Introduction
Ruminal content is a by-product obtained from this organ that, at the moment of the death of the bovine, contains all the material that was not digested. It has a complete balance of amino acids, fat, minerals and vitamins of B complex and C vitamin, besides some unidentified growth factors (Sugiarto 2014). For years, this material has been discarded, although it is, due to its high organic load, of great environmental impact. This residue also has a large number of microorganisms (fungi, protozoa and bacteria) living in symbiosis in the rumen (Dairio et al. 2005). For all the above, it is an alternative for feeding broilers and fattening pigs, rabbits and ruminants, due to its chemical, biological and bromatological characteristics, and a wide availability (Ríos and Ramírez 2012).
On the other hand, fermentation is one of the oldest technologies used for improving food quality (Kim 2012). Nowadays, there is a great variety of fermented products for animal feeding (Beruvides 2019).
The objective of this research was to determine the effect of a dry fermented product (PFS), obtained from ruminal content, on morphological, immunological, health and histological indicators of broilers.
Materials and Methods
Preparation of the dry fermented product (PFS). The PFS was produced with the use of a liquid fermented product (PFL) mixed with corn meal (1:1) weight/volume (w/v), according to Gutiérrez (2005) procedure. It was dried under the sun for 48-72 h and manually removed every 2 hours to avoid the development of undesirable reactions. Once the product was dried, it was packed into two paper bags for later use. Three samples of 500 g were taken from each bag of dry product prepared in July 2015 in the Food Production Laboratory of the Department of Digestive and Biochemical Physiology, Institute of Animal Science. A sample was taken from three different locations in the bag (low, medium, top) and mixed to form a single sample. This process was repeated three times for each bag, to obtain a total of six samples (three from each bag). Later, the PFS was taken to a hammer mill to obtain a particle size of 1 m.
Chemical analysis. The chemical analysis was performed according to the methodology of AOAC (2010). True protein determinations were carried out according to Bernstein (1924), modified by Meir (1986). Fiber fractioning (neutral detergent fiber, acid detergent fiber, lignin and cellulose) was determined according to the procedure of van Soest et al. (1991). Calcium, magnesium and potassium were determined by atomic absorption spectrophotometry, and phosphorus was determined by Amaral (1972).
Animals and diets. An amount of 32 male broilers (EB24), of 35 days old, were used, which came from an experiment of productive performance. Animals were distributed in four experimental treatments: 1) control treatment (without the fermented food) and treatment II, III and IV with 1, 2 and 3% of PFS, respectively. During the time of experimentation, animals had free access to water and food. The experimental diets were formulated according to the requirements of Rostagno (2011), as it is shown in table 1.
Ingredients | Treatments | |||
---|---|---|---|---|
Corn/soybean | Corn/soybean 1% PFS | Corn/soybean 2%PFS | Corn/soybean 3% PFS | |
Corn meal | 62.4 | 61.523 | 60.068 | 59.068 |
Soybean meal | 30.018 | 29.615 | 29.68 | 29.677 |
Dry Fermented Product | 0 | 1 | 2 | 3 |
Plant oil | 3.6 | 4 | 4.5 | 4.6 |
Monocalcium phosphate | 1 | 0.95 | 0.95 | 0.95 |
Calcium carbonate | 1.2 | 1.11 | 1 | 0.9 |
Salt | 0.45 | 0.45 | 0.45 | 0.45 |
Methionine | 0.152 | 0.157 | 0.162 | 0.165 |
Lysine | 0.05 | 0.065 | 0.06 | 0.06 |
Choline | 0.13 | 0.13 | 0.13 | 0.13 |
Premix2 | 1 | 1 | 1 | 1 |
Calculated contribution (%) | ||||
CP | 18 | 17.99 | 18 | 18 |
CF | 2.68 | 2.64 | 2.62 | 2.61 |
ME1 | 12.97 | 12.95 | 12.94 | 12.84 |
P disp. | 0.326 | 0.32 | 0.32 | 0.32 |
Ca | 0.717 | 0.713 | 0.712 | 0.715 |
Meth+Cyst | 0.708 | 0.706 | 0.707 | 0.707 |
Lis | 0.99 | 0.99 | 0.99 | 0.986 |
Na | 0.2 | 0.2 | 0.2 | 0.2 |
1 it is expressed in Mj/kg
2Each kg contains vitamin A, 13,500 IU; vitamin D3, 3,375 IU; vitamin E, 34 mg; B2, 6 mg; panthotenic acid, 16 mg; nicotinic acid, 56 mg; Cu, 2,000 mg; folic acid, 1.13mg; vitamin B12, 32µg; Mn, 72 mg; Zn, 48mg
At 42 d of age, 8 broilers were selected per treatment for control treatment and 6 for experimental treatments. They were weighed and the indicators shown in the experimental procedure were determined.
