Growth , body characteristics and blood parameters of ostrich chickens receiving commercial probiotics

This study was undertaken to determine the effect of four commercial probiotics on growth, body characteristics and haematological parameters of ostrich chicks. A total of 25 ostrich chicks (937±68.1 g) were individually allocated and fed the experimental diet for six weeks (n=5 per treatment). Experimental diets consisted of a corn/soybean meal-based diet unsupplemented (T1: Control), and four diets supplemented with probiotics according to the recommendations of the manufacturer (T2: 0.04% Bioplus 2B; T3: 0.09% Primalac; T4: 0.1% Thepax; and T5: 0.03% Protexin). Feed intake (FI), body weight (BW) and seven body characteristics (e.g. height) were measured every week. Blood samples and other body characteristics were also taken in the last week. There was an interaction effect between diet and time on all the growth variables and body characteristics (p<0.05). Both the BW and the BW gain of the ostrich chicks were, in general, higher for those fed the diet T2 than those fed the control diet (0.42, 1.07, 0.99, 1.09, 2.51, and 1.66 kg BW gain vs 0.28, 0.41, 0.83, 0.94, 1.15, and 1.15 kg BW gain at 7, 14, 21, 28, 35, and 42 days respectively), while for those fed the other diets containing probiotics differences were only observed at 42 days (p<0.05). Consuming probiotics over an extended period influenced several of the haematological parameters differently compared to those fed the control diet (p<0.05). T2 and T3 increased the concentration of total cholesterol (157 and 210 mg/dL respectively), when compared to those fed the control diet (119 mg/dL), while total cholesterol was slightly reduced (p>0.05) for those fed the diet containing Thepax (T4, 79 mg/ dL). In conclusion, the effects of commercial probiotics on growth performance, body characteristics and haematological parameters varied among probiotics. Additional key words: blood parameters; growth performance; ostrich chicks; poultry nutrition Abbreviations used: BW (body weight); FCR (feed conversion ratio); FI (feed intake); HDL (high density lipoproteins); LDL (low density lipoproteins); VLDL (very low density lipoproteins) Citation: Karimi-Kivi, R.; Dadashbeiki, M.; Seidavi, A. (2015). Growth, body characteristics and blood parameters of ostrich chickens receiving commercial probiotics. Spanish Journal of Agricultural Research, Volume 13, Issue 1, e06-004, 11 pages. http:// dx.doi.org/10.5424/sjar/2015131-6146 Received: 24 Apr 2014. Accepted: 13 Feb 2015 http://dx.doi.org/10.5424/sjar/2015131-6146 Copyright © 2015 INIA. This is an open access article distributed under the Creative Commons Attribution License (CC by 3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Funding: This work was supported by Rasht Branch, Islamic Azad University, Rasht, Iran. Competing interests: The authors have declared that no competing interests exist. Correspondence should be addressed to Alireza Seidavi: alirezaseidavi@iaurasht.ac.ir


Introduction
The use of probiotics in poultry nutrition has gained great importance during the last 20 years due to their useful effects on production, health and quality of carcasses when compared to antibiotics (Hajjaj et al., 2005;Kabir, 2009).Probiotics may stabilise microbiota populations throughout the gastrointestinal tract by producing specific metabolites (e.g.bactericins, hydrogen peroxide, short chain fatty acids) that help overcome the adverse effects of pathogens (Gabriel et al., 2006;Kabir, 2009;Khan & Naz, 2013).In addition, probiotics have been found to improve feed intake (FI) and digestion (Gabriel et al., 2006), reduce blood cholesterol (Mohan et al., 1996;Hajjaj et al., 2005) and triglycerides (Santoso et al., 1995), and improve bone strength (Khan & Naz, 2013).
The effects of probiotics on poultry have been mainly focused on broilers and little information has been reported in the literature regarding its effects on ostriches.Ostriches have a different gastrointestinal tract compared to broilers, which allows them to digest dietary fibre more efficiently (Cilliers et al., 1992(Cilliers et al., , 1997;;Brand et al., 2000;Sales, 2006).The length of the colon in an adult ostrich represents approximately 57% of the intestines compared to only 3% in an adult broiler (Angel, 1996).This may explain the higher apparent metabolizable energy of feed ingredients in adult ostriches compared to cockerels (e.g.barley 15 and 11 MJ/kg respectively) (Cilliers et al., 1997).This impor-2 probiotics described above.The probiotic was added to the basal diet according to the manufacturer's recommendations: 0.04% Bioplus B2, 0.09% Primalac, 0.1% Thepax, and 0.03% Protexin.

