Nutrition and Sexual Development in Bulls.

This manuscript provides an overview of the effects of nutrition during different stages of bull sexual development. Nutrition during the prepubertal period can modulate the hypothalamic GnRH pulse generator. Increased nutrition results in greater LH secretion, earlier puberty, and greater testicular mass in yearling bulls, whereas low nutrition has opposite effects. Targeting average daily gain from birth to 24 weeks of age to > 1.2 kg/d and limiting gain after 24 weeks of age to < 1.6 kg/d is recommended to optimize bull sexual development.

Penicillin and amikacin mixture has bactericidal activity equivalent to gentamicin, tylosin, lincomycin and spectinomycin mixture in equine frozen semen.

Neat stallion semen can contain a variety of microorganisms, some of which may impair sperm quality and/or cause infection of the mares' reproductive tract. For this reason, antibiotics are commonly added to semen extenders. A combination of gentamicin, tylosin, lincomycin and spectinomycin (GTLS) has been recommended for use, but there are no reports on the use of this mixture in equine semen extender. Penicillin and amikacin (PA) are safe for preserving sperm quality while effectively controlling bacterial growth in equine cooled stored semen, but data on frozen semen are scarce. Therefore, a bioequivalence study was performed to assess the bactericidal activity of GTLS and PA in equine frozen semen. Nine mature, healthy stallions were used in the study. Split ejaculates were processed using media without antibiotics (Control) or with different antibiotics. For the GTLS group, centrifugation medium and freezing extender were prepared with gentamicin 250 µg/ml, tylosin 50 µg/ml, lincomycin 150 µg/ml and spectinomycin 300 µg/ml. For the PA group, the centrifugation medium was prepared with potassium penicillin G (PPG) 1200 units/ml and the freezing extender was prepared with PPG 1200 units/ml and amikacin 500 µg/ml. Semen processed in extenders without antibiotics had higher (p < .005) bacterial loads throughout all cryopreservation processing steps than semen samples processed using antibiotics. There were no differences in semen bacterial load after centrifugation, 15 and 30 min after final extension, and after thawing between GTLS and PA groups, but PA had faster (p < .05) kill-time kinetics than GTLS. Only minor differences in sperm kinetic parameters were observed among groups. In conclusion, this study demonstrated bioequivalence between GTLS and PA in mitigating end-point bacterial loads. Prudent concentrations of the antibiotic mixtures evaluated in this study can be considered both effective and sperm-safe for equine frozen semen.

Andrology laboratory review: Evaluation of sperm concentration.

This article is the result of the work of the andrology task-force of the Association of Applied Animal Andrology, American College of Theriogenologists, European College of Animal Reproduction, Society for Theriogenology, and National Association of Animal Breeders. It is intended to serve as a comprehensive reference on methods to evaluate sperm concentration and to contribute to the adoption of best practices in veterinary andrology laboratories. The information covered in the article includes sample preparation and the use of manual counts, spectrophotometers, computer-assisted semen analysis, NucleoCounter, and flow cytometry. Emphasis is given to the principles of the methods and equipment, performing the evaluation, and common mistakes and/or pitfalls. In addition, the precision and accuracy of the different methods are also discussed.

A multilaboratory study on the variability of bovine semen analysis.

