Advances in Animal and Veterinary Sciences
Research Article
Haematological Profile of Marecha Male Camel (Camelus dromedarius) during Rutting and Non-Rutting Season under Farming System
Asim Faraz1*, Abdul Waheed1, Nasir Ali Tauqir2, Riaz Hussain Mirza1, Muhammad Shahid Nabeel3
1Department of Livestock and Poultry Production, Bahauddin Zakariya University Multan, Pakistan; 2Department of Animal Science, University of Sargodha, Pakistan; 3Camel Breeding and Research Station Rakh Mahni, Livestock and Dairy Development Department, Punjab, Pakistan.
Abstract | This study was undertaken to investigate haematological parameters of Pakistani male dromedary camel in rutting and non-rutting conditions. Five clinically healthy male Marecha camels of 5-10 years old having average body weight of 690±40 kg were studied for haematology in non-rut (August) and rut (February) season. The animals were kept in semi-open pens throughout the experiment, fed with concentrate, fodder, gram straw and available jungle grazing/browsing daily for 4-6 hr. Water was provided twice daily. The animals were dewormed and vaccinated regularly after three months, according to routine farm practices. Haematological parameters were analyzed on haematology analyzer. Haemoglobin (Hb), red blood cell (RBC) counts, white blood cell (WBC) counts, granulocyte, lymphocytes, packed cell volume (PCV), mean corpuscular volume (MCV), mean corpuscular haemoglobin (MCH) and mean corpuscular haemoglobin concentration (MCHC) were determined. The Hb concentrations (P>0.05) were found to be 14.86±1.28 and 14.26±1.12 gm/dl respectively in non-rut and rut season, being higher in non-rutting males. The values of RBC, WBC, PCV, MCV and MCHC were found to be differed significantly (P<0.05) among groups being higher in non-rutting males except WBC which was higher in rutting camels. The values of granulocytes, lymphocytes and MCH were found to be non-significantly (P>0.05) varied among groups. The findings are discussed in relation to reported results and could be used as a primary database for future studies.
Keywords | Camel, Haematology, Desert, Pastoral, Physiological condition
Received | June 24, 2020; Accepted | September 04, 2020; Published | January 01, 2021
*Correspondence | Asim Faraz, Department of Livestock and Poultry Production, Bahauddin Zakariya University Multan, Pakistan; Email: drasimfaraz@bzu.edu.pk
Citation | Faraz A, Waheed A, Tauqir NA, Mirza RH, Nabeel MS (2021). Haematological profile of marecha male camel (Camelus dromedarius) during rutting and non-rutting season under farming system. Adv. Anim. Vet. Sci. 9(2): 175-181.
DOI | http://dx.doi.org/10.17582/journal.aavs/2021/9.2.175.181
ISSN (Online) | 2307-8316; ISSN (Print) | 2309-3331
Copyright © 2021 Faraz et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
INTRODUCTION
Like other farm animals camels are classified as ruminants but pseudo-ruminant regarding the variation in stomach anatomy. Unlike other ruminants, they have no horns, have long necks and evenly padded feet with two toes (Al-Harbi, 2012). They are capable of using water very efficiently (Faraz et al., 2019a). Camels are animals with a very high level of red blood cells which are oval in shape and have the capacity to fill up to 230% at the availability of water. Due to this ability, camels have no match in thriving under hostile environments (Osman and Al-Busadah, 2003; Faraz, 2020a).
These mammals are classified as real dervishes i.e. dromedary (single humped), bactrian (double humped), new world (llama and alpaca) and South American (guanaco and vicuna) (Bogin, 2000). As mentioned above, old world camels are basically divided into two groups as Dromedary or Arabian (Camelus dromedarius) and Bactrian (Camelus bactrianus). While Arabian camels have one hump, Bactrian camels have two humps (Faraz et al., 2019b). One-humped or Arabian camels are well-adapted to hot, harsh and arid environments. Cameleers breed them for milk, meat, wool, hair, transport and sometimes for aesthetic preferences (Faraz et al., 2019c).
Investigation of blood constituents could provide valuable outputs and indications about the general health status and physiological condition of animals. Observation of deviation of certain blood parameters from normal range could be an indication for diagnosis/differential diagnosis of a diseased condition (Abebe et al., 2002). Realizing this, it is evident that increased fundamental knowledge of hemogram and blood metabolites contributes greatly to the understanding of physiology in dromedaries.
