Morphological Aspects of the Enteric Nervous System of the Stomach in Artificially Fed Lambs
Natalya Aleksandrovna Vechkanova*, Olga Sergeevna Bushukina, Alexander Sergeyevich Zenkin, Svetlana Anatolievna Lyapina, Natalya Alekseevna Mel'nikova, Nataliya Yurievna Kalyazina
National Research Mordovian State University Named after N. P. Ogarev, Bolshevistskaya Street, 68, 430005, Saransk, Russia
*Corresponding Author E-mail: vechkanova_na@mail.ru
ABSTRACT:
This paper reposts the results of the study of the structural features of the enteric nervous system of a multi-chambered stomach in lambs of the Edilbay breed, which were grown using artificial feeding with the use of a sheep milk substitute. Neuromorphological and morphometric methods of research were used. The paper also presents comparative data on the development of neuronal processes (axons and dendrites) during the early postnatal ontogenesis of ganglia of the rumen, reticulum, omasum, and abomasum in ruminants due to adaptive rearrangement under the influence of the feed and growing conditions. The obtained data provides a better understanding of the specific features of the development of intermuscular ganglia in the multi-chambered stomach and allows to analyze their adaptive rearrangements during artificial feeding of lambs in combination with the traditional way of housing and feeding. This will help to determine the mechanisms of progressive and regressive development, as well as the maximum implementation of the biological capabilities of the body of ruminants.
KEYWORDS: Enteral nervous system, intermuscular ganglia, neuronal processes, early postnatal ontogenesis, multi-chambered stomach, artificial feeding, Kalvo-Start.
INTRODUCTION:
Over the past decade, there has been an increase in the number of studies reporting on the increased sensitivity of the enteric nervous system to the effect of various extreme factors, including the restriction of the diet8,9. Results of these studies have allowed proposing the idea that nerve plexuses of the enteric nervous system are constantly restructuring. This is the basis of the neurotrophic effect of dendrites of intramural neurons on surrounding tissues10. In connection with this, the aim of this research was to study the effect of artificial feeding on the formation of neuronal processes in the ganglia of the enteric nervous system in the multi-chambered stomach.
MATERIALS AND METHODS:
Thirty-five lambs (males) of The Edilbay breed were used in the study, which was carried out on the basis of a peasant farm enterprise. All animals were divided into two groups – control and experimental. Newborn lambs of the control group were fed naturally by their mothers (or ewes). Lambs of the experimental group were artificially fed with Kalvo-Start sheep milk substitute (SMS) according to the standards recommended by the manufacturer “Dutch technologies in animal husbandry” (Russia). According to the aim of the study, the control and experimental animals were slaughtered at the following ages: newborns, 15th day, 2.5 months, 4.5 months (according to the following periods: newborn, milk-fed, transitional, period of adaptation to the definitive diet). The object of the study was the ganglia of the myenteric or intermuscular plexus in the walls of the rumen, reticulum, omasum, and abomasum. For morphological studies, pieces of tissue were taken from the walls of the ventral sac of the rumen, the greater curvature of the reticulum, the base of the omasum, and the fundus of the abomasum. We used an integrated approach, including histological and neuromorphological research methods. Cytoarchitecture of ganglia was studied using impregnated specimens. Treatment with silver nitrate according to the method described by Bilshovsky-Gross allows visualizing the neuronal bodies, glial cells, neuronal processes and synaptic terminals in the myenteric ganglia. Using an Aksioskop microscope, we measured (in 30 fields of view) linear parameters of the ganglia and the thickness of processes in 100 neurons. The relative growth, which shows the growth intensity of the measured object, was calculated using the formula:
В = (V1 – V0) ÷ (V1+V0): 2 х 100%
where В is Brodi coefficient, V0 is the starting point, and V1 is the endpoint11.
The resulting images were analyzed and processed using FIJI software (ImageJ, NIH). Statistical processing of the data was conducted using Microsoft Excel and Statistica. Evaluation of the statistical significance of the measurement results was performed using Student's t-test (p ≤ 0.05). The microscopic examination of the specimens was carried out using a Mictron-400 microscope (Petrolaser). The magnifications shown on the microphotographs correspond to the magnifications used when they were taken.
