The motion characteristics as well as velocity and kinematic attributes of freshly diluted and frozen-thawed sperm of Gir and Murrah bulls were observed by Biovis CASA in control TFYG extender and TFYG fortified with Mifepristone (10 µg/ml), Sericin (5 mg/ml) and Taurine (4 mg/ml). The mean values and per cent change in post-thawed sperm over freshly diluted sperm quality are presented in Table 1 to 3 and Fig 1 and 2.
Motion characteristics/motility rating
The overall mean percentages of total motile spermatozoa observed on dilution and at post-thaw stage were 85.50±0.92 and 48.76±1.69 for Gir bulls and 85.03±0.72 and 52.61±1.46 for Murrah bulls, respectively, without statistical difference between the breeds. Moreover, the values were at par with subjective assessment of sperm motility. The mean percentages of total motile sperm were significantly (p<0.05) higher in Mifepristone fortified extender than in control, while values for Sericin and Taurine fortification were statistically similar to Mifepristone in post-thawed Gir bull and fresh Murrah bull semen, while in post-thawed buffalo semen they were at par with control group. Furthermore, the percent decline in total motile sperm due to freezing-thawing stress was more or less same in both the breeds in all the extender-additives, being lowest in Mifepristone supplemented extender as compared to control in cattle (32.27 vs 48.81%) and buffalo (31.27 vs 43.14%) semen (Table 1).
The overall mean percentages of rapid progressive motile sperm on dilution and at post-thaw stage were 24.64±1.07 and 6.66±0.62 for Gir and 23.48±1.59 and 8.74±0.93 for Murrah bulls, respectively, which also did not differ significantly between the breeds. The mean percentage of rapid progressive motile spermatozoa were comparatively higher in Mifepristone, intermediary in Sericin and Taurine groups and lowest in control, but did not differ statistically at any of the stages in any of the breeds. The per cent decline in rapid progressive motile spermatozoa due to freezing-thawing stress was more or less same in both the breeds in all the extender additives, being lowest in Mifepristone supplemented extender than control in cattle (66.90 vs 79.51%) and buffalo (58.88 vs 68.85%) semen (Table 1; Fig 1).
The overall mean percentages of slow progressive motile spermatozoa on dilution and at post-thaw stage were 36.31±0.83 and 20.68±1.14 for Gir bulls and 39.10±1.16 and 25.67±1.19 for Murrah bulls, respectively, being significantly (p<0.05) higher in Gir than in Murrah bull sperm at both the stages of evaluation. The mean percentage of slow progressive motile sperm were significantly higher in Mifepristone fortified than in control extender, but statistically at par with Sericin and Taurine, particularly at post-thaw stage. Moreover, the percent decline in slow progressive motile spermatozoa due to freezing-thawing stress was apparently low in all the extender-additives in buffalo sperm than the cattle sperm, with significant difference in pooled values (43.05 vs 34.35%) and the decline was much less in Mifepristone supplemented extender compared to others in both the species (Table 1).
The overall mean percentages of non-progressive motile spermatozoa on dilution and at post-thaw stage were 24.56±0.91 and 21.43±0.74 for Gir bulls and 22.45±1.18 and 18.20±0.75 for Murrah bulls, respectively, the values for Gir bulls being higher than in Murrah with significant (p<0.01) difference only at post-thaw stage. The mean percentages of non-progressive motile spermatozoa in control TFYG extender and extender supplemented with Mifepristone, Sericin and Taurine on dilution and at post-thaw stage were statistically similar at both the stages of evaluation and in both the breeds. The overall mean percentages of immotile spermatozoa on dilution and at post-thaw stage were 14.50±0.92 and 51.25±1.69 for Gir bulls and 14.97±0.72 and 47.39±1.46 for Murrah bulls, respectively, which however did not differ between the breeds at any stage. The mean values were lowest in Mifepristone supplemented group than other additives including control at both the stages of evaluation with significant difference only in Murrah at post-thaw stage. The percent increase in immotile sperm due to freezing-thawing was more with all the extender-additives in cattle than the buffalo semen (+253.45 vs. +216.57%) (Table 1, Fig 1).
The Biovis CASA based results of fresh diluted and post-thaw motility rating for Gir and Murrah bull sperm particularly in control TFYG extender were in accordance with the recent observations of
Pathak et al., (2019a, b;
2020) using the same Biovis CASA version and also with
Karthikeya (2003),
Kathiravan et al., (2008), Patel et al., (2012) and
Patel and Dhami (2016) with different versions of CASA. The present findings on kind of sperm motility ratings for fresh diluted and/or frozen-thawed semen observed also concurred with the earlier reports on different farm animals species by various workers using different grades of CASA machines and chambers
(Anderson et al., 1992; Tardif et al., 1997; Abbas et al., 2001; Rasul et al., 2001; Mandal et al., 2003; Hoflack et al., 2007;
Anand and Yadav, 2016;
Rodriguez-Montana and Roa-Guerrero, 2017). Although
Kumar et al., (2018) using same version of Biovis CASA, found much lower values of motility rating for buffalo sperm in fresh diluted, equilibrated and frozen-thawed semen than the present findings in either of the species studied. However, the percentages of total motile and progressive motile sperms varied among different studies due to variation in the breeds of bulls, initial and post-thaw quality of semen, extender used, post-thaw longevity of sperms, software and make of CASA machines used for evaluation such as Biovis CASA, Hamilton Thorne CASA
etc. Moreover, the per cent decline in total and progressive motile sperm and increase in immotile and non-progressive motile sperm in post-thawed semen over freshly diluted semen concurred well with the earlier reports of
Patel and Dhami (2016) and
Pathak et al., (2020).
