Animal Reproduction Science 79 (2003) 1–15
Effects of scrotal insulation on sperm production,
semen quality, and testicular echotexture in Bos
indicus and Bos indicus × Bos taurus bulls
Leonardo F.C. Brito a,∗ , Antonio E.D.F. Silva b ,
Rogerio T. Barbosa c , Maria M. Unanian b , John P. Kastelic d
a
d
Department of Large Animal Clinical Sciences, Western College of Veterinary Medicine,
University of Saskatchewan, 52 Campus Drive, Saskatoon, SK, Canada S7N 5B4
b Empresa Brasileira de Pesquisa Agropecuaria, CENARGEN, Brasilia, DF, Brazil
c Empresa Brasileira de Pesquisa Agropecuaria, CPPSE, Sao Carlos, SP, Brazil
Agriculture and Agri-Food Canada, Lethbridge Research Centre, Lethbridge, AB, Canada
Received 10 December 2002; received in revised form 3 March 2003; accepted 4 March 2003
Abstract
The objectives of the present study were to evaluate the effects of scrotal insulation on sperm
production, semen quality, and testicular echotexture in Bos indicus and Bos indicus × Bos taurus crossbred bulls. In one experiment, B. indicus bulls (n = 12) were allocated to control and
whole-scrotum insulation groups, while in a second experiment, crossbred bulls (n = 21) were
allocated into control, whole-scrotum, and scrotal-neck insulation groups. Insulation was applied
for 4 days (start of insulation = Day 0) and semen collection and testicular ultrasonographic examinations were performed twice weekly until Day 35. Sperm concentration and total sperm output
during the post-insulation period were greater in control groups, but significant differences were
observed only in B. indicus bulls. Overall, sperm motility in scrotal-insulated B. indicus bulls was
lower (P < 0.05) than in the control group. After whole-scrotum insulation in crossbred bulls,
sperm motility was lower (P < 0.05) than pre-insulation levels between Days 21 and 31, and lower
than control levels on Day 24. The proportion of normal sperm after whole-scrotum insulation
was lower than pre-insulation and control values from Day 11 to the end of the experiment in B.
indicus bulls (P < 0.05 from Days 14 to 21 and on Day 27), and from Days 14 to 25 in crossbred
bulls (P < 0.05 on Days 14 and 18). Insulation of the scrotal neck in crossbred bulls did not significantly affect semen quality. Loose sperm heads (Day 11), midpiece defects (Days 11 and 14),
and acrosome defects (Days 27 and 31) increased (P < 0.05) in insulated B. indicus bulls, while
proximal cytoplasmic droplets (Days 14, 18 and 27 in B. indicus; Days 24 and 27 in crossbred
bulls) and sperm vacuoles (Days 18 and 21 in B. indicus; Day 18 in crossbred bulls) increased
∗ Corresponding author. Tel.: +1-306-966-7169; fax: +1-306-966-7159.
E-mail address: [email protected] (L.F.C. Brito).
0378-4320/03/$ – see front matter © 2003 Elsevier Science B.V. All rights reserved.
doi:10.1016/S0378-4320(03)00082-4
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L.F.C. Brito et al. / Animal Reproduction Science 79 (2003) 1–15
(P < 0.05) in whole-scrotum insulation groups in both experiments. There was considerable variation among bulls in the incidence of specific sperm defects. The timing of appearance of sperm
defects after insulation provided insights into the pathogenesis of specific abnormalities. Neither
whole-scrotum nor scrotal-neck insulation affected testicular echotexture in either experiment. In
conclusion, whole-scrotum insulation resulted in decreased sperm production and semen quality in
B. indicus and B. indicus × B. taurus bulls, but those changes were not associated with changes in
testicular echotexture.
© 2003 Elsevier Science B.V. All rights reserved.
Keywords: Bull; Scrotal insulation; Semen; Sperm; Ultrasound
1. Introduction
Testes in bulls are maintained 4–5 ◦ C below body temperature (Kastelic et al., 1995;
Brito et al., 2003). Elevation of testicular temperature, either by exposure to high ambient
temperatures or thermal insulation of the scrotum, disrupts spermatogenesis with a consequent decrease in both sperm production and semen quality (Casady et al., 1953; Austin,
1961). Metabolic rate and oxygen demand increase as a result of augmented temperature,
but the long and extremely coiled testicular artery limits the blood supply to the testes.
