ely Dibakou, Ulrich Maloueki, Barthélémy Ngoubangoye, Larson Boundenga, Stephan Ntie, Thierry-Audrey Tsoumbou, Cyr Moussadji, Rina Obame Zang, Dikenane Kombila, and Didier Basset

Volume-14 | Issue-12 | Article-12 | https://doi.org/10.14202/vetworld.2021.3149-3155

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Gastrointestinal parasites identified in the wild can negatively affect host fitness, lower performance, and growth. On the other side, sympatric mammals that share habitat and resources may also cross-transmit parasites, which are often zoonotic and can contribute to morbidity and mortality. This study aimed to characterize the diversity of gastrointestinal parasites circulating in mammalian hosts in Moukalaba-Doudou National Park. We screened a total of 25 fecal samples collected from nine wild mammalian species, namely, western gorilla (Gorilla gorilla gorilla), chimpanzee (Pan troglodytes), putty-nosed monkey (Cercopithecus nictitans), African forest elephant (Loxodonta cyclotis), African buffalo (Syncerus caffer), blue duiker (Philantomba monticola), bay duiker (Cephalophus dorsalis), and red river hog (Potamochoerus porcus) as well as people working as trackers (Homo sapiens) using direct microscopic observations following a sedimentation technique to concentrate the fecal material. Of the total 25 fecal samples screened, 15 (60%) were positive for parasitic gastrointestinal infection. Based on the morphology of parasite eggs and cysts, we identified a rich diversity of nematodes, protozoans, trematodes, and cestodes, including unidentified strongyles (73%), Oesophagostomum spp. (53%), Ancylostoma spp. (27%), Trichuris spp. (13%), Ascaris spp. (13%), Mammomonogamus spp. (13%), Strongyloides spp. (47%), Balantidium coli (20%), Entamoeba coli (20%), Endolimax nana (6%), Fasciola hepatica (6%), Paramphistomum spp. (13%), and Taenia spp. (6%). All parasites were found at least once in one of the hosts, and most were potentially zoonotic and responsible for several diseases of public health concern. Because of the small sample size, our findings should not be considered conclusive. Nevertheless, they highlight the diversity of gastrointestinal parasites in this area. Keywords: conservation, coprology, gastrointestinal parasites, Moukalaba-Doudou National Park, wildlife mammals.
Research Article | 25 Dec 2021
Establishment of semen collection technique using electroejaculator and semen cryopreservation of Javan leopard (Panthera pardus melas Cuvier, 1809)
Bongot Huaso Mulia, Ardyta Widianti, Jansen Manansang, Dedi Rahmat Setiadi, Vincentia Trisna Yoelinda, Taufiq Purna Nugraha, Ni Wayan Kurniani Karja, and Raden Iis Arifiantini

Volume-14 | Issue-12 | Article-13 | https://doi.org/10.14202/vetworld.2021.3156-3163

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The Javan leopard (Panthera pardus melas Cuvier, 1809) is a subspecies of Panthera pardus spp., spread across the African and Asian regions. Information on reproductive aspects is crucial for wild animals, including the Javan leopard. In this study, we aimed to develop electroejaculator (EE) techniques and evaluate cryopreservation success in Javan leopard semen. The semen of four adult Javan leopards was collected once a week using EE. Placement of the EE probe in the rectum was performed after ultrasound imaging (ultrasonography) to determine the prostate body location. The semen obtained was then evaluated macroscopically and microscopically. Three Javan leopards were used for cryopreservation. The ejaculate was divided into two parts [i.e., one part diluted with AndroMed® (Minitüb, Tiefenbach, Germany) and the other part with Steridyl® (Minitüb)] at a 1:1 ratio immediately after collection and evaluation. The semen was then packed in a 0.25 mL MiniStraw® (Minitüb) then equilibrated at 4°C for 2 h. After equilibration, the straw was then frozen in liquid nitrogen vapor. Frozen semen was then stored in containers until further evaluation. The results showed that ejaculation response occurred at all levels of stimulation, while erections did not always occur. The fastest ejaculation and erection occurred at the fourth voltage. The macroscopic evaluation showed that the semen volume was 0.80±0.26 mL, cloudy white, pH 7.44±0.14, and with watery semen consistency. The microscopic evaluation showed that the sperm motility was 66.98±0.39%, with sperm viability of 75.6±1.79%. Sperm concentration was 62.17±46.95×106 mL–1 with a total concentration of 42.14±23.51×106 cells. Normal sperm morphology is only 40.72±6.26%. This study concluded that the development of a semen collection technique using an EE preceded by imaging of the EE probe location using ultrasound was effective for the ejaculation of Javan leopards. The characteristics of the semen of the Javan leopard showed moderate semen volume, sperm motility, and viability. Javan leopard showed low sperm concentration and normal sperm morphology. Keywords: electroejaculator, Javan leopard, semen characteristics, semen cryopreservation.
Research Article | 26 Dec 2021
Impact of L-carnitine supplementation on the in vitro developmental competence and cryotolerance of buffalo embryos
Mohamed M. M. El-Sokary, Al-Shimaa Al-H. H. El-Naby, Amal R. Abd El Hameed, Karima Gh. M. Mahmoud, and T. H. Scholkamy

