Effect of partial substitution of milk with probiotic yogurt of different pH on performance, body conformation and blood biochemical parameters of Holstein calves

Abstract

The present study was conducted to evaluate the effect of yogurt with different pH values as partial milk replacer on performance, health and blood parameters of Holstein calves. A total of 35 Holstein calves (two males and five females per each treatment) from the fourth day of birth until two weeks after weaning were randomly assigned to the five experimental dietary treatments. Experimental groups consisted of control (milk without yogurt) and groups fed milk containing 15% and 30% probiotic yogurt with pH values of either 3.8 or 4.5. Results showed that starter intake (SI) and dry matter intake (DMI) were not affected by dietary treatments throughout (except 4–7 d) the trial period. Partial substitution of probiotic yogurt resulted in a significant (P < 0.05) increase in SI compared with control calves at seventh and eighth weeks of trial. Also, using probiotic yogurt with pH of 4.5 increased (P < 0.05) SI when compared with pH value of 3.8 at the second trial week. On the other hand, DMI was significantly (P < 0.01) lower in yogurt-fed calves than milk group at seventh week of experiment. Compared to control, inclusion of probiotic yogurt increased (P < 0.001) daily and total weight gain. Feed conversion ratio was lower (P < 0.01) in yogurt-fed groups compared with control calves. Partial substitution of probiotic yogurt for milk increased body length, wither height and hip depth. In addition, inclusion of probiotic yogurt to partially replace milk resulted in increased (P < 0.01) lymphocyte proportion and decreased (P < 0.05) neutrophil to lymphocyte ratio. Although experimental treatments affected serum concentrations of triglyceride (P < 0.01) and cholesterol (P < 0.05), however, had no effect on serum glucose and total protein levels. Results indicate that partial substitution of probiotic yogurt for milk could improve growth performance and modulate immune status of calves as measured by leukocytes alterations.

Keywords:

• Holstein calf
• probiotic yogurt
• growth rate
• immune status
• serum metabolites

1. Introduction

Diarrhoea has been reported as the main reason for mortality and huge economical losses in rearing neonatal calves (Frizzo et al. 2010). Feeding the high levels of lactose to the newborn animals causes a noticeable microbial imbalance, especially the presence of coliforms such as Escherichia coli in intestine, resulting in diarrhoea (Frizzo et al. 2011). Thus, antibiotics have been widely utilized to promote growth and to prevent diarrhoea of newborn calves resulting from pathogenic bacteria (Constable 2004). However, the use of antibiotics in livestock can make serious problems such as expansion of antibiotic-resistant bacteria (Fey et al. 2000; Langford et al. 2003) as well as remaining antibiotics in dairy foods, meat and milk (Phillips et al. 2004). Probiotics are one of the best antibiotic alternatives (Callaway et al. 2004), avoiding the presence of E. coli in intestine, and in turn, they can control or treat diarrhoea in human (Sazawal et al. 2006) and animals (Reid & Friendship 2002). Probiotics have beneficial impacts such as the improvements in intestinal microbial balance and calf enteric environment (Kaur et al. 2002), production of inhibitory metabolites against pathogenic bacterial growth (Voravuthikunchai et al. 2006) and protection against infectious factors due to enhanced immunological responses (Schiffrin & Blum 2002). Lactic acid bacteria, Bifidobacterium, yeast and bacilli are the most widespread and beneficial microbes used as probiotics for prevention of diarrhoea (Abu-Tarboush et al. 1996; Agarwal et al. 2002). Supplementation of probiotics resulted in an improvement in microbial ecosystem, increase in nutrient bioavailability and improvement of growth performance in lambs (Khalid et al. 2011). In addition, Whitley et al. (2009) reported that apparent dry matter and crude protein digestibilities increased in lambs fed on probiotic diet. Moreover, Abo El-Nour and Khalif (1998) showed an increase in blood glucose concentration of cows received diets containing probiotic. However, Antunovic et al. (2006) reported that blood glucose content did not change in lambs fed on a probiotic-supplemented diet.

