Computer assisted sperm analysis of fresh and frozen-thawed buffalo semen and their interrelationship

2016 ◽  
Vol 50 (1) ◽  
Author(s):  
J. B. Patel ◽  
A. J. Dhami

Sixty semen ejaculates from 10 mature bulls, 5 each of Jafarabadi and Mehsana breed, were studied for sperm motility and velocity parameters of fresh and frozen-thawed spermatozoa using computer assisted sperm analyzer (CASA). The mean values of motile and progressively motile spermatozoa observed in fresh semen of Jafarabadi and Mehsana bulls (79.77±1.62 and 61.80±1.85, and 78.90±1.22 and 61.37±1.58%) were highly significantly (P<0.01) reduced (51.20±1.57 and 33.20±1.45, and 52.10±1.70 and 34.30±1.54 %, respectively) in post-thawed semen. The average path velocity, straight line velocity and curvilinear velocity (μm/sec) of spermatozoa of Jafarabadi and Mehsana bulls noted in fresh semen were also reduced highly significantly (P<0.01) in frozen-thawed semen. Among the other velocity parameters, amplitude of lateral head displacement (μm), elongation (%) and medium motile sperm (%) increased, while beat-cross frequency (Hz), straightness (%), linearity (%), sperm area (μm<sup>2</sup>) and rapidly motile sperm (%) decreased significantly in post-thawed sperms when compared with the fresh sperm of both Jafarabadi and Mehsana bulls. The initial motility and live sperm per cent were significantly correlated with CASA traits of fresh and frozen-thawed semen, and all the sperm motility and velocity traits of fresh and frozen-thawed semen assessed by CASA were significantly interrelated among both the breeds. The interrelationships were stronger in Mehsana bulls as compared to Jafarabadi bulls.

Author(s):  
P K Pathak ◽  
A J Dhami ◽  
D V Chaudhari ◽  
K K Hadiya

A study was undertaken on semen of three mature bulls each of Gir, Surti and Murrah breed to evaluate the comparative motion characteristics and kinematics of their fresh and frozen-thawed spermatozoa by Biovis CASA. The ejaculates (n= 24/breed) having >75% initial motility were diluted @ 80 million sperm/ml using TFYG extender and were assessed. Amongst motility traits, the total motile, rapid progressive motile and slow progressive motile spermatozoa percentage decreased significantly by 23.08 - 30.09, 43.57 - 55.18, 9.12 - 22.75 %, respectively, plessthan0.01), while non-progressive motile sperm (4.78 - 21.48%) and immotile sperm (164.38 - 178.38 %) percentage increased significantly ( plessthan0.01) in frozen-thawed semen compared to that of fresh semen. The post-thaw quality of semen of all three breeds was in acceptable range. The mean values of sperm velocity/kinematic parameters observed in frozen-thawed semen of Gir, Surti and Murrah bulls, based on total motile sperms, viz., average path velocity, curvilinear velocity, straight line velocity, linearity, straightness, beat-cross frequency, amplitude of lateral head displacement, dancing frequency and dancing mean decreased significantly by 13.70 - 17.79; 9.76 - 12.95; 13.28 - 21.90; 7.28 - 9.68; 4.36 - 7.79; 15.56 - 25.15; 8.78 - 10.50; 6.16 - 18.67 and 12.98 - 15.96 %, respectively, as compared to that of their fresh semen samples. However, wobbling index remained almost same for both fresh and frozen semen. All motility traits differed but none of kinematics/velocity traits differed significantly between breeds/species. The values of all velocity parameters for progressive motile sperms were higher than total motile sperms in all three breeds. The effect of freezing-thawing on velocity and kinematic attributes was much less compared to absolute sperm motility, and both the fresh and frozen-thawed sperms behaved identically with respect to their velocity and kinematics. The rapid progressive motile sperm in both fresh (r=0.41 to 0.92) and frozen-thawed (r=044 to 0.88) semen had significant correlations with most of their velocity traits, and the later were significantly and positively or negatively inter-related among each other in semen of all three breeds. It was therefore concluded that cryopreservation process significantly reduces the motility and kinematics attributes of bovine spermatozoa and, CASA analysis of fresh semen for motility and velocity traits could predict the post-thawed sperm motility and velocity/ kinematics of spermatozoa.


