CLASS ASSIGNMENT

(Introduction to Chinese Agriculture)

Ali Hassan Nawaz
Student ID: 2022205048

Review
Title: Research progress on the effect of heat stress on spermatogenesis in Bovines
Abstract:
In 21st century, global warming has become a potential threat for existence of life on earth. Heat
stress is a major stressful environmental condition that compromises the reproductive efficiency
of animals. The process of spermatogenesis is temperature-dependent and requires temperature
slightly lower than that of the body. Proper mechanism of thermoregulation is mandatory to
maintain testicular temperature at levels lower than that of the body core. Raised testicular
temperature has a detrimental effect on mammalian spermatogenesis and the resultant
spermatozoa. Therefore, thermoregulatory failure leading to heat stress can compromise sperm
quality and increase the risk of infertility. Bull testis require 2°C to 6°C lower temperature than
normal body temperature in order to produce physiologically and morphologically normal and
fertile sperm. There are number of mechanisms in body to regulate the testicular temperature to
carry out successful spermatogenesis. It is well known that increased testicular temperature has
adverse effects on spermatogenesis, percentages of motile, live and morphologically normal
sperm. In particular, increased testicular temperature increases the percentage of sperm with
abnormal morphology, particularly head defects, which directly compromises the productive
inefficiency of dairy industry. In short, heat stress is lethal for reproductive performance of dairy
bulls. Heat stress is one of the major problems dairy industry is facing in today’s scenario.
Reproductive inefficiency caused by heat stress is widely responsible for great economic damage
in livestock.
Keywords: heat stress, spermatogenesis, economic loss, bovine

1. Introduction
The testes of most mammals are located in a scrotum outside the body cavity, its temperature is
lower than the temperature of core body 2–8 °C (Ivell et al., 2017). This constant temperature is
necessary to maintain normal spermatogenesis (Paul et al., 2015). The increase in temperature
damages spermatogenesis and sperm quality. Semen quality and fertility of male mice were
significantly reduced when exposed to high temperatures (37 °C) (Zhao et al., 2018). In the hot
summer, semen quality of boars declined (Koh et al., 2009). Occupational heat exposure such as
welders has a reversible decline in semen quality (Bonde, 2010). Local testicular heating or
surgical induction of cryptorchidism in mice (Paul et al., 2018), rats (Kanter et al., 2013), dogs
(Henning et al., 2014) and monkeys (Zhang et al., 2013) resulted in increased germ cell
apoptosis. Our previous studies had found that heat stress increased germ cell apoptosis and
decreased semen quality in boars (Fan et al., 2017; Xi et al., 2017). Numerous studies suggested
that germ cell apoptosis of mammalian testes can be aggravated by heat stress and androgen
deprivation, and at normal temperature, exogenous testosterone was capable of inducing germ
cell apoptosis in mice (Vera et al., 2006), rats (Lue et al., 2000) and monkeys (Lue et al., 2006).
There were many researches about germ cell apoptosis can be induced by heat stress and
androgen deprivation, while the intrinsic relationship between heat stress and androgen has not
been reported. Early studies have shown that heat stress had no significant effect on the serum
testosterone concentration in rats (Lue et al., 2012) and lambs (Rasooli et al., 2010). However,
there are conflicted data reported on the serum testosterone concentration in testes exposed to
heat stress. The serum testosterone concentration of boars was not affected by heat stress (Li et
al., 2015; Wettemann and Desjardins, 1979). In another study, however, serum testosterone
levels of boars increased significantly after heat stress and then returned to normal levels (Li et
al., 2017).
Heat stress has been established as a cause of subfertility in animals, having detrimental effects
on the formation and function of gametes, and embryonic and fetal development (Hansen et al.,
2009). Elevations in temperature associated with global climate change are concerning for
animal agriculture, particularly in countries with a moderate climate, and especially when it
relates to bovine fertility given the economic importance of cattle in modern farming systems.
The Netherlands has a mild maritime climate, i.e., summers are typically highly humid and
winters are typically relatively mild. In view of climate change, the dairy industry in the
Netherlands is implementing preventive measures ventilation systems, to minimize the effect of
heat stress on cow fertility, based on knowledge obtained in countries with extreme
temperatures, such as Australia. Australia has a significant portion of its cattle production under
an arid climate, which has a great impact on cattle fertility (Dairy Aus 2009). This impact has
been mainly studied in cows, as female physiology is crucial for correct oocyte production,
fertilization, embryo development and to produce healthy offspring (Sabes-Alsina et al., 2019).
At present, it is known that bulls exposed to natural heat stress experience a decrease in sperm
quality, even in regions with temperate climates (Roth et al,. 2017). Natural heat stress has been
found to induce variations in lipid composition and reduce normal morphology in fresh bovine
semen (Argaman et al., 2013), and reduce very-low-density lipoprotein receptor (VLDLr)
mRNA expression, sperm motility, progressive motility and velocity, and to alter both the
concentration and composition of fatty acids in frozen bovine sperm (Argov et al., 2007). In a
recent study performed on dairy bulls in Northern Spain (Alsino et al., 2017), higher sperm
quality, in terms of kinetics, plasma membrane integrity, acrosome status, mitochondrial
membrane potential, and reactive oxygen species (ROS) production, was observed in semen
samples collected in spring compared to other seasons. In a previous study from our group
(Rahman et al., 2011), artificial scrotal insulation was performed to detect at which period sperm
is mostly affected by heat. This insulation mimicked abnormal high temperatures for the
scrotum, which had dramatic consequences on the sperm quality (Orgal et al., 2012).
Significantly higher abnormal morphology, chromatin protamination, and nuclear shape and a
decrease in motility and viability of sperm were detected in semen ejaculated between 14 and 42
days after scrotal insulation.
Spermatogenesis requires approximately 61 days in total to be complete in the bull: 21 days of
spermatocytogenesis, 23 days of meiosis, and 17 days of spermiogenesis. Therefore, increased
testicular temperature affected mostly those sperm cells that were at meiotic and the beginning of
spermiogenic stages of development at the time of scrotal insulation (Koppers et al., 2007). In
summer, ROS production significantly increases in sperm, leading to lipid peroxidation (LPO)
and major sperm defects (Nichi et al., 2006). During normal cell metabolism, ROS are produced
by mitochondrial respiration (Holland 2011). Sperm cells naturally produce ROS either during
their stay in the male and female reproductive tract (Wathes et al., 2017) or by being handled
during assisted reproductive technologies (ART), such as semen cryopreservation or sperm
centrifugation (Bilodeau et al., 2001)). Unsaturated fatty acids present in the sperm plasma
membrane are vulnerable to ROS, initiating a lipid peroxidation cascade and restricting the
fluidity needed to participate in the membrane fusion events associated with fertilization (Jones
et al., 2003). Hydrogen peroxide has been identified as one of the major ROS involved in LPO,
impairment of protein function, apoptosis, and DNA damage (Tunc et al., 2009; senner et al.,
2011). Reactive oxygen species are not only responsible for DNA fragmentation, but may also
cause lower sperm DNA methylation (Aitken et al., 2011; silva et al., 2011). Finally, this study
will focus especially on heat stressed sperm-mediated epigenetic spermatogenesis and will cover
numerous aspects through which heat stress disturbs the normal spermatogenesis in bovine.