Testicular hypoxia and generation of reactive oxygen species are consequences of elevated
temperature that result in production of abnormal spermatozoa (Setchell, 1978, 1998).
The scrotal insulation model is useful to determine the effects of increased testicular
temperature on sperm production and semen quality, and to provide insights regarding the
pathogenesis of specific sperm defects. The scrotal neck is the warmest spot on the scrotal
surface (Coulter and Kastelic, 1994) and heat loss by irradiation from the scrotal neck seems
important for maintenance of adequate testicular temperature. Insulation of the scrotal neck
results in increased scrotal and testicular temperatures with adverse effects on semen quality
that resemble the effects of whole-scrotum insulation (Kastelic et al., 1996).
Bos indicus and crossbred bulls are more resistant to high ambient temperatures and have
a less pronounced decrease in semen quality than Bos taurus bulls (Johnston et al., 1963;
Skinner and Louw, 1966; Kumi-Diaka et al., 1981). B. indicus cattle are better adapted to
high temperature environments than B. taurus cattle because they have a greater skin surface
to body size ratio, more sweat glands, lower thermogenesis, and usually a smaller frame
(Turner, 1980). Moreover, B. indicus and crossbred bulls have better testicular blood supply
and thermoregulatory capabilities than B. taurus bulls, due to differences in the morphology
of the testicular vascular cones (Brito et al., 2003). However, few scrotal insulation studies
have been done in B. indicus or crossbred bulls (Wildeus and Entwistle, 1983, 1986; Fonseca
and Chow, 1995; Gabaldi, 2000). Ultrasonography is a useful, non-invasive tool that can
be used for evaluating breeding bulls. Testicular ultrasonic echotexture has been associated
with seminiferous tubule area, sperm production and semen quality in bulls (Kastelic et al.,
1997, 2001; Gabor et al., 1998a,b).
The objectives of the present study were to evaluate the effects of whole-scrotum or
scrotal-neck insulation on sperm production, semen quality and testicular echotexture in B.
indicus and crossbred bulls.
L.F.C. Brito et al. / Animal Reproduction Science 79 (2003) 1–15
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2. Materials and methods
2.1. Animals
This study was conducted from June to August at Sao Carlos, SP, Brazil. During the experimental period, mean monthly relative humidity, and average and maximum temperatures
ranged from 59 to 80%, 16.9 to 19.3 ◦ C, and 22.3 to 26.6 ◦ C, respectively. In Experiment 1,
12 B. indicus bulls (Nelore), approximately 3 years old, 665.7 ± 25.3 kg (mean ± S.E.M.),
and scrotal circumference (SC) of 32.7 ± 0.9 cm were used. In Experiment 2, 21 B. indicus × B. taurus (Canchim; 5/8 Charolais, 3/8 B. indicus), approximately 2 years old,
405.7 ± 18.6 kg, and SC of 29.2 ± 0.5 cm were used. Bulls were kept on pasture with
mineral supplementation ad libitum during the experimental period.
2.2. Scrotal insulation and semen evaluation
In both Experiments, bulls were block-randomized on the basis of SC and sperm morphology into treatment groups. In Experiment 1, bulls were allocated into two groups: control
and whole-scrotum insulation (n = 6 per group). In Experiment 2, bulls were allocated
into three groups: control, whole-scrotum insulation, and scrotal-neck insulation (n = 7
per group). For bulls in the whole-scrotum insulation groups, the scrotum was surrounded
by a disposable diaper that was covered by a cloth bag; both were held in place by placing
tape around the neck of the scrotum. For scrotal-neck insulation, an insulating material consisting of plastic-encased air bubbles sandwiched between two foil sheets (approximately
0.5 cm thick and 5 cm wide) was wrapped around the scrotal neck and taped into place.
Scrotal and scrotal-neck insulation were removed after 4 days (day of insulation = Day 0).
