Effect of growth promoters ( probiotics ) supplementation on performance , rumen activity and some blood constituents in growing lambs

The current study examined the effects of probiotics (Pronifer and More-yeast) supplementation to growing lambs. 25 crossbred (Osimi×Rahmani) growing lambs of about 6-8 months age and an average initial live body weight of 25 kg were used. Lambs divided into five groups (5 lambs for each group) and the experimental period lasted for 169 days. The control group T1 received concentrate feed mixture (CFM), group T2 and T3 received 1.5 and 3 kg Pronifer/Mg (megagram) CFM and group T4 and T5 received 2.5 and 5 kg More-yeast/ Mg CFM, respectively. Rumen liquor and blood samples were taken and digestibility trial was conducted at the end of the experiment. Results showed higher digestibility values for crude protein (P<0.05) in T3 and T4 when compared to control. Nutritive ratio was lower (P<0.05) in groups T3 and T4 than in group T2. More-yeast supplementation improved dry matter intake (P<0.001), while a reverse trend was observed with Pronifer supplementation. All treatments had little effect on ruminal liquor except ruminal ammonia concentration in T5 was lower (P<0.05) than other treatments. The differences in most blood plasma parameters due to treatment effect were not significant, except plasma urea and globulin concentrations increased (P<0.05) with T5 treatment. The obtained results appear to indicate that Moreyeast supplementation improved lambs performance and digestibility, whereas, Pronifer supplementation enhanced digestibility.


Introduction
Probiotics are being explored as substitutes of antibiotic feed additives that improve gut health and promote animal performance.Microbial products are known to promote rumen metabolic development by modulating rumen function and fermentation activity of its microflora, which improves ruminant production performance (Tripathi & Karim 2011).Probiotic growth promoters have been used as supplements in animal feeds for more than seven decades to enhance growth and decrease feed:gain ratio (Dawson 1993).Probiotics are viable microorganisms that exhibit a beneficial effect on the health of the host by improving its intestinal microbial balance (Cremonini et al. 2002).
Most evidences for such effects come from results of intensive studies of microbial activities in the rumen of cattle and sheep (Windschitl 1992 andDawson 1993).Pronifer is a fermentation product consisting of a bacterial cocktail of specific lactic acid producing bacteria.As a probiotic agent, it may act through improving the balance of the intestinal microflora (Games 1987).In addition, Dawson et al. (1990) reported a decrease in ruminal pH and an increase in cellulolytic ruminal bacterial numbers in steers fed hay supplemented with lactic acid bacteria (L.acidophilus).Also, Pronifer improves health, performance, minimize diarrhoea and increase growth rates (Bohm & Srour 1995).
Yeast product supplementation has several benefits in ruminant nutrition which have been demonstrated an increase in nutrient digestibility, alteration of the proportion of volatile fatty acids produced in the rumen, reduction in ruminal ammonia, and increase of ruminal microorganism population (Chaucheyras-Durand et al. 2008).Furthermore, yeast culture supplementation in growing lamb has the potential to improve intake and growth and can substitute antibiotics as growth promoting feed additive (Tripathi & Karim 2011).Dawson (1992) mentioned that yeast stimulates rumen bacteria and enhance lactate and ammonia utilization resulting in moderate ruminal pH and increases in microbial population which lead to increased in fibre digestion and protein synthesis in the rumen.Yeast culture improved feed intake, weight gain and feed conversion in growing ruminants (Salem et al. 2000 andEl-Waziry et al. 2000).Yeast also provides vitamins to support the growth of rumen fungi (Chaucheyras-Durand et al. 1995).
The objective of this study was to investigate the effect of adding Pronifer and More-yeast supplements as growth promoters on performance, digestibility, rumen activity and blood parameters in growing crossbred lamb.

