Content uploaded by Michela Toro
Author content
All content in this area was uploaded by Michela Toro on Nov 28, 2023
Content may be subject to copyright.
Journal of Buffalo Science, 2023, 1 2, 111-116 111
ISSN: 1927-5196 / E-ISSN: 1927-520X/23
Hematological and Biochemical Parameters in Prepubescent Water
Buffaloes (Bubalus bubalis)
M. Toro, G.C. Telera, N. D’Aurelio*, F. Sacchini, N. Ferri, E. Rossi, F. Perletta, M. Tittarelli,
D. Giansante and F. De Massis
Experimental Zooprophylactic Institute of Abruzzo and Molise “G.Caporale”, Teramo, Italy
Abstract: Water buffaloes are bred in Europe, Australia, North America, South America, and some African countries.
European water buffaloes are all of the river type and are considered to be of the same breed named the Mediterranean
water buffalo. This species plays a key role in milk production in Italy. Buffalo breeding for milk production has
represented an important economic value in recent years, even in Italian regions where this activity was not historically
rooted. Therefore, it is very important to monitor animal health and welfare by performing periodical clinical examinations
combined with different hematological and biochemical tests. However, there are few studies regarding these aspects,
as well as research aimed at establ ishing the reference ranges for species. This study aims to define hematol ogical and
biochemic al parameters in a population of healthy water buffalo in controlled conditi ons for over a y ear in
Italy.
Keywords: Water buffalo, Bubalus bubalis, hematology, biochemical parameters, prepubescent water buffalo.
1. INTRODUCTION
The domestic water buffalo (Bubalus bubalis)
represents an important source of milk in many
European countries, especially in Italy. Buffaloes' milk
and milk products are particularly appreciated by
consumers [1]. In 2018, buffalo produced 127 million
tons of milk worldwide; in Europe, production was
estimated at 390,000 tons, of which 378,000 tons have
been produced in Italy [2]. From 2015 to 2019, the
buffalo population in Italy increased by 7% [3-5]. The
buffalo breeding, aimed at milk production, represents
an important economic resource, even in regions
where this activity was not historically rooted. The
buffalo mozzarella, recognized under the EU’s
Protected Designation of Origin (PDO) scheme, is
estimated at a sales value of 766 million Euros [6].
Proper management of these animals would produce
individual water buffaloes with high genetic and health
quality. In this context, studies are encouraged to
establish reference values for hematological and
biochemical parameters in this species. This is justified
by the need to investigate possible parameter
variations, such as health, nutrition, and breeding
systems. These data may be used in clinical practice,
as well as a tool for assessing the health and welfare of
these animals. Few studies are available in the
international literature aimed at determining the
hematological and biochemical parameters of water
buffaloes [7]. The study aimed to investigate
hematological and biochemical parameters in
*Address correspondence to this author at the Experimental Zooprophylactic
Institute of Abruzzo and Molise “G.Caporale”, Teramo, I taly;
E-mail: nausicadaurel io@gmail.com
prepubescent water buffaloes to establish reference
values for functional health and welfare evaluations.
2. MATERIALS AND METHODS
2.1. Animals under Study
Twenty-six buffaloes, 4-6 months old (15 females
and 11 males), were enrolled in the study (Table 1). All
individuals were born in captivity and were used to
frequent handling. Clinical evaluation was performed by
general inspection, palpation, and the measurement of
heart and respiratory functions. Cardiac and pulmonary
functions were measured by hearing accurately to
identify sounds of the heart and lungs, using a
stethoscope (Littmann® Classic II, 3M™ USA) at the
apex of the heart and on the whole lung area. The
animals were preliminarily subjected to serological
tests for antibodies against Brucellosis, Chlamydiosis
(Chlamydia psittaci), Parainfluenza 3, Bovine Viral
Diarrhea (BVD), Q fever (Coxiella burnetii) and Yersinia
enterocolitica O:9. The trial was carried out in the
premises of the Istituto Zooprofilattico Sperimentale
dell’Abruzzo e del Molise “G. Caporale”, IZS-Teramo,
Italy) where it is a duly authorized premise pursuant to
Italian law (D. Lgs. 26/2014) regulating the use of
animals in experimental conditions (Figure 1). The
animals were housed in standard rearing
environments, with ad libitum feeding and drinking
water. The buffaloes were fed three times a day with a
mix of complete feed for buffaloes, in addition to hay
(mixed first and second cut), always available ad
libitum (Table 2).
