Research in Veterinary Science 138 (2021) 53–61

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Research in Veterinary Science

journal homepage: www.elsevier.com/locate/rvsc

Weaning at 30, 75 and 180 days: Comparison between immune responses

of beef calves
Odilene de Souza Teixeira a, *, Marcela Kuczynski da Rocha a, Amir Gil Sessim a,
Everton Dezordi Sartori a, Yago Machado da Rosa a, Maria Carolina Muniz de Oliveira a,
Julia Abud Lima a, Maria Eugênia Andrighetto Canozzi b, Jaime Urdapilleta Tarouco a,
Stella de Faria Valle c, Concepta McManus d, Júlio Otávio Jardim Barcellos a
a
Department of Science Animal, Universidade Federal do Rio Grande do Sul, Ave. Bento Gonçalves, 91540000 Porto Alegre, Brazil
b
Instituto Nacional de Investigación Agropecuaria, Programa Producción de Carne y Lana, Estación Experimental INIA La Estanzuela, Ruta 50 km 11, 39173, Colonia,
Uruguay
c
Department of Pathology and Veterinary Clinic, Universidade Federal do Rio Grande do Sul, Ave. Bento Gonçalves, 91540000 Porto Alegre, RS, Brazil
d
Institute of Biological Sciences, Universidade de Brasília, 70910-900 Brasília, DF, Brazil

A R T I C L E I N F O A B S T R A C T

Keywords: The interruption of the maternal-filial bond in young calves can alter the physiological responses due to
Age at weaning increased stress at this moment. Therefore, we aimed to analyze the association between age at weaning and the
Beef cattle immune responses of 64 beef calves weaned abruptly. The animals were distributed in three weaning (W)
Cortisol
treatments: hyper-early (W30), early (W75) and conventional (W180). The treatments agree with the weaning
Hematology
Stress
ages used in cow-calf systems in South America. Blood samples were collected of all experimental animals on the
day of weaning and days 1, 2 and 7 post-weaning day of the three treatments. Blood samples were used to
analyze hematological variables, total plasma proteins (TPP), fibrinogen, serum amyloid A protein (SAA) and
cortisol. The variables analyzed were influenced by the sampling day (P < 0.001) and the calf gender (P < 0.05),
and an interaction between treatment x sampling day was observed. Over the study period, high serum cortisol
levels were observed for W30 calves. The serum amyloid A protein was significantly influenced by sampling day.
When compared to W180 calves, W30 and W75 animals showed prolonged concentrations in plasma fibrinogen.
In addition, they presented neutrophilia and lymphopenia during the 180–187 days period, which resulted in a
greater neutrophil:lymphocyte ratio (N:L ratio). Calf gender resulted in changes in the number of blood leuko­
cytes (P < 0.05). This result provides an alert for weaning at an early age, indicating that different management
strategies must be tested to minimize physiological instabilities in critical periods of the life of animals.

1. Introduction hyper-early weaning, presents itself as one of the most precocious

The intensification of production in the cow-calf system and the
emergence of new nutritional technologies have made it possible to
improve management practices, such as weaning calves at early ages.
These techniques that aim to anticipate the weaning age are adopted to
optimize the reproductive indices in the cow-calf operation, mainly the
indicator of the pregnancy rate of the cow (Oliveira et al., 2018). Once
the maternal-filial bond is broken, the ovarian cycle of the cow can be
reestablished in a shorter time (Orihuela and Galina, 2019). This system
also provides the calves with a differentiated diet to prepare them for
rearing and finishing. Given the above, weaning at 30 days of age, called
techniques in beef cattle and has been used to improve efficiency within
the livestock industry.
Nevertheless, weaning is characterized by multiple stressors for the
calf (Lynch et al., 2010; Lynch et al., 2019). When recognized by the
central nervous system as threatening homeostasis, these stressors result
in a combination of biological defenses, presenting alterations in
behavior and in autonomic nervous, neuroendocrine and immune sys­
tems (Moberg, 2000). Thus, when the stress modifies neuroendocrine
system, consequently, the liberation of glucocorticoids occurs. In
sequence, there can be changes in the hematologic variables and serum
acute-phase proteins (O’Loughlin et al., 2014). These proteins are

* Corresponding author.
E-mail address: odilene.zootecnista@gmail.com (O. de Souza Teixeira).

https://doi.org/10.1016/j.rvsc.2021.06.002
Received 11 January 2021; Received in revised form 10 May 2021; Accepted 1 June 2021
Available online 2 June 2021
0034-5288/© 2021 Elsevier Ltd. All rights reserved.
O. de Souza Teixeira et al.
recognized as physiological indicators of stress, which are also sensitive
to the neuroendocrine-immune network (Murata, 2007).
In these circumstances, it is assumed that weaning younger calves
may result in even more marked changes in the immune system once
these animals experience severe stress conditions. The weaning at 30
days is characterized by increased vocalizations, cross-sucking, walking
activity and respiratory rate, and a lower percentage of time in the
feeder when compared to traditional weaning that occurs at 180 days of
the age of calves (Teixeira et al., 2021). Allied to this, it is also note­
worthy that, at the age of hyper-early weaning, there are important
changes in the development of the immune system. Between the third
and fourth week of life, immunity obtained from maternal antibodies
(passive immunity) is in decrescent levels, and the antibodies produced
by the own organism of calves (active immunity) are initiating their
responses (Hulbert and Moisá, 2016). Therefore, this period is charac­
terized as susceptible to diseases e may be severed by weaning stress
consequences.
In this sense, there is currently a gap in scientific research as it is not
known how these immunological changes occur in calves weaned at
young ages. Most research is based on later weaning from 89 to 250 days
of age (Blanco et al., 2009; Lynch et al., 2010; O’Loughlin et al., 2014).
