Plasma and testicular testosterone levels, volume density and number of Leydig cells and spermatogenic efficiency of rabbits.

Plasma and tissue testosterone concentrations were determined by radioimmunoassay in 12 eight-month-old sexually mature New Zealand White rabbits and evaluated for possible associations with spermatogenic efficiency as well as with volume density and number of Leydig cells. Testicular tissue was processed histologically and histometry was performed in order to quantify germ cells, Sertoli cells and Leydig cells. Spermatogenic efficiency, reported as the ratios among germ cells (spermatogonia, primary spermatocytes and round spermatids) and by the ratio of germ cells to Sertoli cells, was not associated with testosterone levels. However, Leydig cell parameters such as number of Leydig cells per gram of testis, total number of Leydig cells per testis and percent cell volume of Leydig cell nuclei were correlated significantly with testosterone levels. The statistically significant correlation (r = 0.82, P<0.05) observed between testosterone levels and the number of Leydig cells per gram of testis suggests that, in the rabbit, the latter parameter can serve as a criterion for monitoring testosterone levels in this species under normal conditions.

Plasma and testicular testosterone levels, volume density and number of Leydig cells and spermatogenic efficiency of rabbits

Plasma and tissue testosterone concentrations were determined by radioimmunoassay in 12 eight-month-old sexually mature New Zealand White rabbits and evaluated for possible associations with spermatogenic efficiency as well as with volume density and number of Leydig cells. Testicular tissue was processed histologically and histometry was performed in order to quantify germ cells, Sertoli cells and Leydig cells. Spermatogenic efficiency, reported as the ratios among germ cells (spermatogonia, primary spermatocytes and round spermatids) and by the ratio of germ cells to Sertoli cells, was not associated with testosterone levels. However, Leydig cell parameters such as number of Leydig cells per gram of testis, total number of Leydig cells per testis and percent cell volume of Leydig cell nuclei were correlated significantly with testosterone levels. The statistically significant correlation (r = 0.82, P<0.05) observed between testosterone levels and the number of Leydig cells per gram of testis suggests that, in the rabbit, the latter parameter can serve as a criterion for monitoring testosterone levels in this species under normal conditions

Assessing sex-drive in young Bos taurus bulls.

Objectives in this study were to determine the accuracy of different methods of evaluating libido score (L), service rate (SR) and reaction time to service (RTS) in yearling Bos taurus bulls. Using restrained, non-estrus females, 26 yearling Bos taurus bulls were evaluated a total of eight times (four sessions, two tests per session) over 2 consecutive months for L, SR, and RTS. Individual bull variation influenced both L (P<0.0001) and SR (P<0.003). Repeatability was moderate for L (R=0.64) and low for both SR (R=0.12) and RTS (R=0.04). Under the conditions of this study and characteristics of these tests, variance was reduced to 69-73% for L and 26-23% for SR with four to eight repetitions, respectively. Bulls that scored highly in the first session, however, tended to score highly throughout. Although, three bulls did not serve in any test, RTS was independent of effects. However, the tendency of RTS to decrease, and for L and SR to both increase with consecutive tests, suggests influences other than genetic, such as learning and/or environmental factors. These tendencies were most evident in bulls which obtained low L scores at the first session. It was concluded that, despite the important degree of individual variability observed in L and SR, accurate quantitative evaluation of the sex-drive in young Bos taurus beef bulls would benefit from use of test procedures of greater repeatability.

Disruption of spermatogenesis in rabbits consuming ethylene glycol monomethyl ether.

Effects of ethylene glycol monomethyl ether (EGME) on spermatogenesis were examined using groups of six to seven Dutch rabbits that received 0, 12.5, 25.0, 37.5, or 50.0 mg of EGME/kg body weight, respectively, in their drinking water 5d/week. After 12 weeks, animals were euthanized and their testes were removed, weighed, and processed to permit germ cell numbers to be quantified. Spermatogenesis was depressed by EGME in a dose-dependent manner; numbers of round spermatids per Sertoli cell (a measure of spermatogenic efficiency) averaged 8.86, 8.87, 6.20, 2.38, and 7.42 for the 0, 12.5, 25.0, 37.5, and 50.0 mg/kg dosages, respectively. The latter value of 7.42 represents an overestimation of sperm production because it is based on only two unexpected outlier rabbits. Nearly complete destruction of spermatogenesis occurred in the other five animals in this highest dosage group, precluding evaluation by the histometric method. Numbers of homogenization-resistant elongated spermatids per testis, measurable on all animals, averaged 231, 256, 195, 52, and 67 x 10(6), respectively. The correlation between the predicted sperm production, based on the elongated spermatids at necropsy, and the number of sperm ejaculated by the males during week 12 was 0.92. Thus, EGME impaired rabbit spermatogenesis in a dose-dependent manner. Generally, rabbit spermatogenesis was at least 10 times more sensitive to EGME than previously reported for rats and mice.