2. How heat stress affects spermatogenesis?

2.1 What is Spermatogenesis?
The success of male reproduction requires the production of a large number of spermatozoa by a
unique process known as spermatogenesis (Saki et al., 2009). This process is carried out in the
seminiferous epithelium that covers the seminiferous tubules continuously, in close association
with Sertoli cells (Wassarman et al., 2016). In the seminiferous epithelium are located
spermatogonia cells (stem cells), which are undifferentiated diploid germ cells (2n), located at
the base of this epithelium, near the basal lamina. These cells are mitotically divided to produce
more spermatogonia (Clermont et al., 2010)

2.1.1 Epididymal maturation and storage of spermatozoa

Once formed within the seminiferous tubules, the immotile spermatozoa are released into the
seminiferous tubular fluid and transported to the epididymis, where they gain the ability to move
and fertilize the ova (Silva et al., 2011). The epididymis is generally divided into three parts:
caput, corpus, and cauda. In bulls, the transit of spermatozoa through the epididymis usually
takes 8e11 days (Heras et al., 2017). To attain the fertilizing capacity, spermatozoa undergo
many maturational changes during their transit through the epididymal duct (Silva et al., 2011).
These include changes in plasma membrane lipids, proteins and glycosylation, alterations in the
outer acrosomal membrane, gross morphological changes in the acrosome, and cross-linking of
nuclear protamines, proteins of the outer dense fiber and fibrous sheath. The cauda or tail of the
epididymis and proximal portion of the vas deferens are the regions where spermatozoa are
stored before ejaculation (Aponte et al., 2019; Waldner et al., 2010). During ejaculation, the
stored spermatozoa and the surrounding fluid are mixed with the alkaline secretions of the male
accessory sex glands, and the resulting socalled semen is deposited during mating into the
vagina, cervix or uterus depending on the species.