In addition, more prolonged effects have not been described yet, as these
studies are limited to 35 days after weaning. Therefore, our study tests
the hypothesis that calves weaned at an early age may present unsatis­
factory immunological conditions resulting from physiological re­
sponses to stress. Over the long term, we objected to analyze the immune
responses in beef calves weaned at 30, 75 and 180 days once these ages
are adopted in the South American beef industry.
2. Material and methods
2.1. Ethics statement
The Ethics Committee on the Use of Animals from the Faculty of
Agronomy and Department of Animal Science, Federal University of Rio
Grande do Sul, Porto Alegre, Brazil, approved all management proced­
ures performed on calves during this research (license number 33439).
2.2. Animals
This study was carried out at the experimental farm of the Federal
University of Rio Grande do Sul, located in the city of Eldorado do Sul,
Rio Grande do Sul, Brazil (latitude 30◦ 06′ 20,7“ S and longitude 51◦ 41’
21,9” W). We distributed 64 Brangus calves (male and female), born in
the spring, in three groups, according to the age of weaning: i) hyper-
early weaning (W30; n = 22, 10 males and 12 females), the average
weight of 53.45 ± 1.10 kg (mean ± SE) and age of 32 ± 0.89 days; ii)
early weaning (W75; n = 20, 12 males and 8 females), the average
weight of 84.63 ± 2.05 kg and age of 77 ± 0.95 days, and; iii) con­
ventional weaning (W180; n = 22, 13 males and 9 females), the average
weight of 157.62 ± 2.87 kg and age of 183 ± 0.82 days. Calves were
born of cows aged 4.3 ± 0.23 years old and under normal conditions of
parturition. Before weaning, cows and calves were kept under the same
dietary conditions, on native pasture with 2200 kg DM/ha and culti­
vated millet pasture (Pennisetum americanum) with 1830 kg DM/ha.
At weaning, cows and calves were walked to the management center.
After separation, the cows were taken to a picket 2.6 km away from the
calves to prevent them from hearing offspring vocalizations. The calves
were weaned abruptly, with total physical separation between the dam
and offspring. Weaning management was the same for all treatments,
with W30 and W75 performed in the summer (December and February,
respectively) and W180 in the fall (May). For the weaning groups, calves
born from dams of the same mating and calving periods were used in
each treatment as in Blanco et al. (2009).
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Research in Veterinary Science 138 (2021) 53–61
2.3. Experimental design
After weaning, the calves from the same treatment were collectively
allocated in pen (Fig. 1). This system was chosen as it allows calves to
develop their natural behaviors, as playing. Also, it was verified that
maintain animals in groups results in increased body weight by 15%
when compared to calves allocated individually (Valníčková et al.,
2015), improving animal welfare (Bučková et al., 2019). In addition, it
comprehends the management used in farms that adopt cow-calf sys­
tems. The pen was equipped with two feeders, an automatic water
fountain and an artificial shade made of polypropylene (80% shading).
The W30 and W75 groups were allocated in two pens with identical
format until the animals were sent to pasture. After this, one of the pens
was used for weaning the W180 calves.
Birth weight, weight at 30 days of age and calf temperament score
(methodology adapted from Hickey et al., 2003) were similar between
groups at the beginning of the experiment. Two management protocols
were applied for blood collection. The first one refers to the period
before the cow-calf separation. The day before the collections, the cow-
calf pairs were led by two herders on horseback to a paddock near the
management center. The next day (collection day), the pairs were
guided by the herders without using the horse to the management
center. Animals were allowed to rest for 30 min, after which collection
began. The second protocol refers to animals that had already been
weaned. The pen where these animals were kept was within the man­
agement center, so the animals were led to the collection chute. No truck
transport was employed during the experiment.
Blood collections started at 08:00 am and were made through the
puncture of the jugular vein. Blood samples were collected of all
experimental animals, regardless of treatment, on days 30, 31, 32, 37,
75, 76, 77, 82, 180, 181, 182 and 187 of age. These collection moments
correspond to the weaning day and days 1, 2 and 7 post-weaning day of
each treatment (W30, W75 and W180, respectively), a protocol adapted
from Blanco et al. (2009) and O’Loughlin et al. (2014). According to
Blanco et al. (2009), the number of host defense cells returns to normal
up to seven days after weaning, therefore, it was evaluated until seven
days after weaning.
2.4. Nutritional and sanitary management of calves
Calves in the W30 group remained confined from 30 to 110 days of
age (Fig. 2). From 30 to 90 days, they were fed ad libitum with initial
pelleted feed 1 (R1) composed of 200 g/kg of crude protein (CP); 60 g/
kg of crude fiber (CF); and 740 g/kg of total digestible nutrients (TDN),
and alfalfa hay (167 g/kg of CP and 456 g/kg of NDF); average total
consumption of 1.0% of live weight (based on the dry matter) from 30 to
40 days of age; and total average consumption of 2.3% from 41 to 90
days of age. From days 91 to 110, calves received the pelleted com­
mercial growth feed 2 (R2) (180 g/kg of CP; 120 g/kg CF and 720 g/kg
of TDN) and alfalfa hay with an average total consumption of 3.1% of
live weight. Food consumption on the day of weaning was lower for the
W30 calves, as seven calves did not search for food on that day, as well as
five W75 calves and one W180 calf. However, on the day after weaning,
there was an increase in feeding activity for all treatments, and on the
third day, all calves were eating properly. In the three treatments, food
consumption was measured collectively, considering the difference be­
tween the quantity offered and the quantity of leftovers on the following
day. The food uneaten was removed and weighed daily to adjust the
amount to be supplied, providing a 5% surplus of natural matter.
Regardless of the treatment, no animals received supplementation
before weaning.
Calves of W75 were weaned and remained until 110 days of age in
feedlot. From 75 to 90 days of age, they received R1 and alfalfa hay, with
an average consumption of 1.0% of live weight. Between 91 and 110
days, the calves received R2 and alfalfa hay, with consumption of 2.20%
of live weight. The calves of W30 and W75 were fed three times a day
O. de Souza Teixeira et al.
Fig. 1. Diagram of the confinement of beef calves. A) Plant. B) Front plant. C) Cross-section. The stalls were equipped with feeders, water fountains and an arti­
ficial shade.
