Journal of Life Sciences Volume 5 Number (5)
J LS
Journal of Life Sciences
Volume 5, Number 5, May 2011 (Serial Number 37)
Contents
Research Papers
327
Macronutrient Composition and Digestibility of Extruded and Fermented Soya Protein Products
Anthony Ojokoh and Yimin Wei 332
Hormonal (Thyroxin, Cortisol) and Immunological (Leucocytes) Responses to Cistern Size and Heat Stress in Tunisia
Rim Ben Younes, Moez Ayadi, Taha Najar, Margherita Caccamo, Iris Schadt and Moncef Ben M’Rad 339
Occurrence of Extended-Spectrum β-lactamase Producing Enterobacteriaceae (ESBLPE) among
Primary School Pupil in Obafemi-Owode, Nigeria
Akinduti Paul Akinniyi, Akinbo John Adeolu, Adenuga W. Funmilayo, Ejilude Oluwaseun, Umahoin Kingsley Omokhudu and Ogunbileje John Olusegun
344
G×E Interaction Effects on Yield of Twenty-five Genotypes of Bread Wheat (Triticum Aestivum L.) during 2009 Winter in Zimbabwe
Tegwe Soko and Ephrame Havazvidi 352
Mutated Clones of Caladium Humboldtii ‘Phraya Savet’ from in vitro Culture and Occurrence of Variants from Somatic Hybridization between Two Caladium Species
Chockpisit Thepsithar, Aree Thongpukdee, Rungniran Sugaram and Usanisa Somkanae 360
Media Appraisal for Somatic Embryogenesis of Elite Inbred Lines of Maize
Inuwa Shehu Usman, Shehu Garki Ado and Ng Shou Yong 364
Circadian Rhythm of Root’s Apical Meristem Mitosis Cells of Soybean
Margarita Kozak 369
Life History of the Lenkoran Capoeta Capoeta Gracilis (Keyserling, 1981) in the Atrak River, Northern Iran
Rahman Patimar, Abdol-Jalil Hajili Davaji and Aisoltan Jorjani 376
Effect of Pork Meat pH on Iron Release from Heme Molecule during Cooking
Monica Bergamaschi and Angela Pizza 381
A Survey on Morphological Traits of Basset Hound Dogs Raised in Italy
Francesca Cecchi, Giovanna Carlini, Elena Ciani, Assunta Bramante and Roberta Ciampolini 387 Using Morphological Markers to Assess Variations between and within Cultivated and
Non-cultivated Provenances of Moringa Oleifera Lam. in Tanzania
Mariam Godwin Mgendi, Agnes Morris Nyomora and Mkabwa Katambo Manoko 393
Evaluation of New Insecticide (Proteus 170 O - TEQ) for the Control of the Brown Cocoa Mirid (Sahlbergella Singularis) in Nigeria
Evarestus Uche Asogwa, Feyisara Abiodun Okelana, Idongesit Umanah Mokwunye, Joseph Chucks Anikwe and Theophilos Chinyere Nkasiobi Ndubuaku
400
Research on the Inert Dust against Phosphine Resistance of Cryptolestes Ferrugineus
Yanyu Li, Yang Cao and Guangtao Li
Journal of Life Sciences 5 (2011) 327-331
Macronutrient Composition and Digestibility of Extruded and Fermented Soya Protein Products
Anthony Ojokoh and Yimin Wei Institute of Agro-Food Science and Technology, Chinese Academy of Agricultural Sciences, Beijing 100094, China
Received: March 26, 2010 / Accepted: June 01, 2010 / Published: May 30, 2011.
Abstract: The macronutrient composition and in vitro protein digestibility of extruded fermented and unfermented extruded soya protein products (low, medium and high moisture), raw and fermented soya meal and soya kernel were studied. The protein content (g/100g soya dry weight) ranged from 38.20 to 62.98 with the highest content in the high moisture extruded protein product fermented with 5 mL inoculum of Bacillus natto. Contents of carbohydrates ranged from 14.77 to 29.08 while those of crude fibre, fat and ash were generally low. Fermentation better improved protein digestibility in the raw soya meal and kernel than in the unfermented extruded and extruded fermented products. SDS-PAGE electrophoresis revealed some degradation of the protein sub units of fermented samples.
Key words: Fermentation, extrusion, macronutrient, soya protein products, digestibility.
1. Introduction means reduced cooking time and energy [4].
The reduction in protein digestibility of soya kernel Extrusion is one of the most common industrial in humans and animals is caused by antinutritional processes used to make snacks and it is among the most factors such as phytic acid and trypsin inhibitor that versatile technological processes for making food bind to enzymes in the digestive tract and thus inhibit products. Extrusion technology has many advantages, utilization of proteins. This adverse effect can be including its versatility, high productivity, low cost and overcome by fermentation, germination [5, 6] or the ability to produce unique product shapes and high
extrusion [7].
product quality [1-3]. Improved protein digestibility in food is due to Extrusion-cooking is one of the most efficient and degradation of complex storage proteins by versatile food processing technologies that can be used endogenous and microbial proteases during to produce pre-cooked and dehydrated foods. A major fermentation whereas extrusion and other forms of technological advantage of extrusion is that the product cooking improve digestibility by solubilization, is simultaneously cooked and dried, resulting in gelatinization and formation of maltodextrins [8]. low-moisture shelf stable extrudates. This reduces the It is also well established that extrusion cost of post-extrusion drying and guarantees an thermomechanically denatures and reorients proteins improved shelf-life of the product without the need for in foods, leading to changes in digestibility and levels cooling or refrigeration. The pre-cooked extrudate of amino acids [8-10]. However, the magnitude of requires only reconstitution in warm water and this starched protein transformation due to extrusion is a
Anthony Ojokoh, Ph.D., research fields: industrial fermentation function of pre and post processing operations, their and biotechnology. E-mail: tonyojokoh@yahoo.com.
interactions and the type of starch and protein. Most Corresponding author: Yimin Wei, Ph.D., research fields: agro-food processing and food safety. E-mail: weiyimin36@
available literature consider only the individual effects hotmail.com.