Volume-14 | Issue-12 | Article-14 | https://doi.org/10.14202/vetworld.2021.3164-3169

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Despite many trials, buffalo embryos have poor cryosurvivability because of their high lipid content. L-carnitine was found to be a lipid-reducing agent when added to oocyte and embryo culture media. The study aimed to determine the most effective concentration of L-carnitine to improve the oocyte developmental competence and cryotolerance of buffalo embryos. In vitro maturation and embryo culture media were supplemented with four concentrations of L-carnitine: 0 (control), 0.25, 0.5, and 1 mM. Good-quality embryos on 7 days were vitrified using mixtures of dimethyl sulfoxide and ethylene glycol at two concentrations (3.5 and 7 M). The result showed that the cleavage and morula rates were significantly (p<0.05) higher in the 0.5 mM group. Blastocyst rates were significantly (p<0.05) higher at both 0.5 and 1 mM. The rates of viable embryos directly after thawing were significantly (p<0.05) increased in the 0.5 mM group. No significant difference was found in embryos cultured for 24 h after warming among all the groups. The addition of L-carnitine at a concentration of 0.5 mM to the culture media improves the oocyte developmental competence and cryotolerance of buffalo embryos directly after warming but not after 24 h of culture. Nevertheless, further studies must identify how L-carnitine exerts its beneficial micromechanisms. Keywords: antioxidant, buffalo embryos, cryotolerance, L-carnitine.
Research Article | 26 Dec 2021
Analysis of culling reasons during the breeding cycle and lifetime performance: The strategy to remove crossbred Landrace and Large White sows under tropical climate
Ratchadaporn Boripun, Watcharapong Mitsuwan, Pawinee Kulnanan, Thotsapol Thomrongsuwannakij, and Warangkana Kitpipit

Volume-14 | Issue-12 | Article-15 | https://doi.org/10.14202/vetworld.2021.3170-3174

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Sow culling is an important practice in commercial swine production because it is directly associated with the economic efficiency of the breeding herd. This study was conducted to analyze the reasons for sow culling and quantify the factors affecting culling in crossbred Landrace and Large White sows under tropical climate. A total of 4887 culled sows from one parent stock farm located in Ratchaburi province, Western Thailand, were examined in this study. Culling reasons were grouped into the following eight categories according to farm management: (1) Reproductive disorders, (2) old age, (3) low performance, (4) diseases, (5) lameness, (6) udder problems, (7) body condition, and (8) other illnesses. Logistic regression analysis was used to explore the relationship between culling sows and environmental factors. Effects of parity and season of culling were considered as fixed effects in a statistical model. Descriptive statistics indicated the following factors accounting for sow removals: Old age (34.93%, n=1707), reproductive disorders (29.32%, n=1433), low performance (12.62%, n=617), lameness (12.56%, n=614), diseases (4.8%, n=235), body condition (4.68%, n=229), udder problems (0.79%, n=39), and other illnesses (0.26%, n=13). Parity and season of culling were also found to have a significant effect on sow culling (p<0.05). The majority of culling sows in this population were of old age and high parity. This study indicated that the purposeful culling of sows on this farm was within the targeted range. However, the incidence of reproductive disorders was too high and required further investigations. Keywords: culling, parity, season, sow, tropical climate.
Research Article | 28 Dec 2021
In silico molecular docking and in vitro analysis of ethanolic extract Ocimum sanctum Linn.: Inhibitory and apoptotic effects against non-small cell lung cancer
Ulayatul Kustiati, T. S. Dewi Ratih, N. Dwi Aris Agung, Dwi Liliek Kusindarta, and Hevi Wihadmadyatami