(08:00 am, 02:00 pm and 06:00 pm). After 110 days of age, calves of
both treatments were conducted to Urochloa pasture (U. decumbens,
U. brizantha and U. ruziziensis). The bromatological composition of the
pasture was 58 g/kg of CP and 665 g/kg of NDF and forage mass of 2270
kg of DM/ha, with an average feed consumption of 1.5% of the live
weight per day. The calves remained on this pasture until 190 days of
age and were supplemented in the morning once a day (08:00 am). The
W180 calves were weaned at 180 days and received the commercial
pelleted R2 and alfalfa hay, with an average total consumption of 1.4%
of live weight per day.
The calves received a preventive vaccination for clostridiosis at 40
days of age, with a booster dose at 90 days. Also, they were medicated
monthly for endo and ectoparasites. This sanitary protocol was adopted
because the region’s climate is subtropical. Hence, the combination of
high temperature and high humidity contribute to parasitic prolifera­
tion, putting the health of calves at risk. After weaning, the incidence of
nasal and ocular discharge and diarrhea (Larson et al., 1977) were
monitored visually during feeding (daily). Rectal temperature was ob­
tained after blood collection or when there was an abnormal clinical
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Research in Veterinary Science 138 (2021) 53–61
sign. The measurements were taken on all calves, weaned or not. For the
animals that had not been already weaned, the supervision was carried
out twice a week, as the animals were maintained at pasture with their
dams in an extensive production system.
2.5. Blood collection
Blood samples for cortisol and serum amyloid A (SAA) analyses were
collected immediately after the animal immobilization using a 10 mL
vacutainer tube (BD, Franklin Lakes, NJ, USA) with a clot activator.
These samples were centrifuged (2000 ×g for 20 min), conditioned in
microtubes and frozen in a cylinder containing liquid nitrogen
(− 196 ◦ C). For blood count and fibrinogen analyses, blood was collected
in 4 mL vacutainer tubes (BD, Franklin Lakes, NJ, USA) with dipotassic
ethylenediaminetetraacetic acid (EDTA K2) anticoagulant.
2.6. Assays of physiological parameters and blood count
Total plasma proteins (TPP) and plasmatic fibrinogen concentrations
O. de Souza Teixeira et al.
Fig. 2. Nutritional management before and after weaning calves. W30: hyper-early weaning – from 30 to 90 days, animals received feed 1 (R1), and from 91 to 110
days, they received feed 2 (R2) in confinement. W75: early weaning – from 75 to 90 days, animals received R1, and from 91 to 110 days, they received R2 in
confinement. The W30 and W75 calves were conducted to the pasture with supplementation after 110 days of age. W180: conventional weaning – from 180 to 190
days, animals received R2 in confinement.
in EDTA-blood samples were estimated by refractometry and heat pre­
cipitation (Willard and Tvedten, 2004). Cortisol was determined using
the chemiluminescence method and a commercial kit (Beckman
Coulter) and read by Access II (Beckman Coulter). For serum amyloid A
(SAA) protein, it was used the Elisa method (specific commercial kit for
cattle - Bioassay Technology Laboratory) and read in a Thermo Fisher
Scientific machine. Blood samples were processed using an automatic
analyzer (Procyte DX, Idexx Laboratories). White blood cell differential
count was performed manually in a stained smear (Diff-Quick Stain)
using optical microscopy (100 x, under immersion oil). The neutrophil:
lymphocyte ratio (N:L ratio) was obtained for all animals from this
analysis.
2.7. Statistical analysis
In a first moment, data were subjected to a principal component
analysis (PCA; PAST 3.0 program - Hammer et al., 2001) to indicate how
the immunological variables of the calves are ordered in treatments
(weaning groups) and sampling days. Then, a PERMANOVA analysis
was used to test differences in orders of immunological responses,
considering a 5% level of significance (PAST 3.0 program - Hammer
et al., 2001). In this way, it was possible to create a general pattern for
the immunological response.
The mathematical model for PERMANOVA was:
γijkl = τj + ρk + (τ*ρ)jk + ηι + (τ*η)jl + (ρ*η)kl + (τ*ρ*η)jkl + εijkl
where
γijk: dependent variables; τj: effect of the j-th treatment; ρk: effect of
the k-th collection day; (τ*ρ)jk: interaction between the j-th treatment
and k-th collection day; ηι: effect of the l-th gender (male or female);
(τ*η)jl: interaction between the j-th treatment and l-th gender; (ρ*η)kl:
interaction between the k-th collection day and l-th gender; (τ*ρ*η)jkl:
interaction between the j-th treatment, k-th collection day and l-th
gender; εijk: random error (error b).
Changes in blood variables were evaluated over time from 30 to 187
days of age for all the calves. A general linear model was used to analyze
variance with repeated measures, considering the age of the dam as a
covariate, using SPSS version 20.0 software (IBM, Armonk, New York,
2011). The model included the effect of age at weaning (W30, W75 and
W180), days (0, 1, 2 and 7 post-weaning) and gender (male or female) as
fixed factors, as well as the interaction between them. The calf was
included as a random factor. The Greenhouse–Geisser correction was
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Research in Veterinary Science 138 (2021) 53–61
used, which allows testing the repeated measures even if sphericity is
not met. Dunnet’s T3 test was used to compare within factors. The sig­
nificance level was 5%, but cortisol showed a tendency of P ≤ 0.06 on
day 77.
