Search results for: bovine tallow
Commenced in January 2007
Frequency: Monthly
Edition: International
Paper Count: 223

Search results for: bovine tallow

193 Effect of Incorporation of Seaweed Extract in Gelatin Based Film on Physic-Chemical and Bioactive Properties of Film

Authors: Shekhar U. Kadam, S. K. Pankaj, Brijesh K. Tiwari, P. J. Cullen, Colm P. O’Donnell

Abstract:

Brown seaweed L. hyperborea is a rich source of phenolic compounds with antioxidant and antimicrobial properties. The aim of this work was to study the effect of incorporation of L. hyperborea extract to bovine gelatin film on the physicochemical and antioxidant properties of film. Films with fraction of 25% by weight of bovine gelatin sample were cast with addition of glycerol as a plasticizer. The total phenolic content and antioxidant activity of the films showed higher levels with addition of seaweed extract. Also film appearance properties such as film thickness, color and light transparency were evaluated. Film appearance was slightly modified whereas microstructure of films showed rough patches at 50% level of extract in the film. Hydrophilicity and glass transition temperature of the films also increased with increased level of seaweed extract. It was found that seaweed extract can be incorporated within gelatin and casein for development of biofunctional films.

Keywords: Laminaria hyperborea, ultrasound, seaweed extract, bovine gelatin film, antioxidant, phenolic compounds

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192 Comparison of Several Diagnostic Methods for Detecting Bovine Viral Diarrhea Virus Infection in Cattle

Authors: Azizollah Khodakaram- Tafti, Ali Mohammadi, Ghasem Farjanikish

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Bovine viral diarrhea virus (BVDV) is one of the most important viral pathogens of cattle worldwide caused by Pestivirus genus, Flaviviridae family.The aim of the present study was to comparison several diagnostic methods and determine the prevalence of BVDV infection for the first time in dairy herds of Fars province, Iran. For initial screening, a total of 400 blood samples were randomly collected from 12 industrial dairy herds and analyzed using reverse transcription (RT)-PCR on the buffy coat. In the second step, blood samples and also ear notch biopsies were collected from 100 cattle of infected farms and tested by antigen capture ELISA (ACE), RT-PCR and immunohistochemistry (IHC). The results of nested RT-PCR (outer primers 0I100/1400R and inner primers BD1/BD2) was successful in 16 out of 400 buffy coat samples (4%) as acute infection in initial screening. Also, 8 out of 100 samples (2%) were positive as persistent infection (PI) by all of the diagnostic tests similarly including RT-PCR, ACE and IHC on buffy coat, serum and skin samples, respectively. Immunoreactivity for bovine BVDV antigen as brown, coarsely to finely granular was observed within the cytoplasm of epithelial cells of epidermis and hair follicles and also subcutaneous stromal cells. These findings confirm the importance of monitoring BVDV infection in cattle of this region and suggest detection and elimination of PI calves for controlling and eradication of this disease.

Keywords: antigen capture ELISA, bovine viral diarrhea virus, immunohistochemistry, RT-PCR, cattle

Procedia PDF Downloads 337
191 Spectrofluorimetric Investigation of Copper (II), Cobalt (II), Calcium (II), and Ferric (III) Influence on the Ciprofloxacin Binding to Bovine Serum Albumin

Authors: Ahmed K. Youssef, Shawkat M. B. Aly

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The interaction between ciprofloxacin and bovine serum albumin (BSA) was investigated by UV-Visible absorption and fluorescence spectroscopy. The influence of Cu²⁺ Ca²⁺, Co²⁺, and Fe³⁺ on the Cip-BSA interaction was investigated. The quenching of the BSA fluorescence emission in presence of ciprofloxacin as well as the influence of metal ions on the interaction was analyzed using the Stern-Volmer equation. The Stern-Volmer quenching constant, Kₛᵥ was calculated in presence and absence of the metal ions at the physiological pH of 7.4 using phosphate buffer. The experimental results showed that interaction mainly static in nature and quenching rate constant is decreased in presence of the studied metal ions with exception of Cu²⁺ ions. The decrease observed in the Kₛᵥ values in presence of Co²⁺, Ca²⁺, and Fe³⁺ can be understood on basis of competition between these metal and Cip when both of them existed in the BSA solution. Cu²⁺ induces interaction between Cip and BSA at faster quenching rates as inferred from the observed increase in the Kₛᵥ value. This allowed us to propose that copper (II) ions are directly involved in the process of Cip binding to BSA. The binding constant for Cip on BSA was determined and the metal ions effect on it was examined as well and their values were in line with the Kₛᵥ values.

Keywords: bovine serum albumin, ciprofloxacin, fluorescence, metal ions effect

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190 The Pathology of Bovine Rotavirus Infection in Calves That Confirmed by Enzyme Linked Immunosorbant Assay, Reverse Transcription Polymerase Chain Reaction and Real-Time RT-PCR

Authors: Shama Ranjan Barua, Tofazzal M. Rakib, Mohammad Alamgir Hossain, Tania Ferdushy, Sharmin Chowdhury

Abstract:

Rotavirus is one of the main etiologies of neonatal diarrhea in bovine calves that causes significant economic loss in Bangladesh. The present study was carried out to investigate the pathology of neonatal enteritis in calves due to bovine rotavirus infection in south-eastern part of Bangladesh. Rotavirus was identified by using ELISA, RT-PCR (Reverse Transcription Polymerase Chain Reaction), real-time RT-PCR. We examined 12 dead calves with history of diarrhea during necropsy. Among 12 dead calves, in gross examination, 6 were found with pathological changes in intestine, 5 calves had congestion of small intestine and rest one had no distinct pathological changes. Intestinal contents and/or faecal samples of all dead calves were collected and examined to confirm the presence of bovine rotavirus A using Enzyme linked immunosorbant assay (ELISA), RT-PCR and real-time RT-PCR. Out 12 samples, 5 (42%) samples revealed presence of bovine rotavirus A in three diagnostic tests. The histopathological changes were found almost exclusively limited in the small intestine. The lesions of rotaviral enteritis ranged from slight to moderate shortening (atrophy) of villi in the jejunum and ileum with necrotic crypts. The villi were blunt and covered by immature epithelial cells. Infected cells, stained with Haematoxylin and Eosin staining method, showed characteristic syncytia and eosinophilc intracytoplasmic inclusion body. The presence of intracytoplasmic inclusion bodies in enterocytes is the indication of viral etiology. The presence of rotavirus in the affected tissues and/or lesions was confirmed by three different immunological and molecular tests. The findings of histopathological changes will be helpful in future diagnosis of rotaviral infection in dead calves.

