Effect of seasonal variations on the haematochemical profile of Cholistani service bulls

ABSTRACT

This reported study is of Pakistan's indigenous Cholistani cattle breed, a native of the Cholistan desert where it has been reported as having high production potential for nomadic herders. The aim of the present study was to assess haematochemical profile of adult Cholistani service bulls (n = 16), being reared at the Semen Production Unit Karaniwala, Bahawalpur, Cholistan desert, Pakistan, during various seasons as an indicator of their health status. Keeping in view the prevailing climatic conditions of the study area, four seasons of three months duration each were defined as (i) cool dry winter (November, December, January) (ii) temperate spring (February, March, April) (iii) hot dry summer (May, June, July) and (iv) hot humid summer (August, September, October). Blood specimens were collected fortnightly during the study period for haematochemical analyses (automated). The present study revealed that Cholistani service bulls have a tendency to maintain most of their haematochemical parameters more or less at the same level during various seasons which is suggestive of their adaptability under stressful climates without showing any signs of stress.

KEYWORDS:

• Cholistan
• Pakistan
• seasonal variations
• haematochemistry

1. Introduction

Pakistan is blessed with 15 recognized indigenous breeds of cattle which constitute 43% of the total cattle population in the country and belong to Zebu (one-humped) cattle (Bos indicus) (Khan et al. 2008). Extensive work has been carried out on various aspects of Sahiwal and Red Sindhi breeds of cattle from Pakistan under local climatic conditions. However, many other indigenous breeds such as Cholistani cattle being reared by the desert nomads of Cholistan desert, Pakistan, still remain unearthed regarding the manipulation of their productive and reproductive traits, thermo-tolerance and physiological indices.

Following a global lead in upgradation/propagation of the indigenous breeds, our group is the first to describe certain phenotypic, productive and reproductive traits of this formerly neglected breed of cattle from Pakistan (Farooq et al. 2010a, 2012a, 2013a, 2013b, 2015). It is a large-sized flabby breed with small horns, long ears, well-developed hump in males and large dewlap in both sexes. Owing to its speckled red, black or brown body it is termed as ‘flea-bitten’ by the nomads of Cholistan (Figures 1 and 2). The switch of the tail is black and the elite specimen of Cholistani cows produce 15–20 litres of milk daily. The average body weight recorded in a preliminary data obtained from the Government Livestock Farm, Jugait Peer, Bahawalpur, varied from 500 to 600 and 300 to 400 kg in male and female cattle, respectively (Farooq et al. 2010a). This breed has been incorporated in Livestock Census 2006 of Pakistan for the first time to stay as a breed (GOP 2006).

Figure 1. Elite specimen of a brown speckled Cholistani cow with the milk yield of 15–18 L per day maintained at the Govt. Jugaitpir Farm, Bahawalpur, Pakistan (Cholistan).

Figure 2. An elite specimen of a black speckled Cholistani service bull maintained at the Semen Production Unit, Bahawalpur, Pakistan (Cholistan).

The haematochemical profile of an animal provides a reliable diagnostic tool for assessing the level of stress in an animal and its health status (Fadare et al. 2012). Seasonal variations in these attributes have extensively been studied in various breeds and species (Satue & Munoz 2009; Shehab-el-deen et al. 2010; Farooq et al. 2011, 2012b) and alterations in them have been attributed to endogenous adaptive mechanisms (Zecchini et al. 2009). However, to the best of our knowledge, no such work has been reported for zebu service bulls being used for semen collection at artificial insemination centres. We have earlier reported seasonal variations in certain reproductive traits of adult Cholistani service bulls which indicated the innate adaptability of the breed to the harsh desert climate (Farooq et al. 2013a, 2013b, 2013c). The present work is a sequel to our continued research on these service bulls. The specific objective was to assess the effect of seasonal variations under local climatic conditions as an indicator of their health status. Though the number of reference individuals is less, these results could be used as a baseline data until a broad-scaled study on a larger population of this breed is carried out.