To evaluate the variability of semen analysis, five replicates of 10 different bovine frozen semen batches were coded with different identification numbers and submitted to various laboratories for evaluation. Three studies were conducted: study I included eight laboratories in semen processing centers in the United States; study II included one laboratory in one semen processing center and five veterinary university laboratories in the United States; and study III included five veterinary university laboratories in Brazil. Evaluation methodology, sample classification criteria, and reporting format varied considerably among laboratories. There were laboratory effects (P < 0.05) on sperm concentration, motility, and morphology results in all studies. When Bland-Altman plots were evaluated, differences in sperm concentration were approximately between -5 and +5 × 10(6) sperm/mL in study I, when the same method of evaluation was used by all laboratories but ranged between -30 and +30 × 10(6) sperm/mL in studies II and III. Differences in the proportions of motile sperm were approximately -30% to +30%, and differences in the proportion of normal sperm were -15% to +15% in studies I and II; these differences were -15% to +15% and -10% to +10%, respectively, in study III. Mean absolute (one tail) proportional differences in estimates across all laboratories ranged from 9% to 31%, 16% to 37%, and 9% to 14% for sperm concentration, motile sperm, and normal sperm across studies; much larger (48%-86%) differences were observed for sperm abnormality categories. Intralaboratory and interlaboratory precision varied considerably across laboratories and seemed to be at least in part related to methods used for evaluation; precision was better when the NucleoCounter was used for evaluation of sperm concentration, whereas the use of computer-assisted sperm analysis for evaluation of sperm motility resulted in greater precision in some but not all laboratories. None of the laboratories that classified samples as satisfactory or unsatisfactory achieved complete consistency for all replicates within all batches. In addition, consistent classification among laboratories was observed for just three batches in studies II and III. These observations put the reliability of semen analysis in check and make it very difficult, if not impossible, to meaningfully interpret evaluation results.

Testicular vascular cone development and its association with scrotal temperature, semen quality, and sperm production in beef bulls.

The objectives of this study were to characterize development of the testicular vascular cone using ultrasonography and to determine associations of vascular cone morphology with scrotal temperature, semen quality, and sperm production. Beef bulls (n=70) were examined from 10 to 70 wk of age in two years, and a third group of bulls (n=44) was examined only at 74 wk of age. Testicular vascular cone diameter increased until approximately 13.5 mo of age, or until 1 to 8 wk before maximum scrotal circumference was observed. Vascular cone fat thickness also increased with age and followed a pattern similar to that observed for backfat. Testicular artery wall thickness and the distance from the arterial to the venous blood in the vascular cone decreased with proximity to the testis. Vascular cone diameter was negatively correlated with scrotal surface temperatures and with the percentage of sperm head defects and detached sperm heads, but positively correlated with the percentage of normal sperm. The arterial-venous blood distance was negatively correlated with the percentage of normal sperm and positively correlated with percentage of sperm head defects and proximal droplets. In conclusion, testicular vascular cone diameter increased with age following testicular development, whereas vascular cone fat thickness increased similar to a pattern observed for backfat. Increased testicular vascular cone diameter and decreased distance between arterial and venous blood were associated with increased percentage of normal sperm and decreased percentages of sperm defects.

Effect of method and clinician on stallion sperm morphology evaluation.

The objective of this study was to determine the effects of method and clinician on stallion sperm morphology evaluation. Five clinicians evaluated 60 semen samples using wet-mount preparations with phase-contrast, eosin/nigrosin-stained semen smears, and Papanicolaou-stained semen smears. There were significant differences among methods for all sperm morphology categories and most intra-class correlation coefficients were only fair to moderate. The use of wet-mount preparations facilitated detection of acrosome defects, nuclear vacuoles, and cytoplasmic droplets when compared to stained smears. Smearing stallion semen samples onto slides increased the proportion of detached sperm heads. In addition, acrosome defects, nuclear vacuoles, rough/swollen midpieces, and cytoplasmic droplets were difficult to observe with Papanicolaou stain; this method resulted in overestimation of normal sperm when compared to other methods. There were significant differences among clinicians for all sperm morphology classification categories. In conclusion, this study demonstrated that sperm morphology evaluation results varied, depending on the evaluation method and clinician. Wet-mount preparation with phase-contrast microscopy appeared to be more sensitive for identification of abnormal stallion sperm when compared to stained smears. Veterinary andrology laboratories should invest in training, continuing education, proficiency testing, and other quality control measures to minimize the variation of sperm morphology evaluation results among clinicians.

Effects of intratesticular zinc gluconate treatment on testicular dimensions, echodensity, histology, sperm production, and testosterone secretion in American black bears (Ursus americanus).

