Goats are considered as multipurpose animals that produce meat,
milk, skin and fibre. They are kept as a source of protein and they supplement
the family income in rural areas. They are unique in their ability to
adapt and maintain themselves in harsh tropical environments. Furthermore,
goats are relatively resistant to diseases and dehydration and are endowed
with inquisitive feeding habits and high digestive efficiency for cellulose
(Devendra and McLeroy, 1987). Goats may experience considerable blood
loss due to trauma and haemorrhage associated with surgery and gynaecological
manipulation. Also internal parasites and blood sucking insects may induce
blood loss in certain occasions. Such situations may cause anaemia and
influence the productivity of goats, particularly during gestation and
The goat is extensively used as a mammalian research model in various
disciplines, particularly in physiological studies. Haematological investigations
into the responses of mammals to haemorrhage provide useful scientific
knowledge that could be utilized in medicine, surgery and immunology.
Such information is also essential for evaluation of efficiency of haemopoietic
and other compensatory mechanisms involved in restoration of homeostasis.
Acute blood loss is a major haemorrhage that occurs within a few minutes
to several hours. Severe blood loss threatens homeostasis as it acutely
decreases blood volume and can lead to cardiovascular collapse, hypovolaemic
shock and death (Hillman, 1995). The physiological responses to haemorrhage
in mammals are aimed at the preservation of blood pressure and tissue
perfusion. The primary mediator of the blood pressure response in animals
is increased activity of the sympathoadrenal system in sheep (Block et
al., 1987). Haemorrhage resulted in a fall followed by a partial recovery
of arterial blood pressure and significant rise of hepatic artery blood
flow in rats (Darlington and Tehrani, 1997).
After moderate haemorrhage, a low PCV is remarkably well tolerated because
of compensatory mechanisms such as increase in concentration of 2,3-diphosphoglycerate
in red blood cell (Ganong, 2003). Erythropoietin secretion from the kidneys
increases in response to blood loss to stimulate erythropoiesis by the
bone marrow (Hillman, 1995) and the iron supply to the red cell production
usually reflects the severity of the anaemia (Jain, 1993). The reticulocyte
response to acute blood loss is highly variable among species (Tyler and
Cowell, 1996). Bleeding in sheep resulted in the appearance of large erythrocytes
in peripheral blood (Wintour et al., 1995). The neurtophilia and
lymphopenia that are common in anaemias could be attributed to the effects
of both haemolysis and endogenous corticosteroids (Duncan et al.,
1994). Most of the studies have dealt mainly with the acute haemodynamic
and endocrine responses of animals to haemorrhage. Accordingly, the purpose
of this study was to investigate the sequential acute and long term changes
in thermoregulation, heart rate and blood constituents in response to
mild and moderate haemorrhage in goats.
MATERIALS AND METHODS
Animals and diet: Eleven young adult (10 months old), apparently
healthy non gestating and non lactating desert breed goats weighing an
average of 16.5 kg were used in the experiment. The animals were examined
clinically and were kept in the animal house for an adaptation period
of 14 days, followed by an experimental period of 6 weeks. During these
periods, the animals were fed alfalfa hay (CP 18%; ME 7.9 MJ kg-1)
and were offered tap water ad libitum. This study was conducted
at the Department of physiology during March-April 2006.
Experimental design: For all animals, the initial baseline physiological
data were determined. The total blood volume was measured in all animals
using Evans blue dye. The animals were randomly assigned to three groups,
3 animals in group A and 4 in each of groups B and C. Group A served as
control while the treated groups, B and C were subjected to 15 and 30%
bleeding, respectively. Graduated blood collection bags were used to collect
the specific volume of blood from the external jugular vein. The acute
responses were monitored for 9 days following bleeding and the parameters
were determined and blood samples were collected weekly for 6 weeks in
order to determine the long-term responses to bleeding.