2. Materials and methods

2.1. Study area and seasons

The present study was conducted at the Semen Production Unit, Bahawalpur, located in the west of Cholistan Desert, Pakistan. Sprawling over an area of 26,000 Km², this desert is located at latitudes 27°42′and 29°45′North and longitudes 69°52′and 75°24′East and at an altitude of about 112 m above sea level (Farooq et al. 2010a). The climate of this area is arid subtropical and continental with low/sporadic rainfall, high temperature, low relative humidity, high rate of evaporation and strong summer winds. It is considered as one of the driest and hottest areas of Pakistan with an extensively lengthy summer season extending from May till October. The early part (hot dry summer) extends from May to July with maximum temperature exceeding 45°C. In the later part (hot humid summer) which extends from August to October, monsoon winds bring in negligible rains. Thus, the area is least influenced by rains and remains water scarce and drought stricken. November, December and January are the coldest months with the temperature ranging between 17°C and 20°C (Farooq et al. 2013c).

The study was conducted over a year spanning from November 2012 to October 2013. For defining the seasons of the present study in the study area, climatic data (ambient temperature, relative humidity and rainfall) were collected on a daily basis from the Meteorological Observatory located in Bahawalpur at latitude 29°20′ and longitude 71°47′ which comes under the administrative control of the Regional Meteorological Centre, Lahore, Pakistan. Hence, four seasons of three months' duration each were defined as (i) cool dry winter (November, December, January) (ii) temperate spring (February, March, April) (iii) hot dry summer (May, June, July) and (iv) hot humid summer (August, September, October) according to the average monthly climatic data.

2.2. Experimental bulls and sample collection

The study included 16 clinically healthy adult Cholistani service bulls, ranging between 6 and 10 years of age and having a mean body weight of 527.55 ± 4.5 kg (Figures are standard error of mean). They were housed individually in 10 × 12 m separate pens with a 4 × 4 m shelter at the centre. These bulls had clinically normal reproductive tract and were donating semen of acceptable quality for artificial insemination. Their feeding regimen included good-quality seasonal fodder at the rate of 10% of their body weight, with 3 kg of concentrate (Anmol wanda^®) (Table 1) per bull per day as per the recommendations provided by the Livestock & Dairy Development Department, Punjab, Pakistan. Water was provided ad libitum. Vaccination against Haemorrhagic Septicaemia and Foot and Mouth Disease was carried out as per schedule. Preventive measures against worm infestation were undertaken twice in a year or whenever felt necessary (Farooq et al. 2013b).

Table 1. Ingredients and nutritive value of concentrate (Anmol Wanda^®) fed to the Cholistani service bulls.

Ingredients	Inclusion level (%)
Cotton seed cake	18
Maize gluten 30	15
Canola meal	10
Maize grain broken	14
Wheat bran	25
Mollasses	16
Mineral mixture^a	2
Total	100
Nutritive value
CP	16
TDN	68

^a100 kg mineral mixture included: DCP 70.81 kg, NaCl 18.91 kg, MgSO₄ 8.64 kg, FeSO₄ 0.89 kg, ZnSO₄ 0.22 kg, CuSO₄ 0.03 kg, KI 8.77 g, CoCl₂ 0.89 g and NaSiO₃ 1.50 g.

For the purpose of sampling, animals were restrained in the cattle crush by trained personnel and fortnightly blood collection was carried out aseptically from the coccygeal vein, a day before weekly semen collection. A total of 384 blood samples, with 24 samples per animal were collected (6 samples/animal.season). The same technique, timing and personnel for blood collection (04:00 pm) were implemented in order to minimize the stress to the animal. Blood was stored in two aliquots: clotted for harvesting of serum and un-clotted for haematological analysis. Serum samples were stored at −24°C until analysed.

2.3. Haematological studies

An automated Hematology Analyser (Sysmex K21, Kobe, Japan) was used for determination of various haematological indices such as haemoglobin concentration, PCV, Total Erythrocyte Count (TEC) and Total Leukocyte Count (TLC). Blood smears stained with Wright's stain were simultaneously prepared for Differential Leukocytic Count (DLC). The Mean Corpuscular Volume (MCV), Mean Corpuscular Hemoglobin (MCH) and Mean Corpuscular Hemoglobin Concentration (MCHC) were calculated using the formula of Jain (1998).