Yogurt is one of the best and most identified foods containing probiotics and it is made by Lactobacillus bulgaricus and Streptococcus thermophilus as a result of lactic acid fermentation of milk (Bourlioux & Pochart 1988). It has been shown that coliform bacteria were decreased by dietary inclusion of Lactobacillus acidophilus (Bruce et al. 1979) and as a result, Fox (1988) could decrease diarrhoea in calves by administration of L. acidophilus. Furthermore, Kawakami et al. (2010) reported that supplementation of Lactobacillus plantarum at 3.7 × 10¹¹ colony-forming units (cfu) significantly increased weight gain and feed efficiency in calves. Conway et al. (1987) reported that yogurt improved gut health due to the formation of gastrointestinal flora and increasing gut immunity.

Despite these beneficial effects, one of the main problems resulted from using dietary cultures is the low resistance of some probiotic species against lower pH values (high levels of acid production resulting from uncontrolled growth of L. bulgaricus) and refrigerator temperature (Rybka & Kailasapathy 1997). It has been reported that L. acidophilus cannot survive and grow at pH values of <4 (Lankaputhra & Shah 1995).

Since there is limited information about substitution of probiotic yogurt for milk in calves’ nutrition, and the effect of pH on viability of probiotic bacteria is contradictory, the objective of this study was to evaluate the effect of probiotic yogurt with different pH values as partial milk replacer on performance, skeletal growth and blood parameters of Holstein calves.

2. Materials and methods

2.1. Treatments and animal management

The present study was conducted in an industrial dairy farm (Foudeh Sepahan Milk & Meat Co., Isfahan, Iran) and all procedures used were approved by Isfahan University of Technology (Isfahan, Iran) Animal Use and Care Committee. A total of 35 four days old Holstein calves were used and fed with experimental dietary treatments until two weeks after weaning. The calves were randomly attributed into the individual boxes among different experimental dietary treatments. Seven calves (two males and five females) assigned for each of five experimental groups. Dietary treatments consisted of control (milk without yogurt), 15% probiotic yogurt with pH value of 3.8, 15% probiotic yogurt with pH value of 4.5, 30% probiotic yogurt with pH value of 3.8 and 30% probiotic yogurt with pH value of 4.5. Calves received 3–4 litres colostrum from birth to 3 d of age; thereafter, they were fed with 4 litres milk per day until 30 d of age, and 5 litres per day for remaining trial period. Starter diets were formulated to meet all of the nutritional requirements for calves based on NRC (2001) recommendations. Starter diets and water were offered ad libitum throughout the trial period. Starter composition was similar for all calves during trial period (Table 1). From d 4 to 30 d of age, starter 1 was fed. From d 30 of age until the end of trial, the starter 2 was fed to the calves along with alfalfa (10% of alfalfa and 90% of starter 2).

Table 1. Feed ingredients and nutrient composition of starter diets.

Items (% unless stated otherwise)	Starter 1	Starter 2
Ingredients
Corn, yellow	39.28	49.78
Wheat	24.44	24.44
Soybean meal	28.80	18.30
Full-fat soybean	4.36	4.36
Na bicarbonate	1.32	1.32
Calcium carbonate	1.13	1.13
Common salt	0.44	0.44
Mineral and vitamin premix^a	0.23	0.23
Nutrient composition
Dry matter	89.23	89.74
Crude protein	22.82	18.53
Ether extract	2.65	3.20
Neutral-detergent fibre	26.80	20.30
Acid-detergent fibre	7.70	4.20
Calcium	0.40	0.37
Phosphorus	0.38	0.31

^a Premix contained the following composition per kilogram of diet: vitamin A, 4,200 IU; vitamin D₃, 1,575 IU; vitamin E, 42 mg; Fe, 15 mg; Zn, 35 mg; Cu, 11.6 mg; Mn, 16.3 mg; Co, 0.13 mg; I, 0.46 mg; Se, 0.35 mg.

2.2. Preparation of probiotic yogurt with pH values of 3.8 and 4.5

Probiotic yogurt was produced according to the instruction proposed by Chr. Hansen Company (Milwaukee, WI). Stages were included pasteurization at 85°C for 30 min, chilling milk to 30–35°C, inoculation of microbial culture (0.02% of microbial inoculant) containing L. bulgaricus, L. acidophilus, Bifidobacterium, S. thermophillus, and incubation at 43°C to reach pH values of 4.5 (for 5–6 h) and 3.8 (for 18–20 h).

