Effects of Fermented Whey in Treating Bacillary Dysentery and on the Gastrointestinal Flora of Apparently Healthy Albino Rats
Effects of Fermented Whey in Treating Bacillary Dysentery and on the Gastrointestinal Flora of Apparently Healthy Albino Rats
Tinuola Tokunbo Adebolu and Sunday A. Awe Department of Microbiology, Federal University of Technology, Akure 340001, Nigeria
Received: June 23, 2014 / Accepted: July 17, 2014 / Published: July 31, 2014.
Abstract: In this study, the effects of fermented whey (FW) in treating bacillary dysentery caused by Shigella flexneri in albino rats and on the gastrointestinal (GIT) flora of apparently healthy albino rats (AHARs) were investigated. Prior the therapeutic assay, the growth inhibitory activity (GIA) of whey subjected to different fermentation durations at 30 ± 2 °C was first investigated using agar diffusion assay on the test organism, conventional antibiotics served as control. After this, the infectious dose of the organism was determined and used to infect another set of AHARs. The infected rats were grouped into two; group one was treated with 1.0 mL of the FW that exerted the highest GIA in the in vitro assay (FW1), once daily for 7 d while group two was left untreated. The rats were observed for signs of recovery while their large intestine was subjected to histopathological examinations. For the effects of whey on GIT flora of AHARs, another group of AHARs was fed with FW1 for 3months. At 7 d intervals, their faeces were examined for microbial types and load. The in vitro GIA of the FWs on the test organism was superior to that of most of the antibiotics used and the administration of FW1 to infected rats caused them to recover by 72 h while those not treated with FW1 started to recover by 168
h. FW1 did not significantly (p < 0.05) affect the GIT microflora loads but only the types.
Keywords: Bacillary dysentery, fermented whey, gastrointestinal flora, histopathology, non-conventional therapy, Shigella flexneri.
1. Introduction it also has growth inhibitory activity on Shigella flexneri, a common cause of bacillary dysentery in
Bacillary dysentery is still of serious concern in the southwest, Nigeria and also to find out whether it has developing countries. This is because of the any deleterious effect on the gastrointestinal flora of complications such as bacteremia, hemolytic uremic
apparently healthy albino rats.
syndrome and toxic megacolon associated with it [1]. Although it is normally treated with antibiotics,
2. Materials and Methods
however because of lack of quick access to these
2.1 Bacteria Used
drugs in some rural areas, it becomes imperative to search for alternative means of treatment of this
The Shigella flexneri used in this study was disease.
obtained from the Microbiology laboratory, Federal In an earlier work, Adebolu and Ademulegun [2]
Institute of Industrial Research, Oshodi, Lagos, observed that fermented cheese whey has antibacterial
Nigeria.
activities against common diarrhoeagenic bacteria.
2.2 Source of Whey
This present study therefore is to investigate whether Fresh whey was collected from Fulani women who
Corresponding author: Tinuola Tokunbo Adebolu, Ph.D., produce local cheese in a suburb village in Akure, professor, research field: medical microbiology. E-mail:
Ondo State, Nigeria.
ttadebolu01@yahoo.com.
Effects of Fermented Whey in Treating Bacillary Dysentery and on the Gastrointestinal
Flora of Apparently Healthy Albino Rats
2.3 Animals Wiley et al. [4] with slight modifications. AHARs were grouped in three’s and different groups were
Wister albino rats, aged 6-8 weeks with weight orogastrically challenged with different doses of the
averaging 110 g were used. They were bought from test organism. The rats were daily observed for the Animal House, Faculty of Health Sciences, symptoms of illness such as loss of appetite, weakness, Obafemi Awolowo University, Ile-Ife, Osun State, dullness, and unformed stool. The dose that was able Nigeria. to cause illness in all the rats in a particular group was
2.4 Determination of Antibacterial Activity of Whey on taken as the infectious dose. This dose was then Test Organism
administered to another group of AHARs and the rats This was done using agar diffusion method of
were daily observed for signs of illness. Adebolu et al. [3]. The organism was grown in
2.7 Treatment of Infected Rats with Fermented Whey nutrient broth at 37 °C for 18 h. The cells were
harvested at 3000 rpm for 5 min into 20 mL sterile Six AHARs were orogastrically administered the
distilled water. One milliliter of the harvested bacterial ID (2.4 × 10 cfu/mL) of the organism. After infection cells containing approximately 10 6 cfu/mL was
sets in, the infected rats were grouped into two groups introduced into a sterile petridish, 20 mL of nutrient
of three rats each. Group one was administered 1.0 agar was added, the plate was carefully swirled to
mL of FW1 daily, while the second group on the other allow even distribution of the organisms within the
hand was not treated with FW1. This served as control. agar and was allowed to gel before 2 wells (8 mm in
The rats were daily observed for signs of recovery. diameter) were bored in the agar with the aid of a
2.8 Effect of Fermented Whey on the GIT Flora sterile cork borer. Whey (0.1 mL) was introduced into
one of the wells while 0.1 mL of sterile distilled water This was done according to the method of Adebolu was put into the other well, to serve as negative
et al. [5].
control. The plate was incubated at 37 °C for 24 h.
2.9 Histological Assay
The diameter of the zones of inhibition around the wells was measured and recorded. This assay was
This was done using standard histological repeated every 24 h for 5 d, using FW at 30 ± 2 °C in
techniques on the large intestine of rats infected with order to evaluate the effects of fermentation duration
the test organism, rats infected and treated with FW1 on the antibacterial potency of whey on the test
and rats that were not infected and not treated with organism. Each assay was done in triplicate.
FW1.
