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Effect of Melatonin Supplementation on Haematological Parameters in Buffalo Calves under Summer Stress

Vijay D. Domple S. S. Dangi Vijay Yadav M. Sarkar G. Singh V. P. Maurya
Vol 7(5), 266-274

This study was designed to observe the effect of melatonin on haematological parameters in buffalo calves under summer stress. Twelve healthy Murrah buffalo male calves of 6 month to 1 year age group were taken for the study. Buffalo calves were divided into control (CG) and treatment (TG) group. In TG, Melatonin (18mg/50 kg BW) was injected subcutaneously, two times at 1st and 20th day. Blood samples were collected on 0th, 7th, 14th, 21st, 28th, 35th and, 42nd days and analysed by using hematology analyzer. Between the days, in CG the monocytes count, granulocytes count, monocytes percentage, granulocytes percentage, RBC(1012/L) count, HGB(g/dl), HCT(%), MCV(fL), MCH(Pg), RDWCV(%), PLT(109/L) count, MPV(fL), PCT(%) and in TG granulocytes percentage, HGB(g/dl), HCT(%), MCH(Pg), RDWCV(%), RDWSD(fL) was observed to vary significantly (p<0.05) at some time points. Between the groups the granulocytes percentage on day 7th and 28th was observed significantly (p<0.05) high in CG as comparison to melatonin TG. The overall effect of granulocytes percentage during 42 days of buffalo calves were found significantly (p<0.05) higher in control group than the melatonin treated group. In conclusion, melatonin affects the haematological parameter to counteract the summer stress condition in animals.

Keywords : Buffalo Calves Heat Stress Hematology Melatonin


Buffalo (Bubalus bubalis) is known as the world’s second most important milch animal. The importance of buffalo to the dairy industry, food security and rural livelihood by providing milk, meat and draught power in tropical and subtropical countries hardly needs emphasizing as the species is an important livestock resource in Asian continent with over 95% of world’s buffalo population (Kumar et al., 2015). The buffalo can be divided in to subspecies i.e. riverine and swamp buffaloes; both have qualities like adaptation to harsh environment, maintenance on poor quality roughages and resistance to tropical diseases. Buffalo reproduction in tropical and subtropical part of the world is considered as short day breeder and its reproductive efficiency is greatly and adversely influenced by biometeorological factors (Phogat et al., 2016).

Stress is the reaction of animal body that disturbs homeostasis often with detrimental effects. Domestic animals undergo various types of stress such as physical, nutritional, chemical, psychological and thermal stress. Among all the domestic animals, heat stress is most concerning now a day in the ever changing climatic scenario. High environmental temperature is the major constraint on animal production in case of tropic and sub tropic regions (Marai et al., 2007; Nardone et al., 2010). Thermal stress is the perceived discomfort and physiological strain associated with exposure to the uttermost hot temperature. It stimulates sort of complex responses which are fundamentals in the preservation of cell survival.

Melatonin (N-acetyl-5-methoxytryptamine), an indolamine synthesized from tryptophan in the pineal gland, has been shown to be associated with the regulation of seasonal reproduction in photoperiodic species. Melatonin has been shown to be a highly effective antioxidant and free radical scavenger (Mauriz et al., 2013). Melatonin, by virtue of its antioxidant properties, quenches the oxidants including nitric oxide, arrests lipid peroxidation, and acts synergistically with other classic antioxidants such as glutathione peroxidase (GPx), superoxide dismutase (SOD), vitamin E, and selenium (Gitto et al., 2001). The main function of melatonin was to serve as an antioxidant to protect organisms from ubiquitous oxidative stresses. Melatonin is considered to be more effective than glutathione (GSH) and mannitol in scavenging free radicals. It was found that melatonin has the ability to neutralize damaging reactive oxygen species (ROS) and reduce lipid peroxide concentrations and DNA damage thereby improving the viability of germ cells (Wang et al., 2013). Therefore, the present study was designed to investigate the effect of melatonin on haematological parameters in buffalo calves under summer stress condition

Materials and Methods

Animals and Experimental Design

Healthy Murrah buffalo bull calves between 6 to 12 months of age group belonging to tropical region were selected for the study purpose. The experiment trial was conducted up to 6 week (42 days) period. Animals were divided into two groups viz., control (CG) and treatment (TG) group (n= 6). TG received melatonin @ 18 mg/50 kg body weight, subcutaneously (s/c), on 1st and 20thday of experiment (Kumar, et al., 2015). Blood samples were collected by jugular veinipuncture under sterile conditions at 7 days interval on 1st, 8th, 15th, 22nd, 29th, 36th and 42nd days.