Collection of the blood samples: Five milliliter of blood samples
were collected using plastic disposable syringes. Immediately, 1 mL of
blood was transferred to a clean dry test tube containing disodium ethylene
diamine tetra acetate (Na2-EDTA) as an anti-coagulant for blood
analysis. One milliliter of blood was also transferred to another test
tube containing sodium fluoride to inhibit the enzymatic reaction (Kelly,
1984) and was centrifuged at 3000 rpm for 15 min; the plasma samples were
used for glucose determination. The rest of the blood was allowed to stay
for 2 h at room temperature and then centrifuged at 3000 rpm for 15 min
and haemolysis-free serum samples were pipetted into clean vials and immediately
frozen at -20° for subsequent analysis.
Measurement of blood volume: The blood volume was determined using
Evans blue dye (T-1824) according to the method described by Pirkle and
Blood analysis: The Haemoglobin concentration (Hb), Packed Cell
Volume (PCV), total leukocyte count (TLC) and differential leukocyte count
(DLC) were determined according to the standard methods described by Kelly
(1984) and Jain (1993).
Serum and plasma analysis: The serum total protein concentration
was determined using Biuret reagent as described by King and Wooton (1965).
Serum albumin concentration was determined by the colorimeteric method
of Bartholmew and Delaney (1966). Plasma glucose concentration was determined
by the enzymatic colorimetric method using a kit (Spinreact, S.A., Spain).
Serum Na concentration was determined by flame photometer technique as
described by Wooton (1974). Serum Ca concentration was determined by the
colorimetric method using chloranilic acid and ferric nitrate (Trinder,
1960). The concentration of Mg in serum was determined as described by
Neil and Neely (1956).
Statistical analysis: The experiment was performed according to
the complete randomized design (Factorian arrangement). The data collected
were subjected to appropriate General Linear Model (GLM) procedure of
statistical analysis using the SAS (1988). The SAS was used to perform
analysis of variance (ANOVA) to evaluate the effects of bleeding on the
responses of goats. The values of parameters measured are expressed as
Meansμ"Standard Deviation (SD). The separation of means was done
by Duncan multiple range test.
Rectal temperature (Tr): Figure
1 shows that the initial values of Tr of the goats ranged
between 38.2 and 39.2°. The treated groups had higher mean initial
values of Tr compared to the mean value measured for the control
group. Generally, for the treated groups of goats, there was an initial
gradual increase in Tr following bleeding for 2 h and the normal
values of Tr were recovered after about 5 h.
||Effect of bleeding level on rectal temperature (Tr) in goats
||Effect of bleeding level on Respiratory Rate (RR) in
Respiration rate (RR): Figure 2 shows
that the initial mean values of RR of the experimental groups were almost
similar (μ. 25 breaths min-1). The control group maintained
this value until the end of experimental period. Generally, the haemorrhaged
groups of goats maintained higher values of RR following bleeding until
day 4 and there was an increase in RR with increase of bleeding level.
Immediately after bleeding, compared to the values obtained for the control
group, RR was higher (p<0.05) with 30% bleeding. The 30% bleeding group
had higher (p<0.01) RR values compared with control and 15% bleeding
group at 1, 2, 3, 4, 5 and 6 h after bleeding.
Heart rate (HR): Figure 3 shows that the initial
mean value of HR was higher in the control group. Following bleeding,
treated groups had higher mean values of HR compared with the control
until day 2. The values of treated groups returned to normal after 4 days.
There was increase in HR with the increase of bleeding level. Immediately
after bleeding, the 30% bleeding group had higher (p<0.05) HR compared
with control group value. At 5 and 6 h after bleeding, the 30% bleeding
group had higher (p<0.01) HR values compared with control. The
||Effect of bleeding level on Heart Rate (HR) in goats
||Effect of bleeding level on Packed Cell Volume (PCV)
30% bleeding group also had significantly higher HR values compared with
control at 6 h (p<0.05) and at 24 h (p<0.01) following bleeding.
Packed cell volume (PCV): Immediately post-bleeding, the PCV was
steady with 15% bleeding and it showed a slight decrease with 30% bleeding
(Fig. 4). There was a sharp decrease in PCV of treated
groups at 6 h post-bleeding. The groups subjected to bleeding had significantly
(p<0.01) lower values of PCV at 6, 24, 48 and 96 h post- bleeding compared
with the control. The 30% bleeding group had lower (p<0.05) PCV mean
values compared with the control at days 6 and 9 after bleeding. The PCV
recovered the initial normal value at day 6 with 15% bleeding and after
2 weeks with 30% bleeding.