Keywords: calves, diarrhea, pathology, rotavirus

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189 Sensitivity of Staphylococcus aureus Isolated from Subclinical Bovine Mastitis to Ciprofloxacin in Dairy Herd in Tabriz during 2013

Authors: Alireza Jafarzadeh, Samad Mosaferi, Mansour Khakpour

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Mastitis is an inflammation of the parenchyma of mammary gland regardless of the causes. Mastitis is characterized by a range of physical and chemical changes in the glandular tissue. The most important change in milk includes discoloration, the presence of clots and large number of leucocytes. There is swelling, heat, pain and edema in mammary gland in many clinical cases. Positive coagulase S. aureus is a major pathogen of the bovine mammary gland and a common cause of contagious mastitis in cattle. The aim of this study was to evaluate the outbreaks of Staphylococcus aureus mastitis. This study is conducted in ten dairy herds about one thousand cows. After doing CMT and identifying infected cows, the milk samples obtained from infected teats and transported to microbiological laboratories. After microbial culture of milk samples and isolating S. aureus, antimicrobial, sensitivity test was performed with disk diffusion method by ciprofloxacin, co-amoxiclav, erythromycin, penicillin, oxytetracyclin, sulfonamides, lincomycin and cefquinome. The study defined that the outbreak of subclinical positive coagulase Staphylococcus mastitis in dairy herd was 13.11% (5.6% S. aureus and 7.51% S. intermedicus). The antimicrobial sensitivity test shown that 87.23% of Staphylococcus aureus isolated from bovine mastitis in dairy herd was susceptible to ciprofloxacin, 93.9% to cefquinome, 4.67% to co-amoxiclav, 12.16% to erythromycin 86.11% to sulfonamides (co-trimoxazole), 3.35% lincomycin, 12.7% to oxytetracyclin and 5.98% to penicillin. Results of present defined that ciprofloxacin has a great effect on Staphylococcus aureus isolated from subclinical bovine mastitis dairy herd. It seems that cefquinome sulfonamides has a great effect on isolated Staphylococcus aureus in vivo.

Keywords: ciprofloxacin, mastitis, Staphylococcus aureus, dairy herd

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188 Bovine Sperm Capacitation Promoters: The Comparison between Serum and Non-serum Albumin originated from Fish

Authors: Haris Setiawan, Phongsakorn Chuammitri, Korawan Sringarm, Montira Intanon, Anucha Sathanawongs

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Capacitation is a prerequisite to achieving sperm competency to penetrate the oocyte naturally occurring in vivo throughout the female reproductive tract and entangling secretory fluid and epithelial cells. One of the crucial compounds in the oviductal fluid which promotes capacitation is albumin, secreted in major concentrations. However, the difficulties in the collection and the inconsistency of the oviductal fluid composition throughout the estrous cycle have replaced its function with serum-based albumins such as bovine serum albumin (BSA). BSA has been primarily involved and evidenced for their stabilizing effect to maintain the acrosome intact during the capacitation process, modulate hyperactivation, and elevate the number of sperm bound to zona pellucida. Contrary to its benefits, the use of blood-derived products in the culture system is not sustainable and increases the risk of disease transmissions, such as Creutzfeldt-Jakob disease (CJD) and bovine spongiform encephalopathy (BSE). Moreover, it has been asserted that this substance is an aeroallergen that produces allergies and respiratory problems. In an effort to identify an alternative sustainable and non-toxic albumin source, the present work evaluated sperm reactions to a capacitation medium containing albumin derived from the flesh of the snakehead fish (Channa striata). Before examining the ability of this non-serum albumin to promote capacitation in bovine sperm, the presence of albumin was detected using bromocresol purple (BCP) at the level of 25% from snakehead fish extract. Following the SDS-PAGE and densitometric analysis, two major bands at 40 kDa and 47 kDa consisting of 57% and 16% of total protein loaded were detected as the potential albumin-related bands. Significant differences were observed in all kinematic parameters upon incubation in the capacitation medium. Moreover, consistently higher values were shown for the kinematic parameters related to hyperactivation, such as amplitude lateral head (ALH), velocity curve linear (VCL), and linearity (LIN) when sperm were treated with 3 mg/mL of snakehead fish albumin among other treatments. Likewise, substantial differences of higher acrosome intact presented in sperm upon incubation with various concentrations of snakehead fish albumin for 90 minutes, indicating that this level of snakehead fish albumin can be used to replace the bovine serum albumin. However, further study is highly required to purify the albumin from snakehead fish extract for more reliable findings.

Keywords: capacitation promoter, snakehead fish, non-serum albumin, bovine sperm

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187 Determining the Presence of Brucella abortus Antibodies by the Indirect Elisa Method in Bovine Bulk Milk and Risk Factors in the Peri-Urban Zones of Bamenda Cameroon

Authors: Cha-ah C. N., Awah N. J., Mouiche M. M. M.

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Brucellosis is a neglected zoonotic disease of animals and man caused by bacteria of genus Brucella. Though eradicated in some parts of the world, it remains endemic in sub-Saharan Africa including Cameroon. The aim of this study was to contribute to the epidemiology of brucellosis in the North-West region of Cameroon by detecting the presence of anti-Brucella antibodies in bovine bulk milk as this serves as a route of transmission from animals to man. A cross sectional study was conducted to determine the prevalence of Brucella abortus antibodies in bovine bulk milk in the peri-urban zones of Bamenda. One hundred bulk milk samples were collected from 100 herds and tested by milk I-ELISA test. The conducted study revealed the presence of anti-Brucella abortus antibodies in bovine bulk milk. The study revealed that bovine brucellosis is widespread in animal production systems in this area. The animal infection pressure in these systems has remained strong due to movement of livestock in search of pasture, co-existence of animal husbandry, communal sharing of grazing land, concentration of animals around water points, abortions in production systems, locality of production systems and failure to quarantine upon introduction of new animals. The circulation of Brucella abortus antibodies in cattle farms recorded in the study revealed potential public health implication and suggest economic importance of brucellosis to the cattle industry in the Northwest region of Cameroon. The risk for re-emergence and transmission of brucellosis is evident as a result of the co-existence of animal husbandry activities and social-cultural activities that promote brucellosis transmission. Well-designed countrywide, evidence-based studies of brucellosis are needed. These could help to generate reliable frequency and potential impact estimates, to identify Brucella reservoirs, and to propose control strategies of proven efficacy.

Keywords: brucellosis, bulk milk, northwest region Cameroon, prevalence

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186 Ability of Gastric Enzyme Extract of Adult Camel to Clot Bovine Milk

Authors: Boudjenah-Haroun Saliha, Isselnane Souad, Nouani Abdelwahab, Baaissa Babelhadj, Mati Abderrahmane

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Algeria is experiencing significant development of the dairy sector, where consumption of milk and milk products increased by 2.7 million tons in 2008 to 4,400,000 tons in 2013, and cheese production has reached 1640 tons in the year 2014 with average consumption of 0.7 kg/person/year. Although rennet is still the most used coagulating enzyme in cheese, its production has been growing worldwide shortage. This shortage is primarily due to a growing increase in the production and consumption of cheese, and the inability to increase in parallel the production of rennet. This shortage has caused very large fluctuations in its price). To overcome these obstacles, much research has been undertaken to find effective and competitive substitutes used industrially. For this, the selection of a local production of rennet substitute is desirable. It would allow a permanent supply with limited dependence on imports and price fluctuations. Investigations conducted by our research team showed that extracts coagulants from the stomachs of older camels are characterized by a coagulating power than those from younger camels. The objective of this work is to study the possibility of substituting commercial rennet coagulant by gastric enzymes from adult camels for coagulation bovine milk. Excerpts from the raw camel coagulants obtained are characterized through their teneures proteins and clotting and proteolytic activities. Milk clotting conditions by the action of these extracts were optimized. Milk clotting time all treated with enzyme preparations and under different conditions was calculated. Bovine rennet has been used for comparison. The results show that crude extracts from gastric adult camel can be good substituting bovine rennet.