Parameters Investigated

Collected blood samples were analysed for estimation of haematological parameters like count of white blood cells (WBC), lymphocytes (LY#), monocytes (MO#), granulocytes (GR#), red blood cells (RBC), platelets (PLT), percentage of lymphocytes (LY%), monocytes (MO%), granulocytes (GR%), hemoglobin (HGB), hematocrit (HCT/PCV), mean corpuscular volume (MCV), mean corpuscular hemoglobin (MCH), mean corpuscular hemoglobin concentration (MCHC), red blood cell distribution width-coefficient of variation (RDW-CV), red blood cell distribution width-standard deviation (RDW-SD), mean platelet volume (MPV), platelet distribution width (PDW), plateletcrit (PCT) using hematology analyzer (HA-22-CLINDIAG).

Statistical Analyses

Data were analyzed by the one-way analysis of variance followed by Tukey’s b test using SPSS 17.0 software (SPSS, Inc., 1997). All data were presented as means ± standard error of the mean.

Result and Discussion

Parameters Group 0 day 7 day 14 day 21 day 28 day 35 day 42 day Overall effect during 42 days
WBC Control 11.5±0.9a 9.4±1.3a 11.2±0.7a 12.3±0.9a 9.1±0.5a 9.1±0.9a 9.8±0.5a 10.3±0.3a
(109/L) Treatment 12.0±0.7a 10.1±1.3a 12.45±1.7a 12.0±1.7a 10.2±0.7a 9.8±0.9a 10.5±1.2a 11.04±0.4a
LY Control 7.6±1.0a 5.2±0.9a 7.3±0.6a 7.4±0.9a 5.3±0.6a 5.5±0.8a 5.7±0.5a 6.3±0.3a
(109/L) Treatment 7.8±0.9a 6.7±1.1a 7.3±0.7a 7.5±1.1a 7.1±0.7a 6.4±0.7a 6.5±0.9a 7.0±0.3a
MO Control 0.65±0.1a 0.45±0.2ab 0.44±0.2ab 0.75±0.2a 0.68±0.1a 0.49±0.2ab 0.35±0.1b 0.55±0.1a
(109/L) Treatment 0.4±0.2a 0.43±0.2a 0.46±0.9a 0.51±0.5a 0.56±0.3a 0.35±0.2a 0.46±0.1a 0.45±0.1a
GR Control 1.9±0.2a 1.77±0.4a 1.15±0.1b 1.4±0.2ab 0.96±0.2b 1.5±0.1a 1.7±0.3a 1.48±0.1a
(109/L) Treatment 2.0±0.2a 1.43±0.08a 1.2 ±1.1a 1.4±0.6a 1.15±0.1a 1.2±0.1a 1.53±0.3a 1.42±0.2a
LY Control 64.5±4.6a 54.3±2.8a 64.7±2.6a 59.9±3.7a 57.4±4.4a 58.3±3.5a 57.2±3.6a 59.5±1.4a
(%) Treatment 66.3±3.2a 64.6±3.6a 59.1±9.3a 53.4±10.7a 69.2±3.9a 64.6±2.3a 60.9±2.3a 62.6±2.2a
MO Control 5.6±0.8ab 4.8±0.6ab 3.9±0.5ab 6.1±1.0ab 7.5±1.3a 5.3±0.7ab 3.8±0.8b 5.28±0.7a
(%) Treatment 3.3±0.5a 4.3±1.0a 3.7±0.5a 4.2±0.3a 5.5±1.3a 3.6±0.7a 4.4±0.9a 4.14±0.9a
GR Control 16.5±2.3ab 18.7±1.3a* 11.2±1.0b 11.8±2.0b 10.6±0.7b* 14.9±2.2ab 17.3±2.7a 14.43±0.8a*
(%) Treatment 16.2±1.0a 14.0±1.2ab* 10.0±1.6b 11.6±1.8b 11.3±0.5b* 12.7±1.9ab 14.6±1.5ab 12.61±0.4b*