The mathematical model for repeated measures was:
( )
γijkl = μ + τj + φi τj + ρk + (τ*ρ)jk + ηι + (τ*η)jl + (ρ*η)kl + (τ*ρ*η)jkl + εijkl
where
γijk: dependent variables; μ: mean of all observations; τj: effect of the
j-th treatment; φi: repetition within treatment (error a); ρk: effect of the
k-th collection day; (τ*ρ)jk: interaction between the j-th treatment and k-
th collection day; ηι: effect of the l-th gender (male or female); (τ*η)jl:
interaction between the j-th treatment and l-th gender; (ρ*η)kl: interac­
tion between the k-th collection day and l-th gender; (τ*ρ*η)jkl: Inter­
action between the j-th treatment, k-th collection day and l-th gender;
εijk: random error (error b).
3. Results
3.1. Principal component analysis (PCA)
The PCA shows the order of the evaluated factors of component 1
(axis 1) and component 2 (axis 2) (Fig. 3). In axis 1, neutrophils and the
N:L ratio were the main causes of variation, while in axis 2 these causes
were plasma fibrinogen and SAA. From this order, PERMANOVA
showed a difference in the immunological variables per treatment (P <
0.001), per day (P < 0.001), and interaction between treatment×sam­
pling day (P < 0.001). The first two components contributed 42.66% of
the total variation.
3.2. Physiological variables
Interaction between treatment×sampling day (P < 0.001) was seen
for cortisol analysis. The W30 calves showed higher cortisol levels on
days 31, 32, 180 and 181 compared to W75 and W180. Similarities were
observed on day 77 between W30 and W75 (P > 0.05); and on day 187
between W30 and W180 (P > 0.05). On days 77 (P = 0.06) and 82 (P =
0.04), W75 calves showed higher cortisol levels than W180 ones (Fig. 4).
The TPP differed only for sampling day (P < 0.001), despite the lower
level at day 75 of age (6.24 ± 0.07 g/dL) and the higher level in the
subsequent days, with a mean of 7.00 ± 0.13 g/dL at day 187.
O. de Souza Teixeira et al.
Fig. 3. Main component 1 (PC1) versus 2 (PC2) of the principal component
analysis (PCA) of the immunological variables for the different ages at weaning.
Treatments – W30: hyper-early weaning; W75: early weaning and W180: con­
ventional weaning; Days: 30, 31, 32, 37, 75, 76,77, 82, 180, 181, 182 and 187
refer to the age of beef calves. Dots: individual animals; ellipses: treatments.
The influence of treatment×sampling day interaction (P < 0.01) for
plasma fibrinogen was observed for W30 and W75 treatments, which
presented higher levels at 32, 180 and 182 days of age than W180 one.
Calves from W30 demonstrated high fibrinogen levels on day 37
compared to those from W75 and W180, and higher levels on day 75 day
than W75 (463.63 ± 33.25 mg/dL v. 350.00 ± 28.56 mg/dL). The SAA
levels were altered only due to sampling day (P = 0.03), and the peaks
were identified between days 82 and 187, ranging from 32.01 ± 3.70 to
36.96 ± 4.30 mg/L.
3.3. Hematological variables
Interactions between treatment×sampling day (P < 0.001) were
observed for lymphocytes (%), neutrophils (%), N:L ratio and monocytes
(%) (Fig. 5). On the other hand, there was no effect on eosinophils (P >
0.05). Calves from W30 and W75 groups had lower percentages of
lymphocytes on days 180, 182 and 187 compared to W180 calves. The
percentage of neutrophils was lower for W30 calves on day 37 (16.54 ±
1.95%) compared to the W75 (34.15 ± 2.48%) and W180 (40.81 ±
2.84%) ones. There was a higher percentage of neutrophils for W30 and
W75 calves on days 75, 77, 180, 182 and 187 than for W180. Because of
this, W30 and W75 calves obtained a higher N:L ratio (P < 0.05) on days
75, 77, 182 and 187. On day 82, the percentage of monocytes was higher
for calves from early weaning (P < 0.001; 10.35 ± 1.08%) compared to
hyper-early (6.69 ± 0.49%) and conventional (4.72 ± 0.40%) weaning.
On day 180 of age, we observed the highest percentage in monocytes for
calves from W30 (8.14 ± 1.37%) in comparison to those from W75
(2.55 ± 0.48%) and W180 (3.59 ± 0.44%) treatments.
Male calves had higher percentage of lymphocytes than females (P =
0.003; 70.37 ± 0.76% vs. 65.76 ± 0.88%, respectively). However, fe­
males showed a higher concentration of neutrophils than males (P =
0.002; 27.73 ± 0.83% vs. 23.50 ± 0.73%, respectively) and N:L ratio (P
= 0. 02; (0.53 ± 0.04 vs. 0.40 ± 0.02, respectively) (Fig. 6).
There were interactions between treatment×sampling day (P <
0.001) for hematocrit, with higher values after weaning. The W30 calves
showed higher values on days 32 (37.81 ± 0.75%) and 37 (40.72 ±
0.75%) than W75 ones (33.35 ± 1.17% and 31.75 ± 1.24%). Similarly,
the highest hematocrit percentage for W75 was observed on days 77
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Research in Veterinary Science 138 (2021) 53–61
(35.20 ± 1.00%) and 82 (35.30 ± 0.76%) compared to W30 (30.45 ±
0.75% %; 32.32 ± 0.74%, respectively) and W180 (30.86 ± 0.84%;
30.10 ± 0.81%, respectively). For W180 calves, the higher values for
hematocrit were obtained on days 182 (33.50 ± 0.92%) and 187 (33.68
± 0.77%) compared to those from W30 (29.59 ± 0.72 and, 31.00 ±
0.76%, respectively).
4. Discussion
The influence of early (30 or 75 days) or traditional (180 days)
weaning on immune responses in calves was evaluated. Our hypothesis
was that calves weaned at an early age face unsatisfactory immuno­
logical conditions resulting from physiological responses to stress. Our
results showed that the younger the calf is, the greater the immuno­
logical challenge suffered by this animal. These reactions are mainly
characterized by higher levels of cortisol, fibrinogen, neutrophilia and
late lymphopenia, resulting in a higher N:L ratio.