2.5 Antibiotic Sensitivity Pattern of the Test Organism
3. Results
This was done as above except that instead of The whey used exerted growth inhibitory activity making wells on the already seeded plates and pouring
on the test organism. However whey fermented at 30 in whey, commercial antibiotics disc was placed on
± 2 °C for 48 h (FW1) exerted the highest growth the seeded agar plates before the plates were incubated
inhibitory effect on the test organism. The growth at 37 °C for 24 h.
inhibition mediated by this whey was higher than that
2.6 Infection of Rats with the Test Organism of most of the commercial antibiotics used except ciprofloxacin which had growth inhibitory effect of Prior infection of the rats, the infectious dose (ID)
zone diameter averaging 10.6 mm on the organism of the test organism was first determined according to
(Fig. 1). In the therapeutic assay, rats infected with
Effects of Fermented Whey in Treating Bacillary Dysentery and on the Gastrointestinal
Flora of Apparently Healthy Albino Rats
Shigella flexneri and treated with FW1 recovered by dropped as a result of the infection started to increase the 72 h of treatment while the infected rats that were rd by the 3 day of the administration of FW1 but the
not treated with FW1 on other hand did not recover ones not treated with FW1 did not start to gain weight until after 168 h (Table 1). Moreover, the results of the th until after the 5 day of administration of FW1 (Table 3).
histological examination of the large intestine showed normal morphology for the rats infected and treated
with FW1 but ulceration was seen in that of the infected rats not treated with FW1 (Plates 1a and 1b respectively). The following bacteria, Escherichia coli and Staphylococcus aureus (total viable count of 1.17
± 0.52 × 10 7 cfu/mL) were isolated from the stools of AHAR used prior feeding with FW1 for three months.
After feeding with FW1, the following bacteria were isolated: Escherichia coli, Streptococcus lactis, Bacillus
substilis, Lactobacillus acidophilus and Bacillus Antibiotics/whey brevis (viable count of 9.0 ± 1.0 × 10 Fig. 1 Growth inhibitory activities of unfermented whey, 6
cfu/mL) (Table
fermented whey and some conventional antibiotics on
2). Furthermore, the weight of the infected rats that
Shigella flexneri (9.3 × 10 6 cfu/mL).
Table 1 Signs of recovery in the rats infected with Shigella flexneri and treated with 48 h fermented whey.
Treatment
Signs of illness/recovery
A, C, E A: Active rats, W: Weak rats/reduced activity, C: Eat well, D: Loss of appetite, E: Normal stool, F: Bloated/soft stool
7 A, C, E
A, C, E
Plate 1a Histological examination of the large intestine of Plate 1b Histological examination of the surface of the rats with Shigella flexneri infected and treated with normal
large intestine of rats infected with Shigella flexneri morphology.
showing ulceration.
Effects of Fermented Whey in Treating Bacillary Dysentery and on the Gastrointestinal
Flora of Apparently Healthy Albino Rats
Table 2 Types of bacteria isolated from the stool of apparently albino rats before and after feeding with 48 h fermented whey.
Days
Types of Bacteria/(cfu/ml)
Escherichia coli, Staphylococcus aureus (1.17 ± 0.52 × 10 7 cfu/ml)
7 Escherichia coli, Bacillus substilis 14 Escherichia coli, Micrococcus lactis 21 Escherichia coli, Lactobacillus acidophilus 28 Escherichia coli, Lactobacillus acidophilus, Bacillus brevis 35 Escherichia coli, Lactobacillus acidophilus 42 Escherichia coli, Lactobacillus acidophilus 49 Escherichia coli, Micrococcus lactis 56 Escherichia coli, Lactobacillus acidophilus, Micrococcus lactis 63 Escherichia coli, Lactobacillus acidophilus 70 Escherichia coli, Lactobacillus acidophilus 77 Escherichia coli, Lactobacillus acidophilus
84 Escherichia coli, Streptococcus lactis, Bacillus substilis, Lactobacillus acidophilus and Bacillus brevis (9.0 ± 1.0 × 10 6 cfu/ml) * = Types of bacteria isolated before feeding the rats with 48 h fermented whey
Table 3 Effects of the administration of 48 h fermented whey on the weight of rats infected with Shigella flexneri.
Weight (g) ± S. E. M
Days IT INT NINT
122.30 ± 4.10 IT = Infected and treated INT = Infected and not treated NINT = Not infected not treated
treat shigellosis caused by this organism. Moreover, In this study, the superior inhibition exerted by
4. Discussion
the increase in weight in the infected rats that were FW1 on the test organism to that of most of the
treated with FW1 shows that it has rehydrating effect antibiotics used shows that this whey has potent
which can be exploited in treating dehydration bioactive components which had greater effect in
associated with diarrhoea/dysentery. Furthermore, the inhibiting the growth of the test organism. These
presence of lactic acid bacteria in the GIT of AHARs bioactive components according to Olorunfemi et al.
in addition to the bacterial flora after the [6] are organic acids and hydrogen peroxide produced
administration of FW1 for 3 months shows that FW1 by the lactic acid bacteria: Micrococcus lactis, can be used as prebiotic and as a source of probiotics.
Bacillus brevis, Lactobacillus acidophilus and In conclusion, therefore, FW1 has been shown to Pediococcus cerevisiae that are present in FW. In the
have antidiarrhoeal activity in addition to antibacterial in vivo assay, the reduction in the duration of infection
activity against Shigella flexneri in rats. It is therefore from 168 h to 72 h observed in the infected rats that
suggested that people having dysentery caused by this were treated with FW1 showed that it can be used to
organism could drink FW1 to treat the infection
Effects of Fermented Whey in Treating Bacillary Dysentery and on the Gastrointestinal
609
Flora of Apparently Healthy Albino Rats
especially in rural areas where they might not have Journal of Biotechnology 6 (9):1140-1143. [4] Wiley, J. M., Sherwood, L. M., and Woolverton, C. J.
access to quick medical attention. 2008. Prescott,Hurley and Klein’s Microbiology.7th ed.
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Komolafe, B. M., and Ogundare, A. O. 2010. “Effects of Picture of Shigellosis in Southern Vietnam: Shifting
Raw Maize “Ogi” on the Gastrointestinal Flora of Species Bmc.” Infectious Disease 9: 204.
Albino Rats and Its Potential in Controlling Diarrhoea [2] Adebolu, T. T., and Ademulegun, O. H. 2005. “Effect of
Caused by Escherichia Coli.” TWOWS Africa Cheese Whey on Diarrhoea Causing Bacteria in
International Journal of Science and Technology 1 (1): Southwest, Nigeria.” Bioscience Research
39-45.
Communication 16: 57-60. [6] Olorunfemi, O. B., Adebolu, T. T., Adetuyi, F. C. 2006. [3] Adebolu, T. T., Olodun, A. O., and Ihunweze, B. C. 2007.
“Antibacterial Activities of Micrococcus Lactis Strain “Evaluation of ‘Ogi’ Liquor from Different Grains for
Isolated from Nigerian Fermented Cheese Whey Against Antibacterial Activities Against Some Common
Diarrhoea Causing Organisms.” Research Journal of Diarrhoeal Bacterial in Southwest, Nigeria.” African
Biological Sciences 1 (1-4): 24-27.