Semen was collected twice weekly by electroejaculation from Day 0 to 35 (crossbred
bulls were also collected on Day −4). Semen volume was measured in a graduated tube
and sperm concentration was determined using a hemocytometer. For evaluation of sperm
motility, a small drop of semen was placed on a slide, covered with a coverslip and examined with phase-contrast microscopy under 400× magnification. Several microscopic fields
were examined and the percentage of progressively motile spermatozoa was estimated in
increments of 5%. A semen sample was preserved in formol-citrate and subsequently evaluated for sperm morphology. A wet-mount was prepared and 100 spermatozoa per sample
were examined with phase-contrast microscopy under 1200× magnification. Sperm abnormalities were classified according to Barth and Oko (1989).
2.3. Testicular echotexture
Testicular ultrasonography was performed on Day −7 and just before semen collection
from Days 4 to 27 in B. indicus bulls. In crossbred bulls, ultrasonography was performed just
before semen collection from Days −4 to 32. A B-mode diagnostic ultrasound scanner with
a 7.5 MHz linear-array transducer (Scanner 450 VET Echograph; Piemedical, Maastricht,
The Netherlands) was used. Coupling gel was placed on the face of the transducer and
moderate pressure was used to press the transducer against the skin on the caudal scrotal
surface (the testis was supported by a hand placed on the anterior aspect). Each testis was
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examined separately, with the transducer oriented vertically, parallel to the long axis of the
testis, and was aligned so that the mediastinum testis was readily apparent (as a continuous
band across the image). An ultrasound image of each testis was frozen and recorded on
a VHS recorder. Ultrasound images were subsequently digitized and mean and standard
deviation (S.D.) pixel intensity (echotexture) of the testicular parenchyma were determined
in an area below the tunica albuginea (as previously described; Gabor et al., 1998b) on
a scale of 1 (white) to 255 (black) using image analysis software (Image 1.58; National
Institutes of Health, Bethesda, MD, USA). For each bull, mean and S.D. pixel intensity
were determined for each testes and the average of the two testes was used for statistical
analysis.
2.4. Statistical analyses
Statistical analyses were performed using the Statistical Analysis System (SAS Institute,
Cary, NC, USA). Mixed-Models analysis with Tukey’s test was used to detect and locate
group, time, and group by time interaction effects on sperm motility, sperm morphology,
and testicular echotexture. The covariate structure that best fitted each end point was used
(Littell et al., 1998). Pearson’s correlation coefficients between testicular echotexture, sperm
production (semen volume, sperm concentration and total sperm output), and semen quality
(proportion of motile and morphologically normal sperm) were determined.
3. Results
3.1. Experiment 1
There was a day effect (P < 0.01) and a tendency for group and group by day interaction
effects (P ≤ 0.1) on sperm concentration and total sperm output. Mean sperm concentration and total sperm output during the post-insulation period were greater in control bulls
(551.8 and 298.9 × 106 sperm/ml, and 4.6 and 2.9 × 109 sperm per ejaculate in control and
scrotal insulation groups, respectively). Sperm concentration and total sperm output in the
insulation group were lower (P < 0.05) than the pre-insulation evaluation (Day 0) on Day
14, and concentration was also lower than the control group on Day 24 (Fig. 1A). Mean
semen volume was 8.5 ml (no significant group or group by day effects).
There were group, day, and group by day interaction effects (P < 0.05) on sperm motility
and proportion of normal sperm. Motility decreased below pre-insulation levels from Days
11 to 31 in scrotal-insulated bulls, with significant reductions (P < 0.05) on Days 11 and
14 (Fig. 2A). Overall, sperm motility was greater (P < 0.05) in the control group than
in the insulation group (69.0 versus 55.0%, respectively). The proportion of normal sperm
in the scrotal insulation group was lower than the pre-insulation evaluation and than the
control group from Day 11 until the end of the experimental period in B. indicus bulls, with
significant reductions (P < 0.05) from Days 14 to 21 and on Day 27 (Fig. 3).