Extragonadal sperm reserves, sperm-depletion rates, numbers of sperm per mating, and fertility with successive matings by intact or unilaterally vasectomized rats.

Because of the high rates of sperm production and large extragonadal sperm reserves of sexually rested male rats, mating trials are insensitive for detecting test-induced alterations in sperm production rates. Mating trials might be more sensitive if, independently of any experimental treatments under study, the number of sperm per mating was closer to the minimum requirements for normal fertility. The present study was undertaken to assess the impact of unilateral vasectomy and/or matings with up to three females in succession, for 1 hour each, on the number of sperm per mating and fertility, in comparison to corresponding values for males allowed unlimited matings with a receptive female overnight. Unilateral vasectomy did not affect sperm production, extragonadal sperm reserves, or removal of contralateral sperm during ejaculation (P > 0.05) but caused a 50% decrease in sperm numbers per mating. Sperm output, judged from numbers of residual extragonadal sperm in unmated and mated males, was excessive (290 x 10[6]) during conventional overnight mating with intact males and during the first and second hours of restricted mating (105 and 184 x 10(6) respectively, for intact males; one-half of these amounts for unilaterally vasectomized males). In contrast, sperm output during the third successive mating was minimal (nonmeasurable) but adequate, since pregnancy rates were similar for females mated first, second, or third in succession (P > 0.05). Since successive matings reduce the number of sperm per mating by natural methods, this approach may enhance the sensitivity of mating tests when applied for assessing the potential effects of experimental treatments on sperm production.

Inherent variability among measures of fertility of rats and its implications in the design of mating trials.

Mating trials are used extensively to assess the effects of experimental treatments on the fertility of male or female subjects. Such trials are often insensitive because many measures of fertility are associated with large inherent variability, which makes it difficult to confirm, by statistical significance, that observed differences among treatment means are actually due to treatment rather than due to chance. Unfortunately, and despite this insensitivity, most researchers choose replication arbitrarily on the basis of cost, convenience, or conventional practice and interpret results without assessing the actual power and sensitivity of their experiments. This study was undertaken to characterize the relationship between the number of rats used in fertility trials and experimental power and sensitivity. The variables examined included pregnancy rates, numbers of fetuses per mated or per pregnant female, numbers of fetuses per corpus luteum, and numbers of fetal resorptions. The relationship between replication and experimental power and sensitivity was estimated via statistical approaches utilizing data from Sprague-Dawley rats used in a mating trial at 105-109 days of age. Tabular data are presented showing the numbers of rats needed per treatment group as a function of the minimal treatment differences to be detected and type II error probability selected. The application of these data in the planning of future studies and in interpreting their outcome is discussed.

Testicular weight, Sertoli cell number, daily sperm production, and sperm output of sexually mature rabbits after neonatal or prepubertal hemicastration.

The present study was conducted to investigate the influence of hemicastration and age at hemicastration on the subsequent testicular development of male rabbits through sexual maturity. Thirty New Zealand white rabbits were left intact or were hemicastrated on Day 35, 49, 77, or 105 postconception. Beginning at 7 mo of age, ejaculates were collected every other day for 1 mo, and the last ten ejaculates were used to quantify daily sperm output. At 8 mo, the rabbits were killed and their carcases, testes, testicular capsules, and epididymides were weighed. Testicular tissue was processed for quantification of sperm production rates by enumeration of homogenization-resistant spermatids and via histometric evaluation. Regardless of the age at hemicastration, this manipulation did not alter the development of the remaining testis as assessed from testis weight, numbers of Sertoli cells per testis, daily sperm production, or sperm output (p > 0.05). On the basis of these findings, it would appear that the development of the spermatogenic capacity of the remaining testis of the rabbit is not altered appreciably by hemicastration at a young age.

A simple, rapid and reliable method for selecting or assessing the number of replicates for animal experiments.