Volume-14 | Issue-12 | Article-16 | https://doi.org/10.14202/vetworld.2021.3175-3187

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Lung cancer, especially non-small cell lung cancer (NSCLC), has been identified as the leading cause of cancer deaths worldwide. The mortality rate from lung cancer has been estimated to be 18.4%. Until now, conventional treatments have not yielded optimal results, thus necessitating an investigation into the use of traditional herbal plants as potential candidates for its treatment. This study aimed to determine the inhibitory and apoptotic activity of the ethanolic extract from Ocimum sanctum Linn. (EEOS) by in silico molecular docking and through in vitro studies using NSCLC cells (A549 cell line). Dried simplicia of Ocimum sanctum was converted into EEOS using the maceration method. Spectrophotometry was then employed to analyze the EEOS compound. The known main active compounds were further analyzed for inhibitory and apoptotic effects on gene signaling using in silico molecular docking involving the downloading of active compounds from PubChem and target proteins from the Protein Data Bank; the active compounds and proteins were then prepared using the Discovery Studio software v. 19.0.0 and the PyRX 0.8 program, interacted with the HEX 8.0.0 program, and visualized with the Discovery Studio Visualizer v. 19.0. Finally, an in vitro analysis was performed using an antiproliferative-cytotoxic test (3-(4,5-dimethylthiazolyl-2)-2,5-diphenyltetrazolium bromide assay in the NSCLC A549 cell line). The analysis revealed that the active compounds in the ethanolic extract were dominated by quercetin (flavonoids) (47.23% b/b) and eugenol (phenolic) (12.14% b/b). These active compounds interacted with the active sites (residual amino acids) of the αvβ3 integrin, α5β1 integrin, caspase-3, caspase-9, and vascular endothelial growth factor. Hydrogen bonds and Pi-cation and Pi-alkyl interactions were involved in the relationships between the active compounds and the active sites and thus may reveal an antioxidant property of the extract. Furthermore, in vitro analysis showed the inhibitory and antiproliferative effects of the EEOS against non-small cell cancer (A549). Taken together, our data showed the ability of EEOS as an inhibitor and apoptotic agent for lung cancer; however, further research is needed to determine the exact mechanism of EEOS as an herbal medication. Keywords: in vitro, lung cancer, molecular docking, Ocimum sanctum.
Research Article | 28 Dec 2021
Effects of xylazine and adrenaline combinations: Preliminary clinical application for non-surgical protocols of nephrosplenic entrapment in horses
Worakij Cherdchutham, Patskit Sukhong, Kanchanog Sae-oueng, Nithisphat Supanwinijkul, Kittanai Wiangnak, Jirayut Srimuang, Tawanhathai Apichaimongkonkun, Sarocha Limratchapong, and Soontaree Petchdee

Volume-14 | Issue-12 | Article-17 | https://doi.org/10.14202/vetworld.2021.3188-3193