The release of cortisol and immunological changes are due to the
activation of the hypothalamic-pituitary-adrenal axis (HPA) during
stress (Blecha, 2000). Under these conditions, the HPA axis acts by
secreting corticotrophin-releasing hormone (CRH) from the hypothala­
mus, which subsequently binds to receptors on the pituitary gland. This
hormone stimulates the release of the adrenocorticotropic hormone,
which conditions the secretion of cortisol and other hormones into the
bloodstream (Axelrod and Reisine, 1984; Herman and Cullinan, 1997;
Algamal et al., 2021). As a result, these hormones activate the acute-
phase proteins (Murata et al., 2004; Murata, 2007) and inhibit the
cells of the immune system, such as leukocytes (Merlot, 2004). Finally,
the levels of cortisol in the HPA coordinate the immune responses to
protect the organism against stress (Borghetti et al., 2009).
In hematological terms, neutrophils are the main line of defense of
the body (Burton et al., 2005), and lymphocytes act on innate and
cellular immunities as well as on antibody production (Tizard, 2009).
Thus, changes in the concentrations of these cells presuppose an insta­
bility of the immune system. For W30 calves, the lowest percentage of
neutrophils occurred 7 days after weaning, which may be related to the
incidence of diarrhea observed in these animals. The occurrence of
neutropenia resulting from the onset of diarrhea was reported by Novo
et al. (2017) during weaning in milk calves. The change of feeding
regime can generate imbalances in the gastrointestinal tract, mainly in
very young calves.
Although no differences were found between neutrophils and lym­
phocytes for immediate post-weaning sampling, we observed that calves
separated earlier from their dams (W30 and W75) showed a late immune
alert status compared to conventional weaning ones (W180). It was
characterized by neutrophilia and lymphopenia, representative of a
stress leukogram, which is associated with management practices in
farm animals (Kelley, 1980). It can be related to the sensitivity of neu­
trophils under conditions of elevated glucocorticoids (cortisol), as
observed in the present study, showing an imbalance. This evokes a
possible increase in the production and release of neutrophils from the
bone marrow (Burton et al., 2005), along with the retraction of neu­
trophils from other organs into the bloodstream (Merlot, 2004). The
occurrence of it is due to neutrophils need the L-selectin protein
expression to migrate from the bloodstream to the tissues. However,
during stress, glucocorticoids are believed to act by reducing L-selectin
gene expression, so neutrophils decrease their circulation, remaining in
the bloodstream (Weber et al., 2001). Thus, once neutrophils reach the
bloodstream, they are prevented from returning to tissues (diapedesis),
which justifies the increase in neutrophils after stressful events.
The opposite occurs with lymphocytes. Under stress, the action of the
corticotrophin-releasing hormone suppresses the proliferation of lym­
phocytes (Jain et al., 1991). Also, those lymphocytes recruited into the
bloodstream are encouraged to move to the lymphoid tissue (Merlot,
2004), resulting in a decrease in circulating lymphocytes. Therefore, the
lower levels of lymphocytes up to 28 days after weaning demonstrates
O. de Souza Teixeira et al.
Fig. 4. Means (± SEM) of cortisol, plasma proteins, fibrinogen and serum amyloid A in beef calves according to the age at weaning and sampling day. Treatments –
W30: hyper-early weaning; W75: early weaning and W180: conventional weaning.
the prolongation of the immune response to stress caused by weaning
(Lynch, 2010). The comparable higher levels in neutrophils after abrupt
weaning are considered a stress biomarker along with the N:L ratio
(O’Loughlin et al., 2014).
Blanco et al. (2009) tested the influence of age at weaning on beef
calves abruptly separated at 90 and 150 days of age. Although there
were no differences in terms of age, the effects of weaning caused high
levels of neutrophils and low levels of lymphocytes. In another study,
evaluating the influence of weaning at 120 and 210 days of age, re­
searchers found no differences for leukocyte subpopulations or N:L ratio
(Smith et al., 2003). For O’Loughlin et al. (2011), the N:L ratio remained
altered until 11 days after weaning, the maximum limit of the study
evaluation. In our research, the percentage of monocytes was above the
maximum limit for W75 calves at 82 days. This parameter can vary from
2 to 7% for cattle (Jones and Allison, 2007). In addition, the calves also
had higher rectal body temperature on the same day (39.9 ◦ C). However,
this clinical sign was monitored and no disease was diagnosed. There­
fore, the temperature rise may have been caused by the weaning stress
since it was measured 7 days after the separation of the cow-calf pair.
For Johnston et al. (2015), this expression of monocyte levels after the
management practice suggests higher sensitivity to weaning and con­
siders it a critical moment in the life of the calf.
In the present study, a different biological response was found due to
calf gender when considering neutrophils, lymphocytes and N:L ratio.
However, there is no consensus about this subject in the literature. For
O’Loughlin et al. (2011), gender did not change the total number of
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Research in Veterinary Science 138 (2021) 53–61
neutrophils or N:L ratio after weaning. However, Hickey et al. (2003)
found a higher proportion of neutrophils and N:L ratio for male weaned
calves than females, an opposite result to the one found in our research.
Moreover, this lack of clarity is also evidenced in investigations on an­
imal behavior in the post-weaning period. Under natural weaning con­
ditions, the cow interrupts the maternal-filial bond about two months
before in the case of a female offspring compared to a male one, not
allowing suckling behavior to occur (Reinhardt and Reinhardt, 1981;
Castanheira et al., 2013). In addition, males receive greater protection
from the dam (Stěhulová et al., 2013). On the other hand, Stěhulová
et al. (2017) highlighted that females vocalize more than males during
separation, showing that this behavior is one of the most sensitive to
characterize post-weaning stress. Thus, our findings also indicate that
females undergo greater stress, represented by the higher percentage of
neutrophils and N:L ratio. However, the mechanisms of immune re­
sponses to stress vary according to age, nutrition, genetics and gender
(Kim et al., 2011), and therefore different results are likely to be found.