Animals and housing
Approval for the animal trials was obtained from the Animal Ethics Committee, Rasht Branch, Islamic Azad University, Rasht, Iran.A total of 25 two-week-old ostrich chicks, from the blue and black Neck African breed, and with an initial live weight of 937 ± 68.1 g, were used.The chicks were housed individually, in cages of 2.0×1.7 m, with an open area of 1×1 m.Each cage had a single compartment dry feeder and a drinking bowl.

Experimental design
Each treatment group was made up of five chicks, two males and three females.Experimental diets were randomly allocated to 3 repetitions together of each treatment goup, so that the five diets were represented in each group, with gender being equalised across the groups, in a randomized complete block design.All the ostrich chickens were fed ad libitum during 42 experimental days.Body weight (BW), feed intake (FI) and body characteristics [total body height (from head to floor); neck length; circumferences at the shoulder joint (thoracic), abdominal (at the lap), bottom of the neck, hip and tail] were recorded weekly.On day 42, blood samples (10 mL) were collected from the neck vein of three male ostrich chicks from each treatment.In addition on day 42, other body characteristics were also measured (top and middle neck circumference, tibiotarsus, tarsometatarsus, tail and head circumference, and leg, wing, neck and beak length), to build a three-dimensional (3-D) ostrich figure representing the mean of each treatment using the Design Modeler AN-SYS TM 14.5 software (Canonsburg, PA, USA).

Blood sample collection and analysis
Blood samples using a syringe containing heparin were taken without anesthesia.Blood plasma was isolated by centrifugation at 3000 rpm for 20 min at 4ºC and aliquots were stored at -20ºC for analysis.Aliquots were analysed for glucose (Barham & Trinder, 1972), alkaline phosphatase (Bessey et al., 1946), uric acid, blood urea nitrogen, creatinine, total cholesterol, triglycerides, high density lipoproteins (HDL), low density lipoproteins (LDL) and very low density lipoproteins tant difference in gastrointestinal tract physiology may also allow the ostrich to have different microbiota populations in terms of diversity and amount (Ahir et al., 2012;Oakley et al., 2014;Waite & Taylor, 2014).Thus, it may be expected that the effects of probiotics in ostriches may differ to those in broilers.Hasan-Rezaie et al. (2013) reported that inclusion in the basal diet of the Primalac probiotics (included Lactobacillus acidophilus, Lactobacillus casei, Bifidobacterium thermophilum, and Enterococcus faecium) improved body weight (BW), feed conversion ratio (FCR) and blood parameters (glucose, cholesterol, uric acid and urea), and those given 0.135% Primalac had the highest body weight, lowest FCR and lowest amounts of blood cholesterol, uric acid and urea (p<0.05) in ostriches.Xu et al. (2010) reported that the morbidity and mortality in ostriches fed probiotics (15 and 2.6%) was lower than ostriches fed a control diet (24.1 and 3.8%) respectively, while the daily gain was higher (26.0 vs 23.2 g/d, p≤0.01).
Therefore, the aim of this study was to determine the effects of four different commercial probiotics on growth, body characteristics and haematological parameters in ostrich chicks.The commercial probiotics were selected based on their difference in microbiota composition and in previous studies done in both broilers (Kabir et al., 2004;Gunal et al., 2006;Mutus et al., 2006;Nayebpor et al., 2007;Paryad & Mahmoudi, 2008;Boostani et al., 2013) and ostriches (Hasan-Rezaie et al., 2013).A basal diet was formulated based on the nutritional recommendations reported in previous studies of ostrich nutrition (Angel, 1996;Cilliers et al., 1998).The diet (Table 1), used as the basal diet throughout the experimental period, was either unsupplemented (i.e.control diet) or supplemented with one of the four 3 Effects of probiotics on the growth of ostrichs 42, a randomised complete block design analysis was performed, with gender as a block.