July 2014, Vol. 8, No. 7, pp. 610-625
Journal of Life Sciences, ISSN 1934-7391, USA DAVID PUBLISHING
Osteoporosis as A Source of Tissue Mineralization Research on Osteoporosis Therapy and Dissolution of Arterial Mineralization
Maciej Pawlikowski Laboratory of Biomineralogy, Department of Mineralogy, Petrography and Geochemistry, Faculty of Geology, Geophysics and
Environmental Protection, AGH University of Science and Technology, Cracow 30-059, Poland
Received: June 06, 2014 / Accepted: July 21, 2014 / Published: July 31, 2014.
Abstract: Based on research conducted by the author in the last thirty-five years, this article presents the physicochemical mechanisms of the osteoporosis process, transport of substances created as its result, and the phenomena of tissue mineralization resulting from osteoporosis. Examination of bones, joint cartilage, arteries, veins, parts of heart, thyroid, salivary glands, various tumors and others was conducted with the use of biological and polarizing microscopy, SEM, EDS, ASA, IR, Raman spectroscopy, and X-ray diffraction. Several devices of the same kind, e.g. different types of SEM, were used. Specimens used for examination were obtained from post-surgery and post mortem materials. Examination of human bones focused on their mineralization and demineralization (osteoporosis). Examination of the mineralization of other tissues was conducted in terms of the ageing of human body. Obtained results show that the process of osteoporosis leads not just to mechanical degradation of bones, but through the transport of ions (mainly Ca and P), it also causes mineralization of soft tissue. Such mineralization occurs in mineralization centers that have been classified in regard to genetics. Tissue mineralization in its first stage is latent and consists of including atoms, mainly Ca and P, into the biological structures of compounds that build the tissues. Latent mineralization may evolve into the next stage—apparent mineralization. Both types of mineralization cause many health issues and may lead to death. This article also presents initial results of research on dissolution of aortic mineralization.
Key words: Human osteoporosis, physicochemical processes, tissue mineralization
1. Introduction tissues and organs [9, 18-32]. This theory appears to
be confirmed by the increase in the degree of It is commonly known that osteoporosis is a mineralization (“calcification”) of many tissues and destructive process causing degradation of the bone organs as osteoporosis progresses with age [22, 33-37]. structure [1-12]. The results in reduction of bone Therefore, acknowledging the relation between strength parameters are due to the release of minerals osteoporosis and age-related tissue mineralization is from bone and degeneration of the trabeculae, which essential for understanding many phenomena, including increases the risk of fracture [2, 13-16]. However, the various diseases leading to death. Researching these fate of the minerals released from the bone, phenomena may also build a foundation for finding particularly the ions released during the bone apatite new methods of countering the process of dissolution, is less known. Long-term research osteoporosis and mineralization of arteries [3, 24, 29, suggests that at least part of those minerals may create 38-42], heart valves [6, 23, 36, 43, 44], cartilage (in areas of changing crystallinity [2, 17] in different various parts of body), and many other organs [45-49]
Corresponding author: Maciej Pawlikowski, professor, possibly including tumor tissue [4, 46, 50, 51]. research fields: Biomineralogy, Mineralogy. E-mail:
This article describes physicochemical issues mpawlik@uci.agh.edu.pl.
Osteoporosis as A Source of Tissue Mineralization
Research on Osteoporosis Therapy and Dissolution of Arterial Mineralization
connected with osteoporosis, as well as tissue examination of minerals present in tissues, as well as mineralization and attempts of its dissolution.
for determination of atom substitution in biological components. Examination was performed using
2. Methods and Materials
BIO-RAD, model FTS 165, equipped with a package The article presents a wide range of problems
of programs;
concerning mineralization and demineralization of Raman spectroscopy was done using the DXR human tissues. Considering that the research has been
Raman Microscope, made by Thermo Scientific Co. conducted since 1978 (for 36 years), methods that
This method was used to identify small mineral grains were used have evolved over time.
present in various tissues.
Various methods of examination have been used to
3. Results and Discussion
observe phenomena present in the tissue samples:
3.1 Osteoporosis—Biomineralogy Phenomena Binocular digital microscope (Lipzig—made in
Germany) was used for preliminary observation of Densitometry, usually conducted in vivo on an samples prepared for each examination;
entire bone (e.g. forearm), is one of the methods of Biological and polarizing light microscopy (various
observation of bone structure degeneration. Thorough microscopes—Car Zeiss, Palladium, AZ50, and densitometry examination on osteoporosis was others). Biological samples were prepared classically
conducted on one-centimeter thick slices of femur and colored using Alizarin and other pigments. Grains
heads that had been acquired during a total hip of minerals discovered in tissues were prepared using
alloplasty. The examination shows that there are classic methods typical for preparation of thin sections
different degrees of osteoporosis visible in different of rocks and minerals (cut, polished with various
slices (example: Fig. 1a, 1b [48]). In the spongy bone, powders, etc.);
there are areas of severe demineralization and SEM-Jeol 540, 560 (made in Japan) and FEI
trabeculae degeneration, as well as areas with hardly Quanta 200 (made in USA) microscopes combined
any visible evidence of osteoporosis (Fig. 1c). with EDS analyzer (biological samples prepared using
Detailed SEM examination in combination with vacuum dry techniques) were used for observation of
chemical EDS analysis prove that the osteoporosis tissue mineralization as well as determination of their
effect observed as trabeculae degeneration consists not chemical composition;
only of dissolution of the bone apatite, but also X-ray examination (diffraction) was conducted in
atrophy of the collagen strands mineralized with order to observe crystalline phases present in various
apatite (Fig. 2).