Macronutrient Composition and Digestibility of Extruded and Fermented Soya Protein Products
of either fermentation or extrusion on starch and
2.3 Protein Digestibility
protein digestibility. In vitro soluble protein (IVSP) digestibility was In this study, the combined effects of extrusion determined by adding 200 mg sample to a 100 mL and fermentation on the macronutrient composition Erlenmeyer flask containing 35 mL 0.1 M sodium and invitro protein digestibility of soya protein citrate tribasic dihydrate (pH 2.0) with pepsin (1.5 g products were determined. Comparisons were also pepsin/L, Sigma P-7012; activity 2650 units/mg made with (a) unfermented extruded soya protein protein). The mixture was incubated for 2 h in a products and (b) fermented and unfermented soya shaking water bath at 37 ℃ and then centrifuged at meal and kernel. 10,000 rpm for 15 min. The supernatant was decanted
2. Materials and Methods
and the residue was washed, dried at 80 ℃ and analyzed for nitrogen content. Digestibility was
2.1 Preparation of Soya Samples and Fermentation calculated by subtracting residue nitrogen from total
Extruded soya protein samples, soya meal and soya nitrogen, dividing by total nitrogen multiplied by 100. kernel were obtained from the Food and Technology
2.4 Nitrogen Solubility Index (NSI) Laboratory of the Institute of Agro-Food Science and
Technology, Beijing. The soya kernels were cleaned to Nitrogen solubility index was determined by remove broken seeds, dust and other extraneous
weighing 1 g sample into 50 mL centrifuge tube and materials and then soaked in water at room temperature
dispersed in 20 mL distilled water. The dispersion was (25 ± 2 ℃) for 20 h while soya meal and extruded soya
mechanically shaken for 1 h, centrifuged at 10,000 rpm protein products with moisture contents of 20 (low),
for 15 min and the supernatant collected. The residue
32.5 (medium) and 45% (high) were soaked in water was resuspended and centrifuged twice in 10 mL for 45 mins and drained thereafter. The samples were
distilled water. The combined supernatants were inoculated immediately after steaming with 5, 10 and
analysed for soluble nitrogen by the Kjeldhal method.
15 mL of inoculum size of Bacillus natto previously Nitrogen solubility index (NSI) was reported as soluble isolated from a commercial natto product (natto
nitrogen expressed as a percentage of total protein. produced from large soya bean cultivar) and allowed to
2.5 SDS-PAGE
ferment at 37 ℃ for 48 h. Sodium dodecyl sulfate-polyacrylamide gel
2.2 Macronutrient Estimation electrophoresis (SDS-PAGE) was performed with
Moisture content was determined by direct oven 12.5% resolving gel and 50% stacking gel according to drying method; the loss in weight after oven-drying
the method of Wang and Fan [13]. Protein solutions was expressed as % moisture content [11]. Crude
extracted with phosphate buffer containing 8 M urea protein was estimated from the total nitrogen (TN)
and 0.1 M 2-ME were diluted with 5 x sample determined by the micro- Kjeldahl method by
dissolution, and then heated in boiling water bath for 5 min. multiplying the TN by a factor of 6.25. Crude fat was
After centrifugation at 1,000 rpm for 10 min, a total of determined by using the soxhlet extraction method
10 μL solution was loaded into each lane and electrically using petroleum ether as the solvent [11]. Ash was
separated. The gels were strained for 30 min with CBB measured gravimentrically after ashing at 550 ℃ to
staining solution (0.1% coomassie brilliant blue R, constant weight. Carbohydrate was determined by the
45% methanol, 10% acetic acid), and destained with anthrone method according to Plummer [12].
10% methanol and 10% acetic acid. The strained gels
Macronutrient Composition and Digestibility of Extruded and Fermented Soya Protein Products
were analyzed by using Fluro Chem FC2 Imaging The macronutrient profile (g/100g) of soya samples System (Alpha Innotech Cooperation, USA) and the
is shown in Tables 1 and 2. There was significant (P < molecular weight of each subunit was quantified. Low
0.05) increase in the protein content of the fermented molecular weight markers (Amersham Biosciences,
samples over unfermented samples. In this study, the UK) used were rabbit phophorylase b (97.4 kDa),
protein content varied among samples fermented with bovive serum albumin (66.2 kDa), rabbit actin (43.0 kDa),
different inoculum sizes with the highest (62.98 g/100g) bovine carbonic anhydrase (31.0 kDa), trypsin inhibitor
occuring in the high moisture extruded soya sample (20.1 kDa) and hen egg white lysozyme (14.4 kDa).
fermented with 5 mL of Bacillus natto.
2.6 Statistical Analysis Reade and Gregory [14] reported that autolysis is likely to increase with initial inoculum due to
Analysis of variance (anova) of the samples, which disproportionate amount of nutrient and lower was performed with mean ± S.D. values, was
conversion efficiency. At lower inoculum level, cells compared at 5% significant level using Ducan’s
are larger, especially when competition for available multiple-range tests.
nutrients was minimal [15]. Similar findings have been
3. Results and Discussion
reported by Ojokoh and Uzeh [16] in production of S. cerevisiae biomass in papaya extract medium.