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The medical treatment of horses with nephrosplenic entrapment (NSE) of the large colon through administrating phenylephrine and rolling during general anesthesia was effective and less expensive than surgical treatment. However, the selection of drugs for non-surgical treatment of NSE is not a usual method for clinical practice. This study aimed to identify the effects of combined drugs on the cardiac and splenic response in horses and provide information on the NSE of the large colon for clinical application. Six healthy Thai native crossbred horses were enrolled in this study. Horses received two protocols with a withdrawal period of 14 days: Group 1 received xylazine (0.5 mg/kg IV) and adrenaline (1 mcg/kg IV), and Group 2 received xylazine (0.5 mg/kg IV) and adrenaline (3 mcg/kg IV). Heart rate (HR), HR variability (HRV), heart dimensions, and the splenic response of six horses were measured before the sedation, 30 and 60 min later, and 65, 70, 75, 80, 90, and 100 min after adrenaline administration. Doppler was used to obtain systolic blood pressure. The HRV low-frequency and high-frequency power ratios decreased after using xylazine. Hypertension was observed after adrenaline administration. In this study, there were only minimal differences in the HR and respiratory rate between groups. However, overall cardiac and splenic parameters were statistically higher in Group 2. This study suggested that xylazine and three micrograms of adrenaline preserved the cardiac autonomic activity balance and were safe to use non-surgical applicability in horses. Keywords: adrenaline, autonomic nervous activity, horse, spleen contraction.
Research Article | 29 Dec 2021
Validation of Brix refractometer to estimate immunoglobulin G concentration in goat colostrum
Chollada Buranakarl, Sumpun Thammacharoen, Morakot Nuntapaitoon, Sapon Semsirmboon, and Kazuo Katoh

Volume-14 | Issue-12 | Article-18 | https://doi.org/10.14202/vetworld.2021.3194-3199

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Immunoglobulin G (IgG) concentration is high in goat colostrum, particularly in the first few hours after parturition, and this is important for the kid's immunity and growth. IgG levels vary depending on several factors, including breed, disease status, colostrum management, handling, and collection time postpartum. A handheld optical refractometer, an affordable instrument that is simple to use in the field, is used widely in dairy farms to measure total solids. However, it can also be applied to estimate colostrum IgG content on the basis of comparison with standard measurement methods, usually radial immunodiffusion. Studies comparing %Brix values in relation to IgG concentration measured using enzyme-linked immunosorbent assay (ELISA) in goats are limited. The present study aimed to evaluate the use of a handheld optical Brix refractometer for the measurement of IgG concentration in goat colostrum, compare results with those using ELISA, and estimate the %Brix cutoff value equating to low-quality colostrum. Colostrum samples were collected on day 0 from 21 goats (nine Black Bengal, six Saanen, and six of their crossbred offspring) and were frozen. Subsequently, they were analyzed for IgG concentration using a goat-specific ELISA test and Brix percentage using a handheld refractometer. The optimum %Brix cutoff value for the evaluation of colostrum quality was evaluated. The mean IgG concentration and %Brix in colostrum were 10.60±2.92 mg/mL and 25.0±3.9, respectively. There was a significant (p<0.01) correlation between %Brix and IgG concentration. For an IgG concentration of 6.9 mg/dL, the cutoff value for %Brix was 18.5, equating to high specificity (100%) but low sensitivity (50%). A higher %Brix cutoff value of 21.5 showed high specificity (95%) and high sensitivity (100%). A Brix refractometer can be used to estimate goat colostrum quality with a proposed %Brix cutoff value of <18.5%-21.5% for poor-quality colostrum. Keywords: caprine, colostrum quality, optical Brix refractometer.
Research Article | 29 Dec 2021
Therapeutic effect of biosynthetic gold nanoparticles on multidrug-resistant Escherichia coli and Salmonella species isolated from ruminants
Abeer M. Abdalhamed, Alaa A. Ghazy, Eman S. Ibrahim, Amany A. Arafa, and Gamil S. G. Zeedan

Volume-14 | Issue-12 | Article-19 | https://doi.org/10.14202/vetworld.2021.3200-3210