Keywords: Algeria, camel, cheese, coagulation, gastric extracts, milk

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185 Phage Display-Derived Vaccine Candidates for Control of Bovine Anaplasmosis

Authors: Itzel Amaro-Estrada, Eduardo Vergara-Rivera, Virginia Juarez-Flores, Mayra Cobaxin-Cardenas, Rosa Estela Quiroz, Jesus F. Preciado, Sergio Rodriguez-Camarillo

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Bovine anaplasmosis is an infectious, tick-borne disease caused mainly by Anaplasma marginale; typical signs include anemia, fever, abortion, weight loss, decreased milk production, jaundice, and potentially death. Sick bovine can recover when antibiotics are administered; however, it usually remains as carrier for life, being a risk of infection for susceptible cattle. Anaplasma marginale is an obligate intracellular Gram-negative bacterium with genetic composition highly diverse among geographical isolates. There are currently no vaccines fully effective against bovine anaplasmosis; therefore, the economic losses due to disease are present. Vaccine formulation became a hard task for several pathogens as Anaplasma marginale, but peptide-based vaccines are an interesting proposal way to induce specific responses. Phage-displayed peptide libraries have been proved one of the most powerful technologies for identifying specific ligands. Screening of these peptides libraries is also a tool for studying interactions between proteins or peptides. Thus, it has allowed the identification of ligands recognized by polyclonal antiserums, and it has been successful for the identification of relevant epitopes in chronic diseases and toxicological conditions. Protective immune response to bovine anaplasmosis includes high levels of immunoglobulins subclass G2 (IgG2) but not subclass IgG1. Therefore, IgG2 from the serum of protected bovine can be useful to identify ligands, which can be part of an immunogen for cattle. In this work, phage display random peptide library Ph.D. ™ -12 was incubating with IgG2 or blood sera of immunized bovines against A. marginale as targets. After three rounds of biopanning, several candidates were selected for additional analysis. Subsequently, their reactivity with sera immunized against A. marginale, as well as with positive and negative sera to A. marginale was evaluated by immunoassays. A collection of recognized peptides tested by ELISA was generated. More than three hundred phage-peptides were separately evaluated against molecules which were used during panning. At least ten different peptides sequences were determined from their nucleotide composition. In this approach, three phage-peptides were selected by their binding and affinity properties. In the case of the development of vaccines or diagnostic reagents, it is important to evaluate the immunogenic and antigenic properties of the peptides. Immunogenic in vitro and in vivo behavior of peptides will be assayed as synthetic and as phage-peptide for to determinate their vaccine potential. Acknowledgment: This work was supported by grant SEP-CONACYT 252577 given to I. Amaro-Estrada.

Keywords: bovine anaplasmosis, peptides, phage display, veterinary vaccines

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184 Viscoelastic Characterization of Bovine Trabecular Bone Samples

Authors: I. Ramirez D. Edgar, J. Angeles H. José, Ruiz C. Osvaldo, H. Jacobo A. Victor, Ortiz P. Armando

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Knowledge of bone mechanical properties is important for bone substitutes design and fabrication, and more efficient prostheses development. The aim of this study is to characterize the viscoelastic behavior of bone specimens, through stress relaxation and fatigue tests performed to trabecular bone samples from bovine femoral heads. Relaxation tests consisted on preloading the samples at five different magnitudes and evaluate them for 1020 seconds, adjusting the results to a KWW mathematical model. Fatigue tests consisted of 700 load cycles and analyze their status at the end of the tests. As a conclusion we have that between relaxation stress and each preload there is linear relation and for samples with initial Young´s modulus greater than 1.5 GPa showed no effects due fatigue test loading cycles.

Keywords: bone viscoelasticity, fatigue test, stress relaxation test, trabecular bone properties

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183 The Effect of Curcumin on Cryopreserved Bovine Semen

Authors: Eva Tvrdá, Marek Halenár, Hana Greifová, Alica Mackovich, Faridullah Hashim, Norbert Lukáč

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Oxidative stress associated with semen cryopreservation may result in lipid peroxidation (LPO), DNA damage and apoptosis, leading to decreased sperm motility and fertilization ability. Curcumin (CUR), a natural phenol isolated from Curcuma longa Linn. has been presented as a possible supplement for a more effective semen cryopreservation because of its antioxidant properties. This study focused to evaluate the effects of CUR on selected oxidative stress parameters in cryopreserved bovine semen. 20 bovine ejaculates were split into two aliquots and diluted with a commercial semen extender containing CUR (50 μmol/L) or no supplement (control), cooled to 4 °C, frozen and kept in liquid nitrogen. Frozen straws were thawed in a water bath for subsequent experiments. Computer assisted semen analysis was used to evaluate spermatozoa motility, and reactive oxygen species (ROS) generation was quantified by using luminometry. Superoxide generation was evaluated with the NBT test, and LPO was assessed via the TBARS assay. CUR supplementation significantly (P<0.001) increased the spermatozoa motility and provided a significantly higher protection against ROS (P<0.001) or superoxide (P<0.01) overgeneration caused by semen freezing and thawing. Furthermore, CUR administration resulted in a significantly (P<0.01) lower LPO of the experimental semen samples. In conclusion, CUR exhibits significant ROS-scavenging activities which may prevent oxidative insults to cryopreserved spermatozoa and thus may enhance the post-thaw functional activity of male gametes.

Keywords: bulls, cryopreservation, curcumin, lipid peroxidation, reactive oxygen species, spermatozoa

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182 Development of Antimicrobial Properties Nutraceuticals: Gummy Candies with Addition of Bovine Colostrum, Essential Oils and Probiotics

Authors: E. Bartkiene, M. Ruzauskas, V. Lele, P. Zavistanaviciute, J. Bernatoniene, V. Jakstas, L. Ivanauskas, D. Zadeike, D. Klupsaite, P. Viskelis, J. Bendoraitiene, V. Navikaite-Snipaitiene, G. Juodeikiene

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In this study, antimicrobial nutraceuticals; gummy candies (GC) from bovine colostrum (BC), essential oils (EOs), probiotic lactic acid bacteria (PLAB), and their combinations, were developed. For antimicrobial GC preparation, heteropolysaccharide (agar) was used. The antimicrobial properties of EOs (Eugenia caryophyllata, Thymus vulgaris, Citrus reticulata L., Citrus paradisi L.), BC, L. paracasei LUHS244, L. plantarum LUHS135, and their combinations against pathogenic bacteria strains (Streptococcus mutans, Enterococcus faecalis, Staphylococcus aureus, Salmonella enterica, Escherichia coli, Proteus mirabilis, and Pseudomonas aeruginosa) were evaluated. The highest antimicrobial properties by EO’s (Eugenia caryophyllata and Thymus vulgaris) were established. The optimal ingredients composition for antimicrobial GC preparation was established, which incorporate the BC fermented with L. paracasei LUHS244 in combination with Thymus vulgaris or Eugenia caryophyllata. These ingredients showed high inhibition properties of all tested pathogenic strains (except Pseudomonas aeruginosa). Antimicrobial GC formula consisting of thyme EO (up to 0.2%) and fermented BC (up to 3%), and for taste masking, mandarin or grapefruit EOs (up to 0.2%) was used. Developed GC high overall acceptability and antimicrobial properties, thus, antimicrobial GC could be a preferred form of nutraceuticals. This study was fulfilled with the support of the LSMU-KTU joint project.

