Testicular growth and semen quality in peripuberal Brahman bulls

 

Luis Vásquez, Oscar Vera and Jesús Arango

 

Facultad de Ciencias Veterinarias, Universidad Central de Venezuela

In Memoriam Alí Benavides(26-01-2000)

vasquezl@ucv.ve

Abstract

 

To study the relationship between testicular growth and semen quality in peripuberal Brahman bulls, 185 records from 37 randomly selected weaned Brahman bulls at two seed-stock farms in Venezuela were used. Animals were maintained as contemporary groups at each location, and received the same feeding and handling regimes during the study. Testicular measurements and semen samples were obtained at 12, 15, 18, 21 and 24 months of age. Testicular growth was monitored by five measurements: scrotal circumference (SC), and testicular length (TL), width (TW), weight (TM) and volume (TV). Semen samples were collected by electro-stimulation. Microscopic evaluation included: sperm individual motility (IM), concentration (CO) and morphology (SM). Primary (PA) and secondary (SA) sperm abnormalities were reported. Pairwise correlations as well as regression and covariance analyses were performed to study the relationships among variables. Measurements of TM and TV were completely colineal; therefore, only TM was considered in statistical models.

 

Pairwise correlation coefficients between age (A), SC, testicular dimensions (TL, TW, TM, TV), CO, IM and SM were significant (P<0.01). Sperm abnormalities, mainly percentage of proximal droplet, were high in ejaculates of young animals and decreased with age as the animals mature. Regression models to explain semen quality in terms of age and testicular measurements were not significant for IM. CO and PA might be predicted by a function that is quadratic on A and SC, R² = 0.72 and 0.60, respectively. For SA the model was quadratic on age but linear on SC (R² = 0.54).

 

Scrotal circumference is an acceptable predictor of testicular growth and semen quality traits and may be considered as criterion for selecting peripuberal Brahman bulls.

 

Key words: Brahman, bulls, semen, sperm, scrotal circumference.

Resumen

 

Desarrollo testicular y calidad del eyaculado en toretes Brahman peripuberales

 

Con el objetivo de estudiar la relación entre el desarrollo testicular y la calidad del semen en toros Brahman peripuberales, se utilizaron 185 registros de 37 toretes recién destetados y seleccionados al azar en dos centros genéticos en Venezuela. Los animales fueron mantenidos a potrero, como grupos de contemporáneos, en cada explotación, donde recibieron el mismo manejo y alimentación durante el estudio. A partir de los 12 y hasta los 24 meses de edad se obtuvieron las medidas testiculares y se recolectaron muestras de semen a intervalos de 90-d (12, 15, 18, 21 y 24 meses de edad). El desarrollo testicular se evaluó con las medidas de circunferencia escrotal (CE); largo (LT), ancho (AT), peso (PT) y volumen (VT) testicular. Las muestras de semen se recolectaron, mediante el método de electroestimulación. La evaluación microscópica del eyaculado incluyó motilidad individual (MI), concentración (CO) y morfología espermática (ME). Dentro del análisis de ME se reportaron anormalidades primarias (AP) y secundarias (AS). Se utilizaron, análisis de correlación, regresión y covariancia para evaluar la relación entre la edad, medidas testiculares y calidad espermática. PT y VT resultaron ser completamente colineales; así que solo se incluyó PT en los análisis estadísticos.

 

Se encontraron coeficientes de correlación significativos (P < 0.01) entre la edad (E), CE, medidas testiculares (LT, AT, PT y VT) y características del eyaculado (CO, MI y ME). La incidencia de anormalidades espermáticas, principalmente gota citoplasmática proximal, resultó alta en animales jóvenes; sin embargo, disminuyó apreciablemente a medida que los animales maduraron. Los modelos de regresión para explicar la calidad seminal en términos de E y medidas testiculares no resultaron significativos para MI. Por otro lado, CO y AP podrían ser predichas por una función cuadrática en E y CE, R² = 0.72 y 0.60, respectivamente. Para AS, el modelo fue cuadrático en E pero lineal en CE (R² = 0.54).