Eight adult American black bears were used to evaluate the effects of chemical castration by intratesticular zinc gluconate treatment on testicular dimensions, echodensity, histology, sperm production, and testosterone secretion. Treatment did not affect testicular dimensions and did not result in decreased resting or GnRH-stimulated testosterone secretion. Multifocal hyperchoic areas in the testicular parenchyma were observed on ultrasound examination, and white foci were observed on gross pathology examination after zinc gluconate treatment. Histologically, there were normal seminiferous tubules containing either round or elongated spermatids, along with abnormal tubules in all bears after treatment. Vacuolation of the seminiferous epithelium, sloughing of germ cells into the tubules' lumen, presence of multinuclear giant cells, and reduced height of the seminiferous epithelium with missing generations of germ cells were commonly observed. The most severe testicular changes were multifocal and included fibrosis, complete degeneration of the seminiferous epithelium with shrinkage of the tubule, and sperm stasis. Epididymal sperm reserve was 982.74 ± 654.16 × 10(6) sperm (mean ± SEM) and motile sperm were observed in the epididymis of all but one of the bears. In conclusion, although intratesticular zinc gluconate treatment in black bears resulted in testicular degenerative changes detected by ultrasound and histology examinations, sperm production was not completely ablated. We inferred that normal fertility might have been compromised, but treatment unlikely resulted in sterility.

Effect of nutrition during calfhood and peripubertal period on serum metabolic hormones, gonadotropins and testosterone concentrations, and on sexual development in bulls.

The objective of the present study was to characterize the effects of nutrition on circulating concentrations of metabolic hormones, gonadotropins, and testosterone during sexual development in bulls. Nutrition regulated the hypothalamus-pituitary-testes axis through effects on the GnRH pulse generator in the hypothalamus and through direct effects on the testes. Pituitary function (gonadotropin secretion after GnRH challenge) was not affected by nutrition. However, nutrition affected LH pulse frequency and basal LH concentration during the early gonadotropin rise (10-26 weeks of age). There were close temporal associations between changes in insulin-like growth factor-I (IGF-I) concentrations and changes in LH pulse frequency, suggesting a role for IGF-I in regulating the early gonadotropin rise in bulls. The peripubertal increase in testosterone concentration was delayed in bulls with lesser serum IGF-I concentrations (low nutrition), suggesting a role for IGF-I in regulating Leydig cell function. Serum IGF-I concentrations accounted for 72 and 67% of the variation in scrotal circumference and paired-testes volume, respectively (at any given age), indicating that IGF-I may regulate testicular growth. Bulls with a more sustained elevated LH pulse frequency during the early gonadotropin rise (high nutrition) had greater testicular mass at 70 weeks of age relative to the control group (medium nutrition), despite no differences in metabolic hormone concentrations after 26 weeks of age. Therefore, gonadotropin-independent mechanism regulating testicular growth might be dependent on previous gonadotropin milieu.

Follicular dynamics and plasma FSH and progesterone concentrations during follicular deviation in the first post-ovulatory wave in Nelore (Bos indicus) heifers.

The objective of the present study was to characterize ovarian follicular dynamics and hormone concentrations during follicular deviation in the first wave after ovulation in Nelore (Bos indicus) heifers. Ultrasonographic exams were performed and blood samples were collected every 12h from the day of estrus until 120-144 h after ovulation in seven females. Deviation was defined as the point at which the growth rate of the dominant follicle became greater than the growth rate of the largest subordinate follicle. Deviation occurred approximately 65 h after ovulation. Growth rate of the dominant follicle increased (P<0.05) after deviation, while growth rate of the subordinate follicle decreased (P<0.05). Diameter of the dominant follicle did not differ from the subordinate follicle at deviation (approximately 5.4mm). The dominant follicle (7.6mm) was larger (P<0.05) than the subordinate follicle (5.3mm) 96 h after ovulation or 24h after deviation. Plasma FSH concentrations did not change significantly during the post-ovulatory period. The first significant increase in mean plasma progesterone concentration occurred on the day of follicular deviation. In conclusion, the interval from ovulation to follicular deviation (2.7 days) was similar to that previously reported in B. taurus females, but follicles were smaller. Diameters of the dominant follicle and subordinate follicle did not differ before deviation and deviation was characterized by an increase in dominant follicle and decrease in subordinate follicle growth rate. Variations in FSH concentrations within 12-h intervals were not involved in follicular deviation in Nelore heifers.