112 Journal of Buffalo Science, 2023, Vol. 12 Toro et al.
Table 1: Buffaloes Subjected to Blood Sampling and their Gender, Age at the beginning of the Observation, and Age
at the End of the Study Observation
ID
Gender
Date of Birth
Months of age at the beginning
of observatio n
Months of age at the end of
observation
*609
F
27/12/2019
7
19
*610
M
28/12/2019
7
19
*612
F
01/01/2020
7
19
*613
F
02/01/2020
7
19
*615
M
02/01/2020
7
19
*616
M
04/01/2020
7
19
*617
F
05/01/2020
7
19
*618
F
06/01/2020
7
19
*619
F
07/01/2020
7
19
*621
F
07/01/2020
7
19
*622
M
08/01/2020
7
19
*623
M
08/01/2020
7
19
*624
F
10/01/2020
7
19
*626
F
11/01/2020
7
19
*627
M
11/01/2020
7
19
*628
F
13/01/2020
7
19
*629
F
16/01/2020
7
19
*630
M
16/01/2020
7
19
*631
F
17/01/2020
7
19
*632
M
19/01/2020
7
19
*634
M
22/01/2020
7
19
*637
F
25/01/2020
7
19
*641
F
02/02/2020
7
19
*644
M
07/02/2020
7
19
*649
M
17/02/2020
7
19
*653
F
24/02/2020
7
19
Figure 1: Image of the water buffaloes enrolled in the study
standing on the premises where the observation was carried
out.
Table 2: Nutritional Value of Feed Administered to the
Buffaloes under Observation
Crude protein
20.5%
Crude fats
4.3%
Crude fiber
8.6%
Crude ash
7.9%
Sodium
0.4%
2.2. Sampling Methods and Frequency
Animals were observed for 12 months (August
2020-August 2021). Blood samples were taken from
the jugular vein using sterile syringes and needles,
using the Vacutainer TM system (VACUETTE®
Multiple Use Drawing Needles 20G, Greiner Bio-One
International GmbH, Cassina de Pecchi, Italy). Starting
from day T0, samples were collected from all animals
twice a week for the firs t 8 weeks (T0-T58); from week
Hematological and Biochemical Parameters in Prepubescent Water Journal of Buffalo Science, 2023, Vol. 12 113
9 to 16 (T62-T111), the samples were taken weekly.
From week 17 (T125) to the end of the observations,
blood samples were taken every 2 weeks. For
biochemical analysis, blood was collected in vacuum
tubes without anticoagulant, left at room temperature
for clot retraction, and centrifuged at 863 rcf (relative
centrifugal force) for 4 minutes. The sera collected
were frozen at -20 °C until analysis. For the
haemogram, blood was taken on a weekly basis from
T0 until the 16th week after, then every 2 weeks until
the end of observation.
2.3. Hematological Analyses
Blood counts were performed on blood samples
collected in EDTA vacutainer tubes (VACUETTE®
EDTA) and dispatched to the laboratory within 24 hours
after collection, stored at 4 °C ± 2 °C. Hematological
analyses were performed at the IZS Teramo branch
laboratory of Pescara, Italy, using commercial kits
(Advia, Siemens, Bayer Diagnostics, NY, USA) and an
ADVIA 120 hematology system (Siemens, Bayer
Diagnostics, NY, USA), equipped with specific software
for veterinary use. The following values were
determined: red blood cell count (RBC), volume
distribution width (RDW), hemoglobin (HGB), mean
corpuscular hemoglobin concentration (MCHC),
hemoglobin distribution width (HDW), mean
corpuscular hemoglobin (MCH), hematocrit (HCT),
mean corpuscular volume (MCV), white blood cells
count (WBC), lymphocytes, neutrophils, basophils,
eosinophils, monocytes. Hematological parameters
were measured according to Feldman B.F. et al. [8].
2.4. Biochemical Analyses
Biochemical analyses were performed on blood
samples collected in vacutainer tubes without
anticoagulant and dispatched to the laboratory within
24 hours after collection. Biochemical analyses were
carried out at the IZS-Teramo branch laboratory of
Pescara (Italy) and performed using commercial kits
(Quantilab Kits, Werfen Company, Milano, Italy) and an
ILAB 650 automated system (Instrumentation Labora-
tory, MA, USA). The following values were determined:
glucose, blood urea nitrogen (BUN), creatinine, uric
acid, total bilirubin, total protein, albumin, calcium,
cholesterol, triglycerides, amylase, alkaline phos-
phatase (ALP), aspartate-transaminase (GOT/AST),
alanine - aminotransferase (GPT/ALT) and gamma –
glutamyl-transferase (GGT). Biochemical parameters
were measured according to Sodikoff C.H. [9].