Probiotics and dietary treatments
The model diagnostics (e.g.homogeneity of variance) of each parameter were tested combining the Proc Univariate and the ODS Graphics options of SAS.When the model assumptions were not fulfilled for an individual parameter, a transformation of its raw data was conducted to achieve those assumptions.In addition, when only the assumption of homogeneity variances was not fulfilled, an analysis with separated variances was conducted.When the F-value of the analysis of variance was significant for a specific response variable (p<0.05), the means of the diets containing the probiotics were individually compared with the control diet using the adjusted Dunnet's tests.

Statistical analysis
The statistical analyses were performed using the Mixed Model procedure of SAS (SAS/STAT v. 9.3, SAS Inst.Inc., Cary, NC, USA).To examine the effect of diet, time and the interaction between diet and time on BW, FI, BW gain, feed conversion ratio (FCR) and body characteristics, a repeated measure analysis using a randomised complete block design was performed, using each ostrich chicken as an experimental unit and the gender as a block.The most appropriate covariance structure for each parameter was selected based on the smallest Akaike's and Bayesian's information criteria value when the covariance structures were compared (Littell et al., 1998).In addition, to examine the effect of the commercial probiotics on the haematological parameters and the body characteristics at day

Body characteristics
All the body characteristics measured in the ostrich chicks throughout the study were influenced by the interaction between diet and time (p<0.05)(Table 3).
In general, at 35 and 42 days, chicks fed the diets containing Bioplus 2B, Primalac and Protexin were higher with a longer hip circumference than those fed the control diet (p<0.05).In addition, those fed Bioplus 2B also had greater thoracic (at 35 and 42 days) and abdominal (at 35 days) circumferences and neck length (at 35 days) (p<0.05).
Several of the body characteristics (top neck, bottom neck, hip, tibiotarsus, tarsometatarsus and head circumferences, neck, wing and beak lengths) of the chicks at day 42 were influenced by the diet (p<0.05), as shown in the 3-D figure build to represent the 'average' ostrich chicken for each diet (Fig. 2 and Table 5).In general, these body characteristics were greater for the chicks fed the probiotic diets than those fed the control diet (p<0.05).However, other body characteristic variables (thoracic, abdominal, middle neck circumferences, tail and neck lengths and height) were not influenced by the diet (p>0.05).
The estimated volume of the body trunk, obtained from the built 3-D figure, was greater for the chicks fed the diets containing probiotics (11548-13751 cm 3 ) than those fed the control diet (9636 cm 3 ).Similarly, the estimated total body area of chicks fed the diets containing probiotics was greater (4310-4841 cm 2 ) than those fed the control diet (3784 cm 2 ).

Haematological parameters
The haematological profile of the samples collected at day 42 was influenced by the probiotic supplementation (creatinine, total and LDL cholesterol, and HDL/ LDL ratio and albumin) (p<0.05)(Table 4).The creatinine content and the HDL/LDL ratio were lower for the those fed the diets containing Bioplus 2B and Primalac when compared to control (p<0.05).In contrast, their total and LDL cholesterol contents were higher (p<0.05).The albumin content of the chicks fed the control diet was lower than for those fed the diets containing Thepax (p<0.05).
(p<0.05) (Fig. 1 and Table 2).FI and BW gain were higher for the ostrich chicks fed T2 and T5 than for those fed the control diet (p<0.01).However, FCR was better for those fed the diets containing Primalac, Protexin and Thepax when compared to the control diet (p<0.05).
There was a highly significant effect of the interaction between diet and time on BW, FI, BW gain and FCR (p<0.001) (Fig. 1 and Table 2).When compared to the chicks fed the control diet, those fed the diet containing Bioplus 2B had a higher FI across all the time points, while those fed the diet containing Protexin had higher intake at day 7, 28, 35 and 42 (p<0.05).For the chicks fed with the other probiotic diets, FI was higher at 42 days only (p<0.05).Both, BW and BW gain were higher for those fed the diet containing Bioplus 2B when compared to those fed the   Greenhill, 2010).Further studies considering the effect of probiotics on the quality of the carcass, feathers and skin on ostriches are warranted.Improving the FCR in the ostrich chicks should result in a higher profit margin for the producer.Nutrition represents up to 80% of total production costs (Delgado et al., 1999).In this regard, the diets supplemented with the probiotics Primalac, Protexin and Thepax appear to be a potential option to improve the profit margin for producers.However, it is necessary to mention that the production costs and the economic benefits of supplementing the diet with probiotics were not considered in this study.Therefore, an economic evaluation of supplementing the diet with probiotics is needed for the producers.
Previous studies have shown that the inclusion of commercial probiotics (e.g.Thepax and Protexin) in diets for broiler chickens increased several growth performance variables (e.g.BW, FCR) (Mohan et al., 1996;Yeo & Kim, 1997;Kabir et al., 2004;Gunal et al., 2006;Nayebpor et al., 2007;Paryad & Mahmoudi, 2008).In this study, the higher growth performance observed in the ostrich chicks consuming the diets containing probiotics may be due to a change in their gastrointestinal tract microbiota populations, as reported for broiler chickens (Schrezenmier & Vrese, 2001;Gunal et al., 2006;Alloui et al., 2013).This change in the microbiota population may be beneficial in several ways, including reducing pathogenic bacteria, stimulating the immune system and improving bone strength (Gunal et al., 2006;Boostani et al., 2013;Khan & Naz, 2013).A further benefit could also be the effect of the microbiota present in the probiotics on fermenting nutrients, mainly fibre, compared to a probiotic-free diet (Gabriel et al., 2006).Improved fermentation may increase the production and absorption of short-chain fatty acids throughout the gastroin-