Animals ◽  
2020 ◽  
Vol 10 (11) ◽  
pp. 1993
Author(s):  
Sabrina Gacem ◽  
Jaime Catalán ◽  
Anthony Valverde ◽  
Carles Soler ◽  
Jordi Miró

In order to optimize the donkey sperm motility analysis by the CASA (Computer Assisted Sperm Analysis)-Mot system, twelve ejaculates were collected from six jackasses. Capillary loaded chamber (CLC), ISAS®D4C depths 10 and 20 µm, ISAS®D4C Leja 20 and drop displacement chamber (DDC), Spermtrack® (Spk) depths 10 and 20 µm were used. Sperm kinematic variables were evaluated using each chamber and a high-resolution camera capable of capturing a maximum of 500 frames/second (fps). The optimum frame rate (OFR) (defined according to curvilinear velocity—VCL) was dependent on chamber type. The highest OFR obtained was 278.46 fps by Spk20. Values for VCL, straight-line velocity (VSL), straightness (STR), amplitude of lateral head displacement (ALH) and beat cross frequency (BCF) were high in DDC and 10 µm depth. In both DDC 10 and 20 µm, the sperm velocities (VCL, VSL, VAP) and ALH values decreased significantly from the centre to the edges, while Wobble and BCF increased. No defined behavior was observed along the CLC. However, all the kinematic variables had a higher value in a highly concentrated sample, in both chamber types. In conclusion, analyzing a minimum of nine fields at 250 fps from the centre to the edges in Spk10 chamber using a dilution of 30 × 106 sperm/mL offers the best choice for donkey computerised sperm motility analysis.


2019 ◽  
Vol 15 (01) ◽  
pp. 9-13
Author(s):  
P K Pathak ◽  
A J Dhami ◽  
D V Chaudhari

This investigation was carried out on semen of three healthy mature breeding bulls of Gir breed to evaluate the interrelationships among sperm quality attributes of fresh and frozen-thawed semen assessed by Biovis CASA. The ejaculates (n = 24) having >75% initial motility were diluted @80 million sperm/mL using TFYG extender, filled in French mini straws, and were frozen using a programmable bio freezer after 4 hours of equilibration. The straws were thawed in a water bath at 37°C for 30 sec. The freshly diluted and frozenthawed samples were assessed for routine subjective tests and various motion characteristics/kinematics by Biovis CASA. The Pearson’s correlations for sperm motility and velocity/kinematic parameters of total motile sperm as well as of progressively motile sperm were studied in freshly diluted and frozen-thawed semen. In fresh semen, total motile sperm assessed by CASA had significant (p less than 0.05, 01) correlations with rapid progressive motile sperm (r = 0.46), wobbling index (r = 0.52) and dancing frequency (r = -0.43) in fresh semen. In frozen-thawed semen, it was significantly correlated only with linearity (r = 0.46). The rapid progressive motile sperm in both fresh (r = 0.41 to 0.92) and frozen-thawed (r = 044 to 0.88) semen, however, had significant correlations with most of their velocity traits. Further, the average path velocity (VAP), curvilinear velocity (VCL), straight line velocity (VSL), linearity (LIN), straightness (STR), wobbling (WOB), beat-cross frequency (BCF), amplitude of lateral head displacement (ALH), and dancing mean (DNM) of sperm showed significant positive or negative interrelationships among each other in both fresh (r = 0.41 to 0.91) as well as post-thawed (r = 0.44 to 0.90) semen. Moreover, the correlations of motility and kinematics parameters of total motile sperm in both fresh and frozen-thawed semen were highly significant with velocity/kinematics traits of only progressively motile sperm, and the velocity traits among only motile sperm were highly significantly interrelated in both fresh (r = 0.46 to 0.98) and frozen-thawed (r = 0.43 to 0.93) semen of Girbulls, though the magnitudes of correlations were lower in frozen-thawed semen as compared to fresh semen. Thus, CASA analysis offresh semen for motility and velocity traits could predict the post-thawed sperm motility and velocity/kinematics of bovine semen.


