Blood profiles of Etawa crossbreed goats fed concentrates substitution diet with bread waste

This study aims to determine the maximum level of the use of bread waste as a substitute for concentrates on feed utilization by livestock through blood profiles consisting of the amount of hemoglobin, erythrocyte numbers, hematocrit levels and leukocyte numbers. The material used in this study was 15 lactating Etawa crossbreed goats. The experimental design used was a randomized complete block design with 3 treatments and 5 replicates. The feed treatment given was forage and concentrate with a composition of 20:80 with ration treatments namely P1 = Silages 20% + Concentrates 64% + Bread waste 16% + Zeolite 2%, P2 = Silages 20% + Concentrates 56% + Bread waste 24% + Zeolite 2%, P3 = Silages 20% + Concentrates 48% + Bread waste 32% + Zeolite 2%. The observed variables were hemoglobin level (g/dL), erythrocyte count (million/μL), hematocrit level (%) and leukocyte count (thousand/μL). Data were analyzed using analysis of variance. The results showed that the treatment had no significant effect (P>0.05) on the blood profile which included hemoglobin levels, erythrocyte numbers, hematocrit levels and leukocyte numbers. Substitution of 24% bread flour in the ration gave the highest blood profile calculation results among the three treatments. The amount of Hemoglobin (g/dL) of P1, P2 and P3 were 8.88; 10.05 and 8.94. Erythrocyte numbers (million/μL). P1, P2 and P3 were 14.23; 15.92 and 14.59 respectively. Hematocrit levels (%) P1, P2 and P3 were 28.3; 30.98 and 28.72. Leukocyte numbers (thousand/μL) of P1, P2 and P3 were 13.68; 18.00 and 17.3. The conclusion of this study is that bread flour can be used as a substitute for concentrate up to 32% level in the ration and can maintain the blood profile of lactating Ettawa crossbreed goats in a normal state.


Introduction
When provided at a 15% level, bread waste can be used as a concentrate feed ingredient for sheep.There were no significant differences (P>0.05) in mean dry matter (DM) consumption, daily weight gain, feed conversion, rumen NH3 content and rumen microbial protein content between treatments P0, P1, P2, and P3.The average DM consumption during treatments P0, P1, P2, and P3 was 1120; 1097; 1167 or 1133 g/day.The average daily weight gain between treatments P0, P1, P2 and P3 was 371; 336; 363 and 307 g respectively.The average feed conversion ratio between treatments P0, P1, P2, and P3 was 3.09; 3.23; 3.43 and 3.33 respectively.The average ruminal NH3 content during treatments P0, P1, P2 and P3 was 0.58; 0.50; 0.54 and 0.37 mg/ml.The average rumen microbial protein content during treatments P0, P1, P2, and P3 was 38.36; 33.21; 35.68 mg/100 ml and 24.75 mg/100 ml, respectively.Nutrient content was similar in treatments P0, P1, P2, and P3, and palatability was similar as well as aflatoxin content in feed and meat; There was no significant difference, which was still within the normal range of 13.50 µg/kg and 0.15 ppm.The low aflatoxin contamination of feed and meat is also due to the low proportion of bread as an ingredient in concentrate feed.In addition, it contains zeolite minerals that act as absorbents and are able to absorb the aflatoxin content in feeds that are resting within the normal range.The above study results can be interpreted to mean that using expired biscuits up to a proportion of 15% has the same effect as the control diet (P0) and does not affect the metabolic processes of the sheep.The nutritional content of bread waste is 14.84% Crude Protein; 1.00% crude fiber; 5.67% extracted ester; ash 2.42% [1].Bread waste suspected of containing mycotoxins, which are secondary metabolites produced by certain fungi.One type of mycotoxin is aflatoxin.Aflatoxins (Aspergillus flavus) are disease toxins produced by fungi.Aflatoxins contaminate feedstuffs such as corn, cottonseed meal, peanuts, bean meal, rice, soybeans, wheat, and sorghum seeds.The metabolites that molds release during growth are mycotoxins, which are known to be harmful to those who ingest them.The effects for people who consume meat or milk contaminated with aflatoxins can lead to short-and long-term illnesses such as liver damage, decreased reproductive and immune system, and gastrointestinal problems.Therefore, in this study, the research team sought to increase the proportion of bread waste in the composition of concentrate feeds and measure the effect on blood profiles (the amount of hemoglobin, erythrocyte numbers, hematocrit levels and leukocyte numbers).

Materials and methods
All experiments related to animals in this study meet Ethical guidelines and approved by Ethical committee of Animal Science Study Program, Universitas Sebelas Maret.