Morphometric indicators of digestive, accessory and immunological organs. Broilers were sacrificed two hours and thirty minutes after food ingestion, according to the method of jugular vein bleeding, described by Sánchez(1990). Subsequently, the abdominal cavity was opened and accessory organs (liver and pancreas) and digestive tract were removed. For analysis, the latter was divided into crop, proventriculus, gizzard, small intestine, caeca and final portion (colon and rectum). In addition, immunological organs were extracted (thymus, spleen and bursa of Fabricius).
Digestive organs were weighed, full and empty (the digestive content was removed by moving the index and thumb fingers to empty them) in a SARTORIUS technical balance. Data of immune, accessory and digestive organs were expressed as relative weight (g / g LWx100). Liveweight (LW) was established at the time of sacrifice (Giambrone 1996).
Indicators of health and blood biochemistry. Blood samples were taken directly from the jugular vein, tilting the tube slightly, so that the blood could roll down the walls. This avoids serum hemolysis, which will be used later. Two sets of collection tubes were used: one for the determination of total blood, and another for obtaining serum. Total blood was placed at room temperature until clot retraction was achieved. Later, it was centrifuged at 3000 rpm for five minutes to obtain the serum. Hematocrit was directly determined from total blood by Wintrobe method.
Blood biochemistry indicators (cholesterol, glucose, triglycerides, total proteins, albumin and uric acid) were determined in blood serum by an automatic Cobas integra 400 PLUS (Roche Diagnostic System).
Histological analysis. For microscopic examination, eight samples of 1 cm2 were taken from each organ for control treatment, and six from each organ for the remaining treatments (1, 2 and 3% PFS). All samples were stored in formalin at 4% for histological analysis and gently shaken to remove remains of adherent intestinal content. Fixing process consisted of dehydration with staggered ethanol solutions (50, 70, 80, 96 and 100%) and cleaning with xylol. The organs were appropriately sized (CENPALAB 2000) and included in paraffin (POT.05.03.003). Subsequently, they were cut (CENPALAB 2000a) (POT.05.03.005) with the use of equipment designed for this purpose (microtome). Cuts were spread in sheets and then they were stained with hematoxylin-eosin (Scheure and Chalk 1986) (POT.05.03.015) (CENPALAB 2000b). The histological sections were examined with an Axioplan-2 optical microscope (Carl Zeiss Jena GmbH, Jena, Germany).
Statistical analysis. A completely randomized design was used with four treatments, consisting of experimental diets and eight repetitions/treatment. For the analysis of results, the computerized statistical package INFOSTAT 2012 (Di Rienzo et al. 2012) was used. In the necessary cases, mean values were compared using Duncan (1955) test.
Results and Discussion
No differences were observed among treatments applied for liveweight. This result could be related to food intake, which did not differ among treatments. That is, PFS did not influence on this indicator. There were no differences in morphometric indexes of full and empty organs of the gastrointestinal tract (GIT) of broilers that consumed different levels of PFS (tables 2 and 3).
Indicators | PFS levels % | ±SE and Sign | |||
---|---|---|---|---|---|
Control (0) | 1 | 2 | 3 | ||
Live weight, g | 1959.33 | 2048.33 | 1991.61 | 2023.33 | 71.38 P=0.8095 |
GIT | 9.19 | 9.70 | 9.01 | 8.76 | 0.35 P=0.3182 |
Crop | 0.55 0.09 | 0.58 0.08 | 0.51 0.08 | 0.50 0.089 | P=0.9190 |
Proventriculus | 0.63 | 0.60 | 0.56 | 0.54 | 0.03 P=0.4123 |
Gizzard | 3.20 | 2.67 | 2.49 | 2.81 | 0.26 P=0.2891 |
Small intestine | 3.98 0.18 | 4.21 0.21 | 3.96 0.18 | 3.86 0.18 | P=0.6524 |
Caeca | 0.81 | 1.05 | 0.78 | 0.88 | 0.10 P=0.2679 |
Colon-rectum | 0.31 | 0.27 | 0.22 | 0.27 | 0.03 P=0.4695 |
Indicators | PFS levels % | ±SE and Sign | |||
---|---|---|---|---|---|
Control (0) | 1 | 2 | 3 | ||
Live weight, g | 1959.33 | 2048.33 | 1991.61 | 2023.33 | 71.38 P=0.8095 |
GIT | 6.69 | 5.56 | 6.17 | 6.35 | 0.36 P=0.2102 |
Crop | 0.36 0.04 | 0.39 0.04 | 0.37 0.04 | 0.38 0.04 | P=0.9676 |
Proventriculus | 0.59 | 0.54 | 0.53 | 0.49 | 0.39 P=0.4221 |
Gizzard | 2.29 | 1.96 | 1.99 | 2.17 | 0.14 P=0.3161 |
Small intestine | 3.15 0.14 | 3.15 0.15 | 3.00 0.14 | 2.97 0.14 | P=0.6770 |
Caeca | 0.43 | 0.43 | 0.46 | 0.49 | 0.04 P=0.7294 |
Colon-rectum | 0.23 | 0.20 | 0.19 | 0.23 | 0.02 P=0.5995 |
In this regard, Gutiérrez (2005) also found no effect on relative weights of empty digestive organs of the gastrointestinal tract of broilers that received doses of 0.5, 1.0, 1.5 and 2% of a dry fermented product, (Vitafert).