A simple approach was developed for determining the number of replicates needed per treatment group to provide experiments of known power and sensitivity, where power equals the probability that a treatment effect would not go undetected if an effect existed and sensitivity equals the minimal treatment response that will be detectable. This approach, in turn, was used to construct reference tables, applicable across scientific disciplines, from which researchers may read replication requirements directly with ease, speed and reliability. To use the tables, one need only furnish a reliable estimate of the coefficient of variability expected among replicates, which may be obtained from prior observations on similar populations. The tabular data also enable a rapid, reliable assessment of the actual power and sensitivity of completed experiments, such as those contained within the published literature.

Changing relationships between testis size, Sertoli cell number and spermatogenesis in Sprague-Dawley rats.

Relationships between several reproductive characteristics were investigated in 25 Sprague-Dawley rats aged 60, 150, and 240 days (n = 75). Daily sperm production correlated with body weight (r = 0.63), paired testes weight (r = 0.68), testes weight as a percentage of body weight (r = -0.50), the number of spermatids supported per Sertoli cell (r = 0.51) and the number of Sertoli cells per gram (r = 0.89) or per testis (r = 0.95) among rats pooled across age groups. In general, the number and magnitude of significant coefficients of correlation were decreased when calculated within age groups. The latter often appeared to reflect a statistical consequence of relative homogeneity among rats rather than the absence of a biological relationship. However, the total number of Sertoli cells per testis correlated with daily sperm production within age groups, and could account for 85 to 94% of the variability in sperm production at 150 and 240 days, respectively.

Age as a factor influencing the power and sensitivity of experiments for assessing body weight, testis size, and spermatogenesis in rats.

Data from 25 rats aged 60, 150, or 240 days (n = 75) were used to determine the number of observations per rat and or rats per treatment group needed to provide future experiments of known power and sensitivity. End points examined included body weight, testicular weight, number of resistant elongated spermatids, yields of germ cells from their younger progenitors, and germ cell:Sertoli cell ratios. Requirements differed in relation to the experimental power and sensitivity desired. However, the replication needed for detecting equivalent treatment effects on paired testes weight or the number of spermatids per gram of testis increased with age, and was twice as great for 240- vs 60-day-old rats. In contrast, approximately twice as many 60-day-old rats were required for detecting treatment effects on the number of spermatids per Sertoli cell than when 150- or 240-day-old rats were used.

Replication requirements and number of ejaculates needed for assessing treatment effects on sperm output and seminal characteristics of electroejaculated Holstein bulls.

Two ejaculates were harvested by electroejaculation on each of 3 d per week for 14 wk from 14, 12- to 24-mo-old Holstein bulls. Ejaculates during the first 2 wk served to stabilize sperm output. Data for the remaining ejaculates were used in a components of variance approach to determine the number of bulls per treatment and ejaculates per bull that would be needed to provide adequate sensitivity and precision for assessing treatment effects on seminal characteristics. The latter included the number of sperm (total and motile), volume and sperm concentrations of the sperm-rich fraction, and the percentage of progressively motile sperm in first and second ejaculates or per day. Replication requirements declined as the number of ejaculates per bull was increased to about 15 to 25 but declined minimally thereafter. The number of bulls needed per treatment varied in relation to the size of the treatment response to be detected but was much greater than the replication used in typical experiments. Replication for assessing sperm output in first ejaculates or per day was approximately one-half as great for experiments in which pretreatment information was used to adjust post-treatment data. Tables should be useful as a guide for designing efficient, cost-effective studies of known sensitivity and precision.

Numbers of Sertoli cells, quantitative rates of sperm production, and the efficiency of spermatogenesis in relation to the daily sperm output and seminal quality of young beef bulls.

Data from 34 yearling Hereford or Angus bulls were used to investigate relationships of testicular size, quantitative rates of sperm production, Sertoli cell numbers, numbers of germ cells supported per Sertoli cell, and the efficiency of spermatogenesis to daily sperm output and seminal quality. Two ejaculates were collected by electroejaculation from each bull on each of 2 days/week throughout the study. The percentage of progressively motile sperm and the percentage of morphologically normal sperm were determined from aliquots of fresh semen. Additional aliquots of semen were frozen in glass ampules or plastic straws and subsequently evaluated for postthaw motility and percentage of sperm with intact acrosomes. Sertoli cell numbers, the numbers of germ-cells per Sertoli cell, and the efficiency of spermatogenesis were unrelated to the quality of fresh or frozen semen (P greater than 0.05). In first ejaculates, the numbers of sperm and motile sperm were related (P less than 0.05) to testicular parenchymal weight (r = 0.38 and 0.50), daily sperm production (r = 0.45 and 0.53), and spermatids per gram of testicular parenchyma (r = 0.35 and 0.34). Testicular parenchymal weight and daily sperm production also were related to daily sperm output and to the average daily motile sperm output of these bulls (P less than 0.05), but could account for less than 25% of the variability in these end points among bulls.