Haemoglobin concentration (Hb): Figure 5 shows
that the initial values of Hb ranged between 9.8 and 11.0 g dL-1.
There was no marked change in Hb concentration
||Effect of bleeding level on haemoglobin concentration
(Hb) in goats
immediately post-bleeding. However, the treated groups showed a sharp
decrease in Hb level at 6 h post-bleeding and the decrease was more pronounced
with 30% bleeding. Thereafter, the control group showed an almost steady
level and the treated groups showed gradual increase in Hb level. The
15% bleeding group recovered the normal Hb level at day 6. The 30% bleeding
group showed a gradual increase in Hb level until day 9, maintained an
almost steady level at weeks 2, 3 and 4 and then the level increased to
assume levels similar to the control and 15% bleeding group in weeks 5
and 6. At 6, 24 and 48 h, the treated groups had lower (p<0.01) values
of Hb concentration compared to values obtained for the control. The mean
values of Hb with 30% bleeding were significantly (p<0.05) lower compared
with the respective control values at days 4, 6 and 9.
Total leukocyte count (TLC): The initial mean values of TLC for
the experimental groups were similar ( 10x103 μ-L-1)
(Fig. 6). The control group showed fluctuating pattern
for TLC during the experimental period. Both treated groups showed a sharp
decline in TLC after 6 h post-bleeding. The group subjected to 30% bleeding
had lower (p<0.05) TLC compared with the control group at 6 hrs post-bleeding.
Then both treated groups showed gradual increase in TLC; the normal control
values were attained at day 6. Thereafter, the control and treated groups
had almost similar values of TLC until the end of the experimental period.
Lymphocyte ratio: Figure 7 shows that generally,
the treated groups showed lower lymphocyte ratio compared with the control
during the experimental period. The initial values of lymphocyte ratios
ranged between 57 and 62% and there was no change in the ratio immediately
||Effect of bleeding level on total leukocyte count (TLC)
||Effect of bleeding level on lymphocyte ratio (%) in goats
bleeding. The control group maintained an almost steady lymphocyte ratio
(μ. 60%) during the experimental period. The treated groups showed
lower (p<0.01) values at 6 h following bleeding. Thereafter, for both
treated groups, the lymphocyte ratio increased to attain the control group
values at day 2 for 15% bleeding and day 6 for 30% bleeding. The group
subjected to 30% bleeding had lower (p<0.05) values compared to the
control at days 1, 2 and 4. The 30% bleeding group had lower (p<0.01)
values compared with control and 15% bleeding at day 4 post-bleeding.
Neutrophil ratio: The initial values of neutrophil ratio were
close to each other (μ. 33%). Generally, the treated groups showed
higher mean values of neutrophil ratio compared with the control (Fig.
8). There was no change in neutrophil ratios immediately after bleeding.
||Effect of bleeding level on neutrophil ratio (%) in
||Effect of bleeding level on serum total protein concentration
groups showed higher (p<0.01) means values of neutrophil compared
with the control at 6 h. The 30% bleeding group had higher (p<0.05)
neutrophil ratios compared with the control group at days 1 and 2.
Serum total protein: Figure 9 shows the effect
of bleeding level on serum total protein concentration. The initial values
of total protein ranged between 7.30 and 7.73 g dL-1. The control
group showed fluctuations in total protein level during the experimental
period. There was a decrease in total protein values with both bleeding
levels. Immediately after bleeding, the 30% bleeding group showed lower
(p<0.01) total protein value compared with the control group. The normal
control value was re-established at 6 hrs for 15% bleeding and at 24 h
for 30% bleeding.
||Effect of bleeding level on serum albumin concentration
||Effect of bleeding level on serum urea concentration
Serum albumin: Figure 10 shows that there was
no marked difference in serum albumin level immediately after bleeding.