2.1.2 Sperm capacitation and fertilization

Although spermatozoa acquire maturity during epididymal transit, they do not attain complete
fertilizing capacity. To attain maximum fertilizing capability, spermatozoa must travel and reside
in the female reproductive tract for a minimum period. During their stay in the female
reproductive tract, spermatozoa undergo some biochemical changes that are known as sperm
capacitation (Kastelic et al., 2016; Cook et al., 2014)). This includes the removal of a large
portion of the extracellular coating proteins that prevent adhesion (including the decapacitation
factors) during the transit of spermatozoa in the uterus (Brito et al., 2004; Coulter et al., 2007).
The fertilization process involves a series of specific interactions between sperm and oocyte.
Firstly, in the oviduct, the motility pattern of sperm becomes hyperactive which is considered to
facilitate sperm-oocyte interaction. Secondly, some specific plasma membrane proteins
overlying the acrosome are exposed and bind to the zona pellucida (ZP) of oocyte (Ivell et al.,
2007). In mice, ZP comprises of three glycoproteins (ZP1, ZP2 and ZP3) while ZP3 acts as a
primary sperm binding receptor (Stechell et al., 2009).
In contrast, bovine ZP consists of ZP2, ZP3, and ZP4 while ZP3 alone cannot act as a sperm
binding receptor, instead, a heterocomplex of ZP3 and ZP4 is responsible for sperm-zona
binding (Hees et al., 2004). Zona binding probably initiates acrosome reaction (Silva et al.,
2011). The acrosome reaction allows the release of a variety of enzymes mainly acrosin and
hyaluronidase that hydrolyze zona proteins (Kasteloc et al., 2013; Blazquez et al., 2008).
Subsequently, the mechanical force generated by the flagellar action of the tail pushes the
spermatozoon to the perivitelline space of the oocyte. Then, the spermatozoon comes into the
direct contact with the oolemma, the plasma membrane of the oocyte. Now of direct contact,
sperm surface proteins, particularly Izumo (Gunn et al., 2001). Equatorial segment of the sperm
head, are involved in adhesion and fusion with the plasma membrane of the oocyte (Kastelec et
al., 2007). Recently, it has been reported that during sperm and oocyte fusion, Izumo1 binds to a
folate receptor termed Juno on the oocyte membrane and ultimately results in fertilization (Cook
et al., 2014). More recently, Izumo1 has also been identified in bovine spermatozoa and the
aberrant location of Izumo1 in cryopreserved spermatozoa with damaged acrosomes, which have
impaired fertilizing capacity in comparison with freshly ejaculated spermatozoa, indicates its
possible role in fertilization (Wladner et al., 2010). Fusion of sperm and oocyte leads to
depolarization of the oolemma which triggers the release of cortical granules located under the
oocyte surface and results in blocking of polyspermia. Further, it has been reported that, after
fertilization, Juno redistributed in the perivitelline space through extracellular vesicles which
may bind and neutralize acrosome-reacted spermatozoa and possibly block polyspermy (Coulter
et al., 2007). Soon after membrane fusion, the sperm nucleus starts to decondense which is
needed to prepare the paternal chromosomes to pair with the maternal chromosomes. The
decondensation of sperm nucleus starts through reducing disulfide bonds between protamines by
a primary reducing agent, glutathione (Dekrester et al., 2002). After decondensation of nuclear
material, the paternal and maternal pronuclei are lined up to form a zygote (Aponte et al., 2019).
However, the whole events including zona binding and penetration are highly dependent on the
intact plasma membrane, acrosome, mitochondria as well as high quality nuclear material of a
spermatozoon.

2.2 Bovine spermatogenesis under Heat stress environment

In our study, we observed that germ cells at post-meiotic stages of development are more
susceptible to heat stress and the resulting spermatozoa are associated with altered chromatin
conformation possibly due to aberrant DNA-protamination (Rahman et al., 2011). In a review
study, Saacke postulated that sperm chromatin aberrations in morphologically normal or nearly
normal ejaculates may lead to uncompensable effect in field fertility. The notion of
uncompensable effect refers to the defects of spermatozoa where the conception rates cannot be
improved by increasing the number of viable spermatozoa in the insemination dose (Daas et al.,
1998). As a matter of fact, uncompensable defects of spermatozoa might be related to epigenetic
modifications since environmental agents can modify the gene expression profiles without
changing their DNA sequence or copy number and these modifications can be transmitted to the
offspring (Dada et al., 2012). This is well addressed in a recent study where high and low bull
fertility were found to be associated with different sperm epigenetic patterns (DNA methylation)
that resulted in transcriptional differences in the preimplantation embryos (Kropp et al 2017).
Preimplantation embryo development is a dynamic process and is regulated by both genetic and
epigenetic mechanisms. Epigenetic regulation in the embryos is considered to be caused by DNA
methylation, histone modifications, and/or post-transcriptional modifications through microRNA
populations (Shi et al., 2017). In cattle, following fertilization, DNA in the paternal pronucleus is
actively demethylated followed by de novo methylation within the narrow developmental
window of the zygotic stage, whereas DNA methylation in the maternal pronucleus remains
almost unchanged (Heras et al., 2016). The typical characteristics of DNA methylation patterns
are also evident in many other species and are considered utmost important for early embryonic
development (Silva et al., 2011). Until today, little is known about sperm-derived epigenetic
regulations in the resulting embryo. In this review, we will focus on what is already known about
bovine testicular development, scrotal shape, scrotal circumference, scrotal and testicular
thermoregulation, and bovine spermatogenesis in relation to their vulnerability under heat stress
conditions.