The hematology analyser was originally meant for human application. Hence, before analysis of study samples, it was validated against blood samples from 100 dogs and 100 cows which were analysed through the analyser as well as with manual reference methods (cynmethemoglobin photometry, haematocrit analysis and haemocytometry) (Wassmuth et al. 2011). To determine analytical accuracy, agreement was determined by comparing results of the evaluated instrument with those of manual methods and reference intervals.

2.4. Serum biochemical analysis

Regarding the biochemical analysis of serum, sodium (Na⁺), potassium (K⁺), glucose and cholesterol were determined through Metrolab 1600 DR Clinical Analyzer using commercial kits as given below:

• Na⁺: Greiner Diagnostic Kits (GA 200; CAT 171141), Greiner Diagnostic Group, Germany

• K⁺: Greiner Diagnostic Kits (GA 200; CAT 161141), Greiner Diagnostic Group, Germany

• Glucose: Glucose (CAT CS605), Crescent Diagnostics, Saudi Arabia

• Cholesterol: Cholesterol liquicolor (CAT 10017), Human Diagnostics Worldwide, Germany

2.5. Statistical analysis

Statistical analysis was conducted through Statistical Package for Social Science (SPSS for Windows version 12, SPSS Inc., Chicago, IL, USA). Mean values (±SEM) were calculated for haematochemical attributes. Influence of season on various attributes was deducted through one-way ANOVA and the difference between mean values was compared through Duncan's multiple range test.

3. Results and discussion

The data regarding climatic conditions of Cholistan desert, Pakistan, during the study period are presented in Table 2. The variations are typical of dry arid, tropic desert with the highest (42.8°C) and lowest mean temperatures (13.2°C) recorded for hot dry summer and cool dry winter, respectively. Highest mean humidity (63%) was recorded for hot humid summer being lowest in hot dry summer (37%). Similarly, highest mean rainfall (72 mm) was recorded for hot humid summer whereas no rain was noticed in temperate spring.

Table 2. Average seasonal values for climatological data of Cholistan desert during the study period retrieved from the Meteorological Observatory located in Bahawalpur at latitude 29°20′ and longitude 71°47′, Pakistan.

Seasons	Ambient Temperature (°C)			Relative Humidity (%)			Rainfall (mm)
	Mean	Min^a	Max^b	Mean	Min	Max	Mean	Min	Max
Temperate Spring^c	25.4	17.3	31.3	49	37	50	0	0	0
Cool Dry Winter^d	13.2	11.2	17.8	47	38	48	2.5	0	3.5
Hot Dry Summer^e	42.8	35.7	49.8	37	25	41	3	1	4
Hot Humid Summer^f	34.0	28.9	37.2	63	47	67	72	14	87

^aMin – Minimum.

^bMax – Maximum.

^cFebruary, March, April.

^dNovember, December, January.

^eMay, June, July.

^fAugust, September, October.

The result of salient haematological parameters of the study as affected by seasons is presented in Table 3. The red blood cell parameters indicated that none of the indices were altered except for haemoglobin concentration which was significantly decreased (P < .05) during cool dry winter as compared to that in the remaining three hotter seasons. Variable reports have been published regarding the seasonal influence on haemoglobin. Higher concentration during hot dry and hot humid summer seasons has been reported previously (Kumar & Pachauri 2000) for dairy cattle of central Himalayas which is in contrast to the results of the present study. An older work has reported a reduction in haemoglobin concentration during summer (Salah et al. 1990) which was attributed to excessive water intake during summer stress which leads to haemodilution and hence a decreased haemoglobin concentration. Still another study could not assess any effect of season on blood haemoglobin concentration (Aengwanich et al. 2009). The mechanism of effect of seasons on haemoglobin level as affected by seasons is still unclear. However, energy shift during various seasons in order to maintain physiological processes to control heat dissipation might be a plausible justification (Scharf et al. 2010; Fadare et al. 2012).

Table 3. Seasonal variations in haematological parameters of Cholistani service bulls (n = 16).

