Effect of β-carotene on selected indices of in vitro rumen fermentation in goats *

Three goats fi tted with rumen fi stula were used as donors of rumen fl uid. Substrates were incubated in vitro with buffered rumen fl uid for 24 h. Different levels of β-carotene (fi ve groups: 10, 50, 100, 200, 500 mg/l) were added as fi ve treatment groups to determine whether it affected rumen fermentation in vitro as compared with blank samples used as controls. NH3-N concentrations with 50, 100 and 200 mg β-carotene/l group were found signifi cantly lower than that in blank samples (P<0.01). Microbial protein concentrations in all β-carotene-added groups were higher than in blanks, while those found in 200 and 500 mg/l groups were signifi cantly higher (P<0.05 and 0.01, respectively). Total VFAs seemed not to be affected by added β-carotene (P>0.05), although propionate and butyrate concentrations changed. It is concluded that β-carotene can enhance the utilization of NH3-N by rumen microorganisms and thus promote their growth in vitro.


INTRODUCTION
Healthy food has attracted much attention, and many researchers have devoted studies on improving the value of milk by effectively transferring nutrients to milk from feed.It has been shown that carotenoids can be transferred to milk from feedstuff.Despite the large variety of carotenoids in plants, no more than 10 are found in ruminant feeds, and the most quantitatively important are β-carotene and lutein.Since the β-carotene content in feedstuff is limited, adding commercial products is effective.Little attention has been paid to the effect of β-carotene on rumen fermentation and its disappearance in the rumen.The fi rst event in the digestive process of carotenoids is degradation of the vegetable matrix that releases carotenoids into the rumen liquid phase (Mora et al., 1999).The extent of carotenoid degradation by microorganisms in the rumen remains uncertain because of the wide range of results from in vitro and in vivo studies, mostly on β-carotene.Whereas some authors reported no degradation (Dawson and Hemington, 1974;Cohen Fernandez et al., 1976a), others found moderate (10-25%; Davison and Seo, 1963;Potkański et al., 1974;Cohen Fernandez et al., 1976b;Mora et al., 1999) or higher β-carotene disappearance (40-55%; King et al., 1962).In the experiment of Hino et al. (1992) β-carotene could greatly alleviate inhibition by grass lipids, mainly galactolipids and phospholipids, of the growth of rumen microorganisms and fi bre digestion, and could stimulate the growth of cellulolytic bacteria.The reason for this effect is unclear (Hino et al., 1992).In the previous experiment, the effect of β-carotene on rumen fermentation was seldom studied in vitro.Therefore, the objectives of this study were: 1. to observe the effect of β-carotene on selected rumen fermentation indicators, 2. to estimate whether β-carotene at a high concentration (500 mg/l) was safe for rumen microorganisms, and 3. to validate that β-carotene can stimulate the growth of rumen bacteria.

Design and treatments
Three goats fi tted with rumen fi stula were used as donors of rumen fl uid.The diet components are given in Table 1 and the substrates were the same.Goats were fed two times per day for at least 15 d prior to sampling.Ruminal fl uid was collected via the cannula and squeezed through two layers of cheesecloth.Filtered ruminal fl uid (1000 ml) was mixed with 4000 ml of manufactured saliva (Menke and Steingass, 1988).The manufactured saliva was maintained at 39°C during preparation.Each in vitro fl ask contained 20 ml of the inoculum and 40 ml man-made saliva and 1 g of air-dried substrate, fi ve doses of β-carotene (Roche Switzerland, 10% CWS) were added to each fl ask as fi ve treatments and one blank served as the control.The content in each treatment was 0, 10, 50, 100, 200, 500 mg/l in six groups (blank, groups A, B, C, D and E, respectively).Flasks were purged with CO 2 , sealed with vented stoppers, and incubated in an environmental shaker (100 rpm) at 39°C.Substrates for the incubations were similar to the diets fed to donor goats (Table 1).Feedstuffs were ground through a 1-mm screen.
Substrates were incubated for 0, 1, 3, 5, 8, 12 and 24 h.Blanks for each goat and each time point were run concurrently.Each incubation was conducted in triplicate (separate runs) for a total of 126 fl asks [six β-carotene treatments (including the blank) × three triplicates × seven time points].One fl ask was considered an experimental unit.