There were group, day, and group by day interaction effects (P < 0.05) on the proportions
of sperm with midpiece defects, proximal cytoplasmic droplets, vacuoles, and acrosome
defects. Day and group by day effects were also significant (P < 0.05) on the proportion of
L.F.C. Brito et al. / Animal Reproduction Science 79 (2003) 1–15
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Fig. 1. Mean (±S.E.M.) sperm concentration after scrotal insulation for 96 h in Bos indicus bulls (A; n = 6 per
group) in Experiment 1 and crossbred bulls (B; n = 7 per group) in Experiment 2. G: group effect; D: day effect;
G∗ D: group by day interaction effect. (∗ ) Values differ (P < 0.05) from pre-insulation period (Day 0); (∗∗ ) value
tended (P = 0.1) to be lower than control group.
loose sperm heads, with a tendency (P = 0.07) for a group effect. Loose sperm heads (Day
11), sperm midpiece defects (Days 11 and 14), proximal droplets (Days 14, 18 and 27), sperm
vacuoles (Days 18 and 21), and acrosome defects (Days 27 and 31) in scrotal-insulated bulls
were higher (P < 0.05) than both the pre-insulation evaluation and the control group (Figs. 3
and 4). Mean proportions of sperm with abnormal head shapes, distal cytoplasmic droplets
and distal piece defects did not exceed 2% in the post-insulation period (no significant group
or group by day effects).
There were no significant group, day or group by day effects on mean and S.D. testicular
ultrasound pixel intensity. Mean ultrasound pixel intensity was correlated with sperm concentration (r = −0.31, P < 0.05), sperm motility (r = −0.28, P = 0.07) and proportion
of normal sperm (r = −0.32, P < 0.05) only in the control group.
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Fig. 2. Mean (±S.E.M.) sperm motility after scrotal insulation for 96 h in Bos indicus bulls (A; n = 6 per group)
in Experiment 1 and crossbred bulls (B; n = 7 per group) in Experiment 2. G: group effect; D: day effect; G∗ D:
group by day interaction effect. (∗ ) Values differ (P < 0.05) from pre-insulation period (A: Day 0; B: Days −4 and
0); (∗∗ ) value differs (P < 0.05) from pre-insulation period and from control and scrotal-neck insulation groups.
3.2. Experiment 2
Mean concentration and total sperm output during the post-insulation period were greatest
in control bulls (338.2, 293.0 and 233.8 × 106 sperm/ml, and 2.5, 2.0 and 1.6 × 109 sperm
per ejaculate in control, scrotal-neck and whole-scrotum insulation groups, respectively).
There were day and group by day interaction effects on sperm concentration (P < 0.05), but
no significant differences were identified between pre- (Days −4 and 0) and post-insulation
evaluation days within group or among groups on any day (Fig. 1B). Mean semen volume
was 6.7 ml (no significant group or group by day effects).
There were day and group by day interaction effects on sperm motility (P < 0.05), with a
tendency (P < 0.1) for a group effect. Group, day, and group by day effects on the proportion
of normal sperm were all significant (P < 0.05). Sperm motility decreased (P < 0.05) below
L.F.C. Brito et al. / Animal Reproduction Science 79 (2003) 1–15
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Fig. 3. Mean (±S.E.M.) proportion of normal sperm (A), loose sperm heads (B), and sperm midpiece defects (C)
after scrotal insulation for 96 h in Bos indicus bulls (n = 6 per group) in Experiment 1. G: group effect; D: day
effect; G∗ D: group by day interaction effect. (∗ ) Values differ (P < 0.05) from pre-insulation period (Day 0) and
control group.
pre-insulation levels between Days 21 and 31 after whole-scrotum insulation; on Day 24 it
was also lower than control and scrotal-neck insulation groups (Fig. 2B). Insulation of the
scrotal neck did not significantly affect sperm motility in crossbred bulls. The proportion
of normal sperm in the whole-scrotum insulation group was lower than the pre-insulation
evaluations and than the control and scrotal-neck insulation groups from Days 14 to 25,
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Fig. 4. Mean (±S.E.M.) proportion of sperm with proximal cytoplasmic droplets (A), vacuoles (B), and acrosome
defects (C) after scrotal insulation for 96 h in Bos indicus bulls (n = 6 per group) in Experiment 1. G: group effect;
D: day effect; G∗ D: group by day interaction effect. (∗ ) Values differ (P < 0.05) from pre-insulation period (Day
0) and control group.
with significant reductions (P < 0.05) on Days 14 and 18 (Fig. 5). Insulation of the scrotal
neck did not significantly affect sperm morphology.