Male dromedary camels are known as seasonal breeders, where the breeding season is confined to the cool winter months of the year (Marai et al., 2009). In rutting condition, the male exhibits morphological, behavioral and endocrinological changes. In addition to this there are also haematological alterations (Dessouky, 1992). The rut stage in a male can last up to 100 days. However, the marked peak in sexual activity is during the breeding season. It is generally thought that the male is sexually quiescent for the remainder of the year but is still capable of mating and fertilizing the estrous female at any time of the year (Tibary and Anouassi, 1997).
As the haematological picture serves as an indicator of health and physiological status, many researchers studied camel hematology influenced by season, age, health status and lactation stage in Saudi-Arabia, Sudan and Iran (Al-Busadah and Osman, 2000; Babeker et al., 2013; Jalali et al., 2018). However, in Pakistan, only limited information on camel hematology is available, with most of the research work being survey reports under the traditional system (Faraz et al., 2018; Faraz et al., 2019d). The previous studies do not justify the requirements of subject; therefore, this study was planned to investigate haematological indices of the Pakistani Marecha male dromedary camel during rutting and non-rutting season, under the farming system at Thal desert. These results could be useful in building the country’s database for future studies of this field.
MATERIALS AND METHODS
Location of study area
The CBRS is located in the deserted plain of Thal. There is subtropical, continental, and arid to semi-arid climate, having mean summer temperature as 45.6 ºC and winter as 5.5-1.3 ºC. Annual mean rainfall is 150-350 mm increasing from South to North (Rahim et al., 2011).
Experimental animals and management
Five male camels of Marecha breed of 5-10 years old with average body weight of 690±40 kg reared under semi-intensive conditions were used for sampling. All animals were found physically healthy and were dewormed by injection 1% Ivermectin @ 1ml/50 kg body weight after every three months. Animals were sprayed with Ecofleece solution @ 1cc/liter water for animals and 2cc/liter of water for shed. They were vaccinated for Trypnosomiasis by injection Trypamedium-Samorine (Isometamidium chloride) 1 gm sachet for four adult camels after every three months as per farm routine practices. They were housed in semi-open pens at farm.
The animals were fed concentrate @ 4-5 kg/day having ingredients; maize grain (9%), wheat bran (24%), cotton seed cake (25%), rape seed cake (6%), corn gluten 30% (20%), molasses (14%), DCP (1%) and salt (1%). The percent chemical composition of concentrate was; dry matter (90.32), crude protein (18.06), neutral detergent fiber (29.09), acid detergent fiber (14.41), total digestible nutrients (70) and metabolizable energy (2.41 Mcal/kg DM). The animals were sent for jungle grazing/browsing for 4-6 hours daily. They were fed Cicer arientinum (gram straw) 5 kg daily as manger feeding. Twice watering was provided. Salt lumps were placed in mangers while 80-100 gm mineral mixture was fed per camel daily.
Sampling and lab analysis
Blood films from all animals were examined. Camels were properly restrained before collection of blood sample. The jugular vein was cleaned and treated with antiseptic (spirit) before and after collection of the blood. Blood samples were collected from jugular vein into test tubes; EDTA was used as anticoagulant and immediately analyzed. Haematological parameters were analyzed on haematology analyzer (BC 2300, Mindray Germany). Samplings were done weekly for the period of one month and all tests were performed in duplicate. Hb, RBC, WBC, Granulocytes, Lymphocytes, PCV, MCV, MCH and MCHC were determined.
The forage species available for grazing/browsing were Acacia nilotica, Acacia modesta, Ziziphus mauritiana, Albizia labbek, Prosopis cineraria, Tamarix aphylla, Cenchrus ciliaris, Suaeda fruticosa, Cymbopogon schoenanthus, Kochia indica, Tribulus terrestris, Capparis spinosa, Haloxylon salincornicum, Calligonam polygonoides, Capparis decidua and Haloxylon recurvum (Faraz, 2020b).
Statistical analysis
The SPSS (SPSS, 2008) software was used to analyze the data statistically by applying t-test on different parameters (Gecer et al., 2016; Akin et al., 2017).
