Leonardo F.C. Brito

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 × 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.

The effect of nutrition on sexual development of bulls

Most bulls that are managed for sale as yearlings are fed high-energy diets in the post-weaning period to maximize rates of gain in body weight. High-energy diets with adequate protein, vitamins and minerals result in a larger scrotal circumference at 1 y of age, however, part of this increase in size is likely due to scrotal fat. It is unclear whether testis size and spermatogenesis is significantly affected by nutritional intake in the post-weaning period. There are indications of an effect of calfhood nutrition on age at puberty and testis size. Scrotal circumference was smaller in yearling bulls raised by first-parity dams, compared to those raised by older dams. This may have been due to lower milk production by first-parity dams, an in utero effect, or both. The effect of reduced calfhood nutrition may be mediated through gonadotropin secretion. Calves destined to become later maturing bulls with smaller testes had lower amounts of LH secretion during the period of the early gonadotropin rise (8-16 wk of age). Furthermore, augmenting circulating LH concentrations at this time by treating calves with GnRH hastened pubertal development. In addition, FSH treatments in calfhood also increased scrotal circumference and hastened spermatogenesis. In that regard, FSH has been considered a main driver of Sertoli cell proliferation in prepubertal animals. Since Sertoli cell multiplication ceases at 20-25 wk of age in bulls, final testis size in bulls is likely determined in calfhood. Four experiments were done to investigate the effects of calfhood nutrition on pubertal development. These studies confirmed that superior calfhood nutrition augmented gonadotropin secretion (which is probably mediated by metabolic hormones); this resulted in larger testes at 1 y of age and an earlier onset of spermatogenesis.

Testicular ultrasonogram pixel intensity during sexual development and its relationship with semen quality, sperm production, and quantitative testicular histology in beef bulls

This study was conducted to evaluate testicular ultrasonogram pixel intensity during sexual development in bulls and to determine its relationship with semen quality, sperm production, and quantitative testicular histology. Beef bulls (N = 152) were examined from 14 - 26 to 70 - 74 wk of age in four different years. Testicular echogenicity increased during sexual development, but the pattern of change differed among years. Echogenicity increased between 26 and 42 to 46 wk of age in 2 yr, but increased considerably earlier in the other 2 yr, reaching maximum values at 34 wk of age. Because increased echogenicity was likely associated with testicular changes leading to initiation of spermatogenesis, these differences were difficult to explain considering that age at puberty did not differ significantly among years. When data were evaluated according to age normalized to puberty, echogenicity started to increase 16 to 12 wk before puberty and reached maximum values 4 wk before or at puberty. These results indicate that a certain developmental stage of the testicular parenchyma must be reached before puberty and that the composition of the parenchyma remained consistent after puberty. Testicular echogenicity was associated with sperm production, seminiferous tubule and epithelium area, and sperm morphology, but the associations were not consistent. Testicular echogenicity was a good indicator of pubertal and mature status, but was not superior to scrotal circumference. In conclusion, although testicular ultrasonogram pixel intensity analysis might be useful for research purposes, clinical application of this technology in the present form for bull breeding soundness evaluation is not justifiable.

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.

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.