Relative weight of the GIT accessory organs was similar among treatments (table 4). This means that PFS does not increase the specific functions of these organs in the release of enzymes to digest and absorb nutrients and in the excretion of bile and cellular metabolism, respectively. As a consequence, the values of these indicators did not differ from the control. These results also agree with those obtained by Gutiérrez (2005), with the dry fermented product Vitafert, and with reports of Elkafi et al. (2015), who analyzed a dry mixture of bovine blood and fermented ruminal content.
Indicators | PFS levels (%) | ± SE and Sign | |||
---|---|---|---|---|---|
0 (control) | 1 | 2 | 3 | ||
Live weight, g | 1,959.33 | 2,048.33 | 1,991.61 | 2,023.33 | 71.38 P=0.8095 |
Liver | 2.26 | 2.18 | 2.24 | 2.02 | 0.08 P=0.2303 |
Pancreas | 0.22 | 0.23 | 0.22 | 0.20 | 0.02 P=0.8694 |
Immunological indicators. No differences were observed among treatments in the relative weights of the bursa of Fabricius and thymus with the inclusion of PFS (table 5). As it is demonstrated, relative weight of the bursa of Fabricius had values between 0.18 and 0.27, so the animals were immunocompetent. Regarding Giambrione (1996), this condition is reached when the relation between absolute weight of the organ and liveweight is between 0.20 and 0.30. According to this author and Contreras and Fernández (1999), the immune system of broilers undergoes evolution, and the main organs of immunity are the bursa and the thymus, during the first stage of life. In the first, B lymphocytes are produced, responsible for humoral immunity (production of antibodies by B lymphocytes), and the second organ is responsible for cell-mediated immunity (production of cytokines and cells by T lymphocytes). Peripheral tissues of the immune system in broilers are spleen, cecal tonsils, Harder glands and bone marrow. These are populated by B and T lymphocytes during broiler growth. When these animals approach maturity, the bursa and thymus involve, and the immunocompetence of broilers starts depending on the peripheral immune system.
Indicators | PFS levels % | ±SE y Sign | 1-ß Test potency | |||
---|---|---|---|---|---|---|
Control (0) | 1 | 2 | 3 | |||
Live weight, g | 1959.33 | 2048.33 | 1991.61 | 2023.33 | 71.38 P=0.86 95 | 8% |
Bursa of Fabricius | 0.21 | 0.27 | 0.18 | 0.24 | 0.03 P=0.2971 | 30% |
Thymus | 0.50 | 0.51 | 0.55 | 0.49 | 0.07 P=0.9401 | 7% |
Spleen | 0.13 | 0.14 | 0.19 | 0.17 | 0.01 P=0.0693 | 61% |
Hematocrit | 31.17 a | 32.16 ab | 33.66 b | 32.83 ab | 0.59 P=0.045 | 72% |
a,b Different letters within the same line differ significantly (P< 0.05) (Duncan 1955)
*P<0.05
The relative weight of the spleen did not differ statistically among treatments. However, from a biological point of view, it was observed that broilers that received 2% of PFS in the diet, had superior relative weight (P = 0.0693). When analyzing the potency of the test, it was observed to be 61%, which is low so the fact that no statistical differences were found for P <0.05, several factors inherent to sampling could have influenced, such as sample size or errors inherent to sampling that were not controlled, as in the weighing of the organ, due to the adhered fat that was not removed, among other factors. In any case, the highest relative weight of the spleen, confirmed by a high hematocrit content, could indicate an immunostimulatory response to the inclusion of PFS. As it is known, this product, made from fermented ruminal liquor content, contains metabolites called “tertiary metabolites”, such as polysaccharides, bacteriocins and enzymes that could promote this action.
