Introduction
With the frequent occurrence of extreme weather globally (Barnett et al.,
2012), the damage of heat stress (HS) to poultry has become increasingly
evident, resulting in serious economic losses (St-Pierre et al., 2003).
Because the skin of birds has no sweat glands and is coated with feathers,
their heat generation, cooling, and body temperature regulation are more
difficult compared to mammals. When birds are in an environment with a
temperature higher than their appropriate temperature,
their thermoregulatory mechanism cannot cope with the high temperature
change, resulting in HS. HS can cause birds wheezing and can cause long-time
alternating acid and base poisoning in the animal's body, lead to respiratory
mucosal congestion, and increase heart rate. This results in an increased
blood flow at the body surface and muscle but a reduced blood supply to the
internal organs and gonads, affecting the uptake and utilization of nutrients
in the body of poultry. Therefore, how to reasonably avoid HS during the
process of intensive, large-scale, and high-density poultry production and
how to reduce economic losses has become an important issue to solve in
current poultry production. Studies have shown that HS-induced wheezing can
lead to an increased respiratory rate; reduced blood volume in the
gastrointestinal tract, liver, and kidney; a decline in feed conversion rate
(FCR); and raised mortality (Quinteiro-Filho et al., 2010). Furthermore, HS
significantly reduces the marker enzymes of intestinal absorption such as
disaccharidases, alkaline phosphatase (AKPase), and adenosine triphosphatase
(ATPase), causing dysfunction of the intestinal mucosal antioxidant system in
poultry (Chen et al., 2013, 2014). By destroying the integrity of the tight
junctions between the epithelial cells of the gastrointestinal tract,
damaging the gastrointestinal mucosal barrier, and thereby triggering
dysfunction of the barrier and increasing its permeability, HS ultimately
causes an inflammatory response in the body and greatly increases the rate of
susceptibility to acute enteritis in poultry (Quinteiro-Filho et al., 2012).
In addition, high temperatures can also induce endocrine disorders in
poultry, resulting in phenomena such as instability of blood insulin levels;
significantly reduced levels of total plasma protein, serum protein, and
serum T3/ T4 (Liu et al., 2000); significantly increased Ca2+
concentration; and hypokalemia. (Du et al., 2000). Furthermore, under HS, the
amounts of follicle-stimulating hormone (FSH), luteinizing hormone (LH), and
estradiol (E2) declined, and the progesterone (P4) level significantly
reduced, too, after an initial elevation (Donoghue et al., 1989; Li and Cui,
2013). Short- and long-term heat exposure significantly reduces the
egg-laying rate, egg weight, ovarian weight, and the number of large
follicles in egg-laying birds, resulting in declined ovarian function,
disordered follicular development and formation, significantly reduced
reproductive performance, etc. (Rozenboim et al., 2007).
Current studies on the effect of HS in poultry mostly involve the aspects of
nutrition, digestion, immune function, etc., of egg-laying and mature birds,
but relatively few studies focus on the development of endocrine and
reproductive systems in chicks. The present study used 1–6-week old male
Wenchang chicks as subjects, aiming to shed light on the factors
influencing the histological and structural development of the
pituitary and testis in chicks of different weeks of age in order to provide
basic information for a further exploration of the effect of HS on the
development of pituitary and testis in Wenchang chicken.
Materials and methods
The animals
Eighty 1-day-old male Wenchang chicks provided by the Hainan Yongji Poultry
Co., Ltd. (Hainan, China) were randomly divided into control (CK) and HS
groups, with 40 chicks in each group. There was no significant difference in
the body weight, feed intake, etc., between the two groups. All chicks had
water and feed (Yilong Feed Factory, Zhanjiang, China) ad libitum. The basic
daily diet met the NRC standards (1994). Chickens hosted in the
large-capacity cages were placed in the feeding house
(7 m × 3.5 m × 3.5 m), which had natural indoor
ventilation and lighting (14 h of light and 10 h of dark). The house was
cleaned and disinfected regularly. Temperature and humidity of the feeding
house were 27.4±0.9 ∘C and 72.1±8.6 %, respectively,
during the experiments.
HS treatment
Chicks in the HS group were placed in a heat chamber remodeled from a
large-capacity artificial climate incubator at a temperature of 40±0.5 ∘C and relative humidity of 73.6±7.7 % for 2 h
(13:00–15:00 UTC + 8) every day. The birds in
the CK group were placed in another chamber under the same conditions but at
room temperature. Chicks were returned to their cages for feeding at the end
of treatment (Ramnath et al., 2008; Chen et al., 2014). The rectal
temperatures of 10 chicks randomly selected from each group were measured
before and after HS treatment. During the period of heat stress, chickens of
the HS group and the CK group were provided with neither drinking water nor
feed in the artificial climate incubator (Ramnath et al., 2008). The consumed
feed and weight gain were recorded every week and the feed conversion ratio
(FCR; feed-to-weight-gain ratio) was calculated.