Keywords: antimicrobial activity, bovine colostrum, essential oil, gummy candy, probiotic

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181 Prevalence of Eimeria spp in Cattle in Anatolia Region, Turkey

Authors: Nermin Isik, Onur Ceylan

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Bovine coccidiosis is a protozoan infection caused by coccidia parasites of the genus Eimeria which develops in the small and the large intestine. The aim of this study was to determine the prevalence of Eimeria spp. in cattle. This study was conducted between March 2014 and April 2015, involved 624 fecal samples of cattle. Cattle were grouped according to their age as follows: 6-12, 12-24 and >24 months. In a retrospective study from these faecal samples of cattle submitted to the University of Selcuk, Faculty of Veterinary Medicine, Laboratory of Parasitology were evaluated regarding the prevalence of Eimeria spp. In the laboratory, faecal samples were examined by Fulleborn saturated salt flotation technique and examined under a microscope for the presence of protozoan oocysts. Eimeria oocysts were found in 4.8% of all the samples. Eimeria infection was detected in 11.8%, 5.3% and 0.4% of the cattle in the age groups, respectively. This study showed that Eimeria infection was commonly seen in 6-24-month-old cattle. Further epidemiological investigation on economic significance and species composition of bovine coccidiosis needs to be pursued.

Keywords: cattle, diarrhea, Eimeria spp, Turkey

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180 Regulation of Differentiating Intramuscular Stromal Vascular Cells Isolated from Hanwoo Beef Cattle by Retinoic Acid and Calcium

Authors: Seong Gu Hwang, Young Kyoon Oh, Joseph F. dela Cruz

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Marbling, or intramuscular fat, has been consistently identified as one of the top beef quality problems. Intramuscular adipocytes distribute throughout the perimysial connective tissue of skeletal muscle and are the major site for the deposition of intramuscular fat, which is essential for the eating quality of meat. The stromal vascular fraction of the skeletal muscle contains progenitor cells that can be enhanced to differentiate to adipocytes and increase intramuscular fat. Primary cultures of bovine intramuscular stromal vascular cells were used in this study to elucidate the effects of extracellular calcium and retinoic acid concentration on adipocyte differentiation. Cell viability assay revealed that even at different concentrations of calcium and retinoic acid, there was no significant difference on cell viability. Monitoring of the adipocyte differentiation showed that bovine intramuscular stromal vascular cells cultured in a low concentration of extracellular calcium and retinoic acid had a better degree of fat accumulation. The mRNA and protein expressions of PPARγ, C/EBPα, SREBP-1c and aP2 were analyzed and showed a significant upregulation upon the reduction in the level of extracellular calcium and retinoic acid. The upregulation of these adipogenic related genes means that the decreasing concentration of calcium and retinoic acid is able to stimulate the adipogenic differentiation of bovine intramuscular stromal vascular cells. To further elucidate the effect of calcium, the expression level of calreticulin was measured. Calreticulin which is known to be an inhibitor of PPARγ was down regulated by the decreased level of calcium and retinoic acid in the culture media. The same tendency was observed on retinoic acid receptors RARα and CRABP-II. These receptors are recognized as adipogenic inhibitors, and the downregulation of their expression allowed a better level of differentiation in bovine intramuscular stromal vascular cells. In conclusion, data show that decreasing the level of extracellular calcium and retinoic acid can significantly promote adipogenesis in intramuscular stromal vascular cells of Hanwoo beef cattle. These findings may provide new insights in enhancing intramuscular adipogenesis and marbling in beef cattle.

Keywords: calcium, calreticulin, hanwoo beef, retinoic acid

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179 Clinical Presentation and Immune Response to Intramammary Infection of Holstein-Friesian Heifers with Isolates from Two Staphylococcus aureus Lineages

Authors: Dagmara A. Niedziela, Mark P. Murphy, Orla M. Keane, Finola C. Leonard

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Staphylococcus aureus is the most frequent cause of clinical and subclinical bovine mastitis in Ireland. Mastitis caused by S. aureus is often chronic and tends to recur after antibiotic treatment. This may be due to several virulence factors, including attributes that enable the bacterium to internalize into bovine mammary epithelial cells, where it may evade antibiotic treatment, or evade the host immune response. Four bovine-adapted lineages (CC71, CC97, CC151 and ST136) were identified among a collection of Irish S. aureus mastitis isolates. Genotypic variation of mastitis-causing strains may contribute to different presentations of the disease, including differences in milk somatic cell count (SCC), the main method of mastitis detection. The objective of this study was to investigate the influence of bacterial strain and lineage on host immune response, by employing cell culture methods in vitro as well as an in vivo infection model. Twelve bovine adapted S. aureus strains were examined for internalization into bovine mammary epithelial cells (bMEC) and their ability to induce an immune response from bMEC (using qPCR and ELISA). In vitro studies found differences in a variety of virulence traits between the lineages. Strains from lineages CC97 and CC71 internalized more efficiently into bovine mammary epithelial cells (bMEC) than CC151 and ST136. CC97 strains also induced immune genes in bMEC more strongly than strains from the other 3 lineages. One strain each of CC151 and CC97 that differed in their ability to cause an immune response in bMEC were selected on the basis of the above in vitro experiments. Fourteen first-lactation Holstein-Friesian cows were purchased from 2 farms on the basis of low SCC (less than 50 000 cells/ml) and infection free status. Seven cows were infected with 1.73 x 102 c.f.u. of the CC97 strain (Group 1) and another seven with 5.83 x 102 c.f.u. of the CC151 strain (Group 2). The contralateral quarter of each cow was inoculated with PBS (vehicle). Clinical signs of infection (temperature, milk and udder appearance, milk yield) were monitored for 30 days. Blood and milk samples were taken to determine bacterial counts in milk, SCC, white blood cell populations and cytokines. Differences in disease presentation in vivo between groups were observed, with two animals from Group 2 developing clinical mastitis and requiring antibiotic treatment, while one animal from Group 1 did not develop an infection for the duration of the study. Fever (temperature > 39.5⁰C) was observed in 3 animals from Group 2 and in none from Group 1. Significant differences in SCC and bacterial load between groups were observed in the initial stages of infection (week 1). Data is also being collected on cytokines and chemokines secreted during the course of infection. The results of this study suggest that a strain from lineage CC151 may cause more severe clinical mastitis, while a strain from lineage CC97 may cause mild, subclinical mastitis. Diversity between strains of S. aureus may therefore influence the clinical presentation of mastitis, which in turn may influence disease detection and treatment needs.