The hematocrit was maintained in the normal physiological ranges for the species (Meluzzi et al. 1992), although it was higher (P = 0.045) for 2% of PFS with respect to the control treatment (table 5). This result was expected, since if the spleen is a hematopoietic organ, the increase in its relative weight should correspond to the increase in blood counts.
Indicators of blood biochemistry. The study of blood biochemistry showed that serum indicators of protein metabolism: total proteins, albumin and uric acid, as well as the albumin/globulin (A/G) ratio, did not vary with the inclusion of PFS in the diet of broilers (table 6). However, the product influenced significantly on energy metabolism indicators.
Levels of 2 and 3% of PFS increased serum blood glucose (P <0.0091) compared to control and 1% of PFS, which were similar to each other. Increase in serum cholesterol (P <0.0204) was observed with PFS levels. This corresponded with the decrease in triglycerides from 0.84 to 0.54mmol / L.
Related to these results, glucose represents the most important sugar in carbohydrate metabolism in all vertebrates. It is the carbohydrate that circulates through blood toward different organs and body tissues that use it as an energy source (D'Mello1995). In conventional diets for broilers and laying hens, starches (amylose and amylopectin), present in corn, wheat, and soybean constitute the main source of circulating blood glucose (Gunsberg et al. 1998, cited by Miranda López et al. (2007). Circulating glucose values in blood plasma of broilers are in the range of 8.4-10.1mmol/L (Miturka et al. 1977). Table 6 shows that only broilers of the treatments with 2 and 3% of PFS showed glucose levels in the normal ranges. This means, perhaps, that PFS levels of 2% and above provide different organs and body tissues with the energy needed to perform their functions.
On the other hand, the increase obtained in serum cholesterol levels with PFS suggests that this product, despite constituting a mixture of microorganisms mainly composed by yeasts and lactic bacteria, did not show a hypocholesterolemic activity for this species (broilers). Similar results were obtained by Pérez (2000), when doses of 50, 75 and 100 mL of yeast hydrolyzed were tested. With regard to control, this author did not observe a decrease in serum cholesterol in treated broilers. It should be noted that the liver is the main organ of lipid metabolism of broilers. It is the main seat of lipogenesis, where cholesterol is synthesized and triglycerides constitute its main product (Martínez 2004).
Indicators1,2 | PFS levels % | ±SE y Sign | |||
---|---|---|---|---|---|
Control (0) | 1 | 2 | 3 | ||
A/G | 0.71 | 0.73 | 0.65 | 0.67 | 0.04 P=0.6050 |
Albumin, g/L | 12.06 | 11.25 | 11.85 | 12.91 0.80 | 0.75 P=0.5090 |
Cholesterol, mmol/L | 3.11a | 3.15a | 3.62ab | 4.10b 0.25 | 0.23 P=0.0242 |
Glucose, mmol/L | 6.85 a | 6.77 a | 9.52 b | 10.21b 0.69 | 0.64 P=0.0091 |
Total Proteins, g/L | 29.13 | 27.19 | 30.31 | 32.10 1.96 | 1.83 P=0.3300 |
TG mmol/L | 0.84 b | 0.61 a | 0.52 a | 0.54 a | 0.06 P=0.0032 |
AU, mmol/L | 354.25 | 378.25 | 399.38 | 358.00 35.02 | 32.76 P=0.7580 |
1TG = triglycerides; AU= uric acid, A/G= Albumin /Globuline relation
2a,b, Different letters in the same line differ at P<0.05 (Duncan (1955)
Histological analysis. Generally, lymphoid tissue hyperplasia, associated with mucous membranes in the cecum (cecal tonsils) was observed in all treatments with PFS compared to control group. Cecal tonsils, spleen and Harder gland are part of the immune system, and are likely to favor an immunostimulatory action (figures 1 and 2).
With respect to intestine (duodenum), in all the treatments where PFS was included, an apparent elongation of villi was observed. Height of villi and depth of the crypt are important indicators of intestinal health of animals, and are directly related to the absorption capacity at mucus level (Buddle and Bolton 1992) (figures 3 and 4). The increase of villi height, as well as their diameter, means superior surface area for nutrient absorption (Sklan and Noy 2003, Wang and Peng 2008 and Rodríguez 2012). A morphometric indicator that could be related to nutrient absorption surface is relative length of the small intestine. Unfortunately, this indicator was not measured. Results suggest that the inclusion of up to 3% of the dry fermented product in the diet of broilers based on corn/soybean, favors health and histological indicators of these animals.