2.3. Bacterial count

To count the bacterial population, 1 ml of probiotic yogurt with pH values of 3.8 and 4.5 was added to 9 cc phosphate buffer saline solution. Samples were cultured at the concentrations of 10^–6, 10^–7, 10^–8 and 10^–9 cfu on MRS medium (Merck GmbH, Darmstadt, Germany). After enumeration, lactobacillus populations in yogurt with pH values of 4.5 and 3.8 were determined to be 3.895 × 10¹¹ cfu and 8.03 × 10⁷ cfu, respectively.

2.4. Measuring performance and skeletal parameters

The calves were weighed on d 4, 56 and 70 of age. Additionally, they were separately weighed every week using a sensitive digital scale after 5 h of hunger. Feed intake was also measured daily by a sensitive digital scale.

Wither height, body length and hip depth were measured by T ruler on d 14, 34 (one month after beginning the trial) and 56 (weaning) of age.

2.5. Faecal scores

Faecal scores were determined daily according to the method described by Meyer et al. (2001). In this method, faeces scores were described as (1) normal, (2) soft, (3) fluid and (4) aqueous. Then, the days in which calves suffered diarrhoea (scores 3 and 4) were analysed as total number of disease days.

2.6. Blood biochemical parameters and leukocytes subpopulations

Calves were bled from jugular vein at the beginning (as covariate in statistical model) and at the final day of trial. Then, serum samples were separated by centrifugation at 4500 × g for 5 min at room temperature. The serum concentrations of triglycerides, cholesterol, high-density lipoproteins (HDL), glucose and total protein were measured by standard kits (Pars Azmoun, Tehran, Iran) according to procedures proposed by producing company. In addition, heparinized-blood samples were collected to evaluate the leukocyte subpopulations on d 42 of experiment.

2.7. Statistical analysis

All data were subjected to analysis of variance using the general linear models procedure of SAS software (SAS Institute 1999) as completely randomized design. Treatment means were separated by Duncan's multiple range test (Duncan 1955) at P < 0.05 statistical level. The initial weight of calves was used as covariate for performance data. In addition, the measurements of d 0 for blood biochemical indices were considered as covariate in the statistical model. The incidence of diarrhoea was analysed as disease days (that is the number of days in which each calve showed diarrhoea over the trial period).

3. Results and discussion

3.1. Performance parameters

The results on dry mater intake (DMI) from starter diet were presented in Table 2. Experimental dietary treatments had no significant effect on the intake of starter diets by the calves throughout the trial period. However, using probiotic yogurt resulted in a significant (P < 0.05) increase in starter intake (SI) at seventh and eighth weeks of trial when compared with control group. Consistent with our observations in the most trial weeks, Francia et al. (2008) also showed that SI was not affected by dietary supplementation of yeast culture in buffalo calves.

Table 2. Effect of probiotic yogurt with different pH values on DMI from starter diets by calves (g).

Experimental treatments	4–7 d	8–14 d	15–21 d	22–28 d	29–35 d	36–42 d	43–49 d	50–56 d
Control	83.2	214.5	1023	1865	3092	4571	5254	6198
15% probiotic yogurt with pH = 3.8	45.1	244.3	825	1656	2521	4147	6708	7996
15% probiotic yogurt with pH = 4.5	63.4	449.9	1513	2507	3908	5662	7519	8752
30% probiotic yogurt with pH = 3.8	35.9	250.5	1191	1957	3404	4592	7081	8003
30% probiotic yogurt with pH = 4.5	65.2	478.3	1290	2225	3490	4504	6088	7669
Contrast comparisons
Control	83.2	214.5	1023	1865	3092	4571	5254^b	6198^b
Yogurt	52.2	355.7	1205	2086	3331	4726	6849^a	8105^a
Low pH	40.0	247.4^b	1008	1806	2963	4369	6895	7999
High pH	64.3	464.1^a	1401	2366	3699	5083	6803	8213
Probability
Treatments	0.733	0.187	0.192	0.392	0.243	0.443	0.218	0.182
Control vs. yogurt	0.282	0.218	0.453	0.554	0.641	0.817	0.050	0.024
pH values	0.285	0.042	0.087	0.091	0.093	0.225	0.902	0.733

Note: Means with no common superscript alphabets within each column are significantly (P < 0.05) different.

Table 3. Effect of probiotic yogurt with different pH values on total DMI of calves (g).