2.3 Effect of heat stress on reproductive hormones during spermatogenesis

Studies of the effects of HS on the activity of endocrine glands and hormones involved in
reproductive activity show large variability and conflicting results. Genetic and environmental
sources of variability may largely affect these results. Besides difference among species, age, and
gender, a different response to HS may be found among breeds and genotypes that are more or
less heat tolerant. In addition, individuals may be naturally or experimentally exposed to a long‐
or short‐term warming and submitted to different experimental designs that make comparison
difficult. Results that emerged during preliminary studies were later reviewed and supported by
in vitro studies that either confirmed or denied previous results. In males, HS generally causes a
transient decline in testicular endocrine and gametogenesis functions, which are restored a few
weeks later for acclimatization to the hot environment. In most cases, the evidence demonstrated
that treatments with antioxidants can effectively counteract these effects. In bulls, testosterone
plasma concentrations significantly drop during the first two weeks of heat treatment but rose to
near control levels during subsequent weeks (Rhynes & Ewing, 2001). In boars, plasma
testosterone and LH concentrations do not seem to be affected by short ‐term HS for 14 days
(34.5°C for 8 hr and 31°C for 16 hr during each 24 hr period). However, using slides of testicular
tissue incubated in vitro, HS significantly altered androgen biosynthesis, as evidenced by lower
levels of androstenedione, testosterone, and dihydrotestosterone and higher levels of
androsterone and androstenediol (Wettemann & Desjardins, 2009). Long‐term HS for 90 days
under the same conditions as short‐term HS for 14 days caused an unequivocal suppression in
spermatid maturation and provokes marked alterations in testicular androgen biosynthesis
(Wettemann & Desjardins, 2009). Within the epididymis, HS (a) increased the clear cell count
and decreased epithelial thickness in the caput epithelium enhancee glucocorticoid receptor
expression in the nuclei of principal and basal cells, increases malondialdehyde and glutathione
levels. Most of these high‐temperature‐induced impairments in the epididymis were efficiently
counteracted by dietary zinc supplementation (Z. Li, Li, Zhou, Cao, & Li, 2017). In rams,
testosterone blood serum concentrations significantly decreased after HS by scrotal insulation
(32.3 vs 30.6°C); however, animals slowly restored their reproductive performance, showing a
time‐by‐treatment interaction (Alves et al., 2016). In male rats, long ‐term exposure to high
temperatures (43°C for 30 min/day for 15 days) caused a significant reduction in serum
testosterone and generated oxidative stress as assessed by lower levels of testicular superoxide
dismutase, catalase (CAT), and glutathione. In addition, animals under HS showed a significant
rise in the level of serum corticosteroid, testicular lipid peroxidase and the apoptotic enzyme
caspase‐3 along with a substantial increase in testicular heat shock protein (HSP) 72 and heat
shock factor 1, and decrease in 17β‐hydroxysteroid dehydrogenase 3. Treatment with
antioxidants such as red grape juice restored these parameters to normal levels (Halder et al.,
2018).