 

Las medidas de CE pueden ser utilizadas para predecir el desarrollo testicular y la calidad seminal representando un importante criterio para seleccionar reproductores Brahman peripuberales.

 

Palabras clave: Brahman, toros, semen, espermatozoides, circunferencia escrotal.

 

Introduction

 

Reproductive efficiency of cattle managed under short breeding periods is affected by numerous factors. The reproductive capacity of young bulls deserves special attention . Selection of sires should be based on results of a Breeding Soundness Evaluation (BSE) in which, besides the capacity for mount, testicular development and semen quality, with special attention on the abnormal spermiogram associated to sexual immaturity, should be included.

 

Testicular development of bulls, during the post-weaning period, is associated to the age and breed of the animals, environmental conditions, and nutritional regime. A positive correlation between testicular development and semen quality has been documented through numerous studies (Fields et al 1979; Lunstra and Echternkamp 1982; Neely et al 1982; Spitzer et al 1988; Bailey et al 1996; Coe 1999; Arteaga et al 2001). Data reported by Lunstra et al (1978) in Bos taurus bulls, indicated that 52, 74 and 92% reached puberty with 28, 29 and 30 cm of scrotal circumference, respectively. Other reports (Cates et al 1981; Chenoweth et al 1996; Madrid et al 1988) have documented the typical low concentration, poor motility, and presence of high number of sperm abnormalities in the ejaculate of prepuberal bulls of different breeds.

 

A recent study (Arteaga et al 2001) with yearling Bos taurus bulls, reported that sperm concentration and number of normal spermatozoides increased lineally between 11 and 15 months of age, and that the percentage of proximal droplets diminished significantly as the animals aged. Likewise, Amann et al (2000), and Thundathil et al (2001) conducted studies to investigate in vitro fertilizing potential of semen with high incidence of proximal droplets and concluded that this sperm abnormality was associated to low percentage fertilization and cleaved ova. These studies also indicated that the incidence of proximal droplet in the semen of young bulls tended to diminish as the animals reach sexual maturity.

 

The relationship among age, scrotal circumference and semen quality in Bos taurus and Bos indicus bulls has also been documented (Morris et al 1978; Lunstra and Echternkamp 1982; Chenoweth et al 1996; Coe 1999). Results indicated that bulls with scrotal circumference measures below the average for their age and breed groups do not produce as normal semen as those bulls above the average for this trait. Scrotal circumference is intimately correlated to capacity of sperm production, number of sperms ejaculated and sperm reserves (Wildeus and Entwistle 1982; Palasz et al 1994). Prepuberal beef bulls exhibit low sperm count and motility, besides a high number of abnormal spermatozoa (Cates et al 1981; Chenoweth et al 1996; Arteaga et al 2001). Carson and Wenzel (1997) analyzing data from 1276 Bos taurus bulls, reported that 52.1 and 12.5% of animals were rejected due to high number of abnormal sperm or insufficient measure of scrotal circumference, respectively.

 

In Brahman bulls, Fields et al (1979) reported a large increase in testicular size between 16 and 20 months of age, and a positive correlation between testicular volume and sperm concentration. Results from another study (Chase et al 1997) indicate that Brahman bulls reached puberty at older age, and greater weight and corporal height than Bos taurus bulls, evidencing the existence of a genotype-age interaction on the characteristics studied. Scrotal circumference has shown moderate to high heritability and high genetic correlation with other testicular measurements and semen quality traits in Bos índicus (Quirino 1999). Delayed puberty in Bos indicus cattle may be improved through selection of the most precocious animals in the herd. Pre-selection of young Brahman bulls based on testicular growth may produce a correlated response for early puberty, allowing breeders to reach their reproductive goals and to improve sexual maturity in their herds. The objectives of this study were to determine the relationship between testicular growth and semen quality traits in peripuberal Brahman bulls in tropical environment, and to establish the practical value of scrotal circumference as a mean to monitor testicular size to select young animals.

