Indian Journal of Animal Research

  • Chief EditorK.M.L. Pathak

  • Print ISSN 0367-6722

  • Online ISSN 0976-0555

  • NAAS Rating 6.50

  • SJR 0.263

  • Impact Factor 0.5 (2023)

Frequency :
Monthly (January, February, March, April, May, June, July, August, September, October, November and December)
Indexing Services :
Science Citation Index Expanded, BIOSIS Preview, ISI Citation Index, Biological Abstracts, Scopus, AGRICOLA, Google Scholar, CrossRef, CAB Abstracting Journals, Chemical Abstracts, Indian Science Abstracts, EBSCO Indexing Services, Index Copernicus
Indian Journal of Animal Research, volume 56 issue 3 (march 2022) : 255-262

Fortification of Tris Extender with Mifepristone, Sericin and Taurine Improves Velocity and Kinematics of Fresh and Frozen-thawed Bovine Spermatozoa 

Devangana Chaturvedi1, A.J. Dhami1,*, D.V. Chaudhari1
1Department of Veterinary Gynaecology and Obstetrics, College of Veterinary Science and Animal Husbandry, Anand Agricultural University, Anand-388 001, Gujarat, India.
Cite article:- Chaturvedi Devangana, Dhami A.J., Chaudhari D.V. (2022). Fortification of Tris Extender with Mifepristone, Sericin and Taurine Improves Velocity and Kinematics of Fresh and Frozen-thawed Bovine Spermatozoa . Indian Journal of Animal Research. 56(3): 255-262. doi: 10.18805/IJAR.B-4315.
Background: The evaluation of bull fertility essentially involves semen analysis and conception rates among inseminated females. The conventional evaluation is relatively imprecise, time consuming and subjected to individual variations, while computer assisted sperm analysis (CASA) is a quite faster, precise and objective tool to identify differences in sperm motility and velocity/kinematics attributes and avoids subjective errors. The present study was planned to evaluate and compare the CASA traits of fresh and frozen-thawed semen of Gir and Murrah bulls in Tris extender without and with different antioxidant additives by adopting Biovis CASA.

Methods: The semen ejaculates with >75% initial motility from 3 Gir and 3 Murrah bulls were split-diluted at 100 million sperm per ml using Tris-citrate-fructose-yolk-glycerol (TFYG) extender without (control) and with three antioxidant additives, viz., Mifepriston (10 µg/ml), Sericin (5 mg/ml) and Taurine (4 mg/ml), were filled in French mini straws and frozen in liquid nitrogen vapour using a programmable biofreezer. The freshly diluted and frozen-thawed semen samples were assessed for sperm motion characteristics, velocity/kinematics using Biovis CASA. 