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Multidrug-resistant (MDR) pathogenic microorganisms have become a global problem in ruminants as a result of the intensive use of antibiotics, causing the development of resistance among gut microbiota. The antibiotic-resistant microorganisms can be transferred from diseased animals to humans. This study aimed to determine the prevalence of MDR Escherichia coli and Salmonella spp. isolated from cattle, buffaloes, sheep, and goats suffering from respiratory signs, diarrhea, and mastitis and to screen the antibiotic sensitivity of selected isolated bacteria. It also detected antibiotic-resistance genes by polymerase chain reaction (PCR), produced green gold nanoparticles (AuNPs) using plant extracts (Artemisia herba-alba and Morus alba), and evaluated the antimicrobial activities of these biosynthesized nanoparticles on selected pathogens (E. coli and Salmonella spp.). MDR E. coli and Salmonella spp. were investigated using fecal samples (n=408), nasal swabs (n=358), and milk samples (n=227) of cattle, buffaloes, sheep, and goats with or without clinical signs, including respiratory manifestations, pneumonia, diarrhea, and mastitis, from different governorates in Egypt. E. coli and Salmonella spp. were isolated and identified on selective media, which were confirmed by biochemical reactions and PCR. Antimicrobial susceptibility testing against 10 commonly used antibiotics was performed using the Kirby-Bauer disk diffusion method. Antibiotic resistance genes blaTEM, blaSHV, blaOXA, and blaCTX-M were detected by PCR. The antibacterial effect of the biosynthesized AuNPs was evaluated by MIC and well diffusion assay. The biosynthesized AuNPs were also characterized by ultraviolet-visible spectrophotometry and transmission electron microscopy (TEM). Among all fecal samples, the prevalence of E. coli was 18.4% (183/993) and that of Salmonella spp. was 16.7% (66/408), as determined by cultural and molecular tests. All isolates of E. coli and Salmonella spp. were 100% resistant to ampicillin (AM) and amoxicillin and highly resistant to cefoxitin and AM-sulbactam. The total rate of resistance genes in E. coli was 61.2% (112/183), while that in Salmonella was 63.6% (42/66) for pathogens isolated from ruminants with respiratory manifestations, pneumonia, diarrhea, and mastitis. Among the resistance genes, blaTEM had the highest prevalence rate in E. coli (25.9%, 21/81) while blaSHV had the lowest (9.8%, 8/81) in fecal swabs. AuNPs were successfully synthesized using aqueous leaf extract of A. herba-alba and M. alba as bioreducing agents. TEM analysis showed particle size of 10-42 nm for A. herba-alba and M. alba AuNPs. The biosynthesized AuNPs showed antibacterial activity against MDR E. coli and Salmonella spp. Rapid and accurate diagnostic methods are the cornerstone for effective treatment to reduce the risk of antimicrobial-resistant pathogenic microorganisms. This is particularly important for overcoming the increasing rate of MDR in ruminants with respiratory manifestations, pneumonia, diarrhea, and mastitis. This can be complemented by the development of AuNPs synthesized in an environmentally friendly manner AuNPs using natural plant extracts for the treatment of antibiotic-resistant microorganisms. Keywords: Escherichia coli, gold nanoparticle, multidrug-resistant, ruminant, Salmonella species.

Volume-14 | Issue-12 | Article-20 | https://doi.org/10.14202/vetworld.2021.3211-3215

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Rapid tests are routinely used to estimate serum immunoglobulin G (IgG) concentrations in diagnosing a failure of passive transfer (FPT) in calves. The study aimed to compare the Fassisi® Bovine IgG (FB-IgG) immunoassay and an enzyme-linked immunosorbent assay for quantifying bovine IgG in neonatal calf serum. A total of 277 calves of 1-10 days of age were used in this study. Blood samples were obtained, and serum was extracted by centrifuging the samples at 2740× g for 5 min at 20°C. The serum was analyzed using the FB-IgG according to the manufacturer's specifications. Serum IgG concentrations were also determined by enzyme-linked immunosorbent assay (ELISA-IgG). FPT was defined as a serum IgG concentration <10 mg/mL. The mean ELISA-IgG serum concentration was 8.40 mg/mL (SD=7.02, range=0.10-47.50 mg/mL). FPT prevalence based on the ELISA measurements was 66.8%. The prevalence of partial and full FPT based on the FB-IgG was 54.5%. The ELISA-IgG and FB-IgG results were subjected to correlation and regression analysis. Overall sensitivity and specificity of the FB-IgG were 61.1% and 58.7%, respectively. A statistically significant dependence on age was identified in the results. Our findings suggest that the FB-IgG rapid method is less accurate and provides no other advantages over established methods. Keywords: antibody concentration, calves, colostrum, passive transfer, rapid method.
Research Article | 30 Dec 2021
Prevalence and antibiotic resistance of Salmonella isolated from poultry and its environment in the Mekong Delta, Vietnam
Thuan K. Nguyen, Lam T. Nguyen, Trang T. H. Chau, Tam T. Nguyen, Bich N. Tran, Takahide Taniguchi, Hideki Hayashidani, and Khai T. L. Ly