tissues. DRON 2, 5 diffractometer (made in Russia) Therefore, osteoporosis involves releasing not only
4 ions and their derivatives from bone, computer program was used for interpretation of
and Philips PW 3020 X'PERT were used. X-rayan 3- Ca and PO
but also the derivatives of collagen disintegration [1, obtained x-ray d hkl data;
22]. That indicates that to counter osteoporosis, it is ASA method was used to determine the presence of
not enough to supply the body with ions needed for trace elements in mineralized tissues. Philips PU
apatite crystallization, but it is necessary to create 9100Xi Camera SX-100 spectrophotometer and collagen with crystallization centers, where the apatite
atomic emission spectroscopy with inductively can crystallize. coupled plasma (ICP AES) using spectrometer 40
It is interesting to note that classic osteoporosis is PLASMA were both used;
observed almost exclusively in fully formed bones IR spectrophotometry method was used for with complete mineralization. That indicates that
Osteoporosis as A Source of Tissue Mineralization
Research on Osteoporosis Therapy and Dissolution of Arterial Mineralization
(a) (b)
(c)
Fig. 1 Densitometry pictures of slices of a bone head removed during a total hip alloplasty. b: Computerized model of osteoporosis—affected areas in slices shown in 1a. c: Advanced osteoporosis in spongy bone of the head of the femur. 2 × magnification.
physicochemical mechanisms resulting in degeneration apatite mineralizing collagen strands in programmed (osteoporosis) develop only in fully formed and
crystallization centers [52, 53].
mineralized bones. In a fully mineralized bone (where mineralization The process resulting in full bone development
of bone collagen has led to the increase of bone apatite causes a consistent increase in the amount of bone
content), bone cells and biological elements are not as
Osteoporosis as A Source of Tissue Mineralization
Research on Osteoporosis Therapy and Dissolution of Arterial Mineralization
Fig. 2 Area of developing osteoporosis in a trabecula of spongy bone. Visible areas of thinned and spaced, demineralized collagen strands. SEM. 2000 × magnification.
easily permeable for bone fluids as in a bone that isn’t understand the process of that exchange. Both fully developed [34, 29]. It means that in fully
nutrients and oxygen are transported from the arterial developed and mineralized bones, the exchange of
system to bone fluids and next to cells. On the other substances between bone cells and circulatory system
side cell metabolites are transported to the venous is much more difficult than it is in earlier stages, when
system. Migration in both directions occurs through bones are not fully mineralized and are more
the concentration equalization process. permeable for bone fluids.
Due to constant supply through the circulatory Therefore, in fully developed and mineralized
system, the concentration of nutrients and oxygen near bones both nourishing of bone cells by the circulatory
the micro-arteries in Haversian canals is high. Moving system and removing the products of their metabolism
away from these arteries towards bone cells, their into the micro blood vessels in bones are more
concentration decreases. It is caused by continuous difficult because of full mineralization of collagen at
consumption of those substances in the bone cells’ life bone trabeculae.
processes. This way, from arteries to cells, a nutrient Difficulties in nutrient transport don’t appear to
and oxygen concentration gradient occurs, which have important biochemical or biomineralogical causes these elements to be transported from arteries consequences for bone environment. However, to bone cells (Fig. 3a). difficulties in releasing bone cell metabolites change
Bone cells, while processing nutrients and oxygen, the collagen-mineral bone environment in trabeculae.
produce mostly carbon dioxide, water and a few other To understand the effect of exchange of elements
compounds. This results in increased concentration between circulatory system and bone cells, we need to
outside active cells of mainly carbon dioxide, but also
Osteoporosis as A Source of Tissue Mineralization
Research on Osteoporosis Therapy and Dissolution of Arterial Mineralization
other compounds, acid residues and free protons and others around cells, and their low concentration created in processing of carbohydrates, sugars etc.
around bone micro-veins (where cell metabolites are
absorbed into the venous system) result in creating have a vital impact on the environment surrounding
These metabolites, especially CO 2 and protons,
a gradient and consequently, in the transport of the cells, by lowering local pH and causing its acidity.
bone cell metabolites from these cells to micro-veins
The acidity is a result of reactions between CO 2 and
(Fig. 3b).
water from bone fluids, in particular of the creation of Durable as it is, bone apatite does not dissolve even
in a weakly alkaline environment. It does, however, Due to a high concentration of cell metabolites in
carbonic acid that easily dissociates into H + and CO 2- 3 .
dissolve in a weakly acidic one (pH < 6.6) (Fig. 4). trabeculae, a concentration gradient occurs, causing
The environment around bone cells is acidic because their transport. High concentrations of CO 2, H 2 CO 3 of large amounts of metabolic CO 2, H 2 CO 3 , as well as
(a)
(b)
Fig. 3 a: Diagram of nutrients migration in a bone, from arterial blood to bone cells in accordance with the concentration
equalizing process: d 1 —highest concentration of substances, d 3 —lowest concentration of substances. Arrows show the
direction of nutrient migration. b: Diagram of bone cells metabolites from the area of said cells to veins, in accordance with the concentration equalizing process: γ 1 —Highest concentration of substances, γ 3 —Lowest concentration of substances. Arrows show the direction of metabolite migration.
Osteoporosis as A Source of Tissue Mineralization
Research on Osteoporosis Therapy and Dissolution of Arterial Mineralization
Fig. 4 Upper diagram shows fields of stability and dissolution of bone apatite depending on the pH of the environment. Lower diagram shows average distance of bone cells from Haversian canals depending on age and degree of bone mineralization.
free protons present. That acidity destabilizes and It is important to note that the transport of bone cell dissolves the apatite crystallized in collagen strands.
metabolites to venous blood is slower after the bones Ions released from bone apatite in the process are
have fully matured, in comparison to earlier period, absorbed into fluids around bone cells. From due to the fact that migration of these products those fluids, through concentration gradient through the trabeculae areas filled with mineralized equalization, they migrate with other cell metabolites
apatite is more difficult. This results in stabilization of to the venous blood (Fig. 3b). The occurrence of
the lowered pH, strengthening the conditions that are this phenomenon had been confirmed through favorable for apatite dissolution. Furthermore, as experiments [54-56].
collagen demineralization and trabeculae degeneration
Osteoporosis as A Source of Tissue Mineralization
Research on Osteoporosis Therapy and Dissolution of Arterial Mineralization
progress, due to increased permeability of already them to “catch” wandering phosphate, calcium and degraded trabeculae, the process of osteoporosis
other ions from blood and bodily fluids. Ions that are speeds up. Therefore in the first phase of the
electrically charged are bound in crystallization osteoporosis process, the degree of bone destruction
centers with (also electrically charged) atoms that and tissue mineralization is small, but as the body gets
build the tissue. Crystallization centers may have older, osteoporosis and mineralization (calcification of
different origins [21, 22, 24, 27, 49, 57-61]. Research tissue) speed up.
conducted by the author allow for preliminary As the apatite dissolves and crystallization centers
classification of crystallization centers occurring in in bone collagen are destroyed, degeneration starts to
human tissues.
affect the collagen strands themselves. This effect is
3.3 Genetic (preliminary) centers observed generally during research with different
methods as trabeculae atrophy, or osteoporosis (Fig. 2). These centers are defective spots in various tissue Ions released in the process from bone apatite,
structures, where electrical charges, i.e. free ionic along with bone cell metabolites, after being released
bonds, occur. Their occurrence in tissues is a result of to venous blood vessels are transported to lungs and
a genetic error replicated in subsequent generations’ other organs. In lungs, during the gas exchange of
bodies.