2.3.1 Heat stress also disturbs steroidogenic activity

The effects of HS on steroidogenic activity can be further investigated in cells that are primarily
involved in the production of male sex hormones, such as Leydig cells, whose secretory activity
strongly affects spermatogenesis. Short‐term testicular HS (42°C for 15 min/day for 3–5 days)
caused a decrease in 3β‐Hydroxysteroid dehydrogenase and progesterone as well as
steroidogenic enzyme expression. In contrast, tauroursodeoxycholic acid (TUDCA), an
endoplasmic reticulum (ER)‐stress inhibitor, rescued the testosterone levels even under HS
conditions (Kim, Park, Kim, Kim, & Lee, 2016). Morphological evaluation showed that Leydig
cells were randomly scattered and severely damaged after repeated testicular heat treatment. In
addition, immunohistochemical analyses revealed that apoptosis was elevated in the testicular
Leydig cells, and rescued by TUDCA (Kim et al., 2016). In rats, a single local testicular heat
treatment (43°C for 30 min) stimulates Leydig cell hyperplasia, as evidenced by enhanced
expression of cell cycle proteins, but decreased serum and testicular testosterone concentrations.
Consistent with these findings, HS downregulates the expression of two enzymes required for
testosterone biosynthesis in Leydig cells, cytochrome P450 family 17 and StAR protein (Z. Li,
Tian, Cui, Wang, & Yu, 2016). The pituitary gland represents another target organ that is
sensitive to HS with significant effects on reproductive activity. In ovariectomized ewes, induced
hyperthermia (adjusted to result 7in rectal temperature 1.2°C higher than control) caused a
decrease in plasma LH and an increase in plasma PRL on Day 2 and Day 3 after heat exposure,
respectively (Schillo, Alliston, & Malven, 2008). In dairy cows, HS occurring during the summer
in breeding locations with a hot climate decreased the pulsatile secretion of LH (Wise et al.,
2008) and suppressed the preovulatory LH surge (Gilad, Meidan, Berman, Graber, & Wolfenson,
2003), with delay or inhibition of ovulation (Roth & Wolfenson, 2016). In goats, induced HS
(38°C for 48 hr) decreased follicular response to pulsatile LH stimulation, evaluated in vivo via
estradiol secretion after gonadotropin‐releasing hormone (GnRH) administration (Boni et al.,
2019).

3. Testicular development, scrotal anatomy and physiology to cope heat stress

3.1 Testicular development and scrotal shape
Postnatal developmental assembly of the testicular tissue and seminiferous epithelium occurs
over a period of months and developmental rate varies between cattle breeds. For example, in
Bos taurus bull calves, testis weight increases from 9 g at 4 weeks to 180 g at 32 weeks of age
(Rhoades et al., 2013). On the other hand, in Bos indicus bull calves, testis weighs only 20 g at
36 weeks of age (Newton et al., 2013), which indicates slow testicular growth and delay of
puberty in this type of cattle. The scrotal shape has a major influence on further testicular
development and function. For a normal testicular function, the testes should be freely movable
inside the scrotum. A possible cause of low fertility in bulls is abnormal testicular and/or scrotal
sac development (Chen et al., 2016; Niwa et al., 1991). Bulls having a normal scrotum with a
distinct scrotal neck generally have the best field fertility reports (Fortes et al., 2014). On the
other hand, beef bulls such as Belgian Blue typically have a small scrotum without a distinct
scrotal neck (13). The anatomical peculiarity of small scrotum without a distinct scrotal neck
possibly increases the susceptibility of such beef bulls to heat stress leading to low field fertility
(Fukud et al., 2016). Scrotal length is categorized in terms of the relative position between the
bottom of scrotal sac and the hock joint. Short scrotum is classified as virtually neckless and
positioned close to the abdominal wall. Normal scrotum has a distinct scrotal neck and bottom
and is positioned above or at the level of hock joint whereas the bottom is pendulated below the
hock joint in case of long scrotum. Irrespective of breeds, normal scrotal shape has been reported
to be associated with high semen quality (Bianchi et al., 2014).

3.2 Scrotal and testicular approach to regulate temperature

Generally in mammals, for normal spermatogenesis the testes require a temperature of 4 or 5 C
lower than body core temperature (Simon et al., 2011; Tsai et al., 2007). This is achieved in most
mammals by localization of the testes in the scrotum, outside the abdominal cavity. In bulls, the
scrotal skin is thin, devoid of subcutaneous fat and is fairly hairless. The extensive vasculature
and lymphatic arrangement of testes with superficial blood vessels of the scrotum may facilitate
in removing heat from the testes. Smooth muscles in the cutaneous arterioles of the scrotum are
innervated by sympathetic neurons. Stimulation of these neurons by cold causes
vasoconstriction; and on the other hand, heat causes a vasodilation of these arterioles and is
thereby decreasing or increasing blood supply to the scrotum (Gadella et al., 2008). Just beneath
the scrotal skin, there are two important muscles: the tunica dartos and the cremaster that play a
pivotal role in thermoregulation. The tunica dartos is a thin sheet of smooth muscle, which is
under tonic control from nerves in the lumbar sympathetic system that promptly positions the
scrotum towards the abdomen or away from the abdomen in response to cold and warm
environments, respectively (Skinner et al., 1996). Due to the tonic characteristic of this muscle,
the contractile nature can be sustained for a prolonged period especially in cold environment.
The function of the cremaster muscle is also to bring the testes close to the abdomen upon
contraction. However, due to the striated nature of this muscle, it cannot sustain contraction for a
prolonged period of time (Fortes et al., 2014).
Beside these muscular contractions, a major player in cooling the testes is the vascular system.
The testicular artery brings warm blood from the body core to the testes and in all farm animals it
is tortuous in nature. This tortuous form of artery is intimately entangled by a complex venous
network, called the pampiniform plexus and the entire structure (the venous network and artery)
is termed the testicular vascular cone (Balhorn et al., 2007). Due to this characteristic
vasculature, a countercurrent blood circulation system is evident in the testes. As a result, the
arterial blood entering the testes is cooled to some extent by the venous blood leaving the testes
(Chen et al., 2016). Moreover, the sweat glands also have an important role in controlling
testicular temperature since the density of sweat glands is higher in the scrotal skin than the other
body regions in bulls (Koivisto et al., 2009). Beside the protection of the testes, the scrotal skin
has a vital role in testicular thermoregulation. Therefore, breeding bulls having normal scrotum
with adequate scrotal circumference can cope up with heat stress to some extent (Rhoades 2013).