RBC Control 5.7±0.1b 7.6±0.7a 6.0±0.2ab 7.7±0.7a 5.5±0.1b 4.9±0.3b 6.3±0.1ab 6.2±0.2a
(1012/L) Treatment 6.2±0.3a 7.1±0.7a 6.8±0.5a 7.3±0.6a 5.9±0.4a 5.4±0.3a 7.2±0.5a 6.6±0.2a
HGB Control 8.9±0.3b 12.3±1.4a 8.6±0.3b 12.4±1.3a 9.9±0.2b 9.5±0.3b 11.4±0.8a 10.43±0.4a
(g/dl) Treatment 9.2±0.8ab 11.8±1.8ab 10.7±1.1ab 11.8±1.4ab 8.5±0.6b 8.7±0.4b 12.5±1.9a 10.46±0.5a
HCT/PCV (%) Control 28.4±1.0b 34.9±2.6a 27.9±0.9b 35.3±2.2a 24.9±0.8b 22.8±0.7b 35.5±2.0a 29.9±0.9a
Treatment 30.5±1.9ab 34.2±3.7ab 31.3±2.2ab 31.1±2.6ab 26.9±1.7b 24.9±0.9b 39.7±3.9a 31.5±1.1a
MCV Control 46.3±1.3ab 45.1±1.7ab 42.5±1.6b 43.1±1.3b 41.6±1.6b 43.3±1.6b 45.2±1.2a 43.87±0.7a
(fL) Treatment 45.8±1.7ab 45±1.5ab 43±1.8b 43±1.6b 42.5±1.7b 43.5±1.8b 44.5±1.1a 43.9±0.7a
MCH Control 16.5±0.7ab 16.1±0.8ab 14.1±0.4b 16.4±0.4ab 14.1±0.4b 14.7±1.1b 19.0±0.7a 15.8±0.3a
(Pg) Treatment 14.9±0.9ab 16.3±1.3ab 15.7±0.9ab 16.2±1.3ab 14.5±0.6b 14.1±0.7b 19.5±1.4a 15.9±0.4a
MCHC Control 35.2±1.4a 35.3±1.5a 30.6±3.5a 37.8±1.7a 33.5±0.4a 33.6±2.3a 36.7±1.3a 34.7±0.7a
(g/dL) Treatment 32±1.0a 35.6±2.2a 36.2±2.2a 37.0±2.0a 33.5±0.5a 32.1±1.4a 37.1±1.5a 34.8±0.6a
RDW-CV Control 27.1±1.6ab 29.5±2.5ab 31.6±1.9a 32.5±2.0a 32.3±1.9a 30.8±1.8a 23±0.5b 29.5±0.8a
(%) Treatment 28.1±2.4ab 29.6±2.0ab 31.8±2.0a 32.1±2.0a 31.6±1.8a 30.3±2.1ab 22.3±0.9b 29.4±0.8a
RDW-SD Control 92.0±0.7a 93.7±1.5a 94.7±0.9a 95.5±1.1a 94.8±0.9a 94.3±0.9a 76.3±13.3a 91.6±2.0a
(fL) Treatment 92.9±1.2ab 93.7±1.2ab 94.6±1.1a 94.8±1.1a 94.4±0.9a 93.4±1.0ab 89.5±0.7b 93.3±0.4a
PLT Control 273±18.9b 326.5±23.4ab 346.5±31.1ab 527.3±121.7a 314.6±46.2ab 326.5±54.0ab 283±22.1ab 342.5±23.2a
(109/L) Treatment 361.5±69.6a 456.8±75.1a 389.5±48.0a 427.6±89.4a 297±41.3a 264.1±31.9a 276±21.3a 353.2±23.2a
MPV Control 17.4±0.4ab 17.1±0.6ab 18.8±0.4a 19.0±0.3a 19.2±0.4a 18.6±0.5ab 16.6±0.5b 18.1±0.2a
(fL) Treatment 17.1±0.5ab 17.4±0.5ab 18.8±0.5a 19±0.3a 19.0±0.5a 18.6±0.7ab 16.5±0.3b 18.1±0.2a
PDW Control 27.67±0.0a 27.67±0.0a 27.67±0.0a 27.67±0.0a 27.67±0.0a 27.67±0.0a 27.67±0.0a 27.67±0.0a
(fL) Treatment 27.67±0.0a 27.67±0.0a 27.67±0.0a 27.67±0.0a 27.67±0.0a 27.67±0.0a 27.67±0.0a 27.67±0.0a
PCT Control 0.4±0.04b 0.5±0.05ab 0.6±0.07ab 1.0±0.2a 0.6±0.09ab 0.61±0.1ab 0.47±0.05b 0.6±0.04a
(%) Treatment 0.6±0.1a 0.8±0.1a 0.7±0.1a 0.8±0.1a 0.57±0.08a 0.4±0.07a 0.45±0.03a 0.6±0.04a