Samplings, recordings and analysis
At the end of each incubation time, the fl asks were swirled vigorously, and then three 5-ml aliquots of the contents were removed using a wide-tip pipette, one sample used for determining the ammonia nitrogen (NH 3 -N) concentration and the others were frozen (-20°C) until analysis for microbial protein concentration (MCP) and VFA.

Statistical analyses
The effects of β-carotene on NH 3 -N, MCP, VFA were analysed by analysis of variance with treatments.Data were analysed within each time point (0, 1, 3, 5, 8, 12 and 24 h) between groups, and the mean value of each group also analysed.All statistical analyses were done using SAS (1996).

RESULTS
Changes of NH 3 -N concentration during 24 h incubation were similar among groups, tended to be elevated at the beginning and then dropped.The ranges of NH 3 -N concentrations in 24 h were, mg/100 ml: blank 24.03-36.70; group A 18.70-27.99; group B 12.38-27.52; group C 16.37-27.55; group D 14.38-27.40 and group E 28.69-33.64.The average values of each group were 30.65, 23.52, 20.31, 22.45, 20.22 and 29.84 mg/100 ml.In the blank, the NH 3 -N concentration reached the peak value, while all treated groups showed a downward trend.NH 3 -N concentrations in all treated groups were lower than the blank between 3 to 24 h.After 24 h fermentation, the concentration in the 200 mg/l group was lower than all other groups.MCP concentrations in the treatment groups were higher than the blank in the incubation, and the MCP concentration increased as the dose of β-carotene rose, especially the 500 mg/l group was the highest at 5 h compared with the other groups (P<0.01),but was lower than in the 200 mg/l group later.The ranges of MCP concentration in 24 h were, mg/ml: blank 0.024-0.099;group A 0.037-0.117;group B 0.027-0.121;group C 0.034-0.101;group D 0.047-0.117;group E 0.049-0.117.The average values of each group were 0.055, 0.057, 0.076, 0.024, 0.097 and 0.100 mg/ml.

DISCUSSION
The effect of β-carotene on rumen fermentation has seldom been reported.Some studies reported that β-carotene plus α-tocopherol enhanced bacterial cell yield in the presence of saffl ower oil, caprate, stearate, or linoleate in incubation in vitro, suggesting that β-carotene and α-tocopherol increase the utilization of fatty acids.β-carotene plus α-tocopherol also stimulated cellulose digestion in the presence of 100 mg/l of saffl ower oil, evidently through the increased growth of cellulolytic bacteria.The decrease of NH 3 -N concentrations in the treatment groups and the increase of MCP compared with the blank may have resulted from β-carotene stimulating the growth of cellulolytic bacteria, but the optimal concentration of β-carotene was not certain, maybe between 200 and 500 mg/l.The increase in the acetic acid concentration in TVFA may indicate that β-carotene can change the type of rumen fermentation and this was signifi cant in dairy production in dairy cows.Compared with β-carotene, α-tocopherol was more effective in increasing the growth of rumen bacteria, but it inhibited growth at high concentrations, proving that added β-carotene in ruminant feeds was safe and effective (Hino et al., 1992).

CONCLUSIONS
In this study, we concluded that β-carotene can improve the utilization of NH 3 -N and the production of microbial protein concentration, maybe due to the increased the growth of cellulolytic bacteria; the 500 mg/l dose of β-carotene did not inhibit rumen fermentation, suggesting that adding β-carotene to ruminant feed was safe; β-carotene may have changed the acetic acid and propionic acid concentrations in rumen fermentation, but did not infl uence TVFA production.The mechanism of β-carotene action is still unclear and should be studied more in vivo.

Table 1 .
Ingredients and chemical composition of diets, % of DM