3.3 Testicular surface and internal temperature gradient

From the above it is clear that normal testicular function in mammals is related to its specialized
cooling mechanisms. Maintenance of the testicular temperature around 32C is highly important
for normal spermatogenesis. In an elaborate study, Kastelic et al. measured the testicular
temperature in 16 crossbred beef bulls at three different positions of the testicle: top, middle, and
bottom (Dogan et al., 2015). The average temperatures at these three locations were recorded as
30.4C, 29.8C and 28.8C (scrotal surface temperature); 33.3C, 33.0C and 32.9C (scrotal
subcutaneous temperature); and 34.3C, 34.3C and 34.5C (intratesticular temperature),
respectively. From this study it could be concluded that the top-to-bottom temperature
differences were 1.6C, 0.4C and 0.2C for the scrotal surface, scrotal subcutaneous, and
intratesticular temperatures, respectively. Hence, the temperature gradient was most pronounced
on the scrotal surface, trivial in the scrotal subcutaneous tissues, and absent in the testicular
parenchyma. In a subsequent study by the same research group, it was reported that the scrotal
surface and testes have opposite, complementary temperature gradients, resulting in a relatively
uniform intratesticular temperature (Fernandes et al., 2014). This study indicates that the scrotum
has a significant influence on maintaining the testicular temperature but the testes have only little
influence on scrotal temperature. This is because both scrotum and testes have a characteristic
vasculature. The scrotum is vascularized from top to bottom whereas the testicular
vascularization is opposite; it is vascularized from the bottom to the top. The testicular artery
(after leaving the ventral aspect of the testicular vascular cone) progresses over the length of the
testis (under the corpus epididymis), reaches the bottom of the testis, and then diverges into
multiple branches that spread dorsally and laterally across the surface of the testis before entering
the testicular parenchyma (Bianchi et al., 2014).
In a subsequent study, (Kastelic et al., 2007) showed that there was no difference in temperature
of testicular arterial blood at the top of the testis (below the vascular cone) compared to the
bottom of the testis, but it was significantly cooler at the point of entry into the testicular
parenchyma. The temperature of caput epididymis is higher than that of the testicular
parenchyma at the top of the testis, probably because the caput is so close to the testicular
vascular cone (Curtis et al., 2014). However, the cauda epididymis, an important site for sperm
storage, is slightly cooler than the testicular parenchyma (Waldner et al., 2010). Therefore, from
the above discussion it is well established that several preset features contribute to the regulation
of the testicular temperature. If there is any interruption in these preset mechanisms harsh
consequences might be ensued on spermatogenesis (Cook et al., 2014).

4. Effects of heat stress on spermatogenesis and testis

4.1 Effect of heat stress on sperm cells and the testis
Testicular thermoregulation is important to maintain testicular temperature within an optimal
range for spermatogenesis. Increases in scrotal temperature, albeit within physiological range,
negatively affect sperm quality (Hjollund et al., 2010). An increase of 1°C entails a 14% drop in
spermatogenesis, and consequently poorer sperm production (Wang et al., 2009). The effect of
temperature on male fertility is evident with the mean scrotal temperature in infertile men being
higher than fertile men and the quality of sperm deteriorating further with higher increases in
scrotal temperature (Mieusset et al., 2013). Spermatogenesis, especially the differentiation and
maturation of spermatocytes and spermatids, is temperature sensitive. Spermatogenesis should
occur ideally at a minimum of 2°C below core body temperature (Chowdhury and Steinberger,
1970; Thonneau et al., 2009). Elevated scrotal temperature, however, causes testicular germinal
atrophy, spermatogenic arrest (Munkelwitz and Gilbert, 2015) and decreased levels of inhibin B
(a biochemical marker of spermatogenesis) (Jensen et al., 1997), which leads to lower sperm,
counts (Hjollund et al., 2012).

