Body development of weaned foals of Mangalarga Marchador breed fed probiotics or phytase supplemented diets

Mangalarga Marchador is the most prevalent horse breed raised in Brazil. Like other light breeds such foals grow quickly during their first year of life (Rezende et al., 2000a,b; Cabral et al., 2004). In tropical climates, in which the rainy season occurs in the summer, many stud farms allow for the adoption of extensive raising systems, which differ from temperate climate studs in which stalls are necessary. In Brazil, a lot of animals are kept on pastures for the most of the year so deterioration of grasses nutritional values has an influence on them. Foal body growth is generally evaluated on the basis of body weight average gain as well as on changes in whither and hip height, body length, chest girth and cannon circumference (Rezende et al., 2000a; Cabral et al., 2004; Pinto et al., 2005; Santos et al., 2005). Maximum height of adult equine is mostly determined genetically, but body development (size ABSTRACT. The aim of the study was to assess the average daily gain (ADG), the growth rate of linear measurements and cortical index changes of the third metacarpus (ICOR) of foals from 173 ± 8 to 363 ± 12 day of life. Sixteen Mangalarga Marchador weaned colts were divided by age and allotted randomly to one of four dietary treatments (n = 4): A – basal diet (Cynodon pasture + 1.2 kg · 100 kg–1 body weight (BW) of concentrate + water and mineral mix ad libitum); B – basal diet + 5 g · day–1 of yeast culture (109 cfu · g–1 Saccharomyces cerevisiae Sc47); C – basal diet + 2 g · day-1 of bacteria (108 cfu · g–1 Bifidobacterium bifidum, Enterococcus faecium, Lactobacillus acidophilus, Lactobacillus plantarum) and yeast culture (105 cfu · g-1 S. cerevisiae); and D – basal diet + 544 FTU of phytase per kg of concentrate per day. Data was analysed as repeated measures and fixed effects of age, treatment and their interactions accordingly to SAS® procedures. The additives did not affect the ADG or growth rate of linear measurements (P > 0.05); however the effect of age was observed. Furthermore, the ICOR was greater in 273 day-old foals fed diet B, which may suggest an increase in mineral uptake and bone development. So it can be concluded that a 100-day diet supplementation with S. cerevisiae Sc47 may increase bone development of weaned foals, however more studies are necessary to assure the beneficial effect of yeast addition on equine growth rate. Received: 15 September 2014 Revised: 18 November 2015 Accepted: 7 March 2016


Introduction
Mangalarga Marchador is the most prevalent horse breed raised in Brazil.Like other light breeds such foals grow quickly during their first year of life (Rezende et al., 2000a,b;Cabral et al., 2004).In tropical climates, in which the rainy season occurs in the summer, many stud farms allow for the adoption of extensive raising systems, which differ from temperate climate studs in which stalls are necessary.In Brazil, a lot of animals are kept on pastures for the most of the year so deterioration of grasses nutritional values has an influence on them.
Foal body growth is generally evaluated on the basis of body weight average gain as well as on changes in whither and hip height, body length, chest girth and cannon circumference (Rezende et al., 2000a;Cabral et al., 2004;Pinto et al., 2005;Santos et al., 2005).Maximum height of adult equine is mostly determined genetically, but body development (size ABSTRACT.The aim of the study was to assess the average daily gain (ADG), the growth rate of linear measurements and cortical index changes of the third metacarpus (ICOR) of foals from 173 ± 8 to 363 ± 12 day of life.Sixteen Mangalarga Marchador weaned colts were divided by age and allotted randomly to one of four dietary treatments (n = 4): A -basal diet (Cynodon pasture + 1.2 kg • 100 kg -1 body weight (BW) of concentrate + water and mineral mix ad libitum); B -basal diet + 5 g • day -1 of yeast culture (10 9 cfu • g -1 Saccharomyces cerevisiae Sc47); C -basal diet + 2 g • day -1 of bacteria (10 8 cfu • g -1 Bifidobacterium bifidum, Enterococcus faecium, Lactobacillus acidophilus, Lactobacillus plantarum) and yeast culture (10 5 cfu • g -1 S. cerevisiae); and D -basal diet + 544 FTU of phytase per kg of concentrate per day.Data was analysed as repeated measures and fixed effects of age, treatment and their interactions accordingly to SAS ® procedures.The additives did not affect the ADG or growth rate of linear measurements (P > 0.05); however the effect of age was observed.Furthermore, the ICOR was greater in 273 day-old foals fed diet B, which may suggest an increase in mineral uptake and bone development.So it can be concluded that a 100-day diet supplementation with S. cerevisiae Sc47 may increase bone development of weaned foals, however more studies are necessary to assure the beneficial effect of yeast addition on equine growth rate.and growth rate) of young animals may be influenced by sex, date of birth (month), climate, nutrition and management (Saastamoinen, 1990).
When probiotics are administered in adequate amounts, they exert health benefits for the host (FAO/ WHO, 2002).In the equine species, this additive is used to improve diet quality, body growth, physical condition but also for prevention and/or recovery from gastrointestinal diseases.Studies conducted on horses have shown that the use of probiotics improves nutrient digestibility, especially fibre (Kim et al., 1991;Medina et al., 2002;De Fombelle et al., 2003;Jouany et al., 2009;Moura et al., 2009Moura et al., , 2011;;Furtado et al., 2010).However, more studies are needed to assess the indirect effects of these nutritional benefits on foals body growth (Glade, 1991a,b;Perrone et al., 2013), especially in animals raised under particular climatic conditions like in Brazil.
Phytase is an enzyme used throughout the world as a nutritional additive for monogastric animals, especially for poultry and pigs, due to its deleterious effects on mineral utilization (P, Ca, Mg, Zn, Fe, Mn).Few studies have been conducted to examine the effects of this enzyme type supplementation on horses (Patterson et al., 2002;Hainze et al., 2004;Van Doorn et al., 2004;Dunnet et al., 2009).Among them were also Mangalarga Marchador foals, which have higher mineral requirements (Moura et al., 2009(Moura et al., , 2011)).
The objective of this study was to assess the body development (weight, linear measurements and bone development) of weaned Mangalarga Marchador foals fed probiotics or phytase supplemented diets.It was hypothesized that the growth of weaned foals fed diet supplemented with probiotics or phytase would be greater than the growth of foals fed unsupplemented diet.