CO 2 to O 2 , blood pH changes from acidic to alkaline.
3.4 Mechanically induced (secondary) centers It is caused by the release (in the form of carbonic
acid) of CO 2 which makes venous blood acidic. Places in tissue where destruction of biological Substitution of carbon dioxide with oxygen creates
structures occurred due to excessive exploitation – favorable conditions for phosphate crystallization,
physical effort. They are areas where interatomic including apatite crystallization, in the oxidized
bonds in biological structures got broken. arterial blood (with pH slightly over 7). As a result,
Centers created due to mechanical influence of lungs are one of the first organs at risk of
external substances entering the body (Fig. 5). A mineralization (calcification) [4].
factor in creation of such centers may be mineral or For the apatite crystallization to occur in tissue, two
synthetic particles entering the body with air, food and conditions must co-exist. There must be crystallization
fluids. Irritating tissue mechanically, they can cause centers present, and phosphate and calcium ions
its damage, resulting in creation of electrically available. These ions occur in blood. Some of them
charged spots.
may come from bones that are affected by
3.5 Chemically induced (secondary) centers osteoporosis, while some may come from diet. It
means that the “material” for calcification is present in It is caused by chemicals that enter the body with arterial blood and may crystallize in pH above 7.
air, food and fluids. These chemicals include strictly Crystallization may however occur only if there are
industrial substances as well as some other substances crystallization-favorable spots. These are called like food preservatives, stabilizers, pesticides, etc. crystallization centers and they areas where the
Some substances like alcohol, certain beauty products, concentration of ions mineralizing the tissues begins.
etc. can also be linked to tissue damage, including creation of crystallization centers. Such substances
3.2 Crystallization centers affect the tissues, damaging them and causing the
Crystallization centers are spots in different kinds creation of spots where subsequently crystallization of tissue where an electrical field occurs, allowing
and mineralization may set it.
Osteoporosis as A Source of Tissue Mineralization
Research on Osteoporosis Therapy and Dissolution of Arterial Mineralization
(a)
(b)
Fig. 5 Example of a secondary crystallization center created mechanically as a result of destruction of polypeptide structure by a mineral particle (in this case, an asbestos fiber).
a: Polypeptide structure before being destroyed by the asbestos fiber. b: Polypeptide structure after its interatomic bonds had been destroyed by the stuck fiber of actinolite asbestos. Arrows show the
resulting, electrically charged crystallization centers where apatite and other substances, e.g. cholesterol, can crystallize.
Osteoporosis as A Source of Tissue Mineralization
Research on Osteoporosis Therapy and Dissolution of Arterial Mineralization
It can also be caused by infections. Bacteria, viruses, of cations and anions. There are no grains or crystals fungi and other microorganisms infecting human
present in tissues at this stage. It is, however, bodies produce toxins, which can not only cause
noticeable in the results from chemical analysis as symptoms of infection, but also destroy tissue. Areas
elevated levels of certain elements. Such where toxins have destroyed tissue turn into mineralization may stop on the latent level or, due to crystallization centers due to the presence of evolution and further crystallization process, it may electrically charged, broken interatomic bonds.
turn into apparent mineralization. Latent Each of the above crystallization centers may occur
mineralization doesn’t cause significant changes in the sporadically or very often. There may also be just one
macroscopic look of the affected tissue. However, due type of center present, e.g. genetic, or all types may
to the presence of ions in the biological structures (in occur in the tissue at the same time. There may be
crystallization centers), it affects tissue qualities on many centers of one type and a few of another.
different levels. For instance, joint cartilage affected Each of the fore mentioned types of crystallization
by the latent mineralization will be a little harder, less centers that is electrically charged may become
flexible and more susceptible to wear and deformations. mineralized. The level of resulting tissue mineralization
Its effects have been observed, for instance, in depends on the size of the center, i.e. the amount of
cartilage and functioning of hip joints (Fig. 6). bonds that can be formed with the ions passing by.
3.8 Apparent Mineralization
Based on the above description, it is easy to guess which organs and tissues will be preferred in
This is the next, more advanced stage of tissue developing mineralization. For some people, it will be
mineralization. It occurs due to further mineralization coronary arteries (genetic centers), for others, lungs
of places affected by the latent mineralization. It is (after past infections). However, there will also be
visible in tissue and organs as various types of grains people for whom mineralization (e.g. calcification)
and crystals, not exclusively phosphate but also will develop simultaneously or gradually in many
cholesterol, carbonic and others. It hinders or even places and many types of crystallization centers.
prevents both tissue and the whole affected organ from proper functioning. It can develop in various tissues and
3.6 Mineralization of crystallization centers (tissues) organs. As a continuation of latent mineralization, it
As mentioned above, there are two elements may manifest by presence of grains and crystals of necessary for the tissue mineralization to happen: the
various sizes and kinds. It can develop on endothelium mineralizing substance and a crystallization center.
in arteries (Fig. a, b), on cardiac valves, and in many Tissue mineralization will not occur when there are
other organs [4]. Phosphate mineralization has even crystallization centers but no mineralizing substance.
been observed on arterial heart valve prostheses (Fig. There will also be no mineralization when we have
8) and on the pacemaker electrode endings. plenty of ions that could mineralize tissue, but no
Phosphate, cholesterol and mixed crystallization centers. Several stages of tissue cholesterol-phosphate mineralization, as well as other mineralization outside of bone structure have been
types, may also occur under aortic endothelium. In observed.
such cases, it grows in the form of crystals or granular concentrations (Fig. 9).