3.1 Macronutrient Composition
Table 1 Proximate composition of soya samples fermented with different inoculum sizes of Bacillus natto (mean ± SD*).
Samples
Carbohydrate LMESPP
Protein
Crude fibre Ash
0.40 ± 0.00 20.12 ± 0.31 fermented with
5 mL inoculum HMESPP
0.34 ± 0.01 21.94 ± 0.81 fermented with
10 mL inoculum HMESPP
0.37 ± 0.01 21.38 ± 0.74 fermented with
15 mL inoculum HMESPP
21.28 ± 0.10 22.03 ± 0.42 LMESPP: Low Moisture Extruded Soy Protein Product; MMESPP: Medium Moisture Extruded Soy Protein Product; HMESPP: High Moisture Extruded Soy Protein Product; SM: Soy Meal; SK: Soy Kernel. * Values represent means of triplicate determinations. The same with Table 2.
Table 2 Proximate composition of unfermented soya samples (mean ± SD*).
Samples Protein Crude fibre Ash Fat Carbohydrate LMESPP
27.04 ± 0.18 MMESPP
28.58 ± 0.81 HMESPP
29.08 ± 0.29 SM
25.84 ± 0.24 SK
Macronutrient Composition and Digestibility of Extruded and Fermented Soya Protein Products
The increase in protein content after fermentation
Table 3 Protein digestibility of soya samples.
was due to a decrease in carbon ratio in the total mass,
Protein digestibility (mean ± SD*) resulting in redistribution of nutrient percentages.
Microorganisms utilize carbohydrates as an energy
FHMESPP
source and produce carbon dioxide as a by-product.
FSM
This causes the nitrogen in the fermented samples to be
FSK
concentrated and thus the proportion of protein in the
total mass increases. The lower protein content in the
UHMESPP
extruded samples compared to the extruded fermented
USM
samples was possibly due to participation of amino
USK
acids in Maillard reactions. The fat and crude fibre FLMESPP: Fermented Low Moisture Extruded Soy Protein Product; FMMESPP: Fermented Medium Moisture Extruded
contents were generally low while there was no Soy Protein Product; FHMESPP: Fermented High Moisture
significant difference in the ash content. Extruded Soy Protein Product; FSM: Fermented Soy Meal; FSK: Fermented Soy Kernel; ULMESPP: Unfermented Low
3.2 Protein Digestibility Moisture Extruded Soy Protein Product; UMMESPP: Unfermented Medium Moisture Extruded Soy Protein Product;
In vitro protein digestibility is a measure of soluble UHMESPP: Unfermented High Moisture Extruded Soy Protein proteins digested under conditions of the pepsin assay.
Product; USM: Unfermented Soy Meal; USK: Unfermented In vitro protein digestibilities increased after Soy Kernel. * Values represent means of triplicate
fermentation because of partial degradation of complex determinations. The same with Table 4 and Fig. 1.
storage proteins by endogenous and microbial Table 4 Nitrogen solubility index of soya samples.
proteolytic enzymes into soluble products [5, 17].
Samples
NSI (mean ± SD*)
Protein digestibilities increased more when samples 13.20 ± 0.12
FLMESPP
27.25 ± 0.35 were unfermented or extruded and fermented than in
FMMESPP
39.84 ± 0.20 the extruded samples (Table 3). Extrusion is a
FHMESPP
77.35 ± 0.16 high-temperature short-time treatment that improves
FSM
FSK ND ULMESPP
protein digestibility via denaturation which exposes 9.75 ± 0.34
6.44 ± 0.17 enzyme access sites. An increase in protein
UMMESPP
10.20 ± 0.11 digestibility was reported in fermented and extruded uji
UHMESPP
73.64 ± 0.08 from maize-finger millet [4].
USM
USK ND
3.3 Nitrogen Solubility Index (NSI) denatures proteins by opening up their quaternary and tertiary structures [21].
The extent of protein denaturation is assessed by the solubility of protein in water and is measured as NSI
3.4 SDS-PAGE Electrophoresis
[18]. The increase in NSI in the fermented and extruded The result of SDS-PAGE electrophoresis of soya fermented samples (Table 4) may be attributed to the
samples is shown in Fig. 1. The two major components proteolytic activity of endogenous and microbial
in soya bean protein 11S with molecular weight of enzymes. In contrast, the NSI of unfermented extruded
38.99 kDa and 7S with molecular weight of 83.80, samples decreased, indicating polymerization,
78.17 and 54.48 kDa were the same in all the soya cross-linking and reorientation of the native proteins
samples. Disappearance of the protein band subunits in to form new fibrous structures [10, 19, 20]. Extrusion
the fermented samples which appeared lighter may be
Macronutrient Composition and Digestibility of Extruded and Fermented Soya Protein Products
[5] N. Khetarpaul, B.M. Chauhan, Effect of germination and fermentation on in vitro starch and protein digestibility of pearl millet, Journal of Food Science 55 (3) (1990) 883-884.
[6] W. Lorri, U. Svanberg, Lactic fermented gruels with improved in vitro protein digestibility, International Journal of Food Science and Nutrition 44 (1993) 29-36.
[7] K.M. Dahlin, K.J. Lorenz, Carbohydrate digestibility of laboratory extruded cereal grains, Cereal Chemistry 70 (3) (1993) 329-333.
[8] I. Bjork, N.G. Asp, The effect of extrusion cooking on nutritional value - A literature review, Journal of Food Engineering 2 (1983) 281-308.