Animals, treatments and experimental design
All applied experimental procedures were approved by the Ethics Committee for Animal Experimentation of The Federal University of Minas Gerais (UFMG), Brazil.The study was carried out at a private stud farm (Montes Claros, Minas Gerais, Brazil) from 13 April to 16 December.Sixteen colts were used, all from the same stallion and breed lineage ('Catuni'), which is considered to be the one of the first established lineages of the Mangalarga Marchador breed that started to be selected in the early nineteenth century (Costa et al., 2005).
Foals were weaned after 173 ± 8 days weighing 178 ± 20 kg.Prior to weaning, foals were kept on a single pasture with their dams and received only a mineral mix ad libitum.Weaning was conducted in groups of four foals from April to May.Before this trial, all animals were treated for internal and external parasites.Treatment against endoparasites was repeated every two months and against ectoparasites -each month.At weaning, all foals were divided by age, and four foals were allocated randomly to one of four dietary treatment groups: A -basal diet; B -basal diet + 5 g • day -1 of yeast culture (10 9 cfu of Saccharomyces cerevisiae NCYC Sc47; Biosaf © ; Lesaffre Feed Additives, Marcq-en-Baroeul, Nord-Pas-de-Calais, France); C -basal diet + 2 g • day -1 of bacteria and yeast culture (10 8 cfu of four bacteria strain (Bifidobacterium bifidum, Enterococcus faecium, Lactobacillus acidophilus, Lactobacillus plantarum) and 10 5 cfu of Saccharomyces cerevisiae; Florafort © ; Laboratório Vitafort Ind. e com.de Produtos Veterinários Ltd., Ribeirão Preto, São Paulo, Brazil); and D -basal diet + 544 phytase units (FTU) (5000 FTU per g; Natuphos © ; BASF, Ludwigshafen, Germany) per kg of pelleted concentrate per day.
The additives, phytase or probiotics (yeast or yeast with bacteria mixture), were administered directly into the horse mouth at every morning feeding.The probiotic composed of a yeast culture was in the form of powder, dehydrated by lyophilization, from the same batch, stored at room temperature and protected from sunlight and humidity.The probiotic composed of a bacteria and yeast culture was in the form of paste, stored at 4 ºC and used up to four months from its preparation.The phytase supplement was in the form of lyophilized powder from a single batch and stored at 4 ºC.
The adaptation to the facilities, managament and diets lasted the first 14 days of the experiment.Foal body development was monitored in sum for 200 days (from weaning at 173 ± 8 to 363 ± 12 day of life).