Growth performance and body characteristics
In contrast with a previous study conducted on ostrich chicks over 37 days with the Lactosym probiotic (Dube et al., 2009), this study shows a beneficial effect of supplementing the diet with commercial probiotics on several growth performance parameters, over an extended period of 42 days as suggested by Hasan-Rezaie et al. (2013).The chicks were in general able to increase their voluntary FI which was reflected in greater BW gain when compared to the control diet.Interestingly, those fed the probiotic diets increased, in different ways, the size of several body components of commercial interest (e.g.meat, skin, feathers).Bioplus 2B was the only probiotic able to increase the length of the wing and Bioplus 2B and Protexin were able to increase the length of the legs.
The higher trunk volume may indicate higher meat deposition and the higher total body area may indicate more skin from chicks fed the diets containing probiotics compared to the chicks not fed probiotics.The latter may suggest that the probiotic composition (i.e. the microbiota profile) was able to modulate specific body characteristics.Previous studies in broiler chickens showed that adding into diets some of the commercial probiotics studied here (e.g.Bioplus 2B and Protexin), improved carcass yield and several body characteristics (e.g.thickness of the medial and lateral wall of the tibia, weight of the legs) (Kabir et al., 2004;Mutus et al., 2006;Ashayerizadeh et al., 2011).However, few studies with ostrich chicks have shown that commercial probiotics to the diet improved carcass yield and body characteristics (Greenhill, 2007;Juste-Poinapen, 2007;Ebrahimzadeh et al.,  7 Effects of probiotics on the growth of ostrichs  3 A natural logarithm transformation of the raw data was required to achieve the model diagnostic assumptions.The values were obtained after back transformation. 4SEM: standard error of the mean. 5The phenotypic characteristics values, for the overall statistical analysis, were obtained after subtracting the values of the day 0 of the study to those obtained at day 42 for each ostrich chicken.testinal tract (Wong et al., 2006).Short chain fatty acids are a source of energy to the host.They have been associated with intestinal tissue proliferation, enhanced absorption of minerals and water and prevention of diseases (Williams et al., 2001).