Author(s):  
A.J. Dhami ◽  
P.K. Pathak ◽  
D.V. Chaudhari ◽  
K. K. Hadiya

A study was carried out on semen of nine breeding bulls, three each of Gir, Surti and Murrah breeds, to evaluate the comparative motion characteristics and kinematics of their fresh spermatozoa by CASA. The ejaculates (n=72, 24 of each breed) having >75% initial motility were diluted @ 80 million sperm/ml using TFYG extender and were assessed for motion characteristics by CASA. The overall mean values of rapid motile and immotile sperm per cent were observed significantly greater in Gir bulls semen, while total motile and slow motile sperms were apparently higher in buffalo semen. The mean values of sperm velocity/ kinematic parameters observed based on all motile sperms in Gir, Surti and Murrah bulls semen were: average path velocity 50.01±1.25, 48.51±1.03 and 49.14±1.30 μm/s; curvilinear velocity 88.62±1.66, 87.90 ±1.74 amd 88.93±1.69 μm/s, straight line velocity 44.51±1.35, 43.14±1.12 amd 41.73±2.24 μm/s; linearity 50.06±1.42, 48.86±1.32 amd 48.49±1.84 %; straightness 85.17±0.92, 84.97±0.88 and 83.90±1.17 %; wobbling index 57.00±1.17, 55.32±1.05 and 55.30±1.48 %; beat-cross frequency 15.55±0.58, 16.14±0.43 and 14.97±0.54 hz; amplitude of lateral head displacement 2.39±0.18, 2.57±0.12 and 2.31±0.14 μm; dancing frequency 208.34±15.52,225.00 ±10.74 and 211.29±13.03 μm2/s, and dancing mean 5.70±0.46, 6.33±0.35 and 6.33±0.50 μm2/s, respectively. Almost similar trend with little higher values were noted for sperm velocity/kinematics based on only progressively motile sperm, without breed/species variation. The semen of all three breeds behaved identically for sperm kinematics. The bull variation was insignificant for all the traits in all the three breeds.


2021 ◽  
Vol 33 (2) ◽  
pp. 116
Author(s):  
Y. Pirosanto ◽  
A. Molina ◽  
M. Valera ◽  
J. Dorado ◽  
E. Terán ◽  
...  

Reproductive performance is one of the key factors in livestock production. It is well known that reproductive traits are influenced by several genetic factors, such as the increase of individual inbreeding levels, which are associated with changes in sperm motility and shape in several species. In horses, the increase in inbreeding is a common problem because of the reduction in effective population size and the increase in selection intensity observed in several breeds. However, studies assessing the effect of high levels of inbreeding on the sperm quality of stallions are scarce. In the present study, we aimed to determine the effect of increased inbreeding levels and age on the sperm motility patterns of Purebred Spanish horses (PRE). We performed kinetic characterisation of 557 sperm samples of 82 PRE stallions aged between 3 and16 years, using computer-assisted sperm analysis (Androvision™, Minitube). We evaluated 5 parameters in 6 different fields per sample: curved line velocity (VCL, µm/s), velocity average path (VAP, µm/s), velocity straight line (VSL, µm/s), amplitude of lateral head displacement (ALH, µm), and beat-cross frequency (BCF, Hz). We determined the pedigree-based inbreeding coefficient (Fped) based on ∼300,000 PRE pedigree records to evaluate the inbreeding effect. Individuals were separated into 2 groups: highly inbred (n=339) and lowly inbred (n=218) according to an F value of 12.5%. Differences between groups were analysed using a generalized linear model. The analysis did not show significant differences (P&gt;0.05) in the variables analysed with respect to the age of stallions. However, VAP, VCL, and AHL were lower in highly inbred than in lowly inbred animals (P&lt;0.05), suggesting less velocity and amplitude of head displacement. In the case of BCF, no significant differences (P&gt;0.05) were observed between the two study groups. In conclusion, age did not affect sperm quality parameters in the age group of stallions analysed. In addition, we demonstrated that high inbreeding coefficient reduced the mean velocity and trajectory pattern of spermatozoa in PRE.