RESULTS AND DISCUSSION
Haemoglobin
The mean values of Hb (P>0.05) were found to be 14.86±1.28, 14.26±1.12 gm/dl respectively in non-rutting and rutting males being normal but higher in non-rutting camels (Table 1). Hb was found greater in males as compared to females, probably due to increased level of erythropoiesis which is accelerated by erythropoietin in response to the testosterone effects on kidneys (Murphy, 2014). Hb concentrations varied between 13 to 16 gm/dl, slightly higher compared to other domestic species.
Current findings are very near to the reported results of Farooq et al. (2011), Elitok and Cirak (2018), Abdalmula et al. (2018, 2019) and Faye and Bengoumi, 2018. While Hb was observed higher than documented results of Amin et al. (2007), Zaher et al. (2017), Adah et al. (2017), Ghafoor et al. (2018), Ebissy et al. (2019) and Islam et al. (2019). However, Hb was found at greater level in Indian dromedary camel as reported by Narnaware et al. (2016).
Amin et al. (2007) reported Hb concentration as 10.67±0.19 and 10.73±0.18 gm/dl respectively, in dry and green season in Sudanese dromedary camel. Farooq et al. (2011) reported mean concentration and range for Hb as 12.00±0.63, 11.34±0.95; 7-17, 8-17 gm/dl in Pakistani dromedary male and females, respectively in Cholistan Desert. Reported Hb concentration was 14.49±1.46, 14.80±1.15, 15.20±1.40 and 14.20±1.55 gm/dl in Saudi Arabian dromedary male camel during pre-rut, non-rut, post-rut and rut season (Al-Harbi, 2012). The Hb concentration in the present study was also found to be in same pattern, as lower values were reported in rutting season: Consequently, this indication may be used as diagnosis of rutting behavior and condition by testing this important parameter if we know about the normal parameters in non-rutting males.
Reported Hb concentration was 7.80±0.42 gm/dl in Sudanese nomadic dromedary camels (Babeker, 2013). Eltayeb et al. (2015) studied the effect of water deprivation on blood constituents of the Sudanese male dromedary camel and reported mean Hb concentration as 12.8±0.6, 11.7±0.5 and 10.1±0.4 gm/dl in groups not being deprived of water, deprived of water for 48h and 4hr after provision of water, respectively. On the other side, Hb concentration was found to be 14.06±0.24 gm/dl in female dromedary camels (Zaher et al., 2017). Adah et al. (2017) reported Hb concentration as 7.33±0.35 gm% in Nigerian dromedary camel. Reported Hb concentration in Bangladeshi dromedary camels was 10.4 gm/dl (Islam et al., 2019). The reported Hb values were found to be varied in the majority of the references between 9.3-15.5 gm/dl as documented by Faye and Bengoumi (2018).
Ghafoor et al. (2018) reported average Hb concentration as 11.78±0.57 gm/dl in Pakistani dromedary camels in Desert Thal. Elitok and Cirak (2018) reviewed haematological features of camels and reported Hb concentration as 14.8±1.2 and 14.2±1.5 gm/dl during non-rut and rut season in male dromedary camels, respectively. Abdalmula et al. (2018) reported Hb concentration and range as 12.55±0.27 and 7.28-17.70 gm/dl, respectively in Libyan dromedary camel. In a different preview, Abdalmula et al. (2019) reported Hb concentration as 11±0.41 and 13.44±0.27 gm/dl in Libyan dromedary males and females, respectively. While reported Hb concentration was 10.62±0.55 gm/dl in female dromedary camels (Ebissy et al., 2019).
RBC and WBC
The mean values of RBC and WBC were found to be varied significantly (P<0.05) among groups, being a higher concentration of RBC in non-rutting and WBC in rutting males. The granulocytes and lymphocytes concentration were found to be varied non-significantly (P>0.05) among groups (Table 1).
The mean values of RBC counts are comparable to that reported by Al-Busadah and Osman (2000), Al-Harbi (2012), Adah et al. (2017), Zaher et al. (2017), Abdalmula et al. (2018, 2019), Elitok and Cirak (2018), Ebissy et al. (2019) and Islam et al. (2019). While values observed for RBC in present study are greater than reported by Amin et al. (2007), Farooq et al. (2011) and Ghafoor et al. (2018). These differences may be due to different geographical, climatic and genetic variations (Saeed and Hussein, 2008).