RESULTS AND DISCUSSION:
The stomach of the sheep is four-chambered and consists of the rumen, reticulum, omasum, and abomasum. Such a complex anatomical structure developed in the course of long-term development under the influence of environmental factors. In all parts of the stomach, the enteral nervous system is represented by two main plexuses – intermuscular and submucosal. Both plexuses are comprised of ganglia, which consist of neurons, nerve processes, and glial cells. Ganglia have heterogeneous structure in animals from birth to 4.5 months of age, due to the presence of nerve cells at different stages of morphogenesis. These are mature neurons, neuroblasts, and weakly-differentiated cells. During different age periods, the ratio of the nerve cells of different morphogenetic stages is determinative for the morphofunctional state of the enteric nervous system of the lamb stomach. A comparative histological analysis of the stomach wall demonstrated that the ganglia of intermuscular nerve plexus in lambs, that received Kalvo-Start SMS, and lambs naturally fed by ewes, have fundamentally similar structural attributes, as well as specific features caused by adaptive restructuring under the influence of the feed and growing conditions. In comparison with the control group, intermuscular ganglia of the rumen in lambs that received Kalvo-Start SMS reduced in length by 6.3% and in width by 19% during the period from birth to 15 days of age. However, from 2.5 to 4.5 months of age, ganglia morphogenesis was characterized by an increase in linear parameters – by 13% in length and by 6.8% in width (p ≤ 0.05). It is believed that the nervous system is one of the most vulnerable and, at the same time, one of the first to implement the mechanisms of adaptation12-14.
On the basis of neuromorphological and morphometric studies, we obtained data on the formation of the process apparatus in highly differentiated large nerve cells in stomach ganglia of sheep in connection with the means of feeding. During the newborn period, in the rumen ganglia, major neurons had a small number of processes, which either branched within the ganglion or extended beyond its borders as a part of the nerve tracts. Axons, unlike dendrites, lacked collaterals. The axon thickness was 1.92±0.012μm. In addition to the axon, up to 1.27± 0.106 of short dendrites can branch off of large cells. They branched dichotomously. At this age, the index of dendritic branching was 1.00±0.001. During the dairy period, in the rumen ganglia of 15-day-old lambs, the axon thickness differed in control (2.16±0.013μm) and experimental (2.26±0.068μm) animals. The study of the growth dynamics of the axons of large neurons in the rumen ganglia during postnatal ontogenesis revealed a period of increased diameter growth during the transitional period in the control group (B = 20%) and experimental group (B = 26%). At 4.5 months of age, we recorded a decrease in the diameter growth rate of the axons in the rumen ganglia in the experimental animals (B = 4%), compared to control (B = 11%). However, by the period of transition of animals to the definitive feed, the morphometric indicators of axon development in the rumen ganglia in the compared groups did not level-off. Artificial feeding of lambs contributes to the formation of more thick neuronal axons in the rumen ganglia (Fig. 1, 2). It is known that the surrounding tissue is involved in the induction of morphological characteristics of axons15.
|
Figure 1 – Rumen ganglion of a 4.5-month-old lamb. Experimental group. Bilshovsky-Gross. Magnification: x 200. |
Figure 2 – Rumen ganglion of a 4.5-month-old lamb. Control group. Bilshovsky-Gross. Magnification: x 400. |
The formation of dendritic processes in large nerve cells occurred gradually during postnatal ontogenesis. It was established that in the rumen ganglia, the maximum number of dendrites formed at the dairy stage, both in the experimental group (B = 176%) and in the control group (B = 136%) of animals. In lambs that were naturally fed, the relative increase in dendrite number was lower than in the group of animals that received Kalvo-Start SMS. Over the entire study period, the number of dendrites in large neurons in the rumen ganglia increased by 7.9 times – from 1.27±0.106 to 10.00±0.336 in the control animals and 9.4 times in artificially fed lambs. Artificial feeding led to an increase in the number of dendrites in the rumen ganglia neurons by 14% during the dairy stage, by 27.5% during the transition period, and by 16.6% during the transition to the definitive feed (p ≤ 0.05). It was established that the highest rate of formation of secondary branches in dendrites in the rumen of the control animals occurred during the transition stage (B= 100%). During the transition to the definitive feed, a decline occurs (B = 75%). The number of secondary branches in dendrites in the lambs fed by ewes increased from 1.00 ± 0.001 to 3.5 ± 0.115 during the period from the newborn to the transitional stage. The process of formation of secondary branches in neuronal dendrites in the rumen ganglia of lambs fed with Kalvo-Start