Materials
Materials used in the study include: a.The livestock used in this study were 15 lactating Etawa crosbreed goats of the second period; b.The cages used in this study were individual cages where one cage was occupied by one goat.Each cage is equipped with a place to feed and drink; c.The feed given to livestock in the form of forages and concentrates.The ration consisted of silage, bread and concentrate.The treatment ration is in the form of mash (flour).The ration is divided into 3 types according to the treatment given.For more details of feed composition can be seen in Table 1; further the tools used in this study include tarpaulins, feed mills, feed scales with a capacity of 500 kg with a sensitivity of 1 gram, buckets, permanent snowman markers, cool boxes, venoject, alcohol, cotton, scissors, vacutainer, plastic bags, broom sticks, hoses, shovels and stationery.The research design used was a completely randomized design (CRD) with a unidirectional pattern with three treatments repeated five times.The research treatments are as follows: P1 = Silages + Concentrates (16% bread waste); P2 = Silages + Concentrates (24% bread waste) and P3 = Silages + Concentrates (32% bread waste).

Preparation Stage a. Cage preparation
The cages used for this study were given a cage identity as a sign of treatment and replication which was carried out after the livestock were put into the cage.Cages and equipment used in the study were first cleaned using water until clean from livestock feces and feed.b.Determination of cage plots Fifteen Etawa crossbreed goats were put into the available cage plots and randomization of goats in each cage was carried out.c.Feed preparation Feed in the form of corn stover silage and concentrate with the substitution of bread flour and 2% zeolite in each treatment.d.Retrieval of discarded bread The bread used is fresh bread taken from the bread factory, Salatiga, Central Java.

Feed preparation
The bread will be used previously separated from the plastic and then dried in the sun until dry, after which it is ground using a feed grinder "chopper" until it becomes flour.Zeolite was also added to each feed treatment at 2%.Silages was given separately as much as 20% of the total ration.Concentrates consisted of basal concentrate, cull bread mixed homogeneously according to the percentage of feed ingredients used according to the treatments.Rations were made according to feed requirements based on body weight (BW), which is 3.5% of the initial BW of the treatment goats.

Implementation stage
The implementation of this research consists of 2 stages, namely the maintenance stage (adaptation and treatment) and data collection.a. Maintenance stage 1) Adaptation The adaptation stage was carried out for one week.At this stage, the initial body weight is weighed to determine the nutrient needs of the livestock and adaptation to feed and the environment is carried out.

2) Treatment stage
The treatment stage is carried out for eight weeks (2 months) after the adaptation stage.Concentrates were given dry twice a day in the morning and evening according to the proportion of each goat.Drinking water is given ad libitum.b.Data collection stage Blood samples collection was conducted at the end of the study in accordance [2].The method used was stabbing 22G needle size venoject into the jugular vein, previously the hair around the area to be stabbed was cleaned first using alcohol.Blood samples were taken from the veins of animals using vacutainer and needle.Blood samples obtained were then inserted into a purple vacutainer to prevent clotting and then analyzed using a veterinary hematology analyzer automatically.The vacutainers have different colors based on the presence or absence of anticoagulants.Blood samples for hematology examination should not clot, so blood is collected in a purple vacutainer containing the anticoagulant Ethylene Diamine Tetra Acetic Acid (EDTA).Anticoagulants are chemicals that are put into the blood to prevent blood clotting [3].

Research variables
This study consists of four variables, namely the amount of hemoglobin (g/dL), the numbers of erythrocytes (million/µL), hematocrit levels (%) and the number of leukocytes (thousand/µL) in the blood.The way it works is that blood is taken from the jugular vein of the goat using a venoject.Blood was immediately put into a purple vacutainer that already contained EDTA.
The vacutainer containing the blood was shaken first to make it homogeneous and then analyzed with a veterinary hematology analyzer for approximately 1-1.5 minutes.Haematology Analyzer is a tool for complete blood examination that has a fairly good speed and accuracy.This tool can reduce the examination time from 30 minutes using the manual method to 15 seconds and can reduce errors.The working principle of hematology analyzer uses the impedance method, which is based on the detection and measurement of electrical resistance produced by blood cells when crossing a small hole (aperture).The size of blood cells is known from the vibration of the electrode.The number of blood cells is calculated from the number of vibrations and will be read based on the size of the cell [4].

Results and discussion
Based on the results of the study, the average of the amount of hemoglobin, the number of erythrocytes, hematocrit levels and the number of leukocytes is found in Table 2.

The amount of Hemoglobin
The results of the analysis of the substition of bread waste in concentrates had no significant effect (P>0.05) on the amount of hemoglobin.This is due to the quality and quantity of feed given, especially the Nitrogen Free Extract (NFE) and crude protein (CP) content which is almost the same or not significantly different between treatments.Etawa Crossbreed goats fed with Nitrogen Free Extract content of 72.43; 70.83 and 67.64% and PK content of 14.74; 18.17 and 19.32% showed hemoglobin levels were not significantly different between treatments.Nitrogen Free Extract and CP content of rations P1, P2, P3 are 58.28;59.99; 61.70 and 19.44; 19.10; 18.75, respectively [2].Hemoglobin is an erythrocyte pigment consisting of iron, porphyrin and complex proteins [5].The provision of bread waste, which increases up to the highest level of 32%, is thought to provide enough minerals for the synthesis of blood hemoglobin (Table 1).In addition, the bread as an energy source feed through gluconeogenesis provides enough glucose as a substrate for glycolysis to produce pyruvate which is then oxidized to acetyl Co-A as a substrate for the Krebs cycle.The Krebs cycle produces succinyl Co-A which plays a role in the formation of hemoglobin.Heme synthesis mostly occurs in the mitochondria starting with the condensation of glycine and succinyl Co-A [6].
The amount of hemoglobin in treated goats fed P1, P2 and P3 is still normal.The amount of hemoglobin in goats ranges from 8-12 g/dL.Hemoglobin is part erythrocyte numbers that function to transport oxygen to the tissues [7].These conditions indicate that the treated goats are in a healthy condition, the oxygen needs of the tissues can be met so that the metabolic process runs well, and the nutrients contained in the feed can be used for basic living needs and production.

