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Abstract
The objective of this study was to determine the relationship between semen quality and testicular
pixel–intensity derived from image analysis of ultrasonograms after scrotal insulation in bulls. In
addition, the ability to predict semen quality based on testicular pixel–intensity was evaluated.
Sixteen beef bulls were selected on the basis of satisfactory semen quality and normal testicular
ultrasonogram appearance. Bulls were allocated into two groups for scrotal insulation for 4 days
(group 1) or 8 days (group 2). Semen was collected and evaluated twice weekly and testicular
ultrasonograms were evaluated once weekly for 8 weeks after removal of scrotal insulation. In
general, the percentages of motile and morphologically normal spermatozoa decreased below pre-
insulation levels from 1 to 5 weeks after scrotal insulation removal. Overall, group 1 had greater
(P < 0.01) percentages of motile and normal spermatozoa than group 2. Mean testicular pixel–
intensity (PI), and the number of pixels corresponding to the intensity that occurs most frequently
(NP) decreased in the first 2–3 weeks after scrotal insulation, coincidently with the decrease in sperm
motility and normal morphology. When the entire data set was evaluated, there was no association
between testicular PI or NP with semen quality observed at the same week of ultrasound examina-
tions. However, regression models indicated that testicular PI and NP accounted for 13–25% of the
variation in sperm motility and morphology in ejaculates collected 2–4 weeks after ultrasound exam.
Testicular PI and NP had moderate sensitivity and negative predictive values (64.5–82.6%), but low
specificity and positive predictive values (33.3–61.2%) as predictors of satisfactory semen quality
(60% motile spermatozoa and 70% morphologically normal spermatozoa) for ejaculates
* Corresponding author. Tel.: +1 306 966 7169; fax: +1 306 966 7159.
E-mail address: lfcbrito@lycos.com (Leonardo F.C. Brito).
0093-691X/$ – see front matter # 2004 Elsevier Inc. All rights reserved.
doi:10.1016/j.theriogenology.2004.12.008
A.A. Arteaga et al. / Theriogenology 64 (2005) 408–415 409
collected 2–4 weeks after ultrasound exam. In conclusion, the deleterious effects of scrotal insulation
on semen quality were dependent on the length of the period of insulation and were associated with
changes in testicular ultrasonogram pixel–intensity. Testicular ultrasonogram pixel–intensity had a
better association with future semen quality than with present semen quality and was a better
predictor of unsatisfactory semen quality than satisfactory semen quality.
# 2004 Elsevier Inc. All rights reserved.
1. Introduction
Sixteen beef bulls (Hereford, Angus, and Charolais; 18–36-month old) were randomly
allocated into two treatment groups with scrotal insulation applied for 4 days (group 1) or
8 days (group 2). Bags for scrotal insulation were made with single layers of denim,
thinsulate, heat reflecting fabric, and flannelette. Insulation bags were lightly tied closed over
the neck of the scrotum with a purse string and sutured to the skin adjacent to the top of the
scrotum. Semen samples and testicular ultrasonograms were obtained and analyzed once
just prior to insulation. Following removal of the insulation, semen collections were done
twice weekly, while testicular ultrasonograms were obtained once weekly for 8 weeks.
410 A.A. Arteaga et al. / Theriogenology 64 (2005) 408–415
3. Results
There were group (P = 0.007) and time effects (P < 0.0001) on sperm motility. Overall,
group 1 had greater sperm motility than group 2 (66.4% versus 41.5%, respectively).
A.A. Arteaga et al. / Theriogenology 64 (2005) 408–415 411
Fig. 1. Mean (S.E.M.) proportion of motile (A) and normal spermatozoa (B) after scrotal insulation for 4 days
(group 1; n = 8) or 8 days (group 2; n = 8) in bulls (G: group effect; T: time effect; G T: group-by-time
interaction effect). (*) Values within group differ (P < 0.05) from the day before insulation (day 0). (a,b) Values
differ (P < 0.05) between groups within examination day.
Sperm motility decreased (P < 0.05) below pre-insulation levels in both groups 7 days
after insulation removal and returned to the pre-insulation levels after 38 days only in group
1 (Fig. 1A). There were group (P = 0.009), time (P < 0.0001) and group-by-time
interaction effects (P = 0.002) on the proportion of normal spermatozoa. The proportion of
normal spermatozoa decreased (P < 0.05) below pre-insulation levels between 10 and 31
days after insulation removal in group 1 and between 3 and 35 days in group 2. The
proportion of normal spermatozoa was lower (P < 0.05) in group 2 than in group 1
between 3 and 49 days after insulation removal. Despite the lack of significant difference
from pre-insulation, the proportion of normal spermatozoa in group 2 was only 59% at 56
days after insulation removal (Fig. 1B).