However, very few workers have evaluated sperm motion characteristics by CASA following incorporation of various additives and antioxidants to compare the present findings using Mifepristone, Sericin and Taurine.
Tardif et al., (1997) evaluated bull sperm motility using CASA in a modified Tyrode’s solution, in Cornell University extender and in egg-yolk-glycerol-Tris extender and recorded the values as 87.0, 79.0 and 66.0%, respectively, with little change following cooling and storage at 5°C in the latter two extenders. Similarly,
Lackey et al., (1998) observed increase in the percentage of rapidly moving bovine sperm cells in modified Tyrodes’ medium supplemented with insulin-like growth factors (IGFs) @ 100 and 250 ng/mL using CASA at 90, 180 and 360 min as compared to the control medium.
Perumal et al., (2011) found better post-thaw sperm functional parameters of crossbred bull semen in CASA following pre-freeze addition of cysteine and glutathione (GSH) in extender.
Dorji et al., (2016) recorded significant improvement in post-thawed total motile and progressively motile sperm of Thai bull semen cryopreserved with inclusion of 0.1 and 0.5% Sericin, while
Dalal et al., (2019) found significantly improved post-thaw buffalo sperm motility with increasing level of Mifepristone from 5 to 10 μM as compared to control and higher levels, which concurred well with the present findings. Thus, CASA provided instant and objective assessment of sperm motility and its grading especially about the quality of sperm motion.
Sperm velocity and kinematics
The overall mean average path velocity (VAP, µm/s) of sperm on dilution and at post-thaw stage were 43.17±0.65 and 30.89±0.63 for Gir bulls and 41.75±0.92 and 33.35±0.72 for Murrah bulls, respectively, being significantly (p<0.05) higher in Murrah breed at post-thaw stage. The corresponding overall mean curvilinear velocity (VCL, µm/s) of sperm in freshly diluted and post-thawed semen were 87.74±0.88 and 73.68±1.03 for Gir bulls and 83.14±1.22 and 72.26±0.83 for Murrah bulls, respectively, with significantly (p<0.01) higher values in Gir than the Murrah breed on dilution. The overall mean straight-line velocity (VSL, µm/s) of sperm on dilution and at post-thaw stage were 35.74±0.65 and 23.98±0.65 for Gir bulls and 35.48±1.00 and 27.29±0.83 for Murrah bulls, respectively. There was significant (p<0.01) increase in VCL in buffalo sperm compared to cattle sperm at post-thaw stage. The mean values of VAP, VCL and VSL were statistically similar at both the stages in control TFYG extender and TFYG fortified with Mifepristone, Sericin and Taurine. The per cent decline in VAP, VCL and VSL was little lower and more or less similar among extender additives than in control, particularly in buffalo sperm (Table 2, Fig 2).
The overall mean linearity (LIN, %) of sperm movement on dilution and at post-thaw stage were 40.86±0.57 and 32.56±0.76 for Gir bulls and 42.59±0.93 and 37.89±0.98 for Murrah bulls, respectively. The corresponding overall mean straightness (STR, %) of sperm movement on dilution and at post-thaw stage were 77.89±0.52 and 69.42±0.84 for Gir bulls and 79.93±0.74 and 74.65±1.00 for Murrah bulls, respectively. The values of both LIN and STR were significantly (p<0.05) higher in Murrah than in Gir sperm at both the stages, particularly in Mifepristone and Sericin fortified extender, with lesser per cent decline in linearity and straightness following freezing-thawing in all the extenders (Table 2).
The mean wobbling index (WOB, %) of freshly diluted and post-thawed sperm were 49.54±0.49 and 42.70±0.68 for Gir bulls and 50.44±0.74 and 46.91±0.78 for Murrah bulls, respectively. Similarly, the overall mean beat-cross frequency (BCF, hz) of sperm on dilution and at post-thaw stage were 11.71±0.26 and 7.47±0.23 for Gir bulls and 11.71±0.26 and 8.94±0.35 for Murrah bulls, respectively. Significantly (p<0.01) higher WOB and BCF were observed in Murrah than the Gir breed at post-thaw stage, with lesser per cent decline in values due to freezing-thawing insult in Murrahs due to the apparent influence of additives (Table 3).