Experimental treatments	4–7 d	8–14 d	15–21 d	22–28 d	29–35 d	36–42 d	43–49 d	50–56 d
Control	946^b	1937	2748	4161	5791	7872	7539	10,933
15% probiotic yogurt with pH = 3.8	1133^a	1969	2549	3381	4808	6733	9849	11,007
15% probiotic yogurt with pH = 4.5	926^b	2174	3237	4231	6186	8249	10,659	12,201
30% probiotic yogurt with pH = 3.8	1197^a	1974	2914	3680	5770	7177	10,220	11,415
30% probiotic yogurt with pH = 4.5	927^b	2202	3013	3949	5776	7089	9227	10,559
Contrast comparisons
Control	946	1937	2748	4161	5791	7872	9989^a	10,933
Yogurt	1046	2080	2929	3810	5635	7312	7539^b	11,296
Low pH	1165^a	1971^b	2732	3530	5289	6955	10,035	11,088
High pH	926^b	2188^a	3125	4090	5981	7669	9943	11,380
Probability
Treatments	0.001	0.171	0.185	0.289	0.197	0.257	0.078	0.518
Control vs. yogurt	0.140	0.206	0.451	0.323	0.743	0.356	0.008	0.640
pH values	0.001	0.037	0.079	0.096	0.124	0.217	0.896	0.673

Note: Means with no common superscript alphabets within each column are significantly (P < 0.05) different.

In addition, supplementation of probiotic yogurt with pH value of 4.5 significantly (P < 0.05) increased SI in the second week of experiment as compared with those on probiotic yogurt with pH value of 3.8. Also, SI was slightly (P > 0.05) higher in calves supplemented with yogurt of pH = 4.5 than pH = 3.8 during the remaining trial weeks.

As shown in Table 3, DMI was markedly (P < 0.001) affected by experimental dietary treatments at the first week of trial, but not in the remaining trial period. Additionally, DMI was lower (P < 0.01) in calves supplemented with probiotic yogurt compared with those on milk at seventh week of trial. In discrepancy with our findings, Kawakami et al. (2010) and Frizzo et al. (2011) stated that inclusion of lactic acid bacteria had no significant effect on DMI of calves.

Table 4. Effect of probiotic yogurt with different pH values on performance traits of calves.

Experimental treatments	Daily weight gain (kg)	Total weight gain (kg)	Daily DMI (kg)	Total DMI (kg)	Feed conversion ratio (kg DMI: kg gain)	Weight gain after weaning (kg)
Control	0.47^b	24.91^b	0.79	41.93	1.69^a	14.24
15% probiotic yogurt with pH = 3.8	0.55^a	29.19^a	0.78	41.43	1.42^b	12.56
15% probiotic yogurt with pH = 4.5	0.58^a	30.57^a	0.90	47.86	1.57^ab	13.87
30% probiotic yogurt with pH = 3.8	0.60^a	31.84^a	0.84	44.35	1.39^b	13.53
30% probiotic yogurt with pH = 4.5	0.57^a	30.11^a	0.81	42.74	1.42^b	13.53
Contrast comparisons
Control	0.47^b	24.91^b	0.79	41.93	1.69^a	14.24
Yogurt	0.58^a	30.48^a	0.83	44.10	1.45^b	13.37
Low pH	0.58	30.52	0.81	42.77	1.40	13.04
High pH	0.57	30.43	0.85	45.30	1.50	13.70
Probability
Treatments	0.020	0.023	0.352	0.357	0.024	0.871
Control vs. yogurt	0.001	0.001	0.432	0.435	0.005	0.482
pH values	0.953	0.957	0.324	0.326	0.182	0.413

Note: Means with no common superscript alphabets within each column are significantly (P < 0.05) different.

As noticed, partial substitution of probiotic yogurt with pH value of 4.5 resulted in a noticeable (P < 0.001) decrease in DMI at the first week as compared with calves supplemented with yogurt with pH value of 3.8. However, DMI was significantly (P < 0.05) higher in calves supplemented with yogurt with pH value of 4.5 than those on yogurt with pH = 3.8 at the second week of experiment. The reasonable explanation for this observation remains to be elucidated.

Daily and total DMI was not influenced by yogurt substitution (Table 4). Also, there was no significant difference between calves supplemented with probiotic yogurt and milk, or among those on yogurt with pH values of 3.8 and 4.5 in daily and total DMI.

Table 5. Effect of probiotic yogurt with different pH values on skeletal growth of calves at different ages.