[9] G. Della Valle, L. Quillien, J. Gueguen, Relationships
Fig. 1 SDS-PAGE electrophoresis of soya samples.
between processing conditions andstarch andprotein 1. USK; 2. USM; 3. ULMESP; 4. UHMESPP; 5. FSK; 6. FSM;
modifica tions during extrusion-cooking of pea flour, 7. FLMESPP; 8. FMMESPP; 9. FHMESPP; 10. UMMESPP.
Journal of the Science of Food and Agriculture 64 (1994) 509-517.
due to microbial degradation during fermentation. [10] M.O. Iwe, D.J. Van Zuilichem, P.O. Ngoddy, Amino acid Modification of protein SDS-PAGE profile during and protein dispersibility index (PDI) of mixtures of extruded soy and sweet potato flours, Lebensmittel hydrolysis may improve the ability of protein to move
Wissenschaft und Technologie 34 (2001) 71-75. quickly to interface [22].
[11] Association of Official Analytical Chemists, Official Methods of Analysis, Washington DC, 1990.
4. Conclusion [12] D.T. Plummer, An Introduction to Practical Biochemistry,
McGraw Hill, New York, 1971, pp. 112-113. The protein content, protein digestibility and
[13] J.Z. Wang, M. Fan, Handbook of Protein Technique, Science in China Publication, Beijing, 2000, pp. 77-110. nitrogen solubility index of the soya protein products
[14] A.E. Reade, K.E. Gregory, High temperature protein improved after fermentation resulting in better
enriched feed from cassava fungi, Applied Microbiology nutritional value and more effective utilization of the 30 (1975) 897-907. [15] A.E. Chikwendu, Microbial treatment of cassava whey products. SDS-PAGE electrophoresis indicated that the
and single cell protein production, M.Sc. Thesis, Uni. of protein sub units of the fermented samples were
Benin, Benin City, Nigeria, 1987, p. 162. [16] A.O. Ojokoh, R.E. Uzeh, Production of Saccharomyces
affected by fermentation. cerevisiae biomass in papaya extract medium, African Journal of Biotechnology 4 (11) (2005) 1281-1284.
Acknowledgments
[17] U.D. Chavan, J.K. Chavan, S.S. Kadam, Effect of fermentation on soluble proteins and in vitro protein These investigations were supported by the Chinese
digestibility of sorghum, green gram andsorghum-green Academy of Agricultural Sciences.
gram blends, Journal of Food Science 53 (5) (1988) 1574-1575.
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Publishing Co., Cambridge, England, 2001, pp. 109-129. variables and glycerol monostearate on extrusion of maize
[19] L.A.M. Pelembe, C. Erasmus, J.R.N. Taylor, Development grits using two sizes of extruder, Journal of Food
of a protein-rich composite sorghum–cowpea instant Engineering 35 (1998) 91-109.
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Wissenschaft und Technologie 35 (2002) 120-127. phosphate salts on extrusion behaviour of rice, Food
[20] D.W. Stanley, Extrusion cooking, in: C. Mercier, P. Linko, Chemistry 64 (1999) 481-488.
J.M. Harper (Eds.), American Association of Cereal [3] H. Koksel, G.H. Ryu, A. Basman, Effects of variables on
Chemists, St. Paul, MN, 1989, pp. 321-341. the properties of waxy hulless barley extrudates, Nahrung
[21] H. Akdogan, High moisture food extrusion, International 48 (1) (2004) 19-24.
Journal of Food Science and Technology 34 (1999) 195-207. [4] C. Onyango, H. Neotzold, T. Bley, Proximate
[22] J. Stephanie, R. Coralie, B. Jenny, Limited hydrolysis of composition and digestibility of fermented and extruded
soy proteins with endo-and exoproteases, JAOCS 81 (10) uji from maize-finger millet blend, Lebensmitted
(2004) 953-960.
Wissenschaft and Technologie 37 (8) (2004) 827-832.
Journal of Life Sciences 5 (2011) 332-338
Hormonal (Thyroxin, Cortisol) and Immunological (Leucocytes) Responses to Cistern Size and Heat Stress in Tunisia
1 2 1 3 3 Rim Ben Younes 1 , Moez Ayadi , Taha Najar , Margherita Caccamo , Iris Schadt and Moncef Ben M’Rad
1. Ressources Animales, Halieutiques et Technologie Alimentaire, Institut National Agronomique de Tunisie, Tunis 1082, Tunisie 2. Production Animale, Institut Supérieur de Biologie Appliquée de Médenine, Médenine 4100, Tunisie 3. CoRFiLaC, Regione Siciliana, Ragusa 97100, Italy
Received: December 15, 2010 / Accepted: January 25, 2011 / Published: May 30, 2011.
Abstract: This study was designed to determine the effects of heat stress on plasma leucocytes, Thyroxin (T4) and cortisol concentrations in dairy cows with small and large cistern under hot climate. This experiment was carried out in 2006, in North Tunisia, using a randomized block design per udder cistern size, using 60 Holstein cows. Cows were classified according to udder cistern size by ultrasonography as large-cisterned (44 ± 13 cm²; LC) and small-cisterned (21 ± 8 cm²; SC). The experiment was carried out in two different periods: spring (Apr. 5 (D1)) and summer (July 19 (D2), Aug. 19 (D3) and Sept. 19 (D4)). On each test day, temperature and relative humidity data were registered hourly and cows’ blood was sampled from the jugular vein to determine serum concentrates of cortisol and T4. Leucocytes (lymphocytes, eosinophils, neutrophils and monocytes) were counted differentially, and percentages of lymphocytes relative to total counted cells were calculated. Mean temperature-humidity index (THI) values were 62 ± 2, 79 ± 2, 84 ± 2, and 77 ± 1 in D1, D2, D3, and D4, respectively. Lymphocyte incidence relative to total cell counts and T4 concentrations were affected by test day (P < 0.001). Lymphocytes (%) were significantly less in hotter months. During summer, T4 concentration at D2 (87.4 nmol/L) was higher relative to concentrations at D3 (42.8 nmol/L) and D4 (53.5 nmol/L). T4 concentrations were higher (P < 0.01) in SC cows (67.7 ± 0.1 nmol/L) compared to LC cows (52.7 ± 0.1 nmol/L). Cortisol concentration was effected neither by test day nor by cistern size. However, the decrease of lymphocyte concentration during summer compared to spring could be considered as an evidence of the suppression of cows’ immune system under heat stress.