4.2 Effects of heat stress on germ cells

Germ cells are more vulnerable to heat stress as they have high mitotic activity (Shiraishi et al.,
2012). Among the germ cells, the types that are most vulnerable to heat are the pachytene and
diplotene spermatocytes and the early round spermatids in humans (Carlsen et al., 2003) and rats
(Chowdhury and Steinberger, 2010; Lue et al., 2007) alike. The basic mechanisms with which
germ cells incur damage include germ cell apoptosis (Lue et al., 1999, 2002; Yin et al., 1997b)
and autophagy (Eisenberg-Lerner et al., 2009; Zhang et al., 2012), damaged DNA due to altered
synapsis and strand breaks (Shikone et al., 1994; Yin et al., 2013), and generation of reactive
oxygen species (Ahotupa and Huhtaniemi, 2010; Ikeda et al., 2001; Peltola et al., 1995).

4.3 Effects of heat stress on epididymal sperm

Epididymal sperm are affected by heat exposure differently from germ cells. Male mice that
were whole body-exposed to temperatures of 36°C for 12 h on two successive days were found
to have lower sperm count, lower testicular weight, less sperm fertilizing capacity in vivo and
produced smaller litter sizes compared with controls. Epididymal spermatozoa from the heated
mice had lower sperm-zona pellucida binding and oocyte penetration capacity. These effects
were first seen at 1 week, and became more prominent at 2 weeks after heat exposure (Yaeram et
al., 2006). In another study, sperm obtained from the cauda epididymis of mice exposed to
whole-body heat (37°C to 38°C × 8 h × 3 consecutive days) had similar sperm count but lower
motility. These epididymal sperm also exhibited membranous changes, making these sperm
more prone to apoptosis (Wechalekar et al., 2010). In a previous study, when heat shock was
applied to male mice at 42°C for 30 min, the resultant spermatozoa retrieved from the epididymis
demonstrated reduced count, motility and viability. Spermatozoa that resulted from heat-exposed
spermatocytes and spermatids both had damaged DNA, with the spermatozoa from heat-exposed
spermatids showing poorer DNA integrity (Perez-Crespo et al., 2008).

5. Heat stress is responsible for hypoxia during spermatogenesis

Real hypoxia is closely related to vascular changes and an increase in testicular temperature.
Both induce a reduction in sperm count and can be related to the increase in germ cell apoptosis.
On the other hand, change in the temperature, and oxygen levels in the microenvironment have
influence on spermatogonial stem cell function and differentiation. The initial connection
between hypoxia and a factor critical for stem cell maintenance is alteration in Oct-4 expression,
and these data may be a useful strategy for modulating stem cell function. Unilateral testicular
ischemia-induced cell death can be accompanied by an increase in germ cell apoptosis in the
contralateral testis. The injury of contralateral testis following unilateral testicular damage is
controversial, and it can contribute to the reduction in fertility.
It is well known that the mammalian testis operates on the brink of hypoxia (Setchell 2013) and
that testicular hyperthermia increases metabolism and hence O2 consumption. However, blood
flow is reported to remain relatively constant and therefore the resulting hypoxia is regarded as
the basis for decreases in sperm motility and morphology (Paul et al. 2009; Hamilton et al.
2016). Regardless, to the authors’ knowledge, this has never been critically tested. In the present
study, transient testicular hyperthermia caused substantial reductions in both motility and
morphology. It was noteworthy that these changes were not prevented by hyperoxia, nor were
they replicated by hypoxia (paul et al., 2009). Therefore, we concluded that effects on motility
and morphology were due to hyperthermia per se and not due to hyperthermia-induced hypoxia
(Hamilton et al., 2016). Although these findings need to be replicated and refined, including
direct measurments of O2 delivery to the testis and ideally more objective means of assessing
sperm motility and morphology, they call into question the long-standing paradigm that the
effects of testicular hyperthermia on sperm motility and morphology are secondary to hypoxia
(Hamilton et al., 2016).