SMS began earlier, at the dairy stage (B= 25%). In the experimental group, similar to the control, the maximum rate of secondary branching of dendrites occurred during the transition period (B= 114%), and then, by a 4.5-month age, the formation rate of secondary branches decreased (B= 60%). In the rumen ganglia of the artificially fed lambs, the number of secondary dendrite branches increased by 4.27 times. The comparative analysis demonstrated that artificial feeding leads to the formation of more robust dendritic processes, which have more secondary branches than the control – by 20% at the dairy stage, by 25% at the transitional stage, and by 18% during the transition to the definitive feed. A similar pattern was found in the dynamics of the development of the reticulum ganglia in both groups – they increased in width at the dairy stage. However, a decline in relative growth rates was recorded in the lambs, which received Kalvo-Start SMS. Therefore, in the animals of the experimental group, at 15 days and 2.5 months of age, the reticulum ganglia were, 17% and 22% smaller in width, and 7.3% and 28% smaller in length (p ≤ 0.05), respectively, than in the control. During the period of transition of animals to the definitive feed, the metric parameters of the reticulum ganglia in animals of the two groups did not have statistically significant differences, which was due to the high growth rate of ganglia at the age of 4.5 months in the artificially fed lambs. The asynchronous development of axons in large neurons was recorded in the reticulum ganglia of these animals, which happened as a result of the feeding method. In lambs fed by ewes, the axon thickness of nerve cells increased from 2.14±0.06 µm to 2.85±0.09 µm during the period from birth to 4.5 months of age. The relative increase in the axon thickness at the dairy stage in the reticulum ganglia of the control lambs was higher (B= 20%) than in the experimental animals (B= 9%). However, during the transition period, the axon growth rate in the agitator ganglia of lambs fed with Kalvo-Start SMS was two times higher. During the transition of animals to definitive feed, no significant differences in the morphometric parameters of the neuronal axons in the agitator ganglia were found between the groups (Fig. 3, 4). Therefore, artificial feeding leads to the formation of thinner neuronal axons in the reticulum ganglia only during the early stages of postnatal ontogenesis. The process of dendrite formation in neurons of the agitator ganglia of naturally fed lambs was characterized by the maximum growth rate at the dairy stage (B = 85%), after which, during the period from 2.5 to 4.5 months of age, the growth rate of dendrites decreased. By the age of 4.5 months, the number of neuronal dendrites in the reticulum ganglia in lambs of the control group increased by 3.5 times – to 9.00±0.303. The effect of artificial feeding on the process of dendritic formation lead to outrunning growth rate of their number (compared to control) only at the dairy stage, and then, during the period from 2.5 to 4.5 months of age, the rate of dendrite formation decreased. However, by the period of transition of experimental animals to the definitive feed, the growth rates of dendrite formation in large neurons of the reticulum ganglia returned to the control level.
|
Figure 3 – Reticulum ganglion of a 4.5-month-old lamb. Control group. Bilshovsky-Gross. Magnification: x 640. |
Figure 4 – Reticulum ganglion of a 4.5-month-old lamb. Experimental group. Bilshovsky-Gross. Magnification: x 160. |
The maximum rate of branching of the dendritic neuronal processes in the reticulum ganglia was recorded in naturally fed lambs at 2.5 months of age (B= 53%) and at the dairy stage in animals that received Kalvo-Start SMS (B = 40%). During the period from 2.5 to 4.5 months of age, there was a decline in the rate of formation of secondary branches in the dendritic processes. The number of secondary branches of dendrites of large neurons in the reticulum ganglia in the control lambs increased 1.7 times – from 1.78±0.22 to 3.00±0.152, during the period from birth to 4.5 months of age. There was a partial loss (or reduction) of the secondary branches of the dendrites in artificially fed lambs during the transition to the definitive feed. It is known that this generally happens due to restriction of the afferent inflow from the organ tissues to the ganglial structures. The dynamics of the omasum ganglia development were characterized by noticeable differences in the linear parameters in the lambs depending on their feeding type. In artificially fed animals, at the dairy stage, the growth of the omasum ganglia was two times faster in width and four times faster in length than in the control lambs. Later, during the transitional period and during the transition to definitive feed, the growth rate of the omasum ganglia, especially the rate of growth in length, decreased in the experimental animals. Consequently, at the age of 15 days, omasum ganglia in the artificially fed lambs were 18% wider and 20% longer than in the naturally fed animals. At 2.5 months