Key words: Heat stress, cistern size, cortisol, thyroxin, lymphocytes.
1. Introduction temperature-humidity index (THI) is widely used as an index to estimate the intensity of heat stress on dairy
Tunisia is characterized, during summer months, by cows. Heat stress causes changes in the homeostasis persistent intense hot temperature. In Tunisia, heat status of the animals and has been quantified through stress usually begins in June and lasts through measurements of rectal temperature (RT), respiratory September [1]. Moreover, dairy cattle are sensitive to rate (RR), plasma thyroxin (T4) and cortisol high ambient temperatures (AT) and relative humidity
concentrations [2].
(RH). These conditions compromise the ability of Heat production and body temperature regulation are lactating cows to dissipate heat which induces heat effectively controlled by thyroxin (T4) hormone which stress. As a result, the cow activates physiological regulates thermogenesis accounting approximately for mechanisms for coping with the heat stress. The 50% of the basal metabolic rate of animals [3]. The
concentration of T4 in blood plasma declines, under Corresponding author: Rim Ben Younes, research field:
heat stress, by up to 25% [4, 5]. Thyroxin hormone is production animals. E-mail: rim.ouerfelli@gmail.com.
Hormonal (Thyroxin, Cortisol) and Immunological (Leucocytes) Responses to Cistern Size and Heat
Stress in Tunisia
of utmost importance in the heat adaptation process, Diets were defined including ingredients commonly allowing the adjustment of metabolic rates in favour of
used in Northern Tunisia. Crude protein and neutral the body heat balance. Similarly, the secretion of
detergent fiber content in diets ranged from 13.8 to cortisol stimulates physiological adjustments that
15.8% and 39.6 to 42.4% (on dry matter basis), enable an animal to cope with heat stress [6]. Besides,
respectively.
cortisol depresses the activity of the immune system All cows were machine-milked in a herringbone and lowers its resistance to diseases. Kamwanja et al.
parlor (Alpha Laval, The Netherlands) twice daily (2×) [7] have reported a slight decrease in lymphocyte
at 04:00 a.m. and 04:00 p.m. Routine milking included population in heat stressed cows.
udder and teat cleaning as well as teat dipping in an High-yielding dairy cows are the most sensitive to
iodine solution (Iodine, Veto Lab, Tunisia). heat stress because they produce more heat than
At the beginning of the study in summer, cows were low-yielding dairy cows [8]. It has been shown that
classified according to udder cistern size as animals with large cisterns are more efficient producers
large-cisterned (44 ± 13 cm 2 ; LC) and small-cisterned of milk yield than little cistern’s cows [9-11]. As a
(21 ± 8 cm 2 ; SC) (Table 1). Cisternal area was consequence, cistern size could be related to heat stress,
measured according to the methodology described by it becomes important to know hormonal and Ayadi et al. [11]. Udder scans for the right front and
immunological response to heat stress and their effect rear quarters were performed in duplicate 8 to 10 h on cistern size in dairy cows. The present study was
after the a.m. milking by using a real time B-mode designed to determine the effects of heat stress on
ultrasonograph (Ultra Sound Scanner B7v; Noveko plasma leucocytes, T4 and cortisol concentrations in
Echograph Inc., Quebec, Canada) equipped with a dairy cows with small and large cistern under
multi-frequency linear probes (7.5-2.6 MHz 2 dB significant climate changes.
power; 80° scanning angle, 0.5 mm axial and 1.5 mm
2. Materials and Methods
lateral resolution).
The first sampling was carried out during spring (D1)
2.1 Cows, Measurements and Sampling when mean daily THI value was 62 ± 1.9. Further
The study was carried out in 2006 at the OTD samplings were conducted during summer (D2, D3 and Ghezala Farm, Mateur (North Tunisia), which is
D4), when mean daily THI were 79 ± 2.3, 84 ± 2, and located at 37°3 ′ North latitude and 9°39′ East
77 ± 0.5, respectively. At each test day, rectal longitudes.
temperatures (RT) and respiratory rates (RR) were Sixty multiparous lactating Holstein Friesian dairy
recorded. The RT was measured using a medical digital cows (499 ± 19.7 kg BW, 170 ± 15 DIM and 18 ± 5.7
thermometer (precision ± 0.01 ℃). The RR l/d milk yield) were used. Cows were housed in free
(breaths/minute) was counted using a medical stalls with concrete surfaces and bedded with hay.
stethoscope. Measures started at 1 p.m. and finished Farm management and diet composition was typical
around 3 p.m. Ambient temperature and relative for the region with forage ratio of 63%, 53%, 59%, and
humidity were measured using a thermo hygrometer 54% per Apr. 5 (D1), July 19 (D2), Aug. 19 (D3) and
(HI 91610C, Hanna instrument, Portugal). Estimation Sept. 19 (D4), respectively, on dry matter (DM) basis.
of THI was performed using the equation described by The concentrate (8 kg/cow/day) was fed in five equal
Kibler [12]. At the test day blood was collected at meals daily. Food and water were available ad libitum.
approximately 12:30 p.m. by jugular vein puncture into Ingredients and chemical composition of diets fed to
vacuum tubes and immediately placed in a portable animals during the experiment are reported in Table 1.
refrigerator and transferred to the laboratory.