Experimental method and data acquisition
At the end of each week, 6 chicks were randomly selected from each group
(n=6), weighed for fasting weight, and sacrificed by cervical dislocation.
The pituitary and testis were completely dissected and weighed after removing
excess tissue and washing with normal saline. The organs were then
according to a particular
routine sectioned and stained for hematoxylin and eosin (HE).
The histological structure of the pituitary and testis from chicks in both
groups was observed under an Olympus BX50F-3 microscope, and the MiPrd3.1
microscopic image analysis software (Shangdong Yichuang Electronics Ltd.,
China) was used to measure and statistically analyze the diameter and
cross-sectional area of 10 testicular seminiferous tubules randomly selected
from each slide (two random slides for each testis). Digital images were
taken of all sections using a YD400C digital camera (Shandong Yichuang
Electronics Ltd., China) and saved.
Data processing and analysis
The experimental data were presented as mean ± standard deviation (SD)
and analyzed using Excel 2003 and SPSS 16.0 statistical software.
P<0.05 and P<0.01 were considered as significant and extremely significant
differences respectively.
Results
The effect of HS on the body weight, FCR and
rectal temperature
As shown in Fig. 1, with the increase in weeks of age, the mean weight of
chicks in the HS group was significantly lower than that in the CK group, and
there were extremely significant differences (P<0.01) at 3, 4, and
6 weeks of age, indicating a significant effect of HS on the body weight of
chicks. In addition, FCRs (feed intake / chick weight gain) in the HS group
at 2, 4, and 6 weeks of age were significantly higher than those in the CK
group (P<0.05), further demonstrating the significant effect of HS on the
feed conversion efficiency in chicks. After heat stress, the rectal
temperatures of chickens were significantly increased in the HS group, unlike
in the CK group (P<0.05; Table 1).
Effects of HS on FCR and weight of Wenchang chicks.
Note: compared between CK and HS groups at the same age. * P<0.05;
** P<0.01.
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The Effects of HS on the rectal temperature of chicken (n=10).
Week
Control
Heat stress
1
38.52 ± 0.197
40.13 ± 0.618
2
39.12 ± 0.310
40.87 ± 0.574
3
38.91 ± 0.314
41.43 ± 0.359b
4
39.23 ± 0.216
41.52 ± 0.300a
5
39.42 ± 0.482
41.61 ± 0.293a
6
39.28 ± 0.425
41.82 ± 0.246a
Compared between the CK group and the HS group at same age.
a P<0.05, b P<0.01.
The effect of HS on the weight change in pituitary and
testis in chicks
As shown in Fig. 2, the weight of chicks' pituitary gradually increased with
the week of age; however, the weight of pituitary in chicks of the HS group
was lower than that of chicks in the CK group, and the difference became
extremely significant at 4–6 weeks of age (P<0.01).
Similarly, Fig. 3 shows that with the increase in weeks of age, the weight of
chicks' testes increased gradually; however, the mean testis weight of
chickens in the HS group was significantly lower than that of the CK group,
and the difference was extremely significant at 4–6 weeks of age (P<0.01).
The effect of HS on the histological structure of
pituitary and testis in chicks
Observation of the tissue sections revealed that the structure, outline, and
cells in the pituitary of chicks in the CK group were clear; however, compared with the CK group, the pituitary in chicks of the
HS group was smaller and had an incomplete histological structure, displaying
significant thermal damage (Fig. 4a and b).
Effects of HS on the weight of pituitary in Wenchang
chicks (n=6). Note: compared between CK and HS groups at the same age. ** P<0.01.
![]()
Effects of HS on the weight of testis in Wenchang chicks (n=6).
Note: compared between CK and HS groups at the same age. ** P<0.01.
![]()
Effects of HS on the histological structure of pituitary and testis
in 6-week-old chicks (n=6). (a) The pituitary of a chick in the CK
group; (b) the pituitary of a chick in the HS group;
(c) the testis of a chick in the CK group; and (d) the
testis of a chick in the HS group. A. P.: adenohypophysis; S. T.: seminiferous tubule;
I. T.: interstitial tissue; P. S.: spermatogenic cell; S. P.: spermatoblast; and B. M.: basement membrane.
Bar in (a) and (b): 100 µm; in
(c) and (d): 50 µm.