Haematological parameters
In contrast to previous studies of probiotics conducted in broiler chickens (Mohan et al., 1996;Paryad & Mahmoudi, 2008), the total and LDL cholesterol 9 Effects of probiotics on the growth of ostrichs concentrations was higher for the ostrich chicks fed the diets containing Bioplus 2B and Primalac probiotics when compared to those fed the control diet.However, the ostrich chicks fed the diet containing the Thepax probiotic reduced the concentration of total cholesterol and increased the concentration of albumin as reported in broiler chickens fed diets containing the same probiotic (S. cerevisiae) (Onifide, 1997;Paryad & Mahmoudi, 2008).Fat deposition is commonly accepted to be correlated with total cholesterol, LDL and VLDL blood concentration, which mainly depends on the triglycerides removed from the plasma (Hermier, 1997;Musa et al., 2006).In this regard, the results from this study suggest that the ostrich chickens fed the diet containing Bioplus 2B and Primalac may have had higher adipose tissue deposition than those fed the control diet.
A lower concentration of creatinine was observed in the ostrich chickens fed the diets containing the Bioplus 2B and Primalac probiotics when compared to those fed the control diet.A reduction in creatinine concentration in blood is related to a lower muscle metabolism (Brosnan & Brosnan, 2010).Therefore, it is possible that at 42 days the muscle deposition may have been influenced, in an unknown way, for the ostrich chicks fed the diet containing the Bioplus 2B and Primalac probiotics.
While there are studies of biochemical parameters in ostrich chicks (Spinu et al., 1999;Fallah et al., 2014), there are very few reports of the effects of probiotics on blood parameters in this species.Other haematological factors (e.g.triglycerides) were not affected when the diet of the chicks was supplemented with the commercial probiotics, contrary to the results of previous studies in broiler chickens (Isshiki, 1979;Kos & Witner, 1982;Santoso et al., 1995;Zobac & Kumperchov, 2000;Paryad & Mahmoudi, 2008).The contrasting effects of probiotics on haematological parameters between this study and those on broiler chickens could be explained by the probiotic composition (i.e. the microbiota profile), the composition of the diets used in the studies, and anatomical differences (e.g.gastrointestinal tract) between both species.
Further studies investigating the relationship between microbiota populations and haematological parameters after probiotics supplementation on ostrich diets are required.
In conclusion, the probiotics improved several of the growth performance variables (e.g.BW gain, FCR) and influenced several of the body characteristics (e.g.neck length, thoracic circumference) and haematological parameters (e.g.creatinine, total cholesterol) in ostrich chickens when compared to those fed a probiotic-free diet.

Figure 1 .
Figure 1.Body weight changes over time in ostrich chickens fed diets containing different commercial probiotics and a control diet.Values are least square mean ± standard error, n=5.Values with the symbol *,# for a given time point, differ significantly (p<0.05) or non-significantly from the control diet, respectively.

Figure 2 .
Figure 2. A 3D-figure of the ostrich chickens fed diets containing different commercial probiotics and a control diet at 42 days.Values to create the 3D-figures are least square mean, n=5.Body characteristics with symbols differ significantly (p<0.05 to p<0.01) from the control diet for: ¤ head circumference; * top neck circumference; # bottom neck circumference; † hip circumference; ‡ tibiotarsus circumference; § tarsometatarsus circumference; ¶ leg length; & wing length; ¥ beak length.

Table 1 .
Ingredients and calculated chemical composition of the experimental diets.while for those fed the other diets containing probiotics differences were observed at 42 days only (p<0.05).The FCR of the diets containing probiotics was better than the control diet at 14 (Bioplus 2B), 28 (Primalac), 35 (Bioplus 2B and Primalac) and 42 (Protexin) days (p<0.05).

Table 2 .
Growth performance and body characteristic variables in ostrich chickens fed diets containing different commercial probiotics from 7 to 56 days of age.
Labeled least square means within each column for each time point or the overall analyses, differ significantly to the control.Values are least square mean of five replicates. 1 FI: feed intake; BW gain: body weight gain; FCR: feed conversion ratio.All data was recorded and presented weekly. 2 A reciprocal transformation was required of the raw data to achieve the model diagnostic assumptions.The values presented in this table were obtained after back transformation. 3M: standard error of the mean.4TheFCR values, for the overall statistical analysis were obtained after dividing the value of cumulative FI (from day 1 to day 42) / cumulative BW gain (from day 1 to day 42).6 2009;

Table 3 .
Growth performance and body characteristic variables in ostrich chickens fed diets containing different commercial probiotics from 7 to 56 days of age.Labeled least square means within each column for each time point or the overall analyses, differ significantly to the control.Values are least square mean of five replicates. 1 NC, TC, AC and HC: neck, thoracic, abdominal and hip circumference, respectively; NL: neck length. 2 A reciprocal transformation was required of the raw data to achieve the model diagnostic assumptions.The values presented in this table were obtained after back transformation.

Table 4 .
Haematological parameters in ostrich chickens fed diets containing different commercial probiotics from 14 to 56 days of age.Labeled least square means within each row differ significantly to the control.Values are least square mean of three replicates. 1 SEM: standard error of the mean. 2 AST and ALT: aspartate and alanine amino transferase, respectively.

Table 5 .
Body characteristic variables in ostrich chickens fed diets containing different commercial probiotics at 42 days of age.