 

Materials and Methods

 

Data Origin

 

Thirty-seven weaned Brahman bulls, from two seed stocks in Venezuela were randomly selected for the present study. The locations were under dry tropical forest conditions; one is an experimental station (farm 1) from the Facultad de Ciencias Veterinarias, Universidad Central de Venezuela, located at Yaracuy state (09º 50´08", 10º 46´ 31" north latitude, and 68º 14´ 14", 69º 13´ 55" west longitude) at 130 m of altitude, showing an average annual temperature and precipitation of 27º C and 1700 mm, respectively. The other (farm 2) is a privately owned farm (farm 2) located at Portuguesa state (08º 05´ 38·, 09º 50´46" north latitude and 68º 33´ 20", 70º 11´ 17" west longitude), at 170 m of altitude, showing an average annual temperature and precipitation of 28º C and 1150 mm, respectively.

 

Animal Management

 

Animals were weaned at about 225 days of age and maintained as contemporary groups at each farm (19 and 18 animals), receiving a similar handling and feeding programs during the study. All animals were grazing on pastures of: Urochloa humidicola and Cynodon plestostachyus (farm 1), and Panicum maximun, Urochloa mutica and Cynodon plestostachyus (farm 2), to an approximate load of 1 AU/ha, and received a mineral mix supplement ad-libitum. Testicular measures were registered and semen samples were collected by electro-stimulation at 90-d intervals from 12 through 24 months of age.

 

Testicular Measurements

 

Scrotal circumference was obtained using a flexible metallic tape (Lane Manufacturing Inc. Denver, CO) graduated in cm, maintaining the testicles toward the bottom of the scrotum by moderate digital pressure. Individual testicular length (TL) and width (TW) were measured with a calibrated caliber, and used to calculate testicular weight (TM) and volume (TV), considering the testicle as a prolate spheroid, and following the mathematical formulas reported by Bailey et al (1996), where TM = 0.5533*(TL)*(TW)², and TV = 0.5236*(TL)*(TW)².

 

Semen Collection and Laboratory Evaluation

 

Semen samples were collected by electro-stimulation, using an electroejaculator (Standard Precision Electronics Inc., Boulder, CO) and an electrode of three metallic bands, for rectal stimulation on the accessory glands and pelvic urethra. Semen evaluation was performed in accordance with guidelines established by the Society for Theriogenology (Chenoweth et al 1992). To estimate individual motility (IM, percent of sperm with progressive individual motility) a small drop of raw semen (20 μl) was place on a warm slide and observed under coverslip in a light microscope at high magnification (400x); if needed, samples were diluted with a saline solution (0.9% NaCl) and maintained on a heating plate (35º C) during the microscopic evaluation. For assessment of sperm concentration(CO), an aliquot of raw semen was maintained under refrigeration (5º C) and transported to the laboratory for direct count on a hematocytometer (Newbauer Bright Line, Boeco, Western Germany), using a 1:200 dilution of saline hyperosmotic solution (NaCl 3%), when it was necessary. Spermatozoa were counted under a phase contrast microscope at 400x magnification. Double chambers were prepared and counted for each sample and the average of both counts was reported. Another aliquot of raw semen (1 or 2 drops) was diluted in 0.5 ml of saline-buffered solution containing 0.2% glutaraldehyde (Johnson et al 1976), and kept under refrigeration to evaluate sperm morphology (SM). Sperm morphology was evaluated on stained smears under 1000x magnification, using Nomarski Differential Interference Optics on a Zeiss microscope. The Hema 3^R kit Protocol (Fisher Scientific Company) was followed to stain the smears. A total of 200 spermatozoa per sample were evaluated to calculate the percentage of spermatozoa with normal (NS) and abnormal (AS) morphology. Percentages of proximal (PD) and distal droplets (DD), detached head (DH), midpiece and principal piece defects were determined, according to Barth and Oko (1989).