Means bearing different superscript a, b, c, d differ significantly at (p<0.05) in the same group between days and superscript * denotes significant difference on the same day between the groups (Values are given in Mean ± SEM)

Thermal stress is known to alter the homeostatic mechanism of animals resulting in impaired erythropoiesis. High environmental temperature increases oxygen consumption of animals by increasing respiration rate. The higher oxygen intake increases the partial pressure of oxygen in blood, decreases erythropoiesis which in turn reduces the number of circulating erythrocytes and thus PCV and Hb values (Maurya et al., 2007; Temizel et al., 2009; Sivakumar et al., 2010; Kumar et al., 2011). Other explanation of decrease in hemoglobin and PCV levels during thermal stress could be increased attack of free radicals on the erythrocyte membrane, which is rich in lipid content and ultimate lysis of RBC or inadequate nutrient availability for hemoglobin synthesis as the animal consumes less feed or decreases voluntary intake under heat stress. During summer stress a significant depression in PCV may also be due to haemodilution effect where more water is transported into the circulatory system for evaporative cooling (EL-Nouty et al., 1990). The percentage of Leukocyte count value was less during the summer than in winter in both male and female of Indian domestic goat Capra hircus (Singh et al., 2014).

Under the heat stress, increased PCV and hemoglobin count in sheep was reported by Rana et al., 2014. In present study, in CG the HGB (g/dl) count were found significantly (p<0.05) higher at 7th, 21st and 42nd day than the other days of experiment. However melatonin treated group shows significant (p<0.05) increases in HBG on 42nd day in comparison to the 28th and 35th days of the experiment. HCT/PCV count in control group was observed significantly (p<0.05) higher on 7th, 21st and 42nd day than other days. The RBC count of buffalo calves were found significantly (p<0.05) higher at 7th and 21st day than the 0th, 28th and 35th days in control group of experiment. This increase of hemoglobin and PCV levels could be due to either increased attack of free radicals on the RBC membrane, which is rich in lipid content and ultimate lysis of RBC or adequate nutrient availability for hemoglobin synthesis as the animal consumes more feed or decreases voluntary intake under heat stress (Srikandakumar et al., 2003). This higher PCV value was an adaptive mechanism to provide water necessary for evaporative cooling process (Al-Haidary, 2004). Hematocrit (HCT) value, hemoglobin concentration and erythrocyte number rise, especially at the beginning of heat stress, evidenced a rise of blood concentration (Koubkova et al., 2002). Toharmat et al., 1998, described an increase in hematocrit values and hemoglobin concentration in summer. However, in contrast to these findings, reduction of Hb and PCV levels were observed as results of RBC lysis either by increased attack of free radicals on its membrane or inadequate nutrient availability for Hb synthesis as the animal consumes less feed or decreases voluntary intake upon HS (Srikandakumar and Johnson, 2004).

In CG between the days the monocytes count were found significantly (p<0.05) increased at 0th, 21st and 28th day than the 42nd day of the experiment. It is also observed that in control group, between the days the granulocytes count were found significantly (p<0.05) higher on 0th, 7th, 35thand 42nd day than the 14th and 28th days of experiment. Monocytes percentage between the days were found significantly (p<0.05) higher at 28th day in comparison to the day 42nd in control group. Between the days the granulocytes percentage were found significantly (p<0.05) higher at 7th and 42nd day than the 14th, 21st and 28th in control group. In melatonin treated group the granulocytes percentage of buffalo calves were found significantly (p<0.05) higher at 0th day than the 14th, 21st, 28th day of the experiment. Between the groups the granulocytes percentage on day 7th and 28th was observed significantly (p<0.05) high in control as comparison to melatonin treated group. The overall effect on granulocytes percentage in buffalo calves during 42 days of experiment were found significantly (p<0.05) higher in control group than the melatonin treated group. Primary indicators of immunity response include WBC, RBC, Hb and PCV in blood get altered on thermal stress. WBC (leukocytes) count increase by 21-26% (Helal et al., 2010) and RBC count decrease by 12-20% (Habeeb, 1987) in thermally stressed cattle that could be due to thyromolymphatic involution or destruction of erythrocytes. The amount of RBC, PCV%, Hb%, WBC were increased with the increased of heat stress. Neutrophil, eosinophil, lymphocyte and monocyte numbers increased significantly (p<0.05) in heat treated group (Alam et al., 2011). In malpura ewes significant variation of Hb, PCV, plasma glucose, total protein and albumin is reported for the different temperature exposure (Sejian et al., 2013). Altan et al., 2000 and Nadia (2003) reported that, exposure of broilers or quail to heat stress results in decreased lymphocyte percentages. Between the groups the granulocytes count on day 28th and granulocytes percentage on day 7th and 28th was observed significantly (p<0.05) high in control in comparison to melatonin treated group. The overall effect of granulocytes percentage during 42 days of buffalo calves were found significantly (p<0.05) higher in control group than the melatonin treated group. Administration of melatonin increased the total leukocytic count and lymphocyte percentage in broiler chicks (Abozahra et al., 1998), rats (Anwar et al., 1998), immature chicks (Bernan et al., 2002) and squirrels (Rai et al., 2009). Rai and Haldar (2003) reported that daily subcutaneous injection of melatonin at 17:30 to 18:00 h significantly increased the lymphocyte count of blood in adult male squirrels, while pinealectomy decreased total leukocyte count and percent lymphocyte count in peripheral blood and bone marrow. The WBC counts of birds injected with 40 mg melatonin/kg BW subcutaneously per day for 7 days were significantly higher than the WBC counts of saline-injected birds (Brennan et al., 2002).