The control group maintained an almost steady albumin level (μ. 4.0
g dL-1) during the experimental period. Both treated groups
showed gradual decease in albumin level at 6 and 24 h following bleeding.
The 30% bleeding group had lower (p<0.05) albumin level compared with
the control group at 6 h and days 1 and 2.
Figure 11 shows that the control group maintained
an almost steady serum urea level during the experimental period. Both
treated groups showed an immediate rise in serum urea level following
bleeding, that was more pronounced with 30% bleeding and significantly
(p<0.05) higher compared to the control group level at 6 h. The treated
groups recovered the normal control level at day 4 following bleeding.
||Effect of bleeding level on serum sodium concentration
(Na) in goats
Plasma glucose: The initial values of plasma glucose level ranged
between 45 and 55 mg dL-1 and the control group had higher
level compared to treated groups (Fig. 12). For all
groups, there were marked similar fluctuations in glucose level and the
treated groups tended to maintain higher glucose levels occasionally.
The glucose level of treated groups was higher compared to respective
control group values for 4 days.
Serum Na: Figure 13 shows that there were marked
fluctuations in serum Na level for all experimental groups. Immediately
post-bleeding there was decline in Na level in both treated groups, but
the decline was more pronounced with 30% bleeding. At 6 h, the treated
groups had higher Na level and then both groups showed gradual increase
in Na level until day 2.
Serum Ca: The initial values of serum Ca were almost similar (μ.
10 mg dL-1) for the control and treated groups of goats (Fig.
14). The control group maintained this value
||Effect of bleeding level on serum calcium concentration
(Ca) in goats
||Effect of bleeding level on serum magnesium concentration
(Mg) in goats
during the experimental period. Following bleeding, both treated groups
showed gradual decline in Ca level for 6 h and the low values were maintained
until day 4. Thereafter, for both groups, there was progressive increase
in Ca level to attain the high control group level at week 2 for 15% bleeding
and week 3 for 30% bleeding. During the experimental period, the 30% bleeding
group maintained lower Ca level compared to respective values obtained
for 15% bleeding group. The treated groups had significantly (p<0.05)
lower mean values of Ca compared with the control group, immediately after
bleeding and at 6, 24 and 48 h; also the treated groups had lower (p<0.01)
mean values compared with the control at day 4. The 30% bleeding group
had lower (p<0.05) Ca level compared with the control at days 6 and
Serum Mg: The initial values of serum Mg were almost simila(2.5
mg dL-1) for the experimental groups (Fig. 15).
This level was almost maintained by the control group during the experimental
period. Both treated groups showed a sharp decrease in Mg level following
bleeding and at 6 h, both groups showed a sharp increase to attain a value
higher than the respective control group value. Thereafter, both treated
groups showed undulating pattern until the end of the experimental period.
The 30% bleeding group had lower serum Mg level immediately after bleeding
(p<0.01) and after 24 h (p<0.05).
The results provide new information which suggests that haemorrhage
influences thermoregulation. This was indicated by post-haemorrhage increase
in rectal temperature, Tr (Fig. 1). The increase
in Tr could be related to retardation of convective heat transfer
from body-core to the periphery associated with reduction in blood volume.
One of the primary reflex adjustments to haemorrhage involves an increase
in total peripheral resistance in order to maintain blood pressure (Vatner,
1974). Also the post-haemorrhage pyrexia could be attributed to the effect
of calorigenic hormones secreted in response to haemorrhage. The hormones
which assume marked role in haemorrhaged animals include catecholamine
and adrenocorticotrophic hormones (Rose et al., 1987). The calorigenic
effect of bleeding could also be associated with the metabolism of fatty
acids, increase in the activity of the membrane bound Na+-K+-ATPase
and increase in concentration of 2,3-Diphosphoglycerate in erythrocytes
that enhances delivery of oxygen to body tissues.
The two bleeding levels also resulted in an increase in respiratory rate,
RR in goats (Fig. 2). Hyperventilation is a recognized
physiological response to haemorrhage. It is presumed to be stimulated
by hydrogen ions formed in tissues due to lowering of oxygen delivery.