3.7 Latent mineralization The problem of secondary mineralization of tissue
This is an early mineralization process where connected with the Ca 2+ and PO 3- 4 ions released from crystallization centers are filled with a small amount
bones in the osteoporosis process may involve many
Osteoporosis as A Source of Tissue Mineralization
Research on Osteoporosis Therapy and Dissolution of Arterial Mineralization
Fig. 6 Latent mineralization. Head of femur removed during a total hip alloplasty. Cartilage covering the head of the femur affected by latent mineralization (elevated amounts of Ca, P and others) had been worn down to the bone. The arrow shows where the remainder of mineralized cartilage meets the worn down to the bone area.
(a) (b)
Fig. 7 Apparent mineralization. a: Phosphate (apatite) crystals in a crystallization center on the endothelium surface in the cartoid. b: Phosphate grains and cholesterol plaque building up on a damaged surface of an aortic valve leaflet. SEM. Magnification according to the scale.
organs and deepen with age, i.e. with progressing
various substances in aortas.
osteoporosis. It is a cause of many irregularities in Research on tumor mineralization [46, 50, 51] organ functioning and serious health problems, suggests that it may probably favor the cancer growth including aortic hypertension due to crystallization of
processes.
Osteoporosis as A Source of Tissue Mineralization
Research on Osteoporosis Therapy and Dissolution of Arterial Mineralization
Fig. 8 Apparent mineralization. Artificial aortic valve, removed. Visible phosphate clusters on the valve leaflets.
Fig. 9 Grainy cholesterol mineralization with added calcium phosphate in a cartoid wall. Arrows show grains of cholesterol containing Ca and P. Histological preparation stained with hematoxylin. 120 × magnification.
Osteoporosis as A Source of Tissue Mineralization
Research on Osteoporosis Therapy and Dissolution of Arterial Mineralization
3.9 Crystallization Mechanism to find a way to reduce or completely remove it. Research on dissolving the substances that
Crystallization of various substances that mineralize mineralize arteries in vitro had been conducted in a set
tissue begins from unsaturated solution, i.e. blood shown on Fig. 10. In the spot marked as # 3 (Fig. 10, a
containing ions. Therefore crystallization in the piece of an aorta was placed, affected by a strong
crystallization centers doesn’t happen due to cholesterol mineralization visible as cholesterol
exceeding the solubility equilibrium of the compounds deposits on the intima surface as well as underneath it
that mineralize the tissue. The mechanism that causes
[38, 39-41].
ion migration towards crystallization centers and Beaker with “solvent”, 2—peristaltic pump,
creation of not visible and then visible mineralization is lack of physicochemical balance between the
3—fragment of a mineralized aorta (or a tube filled chemical composition of arterial blood and the place
with synthetic calcium hydroxyapatite), 4—beaker for (center) of mineralization. Due to this phenomenon,
collecting the “solvent” after being run through the particularly the presence of calcium and phosphorus
aorta (or tube) shown in point 3.
ions in blood as well as other electrically charged Various media were run through the aorta, sucked particles, those ions are caught by the crystallization
in by a peristaltic pump (Fig. 10, 2) from a beaker (Fig. centers. Thus, despite low ion saturation, tissue
10, 1). Water and alcohol extracts of various plants mineralization is possible.
were used for dissolving the deposits. After running these fluids through the aorta, they were collected in
3.10 Dissolving Arterial Mineralization the beaker (Fig. 10, 4) and evaporated.
It would be the most beneficial if osteoporosis or In this experiment, after complete evaporation of soft tissue mineralization could be prevented certain solvents, little crystals of cholesterol had altogether. However, if it already exists, it is essential
formed. Their size didn’t exceed 1 mm (Fig. 11a).
Fig. 10 Diagram of the set used in in vitro experiments on dissolution of mineralization in human aortas.
Osteoporosis as A Source of Tissue Mineralization
Research on Osteoporosis Therapy and Dissolution of Arterial Mineralization
(a) (b)
Fig. 11 Cholesterol crystal (about 1 mm tall) grown from the “solvent” run through an aorta mineralized with cholesterol (Fig. 10). b: Dissolved surface of a grain of synthetic apatite placed in a polyester tube (Fig. 10, point 3) and subjected to dissolution. SEM. magnification according to scale.
Another type of experiment involved dissolving tissue, and its resulting mineralization, slim people phosphates crystallized in aortas. Due to the difficulty
will be more at risk.
of acquiring aortas mineralized with phosphates
A separate problem is working on a technique of (particularly apatite), the experiments used strengthening bones through elimination of hydroxyapatite synthesized according to the recipe
osteoporosis. To reconstruct bones, particularly given in [61].
taberculae, we need collagen with crystallization Grains of synthetic apatite crumbles had been
centers and a substance from which apatite can analyzed with SEM before and after an attempt to
crystallize (phosphatase alkaline). It is only with these dissolve them. The “solvent” was also analyzed before
elements present that bone structure may be rebuilt and after being run through a tube filled with
and significantly strengthened. Administering calcium crumbled particles of carbonated hydroxyapatite.
and phosphor compounds without a collagen matrix Observations of the morphology of apatite particles
(in the bones) may result in those elements being built subjected to in vitro dissolution attempts indicate that
into centers in tissues outside of bone system. It may despite its poor solubility, carbonated hydroxyapatite
lead to various new health problems. does dissolve in certain “solvents” (Fig. 11b).
4. Conclusions
3.11 Preventing Osteoporosis Acquired results suggest that there is a link between
Research shows that weightless environment, such tissue and organ mineralization and bone as the one that astronauts live in, creates favorable
demineralization observed as osteoporosis. The link is conditions for development of osteoporosis. Therefore
confirmed by the age-related connection in humans it seems that a heavier load on the bone structure may
between progressing osteoporosis and progressing slow down the osteoporosis process. It means that in
tissue calcification.
people of larger weight the process of osteoporosis Therefore eliminating or slowing down the process may develop slower than in those who are slim.
of osteoporosis will result not only in better Furthermore, taking into consideration the functioning of the bone structure in old age, but it will osteoporosis-related ion transfer from bones to soft
reduce the transfer of elements from bones to
Osteoporosis as A Source of Tissue Mineralization
Research on Osteoporosis Therapy and Dissolution of Arterial Mineralization
mineralized tissues and organs. Human Tissues. Mineralization of Heart Structures].” Przegl. Lekarski 52 (4): 24-27.
It’s essential that we act to reduce the amount of [7] Pawlikowski, M. 2003. “Minerals in Human Blood
crystallization centers where mineralization may Vessels and their Dissolution in Vitro.” In Health edited develop while reducing the amounts of various
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Further research should focus on experiments
Osteoporoza. Osteoprint.
