Introduction

Motility of spermatozoa is one of the most important parameter used for sperm quality evaluation. It also provides important information on the energy status of the mammalian sperm (Quintero et al., 2004) to reach the uterotuberal junction and cause fertilization. Subjective semen motility grading is routinely followed in many of the bull semen stations as it can be performed anywhere with less cost and time. But these form of analysis have unfortunately yielded results with wide discrepancies between laboratories and technicians performing them (Christensen, 2005). With time the motility analysis have been replaced with Computer Assisted Semen Analyser (CASA). This device has been taught how a sperm looks like and how they move. However, these machines are not ready to use devices and require good expertise of the user and technical settings. Among the numerous variables affecting motility results, semen concentration is one of them. At higher concentration, multiple collisions, clumping and incomplete sperm tracks have been encountered (Leonardo, 2010).   Alternatively, ‘dilution effect’ at lower concentrations results in inaccurate motility parameter of the spermatozoa. Since then the motility patterns of spermatozoa in different species have been studied both for research and practical purpose. Hence, the present work was conducted with the aim to evaluate and quantify the optimal concentration of spermatozoa suitable for motility study using CASA device.

Materials and Methods

Semen Collection

Eight Frieswal bulls of uniform age and weight maintained at Bull Rearing Unit of Central Institute for Research on Cattle (CIRC), Uttar Pradesh, India was used for the study. The semen was collected by artificial vagina method in the morning and evaluated for its volume, concentration with a photometer, percent motility by subjective evaluation on placing a small drop of semen sample on a pre-warmed glass at 37°C and examining five different microscopic fields under 200X phase contrast microscope. Samples having motility, and concentration above 70% and 600 millions/ml respectively was considered fit for further dilution in egg yolk-glycerol-Tris (EYGT) buffer. In order to verify the concentration, manual estimation was done by a haemocytometer.

Sperm motility Evaluation by Computer Assisted Semen Analysis (CASA)

Motility is evaluated for the respective concentration using CASA IVOS 13 (Hamilton Thorne Biosience, MA, USA) and the settings mentioned in Table 1 are also as per the machine settings mentioned for bull. All CASA characteristics were obtained in phase illumination. Motility studies were conducted in Maklers chamber (Makler counting chamber, Sefi-Medical Instruments ltd, Israel) having a depth of 10mm. To avoid any repetition, the field was selected starting in the left upper corner and ending in the right lower corner of the chamber. Each field was visually examined for motile and non motile sperms while the static particles were ignored. With the help of playback facilities each screen showing the video sequence of the previous field analyzed allowing verification of sperm and non sperm particles along with its trajectories by the click of mouse and removing the non sperm particles.

Table 1: Settings of the Hamilton Thorne IVOS 13 for the present study

Fig.1 represents the different motility parameters analysed by CASA.

Fig. 1:  Sperm Kinetic parameters by CASA- average path velocity (VAP) = the average velocity of the smoothed cell path (mm/sec); straight line velocity (VSL) = the average velocity measured in a straight line from the beginning to the end of the track (mm/sec); curvilinear velocity VCL(mm/sec), the amplitude of the lateral head displacement (ALH) = the mean width of the head oscillations as the sperm cells swim (mm); the beat cross frequency (BCF) = frequency of the sperm heads crossing the average path in either direction (Hz); the straightness (STR) = average value of the ratio VSL/VAP (%)the percentage of totally motile spermatozoa (MOT, %) and the percentage of progressive spermatozoa (PMOT; %) (Mortimer, 2000)

Statistical Analysis

In this study, all data are reported as Mean ± Standard Error (SE). Differences in the sperm kinetic values due to different concentration of 15-20, 20-30 and more than 30 x 10⁶ spermatozoa/ml were tested by one way ANOVA. Difference are considered significant when p≤0.05 (Steel and Torrie , 1980).