Result: The mean percentages of total motile spermatozoa, irrespective of additives, in freshly diluted and frozen-thawed semen 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. The mean total motile as well as rapid and slow progressive motile sperm percentage were significantly enhanced by fortification of TFYG extender with Mifepristone than in control extender, while values for Sericin and Taurine were intermediary and statistically similar to Mifepristone. The per cent decline in total motile sperm due to freezing-thawing was more or less same in both the breeds for all the extender additives, being lowest in Mifepristone supplemented extender in cattle (32.27 vs 48.81%) and buffalo (31.27 vs 43.14%) semen. The rapid and slow progressive motile sperm also followed the same pattern. The values of VAP and VCL were significantly (p<0.05) higher and VSL lower in Murrah than Gir breed at post-thaw stage. The overall mean linearity (%), straightness (%), beat-cross frequency (hz), lateral head displacement (µm), wobbling index (%), dancing velocity (µm2/s) and dancing mean (µm2/s) of sperm were higher in Murrahs than in Gir semen with significant (p<0.01) difference at post-thaw stage. The fortification of extender with Mifepristone improved all these traits at post-thaw stage compared to other additives and control TFYG extender. The per cent decline in these traits due to freezing stress was much lower in buffalo sperm than the cattle sperm particularly with Mifepristone fortification. It was concluded that freezing-thawing stress adversely affected the motion and kinematics of both cattle and buffalo sperm and that Tris extender fortified with antioxidant additives, particularly Mifepristone @ 10 µg/ml, protected sperm from adverse effect of dilution and cryopreservation with improved post-thaw sperm quality.
The evaluation of male fertility potential essentially involves semen analysis and conception rates in inseminated females (Verstegen et al., 2002). However, there is no single test or a combination of tests that has been proved to be totally reliable for accurate prediction of semen quality and fertility. The conventional evaluation of bovine semen is relatively imprecise, time consuming and subjected to individual variations (Christensen et al., 2005; Patel et al., 2012; Pathak et al., 2020), while computer assisted sperm analysis (CASA) is a quite faster, precise and objective tool to identify differences in sperm motility and velocity/ kinematics attributes and avoids subjective errors (Johnson et al., 1996; Amann and Waberski, 2014; Patel and Dhami, 2016; Kumar et al., 2018). In recent years, there is increasing interest in evaluating sperm motion characteristics and kinematics by CASA (Amann and Waberski, 2014; Rodriguez-Montana and Roa-Guerrero, 2017), which covers different velocities of sperm movement (Hoflack et al., 2007; Pathak et al., 2020) and thus providing quantitative assessment of sperms. However, the literature on use and application of CASA in bovine semen laboratory is meager in India (Ramachandran et al., 2007; Patel et al., 2010), particularly with respect to simultaneous evaluation of fresh and frozen-thawed semen (Mandal et al., 2003; Patel and Dhami, 2013, 2016; Kumar et al., 2018; Patel et al., 2020; Pathak et al., 2020), following the use of different extender-additives (Tardif et al., 1997; Lackey et al., 1998; Perumal et al., 2011; Dorji et al., 2016; Dalal et al., 2019). Hence, this study was planned to evaluate and compare the motion characteristics/kinematics of spermatozoa of fresh and frozen-thawed semen of Gir and Murrah bulls using Biovis CASA in standard TFYG extender without and with antioxidant additives Mifepristone, Sericin and Taurine to assess the influence of cryopreservation process and extender additives on these traits.
This study was carried out on semen of three healthy mature breeding bulls each of Gir and murrah breed, aged between 5-8 years, at the College of Veterinary Science, AAU, Anand, Gujarat from September to March 2019-20. All the bulls were in good health, dewormed and vaccinated against common contagious diseases. They were maintained in nearly identical nutritional and managerial conditions with twice a week semen collection schedule. Semen was collected using artificial vagina from each bull in the morning hours over a dummy bull. Ejaculates (n=21 per breed) with >75% initial motility were split into four aliquots and were extended at 34°C @ 100 million sperm per ml with Tris-citric acid-fructose-egg yolk-glycerol (TFYG) extender without and with Mifepristone (10 µg/ml, RU-486@, Sigma-Aldrich, USA), Sericin (5 mg/ml, Sigma-Aldrich, USA) and Taurine (4 mg/ml, Central Drug House Ltd., India) as antioxidant additives.
       
The extended aliquots were soon filled and sealed in French mini straws by IS4 machine, cooled to 5°C, equilibrated for 4 hrs and frozen in liquid nitrogen vapour using a bio-freezer (IMV, France) employing standard freezing protocol. The straws were thawed in water bath at 37°C for 30 sec to obtain frozen-thawed samples. The freshly diluted as well as frozen-thawed samples of each ejaculate were assessed for sperm motion and kinematics characteristics by Biovis CASA (Expert Vision Lab Pvt Ltd, Mumbai) as per the manufacturer’s instruction and procedure adopted by Pathak et al., (2020). The CASA traits studied included total motile sperm as well as rapid, slow and non-progressive motile sperm, immotile sperm, average path velocity (VAP), curvilinear velocity (VCL), straight line velocity (VSL), straightness (STR), linearity (LIN), beat-cross frequency (BCF), amplitude of lateral head displacement (ALH), wobbling index (WOB), dancing frequency (DNC) and dancing mean (DNM). 
       
The data on fresh and frozen-thawed semen were analyzed statistically to derive mean±SE and one way ANOVA was performed using SPSS software version 20.00 for each breed. The means were compared between extender-additives by Duncan’s NMRT and those between breed and between fresh and post-thaw stage within breed by paired ‘t’ test. The per cent changes in post-thawed samples over pre-freeze values (plus or minus) were calculated using descriptive statistics for significance at p<0.05 (Snedecor and Cochran, 1994).
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.
 

Table 1: Sperm motility parameters (Mean±SE) of fresh and frozen-thawed semen of Gir cattle and Murrah buffalo bulls assessed by Biovis CASA in Tris extender without and with different additives.


 

Table 2: Sperm path velocity (Mean±SE) parameters of total motile sperms of fresh and frozen-thawed semen of Gir cattle and Murrah buffalo bulls assessed by Biovis CASA in Tris extender without and with different additives.


 

Table 3: Sperm kinematics parameters (Mean±SE) of total motile sperms of fresh and frozen-thawed semen of Gir cattle and Murrah buffalo bulls assessed by Biovis CASA in Tris extender without and with different additives.


 

Fig 1: Motility rating of freshly diluted and frozen-thawed sperm of Gir and Murrah bulls semen in control TFYG extender and TFYG supplemented with Mifepristone, Sericin and Taurine assessed by biovis CASA.