Hormonal (Thyroxin, Cortisol) and Immunological (Leucocytes) Responses to Cistern Size and Heat
Stress in Tunisia
Table 1 Ingredients and chemical composition of the total mixed ration diet (on dry matter basis) and means and standard
deviation of production performance of cows during the experiment. Item
Sept. (D4) Feed ingredient % Oat silage
Apr. (D1)
July (D2)
Aug. (D3)
Corn silage
Alfalfa forage
Tritical ground green forage
Bersim green forage
Corn green forage
Sorghum green forage
Corn grain grind
Soybean meal
Barley grain
Wheat bran
Corn grain
Calcium phosphate
Sodium bicarbonate
Chemical composition DM% 29.2 33.4 31.0 28.2 CP, % of DM
NDF, % of DM
NDF forages, % of DM
Starch, % of DM
NE L , Mcal/kg 1.5 1.5 1.5 1.4 Animals
LC SC LC SC LC SC Cows (n)
60 30 30 30 30 30 30 Age (m)
67 (25) DIM (d)
170 (15) 169 (16) 200 (15) 199 (16) 230 (15) 229 (16) Under Cisterns Size (cm 2 )
13(6) ( ) = Standard deviation; LC = Large cistern, SC = Small cistern.
Milk yield (L/d)
using RIA kit (Immunotech, IM 1447). The total
2.2 Laboratory Analysis cortisol concentration was determined using the 125 I As soon as blood was collected, smears were
RIA kit (IM 1841).
prepared using wrights- Giesma method (Fisher
2.3 Data Analysis
Scientific Company) for differential leucocytes profile. Total leucocytes number was determined for each
To estimate the effect of test day on RT, RR, T4, smears blood by light microscope. Every test day,
cortisol and leucocytes a mixed model was used: collected blood was centrifuged at 3,000 rpm for 15
Y ijk = μ+c i +t j + (c × t) ij +a k +e ijk minutes and sera were collected and frozen in vials at
where y ijk is the measured values of RT, RR, -20 ℃. Blood plasma was analyzed for total thyroxin
neutrophils, eosinophils, lymphocytes, monocytes,
Hormonal (Thyroxin, Cortisol) and Immunological (Leucocytes) Responses to Cistern Size and Heat
Stress in Tunisia
thyroxin, and cortisol; μ is the model mean value, c i for total T4 and cortisol concentrations are presented in fixed effect of cistern size, t j fixed effect of test day, (c
Table 2. During summer, T4 concentration at D2 (87.4 × t) ij interaction cistern size-test day, a k effect of animal
nmol/L) was higher relative to concentrations at D3 and e ijk is the residual error. Observations within cows
(42.8 nmol/L) and D4 (53.5 nmol/L). Thyroid were considered random as repeated measurements.
hormones are of utmost importance in the heat Eosinophil, monocyte, thyroxin and cortisol were
adaptation process, allowing the adjustment of the transformed using the logarithmic link function, since
metabolic rates to favor body heat balance. Lu [15] the distributions of data were not normal.
reported reduced metabolism in cattle under heat stress, All analyses were conducted using Jmp V 8.0.1
which was associated with reduced T4 concentration. (SAS Institute Inc., 2004). Differences were Working with lactating cows, Johnson et al. [16] found considered significant at P < 0.05.
a decline in triiodothyronine (T3) and T4 in response to heat stress. They attributed this decline in attempts, by
3. Results and Discussion
the cow, to reduce metabolic heat production. According to Armstrong [13] and Johnson [14], heat
Thompson [17] concluded that adaptation to high stress is considered absent when THI ≤ 72, mild when
temperatures is followed by an increase of body THI is between 73 and 77, moderate when THI is
temperature and a decreased thyroid activity. Thyroxin between 78 and 88 and severe when THI > 88. In the
concentrations were significantly higher (P < 0.05) in present study, recorded THI in D1 of 62 (± 1.9)
cows with small cisterns (67.7 nmol/L) compared to indicated absence of heat stress. As expected, heat
cows with large cisterns (52.7 nmol/L). Ayadi et al. [11] stress occurred during D2, D3 and, D4 with THI values
reported a positive correlation of cistern size to milk of 79 (± 2.3), 84 (± 2), and 77 (± 0.5), respectively
production. High yielding dairy cows might need to (Table 2).
reduce body heat production to a large extent relative to At high environmental temperatures cows attempted
low producing cows [8], because they produce more to restore their thermal balance. In this study, heat
heat than low yielding cows. However, T4 stress altered (P < 0.001) RT and RR (Table 2). Rectal
concentrations are also negatively correlated to milk temperature increased from D1 with 38.5 ℃ to D3 with
production [18], and response in T4 to cistern size
39.6 ℃. Recorded RT in D4 (38.6 ℃) was not different could be explained by both, different susceptibility to (P > 0.05) from D1. The decrease of RT in D4
heat stress and different milk production. compared to D2 and D3 could be explained, at least in
In the present study, cortisol concentrations of 25.0, part, by the lower THI values in D4 compared to D2
37.3, 28.9, and 31.0 nmol/L in D1, D2, D3 and D4, and D3 and by the fact that cows in D4 were adapted to
respectively, were neither affected by test day nor by high temperatures. Respiratory rates increased from D1
cistern size (P > 0.05). These results are in accordance with 55.7 breaths/min to D3 with 90.6 breaths/min.