Lambs, treatments and samples
Twenty five crossbred (Osimi×Rahmani) growing lambs, about 6-8 months old with an average initial live body weight (LBW) of 25.04±0.36kg were used in this experiment.The experiment was carried out at Moshtohor Animal Farm, Ministry of Agriculture, Egypt.The animals were divided into five groups on the basis of initial LBW and age.Each group contained five animals.The feeding trial lasted for 168 days and animals were fed individually.Lambs were assigned at random to one of the five experimental diets as shown in Table 1.The diets used in this study consisted of wheat straw, concentrate feed mixture (CFM) and Pronifer or More-yeast.
The chemical analysis of the experimental feeds used is presented in Table 2. Pronifer is a probiotic feed additive which contains: -viable lactic acid bacteria (approx.10 6 CFU/g) (Lactobacillus planetarium, L. brevis, L. fermeritum, L. casei, and Pediococcus acidilacticii) -lactic acid fermentation metabolites and enzymes (organic acids, glucosidase, and peptidase enzymes) -free (soluble) amino acids and short-chain peptides (Srour et al. 2000).More-yeast is a probiotic including live yeast (saccharomyces cerevisiae) and other directfed microbials including strains of lactobacillus, streptococcus, aspergillus and several others.The live yeast contained pure strains of saccharomyces cerevisiae grown in purified cane and beet molasses solutions under the most stringently controlled chemical, bacteriological and sanitary conditions.More-yeast was imported from Norchem, Inc. (Hauppauge, NY, USA) Company by International Marketing Center (Cairo, Egypt).Lambs were adapted to the experimental diets for two weeks before the start of the feeding trial.Wheat straw was offered ad libitum, while CFM was allowed according to the NRC (1985) recommendations based on LBW.Diets were offered twice daily in two equal portions at 8.00 a.m. and 4.00 p.m. Feed intake and refusals (if any) were recorded daily.Lambs were weighed every second week in the morning before feeding.At the end of the experiment, a digestibility trial was conducted using three animals from each experimental group assigned at random and maintained in individual digestibility cages.Lambs were weighed at the start and end of each trial.
The digestibility trial consisted of 15 days as a preliminary period, followed by 10 days as the collection period.The experimental rations were weighed and fed according to NRC (1985).Wheat straw was offered ad libitum.Feed intake was recorded daily once at 8.00 a.m.Faeces were collected from each individual animal once daily before the morning meal, the refusal of feeds (if any) were collected and recorded daily before offering the new feed.Daily faeces excreted from each lamb was weighed and 10 % of the fresh faeces was taken and dried at 60 °C for 24-72 h to determine dry matter of the faeces.Composite samples from the daily dried faeces, for each lamb, were mixed, ground and stored in refrigerator for subsequent chemical analysis.Representative samples of experimental diets, refusals and faeces were analysed for dry matter (DM), organic matter (OM), crude protein (CP), ether extract (EE), crude fibre (CF) and ash contents according to AOAC (1999) methods, while nitrogen-free extract (NFE) was calculated by differences.
Rumen liquor samples were taken individually from three lambs of each experimental group at four times during the feeding trial period (0, 8, 16 and 24 weeks).Rumen samples were taken using a rubber stomach tube connected to a vacuum pump (Nagah 2002), before feeding and at 3 and 6 h post-feeding.Rumen samples were strained through four layers of cheese cloth.Rumen liquor analysis included ruminal pH value (pH meter), Ammonia nitrogen (N-NH 3 ) concentration determined immediately according to Conway (1962).Total volatile fatty acid (TVFA) concentrations were determined by the steam distillation method (Abou-Akkada & El-Shazly 1964).
Blood samples were taken at 0, 8, 16 and 24 weeks throughout the feeding period.The blood samples were taken from jugular vein of three lambs from each experimental group before morning feeding by vein puncture and were put into 10 ml heparinized test tubes.Blood samples were immediately centrifuged at 3 500 rpm for 15 min to separate plasma.Blood plasma samples were immediately frozen at −20 °C and stored until analysis.Commercial kits (Diamond Diagnostics Co., Egypt) were used to determine total protein (TP), albumin (Al), urea (U), cholesterol (Ch), creatinine and plasma transaminases; glutamicoxaloacetic-transaminase (GOT) and glutamic-pyruvic-transaminase (GPT), according to Armstrong & Carr (1964), Doumas et al. (1971), Patton & Crouch (1977), Allain et al. (1974), Henry (1965) and Reitman & Frankel (1957), respectively.While, globulin (G) was calculated by subtracting the Al concentration from the TP value and Al:G ratio was measured by dividing albumin value by its corresponding globulin value.