Keywords: Bovine mastitis, host immune response, host-pathogen interactions, Staphylococcus aureus

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178 Fungi Isolated from House Flies (Diptera: Muscidae) on Penned Cattle in South Texas

Authors: Cherity A. Ysquierdo, Pia U. Olafson, Donald B. Thomas

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Musca domestica L. were collected from cattle diagnosed with bovine ringworm to evaluate the potential of the house fly to disseminate Trichophyton verrucosum E. Bodin, a fungal dermatophyte that is the causative agent for ringworm in cattle. Fungal isolates were cultured from 45 individual flies on supplemented Sabouraud dextrose agar, and isolates were identified using morphological and microscopic approaches. Each isolate was further identified by PCR amplification of the ribosomal DNA locus with fungal specific primers and subsequent amplicon sequencing. No T. verrucosum were identified using these approaches. However, 36 different fungal species representing 17 genera were cultured from these flies, including several allergenic and pathogenic species. Several species within the fungal orders Hypocreales, Microascales, Onygenales, Saccharomycetales, Xylaniales, and Agaricales were observed for the first time on house flies. The most frequent fungus recovered was Cladosporium cladosporoides, which is known to be a ubiquitous, airborne allergen.

Keywords: bovine ringworm, Cladosporium, dermatophyte, Musca domestica

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177 Phase Diagrams and Liquid-Liquid Extraction in Aqueous Biphasic Systems Formed by Polyethylene Glycol and Potassium Sodium Tartrate at 303.15 K

Authors: Amanda Cristina de Oliveira, Elias de Souza Monteiro Filho, Roberta Ceriani

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Liquid-liquid extraction in aqueous two-phase systems (ATPSs) constitutes a powerful tool for purifying bio-materials, such as cells, organelles, proteins, among others. In this work, the extraction of the bovine serum albumin (BSA) has been studied in systems formed by polyethylene glycol (PEG) (1500, 4000, and 6000 g.mol⁻¹) + potassium sodium tartrate + water at 303.15°K. Phase diagrams were obtained by turbidimetry and Merchuk’s method (1998). The experimental tie-lines were described using the Othmer-Tobias and Bancroft correlations. ATPSs were correlated with the nonrandom two-liquid (NRTL) model. The results were considered excellent according to global root-mean-square deviations found which were between 0,72 and 1,13%. The concentrations of the proteins in each phase were determined by spectrophotometry at 280 nm, finding partition efficiencies greater than 71%.

Keywords: aqueous two phases systems, bovine serum albumin , liquid-liquid extraction, polyethylene glycol

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176 Prevalence and Mechanisms of Antibiotic Resistance in Escherichia coli Isolated from Mastitic Dairy Cattle in Canada

Authors: Satwik Majumder, Dongyun Jung, Jennifer Ronholm, Saji George

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Bovine mastitis is the most common infectious disease in dairy cattle, with major economic implications for the dairy industry worldwide. Continuous monitoring for the emergence of antimicrobial resistance (AMR) among bacterial isolates from dairy farms is vital not only for animal husbandry but also for public health. In this study, the prevalence of AMR in 113 Escherichia coli isolates from cases of bovine clinical mastitis in Canada was investigated. Kirby-Bauer disk diffusion test with 18 antibiotics and microdilution method with three heavy metals (copper, zinc, and silver) was performed to determine the antibiotic and heavy-metal susceptibility. Resistant strains were assessed for efflux and ß-lactamase activities besides assessing biofilm formation and hemolysis. Whole-genome sequences for each of the isolates were examined to detect the presence of genes corresponding to the observed AMR and virulence factors. Phenotypic analysis revealed that 32 isolates were resistant to one or more antibiotics, and 107 showed resistance against at least one heavy metal. Quinolones and silver were the most efficient against the tested isolates. Among the AMR isolates, AcrAB-TolC efflux activity and ß-lactamase enzyme activities were detected in 13 and 14 isolates, respectively. All isolates produced biofilm but with different capacities, and 33 isolates showed α-hemolysin activity. A positive correlation (Pearson r = +0.89) between efflux pump activity and quantity of biofilm was observed. Genes associated with aggregation, adhesion, cyclic di-GMP, quorum sensing were detected in the AMR isolates, corroborating phenotype observations. This investigation showed the prevalence of AMR in E. coli isolates from bovine clinical mastitis. The results also suggest the inadequacy of antimicrobials with a single mode of action to curtail AMR bacteria with multiple mechanisms of resistance and virulence factors. Therefore, it calls for combinatorial therapy for the effective management of AMR infections in dairy farms and combats its potential transmission to the food supply chain through milk and dairy products.

Keywords: antimicrobial resistance, E. coli, bovine mastitis, antibiotics, heavy-metals, efflux pump, ß-lactamase enzyme, biofilm, whole-genome sequencing

Procedia PDF Downloads 178
175 Effects of Li2O Doping on Mechanical and Electrical Properties of Bovine Hydroxyapatite Composites (BHA)

Authors: Sibel Daglilar, Isil Kerti, Murat Karagoz, Fatih Dumludag, Oguzhan Gunduz, Faik Nuzhet Oktar

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Hydroxyapatite (HA) materials have common use in bone repairing due to its ability to accelerate the bone growth around the implant. In spite of being a biocompatible and bioactive material, HA has a limited usage as an implant material because of its weak mechanical properties. HA based composites are required to improve the strength and toughness properties of the implant materials without compromising of biocompatibility. The excellent mechanical properties and higher biocompatibilities are expected from each of biomedical composites. In this study, HA composites were synthesized by using bovine bone reinforced doped with different amount of (wt.%) Li2O. The pressed pellets were sintered at various sintering temperatures between 1000ºC and 1300°C, and mechanical, electrical properties of the obtained products were characterized. In addition to that, in vitro stimulated body fluid (SBF) tests for these samples were conducted. The most suitable composite composition for biomedical applications was discussed among the composites studied.

Keywords: biocomposites, sintering temperature, biocompatibility, electrical property, conductivity, mechanical property

Procedia PDF Downloads 373
174 The Effect of Ethylene Glycol on Cryopreserved Bovine Oocytes

Authors: Sri Wahjuningsih, Nur Ihsan, Hadiah

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In the embryo transfer program, to address the limited production of embryos in vivo, in vitro embryo production has become an alternative approach that is relatively inexpensive. One potential source of embryos that can be developed is to use immature oocytes then conducted in vitro maturation and in vitro fertilization. However, obstacles encountered were oocyte viability mammals have very limited that it cannot be stored for a long time, so we need oocyte cryopreservation. The research was conducted to know the optimal concentration use of ethylene glycol as a cryoprotectant on oocytes freezing.Material use in this research was immature oocytes; taken from abbatoir which was aspirated from follicle with diameter 2-6 mm. Concentration ethylen glycol used were 0,5 M, I M, 1,5 M and 2M. The freezing method used was conventional method combined with a five-step protocol washing oocytes from cryoprotectant after thawing. The result showed that concentration ethylen glycol have the significant effect (P<0.05) on oocytes quality after thawing and in vitro maturation. It was concluded that concentration 1,5 M was the best concentration for freezing oocytes using conventional method.