of age, the omasum ganglia in the experimental lambs were 9% wider and 12% longer (p ≤ 0.05) than in the control animals. However, at the age of 4.5 months, omasum ganglia in the experimental lambs decreased in length by 1.4 times and there were no statistically significant differences in width. It was established that the development of the neuronal process apparatus in omasum ganglia differed depending on the feeding type. It was found that during the period from birth to 4.5 months of age, in the omasum ganglia of lambs fed with Kalvo-Start SMS, the thickness of neuronal axons increased by 1.5 times – from 1.84± 0.012μm to 2.77±0.005μm. The relative diameter growth rate of axons of the omasum ganglia neurons at the dairy stage was higher in the experimental animals (B= 24%), compared to the control lambs (B= 18%). In experimental lambs, the diameter growth rate of axons decreased at the transition stage and stopped during the period from 2.5 to 4.5 months of age, unlike the lambs fed by ewes (Fig. 5,6). The process of dendritic formation in the omasum ganglia occurred before the transition to definitive feed. At 15 days of age, the number of dendrites in the omasum ganglia neurons was 14.8% higher (p ≤ 0.05) in artificially fed animals compared to the naturally fed lambs. The number of neuronal dendrites in the omasum ganglia in the experimental lambs at 4.5 months of age was 9.5% higher than in the control animals. At the dairy stage, the relative increase in the number of secondary branches of dendrites in the omasum ganglia of artificially fed lambs was B = 50%. Comparative analysis showed that the branching of the neuronal dendrites in the omasum ganglia was more intensive in the control than in the experimental group only at the dairy stage (B = 60%). In experimental lambs, the formation of secondary branches of dendrites was the most intensive (B = 67%) at 2.5 months of age. Later, at 4.5 months of age, the rate of dendritic branching decreased. We found that the number of secondary neuronal dendrites in the omasum ganglia of lambs fed with Kalvo-Start SMS was 6% higher at the age of 15 days, and 5% higher at the ages of 2.5 and 4.5 months, compared to the control group. It is well known that dendrites increase the cell surface and, consequently, the surface that receives the impulses16.
|
|
|
|
Figure 5 – Omasum ganglion of a 4.5-month-old lamb. Experimental group. Bilshovsky-Gross. Magnification: x 200. |
Figure 6 – Omasum ganglion of a 4.5-month-old lamb. Control group. Bilshovsky-Gross. Magnification: x 200. |
From birth to 4.5 months of age, artificial feeding of lambs has an adverse effect on the morphogenesis of the abomasum ganglia. The linear dimensions, both length and width, of the abomasum ganglia decreased: by 13% at the age of 15 days, by 8% at the age of 2.5 months, by 22% at the age of 4.5 months (p ≤ 0.05). During the transition to the definitive feed, the abomasum ganglia were 1.27 times smaller in the artificially fed animals than in the control (p ≤ 0.05). In lambs that were naturally fed, dendritic processes of neurons in the abomasum ganglia developed more intensively than axonal processes during the period of early postnatal ontogenesis. As noted in a number of studies, the neuro-reflex mechanism is activated by the stimulation of the mucosal peripheral receptors by chemical substances17, 18. In the experimental animals, the thickness of large nerve cells axons increased from 2.54±0.012μm to 2.80 ±0.007μm, the number of dendrites increased from 2.40 ±0.123 to 8.00±0.439, and the number of their secondary branches from 1.33±0.333 to 2.50±0.177 during the period from birth to 4.5 months of age. During the dairy and transitional stages, artificial feeding slowed the diameter growth of the axons in the abomasum ganglia. However, at the dairy stage, the relative growth of dendritic processes of large neurons (according to the Brodi coefficient) was higher in the experimental group (B = 92%) than in the control group (B = 67%). During the transitional period, by the age of 2.5 months, the growth potential of the dendritic tree in abomasum ganglia neurons in artificially fed lambs was reduced. During the period of transition of animals to the definitive feed, the relative dendritic growth rate became 14.9% higher than the control level (p ≤ 0.05). The comparative analysis demonstrated that artificial feeding at 2.5 and 4.5 months of age contributed to the partial loss of secondary dendritic branches in large neurons of the abomasum ganglia (B = -3%) (Fig. 7,8). Partial reduction of the dendritic tree is considered as one of the factors limiting afferent inflow from the organ tissues to the ganglia19.
|
Figure 7 – Abomasum ganglion of a 4.5-month-old lamb. Experimental group. Bilshovsky-Gross. Magnification: x 200. |
Figure 8 – Abomasum ganglion of a 4.5-month-old lamb. Control group. Bilshovsky-Gross. Magnification: x 200. |
Consequently, adaptive-compensatory changes in the abomasum can be considered as stress to the structural-functional potential that occurs in the nervous tissue of the stomach during the artificial feeding of lambs.