![]()
Effects of HS on the cross-sectional area of seminiferous tubule
in chicks (n=6). Note: compared between CK and HS groups at the same age.
** P<0.01.
![]()
Observation of the histological structure on sections also showed that in
comparison with the CK group, the testes of chicks in the HS group exhibited
a higher degree of fragmentation, significantly reduced integrity, thinner
seminiferous epithelium, occurrence of vacuolar changes, reduced number of
spermatogenic cells, and a loose and disordered arrangement. Moreover, part
of the seminiferous tubules only had a single layer of cells, composed of a
small number of spermatocytes and Sertoli cells, without sperm cells (Fig. 4c
and d). In addition, with the increase in weeks of age, the cross-sectional
area of testicular seminiferous tubule increased in both groups; however,
starting from 3 weeks of age, the difference in cross-sectional area of
seminiferous tubule between the two groups became evident, and the difference
was extremely significant at 5 and 6 weeks of age, reaching its maximum level
at 6 weeks of age (P<0.01). Starting from 4 weeks of age, the diameter of
seminiferous tubule in chicks of the HS group was extremely significantly
lower than that of the CK group (P<0.01). These results indicated that HS
induced seminiferous tubule atrophy and seriously affected the growth of
chicks' testes (Fig. 5).
Discussion
Early studies have shown that high temperature may reduce the ability of the
animal's brain to dissipate heat to the external environment, which affects
the neuronal activity and thereby induces abnormality in the secretion of
relevant hypothalamic hormones, further causing abnormal secretion of
anterior pituitary FSH and LH. Because normally secreted LH and FSH in male
animals promote the development of the seminiferous epithelium and the
proliferation of spermatogonia, abnormal levels of LH and FSH secretion
ultimately impair the normal process of testicular development. In addition,
HS also seriously affects the reproductive performance of male mammals,
drastically damages spermatogenesis, impacts sperm motility, acrosome
integrity, and the fertilization process, thereby reducing the success rate
of animal breeding (Hansen, 2009; Paul et al., 2009; Kim et al., 2013).
Recent studies have shown that under long-term HS, adult chickens displayed a
series of physiological anomalies such as reduced ovarian weight and number
of follicles, deteriorated ovarian function, and blocked development and
formation of follicles. The serum contents of both LH and FSH in 30-week-old
white Leghorn chickens reduced on the second day of HS; meanwhile, the levels
of testosterone, P4, and E2 all significantly decreased (Rozenboim et al.,
2007). HS also hinders the ovarian development and slows down the increase in
the total number of follicles in different varieties of chicken. The contents
of FSH and LH significantly reduced in laying hens under HS. After 7 days of
HS, E2 content reduced significantly, while the content of P4 also
significantly decreased after an initial elevation; meanwhile, HS
significantly reduced the mRNA expression of LH and FSH receptors too (Li and
Cui, 2013). These results indicated that HS seriously affected the normal
developmental process of animal's reproductive organs.
Sertoli cells in testicular stroma are directly affected by certain
environmental conditions or toxins. In male reproductive injuries caused by chemical and
physical factors, the damage to the structure and function of Sertoli cells
are all earlier than those of other tissues (Bergh et al., 1983; Richburg and
Boekelheide, 1996). The number and functional status of Sertoli cells
determine the testicular size and the normal progress of spermatogenesis
(Russell, 1980), playing a decisive role in male reproductive function. The
present study indicated that compared with the CK group, in the HS group, the
integrity of the testicular structure in chicks was damaged significantly,
and the seminiferous epithelium became thinner with vacuolar-like changes;
the spermatogonia also showed a decreased number and loose and disordered
arrangement. These data suggested that HS might have damaged the structure
and function of the stromal Sertoli cells in testes of chicks in the HS group
and thereby caused the thickening of seminiferous tubular epithelium and the
inhibition of the development of different levels of spermatogenic cells,
resulting in the phenomena of dysplasia exemplified by various degrees of
atrophy. Between 3 and 6 weeks of age, the diameter of the seminiferous
tubule in chicks of the HS group was significantly smaller than that of the
CK group; at 5–6 weeks of age, the sectional area of the seminiferous tubule
in chicks of the HS group was extremely significantly smaller than that of
the CK group, further suggesting that 3–6 weeks of age may be one of the
important stages for gonadal development in male chicks. In this period, the
testicular weight and the diameter and sectional area of the seminiferous
tubule increased significantly; however, HS extremely significantly affected
this developmental process.