Keywords: bovine, conventional freezing, ethylen glycol, oocytes

Procedia PDF Downloads 326
173 Effects of Stiffness on Endothelial Cells Behavior

Authors: Forough Ataollahi, Sumit Pramanik, Belinda Pingguan-Murphy, Wan Abu Bakar Bin Wan Abas, Noor Azuan Bin Abu Osman

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Endothelium proliferation is an important process in cardiovascular homeostasis and can be regulated by extracellular environment, as cells can actively sense mechanical environment. In this study, we evaluated endothelial cell proliferation on PDMS/alumina (Al2O3) composites and pure PDMS. The substrates were prepared from pure PDMS and its composites with 5% and 10% Al2O3 at curing temperature 50˚C for 4 h and then characterized by mechanical, structural and morphological analyses. Higher stiffness was found in the composites compared to the pure PDMS substrate. Cell proliferation of the cultured bovine aortic endothelial cells on substrate materials were evaluated via Resazurin assay and 1, 1’-Dioctadecyl-1, 3, 3, 3’, 3’-Tetramethylindocarbocyanine Perchlorate-Acetylated LDL (Dil-Ac-LDL) cell staining, respectively. The results revealed that stiffer substrates promote more endothelial cells proliferation to the less stiff substrates. Therefore, this study firmly hypothesizes that the stiffness elevates endothelial cells proliferation.

Keywords: stiffness, proliferation, bovine aortic endothelial cells, extra cellular matrix, vascular

Procedia PDF Downloads 317
172 Assessment of Mammary Gland Immunity and Therapeutic Potential of Topical Herbal Gel against Bovine Subclinical Mastitis

Authors: Mukesh N. Kher, Anju P. Kunjadia, Dev S. Nauriyal, Chaitanya G. Joshi, Navin R. Sheth, Vaibhav D. Bhatt

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In-vivo immunotherapeutic potential on cytokines production and antibacterial activity of a topical herbal gel was evaluated in two breeds of cattle in bovine subclinical mastitis. The response to treatment was evaluated by enumerating somatic cell count (SCC), determining total bacterial count and studying the expression of different cytokines like (interleukin 6, 8, 12, GMCSF, interferon–γ and TNF‑α). The pre‑ and post‑treatment SCC in mastitic quarters did not differ statistically-significantly. However, total bacterial count declined significantly from day 0 onwards in both the breeds. Significant differences (P < 0.01) were observed in all types of cytokines production on day 0, 5, and 21 post last treatments in both the breeds. The comparison of cytokine expression profiles between crossbred and Gir cattle affirmed a significant difference in expression of IL-6 and TNF-α. The topical herbal gel showed immunomodulatory and antimicrobial activities in subclinical mastitis, and therefore the work supports its use as substitute herbal therapy against subclinical mastitis in bovines.

Keywords: antibacterial activity, immunomodulation, herbal gel, subclinical mastitis

Procedia PDF Downloads 235
171 In vitro Effects of Salvia officinalis on Bovine Spermatozoa

Authors: Eva Tvrdá, Boris Botman, Marek Halenár, Tomáš Slanina, Norbert Lukáč

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In vitro storage and processing of animal semen represents a risk factor to spermatozoa vitality, potentially leading to reduced fertility. A variety of substances isolated from natural sources may exhibit protective or antioxidant properties on the spermatozoon, thus extending the lifespan of stored ejaculates. This study compared the ability of different concentrations of the Salvia officinalis extract on the motility, mitochondrial activity, viability and reactive oxygen species (ROS) production by bovine spermatozoa during different time periods (0, 2, 6 and 24 h) of in vitro culture. Spermatozoa motility was assessed using the Computer-assisted sperm analysis (CASA) system. Cell viability was examined using the metabolic activity MTT assay, the eosin-nigrosin staining technique was used to evaluate the sperm viability and ROS generation was quantified using luminometry. The CASA analysis revealed that the motility in the experimental groups supplemented with 0.5-2 µg/mL Salvia extract was significantly lower in comparison with the control (P<0.05; Time 24 h). At the same time, a long-term exposure of spermatozoa to concentrations ranging between 0.05 µg/mL and 2 µg/mL had a negative impact on the mitochondrial metabolism (P<0.05; Time 24 h). The viability staining revealed that 0.001-1 µg/mL Salvia extract had no effects on bovine male gametes, however 2 µg/mL Salvia had a persisting negative effect on spermatozoa (P<0.05). Furthermore 0.05-2 µg/mL Salvia exhibited an immediate ROS-promoting effect on the sperm culture (P>0.05; Time 0 h and 2 h), which remained significant throughout the entire in vitro culture (P<0.05; Time 24 h). Our results point out to the necessity to examine specific effects the biomolecules present in Salvia officinalis may have individually or collectively on the in vitro sperm vitality and oxidative profile.

Keywords: bulls, CASA, MTT test, reactive oxygen species, sage, Salvia officinalis, spermatozoa

Procedia PDF Downloads 311
170 Selection of Developmental Stages of Bovine in vitro-Derived Blastocysts Prior to Vitrification and Embryo Transfer: Implications for Cattle Breeding Programs

Authors: Van Huong Do, Simon Walton, German Amaya, Madeline Batsiokis, Sally Catt, Andrew Taylor-Robinson

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Identification of the most suitable stages of bovine in vitro-derived blastocysts (early, expanded and hatching) prior to vitrification is a straightforward process that facilitates the decision as to which blastocyst stage to use for transfer of fresh and vitrified embryos. Research on in vitro evaluation of suitable stages has shown that the more advanced developmental stage of blastocysts is recommended for fresh embryo transfer while the earlier stage is proposed for embryo transfer following vitrification. There is, however, limited information on blastocyst stages using in vivo assessment. Hence, the aim of the present study was to determine the optimal stage of a blastocyst for vitrification and embryo transfer through a two-step procedure of embryo transfer followed by pregnancy testing at 35, 60 and 90 days of pregnancy. 410 good quality oocytes aspirated by the ovum pick-up technique from 8 donor cows were subjected to in vitro embryo production, vitrification and embryo transfer. Good quality embryos were selected, subjected to vitrification and embryo transfer. Subsequently, 77 vitrified embryos at different blastocyst stages were transferred to synchronised recipient cows. The overall cleavage and blastocyst rates of oocytes were 68.8% and 41.7%, respectively. In addition, the fertility and blastocyst production of 6 bulls used for in vitro fertilization was examined and shown to be statistically different (P<0.05). Results of ongoing pregnancy trials conducted at 35 days, 60 days and 90 days will be discussed. However, preliminary data indicate that individual bulls demonstrate distinctly different fertility performance in vitro. Findings from conception rates would provide a useful tool to aid selection of bovine in vitro-derived embryos for vitrification and embryo transfer in commercial settings.