2.5. Statistical Analysis
The software used for statistical calculation was
SPSS version 20 (SPSS for Windows, version 20.0;
SPSS Inc., Chicago, IL, USA). Descriptive statistics
were used to summarize the data, including median,
2.5, and 97.5 percentiles and minimal-maximum
values.
Table 3: Hematological Parameters
HAEMATOLOGIC AL VALUES
Reference UNIT
Median
Percentile 2.5
Percentile 97.5
Minimum
Maximu m
Red Blood Cells (RBC)
(x 106/µL)
7.1
5.9
8.2
5.4
8.9
Red Blood Cells Distribution Width (RDW)
(%)
19.1
17.3
21.5
16.4
23.6
Haemoglobin (HGB)
(g/dL)
12.0
9.9
14.5
9.0
16.2
Mean Corpuscolar Haemoglobin
Concentration (MCHC)
(g/dL)
41.7
39.9
43.8
38.5
44.6
Haemoglobin Distribution width (HDW)
(g/dL)
3.0
2.4
5.3
2.2
7.0
Mean Corpuscolar
(pg)
17.0
14.8
19.4
14.3
20.3
Hemoglobin (MCH)
Haematocrit (HCT)
(%)
28.7
23.5
35.1
22.1
40.6
Mean Cells Volume (MCV)
(fL)
40.8
34.9
47.7
33.9
50.1
White Blood Cells (WBC)
(x 103/µl)
12.4
8.0
16.9
6.7
19.7
Lymphocytes
(%)
61.4
50.4
72.1
43.6
82.3
Neutrophils
(%)
32.5
21.0
43.4
11.9
50.3
Basophils
(%)
1.3
0.9
2.2
0.7
4.0
Eosinophils
(%)
1.8
0.7
4.9
0.3
9.8
Monocytes
(%)
2.0
0.9
4.9
0.6
10.7
114 Journal of Buffalo Science, 2023, Vol. 12 Toro et al.
3. RESULTS
3.1. Hematological Analyses
Among the parameters considered, the 12-month
observation did not show significant changes in the
study period (August 2020-August 2021).
Hematological tests carried out during the study did not
highlight important changes during the observation
period. Results are reported in Table 3.
3.2. Biochemical Analyses
Among the parameters considered at the first
observation, no significant changes in the 12-month
study period were observed (August 2020-August
2021). Biochemical tests performed during the study do
not seem to highlight important changes related to
particular stressful situations, both infectious and
environmental. There were no other statistically
significant differences between males and females for
any other parameters considered. Results are reported
in Table 4.
4. DISCUSSION
The hematological and biochemical parameters
found in this study for water buffaloes are in
accordance with those already investigated by several
authors [8-12].
As regards the relation with age, Ciaramella et al.
observed lower levels of red and white blood cell count
from older to younger animals; in this case, this is due
to a reduced hematopoietic action by hormones, which
decreases with age. In fact, in our study, we did not
observe significant changes in these values just
because we focused on prepubescent buffaloes [14].
As regards the effects of gender on hematological
values, our results are in accordance with Patel et al.
study, which demonstrated no significant differences
between males and females in young buffaloes [16].
Furthermore, Patel et al. observed a significant
variation of glucose, total protein, total cholesterol,
creatinine, and BUN levels in relation to age and
gender and/or physiological status in adult buffaloes;
however, in the case of young buffaloes, the authors
found a non-significant variation of this values between
males and females [15].
The twenty-six buffaloes chosen for our study were
4-6 months old at the beginning of the observation
(lasted for one year), so the study's results are focused
on prepubescent animals. According to previous
studies [13-15], it was observed that, in the case of
prepubescent animals, the variations are not significant
between males and females.
Moreover, water buffaloes (like other animal
species) generally have almost the same metabolism
Table 4: Biochemical Parameters
BIOCHEMICAL VALUES
Reference UNIT
Median
Percentile 2.5
Percentile 97.5
Minimum
Maximu m
Glucose
(mg/dL)
61.0
37.0
85.0
2.0
95.0
BUN
(mg/dL)
21.0
10.0
33.0
6.0
47.0
Creatinine
(mg/dL)
1.2
0.8
1.9
0.5
2.5
Uric Acid
(mg/dL)
0.8
0.4
1.2
0.3
1.5
Total Bilirubin
(mg/dL)
0.1
0.0
0.3
0.0
0.4
Total Protein
(g/dL)
6.2
5.6
6.8
5.2
7.5
Albumin
(g/dL)
3.1
2.7
3.7
2.5
3.9
Calcium
(mg/dL)
10.3
9.5
11.5
9.2
12.3
Cholesterol
(mg/dL)
76.0
55.0
100.2
44.0
122.0
Triglycerids
(mg/dL)
33.0
17.0
59.8
12.0
73.0
Amylase
(IU/L)
3.0
0.0
0.3
0.0
0.4
ALP
(IU/L)
198.0
107.0
468.2
77.0
574.0
GOT (AST)
(IU/L)
153.0
111.8
263.0
94.0
528.0
GPT ALT
(IU/L)
62.0
42.0
80.2
30.0
96.0
GGT
(IU/L)
21.6
13.6
30.6
9.4
38.5
Hematological and Biochemical Parameters in Prepubescent Water Journal of Buffalo Science, 2023, Vol. 12 115
between males and females until sexual maturity.