Body growth evaluation and diet analyses
At weaning and every 30 days until the end of the experiment, the foals were weighed after the morning feeding and measured with a Lydthin measuring stick (wither height -WH, hip height -HH, body length -BL and barrel height -BH) and with a tape (chest girth -CG and cannon circumference -CC), as illustrated on For the age effect comparisons, individual ICORs measured at the end of each sampling period were converted to percent change in relation to previous or initial value of each period.The individual mean percentage changes were then averaged across each age period and the means (absolute % values and % changes) between ages compared by orthogonal contrasts.
For the diet effect comparisons, individual ICORs measured at each sampling period (age) were converted to percentage change in relation to the control one.For comparisons the same procedures for age were used.
Feed samples were collected to determine their chemical composition in the Animal Nutrition Laboratory of The Federal University of Minas Gerais (UFMG), Department of Animal Science (Table 1).Diets were analysed for the following parameters: dry matter (DM), crude protein (CP), ether extract (EE), calcium (Ca), phosphorus (P) and oxalate (AOAC International, 1995), starch, neutral detergent fibre (NDF), acid detergent fibre (ADF) and lignin (Van Soest et al., 1991).The digestible energy (DE) of the concentrate and forage harvested in August was determined by Moura et al. (2011)

Statistical analysis
All analyses were performed by the SAS ® package version 6 (1998), SAS Institute Inc., Cary, NC, USA.Data relative to the average daily gains

Results
There was no effect of dietary treatment and its interaction with age (P > 0.05) on body weight (BW) absolute values and body linear measurements (Table 2), neither on average daily/monthly gains (Table 3) of foals from 173 (weaning) to 363 day of life.Average body condition score across the experimental period was 3.0, and no differences were observed between treatment, age or between age interaction treatment throughout the study (P > 0.05).
Average daily gains (ADG) and monthly gains of linear measurements were only influenced by foal age (Table 3).With the exception of WH and CC, the average monthly gains of linear measurements for all foals significantly fit the regression models.
Cortical index was not influenced by dietary treatment but was higher in 368-day old foals (60.61 ± 0.57%) in comparison to 273-day old ones (55.35 ± 0.57), indicating again the age effect (Table 4).The cortical index for all foals over time significantly fit the following regression model: where: Y -cortical index (%) and X -age (day).
The monthly cortical index changes were not dependent on either dietary treatment or age; however an interaction (P < 0.05) between cortical index and age was observed (Table 4).This effect was only observed on certain combinations between age and dietary treatment.Only at 273 day of life, the monthly change on cortical index was higher in foals reciving additional yeast treatment (4.1175 ± 0.8754) when compared to the control group (1.1604 ± 0.8718), to the bacteria + yeast treated group (1.6463 ± 0.8658) and to the phytase supplemented group (-0.1891 ± 0.8673).