In CG the MCV of buffalo calves were significantly (p<0.05) higher at 42nd day than the 14th, 21st, 28th and 35th day of the experiment. Similar trend was observed in melatonin treated group. The MCH of buffalo calves were significantly (p<0.05) higher at 42nd day than the 14th, 28th and 35th day in control group. In CG the HCT/PCV count of buffalo calves were found significantly (p<0.05) higher at 7th, 21st and 42nd day than the 0th, 14, 28th and 35th days of experiment. In melatonin treated group the HCT/PCV count of buffalo calves were significantly (p<0.05) higher at 42nd day than the 28th and 35th day of the experiment. Lee et al., 1976 described a significant decrease in hematocrit value in dairy cows exposed to high temperatures. This hematocrit and red blood cells decrease were perhaps caused by a rise in erythrocyte destruction; haemodilution effect could also participate here, because more water was transported in circulatory system for evaporative cooling. High ambient temperature significantly (p< 0.05) reduced hematocrit and hemoglobin in broiler chickens (Olanrewaju et al., 2010). Nadia (2003) who indicated that, heat stress decreases the mean corpuscle hemoglobin (MCH), increase in MCV value and decrease in MCHC in heat stressed in Japanese quail.

The RDWCV were significantly (p<0.05) lower at 42nd day than the 14th, 21st, 28th, and 35th day of the experiment in CG. However TG shows similar trend except on day 35th the RDWCV of buffalo calves were significantly (p<0.05) lower at 42nd day than the 14th, 21st and 28th day of the experiment. TG RDW-SD values were significantly (p<0.05) lower at 42nd day than the 14th, 21st and 28th day of the experiment. No significant (p<0.05) difference in values of RWD-CV and RDW-SD in broiler chickens under the hot-dry season were recorded by Habibu and coworkers (2014) with conclusion that this is due to enhanced erythropoiesis in broiler chickens under the hot-dry season.

In CG the PLT count of buffalo calves were significantly (p<0.05) higher at 21st day than the 0thday of the experiment. In control group the MPV of buffalo calves were significantly (p<0.05) lower at 42nd day than the 14th, 21st and 28th day of the experiment. Similar trend was found in melatonin treated group. In CG the PCT were significantly (p<0.05) higher at 21st day than the 0th and 42nd day of the experiment. In a study of effects of melatonin application on some haematological parameters, testosterone and thyroid hormones in male goats, it has been found that the number of erythrocyte, leukocyte and haematocrit values were similar in control as well as treatment group (Donmez et al., 2004). Ahmed (2011) reported melatonin administration in chicks improve the health and immune status of the chicks by increasing in RBCs count, PCV, Hb concentration, total leukocytic count and lymphocyte percentage. The increase in RBCs count, PCV and Hb concentration obtained may be attributed either to its direct stimulatory effect on bone marrow. Anwar et al., 1998 found that melatonin treatment in rats numerically increased RBC, Hb and PCV. On the other hand Durotoye and Rodway (1996) proved that subcutaneous implants of melatonin in ewes reduced RBC count and PCV.


It could be concluded from the result of this study that, melatonin affects the haematological parameter to counteract the summer stress condition in animals.


Authors are highly thankful to Director, I.V.R.I. for providing facilities to conduct the research work. Financial support from NICRA and UGC-RGNF fellowship are duly acknowledged.


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