Therefore, this study did not compare the results by
gender and age. Pregnancy and lactation are the two
most important stages in the life of dairy animals that
affect metabolism, resulting in the alteration of the
hematological parameters [16, 17]. However, these
aspects were not investigated, given the age of the
animals under study.
Among the parameters considered, the 12-month
observation (August 2020-August 2021) did not show
significant changes in the considered parameters
during the study period. In Italy, there is temperate
weather, so this may explain the absence of significant
changes across the different seasons. A one-year
monitoring period has allowed a broad observation of
the buffaloes' blood parameters, which made it
possible to evaluate various external factors, possibly
conditioning the metabolism of the animals. To the best
of our knowledge, this is the first study that considered
such long monitoring to identify the trend of blood
values over time in buffaloes.
The values identified by this study for RBC, RDW,
HGB, MCHC, HDW, MCH, HCT, MCV, WBC,
Lymphocytes, Neutrophils, Basophils, Eosinophils and
Monocytes are in accordance with previous reports
[14]. The levels identified for Glucose, BUN, Creatinine,
Uric Acid, Total Bilirubin, Total Protein, Albumin,
Calcium, Cholesterol, Triglycerides, Amylase, ALP,
GOT (AST), GPT (ALT), GGT do not vary significantly
between male and female calves, in agreement with
the observations of other studies [13]. All the
parameters remained stable during the one-year time
observation. As previously reported, some serum
enzyme values after the exercise could rise [18]. Also,
in our study, the value of transaminases (GOT (AST),
GPT (ALT), and GGT) are higher than previously
reported, and this could be related to the free
husbandry practice for buffaloes on the farm. This
condition allows the animals to walk and even run,
generating a muscular activity that may determine a
physiological increase in these enzymes in the blood.
5. CONCLUSIONS
The findings of this study serve as a valuable
reference tool for understanding the health and welfare
of prepubescent water buffaloes. It is important to
acknowledge that these markers were assessed under
specific conditions, and further evaluation under
varying circumstances, including different nutritional
and climatic stresses, is essential. These factors are
crucial in an animal's ability to adapt to its captive
environment. Additionally, variables such as age,
nutrition, management conditions, and housing can
significantly influence the hematological and
biochemical parameters of water buffaloes.
In conclusion, this study underscores the need for
continued research to expand our understanding of
water buffalo health and well-being. Future
investigations should encompass a wider range of
conditions and variables to promote the establishment
of optimal welfare standards and facilitate a more
comprehensive interpretation of hematological and
biochemical values in water buffaloes. Such endeavors
will contribute to the overall betterment of their care
and management.
ACKNOWLEDGEMENT
The Authors would like to thank the Italian Ministry
Of Health for the financial support grant code IZSAM
09/19 RC.
REFERENCES
[1] Borghese A, Moioli B. Buffalo: Mediterranean region. In
Encyclopedia of Dairy Sciences, 2nd ed. (Fuquay JW, Fox
PF, McSweeney PLH, Eds). Academic Press, London, 2011;
pp. 780-784.
https://doi.org/ 10.1016/B978-0-12-374407-4.00230-2
[2] Brescia MA, Monfreda M, Buccolieri A, Carrino C.
Characterisation of the geographical origin of buffalo milk
and m ozzare lla cheese by means of analytical and
spectroscopic determinations. Food Chem 2005; 89: 139-
147.
https://doi.org/ 10.1016/j.foodchem.2004.02.016
[3] Dati Forniti dalla BDN Dell'anagrafe Zootecnica Istituita dal
Ministero della Salute presso il CSN d ell'Is titut o "G .