 

Statistical Analysis

 

Pairwise correlation analyses between variables were used to measure relationships among age, testicular measurements and semen-quality traits. Covariance analysis was used to evaluate the effects of herd, age and scrotal circumference on semen quality traits (the last two used as regressor variables, exploring different polynomial degrees). Regression analyses were used to explore the importance of age, and different testicular measurements (SC, TL, TW and TV), which can be easily taken at farm, on prediction of different semen quality traits (IM, CO, NS, PA and PD), model comparisons were based on goodness of fit through the R-squared (R²) method and the Mallows C(P) statistics (Freund and Littell 1991), using the Statistical Analysis System (SAS 2001). Sperm concentration was included as the log of the number of sperm cells (sperm x 10⁶ /mL) to avoid extreme differences in trait scales.

 

Farm was not a significant source of variation in preliminary analyses, and was excluded from the final statistical models. Therefore pooled results (from both farms) are presented.

 

Results and Discussion

 

Mean, standard deviation and range of values for all variables are presented in Table 1. These data suggest that adequate testicular development is reached around 18 months of age and 30 cm of scrotal circumference for young Brahman bulls. Based on research data on Bos taurus bulls, the Society for Theriogenology (Chenoweth et al 1992) set the minimum scrotal circumference according to the age, such as yearling bulls should have at least 30 cm to be considered as potential sires. According to data from young Brahman bulls, presented here, selection of future sires should be based on a minimum of 30 cm of scrotal circumference at 18 months of age, which agree with Chenoweth et al (1996).

The relationship between scrotal circumference and semen quality traits in bulls has been broadly documented (Palasz et al 1994; Bailey et al 1996; Chase et al 1997; Coe 1999; Arteaga et al 2001; Kastelic et al 2001). Results from the present study corroborate the enunciated by Lunstra and Echternkamp (1982) and Chenoweth et al (1996), that very few animals with measures of scrotal circumference below the average of their contemporaries, produce semen of acceptable quality, when compared with those above the average. Veeramachaneni Rao et al (1986) and Madrid et al (1988) reported a high number of damaged tubules and sperm abnormalities in young Bos Taurus bulls with less than 32 cm of scrotal circumference.

 

Pairwise correlation coefficients between age, testicular measurements and semen quality traits are shown in Table 2. Positive correlations between scrotal circumference, age, body weight and testicular volume have also been previously reported (Coulter and Foote 1977; Carter et al 1980; Ohl et at 1996; Vásquez and Arango 2002). Data from the present study indicated that testicular growth is directly related with the age of the animals and could be use as a criterion to select precocious animals within a population. Fields et al (1982) suggested that scrotal circumference might differ among different populations of Brahman bulls due to various genetic and environmental factors.

Since scrotal circumference showed a high correlation coefficient with all other testicular measurements, it seems appropriated to recommend the use of scrotal circumference as a routine measure to monitor testicular growth in young Brahman bulls. Due to the direct association among scrotal circumference, age and sperm concentration, scrotal circumference might also be a useful trait to select for higher potential fertility. Literature reports indicate that scrotal circumference is the most easily obtainable measure of a bull's ability to produce adequate numbers of normal spermatozoa; it represents a reliable indicator of age to puberty, and it is also directly related to semen quality. Rocha et al (1996) reported a high correlation coefficient (r=0.78) between scrotal circumference and testicular weight in peripuberal Brahman bulls. The high correlation coefficients between age and scrotal circumference (r=0.89), age and morphologically normal sperm (r=0.72), and scrotal circumference and normal sperm (r=0.61) support the conclusion that adequate testicular growth must assure fertility during the first breeding season of young bulls. However, we must be aware that testicular growth also depends on the feeding and body condition during the prepuberal period.