Relationship of intratesticular testosterone content of stallions to age, spermatogenesis, Sertoli cell distribution and germ cell-Sertoli cell ratios.

Testes were obtained from 47 1-20-year-old stallions during the natural breeding season. Total testicular testosterone and testosterone/g testis increased with age (P less than 0.005), and total testicular testosterone was associated with larger testis size (P less than 0.05). Neither testosterone per gram nor per paired testes were related to total Sertoli cell number (P greater than 0.05), but greater testosterone per paired testes was associated with fewer Sertoli cells per unit of seminiferous tubule length (P less than 0.005) or basement membrane area (P less than 0.02) and with a higher number of germ cells supported per Sertoli cell (P less than 0.05). Although values for testosterone per gram and per paired testes were unrelated (P greater than 0.10) to sperm production/g testis or to the yield of spermatids/spermatogonium, testosterone per paired testes was positively related to sperm production per paired testes (P less than 0.05). It is concluded that intratesticular testosterone increases with age, is related in a positive manner to quantitative rates of sperm production, and can account for some of the differences in sperm production among individual stallions within a single breeding season.

Optimal replication for histometric analyses of testicular function in rats or rabbits.

Quantitative evaluations of testicular histology can provide sensitive endpoints for determining toxicity of chemicals to the male reproductive system. But, the numbers of observations per testis or number of animals per treatment group often are selected by tradition or availability, rather than from a statistical basis. Therefore, we studied the number of observations per male (sampling intensity) and number of animals per treatment (replication) needed to detect treatment effects of given magnitude, with predictable error probabilities, using data from Sprague-Dawley rats and Dutch-belted rabbits that received 0.0, 0.94, 1.88, 3.75, 7.5, or 15.0 mg/kg body wt of 1,2-dibromo-3-chloropropane (DBCP). Data for one testis from 102 rats and 34 rabbits were available. For each testis, observations included measurement of the minor diameter of 50 seminiferous tubules, counts of the number of leptotene primary spermatocytes per 250 Sertoli cells, and counts of spherical spermatids within 20 seminiferous tubular cross sections. Tabular data are presented showing optimal numbers of observations per testis and animals per treatment group as a function of the difference to be detected and selected probabilities for Type I and II errors. In general, precise assessments required far fewer observations per testis than are used routinely. However, due to the inherent variability among animals, the number of animals required per treatment tended to be greater for experiments with the rabbit, and increased substantially for both species when detection of small differences had to be ensured. The data presented should enable investigators to design experiments of chosen sensitivity and precision while making cost-effective use of animals and labor.

Sampling intensities and replication requirements for detection of treatment effects on testicular function in bulls and stallions: a statistical assessment.

Data from testes of 16, 2- to 3-yr-old stallions and 34 yearling beef bulls were utilized in a components of variance approach to calculate the number of observations required per testis and(or) the number of animals required per treatment group to provide experiments of known sensitivity and precision, where treatment was to be assessed by one of several endpoints. The latter included paired testes weight, seminiferous tubular diameter, the number of germ cells per seminiferous tubular cross-section, or the number of elongated spermatids per gram of testicular parenchyma or per testis. For all variables for which several observations were available for each testis, precise assessment of any given male required far fewer observations than have been used routinely. However, replication requirements varied substantially in relation to the sensitivity (i.e., size of difference to be detected) and precision (i.e., Type I and II error probabilities) desired. Replication requirements were greater for stallions than for bulls, particularly at higher levels of sensitivity, for which requirements for both species were very large. The data presented should permit future experiments involving assessment of these endpoints to be designed with known sensitivity and precision and with optimal efficiency and cost-effectiveness.

Spermatogenesis, sperm output and seminal quality of Holstein bulls electroejaculated after administration of oxytocin.

The 12- to 24-month-old Holstein bulls were electroejaculated twice on each of 3 days per week throughout the study. After a 2-week stabilization period and subsequent 2-week pre-treatment period, 7 bulls were given 50 i.u. oxytocin via the jugular vein 10 min before each first ejaculate for 10 weeks. The 7 control bulls were handled identically but did not receive oxytocin. All bulls were castrated at the end of the study. Oxytocin was without effect on spermatogenesis (P greater than 0.10). Oxytocin did not alter the total number of spermatozoa harvested per collection day (P greater than 0.10), but increased the number of spermatozoa in first ejaculates by an average of 34.2% (P less than 0.025). Oxytocin did not affect sperm quality (P greater than 0.10) as judged by the motility of spermatozoa in fresh semen or by the motility or percentage of spermatozoa with intact acrosomes in thawed semen. It is concluded that 50 i.u. oxytocin enhanced sperm output in first ejaculates of electroejaculated bulls without altering daily sperm production or seminal quality.