There were group, day, and group by day interaction effects (P < 0.05) on the proportion
of sperm with proximal cytoplasmic droplets. Day and group by day effects were also
significant on the proportion of sperm vacuoles, with a tendency (P = 0.07) for a group
L.F.C. Brito et al. / Animal Reproduction Science 79 (2003) 1–15
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Fig. 5. Mean (±S.E.M.) proportion of normal sperm (A), sperm vacuoles (B), and sperm with proximal cytoplasmic
droplets (C) after scrotal insulation for 96 h in crossbred bulls (n = 7 per group) in Experiment 2. G: group effect;
D: day effect; G∗ D: group by day interaction effect. (∗ ) Values differ (P < 0.05) from pre-insulation period (Days
−4 and 0), and control and scrotal-neck insulation groups; (∗∗ ) values differ from (P < 0.05) from pre-insulation
period and tended (P = 0.08) to be higher than control and scrotal-neck insulation groups.
effect. Sperm vacuoles (Day 18) in the whole-scrotum insulation group were higher (P <
0.05) than both the pre-insulation evaluation and the control group, while proximal droplets
(Days 24 and 27) were higher (P < 0.05) than the pre-insulation evaluation and tended
(P = 0.08) to be higher than the control group (Fig. 5). Although the mean proportion
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of midpiece defects was somewhat elevated in the scrotal insulation group (mean 11.3%
during the post-insulation period), there were no significant group or group by day effects.
Mean proportions of loose sperm heads, and sperm with abnormal head shapes, acrosome
defects, distal cytoplasmic droplets and distal piece defects did not exceed 3.6% in the
post-insulation period (no significant group or group by day effects).
There were no significant group, day or group by day effects on mean and S.D. testicular
ultrasound pixel intensity. Ultrasound pixel intensity S.D. was correlated with the sperm
concentration, total sperm output, and proportion of normal sperm (r = −0.16, P < 0.05
for all).
4. Discussion
A seasonality study indicated that semen quality decreased to a greater extent in crossbred
than in B. indicus bulls during the hot summer months in Brazil (Silva et al., 1991). In Costa
Rica, Chacon et al. (1999) reported that crossbred bulls evaluated at ambient temperatures
around 31 ◦ C had a greater proportion of sperm abnormalities than B. indicus bulls. It was
therefore surprising that the effects of whole-scrotum insulation were generally less severe
in crossbred than in B. indicus bulls in the present study. Effects on sperm production
were moderate and the changes in semen quality occurred later in relation to the start
of the insulation period, were less pronounced, and subsided faster in crossbred bulls.
Another observation was the great individual variation regarding the changes in sperm
quality after insulation, which is consistent with previous reports (Vogler et al., 1993; Barth
and Bowman, 1994). Barth and Oko (1989) indicate that there is a genetic predisposition
for the development of specific sperm abnormalities in response to adverse conditions like
elevated testicular temperature. This individual variation may be due to differences in the
testicular blood supply and biochemical response to the thermal insult (e.g. antioxidant
production).
Different testicular germ cell lines have different sensitivity to thermal insults; spermatocytes and spermatids are particularly sensitive, while spermatogonia are more resistant
(Setchell, 1978). Therefore, after scrotal insulation changes in sperm production and semen
quality are manifested after an interval that varies according to the developmental stage
of the germ cells at the time of insulation and the time required for the damaged cells to
be released into the seminiferous tubules and transported through the epididymis (Barth
and Oko, 1989). The duration of the seminiferous epithelial cycle (SEC) and the time of
epididymal transport (approximately 11 day in bulls) are not altered by scrotal insulation
(Ross and Entwistle, 1979). Furthermore, the duration of the SEC in crossbred and B. indicus bulls is similar to B. taurus bulls (Salim and Entwistle, 1982; Cardoso and Godinho,
1983), allowing inferences about the sensitivity of specific germ cell lines to be drawn from
the time required for changes in sperm production and semen quality to be manifested after
scrotal insulation.