 

Sperm concentration showed positive correlation (P<0.01) of 0.75, 0.74 and 0.47 with scrotal circumference, percentage of normal sperm and individual motility respectively, and a negative correlation of -0.76 and -0.67 with primary abnormalities and proximal droplets, respectively. Sperm motility was positively (P<0.01) correlated with percentage of normal sperm (r=0.47) and sperm concentration (r=0.50). Marked increased in sperm motility in bulls between 15 and 18 months of age, was apparently associated to sexual maturity. Primary sperm abnormalities were negatively associated to all other testicular characteristics, showing a high percentage in young animals, and decreasing as the animals reached sexual maturity. Proximal droplet, in young bulls, is considered as a sign of sexual immaturity associated to decreased fertility (Amman et al 2000). In the present study, percentage of proximal droplet decreased with the age of bulls. This result agrees with reports from Lunstra and Echternkamp (1982) and Arteaga et al (2001). Secondary sperm abnormalities were not as prevalent as primary abnormalities, and did not represent a useful trait for selection in young Brahman bulls.

 

Different models of linear regression, which included age and different testicular measurements (SC,TL,TW and TV) as independent variables to predict semen quality traits were implemented as follow:

 

Sperm concentration

 

When only one variable was included in the model, the best regressor to predict sperm concentration was testicular width (R² = 0.47), closely followed by scrotal circumference (R² = 0.45), which is a more practical and commonly taken measurement at the farm. However, both models had a high C(P) value (22.32 and 25.73) indicating that more complete models were needed. Regression models with two independent variables did not produce important improvements in R². Three-variable models including testicular length, testicular width and testicular volume (R² = 0.56) seemed to be a better approximation, but still showing a relatively high C(P) of 6.47. The best model included four regressor variables (A, TL, TW and TV), R²= 0.58 and C(P)= 4.03. The inclusion of scrotal circumference did not produce additional improvement in R² and C(P).

 

Sperm motility

 

Regression models to explain sperm individual motility using testicular measurements and age were very poor. In fact, the best model including four variables (SC, TL, TW and TV) only reached a R² = 0.11 (P>0.05). It seems that variation in sperm motility cannot be adequately predicted by testicular measurements. That may be explained by the subjectivity involved in the evaluation of this trait.

 

Percentage of normal sperms

 

Age was the best single variable to predict the percentage of normal sperms in the ejaculate (R²= 0.42), which is in agreement with a high correlation between these variables (r= 0.72), indicating a linear relationship between age and the proportion of normal sperm cells produced. That seems to be related to the age at which the animals reach puberty. There was not important increments of R² with the inclusion of others variables, except for the best model that included five variable (A, SC, TL, TW and TV) [R² = 0.52, C(P)= 6.00].

 

Percentage of primary sperm abnormalities

 

Age was the single most important variable to predict the proportion of primary abnormalities (R²= 0.49). All other models increasing the number of variables, also included age, indicated that (as for the proportion of normal sperm), this trait improves as the animal matures. The best model (R²= 0.57, C(P) = 4.16) included four variables: age, testicular length, testicular width and testicular volume.

 

Percentage of proximal droplets

 

Age also was the best single variable to predict the presence of proximal droplet (R²= 0.50). Increasing the number of variables in the regression model improved the R² up to 0.59. The best model included age, testicular length, testicular width and testicular volume, as for the previous three traits.

 

From the previous regression models, it seemed clear that age is an important variable to consider in the models in order to predict semen quality traits. That was expected since in young, maturing and growing animals, most of semen quality traits are indicators of puberty and improve as the animal ages. On the other hand, testicular measurements improved the goodness of fit of the model but the increments of the model R² were only of moderate magnitude. There was a need to further explore regression models, which may allow predicting semen quality traits from age and testicular measurements. Since scrotal circumference is the most easily taken testicular measurement at the farm, and it showed high correlation coefficients with all other testicular measurements; it seemed adequate to include it in prediction models for practical purposes. Therefore, a set of regression models including age and scrotal circumference were implemented forward, increasing the polynomial degrees until the highest order resulted not statistically significant. Table 3 summarizes the results of the regression models for sperm concentration and percentages of normal sperm, primary abnormalities and proximal droplets. Individual motility was not included in this set of models since in the previous regression models it was evident that age and testicular measurements were not efficient predictors of sperm motility (i.e., low R² values) and the corresponding models were not statistically significant.