The respiratory centre located in the medulla is sensitive to hydrogen
ion concentration and accordingly marked increase in respiratory frequency
was triggered in goats immediately after bleeding Loss of red blood cells
decreases the O2 carrying capacity of the blood and the blood
flow in the carotid and aortic bodies is reduced (Ganong, 2003). The resultant
anaemia and stagnant hypoxia, as well as acidosis that may occur, stimulate
the chemoreceptors. Maltz et al. (1984) noted that in Bedouin goats,
23% bleeding was associated with forced breathing and increase in RR from
50 to 100 breaths min-1.
The cardiovascular responses of goats to bleeding, manifested by marked
increase in heart rate, HR (Fig. 3) could be related
to stimulation of autonomic nervous system which increases the sympathetic
activity induced by the baro-receptors. The present results in goats are
in agreement with the tachycardia reported after 20% bleeding in adult
sheep (Rose et al., 1987; Wintour et al., 1995) and rabbits
(Clow et al., 2003).
The results indicate that loss of 15 and 30% of total blood volume resulted
in significantly lower PCV level (Fig. 4) associated
with decrease in Hb concentration (Fig. 5) after 6 h
of bleeding. The immediate post-haemorrhage values of PCV of goats were
apparently normal because erythrocyte and plasma volume were lost in similar
proportions. The drop in PCV and Hb concentration was caused by the shifting
of water from the interstitial fluid compartment to restore blood volume.
Previous studies indicated that the PCV decreased significantly post-haemorrhage
in sheep (Wintour et al., 1995). The return of PCV to pre-haemorrhage
level occurred in approximately 6 days and 2 weeks for 15 and 30% bleeding
groups, respectively. For Hb concentration, recovery occurred in 6 days
and 5 weeks for 15 and 30% bleeding groups, respectively. These findings
illustrate the influence of bleeding level on the capacity of the haemopoietic
system to restitute normal haematologic values. The results are in general
agreement with the findings of Lassen and Swardson (1995) and Tyler and
Cowell (1996) which suggest that return to normal level of PCV occurs
in approximately 2-4 weeks for most species of mammals.
Haemorrhage in goats was associated with decrease in total leukocyte
count, TLC (Fig. 6). The decline in TLC values 6 h post-bleeding
may be attributed to haemodilution. The value returned to normal level
after 2 and 6 days for 15 and 30% bleeding, respectively. Duncan et
al. (1994) noted that immature leukocytes may also appear in the blood,
particularly in cases of severe blood loss.
Bleeding of goats resulted in an increase in the neutrophil ratio associated
with decrease in lymphocyte ratio (Fig. 7, 8).
The post-haemorrhage neutrophilic leukocytosis is related to a shift of
neutrophils from marginal pool and bone marrow reserve to the circulation
(Duncan et al., 1994). Epinephrine released in response to haemorrhage
also mobilizes neutrophils from marginal pool into the circulation. The
lymphopenia observed could be attributed to release of ACTH or cortisol,
usually encountered after bleeding; ACTH induces dissolution of lymphocytes
in tissue and increase in antibody concentration in blood (Swenson, 1993).
The significant decrease in serum total protein and albumin immediately
after bleeding and after 6 h (Fig. 9, 10)
is related to haemodilution. Kovacs et al. (2000) reported that
reduction in total protein and albumin levels occurs in post-operative
blood loss in humans, whereas in dogs, there was an increase in the plasma
proteins in the beginning and gradual decreases in protein values in later
stages after blood loss. The plasma proteins are usually replaced from
mobilized fluid resource or intake (Cope and Litwin, 1962). The serum
total protein level returned to normal values after 6 h and 1 day for
15 and 30% bleeding, respectively. The values of albumin returned to normal
levels after 2 and 4 days for 15 and 30% bleeding, respectively. The current
results for goats are in general agreement with the finding of Jain (1993)
who reported that plasma proteins concentrations returned to pre-bleeding
level within 5-7 days in animals.
