Registration Dossier

Diss Factsheets

Administrative data

toxic effects on livestock and pets
Type of information:
experimental study
Adequacy of study:
supporting study
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
study well documented, meets generally accepted scientific principles, acceptable for assessment

Data source

Reference Type:
Effect of sodium bicarbonate supplementation on feed intake, digestibility, digesta kinetics, nitrogen balance and ruminal fermentation in young fattening lambs.
Bodas, R. Frutos, P., Giraldez, F.J., Hervas, G. and Lopez, S.
Bibliographic source:
Spanish Journal of Agricultural Research 2009, 7 (2), p. 330-341

Materials and methods

Test guideline
no guideline available
Principles of method if other than guideline:
Twenty-two Merino lambs were divided into two experimental groups. One group served as the control group, while the other group received concentrate supplemented with sodium bicarbonate. Urine and faeces were collected after 24 days of dosing in order to determine digestibility and rate of passage. Lambs were slaughtered when they reached a weight of 25 kg. Following sacrifice, samples were taken from the rumen of the lambs, and the content of the rumen was used as inoculum for the batch cultures to examine the rumen microorganisms population.
GLP compliance:
Limit test:

Test material

Constituent 1
Reference substance name:
sodium hydrogencarbonate
Cas Number:
sodium hydrogencarbonate
Details on test material:
No details on test material available.

Test animals

other: sheep (Merino)
Details on test animals and environmental conditions:
Average initial age: 8-9 weeks.
Weight: 15.3 +/- 0.14 kg.
Housing: individual floor pens.

Previously, the lambs had remained stalled with their mothers, with free access to a commercial starter concentrate and alfalfa hay until the commencement of the trial. Immediately after birth, lambs had been treated with Vitasel to prevent white muscle disease, and later on with Miloxan to prevent enterotoxaemia and with albendazol 2.5% Ganadexil® to control internal parasites.

Diet: barley straw + a concentrate containing barley grain, maize grain, soya bean meal, cane molasses and a mineral/vitamin mix, both provided ad libitum once daily in the morning. The amount of feed offered was adjusted daily on the basis of the previous day intake, allowing refusals of 20%. Both concentrate and straw refusals were removed daily, pooled weekly for each animal and weighed and dried to determine dry matter (DM) intake.
Water: free access, ad libitum.

Administration / exposure

Route of exposure:
oral: applied on feed
unchanged (no vehicle)
Details on exposure:
The lambs were divided in an exposed and a control group. The control group received only the diet. In the exposed group, the diet was supplemented with 20 g sodium bicarbonate per kg feed.
Analytical verification of doses or concentrations:
not specified
Duration of treatment / exposure:
Until the lambs weighed 25 kg.
Frequency of treatment:
Post exposure period:
Doses / concentrations
Dose / conc.:
20 000 mg/kg diet
No. of animals per sex per dose:
11 males in the control group and 11 males in the treatment group
Control animals:
yes, plain diet
Further details on study design:
On day 20, lambs (22.5 ± 0.26 kg live weight) were confined in metabolism cages fitted with specific devices to collect faeces and urine separately. After 4 days of adaptation, faeces of each animal were collected daily, weighed, mixed thoroughly and subsampled (10%), for 5 days. Aliquots from each sheep were pooled and stored at -30°C. Pooled samples were dried to constant weight before analysis.

Urine was collected in a solution of H2SO4 (10%; v/v) to maintain the pH<3. Daily urine was weighed, its density was measured and a subsample (20%) was taken for each lamb for 5 days. Daily samples were pooled to form composite samples and stored at -30°C until analysed for total nitrogen and purine derivatives.

Co-EDTA was used to estimate the liquid rate of passage, and was prepared according to the method of Udén et al. (1980). On day 22 of experimental trial, the animals were dosed orally with the marker (1.125 g Co-EDTA dissolved in 30 mL of water). Faeces were collected at 0, 4, 8, 12, 16, 22, 28, 34, 40, 48, 60, 72, 96, 120 and 144 h after marker administration, weighed and a subsample (10%) was taken and stored at -30°C. Samples were dried to constant weight before analysis.


Sacrifice and pathology:
When lambs reached 25 kg of live weight, they were slaughtered following standard procedures. The day before slaughter, feed was withdrawn at 20.00 h. Then, two hours prior to slaughter, feed was offered again to all lambs for 1 hour so that each had a full rumen and active fermentation at the time of slaughter. Each lamb was euthanized with an intravenous injection of barbiturate (Euta-lender®, Normon, Spain), slaughtered by exsanguination from the jugular vein and eviscerated. Total rumen contents from each slaughtered lamb were collected, mixed thoroughly and sampled.