The best regression model including age and scrotal circumference to explain sperm concentration was one that had both variables at linear and quadratic degrees (P < 0.01) (Table 3). The corresponding regression equation was: CO = - 15.392 + 1.630 (A) - 0.034 (A²) + 0.848 (SC) - 0.011 (SC²), R² = 0.72, which indicates that sperm concentration, in the logarithmic scale, increases in a quadratic or curvilinear fashion as either age or scrotal circumference increases. However, when sperm concentration was back transformed to the regular scale, the change of sperm concentration with age showed a sigmoideal trend (Figure 1).

 

Figure 1. Trend for scrotal circumference and sperm concentration in peripuberal Brahman bulls.

 

Regression analysis for percentage of normal sperm (Table 3) indicated that scrotal circumference was not a significant source of variation neither at linear nor at quadratic level; however, age was significant (P < 0.01) at linear and quadratic degree, which indicates that the percentage of normal cells increases with age in a curvilinear fashion in young Brahman bulls. The prediction equation was: NS= - 185.613 + 20.889 (A) - 0.417 (A²); R² = 0.60. Figure 2 shows the curve for percentage of normal sperm.

Figure 2. Trend for percentage of normal sperm, primary abnormalities, and proximal droplet in peripuberal Brahman bulls.

 

The incidence of primary abnormalities was affected at linear and quadratic degree (P < 0.01) by age (Table 3); scrotal circumference was significant at quadratic (P < 0.01) but not at linear level (P > 0.05). The prediction equation was: PA= 316.268 - 8.527 (A) + 0.124 (A²) - 9.819 (SC) + 0.140 (SC²); R²= 0.60. Figure 2 shows how the incidence of primary abnormalities decreased as the animals aged.

 

The occurrence of proximal droplets was best explained by a model that included age at linear (P < 0.01) and quadratic (P < 0.05) degree, and scrotal circumference just at linear degree (P < 0.05), as is shown in Table 3. The prediction equation was: PD=203.866 - 12.642 (A) + 0.252 (A²) - 1.158 (SC); R² = 0.54. Figure 2 shows the trend for proximal droplets on age.

 

Results from the present study indicated that as the bulls reached the age of puberty, scrotal circumference and sperm concentration increased rapidly, which is in agreement with previous reports (Lunstra and Echternkamp 1982, Chenoweth et al 1996, Coe 1999 and Arteaga et al 2001). A similar trend occurred for increased percentage of morphologically normal spermatozoa and for decreased incidence of proximal droplets as reported by Lunstra and Echternkamp (1982), Coe (1999) and Arteaga et al (2001). On the other hand, results indicated that bulls with larger testicles tended to have a lower percentage of abnormal spermatozoa as reported by Palasz et al (1994). Similar results in young beef bulls, reported by Coe (1999) indicated that as scrotal circumference increases, with age, the risk of failing below the threshold of producing at least 70% morphologically normal spermatozoa decrease.

 

 

Conclusions

 

Acknowledgements

 

The study was funded by the "Consejo de Desarrollo Científico y Humanístico" (CDCH), Universidad Central de Venezuela, through project Nº PI-11-31-4235-98. To "La Cumaca" Experimental Station, Facultad de Ciencias Veterinarias, Universidad Central de Venezuela, and to Mr. Carlos Mendoza for allowing the use of the animals for this study. The authors gratefully acknowledge the contribution of Dr. Thais Diaz for the peer review of the manuscript

 

 

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