MATERIALS AND METHODS

This study was done in Isfahan Milk and Meat Company at summer 2004. Forty Holstein calves (Mean BW 43±5) in groups balanced by sex and weight were randomly assigned at 3 days of age to one of four treatments: 1) 4 L of whole milk fed twice a day without probiotic, 2) 4 L of whole milk fed twice a day with probiotic, 3) 4 L of whole milk fed once a day without probiotic and, 4) 4 L of whole milk fed once a day with probiotic. Throughout the 7 week trial, calves were placed in individual calf hutches with ad libitum access to water and starter ration (Table 1). The probiotic was a commercial preparation containing Bacillus subitillis (CH 201) and Bacillus lishniformis (CH 200) (hr. Hansen, Milwaukee, WI). Treatments 2 and 4 were offered 1 g probiotic once a day providing 3.2x10⁹ cfu day^-1 head^-1.

Starter intakes were recorded daily and body weight was measured weekly. Five calves per treatment were blood sampled from the jugular vein in a 10 days interval. Samples were centrifuged (3000 rpm, 20 min) and plasma was frozen at -18 until analyzed for albumin and Immunoglubolins. Fecal samples of 5 calves in each treatment were collected prior to the experiment as well as weeks 2, 4 and 6 for determination of total coliform bacteria. For determination of bacterial count, 10 g of feces were diluted in 90 mL of sterile normal saline.

Table 1:	Ingredient composition of the starter ration

Serial dilutions of the original extract were made to achieve a final volume of 0.1 mL plate in McConkey agar (Antec- Iran) and then incubated. After the time and conditions of incubation were optimized coliforms then were counted according to predefined standard procedures.

The study was performed in a completely randomized design and data were analyzed using the proc. GLM of SAS (1999). Treatment effects were declared significant at p<0.05 and trend were discussed when p<0.10. Means were compared using Duncan’s where F test for the treatment effect was significant.

RESULTS AND DISCUSSION

Feed intake during the trial was higher (p<0.10) for calves Mean Feed intake during the trail was higher (p<0.10) for calves receiving milk twice daily plus probiotic and those receiving milk once daily without probiotic compared to treatment 1 and 4 (Table 2). Probiotic did not influence feed intake in this experiment (p> 0.10) (Table 3). This result is in contrast with those reported by Higginbotham and Bath (1993) and Ruppert et al. (1994).

According to the Ruppert et al. (1994), when the diet was supplemented with a probiotic and when calves kept under stressful conditions, feed intake of calves (2 to 28 days) was higher than that of calves in the negative control group. Jenny et al. (1991) also observed no difference in dry matter intake of solid feed when B. Subitilis was fed to calves.

Average daily starter intake was not affected (p>0.10) by milk feeding frequency (Table 4). Average Daily Gain (ADG) was significantly different (p<0.05) for calves fed milk twice daily plus probiotic than calves fed milk once daily plus probiotic. Windschitl et al. (1991) reported that ADG was no statistically different between the control and probiotic treatments and it 1.94 Ibs/day for both of them.

Average daily gain was unaffected by probiotic (Table 3). Daily gain did not differ among calves receiving lactic acid- producing bacteria vs. no DFM (Krehbiel et al., 2001). Previous reports (Cerna et al., 1991; Higginbothem et al., 1997; Panda et al., 1995.) have noted improvement in gain when Lactobacillus products were added to milk or milk replacer fed to dairy calves, whereas other researchers (Cruywagen et al., 1996; Windschitl et al., 1991) have found no improvement in growth. Abe et al. (1995) reported that BW gain of calves in the group fed probiotic (B. pseudolongum and L.acidophilus) was greater than that of the control group. Bifido bacterium pseudolongum has been isolated from many kinds of animals, including calves, piglets, chickens, dogs and others, suggesting that this bacterial species has a wide host specificity and might be commercially useful as a probiotic (Mitsuoka and Kaneuchi, 1977; Rasic and Kurmann, 1983).