Effect of improved nutrition during calfhood on serum metabolic hormones, gonadotropins, and testosterone concentrations, and on testicular development in bulls.

The objective of the present study was to evaluate the effects of improved nutrition during calfhood on serum metabolic hormones, gonadotropins and testosterone concentrations, and on sexual development in bulls. Bulls received high (n=17) or control nutrition (n=16) diets from 10 to 30 week of age and the same control nutrition diet from 31 to 74 week of age. Improved nutrition during calfhood resulted in a more sustained period of elevated LH secretion (pulse frequency and total secretion in 10h) during the early gonadotropin rise. GnRH-stimulated LH secretion was not affected by diet, indicating that pituitary responsiveness was not altered; therefore, improved nutrition had direct effects on GnRH secretion by the hypothalamus. Insulin and insulin-like growth factor-I (IGF-I) concentrations were greater during calfhood in bulls receiving high nutrition, indicating that these metabolic hormones might be involved in regulating GnRH and LH secretion. Improved nutrition also resulted in increased testosterone secretion that was associated with greater circulating IGF-I concentrations, suggesting a role for this metabolic hormone in regulating Leydig cell number and function. Furthermore, improved nutrition during calfhood resulted in greater testicular weight and sperm production in mature bulls, indicating that increased LH secretion during calfhood, and increased IGF-I and testosterone concentrations during calfhood and peripubertal period were associated with greater testicular cell proliferation and enhanced function.

Relationship between semen quality and pixel-intensity of testicular ultrasonograms after scrotal insulation in beef bulls.

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 examinations. 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 (> or = 60% motile spermatozoa and > or = 70% morphologically normal spermatozoa) for ejaculates 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.

Sexual development in early- and late-maturing Bos indicus and Bos indicus x Bos taurus crossbred bulls in Brazil.

Two experiments were conducted to evaluate sexual development in early- and late-maturing Nelore (Bos indicus) and Canchim (3/8 Bos indicus x 5/8 Bos taurus crossbred) bulls and to determine predictors of sexual precocity, and pubertal and maturity status. In Experiment 1, 12 Nelore bulls where examined from 300 to 900 days of age. Puberty was characterized by an ejaculate containing > or =50 million sperm with > or =10% motile sperm, and maturity by an ejaculate containing > or =70% morphologically normal sperm. In Experiment 2, 28 Canchim bulls where examined from 295 to 488 days of age and puberty was characterized by an ejaculate containing > or =30% motile sperm. In both experiments, bulls were classified as early- or late-maturing based on age at puberty. Early-maturing bulls were younger (P < 0.05) than late-maturing bulls at puberty (527 days versus 673 days in Experiment 1 and 360 days versus 461 days in Experiment 2) and at maturity (660 days versus 768 days in Experiment 1). In general, early-maturing bulls were heavier and had greater scrotal circumference (SC), testes, and testicular vascular cone diameter than late-maturing bulls during the experimental period. Scrotal circumference adjusted for 365 days of age was a good predictor of sexual precocity; minimum yearling SC of 19 and 24 cm for Nelore and Canchim bulls, respectively, had the best predictive values. Early-maturing bulls were lighter and had smaller SC at puberty than late-maturing bulls; therefore, sexual precocity was not related to the attainment of a threshold body weight or testicular size earlier, but to lower thresholds in early-maturing bulls. When predictors of pubertal status were evaluated, SC had the best sensitivity/specificity relationship in Nelore bulls, and high sensitivity and specificity in Canchim bulls. When predictors of sexual maturity were evaluated in Nelore bulls, age, weight, and SC had similar sensitivity, specificity, and predictive values. At puberty, approximately 60% of the sperm present in the ejaculate were morphologically defective. Changes in semen quality after puberty in Nelore bulls were characterized by increased motility and proportion of morphologically normal sperm, with a decrease in the proportion of major sperm defects. In conclusion, early-maturing bulls were more developed in the pre-pubertal period and attained puberty at earlier stages of body and testicular development than late-maturing bulls. Yearling SC could be used to select bulls for sexual precocity and SC was the best predictor of pubertal status. Age, weight, and SC were equally good predictors of sexual maturity in B. indicus bulls.