Volume-14 | Issue-12 | Article-21 | https://doi.org/10.14202/vetworld.2021.3216-3223

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Salmonella is one of the leading causes of zoonotic and foodborne infectious outbreaks in humans and poultry and its associated environment is a potential reservoir of Salmonella. In recent years, the antibiotic resistance of bacteria, including Salmonella, has been increasing. This study aimed to investigate the prevalence and antibiotic resistance of Salmonella isolated from poultry, its environment, and the pest animals found at poultry farms and households of the Mekong Delta, Vietnam. A total of 3,055 samples were collected from the broiler farms and households of the Mekong Delta from 2017 to 2020. Salmonella was isolated using conventional methods (culturing on selective agar – BPLS and biochemical test) and the isolates were examined for antibiotic resistance against 14 antibiotics using the disk diffusion method. Salmonella was isolated from 181 samples (5.92%), which included chicken feces (7.67%), pest animals (5.98%), and environmental samples (4.33%). The environmental samples comprised bedding (5.88%), feed (5.48%), and drinking water (0.70%). The prevalence of Salmonella was the highest in rats (15.63%) and geckos (12.25%) followed by ants (2.83%) and cockroaches (2.44%); however, Salmonella was not isolated from any fly species. Most of the isolates exhibited resistance to 1-9 antibiotics. The isolates were relatively resistant to chloramphenicol (62.98%), tetracycline (55.80%), ampicillin (54.14%), and sulfamethoxazole/trimethoprim (53.04%). Sixty-two multiple resistance patterns were found in the isolates, with ampicillin-cefuroxime-chloramphenicol-tetracycline- sulfamethoxazole/trimethoprim being the most frequent (7.18%). The chickens, husbandry environment, and pest animals at poultry farms and households were found to be important Salmonella sources in the Mekong Delta. Salmonella isolates from these sources also exhibited a wide-ranging resistance to antibiotics as well as several resistance patterns. Hence, biosecurity should be addressed in poultry farms and households to prevent cross-contamination and reduce the spread of Salmonella infections. Keywords: antibiotic resistance, chickens, environment, farms, Salmonella, wild animals.
Research Article | 31 Dec 2021
Melanocortin-4 receptor and leptin as genes for the selection of superior Madrasin cattle
Budi Utomo, Rimayanti Rimayanti, Indah Norma Triana, and Amaq Fadholly

Volume-14 | Issue-12 | Article-22 | https://doi.org/10.14202/vetworld.2021.3224-3228

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The genetic improvement of cattle through livestock section is based on quantitative, qualitative, and molecular characteristics. This study examined polymorphisms of the melanocortin-4 receptor (MC4R) and leptin genes as a reference for the selection of superior breeds in Madrasin cattle. The leptin and MC4R genes of Madrasin cattle were amplified using polymerase chain reaction (PCR); then, restriction fragment length polymorphism of the leptin gene was performed using the restriction enzyme BsaA1, at site 2793 with ACGT point position. The leptin gene was divided into three bands, namely, AA with one fragment (522 bp), CG with two fragments (441 bp and 81 bp), and AG with three fragments (522 bp, 441 bp, and 81 bp). The MCR-4 gene was divided into three bands, namely, 493 bp, 318 bp, and 175 bp. The MC4R and leptin genes can act as molecular markers for growth traits in Madrasin cattle and can be used to genetically optimize and improve growth. The GG allele of the MC4R gene and the AA allele of the leptin gene can be used in Madrasin cattle. Keywords: leptin, Madrasin, melanocortin-4 receptor.