Whole-scrotum insulation diminished both sperm concentration and total sperm output in B. indicus and crossbred bulls. The decline in sperm concentration observed around
Days 11 to 14 in the present study and in other reports (Fonseca and Chow, 1995;
Gabaldi, 2000) indicated that degeneration of spermatids accounted for the initial reduction
L.F.C. Brito et al. / Animal Reproduction Science 79 (2003) 1–15
11
in sperm concentration after scrotal insulation. Wildeus and Entwistle (1983, 1986) did not
detect a significant effect of scrotal insulation on sperm output, but suggested that damaged
spermatids were selectively resorbed in the epididymis, resulting in decreased epididymal
sperm reserves. Reduced sperm production that persists beyond 30 days after insulation
indicates degenerative changes in spermatocytes (Austin et al., 1961; Fonseca and Chow,
1995; Gabaldi, 2000).
The proportion of motile and morphologically normal sperm declined 11 days after
whole-scrotum insulation in B. indicus bulls. Although sperm motility reached control levels by Day 35, the proportion of normal sperm had still not returned to control levels by
that time (although there was consistent improvement after Day 27). In crossbred bulls,
whole-scrotum insulation resulted in reduced sperm motility from Days 21 to 31 and increased proportion of abnormal sperm from Days 18 to 28, with complete recovery by the
end of the experiment. Therefore, spermatids were adversely affected during all phases of
spermiogenesis in B. indicus bulls, but spermatids in maturation and late acrosome phases
were not markedly affected in crossbred bulls. Moreover, the reduced proportion of normal sperm beyond 30 days after insulation in B. indicus bulls indicated that spermatocytes
were also affected in these bulls, but not in crossbred bulls. In previous studies in B. taurus bulls, scrotal insulation also resulted in decreased numbers of normal sperm, which
returned to control levels about 32 to 42 days after insulation (Austin et al., 1961; Vogler
et al., 1993; Barth and Bowman, 1994). However, in other experiments scrotal insulation
in B. indicus bulls for 96 or 168 h resulted in a greater and more prolonged reduction in the
proportions of motile and normal sperm, which required 105 to 148 days to attain control
levels (Fonseca and Chow, 1995; Gabaldi, 2000). Individual variations in sperm motility
and normal morphology were evident. One B. indicus bull had very little variation in sperm
motility (mean 67%, minimum 50%) and normal morphology (mean 84%, minimum 74%)
after scrotal insulation. The lowest levels of sperm motility ranged from 0 to 40% and the
lowest proportion of normal sperm ranged from 12 to 66% in B. indicus and crossbred bulls.
Increased proportion of loose sperm heads after scrotal insulation is a consistent finding
among studies (Austin et al., 1961; Wildeus and Entwistle, 1983, 1986; Barth and Bowman,
1994; Vogler et al., 1993; Fonseca and Chow, 1995; Gabaldi, 2000). Despite the significant
increase in loose sperm heads in B. indicus bulls, maximum levels of only 3–6% were
observed during the post-insulation period in three bulls. The peak incidence of loose heads
was observed on Days 11 and 14 and ranged from 9 to 38% in the three remaining bulls. In
crossbred bulls, despite the lack of a significant group effect, two bulls in the whole-scrotum
insulation group had a substantial increase in loose sperm heads on Day 7 (14%) or Day 18
(75%). The basal plate, a structure of nuclear origin that lines the implantation fossa and
connects the head to the capitulum of the tail, does not form completely in these abnormal
sperm. Therefore, the head and the tail are kept together only by the plasmalemma and the
head is subsequently broken loose from the tail, probably as a consequence of initiation
of motility and cytoplasmic droplet migration in the epididymis (Barth and Oko, 1989).
The increased proportion of loose sperm heads around 11–14 days after scrotal insulation
suggested interference with basal plate formation in spermatids during the maturation phase.
However, malformation of the basal plate may also occur earlier, during the late acrosome
phase, as suggested by the increase in loose heads 18 days after scrotal insulation in one
bull. The manifestation of this abnormality in sperm that would have been in the epididymis
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during the period of insulation (i.e. sperm collected before 10 days after insulation) in
the present study and in other reports (Wildeus and Entwistle, 1983; Barth and Bowman,
1994; Gabaldi, 2000) also indicated that altered epididymal function may have a role in the
pathogenesis of this defect.