5.1 How Hypoxia due to heat stress causes abnormal spermatogenesis?

Effects of hyperthermia on testicular function and fertility have been studied in laboratory and
farm animals, whereas effects of hypoxia have been studied within the contexts of disrupted
blood flow and hypobaric hypoxia, as models of testicular torsion and living at high altitudes,
respectively (Rockket et al., 2001). Despite an apparent paucity of studies concurrently
examining both conditions, there is impetus for doing so. Male mice exposed to an ambient
temperature of 36 C had increases of ~1C in body temperature and ~5C in testicular temperature
at 12 h after the onset of exposure (Wechalekar et al., 2010). In a subsequent study, male mice
exposed to 36 ◦C for two 12-h intervals (on successive days) and mated to females 10 or 14 d
after exposure had significantly lower pregnancy rates and litter sizes. Regarding O2
concentrations, atmospheric air is ~21% O2, whereas 10.8 and 16.0% O2 in inspired air
constituted hypoxia and mild hypoxia, respectively, in rats (12). Mice breathing air with 12.5,
15.0, 21.0, and 100% O2 had testicular O2 concentrations of 16, 24, 36, and 102 mmol/L
(Wechalekar et al., 2016). Similarly, breathing 100% O2 reportedly doubled O2 saturation in rat
testes. Based on those data, there is an apparent association between O2 content of inspired air
and O2 content of the testes, enabling testicular O2 content to be varied from approximately 50
to 200% of physiologic concentrations.
The ability to independently alter testicular temperature and testicular O2 content provided a
novel opportunity to critically test effects of hyperthermia and hypoxia on spermatogenesis. In a
recent ram study (Shen et al., 2019), insulating the scrotum (testicular hyperthermia) caused
expected decreases in motile and morphologically normal sperm. However, it was noteworthy
that these effects were neither replicated by hypoxia nor prevented by hyperoxia. To our
knowledge, a similar study has apparently never been conducted in rodents. The objective was to
determine relative effects of hypoxia versus hyperthermia on sperm quality and production.
Hypoxia replicates effects of hyperthermia on reducing number and quality of sperm produced,
whereas hyperoxia mitigates effects of hyperthermia (Kastelic et al., 2019).

5.2 Heat stress induces hypoxia and damages spermatogenic cells

Hypobaric hypoxia is a stress factor that generates a series of physiological changes in order to
compensate for environmental low partial oxygen pressure. Exposure to low levels of
environmental oxygen and high altitude tend to trigger chronic mountain sickness (CMS) in
humans and animals not genetically adapted (GA) to high altitude, for example, llamas or
alpacas. The absence of CMS in GA or nonGA animals or humans appears to be linked to
various adaptations involving certain patterns of gene expression (Aziz et al., 2004).
With regard to the effects of environmental hypoxia on male infertility, chronic hypoxia induces
a state of reversible oligozoospermia in healthy men (Paul et al., 2008). Previous studies on
nonGA male rats indicated that chronic intermittent hypoxia reduces sperm motility and the
sperm count in semen (Halder et al., 2018). A reduced sperm count can be related to the increase
in germ cell apoptosis promoted by this hypoxic condition (Feil et al., 2012; Suarez et al., 2007).
The same results were observed in male rhesus monkeys (Flesch 200). Morphological studies
have revealed that chronic hypoxia causes degeneration of the germinal epithelium, folding of
the basement membrane, degeneration and detachment of germ cells, changes in lipid droplets in
Sertoli cells, and an increase in lipoperoxidation (Yanagimachi et al., 2014). Other local changes
in the testicles have also been observed, including an increase in vascularization, an increase in
testicular temperature, a decrease in testicular mass, and an increase in interstitial space (Lue et
al., 1999; Halder et al., 2018).

6. Conclusion and future prospectives

In light of the preceding discussion, it has become obvious that heat stress can have severe
effects on bull's fertility, and the severity of the effects may be related to the bull's breed. If heat
stress affects sperm quality it may exert a subtle effect that remains largely unnoticed by
conventional semen analysis but which may have prolonged effects on subsequent field fertility.
Our in vivo heat stress study signifies that heat stress can induce changes in sperm chromatin
conformation, which lead to sperm abnormalities and low fertility. The data can practically be
implemented to avoid wrongful culling of young bulls by commercial semen producers.
This culling is often based on the increased presence of morphologically abnormal spermatozoa
which might be due to heat stress exposure. In commercial AI centers, young bulls (~11 months
old) are culled when their ejaculates have less than 75% normal spermatozoa (Gholami et al.,
2011). If these morphologically abnormal spermatozoa are due to heat stress, then the wrongful
culling of these young bulls will lead to higher economic losses. There is a higher chance of
wrongful culling of young Belgian Blue bulls since this breed is more susceptible to heat stress.
Although a few published reports are available, this problem may happen in tropical countries
since the bulls are more susceptible to suffer from heat stress. When heat stress is suspected even
for a shorter period of time, a prolonged testing period for young bulls should be considered to
prevent unreasonable and early culling of valuable bulls. This will optimize profitability of the
breeding station, notwithstanding the extra costs for the prolonged testing period.
Supplementation of bull ration with commercially available 100e160 g Omega-3 daily for each
bull can be a mitigation option to some extent under heat stressed environments (Gholami et al.,
2011) as it has also been reported to protect sperm membrane during cold shock or
cryopreservation (Van tran et al 2017). When the heat stress cannot be avoided, even by proper
cooling systems in the stable of breeding bulls located especially in tropical or subtropical
countries, stopping of semen collection until it cools down would be a practical solution.
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