Parameters	Temperate Spring	Cool Dry Winter	Hot Dry Summer	Hot Humid Summer	Overall Mean
Haemoglobin (g/L)	120.6 ± 0.2^b	110.4 ± 0.3^a	120.0 ± 0.2^a,b	120.7 ± 0.2^b	120.2 ± 0.1
Total erythrocytic count (10^12/L)	6.5 ± 0.1^a	6.0 ± 0.1^a	6.1 ± 0.1^a	6.4 ± 0.14^a	6.2 ± 0.8
Packed cell volume (%)	37.7 ± 0.8^a	36.9 ± 0.8^a	36.6 ± 0.8^a	37.8 ± 0.6^a	37.3 ± 0.4
Mean corpuscular volume (fL)	58.0 ± 0.7^a	61.6 ± 1.7^a	59.9 ± 1.0^a	59.4 ± 1.1^a	59.7 ± 0.6
Mean corpuscular haemoglobin (pg)	19.5 ± 0.2^a	19.1 ± 0.6^a	19.7 ± 0.2^a	19.9 ± 0.2^a	19.5 ± 0.2
Mean corpuscular haemoglobin concentration (g/L)	330.6 ± 0.1^b	310.2 ± 1.1^a	330.0 ± 0.3^a,b	330.6 ± 0.3^b	320.8 ± 0.3
Total leukocytic count (10^9/L)	7.7 ± 0.3^a,b	7.0 ± 0.3^a	8.1 ± 0.2^b	7.3 ± 0.3^a,b	7.5 ± 0.1
Neutrophils (%)	44.4 ± 1.4^b	45.6 ± 0.8^b	29.7 ± 2.0^a	42.0 ± 1.7^b	40.4 ± 1.0
Lymphocytes (%)	51.0 ± 1.2^a	51.9 ± 0.8^a	57.7 ± 1.7^b	52.1 ± 2.3^a	53.2 ± 0.8
Monocytes (%)	2.9 ± 0.2^a,b	1.4 ± 0.2^a	7.8 ± 0.9^c	3.9 ± 1.1^b	4.0 ± 0.4
Eosinophils (%)	1.6 ± 0.2^a	1.0 ± 0.1^a	4.6 ± 0.6^b	1.8 ± 0.5^a	2.3 ± 0.2

Figures are standard error of the mean.

^a,bwithin rows means with different superscript letters differ (P < .05).

No significant effect (P > .05) of season was noticed on TEC, PCV, MCV and MCH. On the contrary, a few earlier published reports have demonstrated an increase in TEC due to heat stress in sheep (Fadare et al. 2012). This has been attributed to an adaptive mechanism in response to heat stress. The non-significant change in TEC, PCV, MCV and MCH during various seasons in the present study is suggestive of adaptability of Cholistani bulls to the harsh climatic conditions without being under stress (Farooq et al. 2010b).

Seasonal variation had a significant effect (P < .05) on all of the WBC parameters in the present study (Table 3). Significantly higher TLC was observed in hot dry summer compared to that in cool dry winter which is in line with a previous report for African buffaloes (Beechler et al. 2009). This increase could either be a stress-induced leukocytosis (owing to high summer temperature) which causes epinephrine release and consequently leukocytosis in cattle or an indication of ‘capture stress’ (Sajjad et al. 2012) inferred upon the bulls while being brought from their pens to the blood collection site. Other reports have described a significant decrease in TLC during summer season in Hassawi cattle. (Al-Shami 2003). Yet another work revealed no influence of season on TLC in Brazilian cattle (McManus et al. 2009).

Differential leukocytic count revealed that lymphocytes were the predominant leukocytes, followed by neutrophils, which is typical for ruminants (Kumar & Pachauri 2000). Neutrophils were lower, while lymphocytes and eosinophils were significantly higher (P < .05) during hot dry summer compared to that in other three seasons; the latter three seasons differed non-significantly from one another. Monocytes were also higher during hot dry summer compared to that in other seasons; moreover, monocytes were also higher during hot humid summer than in winter (Table 3). This suggests that overall increase in TLC during dry summer was due to increased lymphocytes, monocytes and eosinophils.