Statistical analysis
The data were statistically analysed according to SAS (1999).The statistical models used in this study were as follows: where Y ij is the observed response, U is the overall mean, A i is the effect of treatment (growth promoters), E ij is the experimental error.The differences among groups were tested by Duncan's multiple rang test (Duncan 1955).

Nutrients digestibility and feeding values
The average digestion coefficients and feeding values of experimental diets are shown in Table 3.The present results showed that lambs fed T 1 (control) recorded numerically lowest digestibility values for all feed nutrients (DM, OM, CP, CF, EE and NFE).The differences in nutrients digestibility due to treatment effect were not significant except for CP digestibility.

Lamb performance
Average LBW, daily weight gain (DWG), dry matter intake (DMI) and feed conversion (FC) of lambs fed experimental diets are illustrated in Table 4. Lambs of More-yeast supplementations T 4 and T 5 recorded the highest values of LBW, DWG without significant, also, lambs of T 4 and T 5 consumed the highest amount of daily DMI (P<0.001),whereas, those of T 2 and T 3 (Pronifer supplemented groups) recorded the lowest amount compared with the control.Data for feed conversion did not show any diet effects.

Ruminal liquor and blood parameters
Values of ruminal pH, NH 3 -N and TVFA concentrations of lambs in the different experimental treatments are shown in Table 5. Results showed that the differences in pH and TVFA values at the different experimental treatment were very limited and without any significant effect.However, differences in NH 3 -N concentration due to treatments effect were significant (P<0.05).Differences in blood plasma concentrations due to treatment effects were seen for globulin (G) and urea (U) concentrations (P<0.05)(Table 5).

Nutrients digestibility and feeding values
The improvement in nutrients digestibility is possibly explained on the basis that yeast culture enhanced microbial activity and also due to that some beneficial activities of lactic acid bacteria in the gastrointestinal i.e., its anti diarrhoea activities, anti tumour activities and its ability to alter enzyme activities (Dawson 1988).
The main effect of yeast culture supplementation on ruminants include improvement of gut health and ecology through rumen maturity by favouring microbial establishment, stabilisation of rumen pH and interaction with lactate utilizing bacteria (Yang et al. 2004).The establishment of complex rumen microbial ecosystem subsequently improve rumen function that promotes absorption ability and feed digestion (Hopper et al. 2001), thus live yeast additives improve gut health.
These results are in agreement with those reported by Bohm & Srour (1995) who reported that supplementing the diet of crossbred calves with 5 g Pronifer/(kg concentrate×d) improved the digestibility of most feed nutrients.El-Ashry et al. (2003) with sheep and goats, Nagah (2002) and Khattab et al. (2003) with crossbred lambs and Ragheb et al. (2003) with Friesian calves indicated that the digestibility of most feed nutrients improved by yeast culture (YC), yea-sac (YS) or lacto-sac (LS) supplementation.Feeding values have improved with supplementations due to positive effect on digestible tract.Nagah (2002) and Khattab et al. (2003) reported that the use of either 3 g YS or 3 g LS/h/d improved SV and TDN values of crossbred lambs.Also, Gado et al. (1998), Salem et al. (2000) and El-Ashry et al. (2003) found that yeast culture supplementations had positive effect on TDN and DCP values of growing lambs.

Lamb performance
Results showed significant increase in DM intake due to More-yeast supplementation (T 4 and T 5 ).In addition, LBW and DWG are enhanced slightly with growth promoter supplementations probably due to enhance in rumen activity and digestibility.
The active yeast supplementation has positive effects in young ruminant's performance through increased DM intake and daily gain, Thus, The performance promoting effects of live yeast additives could be correlated to an improvement in rumen development parameters such as papillae length and width, and rumen thickness (Lesmeister et al. 2004), early establishment and stabilization of rumen microbial communities (Chaucheyras-Durand & Fonty 2001, 2002) and reduced number of days of diarrhoea (Galvano et al. 2005).Moreover, YC, YS and LS supplements improved the growth performance of growing lambs (Salem et al. 2000, Fayed 2001, Nagah 2002and El-Ashry et al. 2003).
On the other hand, supplements had no significant effects on FC agreement with Nagah (2002) and El-Ashry et al. (2003), they indicated that YC, YS and LS supplements had no effect on feed conversion values (as DM, TDN and DCP/kg gain) of growing lambs.Similarly, Mir & Mir (1994) with steers and Ragheb et al. (2003) with Friesian calves found that live yeast and LS had no positive effect on feed utilization values (kg gain/kg DM, TDN and DCP).

