Cheryl A. Thomas,¹ Duane L. Garner,¹ J. Mel DeJarnette² and Clifton E. Marshall²
School of Veterinary Medicine, University of Nevada,¹ Reno, NV 89557
and Select Sires,² Plain City, OH 43064

Bulls vary greatly in semen quality, both among bulls and among ejaculates within a bull. Therefore, semen is evaluated routinely using those characteristics that are known to affect fertility. These include general appearance of the semen, volume of the ejaculate, concentration of sperm, motility, metabolic activity, percent live/dead and morphological abnormalities. The minimum levels for a sample of probable fertility include about 500 million sperm/ml, at least 50% progressively motile sperm, and 80% with normal morphology.

The University of Nevada, Reno, has developed techniques for semen evaluation that are precise and repeatable, yet faster and easier than microscopic methods. The sperm are labeled with fluorescent stains specific for some property of the cell, such as live or dead, the intactness of the acrosomes that are needed for fertilization, or the metabolic activity of the sperm (a biochemical indicator of the ability of the sperm to be motile).

Ejaculated semen was collected from 12 bulls housed at Select Sires, Inc. in Plain City, Ohio. The ejaculates were diluted, processed and cryopreserved in a 20% egg yolk, 2.9% sodium citrate medium, and packaged in 0.5-ml French straws at a concentration of 35 x 10⁶ sperm/ml.

At Select Sires, the percentage of sperm with intact acrosomes (IA) was determined. These were analyzed in triplicate on 300 sperm from the thawed, cryopreserved samples using differential interference contrast microscopy (DIC). These assessments were made after thawing the straws and incubating them for 3 hr at 37°C. The percentage of progressively motile sperm (MOT), also in triplicate, was estimated visually using a thermostatically warmed stage (37°C) and light microscopy at 10X magnification. Motility estimates were made immediately after thawing the straws in 37°C water and after 3 hr of incubation at 37°C.

Intact Acrosomes. In this experiment, acrosomal integrity was examined by using two stains to quantify the green population of sperm with intact acrosomes and those dead sperm that stained red with propidium iodide (PI). LysoTracker (LYSO-G), which is a probe that stains acidic organelles green, was used to identify living sperm with intact acrosomes. Propidium iodide was used to stain the nuclei of sperm having degenerate membranes (dead sperm). A stock solution of HEPES/BSA containing PI and LysoTracker was prepared and warmed to 37°C. After semen addition, samples were incubated at 37° C for 30 min before analyses.

Metabolic Activity. The ability of sperm to move depends on the activity of the sperm mitochondria. This was determined using three dual-staining systems: PI and rhodamine 123 (R123), PI with JC-1, and PI with MitoTrackerÔ Green (MITO). These stains resolved populations of sperm with actively respiring mitochondria (motile sperm), a population of dead sperm, and two populations of dying sperm. The nuclei of dead sperm stained red with PI while living sperm with functioning mitochondria stained green with the mitochondrial stains. Three stock solutions containing the stains were prepared and aliquots of semen were added to each.

Sperm Viability. In this experiment, the percentages of viable sperm were quantified using the green fluorescent stain, SYBR-14, to identify living sperm. Propidium iodide stained dead sperm red, while a small portion stained doubly with both SYBR-14 and PI. This combination was developed in our laboratory and is commercially available as LIVE/DEAD® Sperm Viability Kit (Molecular Probes, Eugene, OR; L-7011). The stock solution was warmed to 37° C, and used to dilute semen 1 part to 3 parts. Stained sperm were incubated at 37° C for 15 min before flow cytometric analyses.

The fluorescently stained sperm are then quantified using a flow cytometer. This is an instrument in which a suspension of sperm flow single file past a beam of light at a rate of about 200-600 sperm/sec. The resulting data are reported as percentages.

For all analyses, variables were randomized. However, for clarity of presentation, bulls have been rank ordered by percentage of sperm with intact acrosomes as determined by DIC microscopy. All other methods of assessing acrosomal status and viability were compared against this standard. Mean percentage IA, initial motility (MOT) and motility after 3 hr (MOT3) for each bull, along with overall means and standard errors, are listed in Table 1. Bulls 10, 11 and 12 consistently ranked lowest in sperm quality across all methods. Bulls 1 and 4 most frequently ranked highest. The remaining bulls ranked in varying order among methods. Duncan’s Multiple Range test of mean MOT indicated that Bulls 1-9 did not differ, nor did Bulls 10-12. Within other methods, more complex groupings were observed.

All microscopic methods showed a high degree of correlation with fluorometric methods (Table 2). Among microscopic methods, IA was most highly correlated with motility after 3 hr (r = 0.99; data not shown).

Analysis of variance performed on the three mitochondrial stains showed that they were not statistically different. The variability among the 3 observations for each bull was generally less in the flow cytometric evaluations than in the microscopic evaluations. The decreased variability was probably due to counting 10,000 sperm flow cytometrically versus 300 sperm microscopically.

The new MitoTracker and LysoTracker stains reflected the percentages of motile sperm and those with intact acrosomes, respectively, as shown in Figure 1.

The microscopic method of evaluating the percentage of sperm with intact acrosomes and progressive forward motility produced values that were similar, but statistically different. LysoTracker™ Green, which is specific for acidotropic organelles such as the acrosome, can 1) be easily used in a batch staining method; 2) showed the highest correlation among methods with IA; and 3) labels sperm with intact acrosomes. Since it is highly correlated with post-thaw motility, as well (r = 0.97), it can be used with or without PI to simultaneously quantify the percentage of viable sperm with intact acrosomes.

The three mitochondrial probes did not differ and were of equal ease in use. Therefore, both LysoTracker™ Green and MitoTrackerÔ Green appear to be useful in assessing seminal quality in bulls.

Table 1. The mean percentages (n = 3) of microscopic and selected fluorometric methods of assessing semen quality in 12 bulls. Microscopic examinations were done using differential interference contrast (DIC) to assess the normalcy of the acrosomes (IA) on 300 sperm and visual estimations of motility at 0 and 3 hr incubation at 37°C. Fluorometric evaluations utilized fluorescent probes and flow cytometry to evaluate 10,000 sperm.

^a The proportion of sperm with intact acrosomes (IA).
^b The proportion of sperm that were stained with LysoTracker™ Green.
^c The proportion of sperm that displayed progressive forward motility.
^d The proportion of viable sperm as indicated by staining with SYBR-14.
^e The proportion of sperm that were stained with MitoTrackerÔ Green.

Figure 1. Graphic illustrations showing the predicted percentages of sperm with intact acrosomes as reflected by the percentages of sperm labeling with the fluorescent acrosomal stain, LysoTrackerÔ Green (A) and the predicted percentages of motile sperm as reflected by the percentages of sperm that labeled with MitoTrackerÔ Green (B). Both regression lines and actual data points are given for (A) where the vertical axis equals the predicted percentage of sperm with intact acrosomes (IA) and the horizontal axis equals the percentage of sperm that stained with LysoTracker; and (B) where the vertical axis equals the predicted percentage of motile sperm and horizontal axis equals the percentage of sperm that stained with MitoTracker.