High concentrations of TPP were observed from 75 to 180 days of
age. As age advances, there is an increase in total proteins (Bueno et al.,
2003) due to a higher concentration of immunoglobulins in adulthood.
Plasma fibrinogen, an acute-phase serum protein, presented a rise in
blood concentrations for calves in the W30 and W75 treatments. How­
ever, even though these concentrations are higher in the W30 and W75
groups than in the W180 one, they are close to the reference values for
beef calves (Edmonds et al., 1975). According to Qiu et al. (2007),
fluctuations in the concentration of acute-phase proteins may be due to
O. de Souza Teixeira et al.
Fig. 5. Percentage (± SEM) of hematologic variables: neutrophils, lymphocytes, N:L ratio, monocytes and eosinophils in beef calves according to age at weaning and
sampling day. Treatments – W30: hyper-early weaning; W75: early weaning and W180: conventional weaning.
handling, such as weaning and animal transport. For W30 calves, the
high values obtained for fibrinogen after weaning are related to the
impulses of the neuroendocrine-immune network, which induces the
synthesis of this protein in the liver (Murata, 2007). In the literature, the
maximum period of assessment of fibrinogen in calves after weaning
corresponded to 35 days, showing increased responses and demon­
strating the prolongation of stress (Lynch, 2010).
The results presented for the serum amyloid A protein were not ex­
pected because, as an acute-phase protein, there should be a rise at the
weaning moment, which did not happen. The inaccuracy of SAA was
reported by O’Loughlin et al. (2014), which indicated that this protein
seems not to be a good indicator for post-weaning calf stress. Other
studies are needed (biochemical, molecular, and cellular biology) to
elucidate its effect under stress conditions. On the other hand, the late
high (187 days) in SAA levels observed in this study should not be dis­
regarded, as the peak of this protein usually occurs at 7 days of age and,
subsequently, the levels decrease and stabilize at 24 days of age (Orro
et al., 2008). As this did not occur, it is assumed that the repeated blood
collections may have caused a high level of this protein, as SAA is sen­
sitive to handling stress calves (Alsemgeest et al., 1995).
Given the late changes in fibrinogen, cortisol and leukocyte cells in
calves at younger ages, we hypothesized that early exposure to stress
may generate suffering memories for the management events to come.
Consequently, it may trigger the complex neuroendocrine network with
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Research in Veterinary Science 138 (2021) 53–61
immune responses. Studies of this nature are carried out in rats. Their
exposition to stressors affects several brain areas and may lead to post-
traumatic disorders due to distressing or aversive memories (Camp and
Johnson, 2015). In other words, after the rats have experienced a
moment of stress, contextual memories of fear are observed in subse­
quent exposures since the mechanisms that promote the formation and
storage of memory are mediated by glucocorticoids with different re­
sponses for males and females (Kulp et al., 2020). However, this hy­
pothesis needs to be tested by assessing brain areas affected by weaning
stress in very young calves.
The rise in the hematocrit immediately after weaning is a relevant
observation in all three weaning ages. According to Wright et al. (2000),
this increment is indicative of dehydration. Thus, even though we did
not measure water intake by the calves, ingestive behavior was evalu­
ated. Among the activities analyzed, visits to the drinking fountain
represented the shortest occupational time of the animals, regardless of
age at weaning (Teixeira et al., 2021). Also, despite the treatment, it is
evident that on the day after weaning, there was a decrease of 53% in the
frequency of visits to the drinking fountain when compared to the
weaning day. Dehydration can have serious consequences, especially in
calves, which demonstrates the importance of monitoring these animals
and treating any expressing clinical signs that may occur, which was not
diagnosed in this study. In the following days, the animals reestablished
water intake. However, the consumption decrease on the second day
O. de Souza Teixeira et al.
Fig. 6. Percentage (± SEM) of lymphocytes, neutrophils and N:L ratio by calf
gender (male and female).
was sufficient to interfere with the hematocrit result.
In summary, weaning at young ages generated greater potential for
damage to the immune system, which at times could have been inter­
preted as a possible diagnosis of disease due to the multiple variations
presented. More research is necessary to associate the effects of age at
weaning to the long-term immunological effects on beef calves, as they
are little-known or understood. It is explained by the limitation of
studies on the analysis of these parameters at weaning. Studies should
prioritize the comparison of animals at all ages, regardless of the
weaning strategy, as performed in the present study, since fluctuations
in immunological parameters are also conditioned to animal growth.
Thus, blood samples at different ages may not accurately represent what
is being tested. In addition, contradictory and inconsistent results
compose the nature of this theme, which is partly understood by the
different perceptions of animals to stressors, as they are dependent on
life experiences, breed, age and psychological state (Moberg, 2000).
5. Conclusion
Calf weaning at 30 or 75 days of age results in different immuno­
logical responses compared to calves weaned at 180 days. Weaning at
younger ages triggers changes in the defense cells, resulting from
physiological responses to stress. These changes are associated with a
higher level of cortisol in the bloodstream, neutrophilia and late lym­
phopenia in animals weaned at 30 or 75 days. Also, there is a differen­
tiation between genres in the primary defense cells contradictory to
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Research in Veterinary Science 138 (2021) 53–61
previous studies. This evidence provides opportunities for weaning
management changes to avoid physiological instabilities in critical pe­
riods in the life of the animal. Therefore, the influences of weaning on
the immune system in young calves is an emerging and necessary theme
in the search for practices with a lower impact on calf welfare.
Declaration of Competing Interest
The authors declare that there is no conflict of interest.
Acknowledgments
We thank the Research Support Foundation of the State of Rio
Grande do Sul, Brazil (FAPERGS - protocol 17/2551-0001277-1), the
National Council for Scientific and Technological Development, Brazil
(CNPq - protocol 431277/2018-4 and 307891/2019-4), the Coordina­
tion for the Improvement of Higher Education Personnel; Brazil (CAPES
- protocol 001 – PROEX PPG-ZOOTECNIA/UFRGS program) and the
company Animal nutrition company Puro Trato, Brazil, which supported
this research. We are grateful to all participants of the Center for
Research on Systems of Beef Cattle Production and Supply Chain
(NESPro), Brazil and the experimental farm team at the Federal Uni­
versity of Rio Grande do Sul for the development of this study.
