attempts to modify the serving capacity test to enable the collection of semen involved having a person deflect the bull's penis away from the cow and into an AV. Most untrained bulls were unwilling to mount with a person standing close by, so attempts were made to conceal the operator behind drapes while the bull mounted and semen was collected [12]. High libido bulls were undeterred by this system, but medium to low libido bulls seemed to be distracted by the drapes or the presence of humans behind the drapes and refused to serve. [12]. The next modification was to design an AV that could be fitted inside the cow's vagina, called the internal artificial vagina (IAV). The IAV consisted of a wire frame supporting a 7.5‐cm length of rubber tubing with a plastic specimen bag attached to the end placed closest to the cervix [23]. Once the bull had mounted the cow and completed service the IAV could be quickly removed and the semen sample recovered. Over a two‐year period, the IAV was compared to EEJ as a method of collecting semen from 165 range bulls. Fifty four and seventy percent of the bulls served the IAV in years one and two, respectively versus a 100% success rate when EEJ was used. Five percent of the bulls were found to be unable to serve cows due to physical abnormalities. Bulls that were unwilling to serve the IAV in year 2 were given a second opportunity, but the result remained the same. Over the subsequent breeding season, the breeding behavior of 15 of the bulls that did not serve the IAV was compared with 15 that did. Most of the bulls that would not serve the IAV were observed breeding cows but mounted less frequently than their counterparts [23]. The IAV proved useful for identifying bulls with physical abnormalities but was not a viable substitute for EEJ. Other concerns included the spread of venereal disease and the welfare of the restrained mount animals [12].
Transrectal massage of the seminal vesicles, ampullae, and pelvic urethra has been used as a method of obtaining a semen sample but is not as reliable as EEJ. In one experiment, TM was shown to be nearly as effective as EEJ for obtaining a semen sample [24], but the percentages of motile and live sperm were less than those of EEJ samples. This was attributed to the tendency of massaged bulls to dribble semen slowly, thereby exposing the sperm to cool temperatures for a longer period of time [24]. Two more experiments were conducted comparing TM with EEJ in infrequently handled, mature breeding bulls (range bulls) and yearling bulls. In an attempt to prevent chilling of the sperm, semen collection tubes were suspended in a 37 °C water jacket during the collection. Despite this effort, fewer motile and live sperm were recovered from TM samples in the range bulls, but there was no difference in percentages of motile sperm in the yearling bulls [25]. These differences were attributed to lack of penile protrusion during semen collection with TM – a problem that was particularly evident with the range bulls [25]. The differences in live and motile sperm percentages were not large, but these authors cautioned that poor semen sample quality and an inability to examine the penis could jeopardize the quality of a BBSE [25]. Transrectal massage of the seminal vesicles and ampullae can be useful following an unsuccessful attempt to electroejaculate a bull. In these cases, the maximum voltage has been reached but only preseminal fluid has been emitted. One useful trick is to rest the bull for at least 30 seconds and restart the collection at a much lower setting. If this quick fix fails, then the probe can be removed and TM may be effective.
Aspiration of semen from the vagina of a recently bred female is a last resort method of obtaining a sample as it is time consuming and requires a female in estrus that must be restrained to allow a sample to be obtained. A syringe and infusion pipette will facilitate the collection of mucus and semen from the anterior vagina.
Semen Evaluation
Once the semen sample has been collected the vial can be easily detached from the semen collection cone. To avoid spillage it works best to pull the cone sideways and slightly downward away from the vial, which has the effect of stretching and shearing the cone off on the edges of the vial. Always keep the semen vial protected from the cold prior to evaluation. A simple and effective method is to hold the vial in a warm hand with the fingers completely encircling it. If evaluation is not going to occur right away the sample should be transferred to a 37 °C water bath. The chest pocket of a close‐fitting pair of coveralls has also worked well for short‐term storage, especially if the hands are needed for other tasks.
Although not always recorded, the skilled examiner should know how to assess each of the following when examining a semen sample:
Color and consistency
Volume
Concentration
Gross motility
Individual motility
Sperm morphology
Percentage staining alive
Color and Consistency
A good quality ejaculate should be opaque, creamy‐white in color. A yellow, buttery hue may also be seen and is a variation of normal when it occurs in concentrated samples with no apparent aberrations. The yellow coloring may be associated with riboflavin in the diet [26] and is different from a yellow tinge that may be evident with urine contamination. A urine‐contaminated semen sample will usually be variably diluted and will have an odor of urine. (Editor's note: if unsure, utilization of a “BUN strip” [blood urea nitrogen] will confirm or rule out.) Preseminal fluid is clear, watery, and odorless. Semen samples diluted with preseminal fluid will not be opaque but rather have a degree of clarity on a spectrum, comparable with skim‐milk, watered‐down juice, or even clear fluid. Generally, preseminal fluid emission is noted during the EEJ process, but it is not collected in favor of waiting for the appearance of the sperm‐rich fraction. Especially in difficult collections, a variable amount of preseminal fluid will be collected, thereby diluting the sample. A pink or red hue or even streaks of red in the semen sample are usually indicative of blood contamination. The source of the blood should be addressed. Blood‐contaminated samples are common when semen collection follows preputial scraping for Tritrichomonas foetus and Campylobacter foetus, especially if penile protrusion did not occur during EEJ. Clumps of white cellular material in the vial are highly suggestive of a high neutrophil content.
Volume
Always note the volume as it is an indication that a representative sample was collected; however, volume is influenced by the collection process and is therefore not a relevant measure. In other words, the volume of an EEJ‐derived semen sample should not be compared with ejaculates from the same or different bulls. The one exception is in cases of sperm accumulation, the “rusty load phenomenon,” where 25–40 ml of concentrated semen may be collected easily during a single ejaculation [2]. A typical EEJ‐derived semen sample is 1–5 ml.
Concentration
A subjective evaluation of concentration should be made by examining the tube containing the semen to ensure that a representative sample has been obtained. A poorly concentrated sample will be watery and translucent containing <250 million sperm per milliliter, whereas a concentrated sample, typical of a mature bull, will appear creamy and grainy when examined against a dark background containing at least one billion sperm per milliliter. Very concentrated, high‐volume ejaculates occur in cases of sperm accumulation. A watery, poorly concentrated sample from a mature bull is typical in cases of testicular degeneration. Closely linked to volume, the concentration of a semen sample also varies with the collection technique. EEJ is not an appropriate method of determining the sperm‐producing capability of a bull.
Gross Motility
Gross motility or gross wave motion is influenced by the percentage of progressively motile sperm, the sperm concentration, and the vigor or rate of speed of the motile sperm. Determining gross