Sperm midpiece defects increased substantially after whole-scrotum insulation in all but
one B. indicus and two crossbred bulls. Lack of a significant group or group by time effect
in crossbred bulls was probably due to the relatively high levels of midpiece defects in some
bulls in both control and scrotal-neck insulation groups. The peak incidence of midpiece
defects ranged from 11 to 43% and was attained between Days 7 and 14 in five B. indicus
bulls and in three crossbred bulls. In the remaining two crossbred bulls, peak levels were
observed on Days 18 or 21. The predominant defective form was the distal midpiece reflex
(DMR). The timing of appearance of sperm with DMR in this study and in a previous
report (Barth and Bowman, 1994) reflects the pathogenesis of this defect, which develops
as sperm migrate to the distal half of the epididymal tail, probably in association with
altered ion concentrations (Barth and Oko, 1989). High proportions of DMR 18–21 days
after insulation also indicate that injured spermatids may result in sperm predisposed to this
defect. A large number of other midpiece defects (disrupted mitochondrial sheet, segmental
aplasia) were observed on Day 24 in one B. indicus bull (accounting for 14% out of a 19%
total midpiece defects) and on Day 32 in one crossbred bull (accounting for 14% out of a
25% total midpiece defects).
Although mean peak levels of proximal cytoplasmic droplets were attained on Days 14
and 18 in B. indicus bulls and on Days 24 and 27 in crossbred bulls, there was wide individual
variation in the interval between whole-scrotum insulation and maximal numbers of proximal droplets. Peak levels occurred between Days 11 and 35 and ranged from 16 to 55%.
The number of sperm with proximal droplets did not increase during the post-insulation
period in just one B. indicus bull (max. 3%). Other studies have reported that the time to
peak levels of proximal cytoplasmic droplets after scrotal insulation is 11–14 days (Barth
and Bowman, 1994; Fonseca and Chow, 1995), 17–20 days (Wildeus and Entwistle, 1983,
1986) or 38 days (Gabaldi, 2000). Taken together, these observations indicate that proximal
droplets can develop as the result of deleterious effects of elevated testicular temperature
on spermatids in any stage of spermiogenesis and even on spermatocytes. The increase
in proximal droplets numbers observed 7 and 10 day after insulation by Gabaldi (2000)
also suggest that altered epididymal function may be involved in the pathogenesis of this
abnormality.
The proportion of sperm vacuoles increased in all B. indicus bulls and in four crossbred
bulls after whole-scrotum insulation. In B. indicus bulls, peak levels ranged from 5 to 10% in
three bulls, but ranged from 22 to 47% in the remaining bulls. The proportion of sperm with
vacuoles was less in crossbred bulls, ranging from 5 to 14%. The peak of sperm vacuoles
was the least variable among all sperm abnormalities, i.e. Days 18 and 21, with high levels
observed only on the peak day, in agreement with previous reports (Vogler et al., 1993; Barth
and Bowman, 1994). According to Barth and Oko (1989), spontaneous vacuole formation
in bulls begins in spermatids during the late cap phase. However, the presence of high levels
of vacuoles in ejaculates collected 18 to 21 days after scrotal insulation indicate that the
adverse effect of elevated testicular temperature causing this abnormality is exerted only on
spermatids during the acrosome phase.
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Acrosome defects did not exceed 2% in one B. indicus bull after scrotal insulation.
The maximum proportion of acrosome defects (mainly knobbed acrosome) was 9% in
two bulls and ranged from 17 to 22% in the three remaining B. indicus bulls, registered
between Days 24 and 35. Despite the lack of a significant group effect in crossbred bulls,
the proportion of acrosome defects was substantially increased on Days 21 or 25 (6 and
8%) after whole-scrotum insulation in two bulls. In a third bull in this group, there were
two peaks of acrosome defects, the first on Day 11 (15%) and the second on Day 35 (11%;
preceded by 7% on Day 32). Variable results have been reported in the literature regarding
the appearance of acrosome defects following scrotal insulation. Some authors reported an
increase in the proportion of acrosome defects 17–18 days after scrotal insulation (Wildeus
and Entwistle, 1983; Barth and Bowman, 1994), while others detected substantial levels
of acrosome defects only after 27 to 28 days (Vogler et al., 1993; Gabaldi, 2000). These
apparent contrasts among studies are consequences of the pathogenesis of the knobbed
acrosome defect, which can develop in spermatids either during the late acrosome phase or
during the cap phase (Barth and Oko, 1989). The rise of acrosome defects on Days 11 and
35 in one bull in the present experiment indicated that spermatids in the maturation phase
and spermatocytes could also be involved in the pathogenesis of this sperm defect.