Other examinations:
Batch cultures of rumen microorganisms:
About 400 g of the mixture of rumen contents were strained through two layers of cheesecloth and the pH was measured immediately. After centrifugation (600 g at 4°C for 10 min), a sample of 5 mL of the supernatant was acidified with 5 mL 0.2 N HCl for ammonia determination. Another 0.8 mL of the supernatant was added to 0.5 mL of a deproteinising solution (2% metaphosphoric and 0.4% crotonic acids, w/v, in 0.5 N HCl) for determination of volatile fatty acids (VFA). The samples for NH3 and VFA were stored at -30°C until analysed. Remaining filtered rumen fluid from twelve lambs (six from the control group and six from the dosed group) was used as rumen inoculum for the batch cultures.

In vitro fermentation kinetics and substrate disappearance (as indicators of the degradative activity of rumen contents) of two feedstuffs, namely barley straw and barley grain, were studied by following the in vitro gas production technique as described by Theodorou et al. (1994).
Rumen fluid inocula were obtained separately from 12 lambs (six from the Control group and six from the Bic group), selected randomly among the lambs of each experimental group. Rumen contents were immediately transferred to the laboratory in pre-warmed thermos flasks, strained again through a double layer of muslin and kept under a flushing of CO2.
Each feedstuff used as fermentation substrate (barley straw and barley grain) was incubated in duplicate in each of the 12 inocula. Incubations were carried out in 120 mL serum bottles in which 500 mg DM of the appropriate feedstuff was weighed out, and 10 mL strained rumen fluid and 40 mL medium were dispensed anaerobically. The medium contained buffer, macro- and micro-mineral, resazurin and reducing solutions according to Menke and Steingass (1988). Then the bot- tles were sealed and placed in an incubator at 39oC. Accumulated head-space pressures and gas volumes were measured, using a pressure transducer (Bailey & Mackey, UK) and a graduated syringe, throughout the incubation period (at 2, 4, 6, 8, 10, 12, 15, 19, 24, 30, 36, 48, 72, 96, 120 and 144 h post-inoculation). The va- lues were corrected for the quantity of substrate organic matter (OM) incubated and gas released from blank cul- tures (i.e., rumen fluid plus buffered medium, without substrate, with and without sodium bicarbonate).
After 144 h of incubation, fermentations were stopped by swirling the flasks on ice. In vitro dry matter disappearance (DMD; g kg-1) at this end-point was estimated by filtering residues using pre-weighed sintered glass crucibles (100-160 μm pore size; Pyrex, UK). Residues were then analysed for neutral-detergent fibre (NDF) content to determine in vitro NDF degradation (NDFD).

Ammonia, VFA and DM and NDF degradation:
Twenty-four more cultures per substrate (2 treatments × 6 inocula [replicates] per treatment × 2 flasks per inoculum) were incubated to compare fermentation patterns when rumen fluid from either dosed or control lambs was used as inoculum. In this case, end-point in vitro incubations at either 8 h (barley grain) or 24 h (bar- ley straw) were carried out. Immediately after termination of the incubation, headspace pressure and gas volume were measured, and bottle contents were centrifuged (600 g, 4°C, 15 min). Samples of the supernatant were taken and stored at -30°C for ammonia and VFA determination as described above. Remaining contents of the centrifuge tube were filtered through sintered glass crucibles for subsequent determination of in vitro DM and NDF disappearance, as described above.

Results and discussion

Any other information on results incl. tables

There were no significant differences between groups in the dry matter intake of concentrate (P>0.10). On the other hand, lambs supplemented with sodium bicarbonate consumed a higher amount of dry matter of barley straw (P=0.015). No treatment effects (P>0.10) were observed on the digestibility of DM and crude protein. However, NDF digestibility tended to be significantly higher (15%) in the dosed group than in the control lambs (0.533vs0.462;P=0.080).

Total dry matter intake (809 g/day (control) vs 844 g/day (dosed) DM), average daily gain (295 g/day (control) vs 316 g/day (dosed)) and feed to gain ratio (2.76 (control) vs 2.72 (dosed)) were not different between treatments (P>0.10).

There were no differences between treatments for any of the parameters related to the rate of passage (k1, k2, TT and TMRT;P>0.10) or nitrogen balance. No differences, due to the supplementation with sodium bicarbonate, were found for ammonia and VFA concentrations,

with the exception of isobutyrate concentration, which was significantly higher in the control group (1.29 vs 0.66 mmol/L,P<0.022). Molar proportions of VFA referred to as “others” (sum of valerate + isobutyrate + isovalerate + + caproate) were also significantly higher in the control than in the dosed lambs (0.0612 vs 0.0412, respectively;P=0.027).

Applicant's summary and conclusion