	Body length (cm)			Wither height (cm)			Hip depth (cm)
Experimental treatments	d 14	d 34	d 56	d 14	d 34	d 56	d 14	d 34	d 56
Control	59.6	64.1^c	75.1	76.6	80.0	85.3^b	27.4	31.7	32.3
15% probiotic yogurt with pH = 3.8	62.4	71.6^a	76.7	78.7	80.9	89.0^a	29.0	31.6	33.0
15% probiotic yogurt with pH = 4.5	59.1	66.1^bc	77.6	77.0	79.9	89.9^a	28.7	33.0	34.7
30% probiotic yogurt with pH = 3.8	62.7	70.0^a	77.3	77.6	82.6	89.0^a	29.6	31.1	33.4
30% probiotic yogurt with pH = 4.5	60.3	69.0^ab	73.9	77.9	81.7	86.6^ab	29.7	31.0	33.4
Contrast comparisons
Control	59.6	64.1^b	75.1	76.6	80.0	85.3^b	27.4^b	31.7	32.3^b
Yogurt	61.1	69.2^a	76.4	77.8	81.3	88.6^a	29.3^a	31.7	33.6^a
Low pH	62.6^a	70.8^a	77.0	78.1	81.7	89.0	29.3	31.4	33.2
High pH	59.7^b	67.6^b	75.7	77.4	80.8	88.2	29.2	32.0	34.1
Probability
Treatments	0.061	0.001	0.102	0.765	0.623	0.044	0.092	0.137	0.059
Control vs. yogurt	0.219	0.002	0.354	0.362	0.436	0.021	0.017	0.961	0.042
pH values	0.006	0.018	0.233	0.505	0.514	0.507	0.901	0.334	0.131

Note: Means with no common superscript alphabets within each column are significantly (P < 0.05) different.

Utilization of probiotic yogurt to partially replace milk increased (P < 0.05) daily and total weight gain (Table 4). This could be attributed to the increases in DMI from starter diet (Table 2), early establishment and stabilization of ruminal microbial balance (Chaucheyras-Durand & Fonty 2002), increase in nutrient digestibility and absorption (Khuntia & Chaudhary 2002) due to increase in enzyme activities such as xylanase, protease α-amylase and β-glucosidase (Tripathi & Karim 2010), changes in volatile fatty acids resulting from increase in ruminal fermentation rate and bacterial count (Thurne et al. 2009), improvement in ruminal development parameters including papillae length and width as well as rumen thickness (Lesmeister et al. 2004) and probably reduction of diarrhoea duration (Galvano et al. 2005). Consistent with our results, Krehbiel et al. (2003) and Kawakami et al. (2010) indicated that inclusion of probiotic caused an increase in weight gain compared with control group.

Experimental dietary treatments had no significant effect on weight gain after weaning (Table 4), which could be due to this fact that beneficial effects of probiotics on performance usually occur during the early growth stages due to the replacement of useful bacteria with pathogenic ones (Heyman & Ménard 2002; Rosmini et al. 2004).

As shown in Table 4, a significant (P < 0.01) improvement in feed conversion ratio (FCR) was occurred in calves received probiotic yogurt compared with control calves. An increase in weight gain resulting from an improvement in microbial ecology (Chaucheyras-Durand & Fonty 2002) and increasing in nutrient absorption (Khuntia and Chaudhary 2002) might be responsible for the improvement of FCR values. Consistent with our findings, Sun et al. (2010) and Frizzo et al. (2011) exhibited that dietary supplementation of lactobacillus improved FCR in calves. However, there was no significant difference in FCR values between calves received yogurt with either pH values of 3.8 or 4.5.

3.2. Body conformation and skeletal parameters

There were significant differences between experimental groups for body length at d 34 (P < 0.001) and wither height at d 56 (P < 0.05) of age (Table 5). In addition, body length at d 34 (P < 0.01), wither height at d 56 (P < 0.05) and hip depth at d 14 and 56 (P < 0.05) of age were significantly increased by partial substitution of yogurt for milk. The increases in hip depth and wither height in yogurt-fed calves can be attributed to increase in mineral bioavailability such as calcium, phosphorus and magnesium (Khuntia & Chaudhary 2002) as well as higher DMI and weight gain in these valves (Table 4). Consistent with our findings, Chandra et al. (2009) found that dietary supplementation of probiotics resulted in an increase in wither height as compared with control calves.