There were time effects (P < 0.001) on PI and NP. Testicular PI decreased (P < 0.001)
below pre-insulation levels at 14 and 28 days after insulation removal and tended
(P = 0.08) to be lower than pre-insulation on day 35 (Fig. 2A). Testicular NP decreased
below pre-insulation levels 21 days after insulation removal (P < 0.05 on Days 21, 49, and
56; Fig. 2B). There was no significant correlation between PI and NP, and sperm motility
412 A.A. Arteaga et al. / Theriogenology 64 (2005) 408–415
Fig. 2. Mean (S.E.M.) testicular ultrasonogram pixel–intensity level (PI; A) and number of pixels of the most
frequent intensity level of the area (NP; B) observed after scrotal insulation for 4 or 8 days in bulls (n = 16) (T: time
effect). (*) Values differ (P < 0.05) from the day before insulation (day 0).
and morphology at the same week that ultrasound exams were performed. However,
significant correlations between PI and NP, and the percentages of motile and
morphologically normal spermatozoa were observed when ejaculates collected at
successive weeks after ultrasound exams were considered. In general, correlations
increased for ejaculates obtained between 1 and 3 weeks after the ultrasound examination
(data not shown). Regression models indicated that PI and NP accounted for 13–25% of the
variation in sperm motility and morphology observed between 2 and 4 weeks after the
ultrasound examination (Table 1); only 6–7% of the variation was accounted for in the
ejaculate collected 1 week after ultrasound examination. There were moderate sensitivity
(64.5–74.2%) and negative predictive values (75.0–82.6%) when PI 26 and NP 150
were used as predictors of satisfactory semen quality for ejaculates collected 2–4 weeks
after ultrasound exam, but specificity (45.7–61.2%) and positive predictive values (33.3–
51.3%) were low (Table 2).
4. Discussion
The timing of decline in semen quality observed after scrotal insulation in the present
experiment was similar to previous studies [2–6]. Sperm motility and morphology reached
A.A. Arteaga et al. / Theriogenology 64 (2005) 408–415 413
Table 1
Regression models for the percentages of motile and morphologically normal sperm observed weeks after
ultrasound exam using mean testicular ultrasonogram pixel–intensity level (PI) and number of pixels correspond-
ing to the intensity level that occurred most frequently (NP) as independent variables
Slope R2 Probability
Motile sperm
P
2 weeks ( R2 = 0.19; y-intercept = 259.3)
PI 3.02 0.1 0.0001
NP 0.83 0.09 0.0005
P
3 weeks ( R2 = 0.18; y-intercept = 242.7)
PI 3.14 0.12 0.0001
NP 0.68 0.06 0.005
P
4 weeks ( R2 = 0.16; y-intercept = 144.1)
PI 3.08 0.16 0.0001
Normal spermP
2 weeks ( R2 = 0.18; y-intercept = 254.6)
PI 2.0 0.03 0.008
NP P 1.04 0.15 < 0.0001
3 weeks ( R2 = 0.25; y-intercept = 284.4)
PI 3.11 0.1 0.0001
NP P 1.02 0.15 < 0.0001
4 weeks ( R2 = 0.13; y-intercept = 132.5)
PI 2.89 0.13 0.0005
P 2
R : total model R2.
the lowest levels 2–3 weeks after insulation removal in both groups and started to improve
after 4 weeks. Eight days of scrotal insulation appeared to have a more severe effect on
semen quality than insulation for 4 days, suggesting that scrotal insulation not only have a
detrimental effect on semen quality, but also that the severity of this detrimental effect is
related to the length of the period of insulation.
Visual assessment of testicular ultrasonograms in the absence of gross pathological
conditions has very limited diagnostic value, since there were no significant correlations
between visual analysis and semen quality in bulls [13]. Furthermore, one study found no
Table 2
Sensitivity, specificity, and predictive values (PV) of mean testicular ultrasonogram pixel–intensity level (PI) and
number of pixels corresponding to the intensity level that occurred most frequently (NP) to predict satisfactory
semen quality (60% motile spermatozoa and 70% morphologically normal spermatozoa) weeks after the
ultrasound examination
Sensitivity (%) Specificity (%) Positive PV (%) Negative PV (%)
PI 26
2 weeks 67.7 53.1 35.6 81.1
3 weeks 74.2 58.5 46.0 82.6
4 weeks 64.5 61.2 51.3 73.2
NP 150
2 weeks 71.0 45.7 33.3 80.4
3 weeks 74.2 50.8 41.8 80.5
4 weeks 71.0 55.1 50.0 75.0
414 A.A. Arteaga et al. / Theriogenology 64 (2005) 408–415
Acknowledgement
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