- In Vitro Evaluation of Frozen-Thawed Stallion Semen: A Review

Sperm motility is important because it is readily identifiable and reflects several essential aspects of sperm metabolism. Therefore, motility should be evaluated together with other parameters when estimating the fertilizing potential of spermatozoa. Usually total motility (any type of motility) and progressive motility (spermatozoa moving actively forward) are estimated as percentages. Motility can also be described as circling, oscillating and serpentine [46]. Often also the speed of spermatozoal motion is assessed. If semen is exposed to low temperatures or it dries on the slide, motility diminishes rapidly.

Stallion spermatozoa have some species-specific characteristics: an asymmetrical head, an abaxial position of the tail, an acrosome of small volume and the presence of microtubules in the neck [8]. The large, circular motion of normal sperm is due to a high incidence of abaxial connections between the sperm head and neck [46]. Estimating only the progressive motility may underestimate good motility of some stallions.

Light microscopy

To obtain an accurate estimate, environmental conditions should be standardized and optimal for semen. All equipment should be clean (preferably disposable) and before use, kept at body temperature by storing in an incubator. If the semen sample is too thick, spermatozoa are in layers and motility cannot be reliably estimated. Samples of a higher concentration are usually judged by the human eye as having higher motility [39]. Semen should be extended to (25 to 50) × 106 spermatozoa/ml, but not with a diluent that influences motility. Temperature of the slide should be controlled (+37°C) by using a stage warmer on a phase-contrast microscope, the depth of suspension on the slide should be standardized and multiple fields near the centre of the slide examined. Motility at the edges declines more rapidly than in the centre as a result of drying and exposure to air. [39]. The light microscopic evaluation does not require expensive equipment and is easy to perform. However, the greatest variation is caused by a variation between examiners, since the evaluation is subjective and requires experience.

When fresh stallion semen was subjectively evaluated, low correlations were found between fertility and the percentage of motile (r = 0.40) and progressively motile (r = 0.46) spermatozoa [42]. The number of mares inseminated with frozen semen has, in most experiments, been so small that statistical evaluation of the data has not been feasible. This may be one reason why very little published data exist on the correlation of motility evaluated by light microscopy and fertility of frozen-thawed stallion semen. In a study where 177 mares (on average 19 mares/stallion; min 6, max 51) were inseminated with frozen semen from 9 stallions, the correlation coefficient of the visually estimated percentage of motile cells to the first-cycle pregnancy rate was only 0.32 [78]. Good motility of frozen-thawed semen was a poor indicator for pregnancy rates in pigs [37]. Similarly, in the horse, the percentage of progressively motile, post-thaw spermatozoa is considered to be a poor predictor of pregnancy rates in mares [71,80,5,90]. Female genital fluids exert an influence on sperm motility. Some sperm that are immotile in vitro might regain motility in vivo, and vice versa [9]. A very low motility would probably be an indication not to use the semen, but a good motility does not necessarily indicate that the fertilizing capacity of spermatozoa has been maintained.

Computer-aided sperm analysis (CASA)

Subjective visual evaluation of motility is prone to human error and bias. Therefore, objective methods have been developed. Methods based on microscopic images include time-lapse photomicrography [82], multiple-exposure photomicrography, frame-by-frame playback videomicrography and cinematography [81], whereas turbidimetry, spectrophotometry and laser Doppler technology are based on physical principles [23]. Because photographs are tedious to analyse, computer-assisted technologies were the next step in the development of automated motility analysis. Due to the high cost of the instrument, computerized sperm image analysis systems are used primarily for research applications. The first systems available were the CellSoft Automated Semen Analyser and the Hamilton Thorn Motility Analyzer (HTM), others have since been introduced to the market.

Video images for computerized sperm motion analysis are obtained from viewing fields of motile sperm using a microscope. A set number (usually 20 to 30) of successive video frames is analysed at a constant rate, typically 30–60 frames per second. When all frames for a given field have been analysed, computer algorithms are used to distinquish sperm from non-sperm objects and to reconstruct sperm tracks [39]. Each sperm is classified as either motile or nonmotile, and the concentration of both is calculated. Motility data is further characterized as follows: mean curvilinear velocity (VCL), path velocity (VAP), mean straight-line velocity (VSL), straightness (STR = VSL/VAP), linearity (LIN = VSL/VCL), percentages of total motility (MOT), progressive motility (PMOT), amplitude of lateral head displacement (ALH) and beat cross frequency (BCF). The newest models also provide morphological measurements for certain species, although limited to sperm head morphology. Automated morphology systems have been validated for human sperm but not for horse. What all these specific motility characteristics tell us about the quality of fresh or frozen stallion semen is somewhat unclear because standard values have not been defined for normal or abnormal sperm motion. No international standardization in equipment settings has yet been implemented. The selection of gates, minimum and maximum values for head size and brightness, minimal velocities, straightness, frame rate, etc. influence results accordingly, and thus, do not allow comparison of results between laboratories. There is an urgent need for users of CASA to agree on standard analysis parameters within a given species.