 

Fig 2: Velocity parameters of sperm assessed by Biovis CASA in freshly diluted and frozen-thawed semen of Gir and Murrah bulls in control TFYG extender and TFYG supplemented with Mifepristone, Sericin and Taurine.


 
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, µm2/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, µm2/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.
From the results of the present study based on evaluation by Biovis CASA of freshly diluted and frozen-thawed semen of Gir and Murrah bulls cryopreserved with TFYG extender without and with Mifepristone (10 µg/ml), Sericin (5 mg/ml) and Taurine (4 mg/ml), it was concluded that freezing-thawing stress adversely affected the motion and kinematics/velocity of both cattle and buffalo sperm and that Tris extender fortified with antioxidant additives, particularly Mifepristone @ 10 µg/ml, protected the sperm from adverse effect of dilution and cryopreservation with improved post-thaw sperm quality.
Authors are thankful to the Dean of the College and authorities of Anand Agricultural University, Anand for providing the necessary facilities for conducting the trials.

  1. Abbas, A. andrabian, S.M.H. and Ahmad, N. (2001). Effect of reducing the number of sperm cells (per insemination), increasing energy and cryoprotecting concentrations on motion characteristics and membrane integrity in frozen thawed buffalo spermatozoa. Pakistan Veterinary Journal. 21(3): 131-135.

  2. Amann, R.P. and Waberski, D. (2014). Computer-assisted sperm analysis (CASA): Capabilities and potential developments. Theriogenology. 81: 5-17.

  3. Anand, M. and Yadav, S. (2016). Assessment of motion and kinematic characteristics of frozen-thawed Sirohi goat semen using computer-assisted semen analysis. Veterinary World. 9(2): 203-206.

  4. Anderson, M., Hellman, T., Holmstrom, B.G. and Jokinen, L. (1992). Computerized and subjective assessments of post-thaw motility of semen from Finnish Ayrshire AI bulls in relation to non-return rates. Acta Veterinaiar Scandinavica. 33(1): 89-93.

  5. Arakeri, S., Tandle, M.K., Vinay, P.T., Bijurkar, R.G., Suranagi, M.D., Rao, J. and Kulkarni, S. (2020). Evaluation of sperm velocity parameters with glutathione and honey in skim milk based extenders by CASA on Boer buck. Indian Journal of Veterinary Science and Biotechnology. 15(4): 34-37.

  6. Christensen, P., Boelling, D., Pedersen, K.M., Korsgaard, I.R. and Jensen, J. (2005). Relationship between sperm viability as determined by flow cytometry and nonreturn rate of dairy bulls. Journal of Andrology. 26: 98-106.

  7. Dalal, J., Kumar, P., Chandolia, R.K., Pawaria, S., Rajendran, R., Sheoran, S. and Kumar, D. (2019). A new role for RU486 (mifepristone): It protects sperm from premature capacitation during cryopreservation in buffalo. Scientific Reports. 9(1): 1-10.

  8. Dorji, P., Vongprolub, T., Pattarajinda, V., Chankitisakul, V. and Chuawongboon, P. (2016). Effect of sericin supplement in Tris-egg yolk citrate extender on quality of cryopreserved semen from Thai native bulls. Bhutan Journal of Natural Resources and Development. 2(1): 11-18.

  9. Hoflack, G., Opsomer, G., Rijsselaere, T., Van Soom, A., Maes, D., Kruif de, A. and Duchateau, L. (2007). Comparison of computer assisted sperm motility analysis parameters in semen from Belgian Blue and Holstein-Friesian bulls. Reproduction in Domestic Animals. 42: 153-161.

  10. Johnson, J., Boone, W. and Blackhurst, D. (1996). Manual versus computer-automated semen analysis. Part I. Comparison of counting chambers. Fertility and Sterility. 65: 150-155.

  11. Karthikeya, G. (2003). Computer Automated Motility and Morphometric Analysis of Bull and Buck Spermatozoa. MVSc Thesis, Tamil Nadu Veterinary and Animal Science University, Chennai, India.

  12. Kathiravan, P., Kalatharan, J., Edwin, M.J. and Veerapandian, C. (2008). Computer automated motion analysis of crossbred bull spermatozoa and its relationship with in vitro fertility in zona-free hamster oocytes. Animal Reproduction Science. 104: 9-17.

  13. Kumar, S., Kumar, A., Singh, A.K., Honparkhe, M., Singh, P. and Malhotra, P. (2018). Improvement in post-thaw semen quality by minimizing the lipid peroxidation following herbal treatment in sub fertile buffalo bulls. The Pharma Innovation. 7(5, Part D): 240.