Reported RBC concentration in Sudanese dromedary camel was 6.41±0.15 and 5.79±0.14 106/µl respectively, in dry and green season (Amin et al., 2007). In Pakistan, Farooq et al. (2011) reported mean concentration and range for total erythrocytic and leucocytic count as 6.83±0.38, 7.31±0.58; 4.3-12.4, 4.25-12.9 106/µl and 12.38±0.97, 12.97±0.99; 4-22.3, 4.2-20.9 103/µl in male and females, respectively, in dromedary camel in Desert Cholistan. Reported concentration and range of lymphocytes was 47.50±1.40, 48.60±1.50 and 40-55, 43-63 % in male and females, respectively. Reported RBC (106/µl), WBC (103/µl) and lymphocytes (%) concentrations were to be 3.03±0.08, 7.70±0.22 and 40.16±0.70 in Sudanese nomadic dromedary camel (Babeker, 2013).
Adah et al. (2017) reported erythrocytes and leukocytes concentration as 15.05±2.10 1012/l and 16.20±0.32 109/l respectively, in Nigerian dromedary camel. Reported erythrocytic and leukocytic count of dromedary camels in Bangladesh was 5.3 million/cumm and 8.9 thousand/cumm respectively (Islam et al., 2019). However, total erythrocytic count was found at a higher level in Indian dromedary camel, as reported by Narnaware et al. (2016). Ghafoor et al. (2018) reported total erythrocyte count as 7.25±0.08 106/µl in Pakistani dromedary camel in Desert Thal.
Reported concentrations of RBC (106/µl) and WBC (103/µl) were 10.55±1.39, 10.30±1.68; 10.90±1.04, 10.10±1.20; 9.87±1.33, 9.70±1.48; 8.90±1.45, 10.5±1.89 during pre-rut, non-rut, post-rut and rut season in male dromedary camels (Al-Harbi, 2012). Current study results are in agreement with these findings as RBC concentration was observed lower in rut season while the WBC concentration was found higher in rut season. The lymphocytes and granulocytes percentages in present study were also found to be higher in rutting males. These measurements could be used as indication of rut, so the breeding season of dromedary male can be diagnosed by testing these parameters.
While reported RBC concentration was 9.48±0.16 106/µl in female dromedary camels (Zaher et al., 2017). Elitok and Cirak (2018) reviewed haematological features of camels and reported RBC concentration as 10.90±1.04, 8.90±1.45 106/µl in male dromedary camels during non-rut and rut season, respectively. White blood cells were found higher in number as compared to previous results reported by Rezakhani et al. (1997), Singh et al. (1997) and Mohamed and Hussein (1999). The mean Lymphocyte percentage was found to be higher than the findings of Al-Busadah and Osman (2000), while lower than of Al-Busadah (2007). The mean Granulocytes percentages are lower than reported concentration by Al-Busadah and Osman (2000), while higher than that of Al-Busadah (2007).
Amin et al. (2007) reported lymphocytes concentration as 56.24±1.57 and 34.93±1.54 percent respectively, in dry and green season in Sudanese dromedary camel. Reported Lymphocytes concentrations were 40±1.32, 45±1.05, 50±1.19 and 54±1.48 % during pre-rut, non-rut, post-rut and rut season in male dromedary camels, respectively (Al-Harbi, 2012). Eltayeb et al. (2015) studied the effect of water deprivation on blood constituents of the Sudanese male dromedary camel and reported mean concentrations of RBC (106/µl) and WBC (103/µl) as 8.6±0.5, 10.8±2.4; 11.6±1.5, 12.7±1.9 and 7.9±0.6, 10.2±2.0 respectively, in groups not being deprived of water, deprived of water for 48hr and 4hr after provision of water.
The reported value for lymphocytes in Nigerian dromedary camel was 6.05±0.3 109/l (Adah et al., 2017). Elitok and Cirak (2018) reported WBC concentration as 10.10±1.20 and 10.5±1.89 (103/µl) in males of non-rut and rut season, respectively. The reported range was 7.5-22 in adult camels, while lymphocytes concentration was 45±1.05 and 54±1.48 % in males of non-rut and rut season, respectively. Reported lymphocytes concentration for normal camels was 41%. On the other side, reported concentrations of RBC and WBC were found to be 10.74±0.42 106/µl and 9.96±1.22 103/µl in female dromedary camels during transition period (Ebissy et al., 2019).