In the analysis of frozen semen, particularly, non-spermatozoal particles (e.g. egg yolk) can mistakenly be identified as spermatozoa, causing "background noise". As a result, not only will the sperm concentration be overestimated, but the proportion of motile spermatozoa will be miscalculated [23]. The effect of egg yolk particles on many motion characteristics has been shown by [93]. If thawed semen is greatly diluted with a clear extender, the number of egg yolk particles and the concentration of viscous glycerol decrease. [85] used nonfat dry milk-glucose extender to dilute frozen-thawed semen samples before CASA evaluation. One approach to analysing frozen semen is to use clarified freezing extender which is prepared by centrifuging egg yolk with extender at 10 000 × g for 15 min. The supernatant including the lipid on the surface is then mixed with the freezing extender [15]. Filtering of extender through a 0.2-μm membrane filter removes larger particles that could interfere with measurements [14]. Recently, fluorescence dyes that do not affect motility (Hoechst 33342) have been used to differentiate sperm cells from egg yolk particles in CASA systems equipped with the epifluorescent illumination (Hamilton Thorne IVOS) [32].

The maximum sperm concentration in CASA-systems is usually 50 × 106/ml. A dilution of stallion semen to 25 × 106/ml has been recommended [85]. [85] used Makler-chambers and videotaped the semen samples. This considerably shortens the time that semen samples have to stand in the Makler-chamber as compared to performing the analyses right away. Sperm dries quickly in a Makler-chamber at 37°C which is a problem in the older, slower, analyzers, but the newest CASA-instruments are able to analyse 400 cells in 2 min. In the study of [85], the most highly variable component was field within chamber. They recommended that 3 chambers/ejaculate and 3 fields/chamber be evaluated which would yield a mean spermatozoal number of approximately 500 evaluated per sample.

No significant correlations were found when ca 20 000 cows were inseminated with frozen semen from 10 bulls and the 75-day nonreturn rate was compared with motility characteristics obtained by CellSoft Analyser [14]. In another fertility trial, the competitive fertility index for 9 bulls was correlated (r > 0.68) with MOT, VCL and VSL [14]. MOT had a low (0.45) but significant correlation with the first-cycle pregnancy rate of 177 mares inseminated with frozen semen from 9 stallions [78]. In a French study, in which 60 ejaculates were frozen from 7 stallions, batches with a post-thaw motility of >35% accepted for use, and 334 mares inseminated, there was no correlation between fertility and subjective post-thaw motility or percentage of sperm moving >30 μm/sec (RAP) analysed by CASA [5]. In another French study, 766 mares were inseminated with frozen semen, but none of the criteria measured by CASA (VCL, LIN, ALH, MOT, RAP) had a significant correlation with fertility [67].

It is not surprising that CASA-systems have been unable to detect differences between "good" and "poor" frozen semen when the ejaculates have been selected to include only those with a post-thaw progressive motility >30% to 35%. The fertility of mares varies widely and one has to bear in mind that a single ejaculate can only be used for 5 to 15 mares. If low-quality semen had not been rejected before freezing, CASA would probably have detected significant differences more readily. On the other hand, computers are not needed to detect large differences. The much less expensive way of subjectively evaluating total or progressive motility using a light microscope yields similar results to automatic analysers [78,67,47]. To date, the superiority of the automatic analyser in the evaluation of frozen semen has not been proven, although it is the only way to accurately assess velocity and linearity. It should be emphasized that automated analysis presents risks of artifacts that must be controlled for and that the apparatus must be correctly set [67]. It is worrying that the mean motility values obtained from the same semen samples sometimes differed by as much as 30% when analysed simultaneously by CellSoft and HTM [41]. Further, when the same semen specimens were analysed by 2 identical HTM analysers, significant differences were seen in sperm counts, ALH, LIN and BCF, which shows that the reproducibility was poor [2].

Longevity of motility (survival tests)

For estimating the longevity of motility, an aliquot of well-mixed – typically extended – semen is used to fill a warm sterile tube which is kept in a draft-free, preferably dark environment. The semen is mixed and an aliquot examined at regular time intervals until [46].

The incubation temperatures and times have varied considerably. Longevity of motility increases with decreasing temperature. [60,61] used survival for >120 h at 2–4°C or at 1–4°C as a criterion for accepting frozen semen for field use. The average time for accepted semen was 202 hours, with a range from 120 to 312 h. Survival tests are in routine use in some stallion stations: 37°C for 4 h (threshold motility 15%), 20°C for 12 to 48 h (threshold 5–10%) and 5°C for 7 days (threshold 5%) [86]. Other laboratories employ shorter incubation times at 37°C, e.g., only 0.5 h [50]. In a retrospective study on commercially used frozen semen with 31 stallions and 1023 mares the thawed semen was kept at 37°C. A significant correlation was demonstrated between the foaling rate and motility evaluated by light microscopy after an incubation of 2 and 4 h [43].

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