The numbers of Sertoli cells in mature Holstein bulls and their relationship to quantitative aspects of spermatogenesis.

Testes from 37 Holstein bulls, 38-99 mo of age, were used to investigate the relationship of Sertoli cell number, Sertoli cell-germ cell ratios and other related factors to daily sperm production (DSP). DSP was assessed by enumeration of spermatids in testicular homogenates, whereas Sertoli cell and germ cell ratios were based on direct counts in 20 round Stage VIII seminiferous tubular cross sections per bull. Numbers of Sertoli cells were calculated as (total homogenization resistant spermatids:spermatid:Sertoli cell ratio)/0.394; the factor of 0.394 adjusted for the presence of homogenization resistant spermatids during only 39.4% of the spermatogenic cycle. Data were subjected to simple linear and second-order regression analyses. Positive linear relationships were observed between DSP and testicular parenchymal weight (p less than 0.005, R = +0.71), DSP per gram (p less than 0.005, R = +0.79), total Sertoli cells (p less than 0.005, R = +0.83), Sertoli cells per gram (p less than 0.01, R = +0.47) and the yield of Step 8 spermatids per Type A spermatogonium (p less than 0.05, R = +0.34). DSP was not related (p greater than 0.10) to the number of germ cells supported per Sertoli cell. Testicular parenchymal weight and DSP per gram were unrelated to each other (p greater than 0.10), but both were related (p less than 0.005) to the total Sertoli cell number (R = +0.61 and +0.62, respectively). Total number of Sertoli cells accounted for more of the variation in DSP between bulls (R2 = 68.2%) than did any other factor examined. It was suggested that total Sertoli cell number may be an important determinant of a bull's spermatogenic potential.

Relationship of absolute numbers of Sertoli cells to testicular size and spermatogenesis in young beef bulls.

Testes were obtained from 34 Hereford or Angus bulls at about 1.5 yr of age and were used to investigate the relationship between the absolute number of Sertoli cells vs testicular size and daily spermatozoal production (DSP). Quantitative determination of DSP was based upon enumeration of elongated spermatids in testicular homogenates. The ratio of step 8 spermatids to Sertoli cells (S:SC) was established by direct counts of these cells in each of 20 round stage VIII seminiferous tubular cross sections for each bull. The number of Sertoli cells per paired testes was calculated as (total spermatids divided by S:SC)/.394, where total spermatids equalled the number of homogenization-resistant spermatids. The factor of .394 adjusted for the fact that the latter cells are present for only 39.4% of the spermatogenic cycle. All data were subjected to simple linear and second-order regression analyses. A positive linear relationship (P less than .005) was found between testicular weight (Y, in grams) and the absolute number of Sertoli cells per paired testes (X, in billions), which was characterized by the equation Y = 315.2 + 10.74X and a coefficient of correlation (r) of .56 (P less than .01). A similar relationship was observed between DSP (Y, in billions) and Sertoli cell numbers (X, in billions). This was characterized by the equation Y = 1.36 + .222X (P less than .005) and a coefficient of correlation of .70 (P less than .01). Daily sperm production was unrelated to the S:SC ratio (P greater than .05).(ABSTRACT TRUNCATED AT 250 WORDS)

A quantitative study of Sertoli cell and germ cell populations as related to sexual development and aging in the stallion.

Testes from 47 stallions, 1-20 yr of age, were used to examine the influence of age on Sertoli and germ cell populations as well as on functional activity of Sertoli cells. For these stallions, the number of Sertoli cells per paired testes declined linearly with age, and was only 41.7% as great at age 20 as at age 2. However, development of reproductive organs proceeded until age 12-13, as evident from increases in paired testes weight and quantitative rates of spermatozoal production. Although the absolute number of Sertoli cells declined during this period of development, individual Sertoli cells displayed a remarkable capacity to accommodate greater numbers of developing germ cells. Between age 2 and age 12, the mean numbers of developing spermatogonia, young primary spermatocytes, old primary spermatocytes, and round spermatids supported by each Sertoli cell at Stage I of spermatogenesis increased by 49, 176, 153, and 161%, respectively.