Keywords: blastocyst, embryo transfer, in vitro-derived embryos, ovum pick-up, vitrification

Procedia PDF Downloads 265
169 Reduction of the Microbial Load of Biocontaminated Bovine Milk Using Grounding with Copper Wire

Authors: Claudivan Costa de Lima, Angelo da Silva Monteiro

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With the aim of evaluating the effects of grounding with copper wire on the reduction of the microbial load of biocontaminated milk samples and on their acidification over time, two complementary experiments were carried out. In the first, the treatments consisted of: i) raw milk sample (control), ii) slow pasteurization, iii) grounding with copper wire and, iv) contact with copper ring. Analyzes of total, thermoresistant and mesophilic coliforms were performed 30 minutes after the application of these treatments. In the second experiment, under the same conditions as the first, measurements of pH and Dornic acidity were performed at 0, 0.5, 2, 4, 8, 12, and 24 h from the installation of the experiment. Pasteurization eliminated almost all groups of bacteria present in the milk samples while grounding only allowed reductions in the population of thermotolerant coliforms and mesophiles, both greater than 95%, maintaining, however, unchanged the amounts of total coliforms. The copper ring, in turn, had no effect on the microbiological parameters studied. The reduction in the population of mesophiles in grounded milk samples, contrary to what happened with pasteurized milk, was not enough to inhibit the acidification process over the experimental period.

Keywords: pasteurization, low frequency electric current, thermotolerant coliforms, mesophiles in bovine milk

Procedia PDF Downloads 73
168 In vitro Antimicrobial Resistance Pattern of Bovine Mastitis Bacteria in Ethiopia

Authors: Befekadu Urga Wakayo

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Introduction: Bacterial infections represent major human and animal health problems in Ethiopia. In the face of poor antibiotic regulatory mechanisms, development of antimicrobial resistance (AMR) to commonly used drugs has become a growing health and livelihood threat in the country. Monitoring and control of AMR demand close coloration between human and veterinary services as well as other relevant stakeholders. However, risk of AMR transfer from animal to human population’s remains poorly explored in Ethiopia. This systematic research literature review attempted to give an overview on AMR challenges of bovine mastitis bacteria in Ethiopia. Methodology: A web based research literature search and analysis strategy was used. Databases are considered including; PubMed, Google Scholar, Ethiopian Veterinary Association (EVA) and Ethiopian Society of Animal Production (ESAP). The key search terms and phrases were; Ethiopia, dairy, cattle, mastitis, bacteria isolation, antibiotic sensitivity and antimicrobial resistance. Ultimately, 15 research reports were used for the current analysis. Data extraction was performed using a structured Microsoft Excel format. Frequency AMR prevalence (%) was registered directly or calculated from reported values. Statistical analysis was performed on SPSS – 16. Variables were summarized by giving frequencies (n or %), Mean ± SE and demonstrative box plots. One way ANOVA and independent t test were used to evaluate variations in AMR prevalence estimates (Ln transformed). Statistical significance was determined at p < 0.050). Results: AMR in bovine mastitis bacteria was investigated in a total of 592 in vitro antibiotic sensitivity trials involving 12 different mastitis bacteria (including 1126 Gram positive and 77 Gram negative isolates) and 14 antibiotics. Bovine mastitis bacteria exhibited AMR to most of the antibiotics tested. Gentamycin had the lowest average AMR in both Gram positive (2%) and negative (1.8%) bacteria. Gram negative mastitis bacteria showed higher mean in vitro resistance levels to; Erythromycin (72.6%), Tetracycline (56.65%), Amoxicillin (49.6%), Ampicillin (47.6%), Clindamycin (47.2%) and Penicillin (40.6%). Among Gram positive mastitis bacteria higher mean in vitro resistance was observed in; Ampicillin (32.8%), Amoxicillin (32.6%), Penicillin (24.9%), Streptomycin (20.2%), Penicillinase Resistant Penicillin’s (15.4%) and Tetracycline (14.9%). More specifically, S. aurues exhibited high mean AMR against Penicillin (76.3%) and Ampicillin (70.3%) followed by Amoxicillin (45%), Streptomycin (40.6%), Tetracycline (24.5%) and Clindamycin (23.5%). E. coli showed high mean AMR to Erythromycin (78.7%), Tetracycline (51.5%), Ampicillin (49.25%), Amoxicillin (43.3%), Clindamycin (38.4%) and Penicillin (33.8%). Streptococcus spp. demonstrated higher (p =0.005) mean AMR against Kanamycin (> 20%) and full sensitivity (100%) to Clindamycin. Overall, mean Tetracycline (p = 0.013), Gentamycin (p = 0.001), Polymixin (p = 0.034), Erythromycin (p = 0.011) and Ampicillin (p = 0.009) resistance increased from the 2010’s than the 2000’s. Conclusion; the review indicated a rising AMR challenge among bovine mastitis bacteria in Ethiopia. Corresponding, public health implications demand a deeper, integrated investigation.

Keywords: antimicrobial resistance, dairy cattle, Ethiopia, Mastitis bacteria

Procedia PDF Downloads 212
167 Development and Utilization of Keratin-Fibrin-Gelatin Composite Films as Potential Material for Skin Tissue Engineering Application

Authors: Sivakumar Singaravelu, Giriprasath Ramanathan, M. D. Raja, Uma Tirichurapalli Sivagnanam

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The goal of the present study was to develop and evaluate composite film for tissue engineering application. The keratin was extracted from bovine horn and used for preparation of keratin (HK), physiologically clotted fibrin (PCF) and gelatin (G) blend films in different stoichiometric ratios (1:1:1, 1:1:2 and 1:1:3) by using solvent casting method. The composite films (HK-PCF-G) were characterized physiochemically using Fourier Transform Infrared Spectroscopy (FTIR), Differential Scanning Calorimetry (DSC), Thermogravimetric Analysis (TGA) and Scanning Electron Microscopy (SEM). The mechanical properties of the composite films were analyzed. The results of tensile strength show that ultimate strength and elongation were 10.72 Mpa and 4.83 MPA respectively for 1:1:3 ratio combination. The SEM image showed a slight smooth surface for 1:1:3 ratio combination compared to other films. In order to impart antibacterial activities, the composite films were loaded with Mupirocin (MP) to act against infection. The composite films acted as a suitable carrier to protect and release the drug in a controlled manner. This developed composite film would be a suitable alternative material for tissue engineering application.

Keywords: bovine horn, keratin, fibrin, gelatin, tensile strength

Procedia PDF Downloads 421
166 Electrochemical Bioassay for Haptoglobin Quantification: Application in Bovine Mastitis Diagnosis

Authors: Soledad Carinelli, Iñigo Fernández, José Luis González-Mora, Pedro A. Salazar-Carballo

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Mastitis is the most relevant inflammatory disease in cattle, affecting the animal health and causing important economic losses on dairy farms. This disease takes place in the mammary gland or udder when some opportunistic microorganisms, such as Staphylococcus aureus, Streptococcus agalactiae, Corynebacterium bovis, etc., invade the teat canal. According to the severity of the inflammation, mastitis can be classified as sub-clinical, clinical and chronic. Standard methods for mastitis detection include counts of somatic cells, cell culture, electrical conductivity of the milk, and California test (evaluation of “gel-like” matrix consistency after cell lysed with detergents). However, these assays present some limitations for accurate detection of subclinical mastitis. Currently, haptoglobin, an acute phase protein, has been proposed as novel and effective biomarker for mastitis detection. In this work, an electrochemical biosensor based on polydopamine-modified magnetic nanoparticles (MNPs@pDA) for haptoglobin detection is reported. Thus, MNPs@pDA has been synthesized by our group and functionalized with hemoglobin due to its high affinity to haptoglobin protein. The protein was labeled with specific antibodies modified with alkaline phosphatase enzyme for its electrochemical detection using an electroactive substrate (1-naphthyl phosphate) by differential pulse voltammetry. After the optimization of assay parameters, the haptoglobin determination was evaluated in milk. The strategy presented in this work shows a wide range of detection, achieving a limit of detection of 43 ng/mL. The accuracy of the strategy was determined by recovery assays, being of 84 and 94.5% for two Hp levels around the cut off value. Milk real samples were tested and the prediction capacity of the electrochemical biosensor was compared with a Haptoglobin commercial ELISA kit. The performance of the assay has demonstrated this strategy is an excellent and real alternative as screen method for sub-clinical bovine mastitis detection.