Discussion
The growth of all foals in presented study was similar to that observed in other studies conducted on Mangalarga Marchador foals (Rezende et al., 2000a;Cabral et al., 2004) and general obtained means were as follows: 0.48 kg • day -1 for average daily gain (ADG); 0.07 cm • day -1 for wither height (WH) and hip height (HH); 0.11 cm• day -1 for body length (BL); 0.03 cm • day -1 for barrel height (BH); 0.09 cm • day -1 for chest girth (CG) and 0.01 cm• day -1 for cannon circumference (CC).
Although probiotic or phytase may be beneficial for horse nutrition and health, such effects related to ADG and body linear growth were not observed in this study after 200 days of supplementation.These findings agreed with those stated by Bennett et al. (1991), who found no difference in ADG and WH of yearlings fed diets supplemented with yeast culture.On the other hand, the current findings are in disagreement with Mason (1993) and de Léon and Samudio (2002), who observed higher body weight, ADG and WH in weaned foals fed diet supplemented with Saccharomyces cerevisiae for 120 days compared to animals fed basal diet.
These contradictory results can be explained by the difference in growth rate of examined breeds, age of foals, composition of commercial products and the quality of the feedstuffs used in related studies.Glade (1991a,b) observed possitive changes in growth estimated by WH measurements in foals from mares supplemented with S. cerevisiae, which showed superior values from 42 to 56 days in comparison to foals from control mares.The use of additive probiotic composed of species-specific microorganisms (Yuyama et al., 2004) and in minimal viable concentration superior to 7 log10 CFU • g -1 for each microorganism present in the product (Vanbelle et al., 1990) may be relevant in the attempt to obtain results with probiotic supplementation.
The lowest rate of ADG was 0.36 kg • day -1 which was observed for 299-day old foals, coinciding with the harshest period of the regional dry season.The climate of the northern region of Minas Gerais, where Montes Claros is located, is mild semiarid with total absence of rains from May to August.Despite the positive effects of probiotics or phytase supplementation on nutrient digestibility, especially fibre, previously observed in foals from the same species (Moura et al., 2009(Moura et al., , 2011)), growth rates remained low due to the overall lower quality of dry season pastures (3.67% crude protein and 9.99% lignin; Table 1).The negative effects of nutrient deficiency on foal growth were most pronounced in weight gain, followed by CG, CC and, finally, WH (Rezende et al., 2000a).So, the positive response to probiotic supplementation, when forages with poor quality were offered in the diet, was questionable.
According to the NRC (2007), the use of supplements does not replace basic animal nutrition recommendations.In this sense, the negative influence of diet energy and protein deficiency on foal growth cannot be overcomed by supplementation.If in this study, the addition of other forage types with higher nutritional value, such as lucerne or other tropical legumes (Silva et al., 2012) had been used, normal foal development should have been observed during the dry season.
A temporary improvement in cortical index was confirmed in foals supplemented with S. cerevisae Sc47 after 273 days of life, expressed by the interactive effect between age and diet (Table 3).A permanent effect on bone development had been expected with the two probiotics studied, because, in a previous study conducted on Mangalarga Marchador foals, the probiotic-supplemented foals showed an improvement in the uptake of minerals, but no effect of phytase supplementation was observed (Moura et al., 2011).Additionally, Sindhu and Khetarpaul (2001) observed a sharp reduction in the contents of phytic acid, polyphenols and trypsin inhibitor activity with Lactobacillus casei or Lactobacillus plantarum, alone or associated with Saccharomyces boulardii in vitro inoculations (containing 10 6 cells • ml -1 broth), which should have also lead to some improvement in mineral uptake.
Moreover, the concentration of the microorganisms in supplements must be considered as a factor that interferes with the possible positive effects of probiotics on body growth.The S. cerevisiae concentration in the tested probiotics varied from 10 9 cfu • g -1 in the probiotic composed only of the yeast culture (Sc47 strain) to 10 5 cfu • g -1 in the probiotic composed of the bacteria and yeast culture.These values should have accounted for benefits in bone development, but only a transitory effect was observed in this experiment.
The absence of a phytase effect could be related to the fact that the dietary calcium and the calcium:phosphorus ratio were balanced to meet the nutritional requirements for foals.Therefore, the phytase catalytic action could have been limited by the deleterious effects of calcium and inorganic phosphorus (Singh, 2008).Further studies in which horse diets will be balanced according to the expected quantity of phosphorus released by enzyme supplements (Dunnett et al., 2009) are needed.

Conclusions
The probiotics and phytase tested under the presented experimental conditions did not influence the body weight and linear measurement growth rates in Mangalarga Marchador foals.Bone development was increased only temporarily by the Saccharomyces cerevisiae culture (Sc47 strain) supplementation indicating a possible beneficial effect of this probiotic on mineral balance.

Figure 1 .
Data published by Gonçalves et al. (2012) was used to estimate the percentage of the values observed for all body measurements, except for barrel height, in relation to the expected values for Mangalarga Marchador adult males.Body condition was scored through the entire study, using the method of Carrol and Huntington (1988): 0 -very thin to 5 -obese.Foals at 235, 273, 308, 339 and 368 days of life were radiographed in the dorso-palmar position of the right metacarpus (Mtc III).The cortical index (ICOR) was calculated in accordance to Rezende et al. (2000b): ICOR = [(total bone width -medular cavity witdh) / total bone width] × 100.

Table 1 .
Chemical composition of feedstuffs

Table 2 .
Absolute values of body weight and linear measurements for weaned Mangalarga Marchador foals fed unsupplemented or probiotics/ phytase supplemented diets

Table 3 .
Average daily gain of body weight and average montly gains of linear measurements for weaned Mangalarga Marchador foals fed unsupplemented or probiotics/phytase supplemented diets Parameter 1 A-Fvalues with different superscripts for each main effect (diet or age) are significantly differerent at P ≤ 0.05

Table 4 .
Absolute values and changes on cortical index of third metacarpus of weaned Mangalarga Marchador foals fed unsupplemented or probiotics/phytase supplemented diets Parameter

Table 2 ;
cortical index change calculated for diet as main effect, % -values were calculated as percentage change in relation to control diet value at each sampling period (diet means (overall from all sampling periods) were compared by orthogonal contrasts); cortical index change calculated for age as main effect, % -values were calculated by subtracting each value at the end of each period (age) by initial value within that period.Values are expressed as percentage change; A-D -values with different superscripts for each main effect (diet or age) are significantly differerent at P ≤ 0.05