Ruminal liquor parameters
The significant differences in the values of ruminal NH 3 -N showed the lowest value T 5 .In accordance with the previous results, Nagah (2002) and Mussa (2001) reported that the ruminal NH 3 -N concentrations for YS and LS supplementations were lower than the control but the differences were not significant.
The reduction in ruminal NH 3 -N of More-yeast treated animals in the present study may be probably attributed to the inhibitory effect of this growth promoter on proteolysis amino acid deamination and ruminal urease activity (Fuller & Johnson 1981, Starnes et al. 1984and Gomez et al. 1991).
The mechanism of yeast action may be due to competition between yeast cells and the bacteria for energy supply and by a direct inhibitory effect of yeast on small peptides and bacterial peptidases (Chaucheyras-Durand et al. 2008).Yeast culture supplementation is reported to enhance microbial growth and decrease N loss by incorporating more digestible carbohydrates into microbial mass (Sniffen et al. 2004).

Blood parameters
The differences in plasma globulin concentrations between T 1 and T 2 or T 5 were significant, whereas no significant differences were observed among other treatments.Sadik (1989) found that lactobacillus concentrate supplement significantly increased plasma globulin concentrations for buffalo heifers.Also, Mohanna (2000) with lactating buffaloes, Salem et al. (2000) with growing crossbred sheep, and El-Ashry et al. (2003) with Barki lambs, reported that YC supplementation significantly increased plasma G values.
Result obtained revealed that plasma urea concentration in blood plasma of lambs fed the experimental diets ranged from 21.52 mg/dl for T 2 (1.5 kg Pronifer/Mg CFM supplemented group) and 27.98 mg/dl for T 5 (5 kg More-yeast/Mg CFM supplemented group).The differences in plasma urea concentrations were only significant between T 5 and those of T 1 and T 2 , whereas no significant differences were detected between other treatments.Ragheb et al. (2003) who reported that dietary YS and LS supplementation increased blood plasma urea concentrations compared with the control group.
In general results of blood plasma parameters for lambs fed the experimental diets indicated that all treatments had little effect on blood plasma parameters estimated in the present study as the differences in all parameters due to treatment effect were not significant except for globulin and urea values.
However, values of most blood plasma parameters estimated in the present study are within the normal ranges for ruminants published by several workers in the literatures (Owen et al. 1954, Mahmoud 1993, Nagah 2002, EL-Ashry et al. 2003and Ragheb et al. 2003), and suggested that the experimental growth promoters are safe for physiological and healthy status of all experimental lambs.
In conclusion, supplementation of probiotics as growth promoters (Pronifer and Moreyeast) in diets of growing lambs enhanced protein digestibility and dry matter intake, whereas these supplements had little effect on feed conversion and rumen activity.Both, Pronifer and More-yeast supplementation in diet improved plasma globulin which may be related to immunity in these animals.
Lambs of T 3 (3 kg Pronifer/Mg CFM) and T 4 (2.5 kg More-yeast/Mg CFM) showed the highest CP digestibility values.The lowest digestible crude protein (DCP) values were shown by T 2 and T 1 (P<0.05)while the highest ones were recorded by T 3 and T 4 (P<0.05).In general, T 3 (3 kg Pronifer/Mg CFM) and T 4 (2.5 kg More-yeast/Mg CFM) showed the best feeding values (DCP) and narrowest nutritive ratio (NR) values, compared with either the control or other treatments.

Table 4
Averages of lamb performance and feed conversion values of lambs fed the experimental diets a,b,c Averages with different superscripts in the same column are significantly (P<0.05)different.

Table 5
Averages of ruminal pH and blood parameters values of lambs fed the experimental diets a,b Averages with different superscripts in the same column are significantly (P<0.05)different.