In the present study, certain serum biochemical metabolites were also studied to identify the potential markers of health status across various seasons in Cholistani service bulls and the results are presented in Table 4. Sodium revealed significantly higher (P < .05) levels during the temperate spring season being lower in the remaining three seasons. This is in line with the previous work published for sheep (Fadare et al. 2012). Summer and winter stress leads to an increased water intake, expanded blood volume and hence decreased cation (Na⁺) levels (Farooq et al. 2010b; Fadare et al. 2012). On the other hand, serum K⁺ was significantly higher (P < .05) in cool dry winter, hot dry summer and hot humid summer compared to that in temperate spring which is not in accordance with previously reported work (El-Nouty et al. 1980; Salah et al. 1990; Srikandakumar & Johnson 2004). In another study on Australian milking zebu, a lower serum K⁺ concentration was noticed during hot dry summer (Srikandakumar & Johnson 2004) which was attributed to loss of K⁺ in sweat during dry summer (Salah et al. 1990). As the cattle contain apocrine glands, the secretions from the skin of cattle contain relatively more K⁺ than Na⁺ (El-Nouty et al. 1980; Farooq et al. 2010b). The present results could be an endogenous adaptive mechanism which prepares the animals for environmental alterations associated with various seasons (Casella et al. 2013). Further detailed studies need to be carried out for a clarified answer.

Table 4. Seasonal variations in blood serum constituents of Cholistani service bulls (n = 16).

Parameters	Temperate Spring	Cool Dry Winter	Hot Dry Summer	Hot Humid Summer	Overall Mean
Sodium (mEq/L)	137.7 ± 1.3^b	131.1 ± 0.8^a	127.4 ± 1.5^a	126.7 ± 2.2^a	130.7 ± 0.9
Potassium (mEq/L)	6.9 ± 0.2^a	7.7 ± 0.1^b	7.8 ± 0.2^b	7.5 ± 0.09^b	7.3 ± 0.1
Cholesterol (mEq/L)	2.0 ± 1.8^a,b	2.3 ± 1.3^c	1.9 ± 4.3^a	2.2 ± 2.7^b,c	2.1 ± 1.5
Glucose (mEq/L)	1.93 ± 2.8^b	2.4 ± 4.5^c	1.54 ± 2.2^a	1.85 ± 1.2^b	1.94 ± 2.0

Figures are standard error of the mean.

^a,b,cwithin rows means with different superscript letters differ (P < .05).

In the present study, serum cholesterol was significantly higher (P < .05) during cool dry winter compared to the remaining three seasons. An increased level of serum cholesterol in cattle under stressful situations, being influenced by the degree of stress has been reported earlier (Shaffer et al. 1981; Fadare et al. 2012). The results of the present study regarding lower serum cholesterol during hot dry summer are consistent with the hypothesis that the experimental animals were adequately adapted to the harsh summer environment and hence did not show elevated serum cholesterol as mostly seen in stressful summer situations (Scharf et al. 2010). However, increased serum cholesterol was also observed during cool dry winter, the physiological significance of which remains unclear. Similar results were noticed for glucose level in the present study, which was significantly higher (P < .05) in cool dry winter season and lowest in hot dry summer. No effect of season on glucose level has been reported in heat-stressed Bos indicus cattle, suggesting it as a caloric balance in thermally adapted breeds (Scharf et al. 2010).

4. Conclusion

In a nutshell, the present study revealed that the Cholistani service bulls have a potential to maintain most of their haematochemical parameters more or less at the same level without showing much variation during various seasons. This is indicative of their adaptability to stressful climatic conditions without showing any signs of stress. Furthermore, the values for haematochemical attributes studied in this work can be considered as baseline reference values for breeding Bos indicus bulls as a monitor of their health status. However, only 16 Cholistani bulls were included in the present study. The haematochemical profile needs further elaboration with a larger population and sample size in relation to age and sex differences with special consideration to the reproductive attributes of this breed.

Acknowledgements

This study was part of a PhD research work of two of the co-authors and was funded by Higher Education Commission (HEC) of Pakistan under its Indigenous PhD Scholarship Scheme.