Scrotal insulation reportedly causes an increase in the proportion of sperm with abnormal
head shapes 21 to 28 days after the insult (Vogler et al., 1993; Barth and Bowman, 1994;
Fonseca and Chow, 1995). However, only one B. indicus and two crossbred bulls had
increased numbers of abnormal sperm heads after whole-scrotum insulation in the present
study; this was responsible for the overall lack of a significant group or group by time effect
on this abnormality. The predominant sperm head shape abnormalities were pyriform and
microcephalic heads. The proportion of abnormal heads increased between Days 24 and 35
after insulation, reaching a peak of 28% on Day 31 in the B. indicus bull. In the crossbred
bulls, the proportion of sperm with abnormal heads increased on Days 18 and 21 (12 and
16%) in one bull, and on Days 21 and 35 (12%) in the other bull. The head shape is dictated
primarily by the nucleus shape, which is in turn determined by extrinsic forces from the
Sertoli cell or the caudal manchette of the spermatid, or by intrinsic factors that affect
nuclear chromatin condensation (Barth and Oko, 1989). The timing of the appearance of
these defects indicates that they were consequences of adverse effects on spermatids not
only during the acrosome phase, when visible nuclear flattening and elongation occur, but
also during the Golgi and cap phases, when morphological changes in the nuclear shape
are still not apparent. Moreover, primary spermatocytes in late pachytene and secondary
spermatocytes were also affected, likely resulting in the formation of microcephalic sperm,
which are probably a consequence of deficient nuclear chromatin content ensuing from
abnormal cell division (Barth and Oko, 1989).
In a previous study, insulation of the scrotal neck in B. taurus bulls increased scrotal
and testicular temperatures, with adverse effects on semen quality reflected by a increased
proportion of abnormal sperm (Kastelic et al., 1996). However, insulation of the scrotal neck
in crossbred bulls in the present experiment did not significantly affect sperm production
and semen quality. Perhaps differences in the duration of insulation (8 days in the previous
study) or ambient temperature were responsible for these contrasting results. Moreover, the
shorter distance between the arterial and the venous blood in the testicular vascular cones
contributes to a more efficient counter-current heat exchange mechanism in crossbred bulls
14
L.F.C. Brito et al. / Animal Reproduction Science 79 (2003) 1–15
compared to B. taurus bulls (Brito et al., 2003). Therefore, heat loss by irradiation from the
scrotal neck may be less important in crossbred bulls.
Significant correlations between testicular echotexture and sperm production and semen
quality have been previously reported in bulls (Kastelic et al., 1997, 2001; Gabor et al.,
1998a). In the present study however, there was no significant change in testicular echotexture associated with scrotal insulation, despite the decrease in sperm production and
semen quality, demonstrating that the mild and transitory effects induced by insulation
were not sufficient to cause morphological changes that could be detected by ultrasonography. Significant correlations of mean testicular pixel intensity with sperm concentration,
motility and morphology production were observed in B. indicus bulls only in the control
group. In crossbred bulls, the negative correlations of testicular pixel intensity S.D. with
sperm concentration, total sperm output, and proportion of normal sperm indicated that a
less homogeneous testicular parenchyma was associated with decreased sperm production
and semen quality.
In conclusion, whole-scrotum insulation resulted in decreased sperm production and
semen quality in B. indicus and crossbred bulls. Decreased semen quality was characterized
by a decline in sperm motility and by a specific temporal appearance of sperm defects,
though great individual variations were observed. The changes in sperm production and
semen quality were not associated with changes in testicular echotexture.
Acknowledgements
Research sponsored by EMBRAPA-CPPSE and Agriculture and Agri-Food Canada. The
authors are thankful to: EMBRAPA-CNPGC for supplying Nelore bulls; EMBRAPA-CPPSE
staff for managing the animals and for technical assistance; and Dr. A.D. Barth for critical
review of this manuscript.
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