References
Algamal, M., Pearson, A.J., Hahn-Townsend, C., Burca, I., Mullan, M., Crawford, F.,
Ojo, J.O., 2021. Repeated unpredictable stress and social isolation induce chronic
HPA axis dysfunction and persistent abnormal fear memory. Prog. Neuro-
Psychopharmacol. Biol. Psychiatry 104, 110035.
Alsemgeest, S.P.M., Lambooy, I.E., Wierenga, H.K., Dieleman, S.J., Meerkerk, B., van
Ederen, A.M., Niewold, T.A., 1995. Influence of physical stress on the plasma
concentration of serum amyloid-a (SAA) and haptoglobin (HP) in calves. Vet. Q. 17,
9–12.
Axelrod, J., Reisine, T., 1984. Stress hormones: their interaction and regulation. Science
224, 452–459.
Blanco, M., Casasús, I., Palacio, J., 2009. Effect of age at weaning on the physiological
stress response and temperament of two beef cattle breeds. Animal 3, 108–117.
Blecha, F., 2000. Immune system response to stress. In: Moberg, G.P., Mench, J.A. (Eds.),
The Biology of Animal Stress: Basic Principles and Implications for Animal Welfare.
CABI Publishing, New York, pp. 111–121.
Borghetti, P., Saleri, R., Mocchegiani, E., Corradi, A., Martelli, P., 2009. Infection,
immunity and the neuroendocrine response. Vet. Immunol. Immunopathol. 130,
141–162.
Bučková, K., Špinka, M., Hintze, S., 2019. Pair housing makes calves more optimistic. Sci.
Rep. 9, 20246.
Bueno, A.R., Rasby, R., Clemens, E.T., 2003. Age at weaning and the endocrine response
to stress. Arq. Bras. Med. Vet. Zootec. 55, 1–7.
Burton, J.L., Madsen, S.A., Chang, L.C., Weber, P.S.D., Buckham, K.R., van Dorp, R.,
Hickey, M.C., Earley, B., 2005. Gene expression signatures in neutrophils exposed to
glucocorticoids: a new paradigm to help explain “neutrophil dysfunction” in
parturient dairy cows. Vet. Immunol. Immunopathol. 105, 197–219.
Camp, R.M., Johnson, J.D., 2015. Repeated stressor exposure enhances contextual fear
memory in a beta-adrenergic receptor-dependent process and increases impulsivity
in a non-beta receptor-dependent fashion. Physiol. Behav. 150, 64–68.
Castanheira, M., McManus, C.M., Paula Neto, J.B., Costa, M.J.R.P., Mendes, F.D.C.,
Sereno, J.R.B., Bértoli, C.D., Fioravanti, M.C.S., 2013. Maternal offspring behaviour
in Curraleiro Pé Duro naturalized cattle in Brazil. Bras. Zootec. 42, 584–591.
Edmonds, C.H., McMillan, M.K., Hughes, D.A., Milam, J.D., 1975. Biochemical,
hematologic and coagulation values in the Hereford calf. Cardiovasc. Dis. 2,
417–424.
Hammer, O., Harper, D.A.T., Ryan, P.D., 2001. PAST: paleontological statistics software
package for education and data analysis. Palaeontol. Electron. 4, 1–9.
Herman, J.P., Cullinan, W.E., 1997. Neurocircuitry of stress: central control of the
hypothalamo-pituitary–adrenocortical axis. Trends Neurosci. 20, 78–84.
Hickey, M.C., Drennan, M., Earley, B., 2003. The effect of abrupt weaning of suckler
calves on the plasma concentrations of cortisol, catecholamines, leukocytes, acute-
phase proteins and in vitro interferon-gamma production. J. Anim. Sci. 81,
2847–2855.
Hulbert, L.E., Moisá, S.J., 2016. Stress, immunity, and the management of calves.
J. Dairy Sci. 99, 3199–3216.
IBM, Corp, 2011. IBM SPSS Statistics for Windows, Version 20.0. Released. Armonk,
Nova York.
Jain, R., Zwickler, D., Hollander, C.S., Brand, H., Saperstein, A., Hutchinson, B.,
Brown, C., Audhya, T., 1991. Corticotropin-releasing factor modulates the immune
response to stress in the rat. Endocrinol. 128, 1329–1336.
Johnston, D., Kenny, D.A., Kelly, A.K., McCabe, M.S., McGee, M., Waters, S.M.,
Earley, B., 2015. Characterisation of haematological profiles and whole blood
O. de Souza Teixeira et al.
relative gene expression levels in Holstein-Friesian and Jersey bull calves undergoing
gradual weaning. Animal 10, 1547–1556.
Jones, M.L., Allison, R.W., 2007. Evaluation of the ruminant complete blood cell count.
Vet. Clin. N. Am. Food A. 23, 377–402.
Kelley, K.W., 1980. Stress and immune function: a bibliographic review. Ann. Rech. Vet.
11, 445–478.
Kim, M.H., Yang, J.Y., Upadhaya, S.D., Lee, H.J., Yun, C.H., Ha, J.K., 2011. The stress of
weaning influences serum levels of acute-phase proteins, iron-binding proteins,
inflammatory cytokines, cortisol, and leukocyte subsets in Holstein calves. J. Vet.
Sci. 12, 151–157.
Kulp, A.C., Lowden, B.M., Chaudhari, S., Ridley, C.A., Krzoska, J.C., Barnard, D.F.,
Mehta, D.M., Johnson, J.D., 2020. Sensitized corticosterone responses do not
mediate the enhanced fear memories in chronically stressed rats. Behav. Brain Res.