The increase in serum urea level that was more pronounced with 30% bleeding
(Fig. 11) may be attributed to decrease in renal plasma
level. Also it could be associated with depression of glomerular filtration
rate induced by reduction in blood volume. Similarly, in sheep, 20% haemorrhage
caused an increase in blood urea nitrogen concentration (Wintour et
al., 1995). Ware et al. (1982) noted that rats had showed an
increase in plasma urea concentration as a result of 20% bleeding.
The haemorrhage-evoked elevation of plasma glucose concentration reported
in this study that was influenced by level of blood loss (Fig.
12) is related to sympathoadrenergic activity and secretion of epinephrine,
ACTH and cortisol which cause increase in glycogenolysis (Jarhult, 1975).
In addition, Seredycz and Lautt (2006) indicated that in acute haemorrhage,
insulin secretion is suppressed and insulin resistance is accounted for
by elimination of the hepatic insulin sensitizing substance component
of insulin action. This finding confirms previous results reported in
sheep (Block et al., 1987) and in rats (Kadekaro et al.,
The current results indicate that bleeding in goats resulted in a decrease
in serum Na level (Fig. 13). This response indicates
haemodilution during acute haemorrhage which involves entry of extravascular
fluid into the vascular space (Hjelmqvist et al., 1991). Similarly
decrease in Na concentration has been reported in sheep subjected to 20%
bleeding (Wintour et al., 1995). Walsh et al. (1980) reported
that blood loss of 25 mL kg-1 resulted in a decrease in Na
level suggesting that compensatory fluid replacement originated in cells
as well as interstitum. The marked post-haemorrhage fluctuations in serum
Na level could indicate that feedback compensatory mechanisms related
to renin-angiotensin system were intermittently stimulated.
Bleeding in goats resulted in a decrease in serum Ca concentration immediately
post-haemorrhage; low values were maintained for 6 days and the normal
control level was recovered after 2 and 3 weeks for 15 and 30% bleeding,
respectively (Fig. 14). Hypocalcaemia observed after
blood loss may be associated with haemodilution and vasodilatation which
is influenced by haemorrhage. The decrease in Ca level may also be associated
with hypoalbuminaemia (Kovacs et al., 2000), as albumin has an
important role in transport of many cations such as Ca (Jain, 1993). About
half of the plasma Ca is bound to albumin and it is not filtered at the
glomerulus (Reece, 1993). In sheep subjected to 20% bleeding, Wintour
et al. (1995) reported a decline in Ca level after haemorrhage.
However, Ware et al. (1982) noted that Ca level was not changed
within 90 min. after haemorrhage in rats subjected to 20% bleeding.
Bleeding was associated with a decrease in serum Mg concentration (Fig.
15). The decrease in Mg concentration may be attributed to vasodilatation
which is caused by haemorrhage. The subsequent sharp increase after 6
h could be related to an increase in Na+- K+-ATPase.
Mg acts as a co-factor of cellular ATPase including the Na+-K+-ATPase
(Hays and Swenson, 1993). A decrease in Mg concentration after bleeding
has been reported by Kovacs et al. (2000) in humans.
For most of the parameters investigated in this study, return to normal
values occurred within 2 weeks for 15% bleeding, whereas the recovery
occurred in 5 weeks for 30% bleeding. This pattern indicates that the
recovery period was influenced by the bleeding level.
The goat model can be adopted for evaluation of the effects of acute
blood loss in mammals. The most sensitive indicators of blood loss and
recovery pattern seem to be respiratory rate, heart rate and erythroid
values. Recovery from acute blood loss is related to the level of bleeding.
We assume that the findings reported in this study have valuable implications
regarding haematology and surgery in both veterinary and medical sciences.
Further investigations are needed to examine the effects of haemorrhagic
shock in the goat model and to investigate the effects of dietary factors
on recovery pattern from acute blood loss anaemia. Change were in acid-base
parameters validated as accurate predictors of blood volume changes and
therefore may be utilized in the assessment of conditions of ongoing haemorrhage.
The authors would like to thank the members of the Department of Physiology,
Faculty of Veterinary Medicine, University of Khartoum for management
and care for experimental animals and technical assistance. The present
study received financial support from the Ministry of Higher Education
and Scientific Research.