3.3. Number of disease days

Data on the number of disease (diarrhoea) days were summarized in Table 6. Dietary treatments had no significant effect on the number of disease days. However, the number of disease days was numerically (P > 0.05) lower in the calves received probiotic yogurt compared with those on milk, and also was lower in calves fed yogurt with pH value of 4.5 compared with probiotic yogurt of pH = 3.8. Abu-Tarboush et al. (1996) found that diarrhoea, particularly neonatal diarrhoea, is the main cause of higher mortality rate in calves during the first four weeks of age, partly because of the low levels of immunoglobulin G in plasma of neonatal animals (Rodríguez et al. 2009). Numerical reduction in the occurrence of diarrhoea and disease days might be related to increase in immunological responses against pathogenic bacteria arising from the addition of probiotics (Emmanuel et al. 2007), because they improve ruminal microbial balance (Abu-Tarboush et al. 1996; Frizzo et al. 2011) and promote the excretion of coliforms into the faeces (Agarwal et al. 2002); consequently increase calve health (Frizzo et al. 2010). Consistent with these observations, no significant effect of probiotics on diarrhoea of calves was obtained by Lesmeister et al. (2004).

Table 6. Effect of probiotic yogurt with different pH values on the number of disease days and differential leukocyte counts of calves.

Experimental treatments	Disease^1 (days)	Neutrophils (%)	Lymphocytes (%)	Neutrophil to lymphocyte ratio
Control	2.43	29.42	66.59^c	0.44^a
15% probiotic yogurt with pH = 3.8	2.14	28.28	69.28^ab	0.41^bc
15% probiotic yogurt with pH = 4.5	1.57	29.55	67.92^bc	0.44^ab
30% probiotic yogurt with pH = 3.8	1.86	27.56	70.12^a	0.39^c
30% probiotic yogurt with pH = 4.5	2.14	28.22	69.31^ab	0.41^bc
Contrast comparisons
Control	2.43	29.42	66.59^b	0.44^a
Yogurt	1.93	28.40	69.16^a	0.41^b
Low pH	2.00	27.92	69.70	0.40
High pH	1.86	28.88	68.61	0.42
Probability
Treatments	0.946	0.166	0.007	0.016
Control vs. yogurt	0.545	0.179	0.005	0.023
pH values	0.845	0.160	0.058	0.084

Note: Means with no common superscript alphabets within each column are significantly (P < 0.05) different.

¹ Disease days shows the number of days which calves suffered diarrhoea (faecal scores of 3 and 4).

3.4. Differential leukocyte count

As shown in Table 6, the proportion of neutrophils was not affected by experimental dietary treatments. Similarly, probiotic yogurt had no significant influence on the neutrophil proportion as compared with control (milk) calves. Additionally, substitution of 30% probiotic yogurt with pH value of 3.8 markedly (P < 0.01) increased the lymphocyte population. Moreover, the proportion of lymphocytes was noticeably (P < 0.001) higher in calves supplemented with probiotic yogurt than that of milk group. It has been demonstrated that probiotics stimulate and promote immunological responses in calves (Schiffrin & Blum 2002). The present results are in contrast to Mohamadi Roodposhti and Dabiri (2012), who observed that supplementation of probiotics did not affect leukocytes subpopulations.

Neutrophil to lymphocyte ratio was significantly (P < 0.05) influenced by dietary treatments. Supplementation of yogurt with pH value of 4.5 tended (P = 0.084) to increase the neutrophil to lymphocyte ratio when compared with yogurt of pH = 3.8. The increase in lymphocytes without changing in neutrophil proportion might be responsible for decrease in neutrophil to lymphocyte ratio in the experimental (yogurt-fed) groups as seen in the present study. Consistent with the present findings, it has been exhibited that lymphocyte proliferation increased in yogurt-supplemented rats compared with control rats (Adolfsson et al. 2004).