Based on a comparative analysis, it was found that in the enteric nervous system of the wall of the multi-chambered stomach of sheep of the Edilbay breed grown with artificial feeding technology using Kalvo-Start SMS, pronounced changes in the morphogenesis of the intermuscular nervous plexus occur, even in the absence of external manifestations of pathology. During the early postnatal ontogenesis, due to the changing nature of feeding, changes in the growth, and development dynamics of the rumen, reticulum, omasum, and abomasum of sheep act as a reflection of the complex and dynamic processes of compensatory adaptation of the nervous system. Adaptive-compensatory changes in the processes of large neurons in conditions of artificial feeding are associated with the formation of thinner axons in the reticulum, omasum, and abomasum and thicker axons in the rumen, as well as with the formation and increase of the number of dendrites and their secondary branches during the dairy and transitional stages. Additionally, during the transition of the animals to the definitive feed, a partial loss of secondary dendrites occurs.
REFERENCES:
1. Bodrova LF. Vliyanie nizkopitatelnykh kormosmesei na morfogistokhimicheskuyu kharakteristiku zhelezistogo otdela zheludka kur [The effect of low-nutritional feed mixtures on the morphohistochemical characteristics of the glandular section of the chickens stomach]. Veterinarnyi vrach [Veterinarian]. 2009; 1: 41-43.
2. Kondratenko YuN et al. Morfologicheskie osobennosti myshechnoi obolochki zheludka belykh krys pri pitanii pishchei s izmenennymi fizicheskimi svoistvami [Morphological features of the muscular layer of the stomach of white rats fed with feed with altered physical properties]. Morfologiya [Morphology]. 2009; 136(4): 80.
3. Sych VF et al. Konsistentsiya pishchi kak faktor postnatalnogo morfogeneza muskulatury zheludka [The consistency of food as a factor in postnatal morphogenesis of the muscles of the stomach]. Vestnik novykh meditsinskikh tekhnologii [Bulletin of new medical technologies]. 2009; 16(2): 180-182.
4. Sanzhapova AF et al. Sravnitelno-morfologicheskii analiz osobennostei morfogeneza gladkoi myshechnoi tkani zheludka belykh krys pri pitanii dispergirovannoi pishchei [Comparative-morphological analysis of the morphogenesis of unstriated muscle tissue of the stomach of white rats fed with dispersed food]. Morfologicheskie vedomosti [Morphological bulletin]. 2011; 1: 46-51.
5. Valkova VV et al. Morfologiya mnogokamernogo zheludka ovets edilbaevskoi porody pri iskusstvennom vyrashchivanii [The morphology of a multi-chambered stomach of sheep of the Edilbay breed during artificial feeding]. Uchenye zapiski Kazanskoi gosudarstvennoi akademii veterinarnoi meditsiny im. N.E. Baumana [Proceedings of the Bauman Kazan State Academy of Veterinary Medicine]. 2014; 218(2): 33-37.
6. Charykova AF. Fizicheskie Svoistva Pishchi Kak Faktor Znachitelnykh Strukturnykh Preobrazovanii i Otklonenii v Razvitii Fundalnykh Zhelez Zheludka Belykh Krys [Physical properties of food as a Factor of significant structural changes and abnormalities in the development of the fundic glands of the white rats' stomach]. Nauchno-metodicheskii elektronnyi zhurnal Kontsept [Concept. Scientific-methodical electronic journal]. 2015; 13: 1621-1625. Avaliable from: https://e-koncept.ru/2015/85325.htm
7. Moldavskaya AA. and Kalaev AA. Morfofunktsionalnye osobennosti stroeniya stenki zheludka u eksperimentalnykh zhivotnykh v zavisimosti ot kharaktera vskarmlivaniya (estestvennoe, smeshannoe, iskusstvennoe) [Morphofunctional features of the structure of the stomach wall in experimental animals, depending on the feeding pattern (natural, mixed, artificial)]. Fundamentalnye issledovaniya [Fundamental research]. 2005; 5: 21-23.