Table 2:	Effect of probiotic and milk feeding frequency on performance and coliform numbers in the feces of calves
Standard error, b,c Within a row, means without a common superscript letter differ (p<0.05), d,e Within a row, means without a common superscript letter differ (p<0.10)

Table 3:	Effect of probiotic on performance and coliform numbers in the feces of calves
aStandard error

Table 4:	Effect of milk feeding frequency on performance and coliform numbers in the feces of calves
Standard error., b,c Within a row, means without a common superscript letter differ (p<0.05)

But average daily gain was not different significantly when calves fed replacer containing the B. subitilis concentrate (Jenny et al., 1991). It has been suggested that at time of stress, such as weaning, the balance of intestinal bacteria may become disturbed and performance reduced (Fuller, 1989; Schwab et al., 1980).

Average daily gain was affected (p<0.05)by milk feeding frequency. Calves that fed milk twice daily showed the highest gain during the trial (Table 4). Stanley et al. (2002) however, reported that milk replacer feeding frequency did not affect BW.

Researchers in the UK found no effect of feeding frequency (1, 2, 4 or 6 times daily) on growth rates or energy balance when calves were less than 28 days of age (Quigley, 2001). In addition Hopkins (1997) reported that rates of body weight gain were excellent when calves were fed 3.8 L of whole milk once daily to weaning at 28 days.

Feed efficiency was different among treatments and calves fed milk once daily without probiotic have the largest FE (Table 2). Probiotic did not influence (p>0.10) feed efficiency in this study (Table 3). Abe et al. (1995) however, reported that feed conversion of the calves fed bifidobacteria or lactobacilli was superior to that of the control group.

Schwab et al. (1980) reported that when calves were fed a Lactobacillus fermentation product there was a trend toward improved feed efficiency. But Jenny et al. (1991) observed no differences in Feed efficiency when B. subitilis was fed to calves.

Variability among calves in early growth and acceptance of dry feeds make it difficult to show benefit of probiotics when effects are likely only in animals in which the gut microflora is out of balance. Feed efficiency was affected by (p<0.05) milk feeding frequency and it was greater for those fed milk twice daily than for calves fed milk once daily (Table 4).

Table 5:	Effect of probiotic and milk feeding frequency on blood parameters
a Standard error., b,c Within a row, means without a common superscript letter differ (p<0.05), d,e Within a row, means without a common superscript letter differ (p<0.10)

Treatments had no effect on the occurrence of diarrhea and fecal counts of coliforms (Table 2). Fecal counts of coliform were affected (p>0.10) neither by probiotic (Table 3) nor milk feeding frequency (Table 4). Previous researchers (Gilliland et al., 1980; Abu et al., 1996) have suggested that animals experiencing normal stools are less likely to be shedding coliform in feces. Fecal shedding of coliform has generally not increased when calves were not experiencing diarrheas (Gilliland et al., 1980; Abu et al., 1996). Schwab et al. (1980) observed no apparent effect of L. bulgaricus fermentation product on numbers of fecal Latobacilli and coliforms. Abe et al. (1995) reported that the addition of bifidobacteria to the diets of calves reduced fecal scours. In this study it seems, the gut microflora was in balance, therefore, not as readily affected by the use of probiotic.

Blood plasma immunoglobulins were not affected by treatments and only in calves fed milk once daily without probiotic. Albumin was lower than other groups (p<0.05, p<0.10) (Table 5). Albumin was influenced by probiotic treatment in the group which was under one time milk feeding (p< 0.05). In addition the group which received milk frequently showed change in plasma albumin level (p<0.05). Mcknight et al. (1991) and Morril et al. (1995) observed no apparent effect of probiotic on blood parameters.

The effect of feeding regimen and stress appeared to complicate the observation of absolute effects of probiotics on calf performance and, according to Fuller (1989), probiotics are only effective when animals are stressed by the presence of a microbial population that depresses growth. Management and feeding condition in our study were good and probiotic might have had different effects under sub optimal conditions.