3.5. Blood measurements

Serum triglycerides concentration was significantly (P < 0.01) influenced by inclusion of yogurt, especially 30% probiotic yogurt with pH value of 3.8 (Table 7). In addition, a numerical increase in serum triglycerides concentration was seen in calves fed probiotic yogurt compared with those on milk. In overall, inclusion of yogurt with pH value of 3.8 resulted in a significant (P < 0.01) increase in serum triglycerides concentration. This might be due to the effect of probiotics and lower pH values on the reduction of pathogenic bacteria in gastrointestinal tract. Decrease in pathogenic bacteria could reduce the conversion of primary bile acids to secondary ones, and in turn, increase fat absorption. In contrast to our results, Chiofalo et al. (2004) indicated that feeding lactobacillus caused a significant decrease in serum triglycerides level of kids. However, Panda et al. (2000) observed no change in serum concentration of triglycerides in pigs supplemented with dietary probiotics.

Table 7. Effect of probiotic yogurt with different pH values on blood parameters of calves (mg/dl).

Experimental treatments	Triglycerides	Cholesterol	High-density lipoproteins	Glucose	Total protein
Control	16.60^b	102.75^a	59.13	114.00	8.16
15% probiotic yogurt with pH = 3.8	23.30^b	80.50^b	56.15	105.50	8.68
15% probiotic yogurt with pH = 4.5	13.85^b	82.50^b	52.15	117.75	8.50
30% probiotic yogurt with pH = 3.8	40.20^a	92.00^ab	62.73	101.25	8.23
30% probiotic yogurt with pH = 4.5	16.88^b	85.00^b	54.90	108.50	8.08
Contrast comparisons
Control	16.60	102.75^a	59.13	114.00	8.16
Yogurt	23.56	85.00^b	56.48	108.25	8.37
Low pH	31.75^a	86.25	59.44	103.38	8.45
High pH	15.36^b	83.75	53.53	113.13	8.29
Probability
Treatments	0.009	0.032	0.562	0.727	0.782
Control vs. yogurt	0.348	0.004	0.614	0.569	0.616
pH values	0.008	0.621	0.155	0.191	0.570

Note: Means with no common superscript alphabets within each column are significantly (P < 0.05) different.

Inclusion of yogurt especially 15% probiotic yogurt with both pH values of 3.8 and 4.5 resulted in a significant (P < 0.05) decrease in serum cholesterol concentration (Table 7). Compared with milk (control) group, cholesterol concentration was significantly (P < 0.01) lower in yogurt-fed groups. In addition, a numerical decline was observed in serum cholesterol level of calves received yogurt with pH value of 4.5 compared with those on yogurt of pH = 3.8. There are two proposed mechanisms for the reduction of blood cholesterol concentration in animals fed on probiotics: (1) increase in degradation of cholesterol across the gastrointestinal tract (Zarate et al. 2002) and (2) simultaneous sediment of cholesterol and deconjugation of bile acids (Fernades et al. 1987). Consistent with these results, Deroos and Katan (2000) reported that dietary inclusion of probiotic resulted in a decrease in blood cholesterol concentration. However, Beena and Prasad (1997) observed that addition of milk, ordinary milk and bifidous yogurt had no marked effect on serum concentration of cholesterol in rats.

As given in Table 7, serum HDL concentration was not influenced by experimental dietary treatments. Also, feeding probiotic yogurt had no significant impact on HDL concentration when compared with control group. Similarly, there was no significant difference in HDL concentration between groups fed yogurt with either pH values of 4.5 or 3.8. Our results are in agreement with those of Panda et al. (2000), who showed that the addition of probiotics had no significant effect on serum HDL concentration. Nevertheless, Deroos and Katan (2000) observed that dietary inclusion of probiotic resulted in an increase in blood HDL concentration.

Serum concentrations of glucose and total protein were not affected by experimental dietary treatments (Table 7). Also, substitution of probiotic yogurt with different pH values had no significant effect on their concentration. These results are in agreement with Frizzo et al. (2010), who exhibited that inclusion of lactobacillus had no effect on serum protein and glucose concentrations in probiotic-fed calves. Similarly, Antunovic et al. (2006) reported no changes in blood glucose concentration in lambs supplemented with probiotics.

4. Conclusions

Results showed that partial replacement of probiotic yogurt for milk increased DMI, resulting in improvements in body weight gain and feed conversion efficiency. On the other hand, substitution of yogurt increased body length, wither height and hip depth. Inclusion of probiotic yogurt to partially replace milk resulted in increased lymphocyte proportion and decreased neutrophil to lymphocyte ratio, indicating yogurt (especially probiotic yogurt) could modulate immune responses.

Disclosure statement

No potential conflict of interest was reported by the authors.