Abdalmula et al. (2018) reported RBC (106/µl) and WBC (103/µl) concentration and range as 11.79±0.36, 10.98±0.50 and 7.53-30.88, 4.75-21.20, respectively in Libyan dromedary camels while lymphocytes concentration and range was found to be 6.73±0.38 and 2.60-18.05 103/µl, respectively. Islam et al. (2019) reported the lymphocytes percentage as 48.6 in Bangladeshi dromedary camels. Reported RBC and WBC concentration were to be 12.27±0.89 106/µl and 10.45±0.86 103/µl respectively, in Libyan male dromedary camels.
PCV, MCV, MCH and MCHC
The mean values of PCV, MCV and MCHC were found to be varied significantly (P<0.05) while MCH non-significantly (P>0.05) among groups. All values were found to be normal but on the higher side in non-rutting camels (Table 1).
Table 1: Mean haematological profile of male Marecha camel (n=5) during rutting and non-rutting season.
Parameters | Non-rut (August) | Rut (February) |
Haemoglobin (gm/dl) |
14.86±1.28 a |
14.26±1.12 b |
RBC (106/µl) |
10.55±0.96 a |
8.80±0.88 b |
WBC (103/µl) |
26.35±4.18 a |
29.31±2.93 b |
Granulocytes (%) |
56.47±2.66 a |
57.78±2.32 a |
Lymphocytes (%) |
31.46±1.50 a |
32.66±2.04 a |
PCV (%) |
38.36±2.34 a |
35.24±1.45 b |
MCV (fl) |
38.47±1.62 a |
36.26±1.44 b |
MCH (pg) |
21.49±1.69 a |
20.89±1.28 a |
MCHC (gm/dl) |
54.53±2.48 a |
51.98±2.78 b |
Means having different superscript in columns are significantly different (P<0.05); RBC: Red Blood Cell Count; WBC: White Blood Cell Count; PCV: Packed Cell Volume; MCV: Mean Corpuscular Volume; MCH: Mean Corpuscular Haemoglobin; MCHC: Mean Corpuscular Haemoglobin Concentration.
In the present study, the values found are very close to the observed results by Al-Harbi (2012), Adah et al. (2017), Zaher et al. (2017), Abdalmula et al. (2018, 2019), Elitok and Cirak (2018), Ebissy et al. (2019) and Islam et al. (2019). Horse, cattle and camel showed higher number of RBC so MCV values are lower as compared to other species (Sarwar and Majeed, 1997). The reported MCH concentrations were higher than reported values by Al Busadah and Osman (2000), Amin et al. (2007), Al Busadah (2007) and Saeed and Hussein (2008). MCHC values are greater in the current study as compared to the values reported by (Al-Busadah, 2004; Saeed and Hussein, 2008). These higher values showed that the oxygen carrying capacity of camel blood is more, as reported by (Rezakhani et al., 1997; Al-Busadah and Osman, 2000).
Amin et al. (2007) reported mean concentration of PCV (%), MCV (fl), MCH (pg), MCHC (gm/dl) as 25.14±0.33, 25.95±0.32; 40.09±0.81, 46.43±0.79; 16.99±0.43, 19.44±0.42; 42.49±0.63, 41.62±0.61 respectively, in dry and green season in Sudanese dromedary camel. In Pakistan, Farooq et al. (2011) reported mean concentration and range for PCV (%), MCV (fl), MCH (pg), MCHC (gm/dl) as 37.21±2.48, 32.83±3.76 and 22-67, 24-50; 57.13±5.09, 50.24±7.72 and 27-140, 26-103; 18.69±1.45, 17.80±2.21 and 11-35, 7-35; 33.61±1.42, 34.81±2.16 and 22-46, 27-42 in dromedary male and females, respectively in Desert Cholistan.
