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We described a protocol for prevention of heat stress effects in rats by oral pre-treatment with beneficial bacteria. This protocol can be modified and used for various routes of administration and for analysis of different compounds.
This study was designed to evaluate the protective effect of the Bacillus subtilis strain against complications related to heat stress. Thirty-two Sprague-Dawley rats were used in this study. Animals were orally treated twice a day for two days with B. subtilis BSB3 strain or PBS. The next day after the last treatment, each group was divided and two experimental groups (one treated with PBS and one treated with B. subtilis) were placed at 45 oC for 25 min. Two control groups stayed for 25 min at room temperature. All rats were euthanized and different parameters were analyzed in all groups. Adverse effects of heat stress are registered by the decrease of villi height and total mucosal thickness in the intestinal epithelium; translocation of bacteria from the lumen; increased vesiculation of erythrocytes and elevation of the lipopolysaccharides (LPS) level in the blood. The protective efficacy of treatment is evaluated by prevention of these side effects. The protocol was set up for the oral treatment of rats with bacteria for prevention of heat stress complications, but this protocol can be modified and used for other routes of administration and for analysis of different compounds.
Different stressors affect human and animal health. Temperature is one of the most stressful factors, causing chronic, acute and even lethal illnesses1. Changes in intestinal morphology and a loss of gut barrier integrity after heat stress were documented in many cases2,3. This protective barrier is responsible for defense against translocation of gut bacteria and their toxins, in particular lipopolysaccharides (LPS), from the lumen to the internal circulation4,5. Stability of the gut microbiota significantly influences intestinal barrier function, the immune response of the host, and tolerance to stress conditions6. Thus, m....
All experimental procedures were approved by IACUC of Auburn University, Auburn, Alabama.
1. Preparation of Culture Media, Dishes and Bacterial Culture
The mean body temperature of animals before and immediately after heat stress was 36.7 ± 0.07 oC and 40.3 ± 0.17 oC, respectively (P < 0.05). Exposure of rats to heat (group 3) resulted in significant inhibition of villi height and total mucosal thickness (Figures 2, 3). Oral administration of BSB3 strain before stress protected intestine from the harmful effect of heat.
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Exposure to high temperature results in serious health conditions29. Prevention and early detection of complications related to heat stress are of vital importance30. The presented protocol can be used to evaluate the efficacy of various approaches for the prevention of heat stress adverse effects.
Critical steps within this protocol include: the heat treatment procedure (45 oC, 25 min); the usage of apyrogenic materials during collection and p.......
The authors declare that they have no competing financial interests.
This work was supported by the Auburn University fund FOP 101002-139294-2050.
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Name | Company | Catalog Number | Comments |
Phosphate buffered saline (PBS) | Sigma-Aldrich, St. Louis, MO | P4417 | |
Ethyl Alcohol | Pharmco products Inc. Brookfield, CT, USA | 64-17-5 | |
Agar | VWR | 97064-334 | |
MacConkey agar plates | VWR | 470180-742 | |
5% blood agar plates | VWR | 89405-024 | |
Brucella blood with Hemin, and Vitamin K, plates | VWR | 89405-032 | |
Bouin’s Fixative | Electron Microscopy Sciences, Hatfield, PA, USA | 15990 | |
IL-10 Rat ELISA kit | Invitrogen, Camarillo, CA, USA | KRC0101 | |
IL-1beta Rat ELISA Kit | Invitrogen, Camarillo, CA, USA | KRC0011 | |
IL-6 Rat ELISA Kit | Invitrogen, Camarillo, CA, USA | KRC0061 | |
INF-gamma Rat ELISA Kit | Invitrogen, Camarillo, CA, USA | KRC4021 | |
TNF-alpha Rat ELISA Kit | Invitrogen, Camarillo, CA, USA | KRC3011 | |
Rat LPS ELISA Kit | NeoBioLab, MA, USA | RL0275 | |
Environmental Chamber 6020-1 | Caron, Marietta, OH, USA | 6020-1 | |
Centrifuge | Beckman Coulter, Indianapolis, IN, USA | Optima L-90K | |
Ultra Centrifuge | |||
Light microscope optical system | CitoViva Technology Inc., Auburn, AL | ||
Colony counter | Fisher Scientific , Pittsburgh, PA, USA | RE-3325 | |
Freezer | Haier, Brooklyn, NY, USA | HCMO50LA | |
Ultra microplate reader | Bio-Tek Instrument, Winooski, VT, USA | ELx 808 | |
Auto Strip Washer | Bio-Tek Instrument, Winooski, VT, USA | ELx50 | |
Pipettes | Gilson, Pipetman, France | P100, P200, P1000 | |
C24 Incubator Shaker | New Brunswick Scientific, Enfield, CT, USA | Classic C24 | |
Rocking Shaker | Reliable Scientific, Inc., Nesbit, MS, USA | 55 | |
Petri dishes | Fisher Scientific, Pittsburgh, PA, USA | 875713 | 100mmX15mm |
SterilGard III Advance | The Baker Company, Sanford, ME, USA | SG403 | |
Culture Growing Flasks | Corning Incorporated, Corning, NY, USA | 4995 | PYREX 250mL Erlenmeyer flasks |
Optical Spectrometer Genesys 20 | Thermo Scientific, Waltham, MA, USA. | 4001 | |
Sony DXC-33 Video camera | Sony | ||
Richardson test slide | Electron Microscopy Science, Hatfield, PA, USA | 80303 | |
Millipore water purification system | Millipore | Direct-Q | |
Image-Pro Plus software | Media Cybernetics, MD, USA | ||
Triple Beam Balance | OHAUS Corporation, Parsippany, NJ, USA | ||
Tissue Homogenizer, Dounce | VWR | 71000-518 | |
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