with Johnson et al. [19] who reported no effect of Measured RR in D4 (65.1 breaths/min) was not
exposure to heat stress on plasma cortisol different (P > 0.05) from D2. THI values in D2 and D4
concentrations. However, there are also conflicting were alike. When THI increased from 62 to 79, RT and
data in the literature regarding the effect of heat stress RR raised by 0.4 ℃ and 14 breaths/min, respectively.
on blood cortisol concentration in lactating cows. One In particular, RT and RR raised by 0.2 ℃ and 5
report indicated that blood cortisol concentration breaths/min respectively per increase of THI unit.
increases after 4 h of exposure to heat stress [6]. Cistern size did not affect RT and RR (P > 0.05).
Chronic exposure to heat stress over several days or Least square means and standard deviation values
longer either increased [20] or decreased [6-21] blood
Hormonal (Thyroxin, Cortisol) and Immunological (Leucocytes) Responses to Cistern Size and Heat
Stress in Tunisia
Table 2 Heat stress and cistern size (LC: Large cistern, SC: Small cistern) effects on rectal temperature (RT), respiration rates (RR), plasma thyroxin (T4) and cortisol concentrations and Leucocytes percentages.
Cistern Size Effect Item
Test Day (TD)
Apr. 5 (D1) July 19 (D2) Aug. 19 (D3) Sept. 19 (D4) LC
SC
TD Cis TD × Cis
LSM SE LSM SE Environment Ambient temperature (℃) 16.9 1.3
LSM SE 2 LSM SE LSM SE LSM SE
0.7 - - - - - - - Relative humidity (%)
6.9 59.1 10.8 51.7 3.2 - - - - - - -
0.5 - - - - - - - Animal response Rectal temperature (℃) 38.5 c 0.1 38.9 b 0.1 39.6 a 0.1 38.6 c 0.1 38.8 a 0.1 39.0 a 0.1 *** ns ns Respiratory rate (breaths/min) 55.7 c 3.3 70.0 b 2.8 90.6 a 2.7 65.1 b 2.7 68.4 a 2.4 72.3 a 2.5 *** ns ns
THI 3 62.0 1.9
Heart rates (beats/min) 97.7 a 2.3 80.3 c 2.0 86.2 b 1.9 84.5 bc 1.9 84.5 a 1.6 89.9 b 1.6 *** * ns Hormones Thyroxin (T4) (nmol/L)
87.4 a 0.1 42.8 b 0.1 53.5 b 0.1 52.7 a 0.7 67.7 b 0.1 *** ** ns Cortisol (nmol/L)
25.0 a 0.3 37.3 a 0.1 28.9 a 0.1 31.0 a 0.1 28.4 a 0.1 32.3 a 0.1 ns ns ns Leucocytes Lymphocyte (%)
73.6 a 2.7 64.7 b 1.1 65.6 b 1.3 60.4 c 1.1 65.4 a 1.2 66.8 a 1.2 *** ns ns Neutrophil (%)
23.4 ab 2.7 20.0 b 1.0 22.8 b 1.3 26.8 a 1.1 23.9 a 1.2 22.6 a 1.2 *** ns ns Eosinophil (%)
1.2 c 2.2 11.5 a 0.9 4.7 c 1.1 8.2 b 0.9 6.3 a 1.0 6.5 a 0.9 *** ns ns Monocyte (%)
1.8 c 1.0 3.8 b 0.4 5.1 a 0.5 4.6 ab 0.4 3.5 a 0.4 4.1 a 0.4 * ns ns *** P < 0.001; ** P < 0.01; * P < 0.05; ns = not significant (P > 0.05); Cis = Cistern;
a,b,c Means within row per Test day and Cistern size not sharing the same superscript are significantly different; 1 LSM = Least square means; 2 SE = Standard error; 3 THI (Temperature Humidity Index) = 1.8 × T – (T – 14.3) × (100 – H) / 100 + 32.
cortisol concentration. count depression during summer suggested decrease of High variability in response of cortisol to heat stress
the immune system activity. Both factors, the duration reported in literature, as well as the lack of response in
of exposure, and the severity of heat stress, decrease the present study, might be explained at least in part, by
immune activity [23]. In the present study THI values the fact that other parameters than heat stress might be
in D4 were lower relative to D2 and D3, but able to alter cortisol levels. As well, stress in general
lymphocytes were lower in D4 compared to D2 and D3. can alter cortisol concentration. Other stress factors
These results are in accordance with Kamwanja [7]. occurred in this trial might have influenced individual
This study reported that in vitro exposure of bovine cow responses, such as the experiment procedure itself
lymphocytes to an ambient temperature of 45 ℃ for 3 (e.g. blood sampling) could have influenced cortisol
hours decreased the number of viable cells. However, response of cows [22].
our results are conflicting with those studies where no
Lymphocytes, neutrophils, eosinophils and effect [24] or an improvement [25, 26] were reported. monocytes, relative to total number of leucocytes, were
Lee et al. [26] showed that high ambient temperature affected by period (P < 0.001). Percentage of
caused leucocytosis in cattle. The percentage of white lymphocytes decreased from D1 (73.6%) to D4
blood cells types varied at high ambient temperatures. (60.4%), whereas in D2 and D3 were intermediate,
In the present study, Neutrophils (%) least square with 64.7% and 65.6%, respectively.