Use of internal artificial vaginas for breeding soundness evaluation in range bulls: an alternative for electroejaculation allowing observation of sex drive and mating ability.

The objective of this study was to test the efficacy of an inexpensive, reusable internal artificial vagina (IAV) developed for breeding soundness evaluation of range beef bulls. In addition, sexual behavior during semen collection by IAV was compared to behavior during pasture breeding. Breeding soundness exams (BSEs) were conducted on 165 bulls in two consecutive years (96 in Year 1 and 69 in Year 2). In Year 1, BSEs were done twice in all bulls, once by a conventional protocol using electroejaculation (EEJ), followed by the IAV method, one week later. In Year 2, all BSEs were done on one day; 69 bulls by the IAV method followed by EEJ in 21 bulls that failed to serve the IAV. For semen collection using an IAV, mount cows were restrained in breeding crates and an IAV was inserted into the vagina just beyond the depth of the vestibular sphincter. After each copulation, the IAV was replaced for the next bull to be tested. Semen collection by IAV was successful for all bulls that mounted and penetrated cows during the testing period (54.3 and 69.6% of the bulls served the cows with IAVs in Year 1 and 2, respectively). Semen was collected successfully by EEJ from all bulls in both years. Differences were observed between semen collection methods in semen volume and percentage of sperm staining alive; however, the differences were opposite in Year 1 and 2 and, therefore, were probably due to natural variations in time and within bull rather than the method of semen collection. Semen collection by IAV allowed the detection of problems that prevented copulation in 8 bulls (4.8%) that were determined to be satisfactory potential breeders when semen was collected by EEJ. In Year 1, breeding observations were made at pasture for 15 bulls that served, and 15 bulls that did not serve cows with an IAV. Bulls that did not serve the IAV during the test period had fewer mounts, attempts to mount, and completed services at pasture than bulls that had served the IAV, indicating that such bulls are likely to be less efficient breeders. In conclusion, IAVs might be used as an alternative to EEJ for breeding soundness evaluation in about 50-70% of range bulls. Semen collection by IAV also allows observation of sex drive and mating ability thus improves the ability to select sound breeding bulls.

Testicular thermoregulation in Bos indicus, crossbred and Bos taurus bulls: relationship with scrotal, testicular vascular cone and testicular morphology, and effects on semen quality and sperm production.