2018 ◽  
Vol 63 (No. 11) ◽  
pp. 429-434
Author(s):  
Zoltán Bokor ◽  
Balázs Csorbai ◽  
Levente Várkonyi ◽  
Zsolt Szári ◽  
Ferenc Fodor ◽  
...  

The effects of a simple saline solution prepared using two different pH (4.4 and 8.5) on sperm motility in burbot were investigated. Results were recorded during a 96-hour chilled storage (4°C) in 24-hour intervals. Measurements were focused on the detailed characteristics of motility using 12 parameters obtained from the Computer-assisted Sperm Analysis (CASA). Significantly higher progressive motility (pMOT), distance average path (DAP), distance curved line, distance straight line (DSL), average path velocity (VAP), curvilinear velocity, straight line velocity, and beat cross frequency (BCF) were observed with the activating solution buffered at pH 8.5 in comparison with pH 4.4. Already after 24 h a significant reduction was measured in pMOT (0 h: 49 ± 24%, 24 h: 12 ± 7%). Similar decreasing tendency was recorded only after 72 h in DAP (0 h: 26 ± 4 µm/s, 72 h: 19 ± 9 µm/s), DSL (0 h: 21 ± 5 µm/s, 72 h: 17 ± 8 µm/s), VAP (0 h: 59 ± 9 µm/s, 72 h: 43 ± 21 µm/s), and BCF (0 h: 28 ± 2 Hz, 72 h: 18 ± 10 Hz). The response of different investigated CASA parameters to different treatments varied in our experiments. According to our studies, numerous burbot sperm motility parameters are sensitive to chilled storage and to low pH of the activating solution. Our results could support the effective sperm quality assessment and successful artificial propagation process in burbot.


2013 ◽  
Vol 25 (1) ◽  
pp. 183 ◽  
Author(s):  
I. Ortiz ◽  
J. Dorado ◽  
D. Acha ◽  
L. Ramirez ◽  
M. Urbano ◽  
...  