The overall mean lateral head displacement (ALH, µm) values of sperm on dilution and at post-thaw stage were 2.21±0.05 and 1.86±0.04 for Gir bulls and 2.13±0.06 and 1.90±0.04 for Murrah bulls, respectively, without significant difference between the breeds at any of the stages. Yet the per cent decline in ALH was lesser in Murrah than the Gir sperm following freezing-thawing of semen. Similarly, the overall pooled mean dancing velocity (DNC, µm
2/s) of sperm on dilution and at post-thaw stage were 199.33±4.40 and 142.09±4.05 for Gir bulls and 178.54±3.89 and 143.34±3.51 for Murrah bulls, respectively, with significant (p<0.05) difference between the breeds only at dilution stage and thus, the per cent decline in DNC was lesser in Murrah than the Gir sperm following freezing-thawing of the semen. Further, the overall values of dancing mean (DNM, µm
2/s) of sperm on dilution and at post-thaw stage were 7.53±0.22 and 7.67±0.20 for Gir bulls and 6.95±0.28 and 6.71±0.18 for Murrah bulls, respectively. The values were higher for Gir than Murrah with significant (p<0.05) difference only at post-thaw stage and more or less similar values at pre- and post-stage stages in both the breeds in all the extender additives (Table 3).
The present findings on different velocity and kinematics attributes and their values of fresh diluted and/or frozen-thawed spermatozoa of Gir and Murrah bulls more or less concurred with the earlier reports of several workers, particularly of
Karthikeya (2003),
Mandal et al., (2003), Patel et al., (2012), Patel and Dhami (2016) and
Pathak et al., (2019a,b, 2020) using different versions of Hamilton Thorne CASA, Ovis CASA or Biovis CASA systems. However, the absolute values of these velocity and kinematics traits varied among different studies probably for reasons already mentioned earlier. Present results with Biovis CASA concurred very well with
Patel and Dhami (2016) and
Pathak et al., (2019a, b,
2020) and were far higher than those reported for buffalo sperm by
Kumar et al., (2018) using same version of CASA and were within the range of results of most of the other studies cited above. The percent decline or increase in velocity and kinematic attributes of both cattle and buffalo sperm in post-thawed semen over freshly diluted one in TFYG extender closely coincided with the recent reports of
Patel and Dhami (2016) and
Pathak et al., (2020). Rasul et al., (2001) reported linear decline (p<0.05) in sperm motility as well as VCL from 112.4±5.3 µm/sec after dilution to 69.4±2.0 µm/s after freezing and thawing and the ALH (p<0.05) from 3.9±0.2 µm to 1.7±0.2 µm.
Ramachandran et al., (2007) reported the mean CASA parameters of frozen-semen of Sahiwal bulls which compared well with the present findings for Gir bull semen.
Dorji et al., (2016) recorded significant improvement in post-thawed VAP and VCL of Thai bull sperm cryopreserved with inclusion of 0.1 and 0.5% Sericin as compared to control and higher levels of Sericin.
Perumal et al., (2011) found better post-thaw VCL and ALH values as well as significantly (p<0.05) higher conception rates for crossbred bulls sperm with glutathione fortified than the cysteine added and control extender. Similarly, significantly better VAP, VSL and VCL values were also reported in buck semen by
Arakeri et al., (2020) in skim milk fortified with 5 mM glutathione and 1% honey after 72 hrs of refrigeration.
Lackey et al., (1998) recorded increased VAP, VSL, VCL, ALH and BCF of bovine sperm using CASA in modified Tyrodes’ medium supplemented with insulin-like growth factor @ 100 and 250 ng/mL as compared to the control extender.
Dalal et al., (2019), however, did not find any change in velocity and kinematic parameters of buffalo sperm cryopreserved using tris extender fortified with different levels of Mifepristone (5, 10, 20 µM) compared to non-fortified control extender, though it reduced cryocapacitation.
The present findings on percent decline in the sperm quality due to freezing-thawing stress over fresh sperm concurred well with those of
Pathak et al., (2020), who found significant reduction in VAP, VCL, VSL, linearity and straightness of sperms with little variations between Gir, Surti and Murrah breeds with similar TFYG dilutor and version of Biovis CASA system, while wobbling index increased marginally by 1.51 to 2.63%. Moreover, the ALH, BCF, DNC and DNM of frozen-thawed sperm declined insignificantly over fresh diluted semen. The present observations further showed that the fresh diluted and frozen-thawed sperms behaved identically with respect to their velocity and kinematics, although differed in their motility ratings. These findings of change over in post-thaw quality of sperm following cryopreservation concurred well with the reports of
Patel et al., (2012) and
Patel and Dhami (2013,
2016) in Jafarabadi, Mehsana and HF × K crossbred bulls semen by using Leja slides on Hamilton Thorne CASA and with the observations of
Pathak et al., (2020) who had used ordinary plain slides on Biovis CASA as also used in the present study.