Mechanisms of testicular thermoregulation, the relationship of scrotal, testicular vascular cone (TVC), and testicular morphology with thermoregulatory capability, and their effects on semen quality and sperm production were studied in 20 Bos indicus, 28 crossbred, and 26 Bos taurus bulls. The ratio of testicular artery length and volume to testicular volume were larger (P<0.05) in B. indicus and crossbred bulls than in B. taurus bulls (1.03 and 0.94 cm/cm3 versus 0.48 cm/cm3; 0.034 and 0.047 ml/cm3 versus 0.017 ml/cm3, respectively). Testicular artery wall thickness (average 192.5, 229.0, and 290.0 microm, respectively) and arterial-venous blood distance in the TVC (average 330.5, 373.7, and 609.4 microm, respectively) were smallest in B. indicus, intermediary in crossbred, and greatest in B. taurus bulls (P<0.05); the proximity between arterial and venous blood was consistent with the estimated decrease in arterial blood temperature after passage through the TVC (5.9, 5.0, and 2.9 degrees C, in B. indicus, crossbred, and B. taurus bulls, respectively). In crossbred and B. taurus bulls, there was a positive top-to-bottom scrotal temperature gradient and a negative testicular subtunic temperature gradient. However, in B. indicus bulls, both scrotal and testicular subtunic temperatures gradients were positive. Differences in the vascular arrangement, characteristics of the artery (e.g. wall thickness) or thickness of the tunica albuginea may have affected the testicular arterial blood and subtunic temperatures in B. indicus bulls. Better testicular thermoregulatory capability was associated with increased scrotal shape (pendulosity), testicular artery length and volume, and top-to-bottom gradient of the distance between the artery wall and the veins in the TVC. Increased semen quality was associated with increased testicular volume and scrotal subcutaneous (SQT) temperature gradient, and with decreased scrotal surface and testicular temperatures. Increased sperm production was associated with increased testicular artery volume, testicular volume, and SQT temperature gradient, and with decreased testicular artery wall thickness, scrotal circumference (SC), and scrotal surface, testicular subtunic, and epididymal temperatures. In conclusion, morphology of the TVC may contribute to the greater resistance of B. indicus bulls to high ambient temperatures by conferring a better testicular blood supply and by facilitating heat transfer between the testicular artery and veins. Testicular thermoregulation was associated with opposing scrotal and testicular subtunic temperatures gradients only in crossbred and B. taurus bulls. Scrotal, TVC, and testicular morphology influence testicular thermoregulatory capability and were associated with differences in semen quality and sperm production.

Comparison of methods to evaluate the plasmalemma of bovine sperm and their relationship with in vitro fertilization rate.

The objectives of this study were to compare different methods of evaluating sperm plasmalemma and to determine their relationship with in vitro fertilization rate. A single batch of frozen semen from each of eight beef bulls was used for assessment of sperm viability and for in vitro fertilization. Conventional viability tests included sperm morphology, motility, acrosome integrity, and abnormal DNA condensation. Methods for evaluation of the sperm plasmalemma included eosin/nigrosin (EN) and trypan-blue (TB) vital stains, propidium iodide (PI) in combination with carboxyfluorescein diacetate (CFDA) or SYBR-14 (SYBR) fluorescent vital stains, and the hypoosmotic swelling test (HOST). A total of 133-150 oocytes were fertilized in vitro with sperm from each bull and cleavage rates were determined. There were high correlations between the results obtained with vital stains and good to excellent interclass correlation coefficients of agreement, indicating that these stains provide measures of the same sperm attribute, i.e. plasmalemma integrity. However, the proportions of membrane-intact sperm identified by EN or TB stains were greater (P<0.0001) than identified by CFDA/PI or SYBR/PI fluorescent stains. The results obtained with the HOST had moderate correlations but poor agreement with the results of the vital stains. The proportion of viable sperm identified by the HOST was lower (P<0.05) than the proportion identified by vital stains, indicating that response to the HOST did not depend only on the integrity of the plasmalemma. Although there were significant differences in fertilization rates and sperm viability among bulls, there was no sharp distinction for the results of sperm viability tests from bulls producing different in vitro fertilization rates. Proportions of normal, motile, acrosome-intact, and HOST-responsive sperm were identified as significant predictors of in vitro fertilizing potential; each of these endpoints explained 12-18% of the variation when evaluated separately (linear regression) and 48% when evaluated collectively (stepwise regression). In conclusion, EN and TB stains overestimated the proportion of plasmalemma-intact sperm compared to PI-based fluorescent stains. Vital stains evaluated the morphological integrity of the plasmalemma, whereas the HOST assessed plasmalemma function. In that regard, the HOST was the only plasmalemma evaluation method that significantly contributed to conventional sperm quality tests in predicting in vitro fertilization rate, indicating that the test could be incorporated to the routine of semen analysis.

Effects of scrotal insulation on sperm production, semen quality, and testicular echotexture in Bos indicus and Bos indicus x Bos taurus bulls.

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 x 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 (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 x B. taurus bulls, but those changes were not associated with changes in testicular echotexture.