Single-layer centrifugation (SLC) with EquipureTM Bottom Layer has been used to enhance the quality of stallion semen samples; however, no studies have been performed on donkeys. The aim of this study was to determine if SLC with EquipureTM Bottom Layer improves kinematic parameters on frozen–thawed donkey sperm. Semen was collected from 4 Andalusian donkeys by artificial vagina. Three ejaculates from each donkey were centrifuged with EquiproTM, supernatant was removed, and pellet was re-extended in the freezing medium GentTM to a final concentration of 200 × 106 spermatozoa per milliliter. Sperm were slowly cooled to 5°C for 2 h, loaded in 0.5-mL plastic straws, and frozen in liquid-nitrogen vapors. After at least one week of storage, straws were thawed in a water bath at 37°C for 30 s. After thawing, semen samples were divided in 2 aliquots: aliquot 1 was used as such (control) and aliquot 2 was processed by SLC using EquipureTM Bottom Layer. Computer-assisted sperm analysis was performed, and sperm kinematics total motility (%), progressive motility (%), curvilinear velocity (VCL; µm s–1), velocity straight line (VSL; µm s–1), velocity average path (VAP; µm s–1), linearity (LIN; %), straightness (STR; %), wobble (WOB; %), lateral head displacement (ALH; µm), and beat cross frequency (BCF; Hz) were statistically compared using GLM model between frozen–thawed semen samples processed or not with EquipureTM. Results were expressed as mean ± standard error. Significant differences (P < 0.05) were found between SLC-selected and unselected semen for total motility (77.44 ± 5.83 v. 58.89 ± 6.07), progressive motility (76.88 ± 4.52 v. 56.59 ± 5.44), VCL (137.50 ± 0.75 v. 133.0 ± 0.99), LIN (69.43 ± 0.31 v. 68.23 ± 0.41), STR (78.45 ± 0.29 v. 76.90 ± 0.37), WOB (85.06 ± 0.18 v. 83.91 ± 0.26), ALH (2.76 ± 0.01 v. 2.44 ± 0.01), and BCF (9.13 ± 0.05 v. 8.53 ± 0.06), respectively. No significant differences were observed for VSL (102.89 ± 0.70 v. 104.32 ± 0.95) and VAP (123.21 ± 0.71 v. 121.50 ± 0.98). Most of the computer-assisted sperm analysis parameters used in the present study have been previously identified as reliable markers of sperm motility in relation to sperm quality and fertility. It has also been reported that VCL appears to be critical for the formation of the sperm reservoir and penetration of the zona pellucida. In addition, other variables improved in the SLC-selected samples have been described as measure of progressivity (LIN, STR) and spermatozoa vigor (BCF, ALH). These preliminary results suggest an additional option for improving sperm quality in donkey semen doses. In conclusion, SLC with EquipureTM can be used to enhance kinematic parameters on frozen–thawed donkey sperm.


2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Nikolas Zuchowicz ◽  
Jonathan Daly ◽  
Jessica Bouwmeester ◽  
Claire Lager ◽  
E. Michael Henley ◽  
...  

AbstractThe declining reproductive viability of corals threatens their ability to adapt to changing ocean conditions. It is vital that we monitor this viability quantitatively and comparatively. Computer-assisted sperm analysis (CASA) systems offer in-depth analysis used regularly for domestic and wildlife species, but not yet for coral. This study proposes quality control procedures and CASA settings that are effective for coral sperm analysis. To resolve disparities between CASA measurements and evaluations by eye, two negative effects on motility had to be resolved, slide adhesion (procedural) and sperm dilution (biological). We showed that the addition of bovine serum albumin, or caffeine, or both to fresh sperm reduced adhesion in the CASA cassettes, improved motility and motile sperm concentration (P < 0.0001), yet these additions did not affect measurements of total sperm concentration. Diluting coral sperm reduced sperm motility (P = 0.039), especially from heat-stressed corals. We found CASA concentration counts comparable to haemocytometer and flow cytometer measures (P = 0.54). We also found that motile sperm per egg is a useful predictor of fertilisation success, using cryopreserved sperm. Standard measurements of coral reproductive characteristics inform our understanding of the impacts of climate change on reef populations; this study provides a benchmark to begin this comparative work.


2019 ◽  
Vol 31 (1) ◽  
pp. 142
Author(s):  
M. A. Lagares ◽  
N. C. Alves ◽  
A. L. A. Guimaraes ◽  
S. B. Luz ◽  
S. A. Diniz ◽  
...  