Volume-14 | Issue-12 | Article-23 | https://doi.org/10.14202/vetworld.2021.3229-3234

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Metaflammation plays a significant role in the pathogenesis, development, and complication of diabetes mellitus (DM). This inflammation is associated with insulin resistance. Therefore, the inflammatory pathways have been targeted for pharmacological treatment. Petiveria alliacea can decrease blood glucose levels and has anti-inflammatory and antioxidant activities; however, there are still insufficient data regarding its efficacy for the treatment of DM. This study aimed to investigate the effect of the self-nanoemulsifying drug delivery system (SNEDDS) of P. alliacea leaf extract on the homeostatic model assessment (HOMA)-insulin resistance (IR) value and interleukin (IL)-6 and tumor necrosis factor-α (TNF-α) levels in a streptozotocin (STZ)-induced diabetic rat model. Thirty-five diabetic rat models were randomly divided into five groups. The first group received the SNEDDS of P. alliacea leaf extract at a dose of 50 mg/kg body weight (BW), the second group received it at a dose of 100 mg/kg BW, the third group received it at a dose of 200 mg/kg BW, the fourth group received 18 mg of metformin, and the fifth group only received the SNEDDS formula. The treatment was administered once a day, orally, for 14 days. On the 15th day after treatment, the rats were sacrificed to obtain blood samples for cardiac examination. The IL-6, TNF-α, and insulin levels in the serum were measured using the enzyme-linked immunosorbent assay method. The HOMA-IR value was calculated using a formula. The mean IL-6 and TNF-α levels were low in the group that received the SNEDDS of P. alliacea leaf extract. There was no significant difference in the insulin level in all treatment and control groups. However, a significant difference in the HOMA-IR value was noted between the group that received the SNEDDS of P. alliacea leaf extract and metformin and the group that did not receive treatment (p<0.05). The SNEDDS of P. alliacea leaf extract reduced the HOMA-IR value and suppressed the TNF-α and IL-6 levels in the STZ-induced diabetic rat model. Keywords: diabetes, homeostatic model assessment-insulin resistance, tumor necrosis factor-α, interleukin-6, nanoemulsifying, Petiveria alliacea.
Research Article | 31 Dec 2021
Culture of spermatogonial stem cells and use of surrogate sires as a breeding technology to propagate superior genetics in livestock production: A systematic review
Wilkister Nakami, Ambrose Ng'eno Kipyegon, James Nguhiu-Mwangi, Christian Tiambo, and Stephen Kemp

Volume-14 | Issue-12 | Article-24 | https://doi.org/10.14202/vetworld.2021.3235-3248

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Spermatogonial stem cells (SSCs) have previously been isolated from animals' testes, cultured in vitro, and successfully transplanted into compatible recipients. The SSC unique characteristic has potential for exploitation as a reproductive tool and this can be achieved through SSC intratesticular transplantation to surrogate sires. Here, we aimed at comprehensively analyzing published data on in vitro maintenance of SSC isolated from the testes of livestock animals and their applications. The literature search was performed in PubMed, Science Direct, and Google Scholar electronic databases. Data screening was conducted using Rayyan Intelligent Systematic Review software (https://www.rayyan.ai/). Duplicate papers were excluded from the study. Abstracts were read and relevant full papers were reviewed for data extraction. From a total of 4786 full papers screened, data were extracted from 93 relevant papers. Of these, eight papers reported on long-term culture conditions (>1 month) for SSC in different livestock species, 22 papers on short-term cultures (5-15 days), 10 papers on transfection protocols, 18 papers on transplantation using different methods of preparation of livestock recipients, and five papers on donor-derived spermatogenesis. Optimization of SSC long-term culture systems has renewed the possibilities of utilization of these cells in gene-editing technologies to develop transgenic animals. Further, the development of genetically deficient recipients in the endogenous germline layer lends to a future possibility for the utilization of germ cell transplantation in livestock systems. Keywords: culture, donor-derived spermatogenesis, marker, spermatogonial stem cells, transfection, transplantation.