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Acknowledgment
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July 2014, Vol. 8, No. 7, pp. 626-631
Journal of Life Sciences, ISSN 1934-7391, USA
DAVID PUBLISHING
Economical Analysis of Milk Production in the Farms of Siedlce Region
Sosnowski Jacek, Jankowski Kazimierz, Truba Milena, Kaczorek Anna, Król Justyna, Roma ńczuk Tadeusz and Kolczarek Roman Grassland Department, University of Podlasie, Siedlc08-110, Poland
Received: June 05, 2014 / Accepted: July 21, 2014 / Published: July 31, 2014.
Abstract: Last years the most profitable line of activity in agriculture is considered with milk production. The-effectiveness of this project depends primarily on the direct costs and the obtained of purchase price. Therefore the aim of this work was to analyze the financial results of dairy farms in the east part of Mazovia region. Completing the research in 2010, to the owners of 36 farms were sent a questionnaire containing 18 questions. The obtained data were used to characterize the various research facilities. And then the whole population was devoted on the 6 production groups, depend on the number of physical units of dairy cows. Moreover, based on survey data, an analysis of the profitability of milk production was done. It was the following economic values: direct costs, revenue, gross margin and the index of production profitability. Also the statistical analysis by calculating the Pearson linear
correlation coefficient (r) and coefficient of determination (R 2 ) was done. The study showed that the highest annual yield of milk from a cow at the level of 7500 kg, reached the farmers with the area of 19.7 ha of with the density of 1.82 BFU/ha and with 8 years of cow utilization. Respondents from this group also received the highest value of effectiveness index (184.60%). In addition, the performed regression analysis showed significant positive correlation between the effectiveness index and the surface agricultural lands occurring in the farms, density of agricultural lands, and efficiency from a cow and the price of milk.
Key words: Milk production, effectiveness index, direct costs, economical analysis.
1. Introduction main factors affecting unit costs, while maintaining unchangeable other production parameters.
According to many experts, one of the most On the profitability of this activity also has a profitable lines of agricultural production is milk. decisive influence also has sales price, which largely According to Sowula-Skrzy ńska et al. [1] the depends on the quality and quantity of milk sold from economics of milk production as well as any other the farm. An important in this regard is the financial product, is dependent on the costs and selling prices of condition of the purchaser. Profitability of milk the raw material. Since 2004, the observed production also favors the concentration of dairy improvement in the profitability of milk production in farms, and thus the development of the dairy industry relation to the production of pig or sheep is observed.
and related areas [3].
It’s also noted a significant improvement in the In Poland, milk production has the largest share in economic situation of the whole dairy industry. As commercial agricultural production, and the dairy showed study Okularczyk [2], in this period gained sector (production and processing) involves 25% of most farmers holding a cow with a capacity greater the industry workforce and produces 17% of the total than 6000 liters of milk per year. It should be noted production of the agricultural industry [2]. Therefore, that the scale of production and cow milk yield are the Okularczyk and Sowula-Skrzy ńska [4] passed the
milk market to the basic agri-food markets in the Corresponding author : Sosnowski Jacek, Ph.D., lecturer,
research field: grassland science. E-mail: laki@uph.edu.pl. country. In addition, it is worth noting that in 2008 the
Economical Analysis of Milk Production in the Farms of Siedlce Region
European markets went up 73.6% of Polish exports of On the basis of questionnaire data, an analysis dairy products [5]. You can, therefore, assume that the
taking into account the profitability of milk production Polish dairy sector is gaining importance in the
following the economic size was done: western markets.
(1) Direct costs:
Therefore, the aim of this study was to analyze the
The cost of hard renewing,
financial results of farms producing milk, taking into The cost of feed: concentrated feed from account the production factors, occurring within
purchase, own concentrated feed, own fodder forage, households and market conditions affecting the
own hay meadow, own corn silage, a mineral mixture, profitability of the business.
The costs of specialist: specialist services of livestock production,
2. Materials and Methods
Other costs: veterinary care, artificial Research by direct interview was conducted in 2010
insemination, means of washing—disinfectants. in 36 farms operating in the eastern Mazovia. The
(2) The value of production (excluding subsidies): main criterion for selection of research sample was
Main production—milk,
conducting of milk production and the location of
Calves at the age of 2 weeks,
farms in the following six counties: Soko łów, Siedlce,
Sorted out cows,
W ęgrów, Łosice, Garwolin and Minsk. To owners a
Sorted out cattle.
questionnaire containing 18 questions was sent. The (3) Gross margin (calculated as the difference obtained data were used to characterize the various
between the value of production and direct costs). research facilities, resulting in a breakdown of the
(4) Indicator of profitability—the value of analyzed population into 6 groups depending on the
cost-effectiveness ratio was calculated for each farm number of physical units of dairy cows: group A: 10
and are presented as average for production group, cows, B: from 11 to 15, C: from 16 to 20, D: from 21
according to the following formula [6]: to 25, E: 26 to 30, F: over 30 cows.
Wo = (Wp:Wk) × 100%
As the data in Table 1 showed, most of the studied
Where:
farms were in the county Sokolow, Siedlce and Łosice Wo—value of profitability index, (23 from 36 farms), and the lowest in Minsk district,
Wp—value of commodity production, because only 3 farm. Moreover, in study population
Wk—value of direct costs.
was dominated the group A having the 10 units of In addition, statistical analysis was performed by dairy cows. In the other production groups were
calculating the Pearson correlation coefficient (r) and analyzed from 5 to 6 dairy farms.
the coefficient of determination (R 2 ) between the
Table 1 Localization and number of studied farms in individual production groups.