Reported PCV (%), MCV (fl), MCH (pg) and MCHC (gm/dl) concentrations were to be 38.20±2.29, 39.60±3.18, 15.90±2.30 and 49.50±3.60; 39.80±1.99, 40.10±2.86, 16.70±1.97 and 49.30±3.12; 41.20±2.15, 37.20±3.13, 16.40±2.21 and 47.40±3.44; 39.50±2.65, 36.40±3.39, 16±2.37 and 46.20±3.77 respectively during pre-rut, non-rut, post-rut and rut season in male dromedary camels (Al-Harbi, 2012). Current findings are in line with these results as higher concentrations were found in non-rutting males, so these indications could be used as diagnosis of breeding season in rutting dromedary camels.
Reported PCV (%), MCV (fl), MCH (pg) and MCHC (gm/dl) concentrations were to be 19.48±1.28, 48.56±0.36, 26.26±0.15 and 75.54±0.11 respectively, in Sudanese nomadic dromedary camels (Babeker, 2013). Eltayeb et al. (2015) studied effect of water deprivation on blood constituents of the Sudanese male dromedary camel and reported mean concentrations of PCV as 28.7±1.5, 26.2±1.1 and 23.4±0.5 % respectively, in groups not being deprived of water, deprived of water for 48h and 4hr after provision of water. Reported concentrations of PCV (%), MCV (fl), MCH (pg) and MCHC (gm/dl) were to be 28.62±0.31, 36.45±0.51, 14.02±0.17 and 42.23±0.19, respectively in female dromedary camels (Zaher et al., 2017).
The mean value for PCV % was reported as 25.71±0.13 in Nigerian dromedary camel (Adah et al., 2017). Islam et al. (2019) reported PCV concentration as 36.5 % in Bangladeshi dromedary camels while in Sudanese camel as 23.44 % (Ibrahim et al., 2015). Ghafoor et al. (2018) reported average concentration of PCV as 29.25±0.13 % in Pakistani dromedary camels in Desert Thal. Elitok and Cirak (2018) reviewed haematological features of camels and reported PCV (%), MCV (fl), MCH (pg) and MCHC (gm/dl) concentration as 39.80±1.99, 39.50±2.65; 40.10±2.86, 36.40±3.39; 16.70±1.97, 16±2.37 and 49.30±1.39, 46.20±3.77 in males of non-rut and rut season, respectively.
Abdalmula et al. (2018) reported concentration and range of PCV (%), MCV (fl), MCH (pg) and MCHC (gm/dl) as 33.47±1.01, 16.00-50.00; 29.24±0.93, 7.10-48.70; 11.06±0.32, 3.30-18.60 and 39.38±1.30, 21.60-87.30 respectively, in Libyan dromedary camel. Reported PCV (%), MCV (fl), MCH (pg) and MCHC (g/dl) concentrations were 29.71±1.67, 25.45±1.37, 9.68±0.61 and 39.76±2.76 respectively, in male dromedary camels (Abdalmula et al., 2019). On other hand, reported concentrations of PCV (%), MCV (fl), MCH (pg) and MCHC (gm/dl) were to be 32.10±1.44, 30.06±1.53, 8.98±0.54 and 26.65±0.95 in female dromedary camels during transition period (Ebissy et al., 2019).
CONCLUSIONS AND RECOMMENDATIONS
The study of blood parameters could serve as mirror of animal welfare and, consequently of general health status and physiological condition. Indeed, rutting season could affect animal welfare, which is considered related to animal’s needs, influencing on nutrition, behaviour, and reproduction (Previti et al., 2016). This study has documented the haematological indices in Pakistani Marecha male dromedary camels in breeding and non-breeding season. The elevated levels of blood parameters like WBC, granulocytes and lymphocytes affected by rutting condition, while other parameters i.e. RBC, PCV, MCV, MCH and MCHC were found lower in rutting season which affect general physiological state and function including sexual performance. Consequently, these parameters could be used to detect the rutting dromedary males and the results will be a useful addition in primary database of country.
ACKNOWLEDGEMENT
The Authors gratefully acknowledge the cooperation and kind support of the management of Camel Breeding and Research Station (CBRS) Rakh Mahni district Bhakkar, Punjab Pakistan.
AUTHOR’S CONTRIBUTIONS
All authors contributed to make the completion of this manuscript possible. Asim Faraz conducted research and wrote the paper. Muhammad Shahid Nabeel helped in conduct of research. Abdul Waheed and Riaz Hussain Mirza helped in analysis. Nasir Ali Tauqir helped in write up.
CONFLICT OF INTEREST
The authors have declared no conflict of interest.
REFERENCES