Stallions were subjected to a gradually increasing photoper iod beginning on October 15, 1973. The maxi,mum artificial daylength (16 hr) was imposed on February 8, 1974, and maintained until October 6, 1974. Two ejaculates were collected from each of five treated and four control stallions weekly, with an interval of 24 hr between the paired ejaculates on the first and alternate weeks, and an interval of 1 hr on the second and alternate weeks. During summer, stallions subjected to the artificial photoper iod produced less (P<.05) gel-free semen and gel per ejaculate and had more (P<.05) spermatozoa per ml of gel-free semen than untreated stallions. Consistent differences due to artificial photoper iod were not observed for spermatozoa per ejaculate or percentage of motile spermatozoa. Light t reatment did not affect mounts per ejaculate or seminal pH, but stimulation time was reduced (P~.05) in treated stallions during late winter. Mean concentration of LH in the serum of untreated stallions varied (P<.01) over t ime and appeared to be highly seasonal. A corresponding change in concentration of testosterone was not observed (P>.05). Mean concentrations of both LH and testosterone were greater (P<.05) in the serum of treated stallions than in

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.

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.

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 × 106, 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.

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.

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.

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.

Investigators may choose among a variety of different approaches for identifying and quantifying potential treatment effects on sperm production rates. Should a treatment actually alter sperm production, the likelihood that the response will be detected and declared statistically significant depends on several factors. These include the size of the treatment response, the inherent variability associated with the endpoint of interest among normal, untreated males, and the number of replicate animals per treatment group. This study was undertaken to characterize the relative power and sensitivity of several widely used methods for quantifying sperm production, for the purpose of identifying those that would be the most likely to enable detection of actual treatment effects. For this exercise, the typical inherent variability associated with several endpoints of testicular function in rats, rabbits and humans was established by a review of the published literature. Based on this typical variability and a components-of-variance approach, the relative number of replicates needed to provide experiments of equivalent power and sensitivity was determined. Consideration was also given to the likely nature of changes in the testis in response to adverse treatments. Based on all of this information, the methods for quantifying spermatogenesis that would be most likely to detect an actual treatment effect were determined to be as follows (from greatest to least powerful and sensitive): (1) quantification of round spermatids per Sertoli cell or per seminiferous tubular cross-section, (2) the determination of the number of spermatids or daily sperm production (DSP) per gram of testis or per testis via volume density approaches, (3) the determination of the number of elongated spermatids or DSP per gram or per testis via the testicular homogenate approach, and (4) the direct enumeration of degenerating germ cells.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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)

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.