382, 1–4.
Larson, L.L., Owen, F.G., Albright, J.L., Appleman, R.D., Lamb, R.C., Muller, L.D., 1977.
Guidelines toward more uniformity in measuring and reporting and experimental
data. J. Dairy Sci. 60, 989–991.
Lynch, E., Earley, B., McGee, M., Doyle, S., 2010. Effect of abrupt weaning at housing on
leukocyte distribution, functional activity of neutrophils, and acute phase protein
response of beef calves. BMC Vet. Res. 6, 39.
Lynch, E., McGee, M., Earley, B., 2019. Weaning management of beef calves with
implications for animal health and welfare. J. Appl. Anim. Res. 47, 167–175.
Lynch, E.M., 2010. Characterisation of Physiological and Immune-Related Biomarkers of
Weaning Stress in Beef Cattle. Doctoral Thesis. National University of Ireland
Maynooth, Co, Kildare, Ireland.
Merlot, E., 2004. Conséquences du stress sur la function immunitaire chez les animaux
d’élevage. INRAE Prod. Anim. 17, 255–264.
Moberg, G.P., 2000. Biological response to stress: implications for animal welfare. In:
Moberg, G.P., Mench, J.A. (Eds.), The Biology of Animal Stress: Basic Principles and
Implications for Animal Welfare. CABI Publishing, New York, pp. 1–21.
Murata, H., 2007. Stress and acute phase protein response: an inconspicuous but
essential linkage. Vet. J. 173, 473–474.
Murata, H., Shimada, N., Yoshioka, M., 2004. Current research on acute phase proteins in
veterinary diagnosis: an overview. Vet. J. 168, 28–40.
Novo, S.M.F., Costa, J.F.R., Baccili, C.C., Sobreira, N.M., Silva, B.T., Oliveira, P.L.,
Hurley, D.J., Gomes, V., 2017. Effect of maternal cells transferred with colostrum on
the health of neonate calves. Res. Vet. Sci. 112, 97–104.
Oliveira, T.E., Barcellos, J.O.J., Whittier, J., Teixeira, O.S., Freitas, D.S., Oaigen, R.P.,
Dill, M.D., McManus, C., 2018. Risks associated to different methods of increasing
pregnancy rate of cows in cow-calf systems. R. Bras. Zootec. 47, e20180051.
61
Research in Veterinary Science 138 (2021) 53–61
O’Loughlin, A., McGee, M., Waters, S.M., Doyle, S., Earley, B., 2011. Examination of the
bovine leukocyte environment using immunogenetic biomarkers to assess
immunocompetence following exposure to weaning stress. BMC Vet. Res. 7, 45.
O’Loughlin, A., McGee, M., Doyle, S., Earley, B., 2014. Biomarker responses to weaning
stress in beef calves. Res. Vet. Sci. 97, 459–464.
Orihuela, A., Galina, C.S., 2019. Effects of separation of cows and calves on reproductive
performance and animal welfare in tropical beef cattle. Animals 9, 1–13.
Orro, T., Jacobsen, S., LePage, J.P., Niewold, T., Alasuutari, S., Soveri, T., 2008.
Temporal changes in serum concentrations of acute phase proteins in newborn dairy
calves. Vet. J. 176, 182–187.
Qiu, X., Arthington, J.D., Riley, D.G., Chase, C.C., Phillips, W.A., Coleman, S.W.,
Olson, T.A., 2007. Genetic effects on acute phase protein response to the stresses of
weaning and transportation in beef calves. J. Anim. Sci. 85, 2367–2374.
Reinhardt, V., Reinhardt, A., 1981. Natural sucking performance and age at weaning in
zebu cattle (Bos indicus). J. Agric. Sci. 96, 309–312.
Smith, D.L., Wiggers, D.L., Wilson, L.L., Comerford, J.W., Harpster, H.W., Cash, E.H.,
2003. Post-weaning behavior and growth performance of early and conventionally
weaned beef calves. PAS. 19, 23–29.
Stěhulová, I., Spinka, M., Sárová, R., Máchová, L., Knez, R., Firla, P., 2013. Maternal
behaviour in beef cows is individually consistent and sensitive to cow body
condition, calf sex and weight. Appl. Anim. Behav. Sci. 144, 89–97.
Stěhulová, I., Valníčková, B., Šárová, R., Špinka, M., 2017. Weaning reactions in beef
cattle are adaptively adjusted to the state of the cow and the calf. J. Anim. Sci. 95,
1023–1029.
Teixeira, O.S., Rocha, M.K., Alforma, A.M.P., Fernandes, V.S., Feijó, J.O., Corrêa, M.N.,
Canozzi, M.E.A., McManus, C., Barcellos, J.O.J., 2021. Behavioural and
physiological responses of male and female beef cattle to weaning at 30, 75 or 180
days of age. Appl. Anim. Behav. Sci. 240, 105339.
Tizard, I.R., 2009. Veterinary Immunology: An Introduction. Elsevier, St. Louis Missouri,
United States of America.
Valníčková, B., Stěhulová, I., Šárová, R., Špinka, M., 2015. The effect of age at separation
from the dam and presence of social companions on play behavior and weight gain
in dairy calves. J. Dairy Sci. 98, 5545–5556.
Weber, P.S.D., Madsen, S.A., Smith, G.W., Ireland, J.J., Burton, J.L., 2001. Pre-
translational regulation of neutrophil l-selectin in glucocorticoid-challenged cattle.
Vet. Immunol. Immunopathol. 83, 213–240.
Willard, M.D., Tvedten, H., 2004. Small Animal Clinical Diagnosis by Laboratory
Methods. Saunders Company, Philadelphia, Pennsylvania.
Wright, C.L., Corah, L.R., Stokka, G., Blecha, F., 2000. Effects of pre-weaning vitamin E,
selenium, and copper supplementation on the performance, acute phase protein
concentration, and immune function of stressed beef calves. PAS. 16, 111–120.