Caporale" di Teramo. SND National Database of Istituto
Zooprofilattico Sperimentale Dell'Abruzzo e del Molise
"Giuseppe Caporale". https://www.vetinfo.it/ (accessed on
December 31, 2019). (In Italian) Animals 2021, 11, 1737 10
of 10
[4] Naveena BM, Kiran M. Buffalo meat quality, composition,
and processing characterist ics: Contribution to the global
economy and nutritional sec urity. Anim Fr ont 2014; 4: 18-24.
https://doi.org/ 10.2527/af.2014-0029
[5] Cappelli G, Di Vuolo G, Gerini O, Noschese R, Bufano F,
Capacchione R, Rosini S, Limone A, De Carlo E. Italian
Tracing System for Water Buffalo Milk and Processed, Milk
Products. Animals 2021; 11: 1737.
https://doi.org/ 10.3390/ani1106173 7
[6] ISMEA. QUALIVITA sulle produzioni agroalimentari e
vitivinicole italiane DOP, IGP e STG. Ed Qualivita Fond
Qualivita. ISMEA: Istituto de Servizi Per Il Mercato Agricolo
Alimentare (2019). Available online at:
https://www.qualivita.it/ (accessed May 28, 2020)
[7] Rocha TB, et al. Haematology and biochemistry of buffalo
(Bubalus bubalis): influence of sex and age on r eference
values. Tropical Animal Health and Production 2021; 53(2):
273.
https://doi.org/ 10.1007/s112 50-021-0272 7-x
116 Journal of Buffalo Science, 2023, Vol. 12 Toro et al.
[8] Feldman BF, Zinkl JG, Jain CJ, Shalm’s Veterinary
Hematology 7th Edition, Wiley Blackwell, Canada 2022; p.
1393.
[9] Sodikoff CH. Laboratory Profiles of Small Animal Diseases –
A guide to laboratory diagnosis 3rd edition, Mosby Inc., Saint
Louis (US A) 20 01; p. 594.
[10] Torres-Chable OM, et al. Hematologic RIs for healthy water
buffaloes (Bubalus bubalis) in southern Mexico. Vet erinary
Clinical Pathology 2017; 46: 3 436-441.
https://doi.org/ 10.1111/vcp. 12508
[11] Canfield PJ, Best FG, Fairburn AJ, Purdie J, Gilham M.
Normal haematological and biochemical values for the
Swamp buffalo (Bubalus bubalis) in Australia. Aust Vet J
1984; 61: 89-93.
https://doi.org/ 10.1111/j.1751-0813.1984.tb15525.x
[12] Abd Ellah MR, Hamed MI, Ibrahim DR, Rateb HZ. Serum
biochemic al and haematological refere nce intervals for water
buffalo (Bubalus bubalis) heifers. Journal o f t he South
African Veterinary Association 2014; 85(1): e1-e7.
https://doi.org/10.4102/jsava.v85i1.962
[13] Jain NC, Vegad JL, Jain NK, Shrivastava AB. Hematological
studies on normal lactating Indian Water buffaloes. Res Vet
Sci 1982; 32: 52-56.
https://doi.org/ 10.1016/S0034-5288(18)32437 -8
[14] Ciaramella P, Corona M, Ambrosio R, C onsalvo F,
Persechino A. Haematological profile on non-lactating
Mediterranean buffaloes (Bubalus bubalis) ranging in age
from 24 months to 14 years. Res Vet Sci 2005; 79: 77-80.
https://doi.org/ 10.1016/j.rvsc.2004.11.004
[15] Patel MD , Lateef A, Das H, Prajapati MV, Kakati P, Savani
HR. Estimation of blood biochemical parameters of Banni
buffalo (Bubalus Bubalis) at different age, s ex, and
physiological stages. Journal of Livestock Science 2016; 7:
250-255.
[16] Patel MD, Lateef A, Das H, Patel AS, Patel AG, Joshi A B.
Effect of age, sex, and physiological stages on
haematol ogical indices of Banni buffalo (Bubalus bubalis).
Veterinary World 2016; 9(1): 38-42.
https://doi.org/ 10.14202/vetworld.2016.38-42
[17] Cenesiz M, Cenesiz S, Yarim GF, Nisbet C. Changes in
hematolo gic and bioche mical values of water buffalo in
different ages, sexes, and pregnancy bred in Samsun
province. YYU Veterinary Fak ultesi Dergisi 2011; 22(1 ): 1-4.
[18] Yaqub LS, Kawu MU, Ayo JO. Influence of reproductive
cycle, sex, age and season on haematological parameters in
domestic animals: A rev. J C ell Anim Biol 2013; 7(4): 37-43.
https://doi.org/ 10.5897/JCAB 12.063
Received on 09-08-2023 Accepted on 01-11-2023 Published on 20-11-2023
https://doi.org/ 10.6000/1927-520X.2023.12.13
© 2023 Toro et al.; Licensee Lifescience Global.
This is an open-access article licensed under the terms of the Creative Commons Attribution License
(http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium,
provided the work is properly cited.