Result and Discussion

Motility is an important parameter for evaluation of frozen thawed sperm quality in bovine industry. On subjective evaluation the motility of frozen thawed semen above 50% is considered fit for insemination. The results are presented in Table 2. When samples were diluted at 15-20 x 10⁶ sperm/ml, high average path velocity (VAP, p<0.01) and straight line velocity (VSL, p<0.01) was found with optimum progression (BCF, STR, LIN). However, Contri et al. (2010) found high total motility and with poor progression at a dilution of 20 and 30 x 10⁶ sperm/ml. None of the sperm in different concentrations identified by CASA were hyperactive i.e. <30%STR and <15% LIN (Farrell et al., 1996). This may be due to the presence of optimum quality spermatozoa collected from good bulls. At higher concentration of more than 30-40 x 10⁶  spermatozoa/ml, rest of the kinetic parameter showed incongruent motility patterns as supported by Rijsselaere et al. (2003). These spermatozoa demonstrated a lower kinetic efficiency as they moved slower and in less straight forward movement as the velocity (VAP, VSL; p<0.01) and direction measures (STR) was lower than rest of the sample concentration. This may be due to too many sperm cells per field along with the non sperm particles making it difficult to identify. Each of the sperm tracks was incomplete. The same theories have been identified by Mortimer et al. (1988).

Table 2: Effect of different sperm concentrations (15-20, 20-30 and >30 x10⁶spermatozoa/ml) on CASA (Hamilton Thorne IVOS 13) measurements

**p≤0.01

The finding suggests that the best motility results in CASA were produced at a concentration of 15-20 x 10⁶ spermatozoa/ml. However, Contri et al. (2010) observed that a dilution of 20 x 10⁶ spermatozoa/ml was optimum and diluting at a lower concentration lead to the possibility of ‘dilution effect’ (Mann 1964). Also the dilution at 15-20  x 10⁶ spermatozoa/ml yielded good number of sperms per field (40-50 cells) which facilitated quick visualization of sperm and non sperm particles. The non sperm particles were easily removed by the click of the mouse. Therefore, it may be considered that 15-20 x 10⁶ spermatozoa/ml is optimum for motility study using CASA systems.

Conclusion

In conclusion, this study also highlights that without giving attention to the concentration of semen before analyzing in CASA may provide misleading results. However, once the system is standardized, the device has enough potential to offer a rapid and objective analysis of large numbers of spermatozoa and can generate a very detailed characterization of sperm dynamics.

References

1. Christensen P, Boelling D, Pedersen KM, Korsgaad IR, Jensen J. 2005. Relationship between sperm viability as determined by flow cytometry and non return rate of dairy bulls.
2. Contri A, Valorz C, Faustini M, Wegher L, Carluccio A. 2010. Effect of semen preparation on CASA motility results in cryopreserved bull spermatozoa. Theriogenology. 74 (3): 424-35.
3. Farrell PB, Foote RH, Mc Ardle MM, Touern- Trend VL and Tardif AL. 1996. Media and dilution procedures tested to minimize handling effects on Human, Rabbit and Bull sperm for Computer Assisted Sperm Analysis (CASA). Journal of Andrology. 17 (3).
4. Leonardo FCB. 2010. Variation in Laboratory Semen Evaluation Procedures and Testing. In: Artificial Insemination and Reproduction. Proceedings of the 23^rd Technical Conference – Online. researchgate.net/publication/213863735
5. Mann T. 1964. The biochemistry of semen and of the male reproductive tract. Wiley, New York.
6. Mortimer D, Goel N and Shu MA. 1988. Evaluation of the Cell Soft automated semen analysis system in a routine laboratory setting. Fertility Sterility. 50:960–8.
7. Mortimer ST. 2000. CASA- Practical aspects. Journal of Andrology. 21 (4): 515-24.
8. Quintero-Moreno A, Rigau T and Rodriguez-Gil JE. 2004. Regression analyses and motile sperm subpopulation structure study as improving tools in boar semen quality analysis. Theriogenology. 61: 673–90.
9. Rijsselaere T, Ann VS, Dominiek Maes, Aart de Kruif, 2003. Effect of technical settings on canine semen motility parameters measured by the Hamilton – Thorne analyzer. Theriogenology.  60: 1553–1568
10. Steel RGD and Torrie JH. 1980. Principles and Procedures of Statistics- A biometrical approach. 2nd Ed., McGraw Hill Inter. Book Co. Tokyo, Japan.