The number of Erythrocytes
The results of the analysis of variance (ANOVA) of the substitution of bread waste in feed had no significant effect (P>0.05) on the number of erythrocytes of the treated goats.This is because the number of erythrocytes, hemoglobin and hematocrit levels have a close correlation [8].Erythrocyte production is influenced by the concentration of hemoglobin and hematocrit in the blood [9].Hemoglobin levels and the number of erythrocytes in lactating Etawa crossbreed goats both differed not significantly between treatments.Erythrocyte production is also influenced by the hormone erythropoietin which is synthesized according to the functional ability of hemoglobin in meeting tissue oxygen demand [5].The average hemoglobin amount of goats in each treatment is within the normal range, so it does not stimulate the release of erythropoietin hormone to increase erythrocyte production.Erythrocytes are red blood cells that function as carriers of hemoglobin in the blood circulation, so the increase and decrease in the number of erythrocytes and hemoglobin levels are comparable.
The provision of bread waste as an energy source feed up to P3 increases the Nitrogen Free Extract content in the ration, this can support the provision of glucose that can be synthesized into energy for the maintenance of erythrocytes.The Nitrogen Free Extract is a soluble carbohydrate so that the fermentation process produces Volatile Fatty Acid (VFA) with more propionic acid content.Propionic acid through gluconeogenesis in the liver will be synthesized into glucose [3].Erythrocytes do not have mitochondria or other organelles, so additional glucose is needed to maintain their function [6].Glucose is broken down through glycolysis into lactate, for every molecule of glucose used, two molecules of Adenosine Triphosphate (ADP) are produced to provide energy for maintaining the volume, shape and flexibility of erythrocytes.Erythrocyte function continues to run well with the provision of bread waste in the ration.This can be seen from the number of erythrocytes that are still in a normal state which is 8-18 million/µL [7].

Hematocrit levels
The results of ANOVA of the treatments feed substituted with bread waste had no significant effect (P>0.05) on blood hematocrit levels.This is because the amount of hemoglobin is proportional to the number of erythrocytes.Hematocrit levels are proportional to hemoglobin and erythrocytes, if hemoglobin levels are high then most likely the hematocrit levels is also high.This can be caused by the lowest NFE of the P1 ration despite the highest CP content of 19.44%.Hemoglobin levels are affected by the interaction of differences in ration energy and protein balance and time of ration feeding.Nitrogen Free Extract content test results of rations P1, P2 and P3 are 58.28;59.99 and 61.70%.The increase in NFE content from 58.28 to 61.70% and the decrease in Crude Protein from 19.44 to 18.75% did not affect the blood hematocrit levels.This situation indicates that the provision of rations up to P3 does not affect the formation of hemoglobin and erythrocytes which are the constituent components of blood hematocrit.
The Crude Protein and Nitrogen Free Extract content of the treated feed, although diverse, is still within the range of the needs of lactating goats.This is evidenced by the levels of blood hematocrit which is still within the normal range [7] which ranges from 22-38%.The hematocrit levels of P1, P2 and P3 goats are 28.3;30.98 and 28.72% respectively.

The number of Leukocytes
The results of ANOVA of the substitution of bread waste in the ration on the number of leukocytes of goats treated had no significant effect (P>0.05), this could be due to the relatively equal content of aflatoxin in feed P1, P2 and P3.In addition, the addition of mineral zeolite as much as 2% is considered to absorb aflatoxin contained in the ration.zeolite has the ability to absorb aflatoxin [10].
The maximum limit of aflatoxin content allowed in corn as a feed ingredient is 50 parts per billion (ppb), aiming to lower the aflatoxin content of the feed given [11]. .The aflatoxin content of cull bread was 13.77 ppb.Based on this information, bread flour can be used as animal feed because the aflatoxin content is still within the safe limits allowed.Aflatoxin content of 25 ppb in feed can still maintain the number of saanen goat leukocytes in a normal state [12].The number of leukocytes is not too different from the results of research, ranging from 16.60-21.90thousand/µL [13].The decrease and increase in the number of leukocytes is a mechanism of the body's response to pathogens that attack.The physical health of animals can be measured by the number of leukocytes produced, where an increased number of leukocytes indicates an increased body defense ability [14].

Conclusion
The conclusion that can be drawn from this research is that the substitution of concentrates with bread up to 32% gives results within normal limits to the blood profiles of Etawa crossbreed goats

Table 2 .
Effect of bread waste substitution in concentrates on blood profiles of lactating Etawa crossbreed goats