The pattern of sperm transport and survival in the mare’s reproductive tract is different between fresh and frozen-thawed semen. A probable reason for this difference is the biophysiological changes in sperm during cryopreservation of equine semen. These changes can impair motility of stallion sperm after thawing. The aim of the present work was to test the effect of different caffeine concentrations on stallion sperm motility after thawing. One ejaculate of 9 stallions was frozen with the INRA82 frozen extender, and after thawing, different caffeine concentrations were added to the semen samples according to the treatments: control INRA82 without caffeine addition (T1), T1+1mM caffeine (T2), T1+2mM caffeine (T3), T1+3mM caffeine (T4), T1+5mM caffeine (T5), T1+7.5mM caffeine (T6), and T1+10mM caffeine (T7). The analysis of sperm motility parameters was performed with a computer-assisted semen analyser in 4 time periods: immediately after semen samples thawing (t0) and 15min (t15), 30min (t30), and 40min (t40) after semen sample thawing. One semen sample of each treatment was thawed, and an aliquot was analysed for the following computer-assisted semen analysis characteristics: velocity curvilinear (VCL; µm s−1), velocity straight line (µm s−1), velocity average path (µm s−1), linearity (%), straightness (%), wobble (%), amplitude of lateral head displacement (µm), beat cross frequency (BCF; Hz), and percentage of total sperm motility (TM) and progressive sperm motility. The statistical analysis was performed with ANOVA and Duncan’s test. The sperm parameters progressive sperm motility, linearity, wobble, and amplitude of lateral head displacement did not differ among the treatments (P&gt;0.05). Immediately after addition (t0) of 5, 7.5, and 10mM caffeine concentrations, an increase of TM was observed (T5: 53.1%; T6: 45.9%; and T7: 47.4%) compared with the other treatments (T1: 37.5%; T2: 36.0%; T3: 36.6%; and T4: 32.3%; P&lt;0.05). Although after 15min of incubation (t15) the TM decreased compared with t0 in T5, T6, and T7 treatments, the percentage was comparable with the other treatments at t15, t30, and t40. The mean value for TM was higher with 5mM caffeine compared with the control group (38.6% v. 34.7%; P&lt;0.05), whereas for the 10mM caffeine treatment velocity straight line (19.9v. 17.1µm s−1), velocity average path (25.6v. 22.9µm s−1), and straightness (75.4v. 72.3%) were higher than the control (P&lt;0.05). For the 5, 7.5, and 10mM caffeine treatments, VCL and BCF were higher than the control (VCL: 33.9, 34.5, 36.8, and 31.5µm s−1, respectively; BCF: 8.1, 8.6, 9.0, and 7.2Hz, respectively). The remaining motility parameters did not differ until 40min after the treatment (P&lt;0.05). In conclusion, the addition of 5, 7.5, and 10mM caffeine concentrations after semen thawing increased TM and most of the sperm motility characteristics. However, given the complexities of sperm transport, capacitation, and so on, further experiments are needed to test whether caffeine treatments could be used to improve the fertilization rate of frozen-thawed equine semen.


2014 ◽  
Vol 2014 ◽  
pp. 1-6 ◽  
Author(s):  
P. Perumal ◽  
S. K. Srivastava ◽  
S. K. Ghosh ◽  
K. K. Baruah

The present study was undertaken to assess the motility and velocity parameters of sperm of freezable and nonfreezable ejaculates by computer-assisted sperm analyser (CASA) such as Hamilton-Thorne Semen Analyser IVOS 11 in mithun semen. Fifty ejaculates (twenty-five ejaculates each for freezable and nonfreezable semen ejaculates) were collected from ten matured mithun bulls. CASA parameters, motility parameters such as forward progressive motility (FPM) (%), nonprogressive motility (NPM) (%), total motility (TM) (%), and static sperms (SM) (%); velocity parameters such as curvilinear velocity (VCL) (μm/sec), straight line velocity (VSL) (μm/sec), average path velocity (VAP) (μm/sec), linearity (LIN) (%), straightness (STR) (%), wobble (WOB) (%), amplitude of lateral head displacement (ALH) (μm), and beat/cross-frequency (BCF) (Hz) were measured by CASA analyser. The result revealed that these parameters varied significantly (P<0.05) between the freezable and nonfreezable ejaculates and freezable ejaculates have significantly (P<0.05) higher value than nonfreezable ejaculates. It was concluded that most of the CASA parameters were significantly lower in nonfreezable ejaculates than in freezable ejaculates in mithun and confirmed that the CASA was effective for a quick and objective analysis of motility and velocity parameters in mithun semen.


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