8. Malashko VV and Tumilovich GA. Morfometricheskie osobennosti mezhmyshechnogo nervnogo spleteniya predzheludka novorozhdennykh telyat s razlichnoi stepenyu antenatalnogo nedorazvitiya [Morphometric features of the intermuscular nerve plexus of the forestomach of newborn calves with varying degrees of antenatal underdevelopment]. Uchenye zapiski uchrezhdeniya obrazovaniya Vitebskaya ordena Znak pocheta gosudarstvennaya akademiya veterinarnoi meditsiny [Proceedings of the Vitebsk State Veterinary Academy]. 2011; 47(1): 272-274.
9. Vechkanova NA and Bushukina OS. Adaptatsionno-kompensatornaya perestroika nervnoi tkani mnogokamernogo zheludka [Adaptive-compensatory restructuring of the nervous tissue of a multi-chambered stomach]. Vestnik novykh meditsinskikh tekhnologii. Elektronnoe izdanie [Bulletin of new medical technologies. Electronic version]. 2015; 1: 2-5. Avaliable from: http://www.medtsu.tula.ru/VNMT/Bulletin/E2015-1/5092.pdf
10. Sotnikov OS et al. Morfometriya i kineticheskaya traktovka razlichii asinapticheskikh dendritov mestnykh i spinalnykh sensornykh neironov kishechnika [Morphometry and kinetic interpretation of differences in asynaptic dendrites of local and spinal sensory intestinal neurons]. Sensornye sistemy [Sensory systems]. 2008; 22(4): 342-348.
11. Krasota VF et al. Metody izucheniya rosta. Razvedenie s.-kh. zhivotnykh [Breeding of agricultural animals]. Agropromizdat, Moscow. 1990.
12. Vovenko EP and Ivanov KP. Prodolnyi gradient napryazheniya kisloroda vnutri kapillyarov kory golovnogo mozga [Longitudinal gradient of oxygen tension inside the capillaries of the cerebral cortex]. Doklady Rossiiskoi Akademii nauk [Reports of the Russian Academy of Sciences]. 1997: 353(1): 121-123.
13. Fomin NA. Adaptatsiya: obshchebiologicheskie i psikhofiziologicheskie osnovy [Adaptation: general biological and psycho-physiological principles]. Izdatelstvo Teoriya i praktika fizicheskoi kultury [Publishing house – Theory and practice of physical culture], Moscow. 2003.
14. Tsigos C. The neuroendocrinology of the stress response. Stress, Gender, and Alcohol – Seeking Behavior. Research Monograph.1995; 29: 125-139.
15. Z.N. Zhuravleva. Ultrastrukturnoe issledovanie plastichnosti kletochnykh elementov i mezhkletochnykh vzaimodeistvii v transplantatakh nervnoi tkani [Ultrastructural study of the plasticity of cellular elements and intercellular interactions in nerve tissue transplants]. Аvtoreferat dissertaciya doktora biologicheskih nauk: 03.00.11 [Author's abstract of thesis of Dr. of Biol. Sciences: 03.00.11]. Pushchino. 1999.
16. Babmindra VP. Strukturnye osnovy mezhneironnoi integratsii [Structural foundations of interneuron integration]. Nauka, Leningrad. 1982.
17. Blum K and Briggs A. Ethanol ingestive behavior as a function of central neurotransmission. Experiential. 1989; 45(5): 444-452.
18. Hammer RP et al. Development of the brain stern reticular core: An assessment of dendritic state and configuration in the perinatal rat. Develop. Brain. Res. 1981; 1: 179-190.
19. Kasprzak KS and North SL. Reversal by nickel of inhibitory effects of some scavengers of active oxygen species upon hydroxylation of 2’-deoxyguanosine in vitro. Chem. Biol. Interact.1992; 84(1): 11-19.
Received on 30.08.2019 Modified on 25.09.2019
Accepted on 21.10.2019 © RJPT All right reserved
Research J. Pharm. and Tech. 2019; 12(12): 5729-5734.
DOI: 10.5958/0974-360X.2019.00991.0