  14. Lackey, B.R., Boone, W.R., Grayand, S.L.L. and Henricks, D.M (1998). Computer-assisted sperm motion analysis of bovine sperm treated with insulin-like growth factor i and ii: implications as motility regulators and chemokinetic factors. Achieve of Andrology. 41: 115-125.

  15. Mandal, D.K., Nagpaul, P.K. and Gupta, A.K. (2003). Motion characteristics of Murrah buffalo bull spermatozoa in various seasons and its relationship with functional integrity of the plasmallema. Theriogenology. 60: 349-358.

  16. Patel, J.B. and Dhami, A.J. (2013). Computer assisted sperm analysis of fresh and frozen-thawed HF x Kankrej (F1) bulls’ semen and their interrelationship. Indian Journal of Animal Research. 47: 315-320.

  17. Patel, J.B. and Dhami, A.J. (2016). Computer assisted sperm analysis of fresh and frozen-thawed buffalo semen and their interrelationship. Indian Journal of Animal Research. 50(1): 8-13.

  18. Patel, J.B., Dhami, A.J., Patel, P.A. and Savalia, F.P. (2012). Assessment of motility and velocity parameters of freshly ejaculated spermatozoa of Bubalus bubalis and Bos indicus bulls through computer assisted semen analyzer. Indian Journal of Field Veterinarians. 8(1): 18-22.

  19. Patel, N.P., Patel, S.H. and Devanand, C.P. (2010). Comparative studies on the assessment of the bovine sperm motility by conventional method and CASA System (HTM IVOS). Proceedings of XXVI Annual Convention of ISSAR and International Symposium. GBPUA and T, Pantnagar, 10-12 Nov., p. 11.

  20. Patel, T.M., Dhami, A.J., Chaudhari, D.V. and Hadiya, K.K. (2020). Inclusion of sericin in tris egg yolk extender improves bovine sperm quality during cryopreservation (-196oC). International Journal of Livestock Research. 10(7): 118-125.

  21. Pathak, P.K., Dhami, A.J., Chaudhari, D.V. and Patel, J.A. (2019b). Motion and kinematics parameters of frozen-thawed bovine spermatozoa assessed by Biovis CASA. Indian Journal of Dairy Science. 72(3): 302-306.

  22. Pathak, P.K., Dhami, A.J., Chaudhari, D.V. and Hadiya, K.K. (2019a). Motion characteristics and kinematics of fresh spermatozoa of Gir, Surti and Murrah bulls assessed by computer assisted semen analyzer. Indian Journal of Veterinary Science and Biotechnology. 14(3): 14-19.

  23. Pathak, P.K., Dhami, A.J., Chaudhari, D.V. and Hadiya, K.K. (2020). Comparative evaluation of motility and kinematics of fresh versus frozen-thawed spermatozoa of cattle and buffalo bull by CASA. Indian Journal of Animal Research. 54(10): 1188-1194.  

  24. Perumal, P., Selvaraju, S., Selvakumar, S., Barik, A., Mohanty, D., Das, S., Das, R. and Mishra, P. (2011). Effect of pre- freeze addition of cysteine hydrochloride and reduced glutathione in semen of crossbred Jersey bulls on sperm parameters and conception rates. Reproduction in Domestic Animals. 46(4): 636-641.

  25. Ramachandran, N., Verma G.S., Gupta, A.K., Raina, V.S., Mohanty, T.K. and Singh, M.K. (2007). Prediction of fertility based on frozen-thawed seminal traits in Sahiwal bulls. Indian Journal of Animal Sciences. 77(8): 884-887.

  26. Rasul, Z., Ahmad, N. and Anzar, M. (2001). Changes in motion characteristics, plasma membrane integrity and acrosome morphology during cryopreservation of buffalo spermatozoa. Journal of Andrology. 22: 278-283.

  27. Rodriguez-Montana, D. and Roa-Guerrero, E. (2017). Objective assessment of bull sperm motility parameters using computer vision algorithms. African Journal of Biotechnology. 16(37): 1871-1881.

  28. Snedecor, G.W. and Cochran, W.G. (1994). Statistical Methods. 14th edn. Oxford and IBH Publishing House, New Delhi, India.

  29. Tardif, A.L., Farrell, P.B., Trouern-Trend, V. and Foote, R.H. (1997). Computer-assisted sperm analysis for assessing initial semen quality and changes during storage at 5oC. Journal of Dairy Science. 80: 1606-1612.

  30. Verstegen, J., Iguer-Ouada, M. and Onclin, K. (2002). Computer assisted semen analyzers in andrology research and veterinary practice. Theriogenology. 57(1): 149-179.

Editorial Board

View all (0)