means in D1, D2, D3, and D4, respectively, were as
ab Percentage of leucocytes types did not vary b follows (superscripts differ by P < 0.05): 23.4 , 20.0 , according to cistern size (P > 0.05). The lymphocytes
22.8 b , and 26.8 a . Eosinophils (%) least square means in
Hormonal (Thyroxin, Cortisol) and Immunological (Leucocytes) Responses to Cistern Size and Heat
Stress in Tunisia
D1, D2, D3, and D4, respectively, were as follows: 1.2 c ,
normal values. More emphasis should be placed on the
a c 11.5 b , 4.7 , and 8.2 . Moncytes (%)least square means influence of heat stress on the immune system of dairy in D1, D2, D3, and D4, respectively, were as follows:
cows. Moreover, additional studies might also
c b a 1.8 ab , 3.8 , 5.1 , and 4.6 . Brou ček et al. [27] found a determine whether or not it is possible to establish decrease in neutrophil and eosinophil and an increase
upper critical values of THI above which the in lymphocytes and monocytes. In our study, an
immunological functions of dairy cows start to change. increase in neutrophil count from D2 and D3 to D4 and
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Journal of Life Sciences 5 (2011) 339-343
Occurrence of Extended-Spectrum β-lactamase Producing Enterobacteriaceae (ESBLPE) among Primary School Pupil in Obafemi-Owode, Nigeria
1 2 3 Akinduti Paul Akinniyi 4 , Akinbo John Adeolu , Adenuga W. Funmilayo , Ejilude Oluwaseun , Umahoin
5 Kingsley Omokhudu 6 and Ogunbileje John Olusegun 1. Department of Medical Microbiology, Olabisi Onabanjo University, Ago-Iwoye, Ogun State 102107, Nigeria
2. Department of Pathology, Federal Medical Center, Abeokuta, Ogun State 102107, Nigeria 3. Department of Community Medicine, Federal Medical Center, Abeokuta, Ogun State 102107, Nigeria 4. Microbiology Laboratory, Sacred Heart Hospital, Abeokuta, Ogun State 102107, Nigeria 5. Department of Chemical Pathology & Immunology, Olabisi Onabano University, Ago-Iwoye, Ogun State 102107, Nigeria 6. Department of Chemical Pathology, University of Ibadan, Ibadan, Ogun State 102107, Nigeria
Received: March 31, 2010 / Accepted: June 21, 2010 / Published: May 30, 2011.
Abstract: Occurrence of extended- β-lactamase producing enterobacteriaceae (ESBLPE), which has reduced the antibacterial efficacy and potency of many 3rd generation cephalosporins, was investigated among the primary school pupils. 88 primary school pupils in Obafemi-Owode Local Government , Southwestern Nigeria, including 49 males (55.7%) and 39 females (44.3%) (mean age
12 ± 3) were screened for ESBLPE isolates with exclusion criterion of antimicrobial use in the preceding 2 weeks either as therapy for gastro-intestinal complication or prophylaxis. ESBLPE detected include 4.5% of Eschericia coli, 2.3% of Enterobacter cloaca, 0% Proteus mirabilis, 2.3% Pseudomonas aeruginosa, 1.1% Staphylococcus aureus and 4.5% of Klebsiella oxytoca. 10 (76.9%) of ESBLPE isolates were resistant to disc of cefuroxime (30 μg), 8 (61.5%) susceptible to amoxicillin/clavulanic (20/10 μg) and low susceptibility of 7 (53.8%) was recorded for ceftazidime (30 μg). 0% susceptibility was recorded for the ESBLPE isolates to cefuroxime MIC > 8 μg/mL and ampicillin MIC > 8 μg/ mL while E. coli and E. cloca each show 50.0% and P. aeruginosa and K. oxytoca show 100.0% and 75.0% susceptibility to augmentin (MIC ≤ 8). This study has shown a 14.7% proportion of the pupil to harbour ESBLPE from enteric source with increased resistant to most new generation cefuroximes. Therefore, transfer of virulent and antibiotic resistant ESBLPE could be aided by sharing feeding materials while fecal-oral route of transmission cannot be ruled out as hygiene level is very low thereby increasing emergence of virulent resistant enteric strains leading to treatment failure.
Key words: Extended-spectrum β-lactamase producing enterobacteriaceae, cephalosporin, pupil.
important to continuously 1. Introduction monitor emerging
betalactamase resistance mechanisms on a routine Increasing emergence of extended-betalactamase basis mostly among the under-aged school pupil in enterobacteriaceae producer (ESBLPE) in recent years developing countries [1]. Originally K. pneumoniae has reduced the antibacterial efficacy and potency of and E. coli were the most common ESBL-producing many cephalosporin and other antibiotics due to bacteria worldwide. Although in recent years, ESBL various resistance mechanisms. It is, however, production amongst Proteus mirabilis and
AmpC-producing Enterobacteriaceae has become Corresponding author: Akinduti Paul Akinniyi, FMLSCN,
M.Sc., research field: antibiotic resistance. E-mail: niyiakinduti more prevalent [2, 3]. As the genes encoding ESBLs @yahoo.com.
Occurrence of Extended-Spectrum ß-lactamase Producing Enterobacteriaceae (ESBLPE) among
Primary School Pupil in Obafemi-Owode, Nigeria
are contained on plasmids, horizontal gene transfer in Gram staining, oxidase, catalase tests, and the sugar many species of bacteria is as well becoming
fermentation test according to Stoke protocol [13]. prevalent. ESBL production has rarely been