Production groups
District Total AB C D E F
Soko łów 3 2 --- 2 --- 18 Siedlce --- 1 2 2 3 --- 8 W ęgrów 2 1 1 --- --- 1 5 Łosice --- 2 2 --- 127
Garwolin 2 --- 1 1 --- 1 5 Mi ńsk Maz 1 --- --- 1 1 --- 3 Total 8 6 6 6 5 5 36
Source: own research
Economical Analysis of Milk Production in the Farms of Siedlce Region
profitability index and the following variables: density solutions in the course of its production. Generally of cows (GUP)/ha of agricultural lands (AL), AL area
they are characterized by easer of networking, greater in hectares, an average annual milk yield of cow in 1
courage in making investment decisions and intense kg, the price of 1 liter of milk in PZL.
absorption of EU funds. The tested farms in all production groups were conducted mostly by people
3. Results and Discussion
with secondary education (Fig. 2). Only in a few cases, Human resources of farm play an important role,
farmers undertook these activities possess a higher both in making production decisions and for the
education.
development of non-agricultural commercial ventures, An important measure of the production quality in especially those based on interpersonal relations.
dairy farms is the yield of one cow [5]. In the Many authors [7, 8] emphasizes that a very important
analyzed population occurred very large variation in element influencing the decision to undertake a
this respect (Table 2). The obtained data show that in particular activity and how to conduct it, is the age of
farms with a higher number of cows milk yield was the farm owners. Results of this study indicate that
up to twice higher than on farms up to 10 numbers of these activities take mainly people in age 30 to 40
dairy cows. The highest value of this parameter (7500 years (Fig. 1), who are characterized by openness to
kg milk per year from 1 cow) was achieved in farms change and innovation.
with an average 28 numbers of dairy cows (group E), The second group connected with age was people
used for a period of 8 years, with an average of 19 ha between the fiftieth and sixtieth year of life. It is worth
of arable land and about 14 ha of meadows and noting that only in two districts of the analyzed region,
pastures. The obtained result was much higher than worked in the farm owners not more than thirty years
an average for the Poland, which in the years of age.
2007/2008 was 4518 kg of milk per year for cow Another important feature influencing the quality of
(Communication of the Commission of the Europe the management is education of farmers. In the
Council, Eurostat, Brussels 2009). Noteworthy is the literature [7, 9], it is noted that those with secondary
fact that an average herd size in the studied farms was education or higher more interested in expertise and
higher about 15, 56 physical units than the national are more open to implementing new technology
average in 2008 [5].
20% 12.5 16.6 33.2 20 20 0% A B C D E F
to 30 years
from 30 to 40
from 40 to 50
from 50 to 60
over 60 years
Fig. 1 The structure of farms owners age in individual production groups (mean for farms).
Source: own research
Economical Analysis of Milk Production in the Farms of Siedlce Region
Fig. 2 The structure of farmer owners education in individual production groups (mean from farms).
Source: own research
Table 2 The basic values characterized studied farms by production groups.
Productional groups
Specification Unit ABCDE F
Data about farm largeness (mean from farms) Surface of agricultural lands
ha 10.3 9.8 18 29.5 19.7 40.2 Grassland surface
22 14.25 20.35 Data about largeness of milk production (mean from farms) Cows number per one farm
ha 7.5 4.8 10.5
6 12 18 24 28 33 Mean field from cows
Phizical no
3600 3400 4500 40 7500 6500 Years of cows utilization
kg/year
8 10 9 8 8 7 Milk cows density
Lear
0.79 0.68 0.73 0.8 1.82 0.82 Utilization of production (mean from farms) Quantity of selled milk
DJP/ha UR
20000 24000 50000 70000 140000 135000 Quantity of for calves milk
1/year
980 3144 Mean utilization of milk at home 1/day 1 1 0,5 4 0,5 2
Source: own research
The study also showed a significant positive An average, in these farms has paid from 0.48 to correlation between the surface of agricultural land
0.51 $ per liter.
and density of dairy cows and profitability of milk Respondents explained that on this state largely production (Table 3). The correlation coefficient for
influenced achieved their sales volume and quality of this relationship was 0.624 and 0.758 respectively.
raw material, which allowed to negotiate prices with Furthermore, the data in Table 3 show that on the
customers. Price, in turn, according to the analysis of profitability of analyzed production largely influenced
the level of linear dependence between the purchase annual average milk yield of one cow. This feature, up
price and the profitability index of milk obtained in to 98% (R 2 = 98%) determined the variability of
the studied farms, in 43% significantly, positively profitability index achieved in this farms population.
affect on the financial results of business (Table 3). In addition, wide diversity had also the value of
Economic analysis of farms producing milk plays milk purchase prices. Highest unit prices were
an important role. It allows you to state how is the negotiated by farmers with more than 26 dairy
financial result in the business. It also provides
cows—Groups E and F (Fig. 3). valuable information about the size and structure of
Economical Analysis of Milk Production in the Farms of Siedlce Region
Table 3 Statistical analysis for relation of individual diagnostic features.
Relation
S yx S yx% index of milk production profitability x surface
R 2 y = ax ± b
of agricultural lands in studded farms
2.75 3.7 index of milk production profitability x milk cows density
0.39 y = 121.96 + 1.223x
1.86 2.4 index of milk production profitability x mean annual yield 0.989
on agricultural lands
0.59 y = 112.63 + 36.471x
1.29 1.6 index of milk production profitability x value of milk price 0.655
0.98 y = 87.694 + 0.1205x
0.43 y = 12.092 + 103.48x
r: correlation coefficient, R 2 : determination coefficient, S yx : standard deviation,
S yx% : relative estimate error Source: own research
ean price M 0.1
Production groups
Fig. 3 The mean value of milk price of purchase in individual farms by production group.
Source: own research
Table 4 Analyze of financial results in studied farms by production groups (mean from farms).
Index of milk production Production groups [$]
Value of commodity production (receipts)
Direct cost
Direct surplus
profitability% A 1355.53
1528.33 967.33 560.80 157.96 * The all values were given in accenting on one cow Source: own research
expenses [10, 11]. Financial analysis involves many direct surplus was calculated, which in this period subjects and is a complicated tool that requires
received individual farms.
advanced accounting. Farms the accounts does not The data in Table 4 shows that the value of usually lead, therefore, in these studies have been
commodity production within the analyzed groups limited to the calculation of direct costs and incomes
varied widely and ranged from 1520 $ (farms with associated with the milk production, obtained from the
group A) to 3978.3 $ (farms with group E). A similar direct interview. Based on the above economic values
tendency was also in the direct costs of analyzed
Economical Analysis of Milk Production in the Farms of Siedlce Region
631
production. Farms with fewer of cow numbers had the production (98%) and the quality of produced raw lowest costs in business (1158.2 $—Group A), while
material, which affected on the value of the purchase the number of farms with cows more than 25 units, the
prices.
value of direct costs were higher more than 433.3 $.
According to Skar żyńska et al. [12], the authoritative accounting in agricultural production is
References
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