Keywords: bovine mastitis, haptoglobin, electrochemistry, magnetic nanoparticles, polydopamine

Procedia PDF Downloads 132
165 Collection, Cryopreservation, and Fertilizing Potential of Bovine Spermatozoa Collected from the Epididymis Evaluated by Conventional Techniques and by Flow Cytometry

Authors: M. H. Moreira da Silva, L. Valadao, F. Moreira da Silva

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In the present study, the fertilizing capacity of bovine spermatozoa was evaluated before and after its cryopreservation. For this, the testicles of 100 bulls slaughtered on Terceira Island were dissected, the epididymal tails were separated, and semen was recovered by the flotation method and then evaluated by phase contrast microscopy and by flow cytometry. For phase contrast microscopy, a drop of semen was used to evaluate the percentage of motile spermatozoa (from 0 to 100%) and motility (from 0 to 5). After determining the concentration and the abnormal forms, semen was diluted to a final concentration of 50 x 106 spz/ml and evaluated by flow cytometer for membrane and acrosome integrity using the conjugation of fluorescent probes propidium iodide (PI) and Arachis hypogea agglutinin (FITC-PNA). Freezing was carried out in a programmable semen freezer, using 0.25 ml straws, in a total of 20 x 106 viable sperm per straw with glycerol as a cryoprotectant in a final concentration of 0.58 M. It was observed that, on average, a total of 7.25 ml of semen was collected from each bull. The viability and vitality rates were respectively 83.22 ± 7.52% and 3.8 ± 0.4 before freezing, decreasing to 58.81 ± 11.99% and 3.6 ± 0.6, respectively, after thawing. Regarding cytoplasmic droplets, it was observed that a high percentage of spermatozoa had medial cytoplasmic droplets (38.47%), with only 3.32% and 0.15% presenting proximal and distal cytoplasmic drops, respectively. By flow cytometry, it was observed that before freezing, the percentage of sperm with the damaged plasma membrane and intact acrosome was 3.61 ± 0.99%, increasing slightly to 4.21 ± 1.86% after cryopreservation (p<0.05). Regarding spermatozoa with damaged plasma membrane and acrosome, the percentage before freezing was 3.37±1.87%, increasing to 4.34 ±1.16% after thawing, and no significant differences were observed between these two values. For the percentage of sperm with the intact plasma membrane and damaged acrosome, this value was 2.04 ± 2.34% before freezing, decreasing to 0.89 ± 0.48% after thawing (p<0.05). The percentage of sperm with the intact plasma membrane and acrosome before freezing was 90.99±2.75%, with a slight decrease to 90.57±3.15% after thawing (p<0.05). From this study, it can be clearly concluded that, after the slaughtering of bulls, the spermatozoa can be recovered from the epididymis and cryopreserved, maintaining an excellent rate of sperm viability and quality after thawing.

Keywords: bovine semen, epididymis, cryopreservation, fertility assessment

Procedia PDF Downloads 57
164 Insights on the Halal Status of Antineoplastic and Immunomodulating Agents and Nutritional and Dietary Supplements in Malaysia

Authors: Suraiya Abdul Rahman, Perasna M. Varma, Amrahi Buang, Zhari Ismail, Wan Rosalina W. Rosli, Ahmad Rashidi M. Tahir

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Background: Muslims has the obligation to ensure that everything they consume including medicines should be halal. With the growing demands for halal medicines in October 2012, Malaysia has launched the world's first Halal pharmaceutical standards called Malaysian Standard MS 2424:2012 Halal Pharmaceuticals-General Guidelines to serve as a basic requirement for halal pharmaceuticals in Malaysia. However, the biggest challenge faced by pharmaceutical companies to comply is finding the origin or source of the ingredients and determine their halal status. Aim: This study aims to determine the halal status of the antineoplastic and immunomodulating agents, and nutritional and dietary supplements by analysing the origin of their active pharmaceutical ingredients (API) and excipients to provide an insight on the common source and halal status of pharmaceutical ingredients and an indication on adjustment required in order to be halal compliance. Method: The ingredients of each product available in a government hospital in central of Malaysia and their sources were determined from the product package leaflets, information obtained from manufacturer, reliable websites and standard pharmaceutical references. The ingredients were categorised as halal, musbooh or haram based on the definition set in MS2424. Results: There were 162 medications included in the study where 123 (76%) were under the antineoplastic and immunomodulating agents group, while 39 (24%) were nutritional and dietary supplements. In terms of the medication halal status, the proportion of halal, musbooh and haram were 40.1% (n=65), 58.6% (n=95) and 1.2% (n=2) respectively. With regards to the API, there were 89 (52%) different active ingredient identified for antineoplastic and immunomodulating agents with the proportion of 89.9% (n=80) halal and 10.1% (n=9) were mushbooh. There were 83 (48%) active ingredient from the nutritional and dietary supplements group with proportion of halal and masbooh were 89.2% (n=74) and 10.8% (n=9) respectively. No haram APIs were identified in all therapeutic classes. There were a total of 176 excipients identified from the products ranges. It was found that majority of excipients are halal with the proportion of halal, masbooh and haram were at 82.4% (n=145), 17% (n=30) and 0.6% (n=1) respectively. With regards of the sources of the excipeints, most of masbooh excipients (76.7%, n = 23) were classified as masbooh because they have multiple possible origin which consist of animals, plant or others. The remaining 13.3% and 10% were classified as masbooh due to their ethanol and land animal origin respectively. The one haram excipient was gelatine of bovine-porcine origin. Masbooh ingredients found in this research were glycerol, tallow, lactose, polysorbate, dibasic sodium phosphate, stearic acid and magnesium stearate. Ethanol, gelatine, glycerol and magnesium stearate were the most common ingredients classified as mushbooh. Conclusion: This study shows that most API and excipients are halal. However the majority of the medicines in these products categories are mushbooh due to certain excipients only, which could be replaced with halal alternative excipients. This insight should encourage the pharmaceutical products manufacturers to go for halal certification to meet the increasing demand for Halal certified medications for the benefit of mankind.

Keywords: antineoplastic and immunomodulation agents, halal pharmaceutical, MS2424, nutritional and dietary supplements

Procedia PDF Downloads 269